Building the Case that Aging is Controlled from the Brain

Last week, a new study came out fingering the hypothalamus as locus of a clock that modulates aging.  This encourages those of us who entertain the most optimistic scenarios for anti-aging medicine.  Could it be that altering the biochemistry of one tiny control center might effect global rejuvenation?  

First some background….

I believe that aging is governed by an internal biological clock, or several semi-independent and redundant clocks.  There are

  • A telomere clock, counting cell divisions on a flexible schedule, eventually producing cells with short-telomeres that poison us.
  • The thymus, crucial training ground for our white blood cells, shrinks through a lifetime.
  • An epigenetic clock alters gene expression over time in directions that give rise to self-destruction.
  • A neuroendocrine clock in the hypothalamus
  • Perhaps other clocks, yet to be identified.

 

A dream is to be able to reset the hands of the clock.  If we’re lucky, then changing the state of some metabolic subsystem will not just temper the rate at which we age, but actually restore the body to a younger state.  Most of the research in anti-aging medicine is still devoted to ways to engineer fixes for damage the body has allowed to accumulate; but I belong to a wild-eyed contingent that thinks the body can do its own fixing if we understand the signaling language well enough to speak the word “youth” in the body’s native biochemical tongue.

Some of these clocks are more accessible and easier to manipulate than others.  The epigenetic clock is most daunting, because it presents the spectre of a global network of signal molecules circulating in the blood, transcription factors that mutually support one another in a state of slowly-shifting homeostasis.  This system could be so complex that it might take decades to understand, and then hundreds of different signal molecules in the blood would need to be re-balanced in order to recreate homeostasis in a younger condition.  (For several years, the Mike and Irina Conboy have been looking for a small subset of molecules that might control the rest, but in a private conversation they recently told me they are less optimistic that a small number of factors controls all the rest.)

At the other end of the spectrum, the hypothalamic clock presents the most optimistic scenario.  It is tightly localized in a tiny region of the brain, and might be relatively easy to manipulate, with consequences that rejuvenate the entire body.  The hypothalamic clock hypothesis is an attractive target for research because, if correct, it will offer direct and straightforward control over the body’s metabolic age.

That aging unfolds according to an internal clock remains a controversial claim, but what everyone agrees is that the body has some way to know how old it is.  There has to be a clock for development that determines when growth surges and stops, when sex hormones turn on and, if it’s not too great a stretch, when fertility ends and menopause unfolds.

The clock that governs growth and development has yet to be elucidated—a major metabolic mystery by my lights.  The clock that we know about and (sort of) understand is the circadian day-night clock that governs sleep and waking, giving us energy at some times of the day but not others.

Is the life history clock linked to the circadian clock?  Maybe the body just counts days to tell how old it is?  This possibility was eliminated, at least for flies, using experiments with cycles of light and dark that were consistently longer or shorter than 24 hours.  Flies living with fast day-night cycles (less than 24 hours) lived shorter, as predicted; but flies living with long day-night cycles failed to have longer lifetimes,  In fact, deviation from 24 hours in either direction shorten the fly’s lifespan [2005].

But this study suggests the short-term clock and the long-term clock may be linked in a way that is less straightforward.  Melatonin may be another reason to expect a connection.  Melatonin is the body’s cue for sleep, and Russian studies have documented a role for melatonin in aging.  A third motivation comes from the fact that aging disrupts sleep cycles, and (in a downward spiral) disrupted sleep cycles are also a risk factor for mortality and diseases of old age.

Cells seem to have their own, built-in daily rhythms.  I want to say “transcriptional rhythms”, adding the idea that gene transcription is the locus of control; however, red blood cells are the counterexample—they exhibit daily cycles, even though they have no DNA to transcribe [2011].  Individual cycles are designed to be 24 hours, but they would soon drift out of phase with day and night if they weren’t centrally coordinated.  The reference clock that keeps the others in line is in the SCN, the suprachiasmatic nucleus, a handful of nerve cells in a neuroendocrine part of the brain called the hypothalamus.

Think of a million pendulums that are all tuned to swing with a period of 24 hours.  All that it takes is a tiny nudge to all these pendulums each day to keep them in phase with one another, so they are all swinging together.  The SCN provides this nudge in a smart way, based on information from the eyes (light and dark) and endocrine signals that indicate activity and sleep.  The SCN is upstream of the pineal gland, and supplies the signal that tells the pineal gland when it’s time to make melatonthematic index of scarsonatas.  The natural resonances of individual cells become entrained in a body-wide response.

 

What does all this have to do with aging?

Experiments in the 1980s and 90s showed that the SCN is related to annual cycles, but the relationship seems to be not as strong or as simple or as direct.  For example, squirrels in which the SCN was removed had no daily sleep-wake cycles at all, but their annual cycles of fertility and oscillations of weight were affected inconsistently, more in some animals than others.  Transplanting a SCN from young hamsters into old hamsters cut their mortality rate by more than half, and extended their life expectancies by 4 months [1998].

I have written in this column about research from the laboratory of Claudia Cavadas (U of Coimbra, near Lisbon) indicating that inflammation and inflammatory cytokines in the hypothalamus are at the headwaters of a cascade of signals that lead to whole-body aging.  They have emphasized the role of TGFß binding to ALK5 and of the neurotransmitter NPY.  We usually think of inflammation as a source of damage throughout the body, but in the hypothalamus, inflammation seems to have a role that is more insidious than this, with full-body repercussions.  Blocking inflammation in the hypothalamus is a promising anti-aging strategy.

New Paper on micro RNAs from the Hypothalamus

Along with Cavadas, Dongshen Cai (Einstein College of Medicine) has been a leader in exploring neuroendocrine control of aging that originates in the hypothalamus.  Several years ago, Cai’s group demonstrated that aging could be slowed in mice by inhibiting the inflammatory cytokine NF-kB and the related cytokine IKK-ß just in one tiny area of the brain, the hypothalamus.  “In conclusion, the hypothalamus has a programmatic role in ageing development via immune–neuroendocrine integration…”  They summarized findings from their own lab, suggesting that metabolic syndrome, glucose intolerance, weight gain and hypertension could all be exacerbated by signals from the inflamed hypothalamus.  In agreement with Cacadas, they identified GnRH (gonadotropin-releasing hormone) as one downstream target, and were able to delay aging simply by treatment with this one hormone.  IKK-ß is produced by microglial cells in the hypothalamus of old mice but not young mice.  Genetically modified IKK-ß knock-out mice developed normally but lived longer and retained youthful brain performance later in life.

In the new paper, Cai’s group identified micro-RNAs, secreted by the aging hypothalamus and circulating through the spinal fluid, that contribute to aging.  A small number of stem cells in the hypothalamus were found to keep the mouse young, in part by secreting these micro-RNAs.  Mice in which these stem cells were ablated had foreshortened life spans; old mice that were treated with implants of hypothalamic stem cells from younger mice were rejuvenated and lived longer.  A class of neuroendocrine stem cells from the third ventricle wall of the hypothalamus (nt-NSC’s) was identified as having a powerful programmatic effect on aging.  These cells are normally lost with age, and restoring these cells alone in old mice extended their life spans.

Exosomes are little packets of signal chemicals. Micro-RNAs from stem cells in the hypothalamus are collected into exosomes and shipped down through the spinal fluid.  These exosomes seem to constitute a feedback loop.  On the one hand, they are generated by the hypothalamic stem cells.  On the other hand, they play a role in keeping these same cells young, and producing more exosomes.

Life extension of about 12% was impressive given that there was just one intervention when the mice were more than 1½ years old, but of course it’s not what we would hope for if the master aging clock were reset.  For really large increases in lifespan, we will probably need to reset two or even three of the clocks at once.

 

The Bottom Line

The reason the body has multiple, redundant aging clocks is to assure that natural selection can’t defeat aging by throwing a single switch.  That means the clocks must be at least somewhat independent.  Nevertheless, I judge it is likely that there is some crosstalk among clocks, because that’s how biology usually works.  To effect rejuvenation, we will have to address all aging clocks, but we see some benefit from resetting even one, and expect more significant benefit from resetting two or more.

The most challenging target is the epigenetic clock,built on a homeostasis of transcription and signaling among hundreds of hormones that each affect levels of the others.  Reverse engineering this tangle will be a bear.

The idea of a centralized aging clock in the hypothalamus seems far more accessible, and is promising for the medium term.  Still, it does not suggest immediate application to remedies.  The hypothalamus is deep in the brain, and you and I might be reluctant to accept a treatment that required drilling through the skull.  A treatment based on circulating proteins and RNAs from the hypothalamus would be less invasive, but even that might have to be intravenous, and include some chemistry for penetrating the blood-brain barrier.  RNA exosomes seem to be our best opportunity

As Cavadas’s group has already pointed out, it is inflammation in the hypothalamus that is amplified by signaling to become most damaging to the entire body.  This raises the interesting question: could it be that the modest anti-aging power of NSAIDs is entirely due to their action within the brain?  In other words, maybe “inflammaging” is largely localized to the hypothalamus.

Sirtuin 6 Improves Healthspan In Mice

Previous research has demonstrated that overexpressing a protein known as SIRT6 increases lifespan of mice and offers protection against obesity-induced metabolic abnormalities. In contrast mice that lack SIRT6 suffer from various aging-related problems and die before the age of 4 weeks. SIRT6 is part of a class of proteins known as the sirtuins that have been well-studied for their anti-aging effects. Earlier this year we reported on a small molecule drug that could selectively activate SIRT6.

Now, two papers by the Cohen lab further investigate the effect of SIRT6 on life- and healthspan.

In the first paper the researchers developed a SIRT6 transgenic mouse that has extra copies of the SIRT6 gene (termed MOSES mice). Old MOSES mice had improved insulin sensitivity, reduced fasting glucose levels, increased physical activity, reduced inflammation of their fat tissue, and improved hair growth.

What about SIRT6 knockout?

In the second paper the authors investigate the effect of a complete lack (a so called knockout) of SIRT6. Such work had been done before but all mice died before four weeks of age. The authors wanted to investigate the effect of SIRT6 on health parameters later in the mouse lifespan. By changing the genetic background of the mice that carry the SIRT6 knockout the researchers were able to generate SIRT6 knockout mice that reach adulthood. Average lifespan of males was just 140 days while 80% of female mice were still alive after 200 days. Hence the effect of SIRT6 on lifespan is gender-specific. The authors succeeded in investigating the effect of certain health parameters up to 10 months of age.

By 5-6 months of age SIRT6 knockout mice showed severe corneal injury. The cornea is the transparent outer layer of the eye allowing light to pass into the eye. The age-related thinning of the retina, the eye layer where light is sensed, was accelerated in SIRT6 knockout mice. Furthermore, SIRT6 knockout mice also show degradation of retinal function.

Surprisingly the SIRT6 knockout mice had higher glucose uptake in skeletal muscle compared to normal mice. The authors showed that this glucose uptake was insulin-independent. The interpretation of this observation is unknown at this moment.

A link to neurodegeneration

Finally, in a third paper by another lab the effect of brain-specific SIRT6 knockout was investigated. Mice that lacked SIRT6 in the brain showed learning impairment at 4 months of age. The authors further discovered that such mice had higher levels of phosphorylated tau in their brains, a characteristic of several neurodegenerative diseases including Alzheimer’s disease. Hyperphosphorylated tau is believed to be neurotoxic and indeed mice that lacked SIRT6 had higher levels of cell death and DNA damage in their brains. SIRT6 levels in the brain decrease with age. Finally, the authors showed that Alzheimer’s disease patients had lower levels of SIRT6 protein in their brains.

A NAD+/PARP1/SIRT1 axis in Aging

 

NAD+ levels decline with age in diverse animals from C. elegans to mice [1] [2] [3] [4] [5] and raising NAD+ levels has apparent anti‐aging effects [3] [4] [2] [6] [5]. Increasing NAD+ levels by dietary supplementation with NAD+ precursors Nicotinamide Riboside (NR) or Nicotinamide Mononucleotide (NMN) improved mitochondrial function [4], and muscle, neural and melanocyte stem cell function in mice. NR supplementation also increased lifespan modestly in mice [5].

Although the mechanisms of gradual NAD+ decline with age remain unclear, beneficial effects have been linked to mitochondrial rejuvenation via restoration of OXPHOS subunits via sirtuin SIRT1/HIF1a/c‐myc pathway [4], and stem cell function via stimulation of the mitochondrial unfolded protein response (UPSmt) [5] and synthesis of prohibitins, a family of mitochondrial stress response proteins linked to senescence of fibroblasts in mammals. Improvement of stem cell function by NAD+ is SIRT1 dependent[5].

NAD+ levels are significant for metabolic homeostasis in that they are important for redox reactions, and as substrates of sirtuins (SIRT1 to SIRT7) and other enzymes including PARPs ([poly(adenosine diphosphate– ribose) polymerases) as well as yet uncharacterized roles through other NAD+ interacting proteins.

Sirtuins have been implicated in aging, as well as in a large number of key cellular processes including mitochondrial biogenesis, stress resistance, inflammation, transcription via epigenomic modulation of histones, apoptosis [7] [8] and SIRT1 can even affect circadian clocks [9].

PARPs are NAD‐dependent pleiotropic enzymes that play a key role in detecting and responding to single‐stranded DNA break repair as well as in inflammation, apoptosis induction and DNA methylation. Increasing PARP activity has been correlated with differential longevity of 13 mammalian species with humans having about 5 fold more PARP activity than rats [10].

Intuitively, increasing PARP activity would seem to be beneficial to organisms and potentially be of benefit to delay aging by maintaining DNA integrity, but PARPs are major consumers of NAD+ and PARP activity can deplete sufficient NAD+ to detrimentally effect mitochondria function. In fact, in apparent contradiction to the species longevity results, PARP1 knockout in mice increases tissue

NAD+ levels, enhances mitochondrial function and blocks inflammation [11]. Moreover, ectopic expression of human PARP1 in mice, an enzyme which has about 2 fold more intrinsic PARP activity than mouse PARP1, induces a set of aging related phenotypes including cardiomyopathy, hepatitis, pneumonitis, kyphosis, nephropathy, adiposity, dermatitis, anemia, increased glucose resistance, and increased incidence of carcinomas.

Paradoxically human PARP1 transgenic mice even have delayed DNA repair [12]. One possible explanation is that human PARP is consuming more NAD+ in mice than can be compensated for metabolically, causing dysfunction.

Clearly decreased NAD+ levels that have been observed in aging animals could be detrimental by multiple mechanisms, which require careful elucidation.

Decreasing NAD+ levels with aging cause DBC1 to bind and inhibit PARP1 allowing accumulation of DNA damage.

Li et al. [13] have recently established that NAD+ levels control protein‐protein interactions of two NAD+ binding proteins: DBC1/CCAR2 and PARP1. At low NAD+ levels DBC1 binds to PARP1 and inhibits PARP activity. In aging mice, DBC1 increasingly binds to PARP1 causing the accumulation of DNA damage, linking reduced NAD+ levels directly to DNA damage.

DBC1 was already known to bind to and inhibit SIRT1, as well as histone deacetylase HDAC3 and methyltransferase SUV39H1. Since SIRT1 is regulated by NAD+, Li et al explored the hypothesis that DBC1 could also bind and inhibit PARP1, another NAD‐dependent enzyme. In HEK293T cells co‐ immunoprecipitation/Western studies showed that PARP1 and DBC1 physically interact. A catalytically inactive PARP1 also interacted with DBC1 in these studies demonstrating that PARP catalytic activity was not necessary for the interaction with DBC1.

However, unlike the DBC1/SIRT1 interaction, the DBC1/PARP1 interaction was sensitive to and broken by NAD+ in the physiological range (100um‐500um), while similar doses of nicotinamide or its structural analog 3‐AB had no effect. NADH and adenine had only weak effects, suggesting that DBC1/PARP1 interaction requires

low levels or the absence of NAD+. A catalytically inactive PARP1 behaved similarly to wild type suggesting that NAD+ cleavage or covalent attachment was not necessary for NAD+ to inhibit the DBC1/PARP1 interaction[13] .

In HEK293T, an inhibitor of NAD+ biosynthesis or depleting NAD+ by genotoxic stress increased the DBC1/PARP1 interaction. Various treatments that increased NAD+, decreased DBC1/PARP1 interaction. These data are strong evidence that NAD+ inhibits the formation of the DBC1/PARP1 complex [13].

In order to determine which domains of DBC1 and PARP1 were interacting, Li et al. expressed a variety of truncated mutants of DBC1 and PARP1. DBC1 lacking a nudix homology domain (NDH) did not bind to PARP1, but a DBC1 carrying only a NDH domain did bind to PARP1. Moreover, DBC1 interacted with a PARP1 mutant consisting only of a BRCT domain, but not with a PARP1 having only a catalytic domain, suggesting that the NDH domain of DBC1 binds to the BRCT domain of PARP1.

Homology modeling based on the known crystal structures of 5 NDH proteins suggested that NAD+ directly binds the DBC1 NHD, and this was confirmed by competition experiments in which radiolabeled or biotin labeled NAD+ bound to DBC1‐NHD and was then competed off with unlabeled NAD+ [13].

Of significance is that PARP1 activity was inhibited by DBC1 interaction in vitro and stimulated in cell culture by siRNA knockdown of DBC1. DBC1 but not DBC1Q391A, a mutant of DBC1 that binds NAD+ poorly and does not interact well with PARP1, reduced PARP1 activity. Reducing DBC1 increased DNA repair after paraquat treatment, as assessed by lower gammaH2AX, reduced DNA fragmentation, increased NHEJ and HR recombination pathway activity and cell viability. NMN. which raises NAD+ levels. had similar effects on these cells. These data support the hypothesis that NAD+ binding to DBC1‐NHD regulates two key pathways of DNA repair through controlling PARP1 activity [13].

DNA repair capability decreases with increasing age [14]. Similarly PARP1 activity has been reported to decrease with age [10], suggesting a possible connection between PARP1 activity and overall DNA repair. Li et al hypothesize that increased DBC1/PARP1 binding occurs with reduced NAD+ levels during aging to reduce DNA repair and increase DNA damage.

In 22‐month old mice, levels of NAD+ were reduced in the liver compared to young mice. Old mice had increased amounts of DBC1‐PARP and increased staining for DNA‐damage associated gammaH2AX. A week of daily NMN treatment (500 mg/kg) intraperitoneally increased NAD+ levels, reduced the number of DBC1‐PARP1 complexes and decreased DNA damage as reported by decreased levels gammaH2AX in old mice. Reduced PARP1 activity in old mice was restored by NMN treatment.

To show that repair was affected, old mice were subjected to gamma irradiation. Irradiated mice were observed to have reduced DNA damage when treated with NMN either before irradiation or even one hour after irradiation[13]. These data support the hypothesis that reduced NAD+ with aging causes increased DBC1/PARP1 inhibiting PARP1‐mediated repair of DNA damage.

The authors reasonably speculate that DBC1 evolved as a buffer to prevent PARP1 from depleting NAD+ levels to cytotoxic levels [15]. They also point out that DBC1 is often mutated or downregulated in cancer and that increased repair capability and protection from radiation are potential mechanisms by which elimination of DBC1 could help cancer cells evade treatment[13]. Another possible mechanism for the tumor suppressor DBC1 is that the known stabilizing interaction of DBC1 with p53 is important[16].

Medical Implications:

Evidence is accumulating that diminished NAD+ during aging results in mitochondrial and stem cell dysfunction, as well as reduced DNA repair activity. Supplementation with NMN or especially NR at doses of 100, 300 or 1000 mg can increase NAD+ levels systemically in humans [17] and may counteract NAD+ aging‐associated phenotypes, although more preclinical and human studies are needed to establish efficacy of NR or NMN as anti‐aging interventions.

 A key question arises, if NAD+ levels and in particular PARP activity levels are so important to maintaining homeostasis and contribute to the biological processes called aging, why did treatment with NR, although beneficial to stem cell function, only increase mean and maximum longevity modestly in one study: about a 5% increase when treatment was initiated in very old mice (24 months) [5].

One point is that the intervention was started very late, which may not allow reversal of aging associated damage or loss of epigenomic information. On the other hand, the effect of increasing NAD+ is significantly less than that achieved by rapamycin, for example[18]. The simplest explanation is that decreased NAD+ represents only one of a set of key regulatory changes that result in the effects of aging.

However, it is possible that the role of PARP is more profound. Given the apparent correlation of PARP activity with mammalian longevity for diverse species, it is quite possible that increasing PARP1 expression by genetic or pharmaceutical means in the context of increasing NAD+ through supplementation may have profound effects on aging.

One interesting experiment would be to supplement the human PARP1 transgenic mice [12], which express a PARP1 2‐fold more active than mouse PARP1, with NR or NMN from birth to overcome the potential detrimental effects of decreased NAD+ levels from the overactive human PARP1. We hypothesize that these mice would not only not be sick, but would thrive and potentially live longer than wild‐type animals.

The significance of PARP1 activity to aging is at least somewhat tied to its DNA repair activity. It is controversial whether that the accumulated effects of DNA mutations alone are sufficient to drive aging [19], normal cells apparently accumulate hundreds to a few thousand mutations over a lifetime of cell divisions[20].

Non‐dividing cells should possess significantly fewer mutations. While some premature aging syndromes and cancer are driven by increased genome instability, it is unclear that accumulation of ~1000 genetic substitutions would sufficiently degrade cell function. On the other hand, because decreased PARP1 activity is expected to also cause increased chromosomal instability through reduced NHEJ and HR DNA repair, PARP1 activity probably plays a significant role in preventing age‐associated cancer.

If PARP1 plays a central role in aging via its role in DNA repair, it could do so indirectly, through effects on the epigenome. PARP1 and other members of the PARP family have been shown to have profound effects on the epigenome through Poly(ADP‐ribosyl)ation (PARylation), affecting DNA methylation/demethylation via effects on DNMT1 and CTCF, histone acetylation, histone methylation, and organizing chromatin domains [21].

DNA repair events initiated by single‐ stranded DNA breaks could change the balance of PARP1 activities changing the epigenome in defined ways. Animals with higher intrinsic PARP1 activity may have less DNA damage and fewer epigenomic alterations over a given amount of chronological time, resulting in a younger biological “age”.

Superimposed upon the effects of decreased NAD+ levels, it does not escape attention that the NAD+/PARP1/SIRT1 axis could contribute to creating the apparent epigenetic aging “clocks” that are reflected in the tissue‐invariant and tissue‐specific DNA methylation patterns observed to strongly correlate biological age in humans, chimpanzees and mice [22][23] [24] through modulation of DNA methylation through DNMT1 and CTCF.

References

  1. [1]  Braidy N, Guillemin GJ, Mansour H, Chan‐Ling T, Poljak A, Grant R. Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats. PLOS ONE 2011;6:e19194. doi:10.1371/journal.pone.0019194.
  2. [2]  Yoshino J, Mills KF, Yoon MJ, Imai S. Nicotinamide Mononucleotide, a Key NAD+ Intermediate, Treats the Pathophysiology of Diet‐ and Age‐Induced Diabetes in Mice. Cell Metab 2011;14:528–36. doi:10.1016/j.cmet.2011.08.014.
  3. [3]  Mouchiroud L, Houtkooper RH, Moullan N, Katsyuba E, Ryu D, Cantó C, et al. The NAD+/Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling. Cell 2013;154:430–41. doi:10.1016/j.cell.2013.06.016.
  4. [4]  Gomes AP, Price NL, Ling AJY, Moslehi JJ, Montgomery MK, Rajman L, et al. Declining NAD+ Induces a Pseudohypoxic State Disrupting Nuclear‐Mitochondrial Communication during Aging. Cell 2013;155:1624–38. doi:10.1016/j.cell.2013.11.037.
  5. [5]  Zhang H, Ryu D, Wu Y, Gariani K, Wang X, Luan P, et al. NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice. Science 2016;352:1436–43. doi:10.1126/science.aaf2693.
  6. [6]  Bhullar KS, Hubbard BP. Lifespan and healthspan extension by resveratrol. Biochim Biophys Acta BBA ‐ Mol Basis Dis 2015;1852:1209–18. doi:10.1016/j.bbadis.2015.01.012.
  7. [7]  Grabowska W, Sikora E, Bielak‐Zmijewska A. Sirtuins, a promising target in slowing down the ageing process. Biogerontology 2017. doi:10.1007/s10522‐017‐9685‐9.
  8. [8]  Mei Z, Zhang X, Yi J, Huang J, He J, Tao Y. Sirtuins in metabolism, DNA repair and cancer. J Exp Clin Cancer Res CR 2016;35. doi:10.1186/s13046‐016‐0461‐5.
  9. [9]  Masri S, Sassone‐Corsi P. Sirtuins and the circadian clock: bridging chromatin and metabolism. Sci Signal 2014;7:re6. doi:10.1126/scisignal.2005685.

[10] Grube K, Bürkle A. Poly(ADP‐ribose) polymerase activity in mononuclear leukocytes of 13 mammalian species correlates with species‐specific life span. Proc Natl Acad Sci U S A 1992;89:11759–63.

 [11] Bai P, Cantó C, Oudart H, Brunyánszki A, Cen Y, Thomas C, et al. PARP‐1 Inhibition

[12] Mangerich A, Herbach N, Hanf B, Fischbach A, Popp O, Moreno‐Villanueva M, et al.

[13] Li J, Bonkowski MS, Moniot S, Zhang D, Hubbard BP, Ling AJY, et al. A conserved NAD(+) binding pocket that regulates protein‐protein interactions during aging.

[14] Gorbunova V, Seluanov A, Mao Z, Hine C. Changes in DNA repair during aging.

[15] Yang H, Yang T, Baur JA, Perez E, Matsui T, Carmona JJ, et al. Nutrient‐Sensitive

[16] Qin B, Minter‐Dykhouse K, Yu J, Zhang J, Liu T, Zhang H, et al. DBC1 Functions as a

[17] Trammell SAJ, Schmidt MS, Weidemann BJ, Redpath P, Jaksch F, Dellinger RW, et al.

