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Connecting homocysteine and obesity through pyroptosis,

gut microbiome, epigenetics, peroxisome proliferator-activator receptor γ (PPARγ) and zinc finger protein 407

(Zfp407)

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2018-0037.R1

Manuscript Type: Review

Date Submitted by the Author: 29-Mar-2018

Complete List of Authors: Laha , Anwesha ; University of Louisville School of Medicine, Physiology Majumder, Avisek; University of Louisville School of Medicine, Physiology Singh, Mahavir; University of Louisville School of Medicine, Physiology Tyagi , Suresh C. ; University of Louisville School of Medicine, Physiology

Is the invited manuscript for consideration in a Special

Issue: IACS EU Section

Keyword: PGC-1α, Zinc finger protein, DNA methylation, Lipopolysaccharide, Fatty

acid metabolism

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Connecting homocysteine and obesity through pyroptosis, gut microbiome, epigenetics,

peroxisome proliferator@Hactivator receptor γ (PPARγ) and zinc finger protein 407 (Zfp407)

Anwesha Laha, Avisek Majumder, Mahavir Singh, Suresh C.Tyagi

Department of Physiology, University of Louisville, Louisville, Kentucky.

Corresponding authors: Anwesha Laha ([email protected])

or Mahavir Singh ([email protected])

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Abstract: Although homocysteine (Hcy), a part of the epigenome, contributes to cell death

by pyroptosis and decreases PPARγ levels, the mechanisms are unclear. Hcy is found in

high concentrations in the sera of obese individuals which can elicit an immune response as

well by hypermethylating CpG islands of specific gene promoters, a marker of epigenetics.

Hcy has also been established to chelate divalent metal ions like Cu+2 and Zn+2, but this role

of Hcy has not been established in relationship with obesity. It has been known for a while

that PPARγ dysregulation results in various metabolic disorders including glucose and lipid

metabolism. Recently, zinc finger protein 407 (Zfp407) is reported to regulate PPARγ target

gene expression without affecting PPARγ transcript and protein levels by synergistically

working with PPARγ. However, the mechanism(s) of this synergy, as well as other factors

contributing to or inhibiting this synergism have not been proven. This review suggests that

Hcy contributes to pyroptosis; changes gut microbiome, alter PPARγ-dependent

mechanism(s) via Zfp407 mediated upregulated adipogenesis and misbalanced fatty acid

metabolism leading that can predispose to obesity and consequently the obesity-related

metabolic disorders.

Keywords: PGC-1α, zinc finger protein, DNA methylation, lipopolysaccharide, fatty acid

metabolism, type 2 diabetes, insulin.

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Homocysteine and obesity

Obesity is one of the leading causes of health complications like type 2 diabetes (T2D),

stroke, hypertension, cardiovascular diseases, and even some cancers. As of 2014, the

World Health Organization (WHO) stated that 1 in 5 individuals in the United States died due

to obesity-related health disorders. Per Center for Disease Control, 20% of the population in

every state in the U.S. is obese (WHO, Centre for Disease Control). Obesity is responsible

for causing the insulin resistance leading to dysregulated lipid metabolism by reducing

adipogenesis and increasing free fatty acids (FFA) in the blood (Harmancey and Smih 2007;

Kim et al. 2000; Odegaard and Chawla 2013) .

Homocysteine is a non-protein amino acid that is generated during methionine metabolism.

The fate of homocysteine is either remethylation to methionine or conversion to cysteine

through the transsulfuration pathway. Folic acid works as the cofactor essential for

remethylation of homocysteine to methionine (Barshop 2012). The two essential enzymes

required for Hcy to cysteine transsulfuration are cystathionine β-synthase (CBS) and

cystathionine γ-lyase (CSE) (Figure 1). In the rate-limiting step, CBS catalyzes the synthesis

of the intermediate, cystathionine from Hcy and CSE converts cystathionine to cysteine.