[18] Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, Flurkey K, et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature

[19] López‐Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. The Hallmarks of Aging. Cell 2013;153:1194–217. doi:10.1016/j.cell.2013.05.039.
[20] Martincorena I, Campbell PJ. Somatic mutation in cancer and normal cells. Science 2015;349:1483–9. doi:10.1126/science.aab4082.
[21] Ciccarone F, Zampieri M, Caiafa P. PARP1 orchestrates epigenetic events setting up chromatin domains. Semin Cell Dev Biol 2017;63:123–34. doi:10.1016/j.semcdb.2016.11.010.
[22] Horvath S. DNA methylation age of human tissues and cell types. Genome Biol 2013;14:3156. doi:10.1186/gb‐2013‐14‐10‐r115.

[23] Hannum G, Guinney J, Zhao L, Zhang L, Hughes G, Sadda S, et al. Genome‐wide methylation profiles reveal quantitative views of human aging rates. Mol Cell 2013;49:359–67. doi:10.1016/j.molcel.2012.10.016.
[24] Stubbs TM, Bonder MJ, Stark A‐K, Krueger F, von Meyenn F, Stegle O, et al. Multi‐tissue DNA methylation age predictor in mouse. Genome Biol 2017;18. doi:10.1186/s13059‐017‐1203‐5.

Rapamycin,rifampicin, Psora-4 cocktail doubles lifespan

Drug combinations work to synergistically extend the life- and healthspan in worms.

The most successful drug combination tested almost doubled lifespan, a feat never reported before. Furthermore, more than half of the worms receiving the drug combination were still in optimal health after all control animals had died! Finally, the authors show that these drugs also extend the lifespan of fruit flies and in fact the same combination that almost doubled worm lifespan was also the most successful one at extending the lifespan of fruit flies.

In a new paper published on the preprint server bioRxiv Jan Gruber and colleagues test various combinations of life extending drugs in worms. Preprint servers allow scientists to publish their article to the wide world for anyone to criticize before submitting it to a classical journal for publication. Furthermore they allow for rapid dissemination of research results as the publication in a peer-reviewed journal can take months to years between submission and publication.

While around a thousand compounds have been found to extend lifespan in at least one study in a model organism few studies have been conducted using combinations of two or more compounds. A rare example includes the combination of rapamycin with metformin which was shown to outperform either drug alone in mice.

The authors started by identifying well known mechanisms for lifespan extension based on a literature review. Then they looked for drugs that influence these mechanisms and had previously been found to extend lifespan leading to the selection of 12 compounds for testing in this study. Next the researchers tested these compounds in roundworms, a common model organism in aging research. When tested in isolation five drugs significantly extended mean and maximal lifespan: Psora-4, rifampicin, rapamycin, metformin, and allantoin.

Image credit: Sven Bulterijs

Next the researchers tested all pairwise combinations of these five compounds. The combination of metformin and rapamycin, both at optimal doses, did not lead to a further increase in lifespan. However if the combination was tested at suboptimal doses than lifespan was further increased. Given that the optimal dose of metformin and rapamycin in mice is unknown the beneficial effect of the combination observed in the mice study mentioned before could possibly be the result of suboptimal concentrations of both drugs. Two combinations, rifampicin + rapamycin and rifampicin + Psora-4 all at optimal concentrations did result in synergistic increases in lifespan.

Next the researchers tested triple combinations of the various compounds. As testing all combinations would be impractical (220 different lifespan tests would be necessary) the researchers decided to first try the combination of the 3 most successful compounds so far (rapamycin + rifampicin + Psora-4). However this combination resulted in a shorter lifespan than the two successful pairwise combinations. Next, the researchers decided to test the two pairwise combinations from before with allantoin added as the third drug. They chose this combination because allantoin shares no mechanistic overlap with the other drugs. Both of these triple combinations resulted in a significant extension of mean and maximal lifespan with the most successful one (rapamycin + rifampicin + allantoin) resulting in a doubling of mean lifespan! The authors remark that this is the largest lifespan extension ever observed by a drug intervention initiated in adult worms.

Abbreviations: Rif = rifampicin; Rap = rapamycin, Allan = allantoin. Image credit: Sven Bulterijs

Certain interventions that result in lifespan extension reduce fertility but neither of the triple drug combinations reduced total fertility and actually slightly extended the fertile period of life. Furthermore, both drug combinations extended the period of life spend in good health (= the healthspan). In fact, more than half of the worms receiving the triple drug combination were still in optimal health after all control animals had died! Old animals on the triple drug combinations were indistinguishable from young control worms when judged by spontaneous movement. Worms really show a significant decrease in spontaneous movement with age. Finally, the two successful triple drug combinations also significantly increased resistance to oxidative and heat stress.

Total mortality is made up of two distinguishable parts: age-dependent and age-independent mortality. The age-dependent mortality is a measure for the rate of aging. So the authors tested which mortality rate was reduced by the drug combinations. Interestingly, the rifampicin + Psora-4 + allantoin combination significantly reduced the rate of age-dependent and age-independent mortality showing that this drug slows down aging in addition to making them more robust at young ages.

Abbreviations: Rif = rifampicin; Rap = rapamycin, Allan = allantoin. Image credit: Sven Bulterijs

The evolutionary distance between fruit flies and worms is larger than between fruit flies and humans. Hence the fact that the drug combinations worked in both fruit flies and worms offers hope that their lifespan extending effect may be conserved in humans. The two drug combinations (Rap + Rif and Rap + Rif + Allan) significantly extended mean and maximum lifespan in fruit flies.

Dessale T et al. (2017). Slowing ageing using drug synergy in C. elegans. bioRxiv: 153205. http://biorxiv.org/content/early/2017/06/23/153205

Targeting NAD+ in Metabolic Disease

Nicotinamide adenine dinucleotide (NAD+) was discovered more than 100 years ago by Sir Arthur Harden as a low-molecular-weight substance present in boiled yeast extracts [1]. In the late 1920s, Joseph Goldberger fed Brewer’s yeast to dogs with pellagra, a devastating disease characterized by dermatitis, diarrhea, dementia, and death, and their health improved.

At that time, pellagra was endemic in parts of the United States, and so the Red Cross supplemented Brewer’s yeast to its food rations in pellagra-endemic areas; within weeks the disease burden dissipated [2, 3]. The health significance of NAD+ was established in 1937, when Conrad Elvehjem and his colleagues made the major discovery that the factor that prevented and cured pellagra was the NAD+ precursor, nicotinic acid [4, 5].

NAD+ plays a central role in cellular respiration, the cascade of reactions that generate adenosine triphosphate (ATP) from nutrient breakdown, by acting as a coenzyme for oxidoreductases and dehydrogenases [6–9]. As coenzymes, NAD+ and its phosphorylated and reduced forms, including NADP+, NADH, and NADPH, are critical for the activities of cellular metabolism and energy production [1, 10, 11]. NAD+ most commonly functions in energy-generating catabolic reactions (such as glycolysis, fatty oxidation, and citric acid cycle), where it is reduced to NADH, which is then shuttled into the mitochondria to generate ATP.

This generates an NAD+/NADH ratio, which is useful to assess the health and energy charge of the cell. The phosphorylated form, NADP(H)+, participates in anabolic reactions, such as fatty acid and cholesterol synthesis [8, 9, 12].

More recently and as importantly, NAD+ has been studied as a rate-limiting substrate for three classes of enzymatic reactions involved in posttranslational modification (Fig. 1), all of which exhibit breaking of the glycoside bond between nicotinamide and the adenosine 5′-diphosphate (ADP)-ribose moiety, and the latter is then transferred onto an acceptor molecule [6–9, 11]. The first class includes mono- and poly-ADP ribose transferases, among which the poly-ADP ribose polymerases (PARPs) are the most studied and are classically described as DNA repair proteins [13, 14].

The second class is the cyclic ADP ribose synthases (CD38 and CD157), which are membrane-bound ectoenzymes that produce and hydrolyze the Ca2+-mobilizing second messenger cyclic ADP-ribose from NAD+ and are therefore key in calcium homeostasis and signaling [15]. The third and most important class in terms of cellular energy metabolism consists of the sirtuins, named for their similarity to the yeast Sir2 gene-silencing protein. Seven sirtuins exist in mammals (SIRT1 through SIRT7), with diverse enzymatic activities, expression patterns, cellular localizations, and biological functions [16]. Sirtuins have a host of metabolic targets, resulting in profound effects on various cellular processes, such as mitochondrial biogenesis, cellular stress response, lipid metabolism, insulin secretion and sensitivity, apoptosis, circadian clock dynamics, inflammation, and aging [17].

Through these targets, sirtuins translate changes in feeding status, DNA damage, and oxidative stress into metabolic adaptations [18–20]. SIRT1, the most-studied sirtuin, targets multiple transcriptional coactivators, such as the peroxisome proliferator-activated receptor γcoactivator-1α (PGC-1α) and transcription factors, such as the forkhead box protein O1. PGC-1α is a central regulator of energy metabolism and mitochondrial biogenesis [21–24], whereas forkhead box protein O1 regulates mitochondrial fatty acid metabolism and protects against oxidative stress [25–27].

As nutrients influence the NAD+/NADH pool, these NAD+-dependent signaling reactions are recognized as the sensors of metabolism owing to their decisive regulatory roles in cellular metabolism [17]. Appropriate regulation of these NAD+-dependent processes relies on the cellular ability to conserve their NAD+ content. Therefore, inadequate NAD+ homeostasis can be pathologic, linked to impaired cell signaling and mitochondrial function [19, 28, 29].

The dependency of sirtuins on NAD+ [30], and the finding that yeast Sir2 protein is required for the lifespan extension mediated by caloric restriction (CR) [31], led to a renascent interest in NAD+ metabolism research, centered on modifying NAD+ availability to support sirtuin-mediated cellular metabolism to mimic CR. This interest was enhanced by the discovery of contemporary NAD+ precursors that can circumvent issues with existing molecules, which can also increase NAD+ in vivo and human tissues [32–34]. As we review here, these key findings underline the prospect of targeting NAD+ biosynthetic pathways to increase mitochondrial function and sirtuin activity in the combat against metabolic disease. We also highlight the challenges and the knowledge gaps that require investigating before these compounds can find their way to the clinics.

1. NAD+ Biosynthesis and Metabolism

In humans, NAD+ can be synthesized via the de novo/kynurenine pathway from the amino acid tryptophan [35, 36]. However, tryptophan is a poor NAD+ precursor in vivo [37]. Most organisms have alternative NAD+ synthesis pathways (Fig. 2) from the dietary vitamin B3 precursors nicotinic acid (NA), nicotinamide (Nam), and nicotinamide riboside (NR), or from a salvage pathway where the Nam molecule split from NAD+-consuming reactions is recycled into nicotinamide mononucleotide (NMN) via the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT), and NAD+ is regenerated [9, 11, 35, 38–41]. In addition, a more recently described salvage pathway recycles NR to NMN via the nicotinamide riboside kinases (NRKs) [32]. In humans, these different routes to NAD+ synthesis converge at the NAD+ and nicotinic acid adenine dinucleotide formation step catalyzed by the nicotinamide mononucleotide adenylyltransferases. Nicotinic acid adenine dinucleotide is then amidated to form NAD+.

Schematic overview of human NAD+ biosynthesis. NAAD, nicotinic acid adenine dinucleotide; NADS, NAD+ synthase; NAPT, nicotinic acid phosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyltransferase; QAPT, quinolinic acid phosphoribosyltransferase. *NAMPT is the rate limiting step in NAD+ biosynthesis.

Nicotinic acid riboside is an NAD+ biosynthesis intermediate that can be converted in yeast and human cells by NRKs into nicotinic acid mononucleotide and then to NAD+ [42]. It is the least-studied NAD+ precursor and is therefore beyond the scope of this review.

The energy sensor adenosine monophosphate-activated protein kinase (AMPK), which adapts cells to low-energy states in the support of ATP production [43, 44], activates NAMPT, increases NAD+ recycling, and enhances SIRT1 activity [45, 46].

In mammals, the entire NAD+ pool is used and replenished several times a day, balanced by the distinct NAD+ biosynthetic pathways [47]. Owing to its constant utilization, the half-life of NAD+ in mammals is short (up to 10 hours) [36, 48–51], with intracellular levels believed to be 0.4 to 0.7 mM [41]; however, the accuracy of this level depends on the cell type and physiologic state being assessed. It is clear that NAD+ concentrations differ substantially between cellular compartments, with mitochondrial NAD+ concentration being the highest and representing 70% to 75% of cellular NAD+ (10- to 100-fold higher than those in the cytosol) [52, 53]. The NAD+/NADH levels vary to adjust cellular and tissue physiology in response to changes in nutrient availability and energy demand. For instance, NAD+ levels drop in response to high-fat diet (HFD) in mice [33, 54] and with aging, contributing to age-related disorders, such as diabetes, cardiovascular disease, cancer, and neurodegenerative disease [55–58]. Conversely, the renowned health adaptive beneficial effects of CR and exercise have been linked to NAMPT activation and the subsequent rise in NAD+, sirtuins, and mitochondrial activity [46, 59–61].

2. Therapeutic NAD+ Boosting

The recommended daily allowance (RDA) of niacin, a collective term for NA and Nam, is around 15 mg/d and can be met through the consumption of meat, fish, and dairy products [12, 62]. More recently, NR was also detected in milk and yeast [32, 63].

A plethora of evidence suggests that higher rates of NAD+ synthesis can positively affect pathways that require NAD+ as a cosubstrate. The NAD+ pools can be elevated via provision of precursors [33, 54, 64, 65], NAD+ biosynthesis augmentation [45, 46], and inhibition of NAD+ consumers [57, 66–68].

3. NAD+ Precursor Supplementation

The most tractable approach to increase NAD+ would be via the supplementation of the different precursors, all of which increase NAD+ levels in human and animal tissues. This approach is the focus of this review because NAD+ precursors are naturally occurring in food and are readily available in isolated forms, allowing nutritional approaches to be applied to modulate NAD+ metabolism in vivo.

A. Niacin

NA has been used for >50 years in the treatment of hyperlipidemia [69, 70]. Dietary niacin is not associated with side effects because the tolerable upper intake level is not exceeded [62], whereas pharmacologic NA dosing is commonly associated with undesirable effects, thereby decreasing treatment adherence. NA is a ligand for the G-protein–coupled receptor GPR109A and is coexpressed on the epidermal Langerhans cells mediating prostaglandin formation, which induces troublesome flushing and other vasodilatory effects, such as itching, hypotension, and headaches [12, 71–73]. To overcome these problems, the selective antagonist of prostaglandin D2 receptors, laropiprant, was introduced into clinical practice in combination with extended-release NA (extended-release NA-laropiprant) [74]. Extended-release NA-laropiprant failed to prove advantageous in clinical trials; safety concerns arose, and the agent was therefore withdrawn from all markets [75]. A long-acting NA analog, acipimox, is undergoing clinical trials [76–79]. However, acipimox remains a GPR109A receptor ligand [80], thus retaining the potential for undesirable side effects that will limit its clinical utility.

Although Nam is the predominant endogenous precursor of the NAD+ salvage pathway, early reports suggested that it may not be as effective as other biosynthesis precursors in increasing NAD+ levels [41]; however, this likely reflects the relatively small dose of Nam used. Additionally, Nam effects likely depend on cell/tissue type and the pathophysiologic state. For instance, in a nonstressed state, Nam is inferior to NA as an NAD+ precursor in the liver [81], whereas under HFD-induced metabolic challenge, Nam is a more powerful NAD+ precursor and SIRT1 activator than NA [82]. Nam has been used for many years for a variety of therapeutic applications (such as diabetes mellitus) at doses up to 3 g/d, with minimal side effects [83]. Unlike NA, Nam has no GPR109A agonist activity [80], thus escaping the prostaglandin-mediated vasodilatory side effects. Yet, at high doses Nam has a toxic potential (particularly hepatotoxicity), raising health concerns [83] and, as well as with long-term use, can cause negative feedback to inhibit sirtuins [84, 85].

B. NR and NMN

NR has been recognized since the 1950s as an NAD+ precursor in bacteria that lack the enzymes of the de novo and Preiss–Handler pathways [86–88]. This changed in 2004, when Bieganowski and Brenner [32] detected the presence of NR in milk and identified two human NRK enzymes capable of synthesizing NAD+ from NR. Subsequent human and animal studies confirmed that NR can increase intracellular NAD+ in a dose-dependent fashion [34, 89, 90]. Likewise, NMN is an intermediate in the NAD+ salvage pathway. Although less studied than NR, several studies proved that NMN increases NAD+ levels in vitro and in vivo [33, 56, 91–93]. Several recent studies using NR and NMN have attracted major research interest and are discussed later.

4. NAD+ Biosynthesis Augmentation

Several AMPK and NAMPT activators have been studied. Resveratrol is a nonflavonoid polyphenol that is present in red grapes, wine, and pomegranates; activates AMPK and SIRT1; and improves metabolic health status in humans [94–98]. However, conflicting outcomes from clinical studies have questioned the efficacy of resveratrol in treating human metabolic disease [99]. Nonetheless, it remains a compound of substantial interest to many [100].

Various AMPK activators exist [101]. Among them is metformin, which was introduced in the 1950s to treat diabetes, with a multitude of favorable metabolic outcomes that rely on AMPK [102]. Cantó et al. [45] reported that the AMPK activators metformin and 5-aminoimidazole-4-carboxamide ribonucleotide, increase NAD+ and sirtuin activity, thereby regulating energy expenditure .

Other compounds have also been reported to increase NAMPT activity. P7C3, a neuroprotective chemical that enhances neuron formation, can bind NAMPT and increase NAD+ levels [103–105]. Likewise, the antioxidant troxerutin, a trihydroxyethylated derivative of the natural bioflavonoid rutin, markedly increased NAD+ levels and potentiated SIRT1 via NAMPT activation and PARP1 inhibition in HFD-treated mouse liver [106]. Remarkably, leucine supplementation in obese mice also increased NAMPT expression and enhanced intracellular NAD+ levels [107]. Moreover, proanthocyanidins, the most abundant flavonoid polyphenols in human diet, can dose-dependently increase NAD+ levels in rat liver via the increased expression of the de novo pathway enzymes [108], and possibly NAMPT [109]. Targeting microRNA, such as antagonizing hepatic miR-34a, has also been reported to increase NAMPT expression and NAD+ and SIRT1 activity in vivo [110].

5. Inhibition of NAD+ Consumers

Inhibiting the nonsirtuin NAD+ consumers also increases NAD+ levels and favors sirtuin activity. Inhibitors of PARPs or CD38 induce NAD+ levels, upregulate sirtuins, and enhance mitochondrial gene expression [67, 68, 89, 111]. PARP inhibitors are effective anticancer agents through DNA damage repair and improved oxidative metabolism (opposing the Warburg effect) in which the NAD+-sirtuin axis may be implicated [112–115]. The first PARP inhibitor, olaparib, is now licensed in the United States and Europe for the treatment of ovarian cancer [116, 117]. Therefore, PARP inhibitors may undergo further studies as NAD+-sparing agents to improve adaptive metabolism [118]. Interestingly, troxerutin and proanthocyanidins also inhibit PARPs in mice, thereby contributing to increased NAD+ [106, 108].

6. Type 2 Diabetes Mellitus

The global burden of obesity, insulin resistance, and type 2 diabetes mellitus (T2DM) continues to limit population health through increased cardiovascular disease risk and premature death [119].

Several studies support the notion that defective mitochondrial structure and function are strongly linked to insulin resistance and T2DM [120–128]. The most described mechanism is via defective mitochondrial fatty acid oxidation and the resultant accumulation of intracellular fatty acid metabolites and reactive oxygen species decreasing insulin sensitivity [129–133]. In addition, perturbed oxidative phosphorylation (OXPHOS) may be a direct cause of insulin resistance [134]. Supporting this, obesity reduces mitochondrial enzymatic activities [135, 136] and engenders metabolic inflexibility [137]; the inability to limit fatty oxidation and switch to carbohydrate oxidation in response to diet (and therefore insulin stimulation) [138–141].

Impaired NAD+-mediated sirtuin signaling is also implicated in insulin resistance and T2DM. In particular, defective SIRT1 activity is thought to be a factor in impaired insulin sensitivity [142–148]. This is endorsed by the finding that metformin acts through hepatic SIRT1 activation as part of its diabetes ameliorating effects [149]; results similarly observed with resveratrol [150].

Lifestyle manipulations, such as CR and exercise, can reverse insulin resistance and T2DM and share common mechanistic pathways of AMPK activation leading to elevated NAMPT-mediated NAD+ generation and SIRT1 activity to enhance mitochondrial function [46, 61, 151, 152]. Corroborating the link to NAD+, adipocyte-specific NAMPT deletion in mice decreased adiponectin production and resulted in severe multiorgan insulin resistance [92]. Aside from insulin sensitization, NAD+ and SIRT1 regulate glucose-stimulated insulin secretion in pancreatic β cells [153–155]. NAMPT inhibition and the lack of SIRT1 resulted in pancreatic β cell dysfunction [93, 156–159]. Interestingly, SIRT1 regulates the key components of the circadian clock, CLOCK and BMAL1 [160, 161], and when circadian misalignment is induced in mice, reduced hepatic BMAL1 and SIRT1 levels and insulin resistance ensue [150].

These lines of evidence suggest that an alternate strategy is to increase the level of NAD+ available to affected cells and tissues. Indeed, the NAD+ precursors used to enhance target tissue NAD+ availability have demonstrated efficacy to improve insulin sensitivity and reduce diabetic burden and associated metabolic derangements in preclinical models [33, 162].

NMN administration restored β cell glucose-stimulated insulin secretion and hepatic and muscle insulin sensitivity in mouse models of induced glucose intolerance [33, 92, 93]. Furthermore, Nam treatment in obese rats with T2DM promoted sirtuin-induced mitochondrial biogenesis and improved insulin sensitivity [82]. Similarly, NR supplementation attenuated HFD-induced obesity in mice, improved insulin sensitivity and glucose tolerance, and ameliorated the adverse lipid profile [54, 162]. Moreover, leucine supplementation in obese mice increased NAD+, mitochondrial biogenesis, insulin sensitivity, and lipid disposal [107].

Thus far, clinical data are limited to acipimox and resveratrol. Acipimox increased tissue insulin sensitivity in T2DM [79, 163–168] and improved β cell function when combined with dapagliflozin [76]. However, the results have been inconsistent at times. For instance, acipimox treatment in obese nondiabetic persons alleviated free fatty acids and fasting glucose with a trend toward reduced fasting insulin and homeostatic model assessment of insulin resistance [77], whereas van de Weijer et al.[78] did not report similar benefits in individuals with T2DM by using euglycemic hyperinsulinemic clamp studies. However, in the later study, this may have been related to the rebound increase in fatty acids after short-term acipimox administration [169]. Similarly, many describe that resveratrol decreases glucose and insulin levels in patients with impaired glucose tolerance and diabetes [95, 96, 170, 171], whereas others have not observed these findings [172]. The conflicting results among these studies may be explained by the heterogeneity in the selection of study population, dose and duration of treatment, and the methods of assessing insulin sensitivity.

7. Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in the Western world, encompassing the spectrum of liver diseases, including simple steatosis, nonalcoholic steatohepatitis, cirrhosis, liver failure, and hepatocellular carcinoma [173]. Hepatic lipid accumulation, which leads to cellular dysfunction, termed lipotoxicity, forms the basis for the development of NAFLD [174–176]. Consequently, a set of metabolic adaptations supervene, such as increased βoxidation. This adaptation induces metabolic inflexibility and drives the oxidative stress and mitochondrial dysfunction that are apparent in NAFLD [177–180].

Sufficient NAD+ levels are essential for adequate mitochondrial fatty acid oxidation [181, 182], and lipid caloric overload in mice reduces hepatic NAD+ levels and triggers lipotoxicity [183]. Zhou et al. [184] demonstrated that hepatic NAD+ levels decline with age in humans and rodents, which may contribute to NAFLD susceptibility during aging. Likewise, ample evidence suggests that impaired hepatic SIRT1 and SIRT3 signaling contributes to NAFLD [183, 185–188] and that SIRT1overexpression reverses hepatic steatosis [189, 190]. Stressing the significance of adequate hepatic NAD+ homeostasis, aberrant NAD+ metabolism is also implicated in alcoholic hepatic steatosis [191, 192] and hepatocellular carcinoma [193].

Several strategies targeting NAD+ metabolism to enhance sirtuin signaling have proved beneficial in the context of NAFLD. Nam and resveratrol protected hepatocytes in vitro against palmitate-induced endoplasmic reticulum stress [64, 194]. NR attenuated the severe mitochondrial dysfunction present in fatty liver of mice on HFD via NAD+-mediated sirtuin activation [54, 195]. Remarkably, NR was able to target many of the molecular aspects of NAFLD pathogenesis, including decreasing hepatic expression of inflammatory genes, blood tumor necrosis factor-α levels, and the hepatic infiltration by CD45 leukocytes [196]. PARP inhibition in mice with NAFLD can correct NAD+ deficiency, augmenting mitochondrial function and insulin sensitivity and allaying hepatic lipid accumulation and transaminitis [197]. Considering the current data, and in the absence of licensed therapies for NAFLD, replenishing the hepatic NAD+ pool to activate sirtuins and tackle mitochondrial dysfunction is staged for assessment in human clinical studies.

8. Aging and Metabolic Decline

By the year 2050, it is projected that the US population aged ≥65 years will be 83.7 million [198], with other low-mortality countries displaying similar population proportions [199].

Sarcopenia, Greek for “poverty of flesh,” is a consistent manifestation of aging, associated with frailty, metabolic disease, cardiovascular morbidity and mortality, and substantial health care costs [200, 201]. Needless to say, strategies aimed at treating sarcopenia and age-related diseases are needed.

A decline in NAD+ homeostasis contributes to the aging process [202, 203]. Indeed, NAD+ and sirtuins regulate diverse pathways that control aging and longevity [31, 57, 204–206], converging on the ability to defend mitochondrial function [207]. Certainly, mitochondrial dysfunction and defective cellular energy signaling have emerged as critical in aging and age-related metabolic diseases, such as T2DM, NAFLD, and sarcopenia [55]. Specifically, altered mitochondrial homeostasis, through reduced NAD+ and SIRT1 activity, is advocated as a hallmark of muscle aging [56]. In addition, limiting NAD+ in mouse skeletal muscle induced the loss of muscle mass and function (i.e., sarcopenia) [208].

Age-related decline in NAD+ results from several mechanisms, which include accumulating DNA damage (and, consequently, chronic PARPs activation) [209, 210] and increased expression of CD38, clearing NAD+ and inducing mitochondrial dysfunction [211]. Additionally, chronic inflammation [212], a common feature in aging, reduces NAMPT expression and the ability to regenerate adequate NAD+ in multiple tissues [154].

The potential of NAD+ supplementation to support healthy aging is supported by several recent studies. NAMPT overexpression in aged mice matched the NAD+ levels and muscle phenotype of young mice [208]. Furthermore, SIRT1overexpressing mice were protected against the age-related development of diabetes and had a lower incidence of cancer [213]. NMN administration in aged mice restored NAD+ levels and the markers of mitochondrial function that decline with age [56].

Looking from a different angle, NR supplementation enhanced the expression of PGC-1α in the brain of a mouse model of Alzheimer’s disease, significantly attenuating the cognitive decline [214]. These findings affirm that decreased NAD+ levels contribute to the aging process and that NAD+ supplementation may prevent and even treat age-related diseases.

9. Discussion and Future Challenges

It is now well established that NAD+ is involved in metabolic regulation via redox and cell signaling reactions and that insufficient NAD+ is linked to a variety of metabolic and age-related diseases. The evidence reviewed here highlights that NAD+ levels can be therapeutically increased to potentiate sirtuins and mitochondrial function. This is a great opportunity in metabolic research that could conceivably lead to clinical utility.

The long-known lipid-lowering effects of NA may, at least partly, be NAD+ mediated. This hypothesis is favored because the half maximal effective concentration for the GPR109A receptor is in the nanomolar range [215, 216]; however, the therapeutic doses of NA are greatly in excess of this amount [71, 217]. Moreover, NR ameliorated hypercholesterolemia in mice without activating the GPR109A receptor [54]. Additionally, the liver lacks GPR109A receptors [218] but expresses liver X receptors, which regulate whole-body lipid homeostasis, that are upregulated by SIRT1 [219].

Although we have described the different pathways to NAD+ biosynthesis, it must be emphasized that not all tissues are capable of converting each precursor to NAD+ with equal efficacy, owing to the differences in the cell- and tissue-specific enzyme expression. For instance, cells must express the kynurenine pathway for de novoNAD+ synthesis, clearly active in the liver and brain [12], and must possess the Preiss–Handler pathway to use NA, which is active in most organs but less prominent in skeletal muscle. In contrast, the salvage pathways are crucial in all tissues to conserve NAD+ sufficiency [220]. Supporting this notion, the recommended daily allowance for NA is in milligrams, whereas an estimated 6 to 9 g of NAD+ are required daily to match turnover [58]. This is facilitated by the high affinity of NAMPT for Nam; thus, even small amounts of Nam are effectively converted to NMN and then NAD+ [221].

In the absence of head-to-head studies comparing the different compounds under defined conditions, it is currently not possible to identify the optimal NAD+ augmenting agent. The ubiquitous expression of NRKs, makes NR a precursor that can affect whole-body metabolism [162]. The inability of NR to activate the GPR109A receptor mitigates the undesirable NA side effects, and, unlike Nam, NR does not inhibit sirtuins. Furthermore, NAD+ generated from NR can target both nuclear and mitochondrial NAD+ pools, activating the respective compartmental sirtuins (i.e., nuclear SIRT1 and mitochondrial SIRT3) [54]. This may be an advantage over other molecules, such as PARP inhibitors, with effects confined to the nucleus [67]. Similar to NR, NMN metabolism into NAD+ is governed by the salvage pathway. However, NMN availability has not been characterized in the diet [93, 222], unlike the naturally available NR.

In major proof-of-concept studies, therapeutically increasing NAD+ has been used to treat mouse models of mitochondrial diseases. Treatment of cytochrome C oxidase deficiency in mice with NR, PARP inhibition, and the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide reversed the mitochondrial dysfunction and improved muscle performance [223–225], effects attributed to NAD+ and sirtuins activation. Treatment of patients with T2DM by using acipimox resulted in improved skeletal muscle oxidative metabolism and mitochondrial function, measured by high-resolution respirometry [78]. However, this acipimox effect was not observed in obese persons without T2DM when assessed by phosphocreatine recovery magnetic resonance spectroscopy, mitochondrial biogenesis gene expression, and mitochondrial density on electron microscopy [77]. Two differences between these studies may explain the observed discrepancy. First, high-resolution respirometry is the current gold standard for ex vivo assessment of mitochondrial respiration if increased oxidative phosphorylation is the question [226]. Second, whereas mitochondrial dysfunction is evident in patients with T2DM, this is not prominent in obese persons without diabetes. Thus, the effects of NAD+ precursor supplementation may vary depending on the intervention and specific pathophysiologic conditions. Nam acts as an NAD+ precursor, increasing SIRT1 activity (below a threshold of sirtuin inhibition), or, conversely, a SIRT1 inhibitor, depending on the specific pathophysiologic state [84, 85].

We still have a limited understanding of the molecular interconversions of the administered NAD+ precursors. Illustrating this, administered NR is converted to Nam in the circulation before entering the cell [208, 227], whereas NMN is transformed extracellularly into NR, which then enters the cell and converts into NAD+ [227].

Knowledge gaps still persist in the role of sirtuins in different contexts. Some reports suggest that not all beneficial SIRT1 activation is through NAD+ and that cyclic adenosine monophosphate plays a role, independent of NAD+, in low-energy states [228, 229]. Upon pharmacologic NAMPT inhibition, Nam failed to increase NAD+; however, this did not prevent SIRT1 upregulation, which was secondary to Nam-induced increase in intracellular cyclic adenosine monophosphate [64].

An important question is whether amplifying NAD+ and sirtuin activity is always desirable. SIRT1 upregulates T helper 17 cells that contribute to autoimmune disease when hyperactivated [230]. Correspondingly, SIRT1 inhibition supports the development of the regulatory T cells that protect against autoimmunity [231, 232]. Therefore, it is possible that SIRT1 activation places susceptible individuals at increased risk for autoimmune diseases. In the same way, whereas NR supplementation increased muscle stem cell number in aged mice, thereby enhancing mitochondrial function and muscle strength, it reduced the expression of cell senescence and apoptosis markers [233]; the state of senescence is important to protect against carcinogenesis [234]. Also, increased NAMPT expression is reported in some malignancies, calling into question whether increasing NAD+ might support aspects of the tumorigenic process [235].

Given the effect of the NAD+-sirtuin pathway on mitochondrial and metabolic homeostasis, novel supplementation strategies (e.g., using NR or NMN) may be exploited to increase endogenous NAD+ availability in the treatment of metabolic and age-related diseases. This is the time for carefully designed human clinical studies to further examine these compounds before we can propose them as being useful nutraceuticals to counteract metabolic disease.

NAD catabolism, GTP and mitochondrial dynamics

Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics

2. Pathology of NAD+ catabolism and downstream metabolic products
NAD+ is an important cofactor involved in multiple metabolic reactions that have a central role in cellular metabolism and energy production (Belenky et al., 2007; Frederick et al., 2016; Mouchiroud et al., 2013). Normally, NAD+ levels decline with age (CamachoPereira et al., 2016; Massudi et al., 2012; Verdin, 2015; Zhu et al., 2015) and NAD+ pools have been shown to decrease during neurodegenerative diseases and after ischemia-reperfusion or TBI (Kauppinen and Swanson, 2007; Martire et al., 2015; Park et al., 2016; Verdin, 2015; Zhou et al., 2015). This decline could be the result of the increased activity of several enzymes that use NAD+ as their substrate; these include: sirtuins, ADP-ribosyl transferases (ARTs), and the cyclic ADP-ribose synthases/ADP-ribosyl cyclases (CD38 and CD157) (Belenky et al., 2007; Feijs et al., 2013; Jesko et al., 2016; Malavasi et al., 2008; Mayo et al., 2008). These NAD+ dependent enzymes hydrolyze NAD+ to ADP-ribose (ADPr) or an ADPr variation (e.g. cyclic ADPr), and nicotinamide (Nam). The resulting Nam can function in a negative feedback fashion by inhibiting the activity of NAD+ dependent enzymes (Avalos et al., 2005; Long et al., 2016; Suzuki et al., 2010), however levels of Nam are usually too low to inhibit sirtuins activity under physiological conditions (Liu et al., 2013). Nam is also the precursor for nicotinamide mononucleotide (NMN), the immediate precursor to NAD+ via the salvage pathway (Imai and Guarente, 2014). The conversion of NMN to NAD+ is ATP dependent and catalyzed by the NMN adenyl transferases (NMNATs): NMNAT1 (nucleus), NMNAT2 (Golgi apparatus/endosomes), and NMNAT3 (mitochondria)

Additionally, commonly in acidic environments, NAD+ levels can also be depleted by NAD+ kinase, by phosphorylating NAD+ to NADPþ (Zhang et al., 2016a).

3. NAD+/NADH
Other then being a substrate, NAD+ plays a key role in cellular bioenergetic metabolism by reversibly being reduced to NADH and ultimately contributing to ATP generation via the glycolytic pathway and in the mitochondria through oxidative phosphorylation (Srivastava, 2016). The reduction of NAD+ is most apparent in the mitochondria, with liver mitochondrial NAD+/NADH ratios being tightly controlled around 7 to 8, while cytoplasmic ratios being much higher, ranging from 60 to 700 in most cells (Stein and Imai, 2012). Techniques to get more accurate measurements are relatively new but suggest that NAD+ pools and NAD+/NADH ratios can vary based on cell type (Cambronne et al., 2016; Christensen et al., 2014). The reduction of NAD+ to NADH is essential for the glyceraldehyde 3-phosphate (GAPDH) step of glycolysis and multiple steps in the tricarboxylic acid cycle (TCA) (Akram, 2014; Sirover, 1999). NADH generated in the mitochondria will be oxidized to NAD+ and donate electrons to complex I of the electron transport chain (Sazanov, 2015).

4. ARTs/PARP1/mtPARP1
There are 22 known human genes that encode proteins with an ADP-ribosyltransferase (ART) catalytic domain. These proteins transfer ADPr from NAD+ onto targeted amino acid residues of proteins. To generate the ADPr chains, ART’s release Nam from NAD+, and then form an a(1e2)O-glycosidic bond between two ADPr molecules. This post-translational modification is named either monoor poly-ADP-ribosylation (MARylation or PARylation), depending if the ART’s are transferring a single ADPr or are generating a chain (Gibson and Kraus, 2012; Hottiger et al., 2010). In vitro, these chains of APD-ribose can be around 200 residues, with the length and branching being dependent on the concentration of available NAD+ (Alvarez-Gonzalez and Jacobson, 1987; Alvarez-Gonzalez and Mendoza-Alvarez, 1995). This process of PARylation is reversible and is constantly being regulated by poly (ADP-ribose) glycohydrolase (PARG) and the mainly mitochondrial, ADP-ribosyl hydrolase-3 (ARH3) (Di Meglio et al., 2003; Mashimo et al., 2013; Niere et al., 2012). PARG and ARH3 associated degradation of PAR can result in releasing intact PAR chains (endoglycohydrolase) or by releasing free ADR-ribose (exoglycohydrolase) (Gibson and Kraus, 2012).
Poly (ADP-ribose) polymerase 1 (PARP1), also known as ADPribosyltransferase diphtheria toxin-like one (ARTD1), is responsible for the majority of PARylation and is involved in the repair of moderate single stranded DNA damage.

Historically, PARP1 has been reported to be exclusively nuclear, although there is growing evidence for PARP1 associated mitochondrial functions. Several studies report PARP1 can modulate mitochondria from the nucleus through: PAR translocation from the nucleus to the mitochondria, depletion of cellular NAD+ pools, and epigenetic regulating of nuclear genes that are involved in mitochondrial DNA transcription and repair (Alano et al., 2010; Fang et al., 2014; Fatokun et al., 2014; Lapucci et al., 2011). Additionally, several studies report that PARP1 can directly interact with mitochondria and that there is an intramitochondrial localized PARP1 (mtPARP1) (Lai et al., 2008; Rossi et al., 2009; Szczesny et al., 2014; Zhang et al., 2016b). Brunyanszki and colleagues, reviewed the growing body of work suggesting there is a mitochondrial PARP1, in which they hypothesize mtPARP1 is activated prior to nuclear PARP1 in the event of oxidative stress (Brunyanszki et al., 2016).

When discussing the possibility of mtPARP1 it is important to note that mitochondrial and cytosolic NAD+ pools are mostly distinct from each other, with studies showing mitochondrial NAD+ pools are maintained for over 24 h after cytoplasmic NAD+ depletion (Alano et al., 2007; Pittelli et al., 2010; Stein and Imai, 2012). Furthermore, the mechanism of mammalian mitochondrial NAD+ regulation is still unclear and it is unknown whether there is a mitochondrial membrane transporter to shuttle NAD+, NMN, or nicotinamide riboside (NR) between the mitochondria and cytosol (Stein and Imai, 2012). Using a novel NAD+ biosensor, Cambronne and colleagues determined that mitochondrial NAD+ pools appear to be regulated differently in various cell types. They found NMNAT3 depletion caused a significant decrease in mitochondrial NAD+ in HEK293T cells but not HeLa cells (Cambronne et al., 2016).

Additionally, Nikiforov and colleagues, reported using stably transfected 293mitoPARP cells that mitochondrial NAD+ pools are regulated by cytosolic NMN being transported to the mitochondria and then being converted to NAD+ by NMNAT3 (Nikiforov et al., 2011). These studies suggest NMNAT3 is essential for mitochondrial NAD+ production in some cells while other cells might use a yet to be discovered mitochondrial NAD+ shuttle. Furthermore, mitochondrial NAD+ pools can vary greatly depending on the cell type: cardiac myocytes (10.02 ± 1.82 nmol/mg protein), neurons (4.66 ± 0.37 nmol/mg protein), and astrocytes (3.20 ± 1.02 nmol/ mg protein). The study also reported cytoplasmic NAD+ pools were most similar to those in neuronal mitochondria. This suggests NAD+ pools are split between the cytoplasm and mitochondria differently depending on the cell type (Alano et al., 2007). Therefore, it is reasonable to suggest that the pathologic outcome of PARP1-dependent depletion of mitochondrial or cytosolic NAD+ could vary greatly depending on the cell type.

For example, Modis and colleagues reported PARP1 depleted mitochondrial NAD+ pools at a much faster rate than cytosolic. They used PARP1 (shPARP1) silencing in A549 cells, which resulted in a four-fold increase in mitochondria NAD+, while showing no change in total cellular NAD+ (Modis et al., 2012).

Several studies have shown substantial amounts of mitochondrial targeted proteins that are subjected to PARylation (Brunyanszki et al., 2016; Gagne et al., 2012). It has also been reported that mtPARP1 interacts with mitochondrial specific DNA base excision repair enzymes. Interestingly, nuclear PARP1 has a positive effect on DNA repair, while mtPARP1 was observed to negatively affect base excision repair enzymes EXOG and DNA polymerase gamma (Polg) (Rossi et al., 2009; Szczesny et al., 2014). In contrast, Rossi and colleagues reported that mtPARP1 is important for normal mitochondrial DNA ligase III function (Rossi et al., 2009). The same study also showed that mitofilin, a mitochondrial inner membrane (IM) protein, plays an important role in regulating the mitochondrial localization of PARP1 (Rossi et al., 2009).

Normally, mitofilin is involved in keeping the cristae membranes connected to the inner boundary membrane and promoting protein import through the mitochondrial intermembrane space (IMS) assembly pathway via being coupled to the outer membrane (OM) (von der Malsburg et al., 2011). Interestingly, mtPARP1 and DNA ligase III co-occupy the D-loop region in mtDNA, and mitofilin depletion results in decreased mtPARP1 and impaired binding of DNA ligase III to mtDNA (Rossi et al., 2009). These studies have led to Brunyanszki and colleagues hypothesizing that mtPARP1 is normally bound to mitofilin but under oxidative stress is released, consuming mitochondrial NAD+, and PARylating mitochondrial proteins, including the electron transport chain complexes (Brunyanszki et al., 2016).

Conversely, parthanatos is cell death induced by nuclear PARP1 overactivation, as a result of extensive nuclear DNA damage (Fatokun et al., 2014). Excessive PARP1-dependent PARylation causes some PAR chains to translocate to the cytoplasm and bind to apoptosis inducing factor (AIF) on the mitochondrial OM (Wang et al., 2011). AIF is predominately located in the mitochondrial IMS and attached to the IM; although it has been reported that about 30% of AIF is loosely associated with the cytosolic side of the OM (Yu et al., 2009). The binding of PAR to AIF helps induce the release of AIF from the mitochondria and it’s translocation to the nucleus (Fatokun et al., 2014; Wang et al., 2011). This translocation of AIF to the nucleus causes chromatin condensation, and large scale DNA fragmentation resulting in cell death (Yu et al., 2002).

PARP1 overactivation is also associated with NAD+ and ATP depletion (Gerace et al., 2014). PARP1 has been reported to inhibit ATP generation by glycolysis via PAR translocating from the nucleus into the cytoplasm and binding to hexokinase 1 (HK1) on the mitochondrial OM (Andrabi et al., 2014; Fouquerel et al., 2014). The binding of PAR to HK1 can allosterically decrease HK1 activity and cause HK1 to migrate to the cytoplasm, resulting in the inhibition of glycolysis and the reduction of mitochondrial ATP production (Fouquerel et al., 2014).

5. Sirtuins/OAADPR
The sirtuins are class III histone deacetylases (HDACs) and consist of seven isoforms SIRT1-SIRT7, with SIRT3SIRT5 being localized in the mitochondria. Sirtuins require NAD+ for their activity and modulate protein signaling and function by removing acetyl groups attached to lysine residues (Jesko and Strosznajder, 2016; Jesko et al., 2016). In the mitochondria, SIRT3 is the major lysine deacetylase; reports show SIRT3 Knockout (KO) mice and cells have hyperacetylation of mitochondrial proteins, while the same effect was not seen in SIRT4 or SIRT5 KO animals (Lombard et al., 2007; Sol et al., 2012). Sirtuins remove acetyl groups by first cleaving Nam from NAD+ (forming ADPr) and then transferring the acetyl group from the substrate to ADPr, resulting in the formation of O-acetyl-ADP-ribose (OAADPr) (Borra et al., 2004). Uniquely, SIRT5 hydrolyzes malonyl and succinyl lysines, forming O-malonyl-ADPr and O-succinyl-ADPr (Du et al., 2011).

Macro domain (macroD) proteins have been reported to hydrolyze OAADPr to ADPr (Chen et al., 2011). Normally these proteins bind to NAD+ metabolites (e.g. PAR), and are involved in a broad range of biological functions, including DNA repair (Han et al., 2011). ARH3, which normally plays a role in degrading PAR to ADPr, was also found to hydrolyze OAADPr to ADPr in a Mg2þ dependent mechanism in cells (Ono et al., 2006). This finding suggests ARH3 could play a role in both OAADPr and ADPr regulation. Interestingly, compared to PARP1, SIRT1 has a lower affinity (higher Km) for NAD+ and is less effective at breaking down NAD+ (lower Km/Kcat). This suggests PARP1 activation could deplete NAD+ pools at a faster rate then SIRT1 and consequently reduce the function of the NAD+ dependent sirtuins (Canto et al., 2013).

6. CD38/CD157
The membrane proteins CD38 and the less abundant CD157 are multifunctional ecto-enzymes that use NAD+ as a substrate to form cyclic ADP-ribose (cADPr), or under acidic conditions use NADPþ as a substrate to form nicotinamide acid ADP (NAADP). Additionally, CD38 and CD157 can act as glycohydrolases and hydrolyze cADPr to ADPr and under acidic conditions convert NAADP to ADPrphosphate (ADPrP) (Malavasi et al., 2008; Quarona et al., 2013). Normally, cADPr, ADPr, ADPrP, and NAADP play roles in cell signaling pathways and regulating cytoplasmic Ca2þ fluxes (Malavasi et al., 2008; Quarona et al., 2013; Sumoza-Toledo and Penner, 2011). CD38 and CD157 are commonly located on the membranes of immune cells but can be found throughout the body; playing a role in the immune response, hormone secretion, cell activation, egg fertilization, and muscle contraction (Malavasi et al., 2008).
In CD38KO mice, brain tissue NAD+ levels have been observed to be up to 10 fold higher than in wild type mice, although the rate of NAD+ consumption by CD38 may vary greatly depending on the brain region (Aksoy et al., 2006; Long et al., 2016). Interestingly, Camacho-Pereia and colleagues showed CD38 levels not only increase with age but also correlate with a decline of NAD+, suggesting CD38 might be the major cause of the decline of NAD+ during normal aging (Camacho-Pereira et al., 2016). Surprisingly, our lab found CD38KO mice to have dramatically higher PAR levels (particularly in neurons) and decreased PARG activity when compared to WT mice. These findings suggest CD38KO mouse studies might be difficult to interpret due to complexities derived from alterations in several enzymes involved in NAD+ catabolism as a result of knocking out the constitutively expressed CD38 gene (Long et al., 2016).

7. PARG/AHR3
Uncontrolled PARP1 activation has been proposed to deplete intracellular NAD+ and ATP (Fouquerel et al., 2014; Szabo and Dawson, 1998). To regulate PARP1 activation, PARG (nuclear/cytoplasmic) and AHR3 (predominantly mitochondrial) degrade PAR, which can have toxic effects (Dumitriu et al., 2004; Mashimo et al., 2013). It has been suggested that the free PAR polymers is the major toxic molecule generated by the combined activity of PARP1 and PARG (Andrabi et al., 2006). Several reports suggested that many types of PAR structures could play a crucial role in stress-dependent signaling processes in vivo (Dawson and Dawson, 2004; Hong et al., 2004). However, most free or protein-associated PAR polymers are rapidly degraded in vivo to ADPr (Jacobson et al., 1983). In the nucleus and cytoplasm, PARG is the predominant PAR glycohydrolase, reportedly acting as an endoglycohydrolase, hydrolyzing off chunks of PAR from the protein, as well as an exoglycohydrolase, releasing ADPr (Wang et al., 2014).
Recently, Niere and colleagues discovered that ARH3 and not PARG is responsible for degrading PAR to ADPr in the mitochondrial matrix (Niere et al., 2012). ARH3 has also been postulated to play a protective role against parthanatos in the cytoplasm and nucleus (Mashimo et al., 2013). In this theory, PARG acts as an endoglycohydrolase, releasing PAR fragments from proteins in the nucleus, allowing translocation of PAR from the nucleus to the cytoplasm. To counteract this, ARH3 hydrolyzes the detached PAR, releasing ADPr, preventing PAR induced AIF translocation to the nucleus and subsequent cell death via parthanatos (Mashimo et al., 2013).

8. NUDIX hydrolase, enzyme controlling cellular and mitochondrial ADP-ribose levels
The intracellular levels of ADPr are tightly controlled by specific ADPr hydrolases, which hydrolyze ADPr to adenosine monophosphate (AMP) and D-ribose 5-phosphate; thereby, limiting free ADPr accumulation (Fernandez et al., 1996; Ribeiro et al., 1995). This family of enzymes catalyze the hydrolysis of a nucleoside
diphosphate linked to another moiety x, hence the acronym “NUDIX” (Mildvan et al., 2005). For NUDIX hydrolases to be active, Mg2þ or a similar cofactor (e.g. Zn2þ) must bind to NUDIX (Zha et al., 2006). Cloning and expression of human cDNA coding for proteins with NUDIX motifs have revealed two human ADPr hydrolases: NUDT5, an ADP-sugar pyrophosphatase (Gasmi et al., 1999), and NUDT9, an ADPr pyrophosphatase (Perraud et al., 2003). While NUDT9 is highly specific for ADPr, NUDT5 only has a preference for ADPr and can also hydrolyze other ADP-sugar conjugates (Zha et al., 2008).
The human NUDT9 gene gives rise to two alternatively spliced mRNAs, NUDT9a and NUDT9b (Li et al., 2002). NUDT9a possesses a putative mitochondrial targeting sequence and accumulates in mitochondria, while NUDT9b is a cytosolic enzyme (Lin et al., 2002; Perraud et al., 2003). Thus, NUDT9 can play an important role in cellular bioenergetic metabolism, particularly under stressed conditions that can lead to high levels of mitochondrial ADPr.

This is important since free ADPr has been reported to be a competitive inhibitor of NADH oxidation at complex I of the electron transport chain (Zharova and Vinogradov, 1997). Similarly, in rat ventricular myocytes ADPr was reported to inhibit ATP-sensitive Kþ channels (Kwak et al., 1996). Additionally, excessive cytosolic ADPr, cADPr, NAADP, OAADPr, and ADPrP are potential agonists along with ROS, and Ca2þ of the transient receptor potential melastatin 2 (TRPM2) ion channel by binding to the channels NUDT9-H domain. Opening of the TRPM2 ion channel is important for Ca2þ signaling; although, overactivation can cause an influx of Ca2þ, leading to inflammation through chemokine recruitment, and ROS (Grubisha et al., 2006; Shimizu et al., 2013; Toth et al., 2015; Yamamoto and Shimizu, 2016). Interestingly, TRPM2-mediated neuronal death in ischemic brain injury is dimorphic, with TRPM2 knockdown only protecting male brains (Shimizu et al., 2013).

NUDIX hydrolases have also been shown as potential metabolizing enzymes of the byproduct of sirtuin activity, OAADPr, producing AMP and O-acetylated ribose 5’-phosphate (Tong and Denu, 2010). This OAADPr hydrolysis was shown using the NUDIX hydrolases YSA1 (yeast), NUDT5 (mouse), and NUDT9 (human). The same study reported YSA1 and NUDT5 to have a similar affinity to hydrolyzing OAADPr or ADPr, while NUDT9 was 500 fold more efficient hydrolyzing ADPr compared to OAADPr (Rafty et al., 2002).