Hyperhomocysteinemia (HHcy) is a medical condition that is characterized by the

homocysteine's level which is more than 15µmol/L homocysteine (Hcy) in the blood due to

the body’s inability to metabolize homocysteine properly (Guo et al. 2009). HHcy is

responsible for a number of pathophysiological conditions like atherosclerosis, hypertension,

vascular dysfunction leading to coronary heart diseases and myocardial infarction (Sen et al.

2010). Deficiencies in folic acid, vitamins B6 and B12 and mutations in CBS gene have been

attributed to HHcy in infants and adults (Huemer et al. 2015). Dietary supplementation of

Folic acid, Vitamin B6 (pyridoxal-5’-phosphate) and Vitamin 12 (Cobalamine) (Maron and

Loscalzo 2009) are used as therapy for HHcy, a defined therapy for congenital HHcy has not

yet been established.

HHcy is also one of the major contributing factors in obesity. Excess Hcy suppresses

preadipocyte differentiation (Mentese et al. 2016) and lipolysis in primary adipocytes (Wang

et al. 2011). Decreased lipolysis in adipocytes results in reduced levels of FFA for energy

expenditure and accumulation of triglycerides in adipose tissues. Concurrently, plasma

homocysteine levels in obese individuals with hypertension have been found to be much

more than in non-obese individuals with hypertension (Konukoglu et al. 2003). Obese

individuals with high Hcy levels are predisposed to cryptogenic strokes (Vaya et al. 2011)

and insulin resistance (Vaya et al. 2012). Although it is well established that Hcy is a

contributing factor to obesity and obesity-related health risks, the exact role of Hcy in altering

the related molecular mechanisms are not yet studied. We are therefore providing avenues

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for uncovering these intricate molecular mechanisms involving the role of Hcy in obesity

paradigm.

Pyroptosis is an inflammation-mediated programmed cell death characterized by cell

swelling and membrane disruption. Pyroptosis occurs upon activation of Caspase 1 followed

by the assembly and formation of the NLRP3 inflammasome (Lamkanfi 2011). Pyroptosis

mediated inflammatory response has been reported to be elicited not only by infections via

pathogens but also by the overexpression of certain macromolecules. High Hcy levels can

elicit pyroptosis in the presence and absence of lipopolysaccharide (LPS) (Xi et al. 2016).

LPS which is usually present in the membranes of Gram-negative bacteria and can elicit an

immune response. This is found in high concentrations in the plasma of obese individuals

consuming a high-fat diet (Erridge et al. 2007) (Troseid et al. 2013). Obese adipocytes have

been reported to overexpress NLRP3 inflammasome and undergo stress-induced pyroptosis

(Giordano et al. 2013). It is also predicted that pyroptosis may be a mode of cell death in

HHcy mediated age-related macular degeneration (AMD), a disorder also prevalent among

obese individuals (Adams et al. 2011) (Singh and Tyagi 2017).

Interestingly, the gut microbiome has been shown to play a major role in the healthy living of

an individual. The gut microbiome consists of innumerable bacteria and virus and their

numbers, their genomes, and the environment are dynamic with respect to an individual's

diet. This normal gut microbiota composition is heavily altered due to perpetual consumption

of high-fat diet and that can contribute to obesity and type 2 diabetes (Baothman et al. 2016;

Wolf and Lorenz 2012). This allows an increased number of Gram-negative bacteria

expressing LPS and thus, capable of eliciting pyroptosis. The exact mechanism of obese

adipocyte stress induced pyroptosis is not clear allowing us to hypothesize that Hcy/LPS is

mediating pyroptosis in the obese adipocytes due to the gut microbiota imbalance. A

schematic representation of this hypothesis is shown in figure 2. There are several studies

which suggest that gut microbiota transplant (GMT) will be an effective therapy for obesity

and obesity related disorders, there are no concrete evidence of the effectiveness of GMT.