9. Mitochondrial NAD+ catabolism
The presence of NUDT9a in mitochondria suggests that enzymes generating ADPr are also localized in the intra-mitochondrial compartment. As discussed earlier, several studies have reported that PARP1 is localized not only in the nucleus, but also in the mitochondria (Du et al., 2003; Lai et al., 2008; Pankotai et al., 2009; Rossi et al., 2009). Furthermore, ARH3 has been reported to control mitochondrial PAR hydrolysis (Niere et al., 2012). Concerted activity of mitochondrial PARP1 and ARH3 results in increased levels of matrix ADPr. Intra-mitochondrial ADPr is then metabolized by the NUDT9a, generating AMP and D-ribose 5-phosphate (Perraud et al., 2003). This NUDT9 dependent intra-mitochondrial AMP generation from the hydrolysis of ADPr has been hypothesized to trigger an AMP-dependent mitochondrial failure due to inhibition of the adenine nucleotide translocase (ANT) (Formentini et al., 2009). The primary role for ANT is to exchange ADP/ATP across the mitochondrial IM, thus releasing ATP into the cytosol and importing ADP into the matrix to be phosphorylated via oxidative phosphorylation (Liu and Chen, 2013). In the cell, AMP is generally converted to ADP by adenylate kinases (AKs) (Panayiotou et al., 2014).

10. Adenylate kinases
Adenylate kinases (AKs) are found throughout the body and are involved in homeostasis of cellular adenine nucleotides by transferring a phosphate from a donor, usually ATP, to AMP, resulting in ADP. There are 9 known AKs (AK1-AK9), which differ by subcellular localization, tissue distribution, phosphate donor, and substrate. While AK2, AK3, and AK4 are all mitochondrial, only AK3 and AK4 are found in the brain (Panayiotou et al., 2014). Noma and colleagues reported AK3 was specific to the mitochondrial matrix, found in most tissues, and specifically can only use GTP or ITP as a phosphate donor; making AK3 a possible major consumer of mitochondrial GTP. AK4, is also mitochondrial but is found predominately in the kidney and has low enzymatic activity, with a slight preference to GTP over ATP as a phosphate donor (Noma et al., 2001; Panayiotou et al., 2010).

11. Mitochondrial dynamics and downstream NAD+ metabolites
Recently, it was recognized that an imbalance in mitochondrial fusion and fission could result in deterioration of mitochondrial bioenergetics (Knott et al., 2008; Stetler et al., 2013). Both fusion and fission have physiologic functions (Knott and Bossy-Wetzel, 2008). For example, mitochondrial fission can have a protective role by segregation of damaged and inactive mitochondria and through facilitating autophagic clearance (Barsoum et al., 2006). When mitochondria in cells are stressed, they undergo fission, a process that is reversible since small fragmented organelles can fuse and regain the normal pre-insult morphology (Barsoum et al., 2006; Knott and Bossy-Wetzel, 2008; Owens et al., 2015). The fission of mitochondria is frequently observed in neurons exposed to mitochondrial toxins or excitotoxic levels of glutamate (Barsoum et al., 2006).

Reduction of mitochondrial length compared to control tissue was observed in the brain following focal ischemia, suggesting post-insult mitochondrial fragmentation (Barsoum et al., 2006). We detected extensive mitochondrial fission following global ischemic insult in both hippocampal neurons and astrocytes (Owens et al., 2015). Fission and fusion are regulated by dynamin family proteins that act as GTPases. Fusion is controlled by mitofusin-1 and -2 (MFN1 and MFN2), which are localized in the mitochondrial OM, and by the mitochondrial IM optic atrophy protein (OPA1) (Hoppins et al., 2007; Song et al., 2009). Since these enzymes are GTP binding proteins the efficiency of fission and fusion depends on the cytosolic and mitochondrial GTP levels. Therefore, Intra-mitochondrial GTP depletion could inhibit OPA1, while cytosolic GTP depletion could inhibit the MFNs and Drp1. Interestingly IM fusion requires high GTP levels while OM fusion has less demanding GTP requirements (Escobar-Henriques and Anton, 2013). Our previous studies suggests that there is dramatic fragmentation of mitochondria following ischemic insult and only mitochondria in ischemia resistant cells re-fuse at a later recovery time (Owens et al., 2015). Furthermore, genotoxic stress-induced mitochondrial NAD+ catabolism leads to significant reduction of GTP pools in mitochondrial matrix, suggesting a causal link between downstream NAD+ degradation products and GTP metabolism (unpublished data). Similarly, Dagher reported using cell culture that cellular GTP levels are severely depleted during chemical anoxia (Dagher, 2000).

12. Intra-mitochondrial GTP metabolism
GTP molecules in mitochondria are generated by nucleoside diphosphate kinase (NDPK) or succinyl-CoA synthetase (ligase)
(SCS) in the citric acid cycle (Sanadi et al., 1954) (Fig. 1). NDPK converts GDP to GTP by using ATP as a phosphate donor. SCS generates succinate and CoA from succinyl-CoA, and GDP, as a cofactor, is phosphorylated to GTP in the presence of inorganic phosphate. GTP is involved mainly in energy transfer within the cell. In mitochondria, GTP with ATP is utilized during protein translocation into the mitochondrial matrix (Sepuri et al., 1998). As mentioned above, AK3 is located in the mitochondrial matrix and uniquely uses GTP instead of ATP as a phosphate donor to phosphorylate AMP (Noma et al., 2001). Downstream NAD+ catabolism leads to generation of intra-mitochondrial AMP due to ADPr hydrolysis by NUDT9a. AMP is then converted to ADP by transferring high-energy phosphate from intra-mitochondrial GTP by AK3. Concurrently, low levels of intra-mitochondrial NAD+ also compromise TCA cycle enzyme activities, including a-ketoglutarate dehydrogenase that produces succinyl-CoA. Reduced succinyl-CoA compromises GTP production by SCS, which further depletes mitochondrial GTP and can inhibit mitochondrial fusion (Fig. 1).

13. AMPK/MFF/Drp1 mitochondrial fission
AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is activated by the depletion of energy levels and is important in cellular metabolism by phosphorylating numerous cellular targets. AMPK is predominately located in the cytosol and is heterotrimeric, consisting of three subunits, abg; a is catalytic, b is scaffolding, and g is regulatory. AMPK is activated by phosphorylation of Thr172 in the a subunit by liver kinase B1 (LKB1) or calmodulin-dependent protein kinase kinase-b (CaMKKb) (Kim et al., 2016; Li et al., 2015). Cellular AMP: ATP ratios are reported to be the main regulator of AMPK, either with inhibition of mitochondrial ATP production or high levels of AMP acting as activators (Hardie et al., 2012).

Ekholm and company reported that after 15 min of ischemia AMP levels increase 25 fold, while concurrently ATP levels decrease 25 fold (Ekholm et al., 1993). AMP is able to allosterically activate AMPK by binding to the g subunit, which stimulates LKB1 phosphorylation of Thr172 on the a subunit (Fig. 2). Conversely, ATP reduces AMPK activation by competing with AMP for g subunit binding (Gowans et al., 2013). Furthermore, AMPK can also be activated in a Ca2þ dependent pathway by CaMKKb phosphorylation of Thr172 on the a subunit (Woods et al., 2005).

Interestingly, AMPK activation has recently been directly linked to mitochondrial fission (Fig. 1). Toyama and colleagues reported activated AMPK is able to phosphorylate mitochondrial fission factor (MFF) at Ser155 and Ser172 on the mitochondrial OM. MFF is a dominant receptor for the mitochondrial fission protein DRP1 (Toyama et al., 2016). The phosphorylation of MFF by AMPK has been shown to catalyze mitochondrial fission by causing the translocation of DRP1 from the cytoplasm to the mitochondrial OM. Drp1 wraps around constriction sites on the mitochondrial OM, causing fission, and can eventually lead to mitophagy (Toyama et al., 2016; Wang and Youle, 2016).

14. Conclusion
Pathophysiology of neurodegenerative diseases and acute brain injury encompass DNA damage with subsequent increases in nuclear and mitochondrial PARP1 activation and the depletion of cellular and mitochondrial NAD+ pools. Mitochondrial NAD+ depletion can cause inhibition of oxidative phosphorylation and TCA cycle enzymes activity. PAR is degraded in the nucleus and cytoplasm by PARG/AHR3 and by ARH3 in the mitochondria. Consequently, high PARylation will significantly increase in ADPr levels, which could inhibit complex I in the mitochondria or bind the NUDT9-H domain on TRPM2 receptors, causing influxes of Ca2þ
A. Long et al. / Neurochemistry International xxx (2017) 1e9 5

6 A. Long et al. / Neurochemistry International xxx (2017) 1e9
Fig. 1. Schematic diagram linking mitochondrial NAD+ catabolism with the depletion of mitochondrial GTP and mitochondrial fragmentation in a neurodegenerative disease/acute brain injury model. Localization of enzymatic reactions are color-coded: Blue (mitochondrial), red (cytoplasmic), green (nuclear and mitochondrial), purple (insult or outcome). Red arrows show changes in enzymatic activity or substrate levels as a result of the disease/injury insult. Mitochondrial and nuclear poly (ADP-ribose) polymerase 1 (PARP1) leads to generation of Poly (ADP-ribose) (PAR) as a result of disease or insult. ADP-ribose (ADPr) is generated by hydrolyzing PAR by NAD+ glycohydrolases; ADP-ribosyl hydrolase-3 (ARH3) and poly (ADP-ribose) glycohydrolase (PARG), and then is further degraded to AMP by NUDIX hydrolases NUDT9a and NUDT9b.

Accumulated AMP is then phosphorylated by adenylate kinse 3 (AK3) to ADP by transferring high-energy phosphate from GTP. Depletion of NAD+ will inhibit the TCA cycle enzyme succinyl-CoA synthetase (SCS)-linked GTP generation. GTP can also be generated by nucleoside diphosphate kinase (NDPK), which phosphorylates GDP by using ATP as phosphate donor. However, since the TCA cycle and mitochondrial respiration is inhibited due to low NAD+ levels, the NDPK activity will be limited.

This depletion of mitochondrial GTP can inhibit fusion by reducing the functionality of GTP dependent mitochondrial fusion enzymes, such as optic atrophy protein (OPA1). Elevated cytosolic AMP can also shift the cellular AMP: ATP ratio, which leads to liver kinase B1 (LKB1) phosphorylation and activation of AMP-activated protein kinase (AMPK). Once activated, AMPK can phosphorylate mitochondria fission factor (MFF), which causes an increase in cytosolic Drp1 migration to the mitochondrial OM and subsequent fission. Thus, depletion of NAD+, reduction of mitochondrial GTP, and elevated AMP can lead to excessively fragmented mitochondria.

Fig. 2. Simplified schematic diagram of the overall mechanism linking NAD+ catabolism to mitochondrial fragmentation. Insult caused from disease or acute injury causes NAD+ depletion and PARP1 activation. This leads to high levels of ADPr and AMP in the cell and mitochondria. Mitochondrial AMP phosphorylation and low NAD+ levels cause depletion in mitochondrial GTP and inhibition of GTP dependent mitochondrial fusion proteins. High levels of AMP also increase AMP: ATP ratios, which leads to the activation of AMPK and subsequent mitochondrial fission.

Additionally, mitochondrial NUDT9a and cytosolic NUDT9b/NUDT5 regulate ADPr levels by hydrolysis, producing AMP and D-ribose-5-phosphatate.
We propose a possible mechanism for elevated mitochondrial AMP levels to cause a shift to mitochondrial dynamics to fission (Fig. 2). First, high AMP levels have been reported to inhibit ATP production by inhibiting ANT. Decreasing ATP production along with the already high levels of AMP will result in a spike in the AMP: ATP ratio, thus activating AMPK. AMPK has been shown to regulate mitochondrial fission by phosphorylating MFF, resulting in the translocation of Drp1 to the mitochondria and Drp1 induced mitochondrial fission. Additionally, high AMP levels will increase AK3 activity, which consumes mitochondrial GTP. Depletion of mitochondrial GTP levels can disrupt GTPases, such as the mitochondrial fusion proteins.

Thus, in stress conditions, NAD+ depletion can significantly deplete mitochondrial GTP levels by inhibiting the TCA cycle and through high AK3 activity, resulting in excessively fragmented mitochondria (Fig. 1).
The link between NAD+ catabolism and mitochondrial fragmentation is an understudied area with translational potential. Understanding mitochondrial PARP1 activity and finding ways to regulate cellular ADPr and AMP levels in disease/acute injury states through studying the NUDIX hydrolases could be a target to reduce mitochondrial fragmentation. Additionally, better understanding of intra-mitochondrial GTP metabolism and the complexities of AMPK in relation to mitochondrial dynamics could be targets for future studies.

Sulforaphane increases metabolism to ameliorate obesity and insulin resistance

Study published here: Glucoraphanin ameliorates obesity and insulin resistance through adipose tissue browning and reduction of metabolic endotoxemia in mice

Low-grade sustained inflammation, triggered by chronically high levels of proinflammatory cytokines and gut microbiota-derived circulatory lipopolysaccharide (LPS), links obesity with comorbidities such as insulin resistance and nonalcoholic fatty liver disease (NAFLD) (1,2).

Although a number of pharmacological treatments for obesity and NAFLD have been tested, few drugs are clinically available owing to the lack of long- term efficacy and safety concerns (3,4). Thus, a novel therapeutic approach that would improve energy metabolism and reduce chronic inflammation in obesity is sorely needed.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a basic leucine zipper transcription factor, is widely expressed in human and mouse tissues, and serves as a defense response against extrinsic and intrinsic stressors (5). Upon exposure to electrophilic and oxidative stress, Nrf2 detaches from its repressor, Kelch-like ECH-associated protein 1-nuclear factor (Keap1), and is translocated from the cytoplasm into the nucleus.

This translocation leads to the transcriptional activation of genes encoding phase 2 detoxifying and antioxidant enzymes (6).

In addition to the ubiquitous induction of cytoprotective genes, Nrf2 regulates a large number of genes involved in glucose and lipid metabolism. In the liver, the constitutive activation of Nrf2 via Keap1 knockdown represses the expression of genes involved in gluconeogenesis (7) and lipogenesis (8), thereby alleviating obesity, diabetes, and hepatic steatosis.

Accordingly, synthetic Nrf2 inducers such as synthetic triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO)-imidazolide (9), CDDO-methyl ester (known as bardoxolone methyl) (10), and dithiolethione analog, oltipraz (11), have been shown to ameliorate high-fat diet (HFD)-induced obesity and diabetes.

These synthetic Nrf2 inducers also decrease liver and adipose tissue lipogenesis, and enhance glucose uptake in skeletal muscles. However, the mechanisms by which Nrf2 enhances energy metabolism in response to a HFD remain largely unknown.

Although enhanced Nrf2 signaling has shown promising results in several animal studies, the synthetic Nrf2 inducers have caused adverse cardiac events and gastrointestinal toxicities in clinical trials (12,13).

These observations prompted us to explore a safer Nrf2 inducer for the treatment of obesity, insulin resistance, and NAFLD.

Sulforaphane, an isothiocyanate derived from cruciferous vegetables, is one of the most potent naturally occurring Nrf2 inducers; this compound exhibits anticancer activity in cancer cell lines and in carcinogen-induced rodent models (14).

Among the cruciferous vegetables, broccoli sprouts are the best source of glucoraphanin, a stable glucosinolate precursor of sulforaphane (15). In both rodents and humans, glucoraphanin is hydrolyzed by gut microbiota-derived myrosinase into bioactive sulforaphane prior to intestinal absorption (16). A recent clinical study demonstrated the safety of orally administered glucoraphanin (17).

In the present study, we examined the dietary glucoraphanin-mediated modulation of systemic energy balance and the mitigation of chronic inflammation, insulin resistance, and NAFLD in diet-induced obese mice.

Research Design and Methods Glucoraphanin preparation
The sulforaphane precursor, glucoraphanin, was prepared as previously described (17) with minor modifications. Briefly, one day after germination from broccoli seeds (Caudill Seed Company, Louisville, KY), sprouts were boiled in water for 30 min.

The water extract was mixed with dextrinized cornstarch and subsequently spray-dried to yield an extract powder containing 135 mg of glucoraphanin per gram (0.31 mmol/g) (18). The total glucoraphanin titer in the resulting powder was determined by HPLC as previously reported (19).

Mice and diets
Male C57BL/6JSlc mice were purchased from Japan SLC (Hamamatsu, Japan) at 7 weeks of age. Nrf2-knockout (Nrf2-/-) mouse strain (RBRC01390; C57BL/6J background) was provided by RIKEN BRC (Tsukuba, Japan) (6). After a week of acclimation, mice were fed normal chow (NC; containing 2.2% dextrinized cornstarch, 10% kcal from fat, Research Diets, New Brunswick, NJ), NC containing 0.3% glucoraphanin (NC- GR; containing 2.2% extract powder), a high-fat diet (HFD; containing 2.2% dextrinized cornstarch, 60% kcal from fat, #D12492; Research diets), or a HFD containing 0.3% glucoraphanin (HFD-GR; containing 2.2% extract powder) for 14 weeks.

Both the NC and the HFD containing cornstarch or glucoraphanin were prepared by Research Diets. All mice studied were maintained on a 12 h light/dark cycle at 24–26°C with free access to water and food. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at Kanazawa University, Japan.

Indirect calorimetry
After 3 weeks of feeding, mice were individually housed in an indirect calorimeter chamber at 24–26°C (Oxymax; Columbus Instruments, Columbus, OH). Calorimetry, daily body weight, and daily food intake data were acquired during a 3-day acclimation period, followed by a 2-day experimental period. Oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured in each chamber every 20 minutes. The respiratory exchange ratio (RER = VCO2/VO2) was calculated using Oxymax software. Energy expenditure was calculated as shown below and normalized to the body mass of each subject.
Energy expenditure = VO2 × [3.815 + (1.232 ×RER)]

Metabolic measurements and biochemical analyses
Metabolic parameters, body fat composition, insulin sensitivity, and glucose tolerance were assessed as previously described (20). Plasma LPS levels were analyzed using a Limulus amebocyte lysate assay kit (QCL-1000; Lonza, Allendale, NJ). Plasma LPS binding protein (LBP) levels were determined using an ELISA kit (Enzo Life Sciences, Farmingdale, NY).

Immunoblotting was performed with primary antibodies (Supplementary Table 1) as previously described (20). mRNA expression levels were determined by quantitative real-time PCR using SYBR Green with the primers (Supplementary Table 2) as previously described (20).

Isolation and differentiation of inguinal white adipose tissue-derived primary beige adipocytes
Stromal vascular fractions (SVFs) from inguinal white adipose tissue (WAT) of 7- week-old wild-type and Nrf2-/- mice were prepared as previously reported (21). At confluence, SVF cells were induced for 2 days with differentiation medium containing DMEM/F-12 supplemented with 10% FBS, 20 nM insulin, 1 nM T3, 5 μM dexamethasone, 500 μM isobutylmethylxanthine, 125 μM indomethacin, and 0.5 μM rosiglitazone (all from Sigma-Aldrich, St. Louis, MO).

Induced cells were subsequently cultured in maintenance medium (DMEM/F-12 containing 10% FBS, 20 nM insulin, and 1 nM T3) for 5 days and treated with DMSO or sulforaphane (Toronto Research Chemicals, Toronto, Canada) at the indicated concentrations for 48 h.

Fluorescence-activated cell sorting (FACS)
Cells from the liver and epididymal WAT were prepared as previously described (22). Isolated cells were incubated with Fc-Block (BD Bioscience, San Jose, CA), and subsequently incubated with fluorochrome-conjugated antibodies (Supplementary Table 1).

Flow cytometry was performed using a FACSAria II (BD Bioscience), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Analysis of gut microbiota via pyrosequencing of the 16S rRNA gene
Metagenomic DNA was extracted from mouse cecal content with a QIAamp DNA stool kit (Qiagen, Hilden, Germany). The V1–V2 region of the 16S rRNA gene was amplified using primer sets as previously reported (23).

Mixed samples were prepared by pooling approximately equal amounts of PCR amplicons from each sample and subjected to a GS Junior System (Roche Diagnostics, Basel, Switzerland) for subsequent 454 sequencing.

Pre-processing and taxonomic assignment of sequencing reads were conducted as described previously (23) and separated by unique barcodes. The 16S rRNA sequence database was constructed by retrieving 16S sequences of bacterial isolates (1200–2384 bases in length) from the Ribosomal Database Project Release 10.27.

We used 4000 filter- passed reads of 16S sequences for the operational taxonomic unit (OTU) analysis of each sample. Clustering of 16S sequence reads with identity scores > 96% into OTUs was performed using UCLUST (www.drive5.com).

Representative sequences with identity scores > 96% for each OTU were assigned to bacterial species using BLAST. Principal component analysis using EZR software (http://www.jichi.ac.jp/saitama- sct/SaitamaHP.files/statmedEN.html) was applied for assessment of alterations of cecal bacterial phylum associated with diets.

Statistical analyses
Data were expressed as mean ± SEM. P < 0.05 was considered statistically significant. Statistical differences between pairs of groups were determined by a two-tailed Student’s t-test. An overall difference between more than two groups was determined using a one-way ANOVA.

If one-way ANOVAs were significant, differences between individual groups were estimated using a Bonferroni post hoc test All calculations were performed using SPSS Statistics (v19.0, IBM, Armonk, NY).

Results

Glucoraphanin decreases weight gain and adiposity, and increases energy expenditure in high-fat diet (HFD)-fed mice
To investigate the effects of glucoraphanin on systemic energy balance, we examined the body weight of wild-type mice fed normal chow (NC) or a high-fat diet (HFD), supplemented with glucoraphanin or vehicle (i.e., cornstarch only). Glucoraphanin reduced weight gain only in HFD-fed mice without affecting food intake (Fig. 1A and Supplementary Fig. 1A).

This reduction was not accompanied by evidence of gross toxicity. We determined the plasma concentration of sulforaphane in NC-GR and HFD-GR mice, but not in NC or HFD mice, indicating that glucoraphanin was absorbed as a sulforaphane following food consumption (Supplementary Fig. 1B).

The reduction of weight gain in glucoraphanin-treated HFD-fed mice was largely attributed to the decreased fat mass but not lean mass (Fig. 1B). To assess energy expenditure, we placed the mice in indirect calorimetry cages after 3 weeks of feeding, before an evident change in the body mass of HFD-fed mice was observed (HFD: 29.9 ± 0.5 g vs. HFD-GR: 28.5 ± 0.5 g).

Glucoraphanin-treated mice fed the HFD exhibited consistently higher VO2 and VCO2 than vehicle-treated HFD-fed controls (Fig. 1C and D), leading to increased energy expenditure (Fig. 1E); however, they displayed similar RER (Fig. 1F), suggesting that glucoraphanin supplementation enhanced sugar and fat use under HFD conditions. On NC-fed mice, glucoraphanin did not affect these parameters of energy balance (Fig. 1B–F).

Consistent with increased energy expenditure, glucoraphanin increased the core body temperature of HFD-fed mice by approximately 0.5°C (Fig. 1G).

Glucoraphanin improves diet-induced insulin resistance and glucose tolerance
After 14 weeks of feeding, glucoraphanin supplementation did not affect plasma triglycerides, total cholesterol, and free fatty acid (FFA) levels in either NC- or HFD-fed mice (Table 1). On NC, blood glucose levels were not altered by glucoraphanin, but on the HFD, glucoraphanin-treated mice exhibited significantly lower fasted blood glucose compared with vehicle-treated controls (Table 1).

Additionally, glucoraphanin significantly decreased plasma insulin concentrations in HFD-fed mice under both fasted and fed conditions, resulting in lower homeostatic model assessment-insulin resistance (HOMA-IR; Table 1).

During the insulin tolerance test (ITT), glucoraphanin significantly enhanced the reduction in blood glucose levels in HFD-fed mice, but not in NC-fed mice compared with the vehicle-treated controls (Fig. 2A). Glucoraphanin improved glucose tolerance in HFD- fed mice during the glucose tolerance test (GTT), but had no effect on NC-fed mice (Fig. 2B).

Insulin secretion during the GTT was not affected by glucoraphanin (data not shown). In line with increased insulin sensitivity, insulin-stimulated Akt phosphorylation on Ser473 was enhanced by glucoraphanin in the liver, muscle, and epididymal WAT of mice fed the HFD (Fig. 2C).

Glucoraphanin does not exert anti-obesity and insulin-sensitizing effects in Nrf2-/- mice
Although the Keap1-Nrf2 pathway is a well-known target of sulforaphane, this isothiocyanate has also been reported to modulate different biological pathways independent of the Keap1-Nrf2 pathway (24,25).

To determine whether the anti-obesity and insulin-sensitizing effects of glucoraphanin are mediated through Nrf2, the effects of glucoraphanin on energy balance and glucose metabolism were assessed in NC- and HFD- fed Nrf2-/- mice.

Although food intake and plasma concentration of sulforaphane in NC-GR or HFD-GR diet-fed Nrf2-/- nice were comparable that detected in wild-type mice fed NC- GR or HFD-GR diet (Supplementary Fig. 1C and D), the effects of glucoraphanin following HFD feeding on weight gain (Fig. 3A), VO2 (Fig. 3B), VCO2 (Fig. 3C), energy expenditure (Fig. 3D), RER (Fig. 3E), rectal temperature (Fig. 3F), insulin sensitivity (Fig. 3G), and glucose tolerance (Fig. 3H) were abolished by the Nrf2 deficiency.

These data are consistent with comparable plasma metabolic parameters between glucoraphanin-treated and vehicle-treated mice on the HFD. These metabolic parameters include lipids, blood glucose, insulin, HOMA-IR, and liver enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST) (Supplementary Table 3).

Glucoraphanin blocks HFD-induced reduction of Ucp1 expression in WAT of wild- type mice but not in Nrf2-/- mice

The increased energy expenditure and body temperature of glucoraphanin-treated HFD-fed mice suggest an increase in adaptive thermogenesis. However, glucoraphanin supplementation had little effect on the size and number of lipid droplets in the intrascapular brown adipose tissue (BAT) of HFD-fed wild-type mice (Supplementary Fig. 2A).

In addition, the mRNA expression of uncoupling proteins (Ucps), PGC-1α, and deiodinase 2 in BAT and of Ucps in skeletal muscle was not altered by glucoraphanin supplementation in both NC- and HFD-fed wild-type mice (Supplementary Fig. 2B and C). In BAT, HFD increased Ucp1 protein expression, but glucoraphanin did not alter the expression in both wild-type and Nrf2-/- mice (Fig. 4A).

Brown-like adipocytes expressing Ucp1, also known as beige cells, exist in various WAT depots and can contribute to thermogenesis (26). Compared with NC, HFD significantly decreased Ucp1 protein levels in epididymal and inguinal WAT of both wild-type and Nrf2-/- mice (Fig. 4A).

Glucoraphanin supplementation restored HFD-induced reduction in Ucp1 protein levels in epididymal and inguinal WAT of wild-type mice but not those in Nrf2-/- mice.

To examine whether the effects of glucoraphanin were fat cell-autonomous and Nrf2-mediated, we tested the effects of sulforaphane, an active metabolite of glucoraphanin, on the expression of brown fat-selective genes in primary beige adipocytes obtained from inguinal WAT of wild-type and Nrf2-/- mice.

In beige adipocytes derived from wild-type mice, treatment with sulforaphane induced the Nrf2 target gene, NAD(P)H:quinone oxidoreductase 1 (Nqo1; Fig. 4B) and antioxidant genes (Supplementary Fig. 3A).

Concurrently, sulforaphane significantly increased the mRNA expression of brown-fat selective genes, including Ucp1, Prdm16, Cidea, and Elovl3 (Fig. 4B).

In contrast, in Nrf2-deficient beige adipocytes, sulforaphane failed to activate Nrf2 as judged by unaltered mRNA expression of the target genes and to promote the expression of brown-fat selective genes (Supplementary Fig. 3B and Fig. 4C).

Importantly, Nrf2-deficient beige adipocytes exhibited less differentiation levels associated with attenuated lipid accumulation (Supplementary Fig. 3C) and lower mRNA expression of fatty acid binding protein 4 (Supplementary Fig. 3C) and brown-fat selective genes compared with wild-type beige adipocytes (Fig. 4C).

Glucoraphanin reduces hepatic steatosis and oxidative stress in HFD-fed mice
The HFD causes hepatic steatosis and inflammation, eventually leading to steatohepatitis. As shown in Fig. 5A, the increase in liver weight caused by the 14-week- HFD was alleviated by glucoraphanin supplementation.

Glucoraphanin also attenuated HFD-induced hepatic steatosis (Fig. 5B). Additionally, compared with the HFD group, the lower levels of plasma ALT, plasma AST, liver triglycerides, and liver FFAs in the HFD- GR group indicate that glucoraphanin alleviated HFD-induced liver damage (Fig. 5C and D).

The reduction in hepatic steatosis was accompanied by the decreased expression of the following lipogenic genes: sterol regulatory element binding protein-1c (Srebf1), fatty acid synthase (Fasn), and peroxisome proliferator-activated receptor gamma (Pparγ) (Fig. 5E).

Additionally, hepatic levels of malondialdehyde, a marker of lipid peroxidation, were increased by the HFD. Glucoraphanin attenuated lipid peroxidation (Fig. 5F) and decreased gene expression of the NADPH oxidase subunits gp91phox, p22phox, p47phox, and p67phox (Fig. 5E).

The HFD led to a compensatory increase in the expression of genes involved in fatty acid β-oxidation (Ppara and Cpt1a) and anti-oxidative stress (Cat, Gpx1, and Sod1) in the liver. However, glucoraphanin did not increase the expression of these genes in the liver of HFD-fed mice further (Fig. 5E).

Glucoraphanin suppresses HFD-induced proinflammatory activation of macrophages in liver and adipose tissue
In response to the HFD, liver-resident macrophages (Kupffer cells) increase the production of proinflammatory cytokines that promote insulin resistance and NAFLD in mice (27). In particular, chemokine (C-C motif) ligand 2 (Ccl2) promotes the recruitment of chemokine (C-C motif) receptor 2 (Ccr2)-positive monocytic lineages of myeloid cells into the liver (28). These recruited cells produce a large amount of proinflammatory mediators and activate a lipogenic program (28).

Here, we found a prominent induction of tumor necrosis factor-α (Tnf-α), Ccl2, and Ccr2 in the liver of HFD-fed mice, which was markedly reduced in glucoraphanin-treated mice (Fig. 6A).

Glucoraphanin significantly suppressed HFD-induced inflammatory pathways such as c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (Erk) (Fig. 6B).

Glucoraphanin tended to decrease levels of p-NF-κB p65 (Ser536) in HFD-fed mice, although this decrease was not statistically significant (Fig. 6B). Of note, in the liver of Nrf2-/- mice, glucoraphanin failed to suppress HFD-induced inflammatory signal pathways (Supplementary Fig. 4).

In addition, glucoraphanin significantly decreased the HFD-induced hepatic expression of macrophage markers, including F4/80, Cd11b, and Cd68 (Fig. 6C). Tissue macrophages are phenotypically heterogeneous and have been characterized according to their activation/polarization state as M1-like proinflammatory macrophages or M2-like anti- inflammatory macrophages (29).

Consistent with the decreased expression of macrophage markers, glucoraphanin prevented macrophage (F4/80+CD11b+ cell) accumulation in the liver of HFD-fed mice (Fig. 6D).

Additionally, glucoraphanin decreased the number of M1- like liver macrophages expressing surface markers (F4/80+CD11b+CD11c+CD206−) (Fig. 6E).

In contrast, glucoraphanin increased the number of M2-like liver macrophages (F4/80+CD11b+CD11c−CD206+), resulting in a predominantly M2-like macrophage population (Fig. 6E).

Moreover, glucoraphanin decreased the mRNA expression of Tnf-α and NADPH oxidase in the epididymal WAT of HFD-fed mice (Supplementary Fig. 5A).

Although the HFD-induced expression of macrophage markers and macrophage accumulation in epididymal WAT were not altered by glucoraphanin (Supplementary Fig. 5B and C), the number of M1-like macrophages was significantly decreased in the epididymal WAT of glucoraphanin-treated HFD-fed mice (Supplementary Fig. 5D).

Glucoraphanin decreases circulating LPS and the relative abundance of Proteobacteria in the gut microbiomes of HFD-fed mice

Gut microbiota-derived LPS induces chronic inflammation that eventually leads to insulin resistance in obesity, termed metabolic endotoxemia (1,2).

Based on our observation that glucoraphanin alleviates inflammation in the liver and epididymal WAT of HFD-fed mice, we subsequently investigated the effects of glucoraphanin on metabolic endotoxemia and gut microbiota.

In accordance with previous studies (1,2), the HFD induced a 2-fold increase in circulatory LPS levels, which was reduced by glucoraphanin supplementation (Fig. 7A). Furthermore, plasma and hepatic levels of the LPS marker, LBP, were significantly elevated by the HFD and were reduced by glucoraphanin supplementation (Fig. 7B).

A principal component analysis distinguished cecal microbial communities based on diet and treatment, revealing that the metagenomes of HFD-fed mice formed a cluster distinct from the cluster formed by NC-fed mice (Fig. 7C). However, samples from glucoraphanin-treated HFD-fed mice formed a cluster that was indistinguishable from that of vehicle- or glucoraphanin-treated NC-fed mice (Fig. 7C).

Importantly, consistent with previous reports (30,31), further analysis at the phylum level demonstrated that the proportion of Gram-negative Proteobacteria was significantly elevated in the gut microbiomes of HFD-fed mice, which was suppressed by glucoraphanin supplementation (Fig. 7D and E).

The increase in the relative abundance of Proteobacteria in HFD-fed mice is mostly explained by an increase in the relative abundance of bacteria from the family Desulfovibrionaceae (Fig. 7F), key producers of endotoxins in animal models of obesity (30).

In fact, the relative abundance of Desulfovibrionaceae was positively correlated with plasma LPS levels (Fig. 7G). Furthermore, plasma LPS levels were significantly and positively correlated with the hepatic mRNA levels of Tnf-α, gp91phox, and F4/80 (Fig. 7H).

Similarly, the liver expression of other marker genes was both significantly and positively correlated with plasma LPS levels and with one another (Supplementary Table 4).

Discussion
In the present study, we demonstrated that glucoraphanin, a stable precursor of the Nrf2 inducer sulforaphane, mitigated HFD-induced weight gain, insulin resistance, hepatic steatosis, oxidative stress, and chronic inflammation in mice.

The weight-reducing and insulin-sensitizing effects of glucoraphanin were abolished in Nrf2-/- mice. Additionally, glucoraphanin lowered plasma LPS levels in HFD-fed mice, and decreased the relative abundance of Desulfovibrionaceae.

At the molecular level, glucoraphanin increased Ucp1 protein expression in WAT depots, while suppressing the hepatic mRNA expression of genes involved in lipogenesis, NADPH oxidase, and inflammatory cytokines.

Our data suggest that in diet-induced obese mice, glucoraphanin restores energy expenditure and limits gut-derived metabolic endotoxemia, thereby preventing hepatic steatosis, insulin resistance, and chronic inflammation.

Consistent with previous reports demonstrating the anti-obesity effects of synthetic Nrf2 inducers (9–11), we show that the oral administration of glucoraphanin mitigates HFD-induced weight gain (Fig. 1A). The dose of glucoraphanin used in the current study (about 12 μmol/mouse/day) is similar to that used in other experiments investigating its antitumor effects in mice (14,32,33).

Here, we show that the effect of glucoraphanin on whole-body energy expenditure and the protein expression of Ucp1 in WAT were abolished in Nrf2-/- mice (Fig. 3D and 4A).

A recent study using adipocyte-specific PRDM16- deficient mice indicated that adaptive thermogenesis in beige fat also contributes to systemic energy expenditure (26). The mutant mice in the aforementioned study, which exhibited markedly reduced Ucp1 mRNA expression in inguinal WAT and minimal effects on BAT, developed obesity and insulin resistance in response to a HFD.

Thus, we believe that the increased energy expenditure in glucoraphanin-treated HFD-fed mice stems from, at least in part, an increase in beige fat, even though the expression of Ucps in BAT and skeletal muscle is not altered. Further analysis using Ucp1-knockout mice will elucidate the relative contribution of beige fat to the Nrf2-mediated metabolic effects elicited by glucoraphanin.

Several studies indicated that Nrf2-/- mice are partially protected from HFD-induced obesity and associated with milder insulin resistance compared with wild-type counterparts (9,34,35). Recently, Schneider et al. demonstrated that mitigation of HFD-induced obesity in Nrf2-/- mice, which was 25% less body weight than that of wild-type mice after 6 weeks of feeding (35).

They also found that HFD-fed Nrf2-/- mice exhibit a 20–30% increase in energy expenditure that is associated with an approximately 3-fold up-regulation of Ucp1 protein expression in abdominal WAT (35). In the present study, Nrf2-/- mice gained less weight after 6 weeks of HFD feeding compared with HFD-fed wild-type mice (Figure 1A and 3A; Nrf2-/-: 35.9 ± 0.9 g vs. wild-type: 39.0 ± 0.8 g, P < 0.05).

The lower body mass of Nrf2-/- mice raises the possibility that anti-obesity effect of glucoraphanin was completely phenocopied by Nrf2 gene deficiency. However, several observations suggest that Nrf2-/- mice only partially phenocopy the effect of glucoraphanin on weight gain reduction. 1)

In the present study, the weight difference between Nrf2-/- and wild-type mice was only 8%, which is much less than that in the previous study (35). 2) In Nrf2-/- mice, compared with NC, HFD induced significant weight gain (Fig. 3A), glucose intolerance (Fig. 3H), and insulin resistance as judged by increased HOMA-IR (Supplementary Table 3). 3)

Metabolic rate and energy expenditure of Nrf2-/- mice were comparable with those in wild-type mice (Fig. 1C–E and 3B–D). 4) Ucp1 protein levels in both epididymal WAT and inguinal WAT of HFD-fed Nrf2-/- mice were lower than those in HFD-GR-fed wild-type mice (Fig. 4A).

Taken together, these findings suggest that Nrf2 gene deficiency is not sufficient to block HFD-induced obesity by increasing energy expenditure and Ucp1 expression in WAT depots, and to mask the effect of glucoraphanin.

However, we cannot fully exclude the possibility that the effects of glucoraphanin are mediated by Nrf2 independent-mechanisms. Possible reasons for the discordance in metabolic phenotypes of Nrf2-/- between the previous study (35) and ours may be due to differences in knockout mouse lines and experimental conditions (e.g. age of mice at beginning of HFD feeding, composition of HFD, and temperature in the metabolic chamber).

Our in vitro study of primary beige adipocytes revealed that sulforaphane promotes the expression of brown-fat selective genes (Fig. 4B). Importantly, the concentration of sulforaphane used in cell culture (0.2–5 μM) is comparable with that detected in mice fed NC-GR and HFD-GR diet (Supplementary Fig. 1B). Moreover, we determined that Nrf2 acts as a positive regulator of beige adipocyte differentiation (Fig. 4C and Supplementary Fig. 3C).

The less differentiation levels in Nrf2-deficient beige adipocytes are in agreement with previous reports demonstrating Nrf2 induces white adipocyte differentiation through increasing the gene expression of Pparγ (34) and Cebpβ (36), common transcription factors regulating the differentiation of brown, beige, and white adipocytes.

Furthermore, it is noteworthy that Nrf2 has been reported to bind NF-E2-binding sites in the 5′-flanking region of the human and rodent Ucp1 genes (37).

However, we cannot exclude the possibilities that glucoraphanin affects sympathetic nervous activity or that hormonal factors are regulating fat browning (38). In addition, mitochondrial reactive oxidative species facilitate Ucp1-dependent respiration in BAT and whole-body energy expenditure by promoting the sulfenylation of a specific cysteine residue (Cys253) in Ucp1 (39).

The molecular mechanism by which Nrf2 regulates the expression and thermogenic activity of Ucp1 in beige adipocytes requires further investigation.

Glucoraphanin supplementation improved the systemic glucose tolerance and insulin sensitivity of HFD-fed mice. Although the molecular mechanism by which synthetic Nrf2 inducers enhance glucose uptake is unclear, AMP-activated protein kinase (AMPK) activation may mediate this enhancement in mouse skeletal muscle and adipose tissue (9– 11).

The phosphorylation levels of AMPK (Thr172) and acetyl-CoA carboxylase (Ser79) in peripheral insulin target tissues were comparable between glucoraphanin-treated NC- and HFD-fed mice and vehicle-treated controls (Supplementary Fig. 6).

These data suggest that AMPK activation is not necessary for glucoraphanin to exert its insulin-sensitizing effect on HFD-fed mice. Additional studies using the hyperinsulinemic-euglycemic clamp technique are needed to determine which tissues contribute to the insulin-sensitizing effects of glucoraphanin.

The beneficial effects of glucoraphanin on hepatic lipid metabolism were not accompanied by AMPK activation or the increased expression of fatty acid β-oxidation genes (Fig. 5E).

Instead, glucoraphanin mitigated HFD-induced oxidative stress and inflammation in the liver. In obesity, hepatic inflammation mediated by macrophage/monocyte-derived proinflammatory cytokines promotes lipogenesis through the inhibition of insulin signaling and SREBP activation (40,41). In fact, the depletion of Kupffer cells by clodronate liposomes ameliorates hepatic steatosis and insulin sensitivity in HFD-fed mice (27).

Furthermore, Ccl2- or Ccr2-deficient mice are protected from diet- induced hepatic steatosis even though they still become obese (42,43). Moreover, the specific ablation of M1-like macrophages restores insulin sensitivity in diet-induced obese mice (44), while the deletion of Pparδ, which promotes M2 activation, predisposes lean mice to develop insulin resistance (45).

Therefore, decreased hepatic macrophage accumulation and M2-dominant polarization of hepatic and adipose macrophages account, at least in part, for the protection from hepatic steatosis and insulin resistance in glucoraphanin-treated HFD-fed mice.

One of the most important findings of this study is that glucoraphanin decreases the relative abundance of Gram-negative Proteobacteria, particularly family Desulfovibrionaceae, while reducing circulatory LPS levels (Fig. 7).

Recent studies demonstrated that a significant increase in Desulfovibrionaceae, potential endotoxin producers, in the gut microbiomes of both HFD-induced obese mice and obese human subjects compared to lean individuals (30,31,46). We cannot exclude the possibility that other microbiota-derived products, such as bile acids and short chain fatty acids, also mediate the metabolic action of glucoraphanin.

Whether the interaction between sulforaphane and gut microbiota is affected directly or indirectly by altered host physiology remains to be determined.

However, several studies have suggested that sulforaphane can alter the gut microbiota directly, as isothiocyanates (including sulforaphane) have been shown to exhibit antibacterial activity against Proteobacteria (47,48).

This activity may proceed via redox disruption and enzyme denaturation reactions involving the isothiocyanate reactive group, -N=C=S, the thiol group (-SH) of glutathione and proteobacterial proteins (47,48). Additionally, sulforaphane exhibits antibacterial activity against Helicobacter pylori, a member of the phylum Proteobacteria (49).

We are unaware of any previous reports demonstrating that isothiocyanates inhibit the proliferation of Desulfovibrionaceae. The mechanistic underpinnings of this antibacterial activity require elucidation.

In conclusion, the results of the present study indicate that glucoraphanin may be effective in preventing obesity and related metabolic disorders such as NAFLD and type 2 diabetes.

A recent clinical study demonstrated that supplementation with a dietary dose of glucoraphanin (69 μmol/day) for two months significantly decreased the plasma liver enzymes, ALT and AST, although body mass did not change (18).

Long-term treatment with a higher dose of glucoraphanin (800 μmol/day), which can be safely administered without any harmful side effects (17), may be required to achieve an anti-obesity effect in humans.

Enhanced Nutraceutical bioactivity to fight cancer

1. Introduction
Nutraceuticals are biologically active molecules found in foods that may not be essential for maintaining normal human functions, but may enhance human health and wellbeing by inhibiting certain diseases or improving human performance (Gupta, 2016; Wildman & Kelley, 2007). Numerous different classes of nutraceuticals are found in both natural and processed foods including carotenoids, flavonoids, curcuminoids, phytosterols, and certain fatty acids (Gupta, 2016).

Many of these nutraceuticals have the potential to act as anticancer agents, and may therefore be suitable for incorporation into functional or medical foods as a means of preventing or treating certain types of cancer. Nutraceuticals vary considerably in their chemical structures, physiochemical properties, and biological effects (Bagchi, Preuss, & Sarwoop, 2015; Gupta, 2016).

For example, nutraceuticals vary in their molar mass, structure, polarity, charge and functional groups, which influences their chemical reactivity, physical state, solubility characteristics, and biological fate and functions (McClements, 2015b).

Some nutraceuticals are naturally present in whole foods, such as fruits, vegetables, and cereals, and are therefore often consumed in this form. Conversely, other nutraceuticals are isolated from their natural states and converted into additives that can be incorporated into functional foods, dietary supplements, or pharmaceuticals.

In this article, we will mainly focus on the delivery of nutraceuticals using foods, as it has been proposed that increased consumption of foods rich in nutraceuticals may decrease the risk of certain types of chronic diseases, including cancer. However, if consumers are going to benefit from consuming foods containing nutraceuticals, it is important that they have certain characteristics:

There are a number of factors that currently limit the utilization of many types of
anticancer nutraceuticals in functional foods (Gleeson, Ryan, & Brayden, 2016;
McClements, 2015a; McClements, Li, & Xiao, 2015b). Firstly, many of nutraceuticals
cannot easily be incorporated into foods because they have poor-solubility characteristics,
or they cause undesirable changes in appearance, texture, or flavor of foods. Second,
many nutraceuticals are chemically or biochemically unstable and therefore lose their
bioactivity because they are degraded within food products or the human body. Third,
many nutraceuticals have a low bioavailability and therefore only a small fraction of
them are actually absorbed and utilized by the body. Fourth, for some nutraceuticals the
optimum dose has not been established, and therefore it is unclear how much to deliver in
a bioactive form, e.g., the anticancer efficacy of resveratrol actually decreases as the dose
increases (Cai, Scott, Kholghi, Andreadi, Rufini, Karmokar, et al., 2015)
The purpose of this review article is to highlight how food matrices can be designed
to enhance the biological activity of anticancer nutraceuticals. In particular, we focus on
two different approaches that can be utilized for this purpose (Figure 1): very systems: In this approach, nutraceuticals isolated from their original environment are encapsulated within a delivery system that is specifically designed to enhance their bioavailability (Arora & Jaglan, 2016; Gleeson, Ryan, & Brayden, 2016; McClements, Decker, & Weiss, 2007; Sagalowicz & Leser, 2010). An example of this approach would be a carotenoid ingredient isolated from carrots that is encapsulated within an oil-in-water emulsion that could then be added to foods such as soft drinks, yogurts, dressings, or sauces (Salvia- Trujillo, Qian, Martin-Belloso, & McClements, 2013a).

In this approach, a nutraceutical-rich food is co-ingested with an excipient food, which is again specially designed to improve the bioavailability of the nutraceuticals (McClements & Xiao, 2014). An example of this approach would be an oil-in-water excipient emulsion consumed at the same time as carotenoid-rich carrots (Zhang, Zhang, Zou, Xiao, Zhang, Decker, et al., 2016b).
nutraceuticals should initially be present in functional foods at a sufficiently high level to have a beneficial physiological effect. The nutraceuticals should remain stable within the functional foods during manufacturing, storage, and utilization, otherwise they may lose their beneficial health effects.
nutraceuticals should not have an adverse effect on the color, taste, or shelf- life of a food product. After ingestion, the nutraceuticals should be released from the functional foods and delivered to the appropriate site-of-action within the human body.

Initially, a brief overview of some of the most important anticancer nutraceuticals that have been identified in foods is given. Some of the major factors limiting the bioavailability of nutraceuticals is then discussed, and potential approaches to overcome these limitations are highlighted. The design of delivery and excipient systems to enhance the bioavailability of anticancer nutraceuticals is then described.

2. Anticancer nutraceuticals
Accumulating evidence suggests that many nutraceuticals, such as curcumin, resveratrol, tea polyphenols, sulforaphane, anthocyanins, genistein, quercetin and
lycopene, exhibit anticancer activities against various forms of cancer (Arvanitoyannis & van Houwelingen-Koukaliaroglou, 2005; Ullah & Khan, 2008).

One of the important advantages of utilizing nutraceuticals to prevent and treat cancer is that they generally exhibit little or no adverse effects frequently associated with pharmaceutical agents after long-term administration. Nutraceuticals have been found to exert a wide range of cellular effects. The possible mechanisms of action of anticancer nutraceuticals include induction of cell cycle arrest and apoptosis in cancerous cells, detoxification of highly reactive molecules, activation of the host immune system, and sensitization of malignant cells to cytotoxic agents (Kotecha, Takami, & Espinoza, 2016; Pan & Ho, 2008). In this section, we focus on several important anticancer nutraceuticals that have beenintensively investigated with particular emphasis on the clinical evidence supporting the safety and efficacy of these compounds in cancer prevention and treatment.

Curcumin: Curcumin is a polyphenol found in turmeric (Curcuma longa), a member of the ginger family (Zingiberaceae) (Joe, Vijaykumar, & Lokesh, 2004). A large number of in vitro and in vivo studies have shown that curcumin inhibits the development of various cancers by inducing cell cycle arrest and cellular apoptosis, through pleiotropic modulation on several key cancer targets such as Wnt/-catenin, nuclear factor kappa B (NF-  B), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF- ), STAT-3 and cyclin D1(Perrone, Ardito, Giannatempo, Dioguardi, Troiano, Lo Russo, et al., 2015). Several phase I and phase II clinical trials have been conducted and demonstrated the safety and anticancer effects of curcumin in patients with different malignancies including myeloma, pancreatic and colorectal cancer (Kotecha, Takami, & Espinoza, 2016). Since curcumin is preferentially distributed in the colonic mucosa, in comparison to other tissues, many clinical trials have been focused on its chemo-preventive efficacy on colorectal cancer (Bar-Sela, Epelbaum, & Schaffer, 2010).

In a phase IIa clinical trial, patients taking 4 grams of curcumin daily were found to have a 40% decrease in the number of aberrant crypt foci (ACF) lesions, which are one of the early histologic signs seen in the colon that may lead to cancer (Carroll, 2012). One of the most important factors limiting the bioefficacy of curcumin is its poor bioavailability.

In animal studies, various edible delivery systems have been developed to improve the bioavailability and bioactivities of curcumin, including liposomes, phospholipid complexes, organogel-based nanoemulsions, chitosan-based nanoparticles and self-emulsifying drug delivery systems (Ting, Jiang, Ho, & Huang, 2014). For example, polylactide co-glycolide nanocapsulated curcumin suppressed cell proliferation, induced cancer cell apoptosis and improved pathological structures in a hepatocellular carcinoma model, whereas an identical concentration of free curcumin was found to be ineffective (Ghosh, Choudhury, Ghosh, Mandal, Sarkar, Ghosh, et al., 2012).

In a recent crossover study, liquid micellar formulations of curcumin showed a 185-fold enhancement in bioavailability within 24 hours without increased toxicity in healthy subjects, compared to powdered curcuminoids (Schiborr, Kocher, Behnam, Jandasek, stede, & Frank, 2014).

Resveratrol is a natural phenol produced by many fruits and plants such as grapes, blueberries, raspberries and peanuts (Bhat, Kosmeder, & Pezzuto, 2001; Kundu & Surh, 2008; Smoliga, Baur, & Hausenblas, 2011a). Animal studies have shown protective effects of resveratrol against several types of cancer, such as breast, skin, gastric, colon, prostate and pancreatic cancers, by interfering with multiple stages of carcinogenesis (Shukla & Singh, 2011).

Recently, clinical trials have established the safety and potential anticancer effects of resveratrol as both a single agent and a constituent of foods (Smoliga, Baur, & Hausenblas, 2011b). A phase I pilot study in colorectal cancer patients determined the anticancer effects of freeze-dried grape powder containing a low dose of resveratrol in combination with other bioactive components. The results suggest that dietary intake of the dry grape powder inhibited the Wnt signaling pathway in the colon, which may have contributed to the observed inhibitory effects on colon carcinogenesis (Nguyen, Martinez, Stamos, Moyer, Planutis, Hope, et al., 2009).