PPARγ, PGC-1α and their relationship with homocysteine through epigenetics

Peroxisome proliferator-activator receptor-gamma (PPARγ) is a master regulator of lipid

metabolism and adipogenesis. It belongs to the nuclear hormone receptor superfamily and is

highly expressed in adipose tissues (Chawla et al. 1994; Tontonoz et al. 1995). Its

heterodimerization with Retinoic X Receptor (RXR) regulates target gene expressions. In the

absence of a ligand, co-repressors are bound to the PPARγ-RXR complex keeping it

transcriptionally inactive. When specific ligands bind to the ligand binding domain of PPARγ,

a conformational change releases the bound corepressors. This allows co-activators like

PGC-1α and other coactivators to be recruited to the target genes promoting PPARγ-

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mediated transcription (Costa et al. 2010; Murphy and Holder 2000). Lack of PPARγ

functions have been shown to reduce preadipocyte differentiation and adipogenesis both in

vitro and in vivo (Murphy and Holder 2000). Conversely, adipogenesis has been shown to be

downregulated when its expression is reduced, implying that it is essential for normal

adipogenesis facilitating normal lipid metabolism (Kim et al. 2001; Rosen et al. 1999).

Although numerous studies reveal the various pathways and regulators of PPARγ mediated

target gene transcription, several regulators and their roles in transcription are still unknown.

Therefore, it is essential to elucidate the mechanisms by which other regulators of PPARγ

mediated transcription affects the expression of target genes. PPARγ interacts with many

coactivators to carry out the transcription of its target genes, such as coactivator-1α (PGC-

1α) which is a key PPAR coactivator regulating the target gene expressions. The LXXLL

domain of activated PGC-1α directly interacts with parts of PPARγ’s DNA binding domain

and hinge domain (Puigserver et al. 1998). This allows the recruitment of histone

acetyltransferase (HATs) containing proteins maintaining PPARγ’s transcriptional activity

(Puigserver 2005). PGC-1α has been reported to play a vital role in regulating essential

biological processes like mitochondrial biogenesis and fatty acid β-oxidation (Vega et al.

2000)

Homocysteine has been established to play a role in epigenetics, especially DNA

hypermethylation due to increased concentration of S-adenosylmethionine (SAM), a methyl

group donor (Figure 1) (Majumder et al. 2017). This eventually contributes to abnormal

silencing of specific genes involved in essential cellular processes and leads HHcy related

pathophysiologies (Kalani et al. 2013). Previous reports have shown that elevated

concentrations of Hcy results in hypermethylation of the CpG islands of the PPARγ promoter

regions resulting in reduced gene expression (Yideng et al. 2008). Similarly, high Hcy levels

also contribute to cardiomyopathy by attenuating PPARγ gene expression and this effect is

alleviated by its agonists and ligands (Mishra et al. 2010). From these and other reports, we

believe that Hcy is behaving as a PPARγ antagonist. This antagonism could dysregulate

fatty acid metabolism through PPARγ governed genes and hence could promote obesity-

related pathophysiological conditions. This hypothesis will open various avenues for

researchers to identify these factors contributing to the antagonism between PPARγ and

Hcy.

Chromatin remodeling and epigenetics play an important role in the inherent functioning of

both, PPARγ and PGC-1α. PPARγ has been reported to go through extensive chromatin

remodeling during the early stages of adipogenesis (Lee and Ge 2014). Also,

CCAAT/Enhancer Binding Proteins (C/EBPs) are a group of leucine zipper transcrition

factors that have been associated with adipogenesis in the presence of PPARγ (Lane et al.

1999) (Rosen et al. 2002). Evidence suggests that the association of C/EBPα with the

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PPARγ promoter allows increased transciption and that inhibitors disrupt this association.

This in turn stops PPARγ transcription through the recuitment of histone deacetylases

(HDACs) (Zuo et al. 2006) These suggested pathways of chromatin remodelling of the

PPARγ promoter allow transcription factors to interact with it allowing transcription of genes.

Like PPARγ, PGC-1α too has a negative correlation with Hcy. Enhanced PGC-1α

expression upregulates genes involved in Hcy metabolism (Li et al. 2009). Likewise, Hcy

contributes to attenuated PGC-1α expression leading to mitochondrial dysfunction (Veeranki

et al. 2015). It is thus clear that the synergism of PPARγ and PGC-1α is antagonized by

HHcy. Just as the PPARγ-PGC-1α synergism may be regulated by other unknown factors,

the same unknown factors may be antagonized by HHcy and these correlations require

further analyses.

PGC-1α expression too has been shown to be epigenetically regulated at its promoter.

Histone modifications can either be transcriptionally activating (H3K4me3, H3K27ac) or

repressing (H3K27me3) for their targets. In a recent study, PGC-1α promoter b (PGC-1α-b)

has been shown to have increased H3K4me3 in mice skeletal muscles upon exercise

(Lochmann et al. 2015). There is also evidence that exercise can mitigate the effects of Hcy

in skeletal muscles and white adipocytes (Winchester et al. 2014). Therefore, Hcy could

potentially silence PGC-1α expression through epigenetic histone remodeling and exercise

may possibly mitigate this effect of Hcy thereby allowing enhanced PGC-1α expression

however this would further work to investigate these possibilities. Both PGC-1α and PPARγ

have been reported to work in tandem and show close relationships as demonstrated in a

study wherein their expression levels were decreased in conditions such as obesity and

insulin resistance. Interestingly, physical training in form of exercise could restore their levels

proving the beneficial effects of physical activity in humans (Ruschke et al. 2010).

PPARγ and Zfp407

In a recent study, PPARγ target genes critical for lipid metabolism were shown to be

regulated by Zinc Finger Protein 407 (Zfp407) (Buchner et al. 2015). Knockdown of Zfp407

decreased expression of target genes. This was shown in an RNA-seq experiment where

expression of several PPARγ regulated genes involved in lipid metabolism were analyzed

after the knockdown of Zfp407 (Buchner et al. 2015). However, when PPARγ levels were

analyzed after the knockdown of Zfp407, there was no change in either the transcript or

protein levels, indicating that Zfp407 does not regulate PPARγ expression. In a luciferase

reporter assay when HEK239T cells were co-transfected with PPARγ and Zfp407, luciferase

activity of the co-transfected cells increased 2-fold compared to PPARγ or Zfp407

transfections alone suggesting that Zfp407 synergistically works with PPARγ. However, the

underlying mechanisms of this regulation remain unknown. Although PPARγ and Zfp407

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have been shown to work synergistically, other factors may influence their activities

(Buchner et al. 2015) and these other factors are yet to be elucidated. Further studies can

help us better understand the mechanism of Zfp407 to regulate PPARγ target gene

expression and determine the key players involved in regulating their activities. This can

eventually lead to the identification of potential drug targets for diabetes and obesity-related

diseases.

Potential functional similarity between Zfp407 and PRDM16

Positive Regulatory Domain Containing 16 (PRDM16) is a zinc finger-containing protein with

10 zinc finger domains that has been shown to interact with both PPARγ and PGC-1α

(Hondares et al. 2011; Kajimura et al. 2009; Seale et al. 2008). Together they form a

complex triggering the PPAR-mediated transcriptional program required for brown

adipogenesis (Fruhbeck et al. 2009). PRDM16 also binds to the PGC-1α promoter to induce

PGC-1α transcription under increased levels of active PPARα in brown adipocytes

(Hondares et al. 2011). Thus, PRDM16 performs a dual function of a transcription

coactivator for PPARs and a transcription factor for PGC-1α in brown adipogenesis.

Although the mechanisms and proteins involved in white adipocyte differentiation have been

extensively studied, many interacting partners of PPARγ and PGC-1α in the transcriptional

program of white adipogenesis still remain unknown. We hypothesize that Zfp407 could

potentially play the role of PRDM16, interacting with PPARγ and PGC-1α during

adipogenesis along with inducing PGC-1α transcription during adipogenesis in white adipose

tissue. Zfp407 could also possibly induce PGC-1α expression and interacting with PPARγ

and PGC-1α together, playing a role in their complex formation to maintain PPARγ’s

transcriptional activity which needs further analyses (Figure 3). Zfp407, transcribed by