The anticancer effect of resveratrol also has been heavily investigated in breast cancer patients. In a randomized, double-blind placebo-controlled trial, twice daily resveratrol supplement (5 or 50 mg) for 12 weeks decreased the methylation of four cancer-related genes in mammary tissue in women with high risk for developing breast cancer (Zhu, Qin, Zhang, Rottinghaus, Chen, Kliethermes, et al., 2012). Research has been conducted to enhance the bioavailability of resveratrol. Using a prostate cancer mice model, liposome encapsulation of resveratrol and curcumin successfully was shown to increase the oral bioavailablity of both nautraceuticals.

Moreover, encapsulation of both nutraeuticals in the same liposome system resulted in a synergistic reduction of prostate cancer incidence (Narayanan, Nargi, Randolph, & Narayanan, 2009). However, as mentioned earlier, the optimum dose for resveratrol has not been established yet, since some studies have shown that higher levels are less effective at inhibiting cancer than low doses (Cai, et al., 2015).

Sulforaphane: Sulforaphane is an isothiocyanate mainly found in cruciferous vegetables, especially abundant in broccoli and broccoli sprouts (Dinkova- Kostova & Kostov, 2012; Houghton, Fassett, & Coombes, 2013). A large number of cell culture and animal studies have shown that sulforaphane is a potent chemo-preventive agent against various type of cancer.

The molecular targets of sulforaphane vary upon cancer type and stage. The major anticancer mechanism by which sulforaphane protects normal cells from carcinogenesis is through Nrf2- mediated induction of phase II antioxidant and detoxifying enzymes. These enzyme systems enhance cell defense against oxidative damage, and facilitate the removal of carcinogens. Sulforaphane also exerts anticancer activities through various mechanisms of action that are involved in regulating cell proliferation, differentiation, apoptosis, and cell cycle progression (Clarke, Dashwood, & Ho, 2008; Juge, Mithen, & Traka, 2007). To date, only a few clinical trials have been conducted on sulforaphane in cancer patients or high-risk populations. In a phase II study, patients who had recurrent prostate cancer were given 200 moles/day of Sulforaphane-rich extracts for a maximum period of 20 weeks.

Although there was no large decline (by ≥50 %) in prostate-specific antigen (PSA), 7 out of 20 patients experienced moderate PSA declines (by <50 %). Moreover, the on- treatment PSA doubling time (PSADT) was significantly lengthened compared to the pre-treatment PSADT (Alumkal, Slottke, Schwartzman, Cherala, Munar, Graff, et al., 2015). In another clinical trial with Helicobacter pylori-infected humans, daily oral intake of sulforaphane-rich broccoli sprouts (70 gram/day; containing 420 moles of sulforaphane precursor) for 2 months reduced the incidence of infection in humans. This trial suggests that sulforaphane may enhance chemo-protection of the gastric mucosa against H. pylori-induced oxidative stress (Yanaka, Fahey, Fukumoto, Nakayama, Inoue, Zhang, et al., 2009). In a melanoma mouse model, sulforaphane-loaded albumin microspheres demonstrated a significantly stronger suppression on tumor growth as compared to non-encapsulated sulforaphane, and no adverse effects were observed (Do, Pai, Rizvi, & D'Souza, 2010). polyphenols: Tea polyphenols are the major bioactive components in tea leaves, and include epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) (Cabrera, Artacho, & Gimenez, 2006; Crozier, Jaganath, & Clifford, 2009; Nijveldt, van Nood, van Hoorn, Boelens, van Norren, & van Leeuwen, 2001). EGCG and ECG can act as potent antioxidants to prevent DNA damage by scavenging reactive oxygen species (ROS), which in turn prevents carcinogenic mutagenesis in normal cells. In pre-clinical studies, tea polyphenols have been found to regulate multiple key cell signaling pathways, resulting in the suppression of angiogenesis, the modulation of the immune system, and the activation of phase II detoxifying enzymes (Yang, Li, Yang, Guan, Chen, & Ju, 2013). Several clinical trials have suggested that teas and tea polyphenols have the potential to prevent multiple types of cancer, including oral leukoplakia, liver, lung and bladder cancer (Kotecha, Takami, & Espinoza, 2016). In a recent phase II randomized presurgical placebo-controlled trial, bladder cancer patients taking 800 or 1200 mg of polyphenon E (a green tea polyphenol formulation mainly consisting of EGCG) showed a dose-dependent tissue accumulation of EGCG in benign and malignant bladder urothelium, which was associated with reduction of cell proliferation and increase of apoptosis (Gee, Saltzstein, Kim, Kolesar, Huang, Havighurst, et al., 2015). However, many clinical studies showed overall inconsistent results regarding the potential anticancer effects of tea polyphenols in different populations, suggesting different types of teas and variable tea preparations may significantly affect the chemo-preventive properties of teas (Sun, Yuan, Koh, & Yu, 2006). Moreover, the food matrix and gastrointestinal tract are known to impact the bioavailability of tea polyphenols (Manach, Williamson, Morand, Scalbert, & Remesy, 2005). flavonoids: Flavonoids are a large group of polyphenolic secondary metabolites of fruits, vegetables, and other plants with a broad-spectrum of bioactivities, including inhibitory effects on a wide range of human cancers (Tapas, Sakarkar, & Kakde, 2008; Weng & Yen, 2012). Epidemiological
evidence suggests a positive correlation between flavonoids-rich diets and low
risk of colon, breast and prostate cancer. Flavonoids can be categorized into
flavones, flavonols, flavanones, flavanols, anthocyanins and isoflavones based on
their structures. Flavonoids can target multiple signaling pathways during
carcinogenesis to inhibit cancer cell proliferation, suppress tumor angiogenesis,
and induce apoptosis in cancer cells (Batra & Sharma, 2013). Quercetin is one of
the most-studied flavones, and is abundant in onions and apples (Chirumbolo,
2013). Many in vitro and in vivo studies have shown the anticancer potential of
quercetin against a variety of human cancers, such as cervical, breast, colon,
prostate, liver and lung cancer (Yang, Song, Wang, Wang, Xu, & Xing, 2015). A
recent phase I study in patients with chronic Hepatitis C, a major causative factor
of liver cancer, reported that quercetin was safe when consumed at levels up to 5
grams per day, and that there was a modest reduction in viral load, suggesting a
potential for liver cancer prevention (Lu, Crespi, Liu, Vu, Ahmadieh, Wu, et al.,
2016). Quercetin normally exhibits a poor oral bioavailability due to its low
absorption. A microencapsulation approach has been shown to enhance the effects
of quercetin in reducing the oxidative damage and attenuating inflammation in a
mouse colitis model, presumably due to increased absorption (Guazelli, Fattori,
Colombo, Georgetti, Vicentini, Casagrande, et al., 2013). Tangeretin is another
well-studied flavonoid mainly found in citrus fruits (Weng & Yen, 2012). Poor
water-solubility limits the oral bioavailability and efficacy of tangeretin. In a
recent study, an emulsion-based delivery system was shown to significantly
enhance the inhibitory activity of tangeretin on colitis-associated colon
carcinogenesis in mice (Ting, Chiou, Pan, Ho, & Huang, 2015).
3. Factors limiting the bioavailability of anticancer nutraceuticals
The design of functional foods to improve the oral bioavailability of anticancer
nutraceuticals relies on an understanding of their fate within the human gastrointestinal
tract (GIT), as well as of the physicochemical and physiological events that typically
restrict their bioavailability. The factors limiting the oral bioavailability (BA) of
anticancer nutraceuticals can be divided into three main categories, as highlighted
schematically in Figure 2 (Arnott & Planey, 2012; Gleeson, Ryan, & Brayden, 2016;
McClements, 2013; McClements, Li, & Xiao, 2015b; Yao, McClements, & Xiao, 2015):
285 BA = B*  A*  T* (1) 286

The parameters B*, A* and T* represent the fractions of the nutraceuticals that are
bioaccessible, absorbed, and biologically active within the GIT, respectively. These
parameters depend on: (i) the molecular and physicochemical characteristics of the
anticancer nutraceuticals; (ii) the composition and structure of the surrounding food
matrices; and (iii) the complex events occurring within the human GIT. Improved
knowledge of the major factors limiting the BA of specific nutraceuticals will lead to a
more rational design of foods with enhanced beneficial health effects.

3.1. Bioaccessibility
The bioaccessibility (B*) of an anticancer nutraceutical represents the fraction of the total
amount of orally consumed nutraceuticals that is in a form that can be readily absorbed
by the GIT (Figure 2):
B*= mB/mI (2) 300
Here, mB is the mass of nutraceutical that is in a form that can be absorbed, and mI is the
total mass of the nutraceutical that is ingested. In the case of lipophilic nutraceuticals, B*
is usually taken to be the fraction of nutraceuticals solubilized in the mixed micelles in
the small intestinal fluids.
The bioaccessibility may be affected by three main factors in the GIT: liberation;
solubility; and interactions (McClements, 2015c; McClements, Li, & Xiao, 2015b).

 3.2. Absorption 

 After an anticancer nutraceutical is released from any structures containing it, and 

 then solubilized within the gastrointestinal fluids, it has to be transported through the GIT 

 contents and then be absorbed by the epithelial cells lining the GIT (Gleeson, Ryan, & 

 Brayden, 2016). The absorption of nutraceuticals is affected by numerous 

 physicochemical and physiological factors

 3.3. Transformation 

 The bioavailability and bioactivity of anticancer nutraceuticals is a result of their 

 precise chemical structures and mole
cular conformation. Changes in the structure or 

 conformation of these nutraceuticals due to chemical or biochemical reactions within the 

 GIT fluids may therefore alter their bio-efficacy. 

 nutraceuticals by using food components that modulate their chemical or 

 biochemical changes within the GIT.

4. Classification of the bioavailability of anticancer nutraceuticals
Foods can be specifically designed to improve the bioavailability profiles of
anticancer nutraceuticals by modulating their bioaccessibility, absorption, and
transformation in the GIT. Nutraceuticals can be classified according to the major factors
that limit their bioavailability, which means that generic food compositions and structures
can often be developed for a broad range of different nutraceuticals. A brief overview of
a recently developed classification schemes for nutraceuticals is given here (McClements,
Li, & Xiao, 2015b). This system is related to the Biopharmaceutical Classification
Scheme (BCS) commonly used to classify the factors limiting the bioavailability of
pharmaceuticals based on their solubility and permeability characteristics (Dahan, Miller,
& Amidon, 2009; Kawabata, Wada, Nakatani, Yamada, & Onoue, 2011; Lennernas &
Abrahamsson, 2005).

The Nutraceutical Bioavailability Classification Scheme (NuBACS) uses a three-
coordinate system (B*A*T*) to classify a nutraceutical according to the primary factors
that limit its bioavailability (McClements, Li, & Xiao, 2015a). Here, B* is the
Bioaccessibility, A* is the Absorption, and T* is the Transformation of the nutraceutical.
Each coordinate is designated “(+)” if it does not limit bioavailability, and “(-)” if it does.
Additional insights into the precise mechanisms associated with the poor bioavailability
are provided using subscripts, such as limited liberation from the food matrix (L), poor
solubility in the GIT fluids (S), and high susceptibility to metabolism (M) or chemical
degradation (C) (Table 1). As an example, curcumin, which is a highly hydrophobic
anticancer nutraceutical whose bioavailability is limited by poor solubility in GIT fluids,
metabolism, and chemical degradation is classified as B*(-)S A*(+) T*(-)M,C.
One of the major advantages of using this classification scheme is that a common
strategy may be developed to increase the bioavailability of a group of nutraceuticals
with similar properties. For example, food matrices (delivery or excipient systems)
containing digestible lipids could be used to increase the bioavailability of many
lipophilic nutraceuticals, i.e., B*(-)S.

5. Boosting the bioavailability of anticancer nutraceuticals using food
matrix design

Conventionally, the composition and structure of foods is usually only optimized to
enhance their quality attributes, such as appearance, texture, mouthfeel, and taste. More
recently, foods have been designed to improve their nutritional profiles by reducing the
levels of macronutrients believed to have adverse health impacts (such as saturated fats,
digestible carbohydrates, and salt) or to enrich them with food components that are
believed to bring beneficial health effects (such as vitamins, minerals, dietary fibers or
nutraceuticals). In this section, we focus on two different food-based approaches that
may be utilized to enhance the bioavailability, and therefore bioactivity, of anticancer
nutraceuticals (Figure 1).

5.1. Delivery Systems
Delivery systems are specifically designed to contain the anticancer nutraceuticals to
be delivered (Arora & Jaglan, 2016; Gleeson, Ryan, & Brayden, 2016). If the anticancer
nutraceutical is soluble or dispersible in one of the components (e.g., oil, water, or
powder) used to prepare a traditional food then it can often be simply dissolved or
dispersed in this component prior to preparing the final product. Nevertheless, the food
may still have to be carefully formulated to avoid potential adverse effects, such as
undesirable appearance, taste, or aroma caused by the presence of the nutraceutical.
Moreover, the food matrix may have to be carefully designed to avoid the chemical
degradation of the nutraceutical within the product during food manufacturing, storage,
and utilization. In many cases, an anticancer nutraceutical cannot be simply dissolved or
dispersed into a food matrix. Instead, specialized colloidal delivery systems have to be
fabricated that consist of the anticancer nutraceutical encapsulated within a small particle
(Figure 2).

5.1.1. Particle types
Numerous different types of colloidal particles have been developed that could
potentially be used as delivery systems to encapsulate, protect, and deliver anticancer
nutraceuticals (McClements, 2015c; Yao, McClements, & Xiao, 2015) (Figure 3). Some
of the most promising ones for commercial applications are highlighted here.
Microemulsions: Oil-in-water microemulsions contain colloidal particles dispersed in
water that consist of small clusters of surface active molecules (surfactants) that have
their hydrophobic tails located towards the interior and their hydrophilic heads located
towards the exterior (Figure 3A).

Conversely, water-in-oil microemulsions contain
colloidal particles dispersed in oil where the surfactants are organized so that their
hydrophilic heads form the interior and their hydrophobic tails face toward the exterior.
Both types of microemulsions are thermodynamically stable systems that usually contain
relatively small colloidal particles (5 nm < d < 100 nm). O/W microemulsions are most suitable for encapsulating hydrophobic nutraceuticals, whereas W/O microemulsions are most suitable for encapsulating hydrophilic ones (Flanagan & Singh, 2006; Spernath & Aserin, 2006). Because they are thermodynamically favorable, these systems should form spontaneously when the required components are mixed together. Nevertheless, some energy often has to be applied to ensure thorough mixing of the ingredients and to overcome any kinetic energy barriers. Emulsions: Oil-in-water emulsions (d > 200 nm) or nanoemulsions (d < 200 nm) consist of emulsifier-coated oil droplets dispersed within water (Figure 3B), and are therefore most suitable for encapsulating non-polar nutraceuticals inside the hydrophobic interior of the oil droplets (McClements, 2012; McClements, Decker, & Weiss, 2007). Conversely, water-in-oil emulsions or nanoemulsions consist of emulsifier-coated water droplets dispersed in oil, and are therefore more suitable for encapsulating polar nutraceuticals within the hydrophilic interior of the water droplets. Typically, a relatively hydrophilic emulsifier is required to form an O/W emulsion, whereas a relatively hydrophobic one is required to form a W/O emulsion. In practice, the formation, stability, and functional performance of emulsion-based delivery systems is highly dependent on the nature of the emulsifiers used. Emulsions may be made using a variety of high-energy (high pressure homogenization, microfluidization, and sonication) or low- energy (spontaneous emulsification or phase inversion temperature) methods (McClements & Rao, 2011). The nature of the homogenization method utilized to prepare an emulsion depends on many factors, including the type of ingredients used, the required particle size distribution, and the amount of material that should be produced. Solid lipid nanoparticles: Solid lipid nanoparticles (SLN) are similar to oil-in-water emulsions or nanoemulsions, but the oil phase is crystallized (Figure 3C) (Guri, Guelseren, & Corredig, 2013; Mehnert & Mader, 2012). Crystallization of the oil phase may improve the physical stability of the particles, as well as improving the retention and stability of the encapsulated nutraceuticals. SLNs are usually fabricated by forming an O/W emulsion or nanoemulsion using a high melting point lipid, and then cooling the system to promote crystallization of the lipids. Hydrogel beads: Hydrogel beads (microgels) consist of spherical particles, typically in the range of about 1 to 1000 m, which consist of cross-linked biopolymers that trap water (Figure 3D) (Chen, Remondetto, & Subirade, 2006; Joye & McClements, 2014; Shewan & Stokes, 2013; Zhang, Zhang, Chen, Tong, & McClements, 2015). Filled hydrogel beads also contain other types of colloidal particles dispersed within the beads, such as lipid droplets or liposomes. Hydrogel beads can be formed using a wide range of different methods depending on the nature of the biopolymers and cross-linking agents used. Typically, food-grade proteins (such as whey protein, caseinate, or gelatin) or polysaccharides (such as agar, alginate, carrageenan, pectin, or starch) are used as the biopolymers. The gelation mechanism used is largely determined by the nature of the biopolymer utilized and may involve temperature changes (heating or cooling), desolvation (dehydration or solvent removal), or addition of cross-linking agents (such as ions, polyelectrolytes, or enzymes). Certain types of hydrophilic nutraceutical can be directly incorporated into the hydrogel beads interior by mixing them with the biopolymer solution prior to fabrication. Conversely, hydrophobic nutraceuticals may have to be encapsulated into lipid droplets or liposomes prior to being incorporated into the hydrogel beads. The composition, dimensions, pore size and interactions of hydrogel beads can be controlled to create delivery systems with different properties. Liposomes: This type of colloidal particle typically consists of single or multiple lamellar spherical structures fabricated from phospholipids (Desai & Park, 2005; Fathi, Mozafari, & Mohebbi, 2012; Mozafari, Johnson, Hatziantoniou, & Demetzos, 2008). The phospholipids are assembled into bilayers with the non-polar tails of each layer being in contact (Figure 3E). Liposomes have a hydrophilic interior that can be used to encapsulate polar nutraceuticals, and a hydrophobic region in the bilayer that can be used to encapsulate non-polar ones. Liposomes can be formed using a number of different methods, including microfluidization and rehydration of surface deposited layers (Mozafari, Johnson, Hatziantoniou, & Demetzos, 2008). 5.1.2. Particle properties
The colloidal particles used as delivery systems may vary appreciably in their
properties, which determines their efficacy at encapsulating, stabilizing, and delivering
anticancer nutraceuticals (Figure 4). Some of the most important particle properties that
can be controlled to obtain specific functional attributes in delivery systems are
summarized below (McClements, 2015c):

Composition: Food-grade colloidal particles suitable for encapsulating anticancer
nutraceuticals can be fabricated from lipids, proteins, carbohydrates, minerals, surfactants
and/or water. The nature of the components used to fabricate the particles plays a major
role in determining their ability to encapsulate, stabilize, and release the nutraceuticals.
The amount of an anticancer nutraceutical that can be encapsulated within a colloidal
particle largely depends on its solubility within the particle interior. Hydrophobic
nutraceuticals can be solubilized within particle domains comprised of non-polar
components such as lipids, surfactant tails, phospholipid tails, or hydrophobic
biopolymers (e.g., zein). Conversely, hydrophilic nutraceuticals can be solubilized with
particle domains comprised of polar components such as water, hydrophilic proteins, and
polysaccharides. The chemical stability of certain nutraceuticals can be enhanced by
encapsulating them in colloidal particles that restrict the molecular motion of reactants
(such as solid lipid nanoparticles) or by incorporating components that protect the
nutraceutical from degradation, such as antioxidants. The gastrointestinal fate of
anticancer nutraceuticals can also be controlled by manipulating the composition of the
colloidal particles, i.e., the rate and extent of their digestion in different regions of the
GIT. For example, colloidal particles comprised of starch may be initially digested by
amylases in the mouth, those made by proteins or lipids may be digested by gastric or
pancreatic proteases or lipases in the stomach and small intestine, and those made by
dietary fibers may not be digested until they reach the colon due to the action of colonic
bacteria.

Dimensions: Colloidal particles can be fabricated with a wide range of dimensions,
ranging from around 10 nanometers (surfactant micelles) to a few millimeters (hydrogel
beads). The dimensions of colloidal particles influence many of the properties of
delivery systems, including their chemical stability, release rate, optical properties,
physical stability (to gravitational separation and aggregation), rheology, release rate, and
gastrointestinal fate. Anticancer nutraceuticals that are prone to chemical degradation
when they are dispersed in water (such as curcumin under basic conditions) tend to
breakdown more rapidly when they are in small rather than large lipid particles (Zou,
Zheng, Liu, Liu, Xiao, & McClements, 2015). This effect can be attributed to the fact
that the fraction of nutraceutical molecules in close proximity to the oil-water interface
increases with decreasing particle size. Conversely, the rate of lipid particle digestion
and nutraceutical release tends to increase as the particle size decreases because then
there is more oil-water interface available for digestive enzymes to adsorb to (Salvia-
Trujillo, Qian, Martin-Belloso, & McClements, 2013a). The particle size also affects the
incorporation of colloidal particles into food products. Larger particles tend to cream or
sediment more rapidly than smaller ones because the magnitude of the gravitational
forces is proportional to the diameter squared. Gravitational separation of particles may
be an important consideration in functional foods and beverages that have relatively low
viscosities. The optical properties of foods depend on the particle diameter (d) relative to
the wavelength of light (): colloidal dispersions go from being transparent when d <<  to turbid/opaque when d  , and to visibly distinguishable as separate particles when d >> . The dimensions of the particles will also affect their perception within the
mouth: a colloidal dispersion with particles less than about 50 m in diameter will feel
smooth in the mouth, whereas one with larger particles will feel gritty or lumpy.
Obviously, these factors have to be taken into account when designing delivery systems
intended for application in commercial food products that must be perceived favorably by
consumers.

Interfacial properties: The colloidal particles used in the food industry as delivery
systems may have different interfacial properties, e.g., composition, thickness, charge,
and hydrophobicity (McClements, 2015c). The interfacial properties also have a major
impact on the chemical stability, physical stability, release rate, optical properties,
rheology, and gastrointestinal fate. For example, the magnitude and sign of the electrical
charge on the surfaces of the colloidal particles determines their ability to interact with
other electrical substances, such as other charged particles, surfaces, or polymers. In
addition, the charge may impact the chemical stability of encapsulated nutraceuticals by
influencing the attraction or repulsion of pro-oxidants to the particle surfaces (such as
cationic transition metals). The surface chemistry and digestibility of the particle surface
influences the ability of digestive enzymes (such as lipases, proteins, or amylases) to
adsorb to them and hydrolyze the particles. Colloidal particles can often be coated with a
substance that is resistant to digestion in one region of the GIT, but that is digested in
another region. This phenomenon can be used to control the release of encapsulated
nutraceuticals in different parts of the GIT, e.g., mouth, stomach, small intestine or colon.

Physical state: Colloidal particles may be liquid, semi-solid, or solid depending on the
materials used to assemble them and the environmental conditions (such as temperature,
pH, and ionic composition). The oils and water phase used to prepare oil-in-water or
water-in-oil emulsions are typically liquid. However, high melting oils can be used to
create solid lipid nanoparticles that have a crystalline interior. The biopolymers (proteins
and polysaccharides) used to prepare hydrogel beads usually form semi-solid gel-like
particles. The physical state of a particle may change appreciably when environmental
conditions are changed within a food or as it passes through the GIT.

5.1.3. Mechanisms of Action
Appropriately designed delivery systems may enhance the oral bioavailability and
bioactivity of anticancer nutraceuticals due to numerous mechanisms of action:
Enhance Dispersibility: Most non-polar nutraceuticals are so hydrophobic that they
cannot simply be dispersed into aqueous-based foods (such as beverages, sauces,
dressings, yogurts, and desserts) because of their extremely poor water-solubility (Donsi,
Sessa, Mediouni, Mgaidi, & Ferrari, 2011). However, if these nutraceuticals can be
encapsulated within colloidal particles that have a hydrophobic core and a hydrophilic
shell (as in oil-in-water emulsions, nanoemulsions, or microemulsions), then they can
easily be dispersed into these aqueous-based foods (Flanagan & Singh, 2006;

McClements, 2012). Conversely, many polar nutraceuticals are so hydrophilic that they
cannot easily be dispersed into oil-based foods (such as butter, margarine, and spreads).
In this case colloidal particles that consist of a hydrophilic core and hydrophobic shell (as
in water-in-oil emulsions, nanoemulsions, or microemulsions) can be used to encapsulate
them.

Enhance Chemical or Biochemical Stability: Many nutraceuticals are highly
susceptible to chemical or biochemical degradation within food products or inside the
GIT (Braithwaite, Tyagi, Tomar, Kumar, Choonara, & Pillay, 2014; Lu, Kelly, & Miao,
2016; Yallapu, Nagesh, Jaggi, & Chauhan, 2015). Delivery systems can be specifically
designed to protect the encapsulated nutraceuticals from degradation by providing a
physical barrier or by containing specific components (such as antioxidants, buffers or
UV-visible absorbers).

As an example, some nutraceuticals (such as curcumin) are more
prone to chemical degradation when they are dissolved in water than when they are
dissolved in oil. Consequently, their chemical stability can be improved by encapsulating
them in delivery systems that have lipophilic domains, such as vesicles, nanoemulsions,
protein nanoparticles, or solid lipid nanoparticles (Zou, Liu, Liu, Xiao, & McClements,
2015; Zou, Zheng, Zhang, Zhang, Liu, Liu, et al., 2016a, 2016b).

Including antioxidants or chelating agents within a delivery system can inhibit the oxidation of unsaturated
lipids (Davidov-Pardo & McClements, 2015; Qian, Decker, Xiao, & McClements, 2012;
Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2014). Delivery systems can be designed so
that chemically unstable nutraceuticals are trapped within colloidal particles with
coatings that physically separate the nutraceuticals from any other components that may
accelerate their degradation.

Enhance Bioaccessibility: Many nutraceuticals have a low oral bioaccessibility
because of their low solubility in aqueous GIT fluids (Arora & Jaglan, 2016;
McClements, Li, & Xiao, 2015b). Delivery systems can be designed to contain digestible
lipids (such as triglycerides) that form free fatty acids and monoacylglycerols that
enhance the solubilization capacity of the mixed micelles for hydrophobic nutraceuticals
(Ozturk, Argin, Ozilgen, & McClements, 2015; Salvia-Trujillo, Qian, Martin-Belloso, &
McClements, 2013b; Salvia-Trujillo, Sun, Urn, Park, & McClements, 2015; Yao,
McClements, & Xiao, 2015).

The type of lipid used is particularly important since it
determines the rates of lipid digestion and mixed micelle formation, as well as the
solubilization capacity of the mixed micelles. The hydrophobic domains in the mixed
micelles (micelles and vesicles) must be large enough to accommodate the nutraceuticals,
so that they can transport them through the mucus layer to the epithelial cells. There is
therefore considerable interest in designing the lipid phase of foods so that they form
mixed micelles in the small intestine that have an appropriate solubilization capacity for
the nutraceuticals being delivered (Yao, McClements, & Xiao, 2015; Yao, Xiao, &
McClements, 2014).

Enhance Absorption: A delivery system may contain specific components that can
enhance the uptake of the anticancer nutraceuticals, such as components that disrupt the
mucus layer, inhibit efflux inhibitors, stimulate active transporters, or increase the
dimensions of tight junctions (Arora & Jaglan, 2016; Gleeson, Ryan, & Brayden, 2016;
McClements, Li, & Xiao, 2015b; Yao, McClements, & Xiao, 2015). Gleeson and co-
workers have recently highlighted the major strategies for increasing the absorption of
nutraceuticals (Gleeson, Ryan, & Brayden, 2016).

Certain components found in foods,
such as bromelain (an enzyme from pineapple) and papain (an enzyme from papaya),
have the potential to disrupt the mucus layer that normally coats the epithelial cells,
which may lead to enhanced absorption of nanoparticles coated with these substances (de
Sousa, Cattoz, Wilcox, Griffiths, Dalgliesh, Rogers, et al., 2015). Other components
present in foods are able to increase the permeability of epithelial cells, including
medium chain fatty acids like caprylic acid (Gleeson, Ryan, & Brayden, 2016). Other
food components may also have the ability to act as permeation enhancers, such as
cationic biopolymers (chitosan) chelating agents (EDTA), surfactants (polyol or sugar
esters of fatty acids) and phytochemicals (such as piperine) (McClements, Li, & Xiao,
2015b).

These intestinal permeation enhancers are believed to increase absorption by
increasing the fluidity of the phospholipid membranes, increasing the dimensions of the
tight junctions, stimulating active transporters, and/or blocking efflux transporters.
Nevertheless, it is important to realize that there may be adverse health effects associated
with altering the normal absorption mechanisms of nutraceuticals, which should be
considered when designing functional foods based on this principle (McCartney,
Gleeson, & Brayden, 2016).

5.2. Excipient Systems
Unlike delivery systems, excipient systems do not necessarily contain any
nutraceutical components (McClements & Xiao, 2014; McClements, Zou, Zhang, Salvia-
Trujillo, Kumosani, & Xiao, 2015). Instead they are designed to boost the bioavailability
and bioactivity of the nutraceuticals present in other foods that they are co-ingested with,
such as fruits and vegetables. Alternatively, they can be designed to boost the
bioavailability of nutraceuticals in dietary supplements or drugs (Salvia-Trujillo &
McClements, 2016b). Typically, the composition and structure of the excipient food
matrix is carefully controlled so as to improve the bioaccessibility, absorption, or
transformation profile of a nutraceutical-containing substance that is co-ingested with it.
Many of the approaches developed to improve the bioavailability of nutraceuticals using
delivery systems can also be used with excipient systems.

A considerable research effort has recently been carried out with the objective of
developing excipient systems based on emulsions that are suitable for utilization in the
food, dietary supplement, or pharmaceutical industries (McClements & Xiao, 2014;
McClements, Zou, Zhang, Salvia-Trujillo, Kumosani, & Xiao, 2015). These excipient
emulsions consist of small lipid droplets dispersed in an aqueous medium.
The
composition, size, and interfacial properties of the lipid droplets are optimized based on a
number of factors: (i) the ability to extract and solubilize lipophilic nutraceuticals from
food matrices (such as fruits and vegetables); (ii) the ability to be rapidly digested within
the GIT; (iii) the ability to rapidly form mixed micelles that solubilize the lipophilic
nutraceuticals released from the food matrices; (iv) the ability to protect the
nutraceuticals from chemical or biochemical degradation within the GIT. Additional
components may also be added to the aqueous phase of the excipient emulsions to
improve their efficacy, including antioxidants, chelating agents, buffering agents,
biopolymers, or permeation enhancers.

Recent research has shown that excipient emulsions can be used to increase the
bioavailability of carotenoids in a range of produce, including mangoes (Liu, Bi, Xiao, &
McClements, 2016), carrots (Zhang, Zhang, Zou, Xiao, Zhang, Decker, et al., 2015,
2016a; Zhang, et al., 2016b), yellow peppers (Liu, Bi, Xiao, & McClements, 2015), and
tomatoes (Salvia-Trujillo & McClements, 2016a). The composition and structure of the
excipient emulsions has to be carefully optimized to effectively enhance carotenoid
bioavailability (Figure 5). Studies have shown that excipient emulsions containing long
chain triglycerides (LCT) are more effective than those containing medium chain
triglycerides (MCT) at improving the bioaccessibility of carotenoids (Zhang, et al.,
2015). This effect was attributed to the fact that digestion of LCT by lipase leads to the
generation of long chain fatty acids that enhance the solubilization capacity of the mixed
micelle phase by increasing the dimensions of the hydrophobic domains (Cho, Salvia-
Trujillo, Kim, Park, Xiao, & McClements, 2014; Salvia-Trujillo, Qian, Martin-Belloso, &
McClements, 2013b).

Conversely, the hydrophobic domains formed by the medium
chain fatty acids resulting from MCT digestion are too small to accommodate large
carotenoids. Studies with carrots showed that the bioaccessibility of the carotenoids
increased with increasing fat content in the excipient emulsions co-ingested with them
(Zhang, et al., 2016b). Moreover, decreasing the size of the fat droplets in the excipient
emulsions was found to increase carotenoid bioaccessibility, which was attributed to their
higher surface area and rate of digestion, leading to quicker mixed micelle formation and
nutraceutical solubilization (Zhang, et al., 2016a).

Excipient emulsions have also been used to improve the bioavailability of curcumin
from turmeric powder (Zou, Liu, Liu, Liang, Li, Liu, et al., 2014; Zou, Zheng, Liu, Liu,
Xiao, & McClements, 2015). A higher amount of curcumin reached the simulated small
intestine when the turmeric powder was co-ingested with an excipient emulsion, which
was attributed to a number of effects. First, some of the curcumin was transported into
the hydrophobic interior of the small lipid droplets. Second, the chemical stability of the
curcumin was higher within the hydrophobic interior of the lipid droplets than in the
surrounding aqueous phase. Third, the mixed micelles formed after digestion of the lipid
droplets were able to solubilize the non-polar curcumin molecules in the aqueous GIT
fluids.

The concept of excipient foods is fairly new, and a considerable amount of research
is still required in this area. Nevertheless, it is a promising approach for improving the
bioavailability characteristics of many anticancer nutraceuticals in functional foods,
dietary supplements, and pharmacological drugs.

6. Conclusions
787 There is growing interest in using specific nutraceuticals commonly found in foods
as chemo-preventative agents. Cell culture and animal studies suggest that ingestion of
these nutraceuticals may inhibit certain types of cancers. Nevertheless, many anticancer
nutraceuticals cannot simply be incorporated into foods because of their poor solubility,
stability, and bioavailability characteristics. There is a considerable potential to create
functional food products designed to overcome these challenges, and therefore increase
the potential efficacy of anticancer nutraceuticals. These foods may be designed to
contain the nutraceuticals themselves (delivery systems) or they may be designed to boost
the bioavailability of nutraceuticals in other foods (excipient systems). Despite their
potential, a considerable amount of research is still needed to demonstrate the efficacy of
nutraceuticals, and the ability of delivery or excipient systems to enhance their bio-
efficacy.

Unlike pharmaceuticals, which are typically taken in a well-defined dose at a
particular time, nutraceuticals are obtained from numerous sources at levels that vary
from person to person and from time to time as part of a complex diet. In addition, the
beneficial effects of nutraceuticals may arise from taking relatively low levels over an
extended period.

Consequently, it is often difficult to carry out clinical studies (double
blind randomized feeding trials) using humans to prove their efficacy. Nevertheless, it
may be possible to gain some insights into their efficacy using long-term animal feedings
studies where the diet is carefully controlled. Having said this, there is also considerable
skepticism about the bioactivity demonstrated by certain nutraceuticals established using
in vitro and in vivo assays.

For instance, recent articles have criticized the evidence
suggesting that curcumin has a high bioactivity due to its ability to interfere with many
types of assays used to measure biological activity (Baker, 2017; Nelson, Dahlin, Bisson,
Graham, Pauli, & Walters, 2017). This may at least partially account for the lack of
efficacy that curcumin has demonstrated in many clinical trials.

Hydroxycitric Acid Nourishes Protein Synthesis via Altering Metabolic Directions

This study is published here

INTRODUCTION
The world is in health transition, and obesity is greatest threat to human health (Jose Hernandez–Morante et al., 2011). Obesity associates with various lifestyle-related diseases, such as diabetes, cardiovascular disease, hypertension and fatty liver disease (Jebb and Moore, 1999; Nakamura et al., 1994), causing a major health burden in terms of morbidity and mortality (Sturm, 2007). Although there has a few drugs in market to ameliorate or prevent obesity, costs and side effects limited their application (Chuah et al., 2013). It is well known that anti-obesity food ingredients could control and reduce body weight (Kim et al., 2008b).

Garcinia Cambogia extracts are found in northeastern India and Andaman Islands, and it has been extensively used for centuries throughout Southeast Asia as a food preservative, flavoring agent, and carminative (Jena et al., 2002).

Garcinia cambogia extracts is now popularly used as an ingredient of dietary supplements for weight loss (Saito et al., 2005), anti-obesity (Kim et al., 2008a; Kim et al., 2004), hypolipidaemic (Altiner et al., 2012) and anticancer activity (Mazzio and Soliman, 2009).

(-)-Hydroxycitric acid (HCA) is the major active ingredient present in the fruit rind of Garcinia cambogia (Jena et al., 2002; Marquez et al., 2012). The Garcinia cambogia extracts contains approximately 10~30% (-)-HCA, which can be isolated in the free form (Lewis and Neelakantan, 1965). Previous studies had identified (-)-HCA as a potent competitive inhibitor of adenosine triphosphate-citrate lyase (Watson et al., 1969), which is an extra-mitochondrial enzyme catalyzing the cleavage of citrate to oxaloacetate and acetyl-CoA (Watson and Lowenstein, 1970). (-)

Hydroxycitric acid reduced the availability of acetyl coenzyme A for the fatty acids and cholesterol synthesis (Watson et al., 1969). Many studies had reported that supplemental with (-)-HCA can promote weight reduction through suppressing de novo fatty acid synthesis, increasing lipid oxidation and reducing food intake (Chuah et al., 2013; Downs et al., 2005).

Recently, our laboratory also certified that Garcinia cambogia extracts could attenuate fat accumulation through regulating lipolysis gene expression by affecting adiponectin-AMPK signaling pathway in rat obesity model induced with high-fat diet (Liu et al., 2015).

(-)-Hydroxycitric acid is structurally similar to citrate, which is generally known as an allosteric regulator for a number of enzymes involved in carbohydrate and fat metabolism (Munday, 2002). Thus, (-)-HCA supplementation is expected to alter metabolic pathways. Amino acids are the fundamental building blocks of proteins (Jiang et al., 2014). In body, amino acids can be transformed to α-ketoglutarate by transamination reactions associated with multiple metabolic pathways such as glycolysis, gluconeogenesis, and the citric acid cycle (TCA) (Fauth et al., 1990).

Previous study showed that α-ketoglutarate is not only a key intermediate in the TCA cycle but also can replenish the cycle in anaplerotic reactions (Fink, 2008). Thus, amino acids are involved in protein synthesis, the energy production, gluconeogenesis, and lipogenesis (Wang et al., 2013). In addition, some amino acids are important for body development because they are the precursors of hormones.

It had been shown that phenylalanine and tyrosine are the precursors of thyroid hormones, melanin, dopamine, and catecholamine (Wang et al., 2013). Also, it had reports that insulin and growth hormone enhance amino acid uptake and protein synthesis, meanwhile the increase of amino acid contents stimulates glucagon secretion that promotes rapid conversion of amino acids to glucose (Rhoades and Rhoades, 2012).

Although dietary supplements of Garcinia cambogia extracts may be a practical way to reduce excessive fat accumulation in human or animal production, the precise physiological mechanism of HCA has not yet been fully clarified. Therefore, present study was conducted to investigate the effect of a long-term Garcinia cambogia extracts supplement on body weight gain, energy metabolism and the changes of amino acids content in serum, liver, and muscle of rats.

To our knowledge, this is the first report to investigate the impact of Garcinia cambogia extracts on the amino acid profile in different tissue. Our results not only provided information about how Garcinia cambogia extracts exerts its action but also certified the use of Garcinia cambogia extracts to control body weight.

MATERIALS AND METHODS
Garcinia cambogia extracts. Garcinia cambogia extracts was purchased from An Ynn Co. Ltd (Zhengzhou, China). The Garcinia cambogia extracts contain 56.0% ~ 58.0% (-)-Hydroxycitric acid including its free and lactone form, and it also contains 12.0%~14.0% cellulose, 5.5%~6.0% α-Dmelibiose, 2.5% ~ 3.0% β-D-lactin, 1.5% ~ 2.0% D-mannopyranose, 11%~12% oxophenic acid, 2.0% ~3.0% octadecyl alcohol, 3.5%~4.0% Coenzyme A, and 1.5% ~ 2.0% inorganic elements.

Animals and diets. Five-week old male Sprague– Dawley (SD) rats weighing 200 ± 20 g were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences (China). Rats were housed individually under constant temperature of 25 ° C and humidity of 50% ~ 60% and maintained on a 12:12 h light/dark cycle. All animal handling procedures were performed in strict accordance with guidelines established by Institutional Animal Care and Use Committee of Nanjing Agricultural University.

Before initiation of experiment, rats were acclimatized to the environmental conditions for 1 week. A total of 60 rats were randomly assigned to one of four groups: control group, low dose of (-)-HCA-treated group, medium dose of (-)-HCA-treated group, and high dose of (-)HCA-treated group.

Rats were supplemented with Garcinia cambogia extracts at 0, 25, 50, and 75 g/kg diet, and the contents of Garcinia cambogia extracts were equivalent to 0, 1000, 2000, and 3000 mg/kg diet of (-)HCA level. Rats were fed ad libitum with free access to water for 8 weeks, diet was removed for the last 12 h of the experimental term, and then the rats were anesthetized with ether and scarified by decapitation. Rats were weighed at the beginning and the end of experiment to determine average daily gain.

Daily feed consumption per day was recorded; the average daily feed intake and feed conversion ratio were then determined. At the end of the experiment, blood samples were allowed to clot at 4 °C and centrifuged at 1520 × g for 20min before harvesting the serum. Then, the serum, liver, and muscle samples were collected and kept at 70 °C until further analysis. Liver was weighted to determine the liver index, which indicated by the liver weight (mg)/body weight (g).

Measurement of serum glucose and glycogen content.
Serum glucose (catalog#: F006), hepatic glycogen (catalog#: A034), and muscle glycogen contents (catalog#: A034) were measured using commercial kits according to the manufacturers’ protocol (Nanjing Jiancheng Biotechnology Institution, Nanjing, China).
Measurement of protein content in liver and muscle.
Protein content in liver and muscle was measured using commercial kits (catalog#: P0012), which were purchased from the Beyotime Biotechnology Institution Shanghai, China.

Measurement of serum hormone content.
Serum triiodothyronine (T3, catalog#: A01PZC), thyroxine (T4, catalog#: A02PZC), insulin (catalog#: F01PJB), glucagon (catalog#: F03PJB) and Leptin (catalog#: C16DJB) contents were measured using Radioimmunoassay Kit according to the manufacturers’ protocol (Beijing North Institute of Biotechnology, Beijing, China). The intracoefficients of variation for all hormones detection kit were less than 10% and inter-coefficients of variation were less than 15%.

Analyses of amino acids profile by high pressure liquid chromatography
Instrumentation and reagents. High pressure liquid chromatography (HPLC) analyses were carried out on a benchtop Agilent1100 series LC chromatographic system (Agilent Technologies, Waldbronn, Germany) equipped with a vacuum degasser, autosampler, thermostated column compartment, quaternary pump and a diodearray detector. The chromatographic column (XTerra®MS C18, 5 μm, 4.6 × 250 mm) was purchased from Waters (Waters Co., Milford, MA, USA).

The standards of alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), glycine(Gly), glutamine (Gln), leucine (Leu), valine(Val), tryptophan (Trp), phenylalanine (Phe), arginine (Arg), asparagine (Asn), threonine (Thr), tyrosine (Tyr), isoleucine (Ile), serine (Ser), methionine (Met), proline (Pro), cysteine (Cys), histidine (His), and lysine (Lys) were purchased from Sigma. Methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from Tedia Company Inc. (Fairfield, OH, USA). Tetrahydrofuran (HPLC grade) and Ophthaldialdehyde (OPA) were purchased from Merck KGaA (Darmstadt, Germany).

High-purity water was prepared from a Milli-Q gradient water purification system (Millipore, MA, USA) and was used for all protocols in this study.

Chromatographic conditions.
High pressure liquid chromatography was performed according the method described by Shen et al (Shen et al., 2010). Briefly, a ternary system was used as mobile phase: solvent A is methanol, solvent B is acetonitrile, and solvent C is 10 mmol · L 1 Na2HPO4–NaH2PO4 (pH = 7.2, containing 0.3% tetrahydrofuran). A gradient elution program is mobile phase A, B, and C is 9%, 6%, and 85% for 10min, and then change to 12%, 8%, and 80% for 25 min, finally 15%, 15%, and 70% of mobile phase A, B, and C was used for another 25min. Flow rate: 1.0 mL · min 1. Fluorescence: excitation wave length = 340 nm and emission wavelength of 450 nm. Oven temperature: 40 °C. Injection volume: 20 μL.

Samples prepared and analyzed. Approximately 100 mg of liver and muscle was homogenized on ice with 1 mL of saline, and then centrifuged at 3000 g for 15 min before harvesting the supernatant. One hundred microliter of serum or tissue supernatant samples were mixed with 200 μL acetonitrile for 30 min at room temperature and then centrifuged at 12000 g for 30 min to harvest the supernatant for HPLC analysis. High pressure liquid chromatography analysis was performed after automatic pre-column derivatization with O-phthaldialdehyde (OPA) according the method described by Zeng et al.

(Zeng et al., 2013). Briefly, 20μL samples were mixed with 40 μL OPA-solution for 2 min at room temperature, and then 20 μL of the mixture was loaded into column. For identification purposes, the amino acid standards were used by spiking the samples, as well as by comparing the relative retention time. For quantification purposes, calibration curves using external standard methodology were performed. For recovery calculations, peak areas obtained from each sample was compared with the peak areas of standard used for spiking (Di Pierro et al., 2000; Uhe et al., 1991).

Statistical analyses.
Data were analyzed using the Statistical Package for Social Science (SPSS Inc., Chicago, IL, USA) and expressed as mean values ± SE. Treatment differences were subjected to a Duncan’s multiple comparison tests, and the differences were considered significant at P < 0.05 RESULTS
Effect of (-)-HCA on body weight and feed intake in rats
Compared with the control group, the body weight gain was significantly decreased in 2000 mg/kg (-)-HCA treatment group (P < 0.05) (Fig. 1A). No significantly differences were observed on the feed intake (p > 0.05)
No statistical differences were observed on the serum glucose content in (-)-HCA treatment groups at different doses compared with the control group (p>0.05) (Fig. 2A). The hepatic glycogen (Fig. 2B) and muscle glycogen (Fig. 2C) contents were significantly increased in 1000 mg/kg (-)-HCA treatment group compared with the control group (p < 0.05). These results indicated that (-)-HCA supplement could promote the glycogen synthesis in rats. Effect of (-)-HCA on protein content in rats As shown in Fig. 3, the protein contents in liver (Fig. 3A) and muscle (Fig. 3B) were significantly increased in (-)-HCA treatment groups at three doses compared with the control group (p<0.05). These results indicated that (-)-HCA supplement could enhance protein synthesis in rats. Effect of (-)-HCA on serum metabolic hormone content in rats
A significantly increase of serum T4 content was observed in 1000 mg/kg and 2000 mg/kg (-)-HCA treatment groups when compared with the control group (p < 0.05) (Fig. 4A). The T3 content was significantly higher in 1000mg/kg (-)-HCA treatment group than that in control group (p < 0.05) (Fig. 4B). No noticeable changes were observed on glucagon content (p>0.05) (Fig. 4D), while the insulin content was significantly increased in 2000 mg/kg and 3000 mg/kg (-)-HCA treatment groups than that in control group (p < 0.05) (Fig. 4C). In addition, serum Leptin content was significantly increased in 1000 mg/kg (-)-HCA treatment group compared with the control group (p < 0.05) (Fig. 4E). These results indicated that (-)-HCA supplement might regulate the glucose metabolism by regulating serum metabolic hormones content in rats. Effect of (-)-HCA on amino acid profile in rats
A chromatogram of synthetic mixture of amino acid standards was shown in Fig. 5. Each peak represents one of specific amino acid, and 15 amino acids were separated (T<50min) under the experimental conditions used. As shown in Table 1, in 7.8125 ~ 500 μmol/L concentration range, amino acid standard concentration was linear related to the peak area and the correlation coefficients is 0.9983 ~ 0.9999. The Intra-day RSD and Inter-day RSD is between 0.47% ~ 2.37% and 1.68% ~ 5.50%, respectively, which are within 6%. In addition, the recovery rate of 15 amino acid standards was between 93.88% ~ 105.20%. These parameters results indicated that this sensitive procedure could be used for the quantitative analysis of amino acid in tissues. Amino acid content in serum
As shown in Fig. 6, most of the amino acid contents were higher in (-)-HCA treatment groups than that in the control group. Among glucogenic amino acid, the threonine content in 2000 mg/kg (-)-HCA treatment group (p < 0.05) and the threonine, arginine, alanine, valine contents in 3000 mg/kg (-)-HCA treatment group (p < 0.05) were significantly increased than that in the control group (Fig. 6A). Among aromatic amino acid, the tyrosine and phenylalanine contents were significantly increased in 3000 mg/kg (-)-HCA treatment group (p < 0.05) than that in the control group (Fig. 6B). Among branched amino acid, the valine and leucine contents were significantly increased in 3000 mg/kg (-)HCA treatment group (p < 0.05) than that in the control group (Fig. 6C). These results indicated that (-)-HCA supplement could increase the glucogenic amino acid, aromatic amino acid, and branched amino acid in serum of rats. Amino acid content in liver
Similarity, most of the amino acid contents were also higher in (-)-HCA treatment groups than that in the control group (Fig. 7). Among glucogenic amino acid, the asparagine and threonine contents in 1000mg/kg (-)-HCA treatment group (p<0.05) and the aspartic acid, glutamic acid, threonine, and valine contents in 3000 mg/kg (-)-HCA treatment group (p < 0.01) were significantly increased than that in the control group (Fig. 7A). Among aromatic amino acid, the tryptophan and phenylalanine contents in 1000 mg/kg (p < 0.05) and 3000 mg/kg (-)-HCA treatment groups (p < 0.01) were significantly increased (Fig. 7B). Among branched amino acid, the isoleucine content in (-)-HCA treatment group at various doses (p < 0.05) and the valine and leucine contents in 3000 mg/kg (-)-HCA treatment group (p < 0.01) were significantly increased (Fig. 7C). These results indicated that (-)-HCA supplement could increase amino acid contents, especially aromatic amino acid and branched amino acid, in the liver of rats. Amino acid content in muscle
Contrary to serum and liver, most of the amino acid contents were lower in (-)-HCA treatment groups (Fig. 8). Among glucogenic amino acid, the glutamic acid content in (-)-HCA treatment group at all dose (p < 0.05) and the valine and glutamine contents in 1000 mg/kg (-)-HCA treatment group (p < 0.05) and aspartic acid content in 2000mg/kg (-)-HCA treatment group (p < 0.05) were significantly decreased (Fig. 8A). Among aromatic amino acid, 1000 mg/kg (-)-HCA treatment significantly decreased tyrosine and phenylalanine contents in muscle (p < 0.05) when compared with the control group (Fig. 8B). Among branched amino acid, the valine content in 1000mg/kg (-)-HCA treatment group (p < 0.05) and the leucine content in 2000 mg/kg and 3000 mg/kg (-)-HCA treatment groups (p < 0.01) were significantly decreased (Fig. 8C). These results indicated that (-)-HCA supplement could decrease amino acid contents, especially aromatic amino acid and branched amino acid, in the muscle of rats. DISCUSSION

Present results showed that diet supplement with (-)-HCA reduced the body weight gain in male rats, and 2000mg/kg (-)-HCA treatment significantly decreased the body weight gain. This observation was consistent with the study of Leonhardt et al. (2001), who reported that (-)-HCA could promote the body weight loss in male rats. Many studies demonstrate that (-)-HCA reduced the body weight gain in rats (Kim et al., 2008b), human (Marquez et al., 2012) and broilers (Liu et al., 2015), and suggesting feed intake inhibition maybe a major mechanism of how Garcinia cambogia extracts exerts its function in controlling body weight (Leonhardt et al., 2001).
Nevertheless, no differences was observed on feed intake in rats supplemented with (-)-HCA, and this results indicated that the inhibition effect of (-)-HCA on body weight gain is not mainly via regulating feed intake in male rats.

The results presented here were consistent with the previous study, which demonstrated that 2 weeks (-)-HCA supplement has no significant effects on appetite in human (Kovacs et al., 2001). In addition, it is reported that Garcinia cambogia leaf supplementation had no effect in male Ross 308 broiler chickens on feed intake in finisher stage (Sebola et al., 2011).

We presumed that the differences in the preparation of (-)-HCA and the different animal or rat strains used in those study could contribute to such discrepancy. As a weight loss agent, it had presume that an increased fatty acid oxidation and decreased fat accumulation in animal with (-)-HCA-treated contributed to decrease its weight (Sullivan et al., 1972).