ZNF407 on chromosome 18, belongs to the C2H2 class of zinc finger proteins and consists

of 22 zinc finger domains (Kambouris et al. 2014; Ren et al. 2013). The C2H2 (Cys-Cys-His-

His) class of zinc finger proteins are DNA binding proteins. They generally work as

transcription factors due to their higher binding affinity to DNA than to RNA. We therefore

believe that Zfp407 may also function as a transcription factor by directly interacting with

DNA through its C2H2 domain resulting in PPARγ target gene expression (Iuchi 2001; Laity

et al. 2001). The sequences could potentially be the promoters of PPARγ coactivators or

enhancers of the PPARγ target genes and Zfp407 is directly interacting with DNA sequences

that could possibly be associated with transcriptional activity. However, there is no concrete

evidence of Zfp407’s interaction with DNA or the nucleotide sequences interacting with

Zfp407 and we therefore pave the way for researchers to identify these DNA sequences

(Figure 3)

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Homocysteine can potentially regulate zinc finger proteins and contribute to obesity

It has been reported earlier and well established that the divalent cation copper (Cu+2) can

form a complex with Hcy during HHcy. This results in copper homeostasis imbalance due to

an increase in blood Cu+2 concentration and decreased intracellular Cu+2 concentration

(Dong et al. 2013). Zinc, like Cu+2 is a divalent metal ion and an essential mineral which is a

part of a zinc finger proteins working as transcription factors and involved in numerous other

cellular processes. Zn+2’ deficiency has been attributed to DNA instability also (Jurowski et

al. 2014) and can potentially result in their subsequent protein synthesis inhibition. Zn+2

deficiency could also lead to the instability and disintegration of zinc finger proteins since Zn

bonds with cysteine and histidine. As mentioned earlier, cysteine is a product of Hcy

transsulfuration metabolism. Therefore, Zn and cysteine deficiency together can exacerbate

the stability of zinc finger proteins. Similar to Cu+2, previous studies showed that serum Zn+2

and Hcy concentrations are positively correlated and serum Hcy and tissue Zn+2

concentrations are negatively correlated (Hong et al. 2000) (Jing et al. 2015). It is therefore

possible that excess serum Hcy is forcing Zn+2 to conjugate with excess Hcy thereby

reducing the intracellular Zn+2 concentrations. This hinders synthesis of Zn+2 containing

proteins including transcription factors like Zfp407 and PRDM16. In this context, it is not

surprising that HHcy can therefore contribute and hence promote obesity by suppressing

PPARγ target genes by chelating cytosolic Zn+2. This could eventually downregulate Zfp407

expression and that in turn could repress genes which are involved in insulin sensitivity and

fatty acid metabolism. We are providing a platform to researchers to establish further that

Hcy is an inhibiting factor to zinc finger protein functioning. In fact, studies revealed that a

sizable number of zinc finger proteins could function as key transcriptional regulators that

are involved in the process of adipogenesis and as a result of this observations they are

being considered as promising human obesity drug targets (Wei et al. 2013) (Figure 4).

Concluding statement

In our current review, we have proposed different mechanisms by which elevated Hcy levels

can contribute to obesity and obesity-related disorders. We have suggested three different

routes of Hcy to exhibit different obesity like phenotypes. The first route is obesity-related

inflammation through pyroptosis aided by Hcy, the second is Hcy mediated DNA methylation

and therefore, silencing of genes essential for normal FA metabolism and the third route is

by modulating cellular Zn+2 concentration to inhibit PPARγ target genes involved in lipid

metabolism. All three mechanisms either can work in tandem or individually. In this review,

we have opened different avenues for researchers to uncover the mode of actions of Hcy in

obesity and its related disorders as well as design drugs to alleviate these important

metabolic disorders.

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Conflict of interest: None

Acknowledgements: We would like to thank all the members of the laboratory for their

continuous support and encouragement. Part of this work was supported by grants from the

National Institute of Health (Heart, Lung, and Blood Institute; No. HL-74815 and HL-139047).