In addition, our recently study demonstrated that Garcinia Cambogia extracts could attenuated fat accumulation and body weight gain through activating the Adiponectin-AMPK signaling pathway in rat obesity model induced by high-fat diet (Liu et al., 2015). Although we did not investigated the effect of (-)-HCA on fat accumulation in this study, taken our results and other reported, we think that suppression fat accumulation may be a major mechanism of weight loss by (-)HCA-induced.

It is well known that body weight gain depends on the balance between energy intake and energy expenditure. Previous study suggests that Garcinia cambogia extracts could enhance energy expenditure in rats (Vasselli et al., 1998), and the suppressive effect of Garcinia cambogia extracts on body weight gain might also depend on increased thermogenesis, except for the reduction in feed intake (Leonhardt et al., 2001).

Thyroid hormones, including triiodothyronine (T3) and thyroxine (T4), are recognized as the key metabolic hormones in body. Thyroid hormones essentially modulate all metabolic pathways through alterations in oxygen consumption and carbohydrate metabolism (Smith et al., 2002).

Serum thyroid hormones contents are associated with energy expenditure and other effects, such as lipid metabolism and protein synthesis (Hornick et al., 2000).

Present study showed that serum T4 content in 1000 mg/kg and 2000mg/kg (-)-HCA treatment groups and serum T3 content in 1000mg/kg (-)-HCA treatment group were significantly increased in rats. Leptin, the ‘satiety hormone’, is an important hormone that helps to regulate energy balance (Pan et al., 2014).

In present study, it was demonstrated that 1000 mg/kg (-)-HCA treatment significantly increased serum Leptin content in rats.

One of the major functions of Leptin is control energy balance by binding to receptors in hypothalamus, which results in the increase energy expenditure (Lerario et al., 2001; Watson et al., 2000). Thus, the above data suggested that one possible mechanism of (-)-HCA supplement reduced body weight gain via enhancing energy expenditure in rats, which might be associated with the increase of thyroid hormones and Leptin levels.

It had been reported that aromatic amino acids, including phenylalanine, tryptophan, and tyrosine, have important roles in body development because they are the precursors of thyroid hormones, melanin, dopamine, and catecholamine (Wang et al., 2013). Our results showed that (-)-HCA treatment significantly increased serum tyrosine and phenylalanine contents in rats, and the tryptophan and phenylalanine contents also significantly increased in liver. Tyrosine and phenylalanine are premise material for the synthesis of thyroid hormones, and up to 80% of the T4 is then converted to T3 by organs such as the liver, kidney, and spleen (Koehrle and Brabant, 2010).

The changes of aromatic amino acids content were consistent with the significant increase of serum T4 and T3 contents in rats after (-)HCA treatment. In addition, tryptophan is premise material for the synthesis of serotonin (Boopathi and Ramasamy, 2014); thus, an increase of serum tryptophan levels could affect food intake behavior (Lopez et al., 2015). Although the serotonin content was not detected in this study, the significant increase of tryptophan contents indirectly indicated that (-)-HCA treatment might increase the serotonin release and availability.

This results was also consistent with previous reports that shows the increase of serotonin content after (-)-HCA treatment might be the main reason for appetite suppression (Ohia et al., 2002; Preuss et al., 2004; Roy et al., 2004).
Taken together, these results further confirmed that (-)-HCA could reduce body weight gain through promoting energy expenditure via its effect on increasing thyroid hormones levels.

(-)-Hydroxycitric acid inhibits ATP-citrate lyase and increases the cellular pool of citrate, which in turn inhibits glycolysis and thus redirects the carbon sources for glycogen production within the liver or muscle (Cheng et al., 2012; Shara et al., 2003). No changes were observed on the glucose content, while 1000 mg/kg (-)HCA treatment significantly increased the hepatic glycogen and muscle glycogen contents in rats. Our results is similar to previous study results that show glycogen levels in skeletal muscle are increased after (-)-HCA supplementation in animal models (Ishihara et al., 2000) or in human (Cheng et al., 2012). Our results showed that 2000 mg/kg and 3000 mg/kg (-)-HCA treatment significantly increased insulin content in rats. Insulin can promote the storage of glucose and inhibit lipolysis and gluconeogenesis (Bernard et al., 2013; Bernard et al., 2011).

It has been reported that treated with Garcinia cambogia extracts for 4weeks significantly increased plasma insulin content (Hayamizu et al., 2003). In addition, previous study certifies that (-)-HCA can inhibit phosphofructokinase, a key enzyme controlling glycolysis (McCune et al., 1989). Once glucose was absorbed, it is rapidly phosphorylated to glucose-6-phosphate and then converted into glycogen through the glycogen synthesis pathway or lactate through the glycolytic pathway. Although we did not measure phosphofructokinase activity in this study, the inhibitory action of (-)-HCA on phosphofructokinase that results in the inhibition of glycolysis is consistent with the higher glycogen content in liver and muscle reported in here.

Therefore, the mechanism that (-)-HCA could increase the glycogen content in liver and muscle might be due to its inhibitory effect on the glycolytic pathway, and this action might be related to its ability to increase insulin content in rats.

Our results showed that (-)-HCA treatment significantly increased the protein contents in liver and muscle, which indicated that administration of (-)-HCA could promote protein synthesis in rats. This was consistent with the feed conversion ratio, which was significantly increased in rats supplemented with (-)HCA. Amino acids are not only the fundamental building blocks for protein synthesis (Jiang et al., 2014); they are also used for energy dissipation or other metabolic purposes (Conceicao et al., 2003). (-)-HCA has a structure similar to citrate, which is generally known as an allosteric regulator for a number of enzymes involved in carbohydrate and fat metabolism (Munday, 2002).

Thus, the indirect inhibition of cytosolic pool of citrate by (-)-HCA and subsequent reduction in acetyl coenzyme A and oxaloacetate alter the citric acid cycle (TCA) that is expected to alter metabolic pathways. Importantly, oxaloacetate is not only an important intermediate of the TCA cycle but also the first designated substrate of the gluconeogenic pathways of all other cycle intermediates, glycerol, or amino acids (Homem de Bittencourt et al., 1993).

Thus, we presume that (-)-HCA supplement may alter metabolic directions of amino acids, which in turn promoted protein synthesis in rats. In the present study, most of amino acid contents in serum and liver were increased, while its content in muscle were decreased in rats supplemented with (-)-HCA. Amino acids are used in a variety of cellular metabolism pathways, such as provision of energy (Conceicao et al., 2003), protein, and nucleotide precursors (Jiang et al., 2014), signaling molecules (Wu et al., 2000) and protection against oxidative stress (Nasresfahani et al., 1992). As energy requirements of body are met, amino acids will be mainly used for protein synthesis rather than for provision energy.

Our results showed that no changes were observed on serum glucose content, while hepatic glycogen and muscle glycogen contents were significantly increased in rats supplemented with (-)-HCA, which indicated that there was sufficient energy to meet the requirement of body. Under this condition, the increased of amino acid contents in serum and liver might be used to promote protein synthesis. In addition, alanine in muscle can be used to transport the ammonia to liver, and then the liver delivers glucose to muscle through serum, which is called as alanine-glucose cycle, and it can provide the adequate glucose for muscle (Rijkers, 2015).

Present study showed that (-)-HCA treatment obviously increase alanine contents in serum and liver of rats, this indirectly indicated that sufficient glucose in muscle provided an essential prerequisite for the protein synthesis. As mentioned earlier, insulin can enhance amino acid uptake and protein synthesis (Bernard et al., 2013; Bernard et al., 2011). Our result showed that serum insulin content was significantly increased in rats after (-)HCA treatment, which was consistent with the significant increase of the protein content in liver and muscle of rats. Therefore, we conjecture that (-)-HCA treatment could promote protein synthesis via regulating metabolic directions of amino acids in rats.

The amino acids predominantly involved in energy metabolic processes are branched amino acids, which include leucine, isoleucine, and valine (Assenza et al., 2004). Under normal conditions, the branched amino acids are selectively excluded from hepatic uptake and are metabolized predominantly in the skeletal muscle (Adibi, 1980; Tsuchiya et al., 2005; Urata et al., 2007).

Our results showed that (-)-HCA treatment significantly increased the contents of valine and leucine in serum and liver, and decreased their contents in muscle of rats. Previous study shows that excessive oxidation of branched amino acids may inhibit citric acid cycle via depleting the glutamate and ketoglutarate pool (Trottier et al., 2002). The changes of serum glucose and glycogen contents in this study indicated that branched amino acids might be mainly used for protein synthesis in muscle rather than for the source of energy. Muscle is one of the main target organs of insulin action, and insulin can promote the protein synthesis (Rijkers, 2015).

Escobar et al. demonstrated that insulin can stimulate the influx of branched amino acids into the skeletal muscle (Escobar et al., 2006). In addition, previous study suggests that branched amino acids play important role in skeletal muscles, specifically in the regulation of protein synthesis metabolism (Desikan et al., 2010). Wang et al. reports that amino acids can enhance protein synthesis (Wang and Proud, 2008). Considering the increase of serum insulin level and muscle protein content, we suspected that decrease of branched amino acids content after (-)-HCA treatment might be due to its enhance on protein synthesis.

In conclusion, in spite that (-)-HCA could promote protein synthesis, it can also promote weight loss by suppressing de novo of fatty acid synthesis and promoting energy expenditure. This study demonstrated that supplement with (-)-HCA could reduce body weight gain through promoting energy expenditure via its effect on increasing thyroid hormone levels. Meanwhile, (-)-HCA treatment could promote protein synthesis in male rats by altering the metabolic directions of amino acids. The elucidation of the precise mechanism involved in this action of (-)-HCA needs further investigation.

Cardamom supplementation improves inflammatory and oxidative stress biomarkers in hyperlipidemic, overweight, and obese pre-diabetic women

This study is published here

Insulin resistance, which is the key feature of pre-diabetes and T2DM, results mainly from low physical activity and obesity, which are associated with the repletion of lipid into adipocytes and the accumulation of adipose tissue.3-5 An increase in insulin, free fatty acid (FFA), and/or glucose levels can increase reactive oxygen species (ROS) production and oxidative stress.6

Oxidative stress is a direct outcome of hyperglycaemia and may be involved in metabolic complications in these subjects.7
 The release of additional acute phase reactants, including TNF-α, IL-6, and CRP, by white adipose tissue has been shown in obese subjects.4 By diminishing insulin receptor signalling and increasing insulin resistance, IL-6 and TNF-α can cause chronic hyperglycaemia in these subjects, leading to the development of diabetes.8
An increased oxidative stress is present in pre-diabetes stages9, which may result in endothelial damage in these subjects.10 Therefore, it can be assumed that interventions to reduce oxidative stress and inflammation could improve the condition of pre-diabetes and prevent its complications and development to T2DM.

Lately, herbal remedies like spices have been considered because of their phytochemical content, which has a beneficial potent.11 Spices may somewhat have the same effects as functional foods to improve health or reduce risks of diseases. 12,13 Like vegetables and fruits, spices also have antioxidant effects.14 Several studies have shown that some spices have great potential to inhibit chronic inflammation and oxidative stress because of their phytochemicals and free radical scavengers like polyphenols, flavonoids, and phenolic compounds.15,16

It has been suggested that by increasing the consumption of spices, chronic diseases morbidity can be prevented.17
Green cardamom, also known as the Queen of Spices consists of the whole or ground dried fruit of Elettaria Cardamomum (Linn.) Maton, which belongs to the ginger family (Zingiberaceae).18,19

So far, several investigations have shown some advantages of cardamom for teeth, gum and throat infection, lung congestion, pulmonary tuberculosis, and gastrointestinal disorders.20 Various studies suggest that cardamom extracts display antimicrobial, anti-cancer, anti-inflammatory, anti-proliferative, pro-apoptotic and anti-oxidative activities.18,20,21-25 Several animal and cellular model studies have shown anti-inflammatory and anti-oxidative activities of cardamom.18,24-30 A few human studies have been conducted to investigate the effects of cardamom on inflammation and oxidative stress status, which have shown conflicting results.11,25,31In one study, supplementation with Greater cardamom or E. Cardamomum improved the antioxidant status in patients with ischemic heart disease or hypertension.25,31 However, the results of another study did not show any significant effect of cardamom on blood oxidative stress and inflammation in T2DM patients.11

According to the results of an in-vitro study, the key components of the essential oil in cardamom (i.e. 1.8-cineol [eucalyptol], beta-pinene, geraniol), by binding with TNF, IL-1 β; IL-4 and IL-5, show anti-inflammatory activities.18In an animal study, cardamom supplementation improved oxidative stress markers and ameliorated the inflammatory cell infiltration and fibrosis in the liver of rats fed on a high-carbohydrate high-fat (HCHF) diet.27

In a human study, 3g cardamom powder intake for 12 weeks in individuals with primary hypertension significantly increased the total blood antioxidant capacity.25 However, no study has been done yet to evaluate the effects of cardamom supplementation in pre-diabetic subjects who are at risk of oxidative stress and inflammation. Previous studies have suggested the need for well-controlled clinical trials of spices.17 Therefore, there is a hypothesis that cardamom may have beneficial effects on pre-diabetes. So, the present study was designed to investigate the effects of cardamom on blood inflammatory and oxidative stress biomarkers in pre-diabetic subjects.
EXPERIMENTAL STATE
Study design and subjects

This double-blind, placebo-controlled trial study was conducted on 79 newly diagnosed pre- diabetic women from two health care centres of Karaj city in Iran from February to April 2014. The aim of the study was to determine the effect of cardamom supplementation on serum lipids, glycemic indices, blood pressure, oxidative stress, and inflammatory biomarkers in overweight and obese pre-diabetic women. Since the majority of these individuals were female, the investigation was limited to female subjects. This study was performed based on the guidelines laid down in the Declaration of Helsinki. All procedures involving human subjects were approved by the Ethics Committee of Tehran University of Medical Sciences. A written informed consent form was signed and dated by the subjects and investigators. This study was registered on the Iranian Registry of Clinical Trials website (http://www.irct.ir/, IRCT2014060817254N2).
A sample sizeof at least 36 in each group was calculated according to the standard deviation of hs-CRP in a similar study30 in the following order: (α=0.05, power=80%)
= 2.18 / 
d=μ −μ =1.47 =0.47
σ√2 3.07
[(z ∝/ ) + (z )] (1.96 + 0.84)
= d = (0.47) = 36

Considering the loss to follow-up, 40 subjects in each group joined the study. The age of the subjects was in the range of 30 to 70 years with a body mass index (BMI) of 25–39.9 kgm-2and had at least one of the following criteria: Fasting blood sugar (FBS) 100-125 mgdl-1, HbA1c 5.7– 6.4%, two-hour blood glucose 140–199 mgdl-1 identified in the last two months. In addition, they had at least one of these risk factors: 300>triglyceride>150 mgdl-1, total cholesterol>200 mgdl-1, 160>low-density lipoprotein-cholesterol (LDL-c) >100 mgdl-1, high-density lipoprotein- cholesterol (HDL-c)<50 mgdl-1. Exclusion criteria were BMI<25 or ≥40 kgm-2, pregnancy or lactation, professional athletes, allergy to cardamom, smoking, clinical history of peptic ulcers, gall or kidney stones, clinical history of inflammatory diseases like diabetes, cardiovascular diseases, multiple sclerosis (MS), rheumatoid arthritis, cancer, inflammatory diseases of the gastrointestinal tract and respiratory (e.g. asthma, allergies), multivitamin or antioxidant supplement consumption at least two days a week in the past month, taking medications for dyslipidemia, blood glucose disturbance, hypertension, psychiatric disease, thyroid and hormonal disease, following a specific diet over the last three months, having LDL-c≥160 mgdl-1 or triglyceride ≥300 mgdl-1, blood pressure>130/80 mmHg, developing diabetes during the study, and not taking the prescribed supplements more than 10%.

Randomization and intervention
The participants were randomized into two groups using block randomization. The stratified randomization method was used to control the age and BMI (age in the range of ≤40 and 41–70 years, BMI in the range of 25–29.9 and 30–39.9 kgm-2).
Forty subjects were classified into each group to receive one capsule of either cardamom or placebo powder three times a day with meal for eight weeks.

The dose of 3.0 g used in the study was chosen based on the two previous human studies investigating the effects of cardamom supplementation 25, 31. Also, 3.0 g of cardamom powder can be a reasonably large amount to be consumed through a diet. Fruits of E. Cardamom were provided by the Traditional Medicine and Research Centre (TMRC), Shahid Beheshti University of Medical Sciences, Tehran, Iran. Fruits of cardamom and dried breadcrumbs are transferred to this centre. After grinding and sifting the whole green cardamom and breadcrumbs, the capsules were filled with these materials.
Each capsule contained one gram of green cardamom powder or breadcrumbs. They were exactly similar in shape, size, and appearance. All placebo capsules were placed near cardamom so that they could take its smell. Sixty capsules were placed in similar jars, which were labelled A and B to be delivered to the individuals. Each person in the study got three jars every 20 days. To confirm the compliance of subjects, they were called up every week. To evaluate compliance, the remaining capsules in the jars were counted.

Dietary analysis
A 24-hour food recall questionnaire of a typical day was completed by interview to get information on food intake at the beginning and the end of the study. Then, the N4 software was used to estimate the daily dietary intake of nutrients.
Anthropometry and physical activity assessments
Anthropometric characteristics, including height and weight, were measured at the beginning and end of the study. Body weight was measured with minimal clothing and without shoes by using a digital scale with a measurement accuracy of 100g. Standing height was measured without shoes to the nearest 0.5cm using a tape measure. BMI was calculated by dividing the weight by the square of height. Physical activity was measured by a short form of the International Physical Activity Questionnaire (IPAQ).33

Laboratory measurements
After 12 hours of fasting, 10ml of venous blood was drawn by a trained nurse before and after the intervention period. Blood samples were centrifuged at 3000 rpm for 10 minutes to separate blood cells and serum. Blood cells were washed three times with 0.9 gl-1NaCl solution. Cell membranes were removed by centrifugation at 1200g for five minutes at 4°C. The haemolysates were then used to determine erythrocyte antioxidant enzyme activity. All samples were then stored at -80°C. Serum levels of hs-CRP (Diagnostics Biochem Canada Inc., Canada), IL-6, and TNF-α (Diaclone, France) and PC (ZellBio GmbH Ulm, Germany) were measured using ELISA kits. The inter- and intra-assay coefficients of variation for hs-CRP, IL-6, TNF-α, and PC were 9.1% and 9.5%, 7.7% and 4.2%, 9% and 3.3%, and <12% and <10%, respectively.

Serum TAC and MDA levels and erythrocyte activities ofSOD and GR were measured using assay kits (ZellBio GmbH Ulm, Germany). The inter- and intra-assay coefficients of variation for TAC, MDA, SOD, and GR were <4.2% and <3.4%, 7.6% and 5.8%, 7.2% and 5.8%, and 6.6% and 5.2%, respectively. At the time of laboratory assessment, the serum of one subject in the cardamom group was not available. In addition, the number of available haemolysates for measuring erythrocyte SOD and GR activity in the cardamom group was 39 and in the placebo group, 40.
Statistical analysis Analysis was done by intention to treat (ITT) using SPSS version 16 (SPSS Inc., Chicago, IL, USA). The ITT population included all the enrolled and randomized participants. The missing observations were accounted for by using the Last Observation Carried Forward (LOCF) and Last Observation Carried Backward (LOCB) methods. Normality distribution of data was evaluated using the Kolmogorov-Smirnov test. Non-parametric tests were used for TNF-α, TAC, and PC, which were non-normally distributed after transformation. The hs-CRPIL-6-1 ratio was calculated by dividing hs-CRP to IL-6 levels. To compare the data between the two groups, the independent sample t-test and Mann-Whitney was used for normal and non-normal data respectively, considering the normality of data. The analysis of covariance (ANCOVA) was used to identify any differences between the two groups after intervention, adjusting for baseline measurements and covariates. Differences of P<0.05 were considered to be statistically significant.

RESULTS
To evaluate the effects of cardamom supplementation on blood oxidative stress and inflammation status,80 participants were enrolled in the study. All participants consumed 90– 100% of the prescribed supplements and all of them completed the study.

Demographic and anthropometric measurements and dietary intakes
The distribution of weight, BMI, and mean duration of pre-diabetes and physical activity were almost similar between the intervention and the control groups (Table 1). In addition, no significant difference was observed in weight, BMI and physical activity between the two groups at the end of the study. The mean±SD ages of the participants in the cardamom and placebo groups were 48.3±10.4 and 47.5±10.3 years, respectively, while no significant differences were found between the two groups. The mean ± SD energy for the cardamom group was 2107.5 ± 317.0 and 2153.4 ± 198.9 kcald-1 before and after the study respectively. These figures for the placebo group were 2157.7±242.2 and 2181.1±212.2 kcald-1, respectively. Comparison between the two groups showed that after intervention, saturated fatty acid (SFA) intake was significantly higher (p=0.005) and poly unsaturated fatty acid (PUFA) intake was lower (p=0.02) in the cardamom group (Table 2). No significant difference was found between the two groups in daily energy, vitamin A, C, E, and selenium intake.

Effect of cardamom supplementation on inflammatory and oxidative stress biomarkers
After intervention, between-group comparisons showed that in the cardamom group, the mean TNF-α (p<0.001) was significantly higher, and hs-CRP (p=0.04), hs-CRP IL-6-1 ratio (p=0.01), PC(p<0.001) and MDA (p=0.003) were lower than the placebo group. However, after adjustment for SFA and PUFA intake changes and baseline values, between-group differences of hs-CRP (p=0.02), hs-CRP IL-6-1 ratio (p=0.008), and MDA (p=0.009) remained significant (Table 3). DISCUSSION
This randomized clinical trial is the first investigation of green cardamom effects on inflammatory and oxidative stress indices in overweight and obese pre-diabetic women who are at risk of cardiovascular diseases. After eight weeks, cardamom supplementation reduced serum hs-CRP, and hs-CRP IL-6-1 ratio. In addition, cardamom supplementation reduced serum MDA levels. However, a complete improvement of inflammatory and oxidative stress parameters was not achieved. Different results may be obtained with a higher supplement dose and duration of intervention.

Several animal studies have shown beneficial effects of cardamom on blood inflammation and oxidative stress indices. However, a few studies have investigated its effect on humans. In atherosclerotic rats, cardamom-rhizome-ethanolic-extract significantly increased SOD levels and decreased MDA, CRP and IL-6.30 By reducing the synthesis of eicosanoid mediators of inflammation, a dose-dependent anti-inflammatory effect of cardamom oil on rats has been shown.26 In addition, by decreasing cyclooxygenase-2 (COX-2) and inducible nitric oxidesynthase (iNOS) expression, the anti-inflammatory activity of cardamom has been reported.24 In another study, cardamom supplementation for eight weeks prevented oxidative stress and ameliorated the infiltration of inflammatory cell and fibrosis in the liver of rats fed on a HCHF-diet.27 A cellular study by Bhattacharjee et al. has shown that the key components of essential oil in cardamom (i.e. 1.8-cineol [eucalyptol], beta-pinene, geraniol) provide anti- inflammatory activity by binding with TNF-a, IL-1 beta; IL- 4; and IL-5.18

In individuals with stage-1 hypertension, Verma et al. found that the intake of 3g cardamom powder for 12 weeks significantly reduced blood pressure, enhanced fibrinolysis, and improved antioxidant status.25 Among the reasons that could lead to different results between Verma’s study and the present study is the lack of a control group in Verma’s study, a different intervention period (12 weeks vs. 8 weeks) and different target groups (hypertensive vs. pre- diabetic individuals).

In another human study by Verma et al., on patients with ischemic heart disease, the intake of G. Cardamom (Amomum subulatum Roxb.) fruit powder for 12 weeks showed an enhancement in the serum total antioxidant status.31 The conflicting results between the current study and that one may be, again, due to differences in the type of cardamom supplemented (E. Cardamom vs. G. Cardamom), different durations of intervention, and different target groups. However, in a study with 204 T2DM patients, intake of 3g cardamom with tea had no significant effect on serum hs-CRP and F2-isoprostan.11 Negative results observed in the study was explained by the combined effects of cardamom and black tea or the dominant effect of black tea.35

Weight gain and obesity, which increase oxidative stress and inflammatory mediators, are major risk factors for insulin resistance, pre-diabetes, and T2DM.4So, any agent with anti-inflammatory and antioxidant effects, such as cardamom, might interrupt this correlation. Cardamom presumably exerts its anti-inflammatory effect by reducing the synthesis of eicosanoid mediators of inflammation.26The antioxidant effect of cardamom also comes from the fact that it is a potent blocker of lipid peroxide formation and scavenger of superoxide anions and hydroxyl radicals.35 Recent studies have shown that the serum levels of CRP in IGT or IFG patients—in other words, pre-diabetic patients—is higher than that in normoglycaemic people.36-38 CRP is an indicator of systemic inflammation, and increased CRP during obesity is thought to be caused by IL-6 derived from adipose tissue.39Recent studies have shown that lower hs-CRP to IL-6 may reflect decreased inflammation.

So, it can be considered as a marker of the inflammation status.40-42Our results showed that cardamom significantly decreased the serum hs-CRP and hs-CRP IL-6-1 ratio. So, cardamom may have a function in decreasing hs-CRP in pre-diabetic individuals.

Tip:

In the present study, cardamom decreased MDA levels. However, our results did not show a significant influence of cardamom on PC, TAC, SOD, and GR levels. Overweight and obese subjects have more oxidative stress than normal-weight pre-diabetic subjects and may need a greater amount of cardamom or a longer duration of supplementation. So, this is perhaps the reason why, in our findings, cardamom consumption in overweight and obese subjects did not have the profound result on oxidative stress.

This study had some limitations. First, the sample size was small. Second, the intervention duration was too short to understand the real effects of cardamom supplementation. Third, a single 24-hour food recall questionnaire can result in measurement error. Fourth, as the population of the present study comprised only women, the results cannot be generalized to male pre-diabetic subjects. However, this study is one of the first investigations of green cardamom supplementation effects on inflammatory indices and oxidative stress in overweight and obese pre-diabetic subjects. The control group in the present study was another strength of this study.