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List of abbreviations

AMD: age-related macular degeneration

Cu+2: copper

C/EBPs: CCAAT/enhancer binding proteins

CBS: cystathionine β-synthase

CSE: cystathionine γ-lyase

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DNMT: DNA methyl transferase

FFA: free fatty acid

GMT: gut Microbiota transplant

HATs: histone acetyltransferases

Hcy: homocysteine

HHcy: hyperhomocysteinemia

LPS: lipopolysaccharide

NLRP3: nucleotide-binding domain, leucine-rich-containing family, pyrin domain-

containing-3

PPAR: peroxisome proliferator activator receptor:

RXR: retinoic X receptor

T2D: type 2 diabetes

Zn+2’: Zinc

Zfp: zinc finger protein

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Figure legends

Fig. 1. A schematic representation of methionine metabolism depicting the transmethylation,

transsulfuration and remethylation pathways. Dietary methionine is converted to s-adenosyl

methionine (SAM) which is further converted to s-adenosyl homocysteine (SAH) in the

presence of DNMT. SAH is converted to homocysteine (Hcy) which is further remethylated

to methionine in the presence of folic acid (Vitamin B12) and the cycle continues. Under low

concentrations of cysteine, Hcy is transsulfurated to cysteine where CBS and CSE act as

essential catalysts.

Fig. 2. Proposed pathway representing elevated homocysteine levels contributing to the

obesity-related dysbiosis and pyroptosis. Perpetual consumption of high fat diet leads to

dysbiosys of the gut microbiota. This increases the number of Gram-negative bacteria,

expressing LPS on their membrane, in the gut. The high LPS concentration together with

elevated Hcy levels elicit an immune response causing activation of Caspase 1 and

consequently NLRP3 inflammasome assembly. This induces cellular death by pyroptosis.

Fig. 3. A model of zinc finger protein 407 (Zpf407) regulating PPAR-γ target gene expression

under normal conditions. Zfp407, upon maturation and entry into the nucleus, may interact

with either the PPAR-γ coactivator-1α (PGC-1α) promoter to increase PGC-1α transcription

which can regulate PPAR-γ target gene expression. Zfp407 can also interact with PPAR-γ

directly or by scaffolding proteins to form a PPAR-γ-PGC-1α complex on PPAR-γ responsive

elements (PPREs) and regulate PPAR-γ target gene expression. In a third proposed

mechanism, Zfp407 itself can work as a transcription factor and interact with its own target

genes which are yet to be studied.

Fig. 4. Model representing the mechanistic aspects of Zfp407 regulation of PPARγ target genes under high serum homocysteine concentrations via Zn+2 sequestration. High Hcy concentration in the serum can sequester free Zn+2 ions forming a Zn-Hcy complex which can be transported to the extracellular milieu. Thus, the Zn+2 ions necessary to be incorporated into the Zfp407 is unavailable and the synthesis of Zfp407 is inhibited through its pre-mature degradation. This could potentially reduce the proposed gene regulatory mechanisms of Zfp407 either by reducing PGC-1α synthesis or weakening the interaction between PPAR-γ-PGC-1α complex.

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Methionine

Homocysteine

S-Adenosyl Methionine (SAM)

S-Adenosyl Homocysteine (SAH)

Diet

THF

B12

CH3-THF

Cystathionine

Cysteine

CSE

DNMT

CH3-Cytosine

CBS

Adenosine

Adenosine

Remethylation

Tra

nssulfura

tion

Transmethylation

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Gut Microbiota disorganization

LPS

Gram- Negative Bacteria

+ Homocysteine

Caspase 1 Activation NLRP3 Inflammosome Pyroptosis

High-Fat Diet

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Zfp407

PPREs

Other Target Genes

PGC-1α

PGC-1α Promoter

Cytoplasm

Nucleus

PPAR-γ?

?

?

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Zfp407

PPREs

Other Target Genes

PGC-1α

PGC-1α Promoter

Cytoplasm

Nucleus

HcyZn

PPAR-γ?

?

?

?

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