Acetylation of Histone and Modification of Gene Expression via HDAC Inhibitors Affects the Obesity

1Department of Pharmacology, Abhilashi College of Pharmacy, Nerchowk, Distt. Mandi, H.P. 175008, India. 2Department of Pharmaceutical Chemistry, Abhilashi College of Pharmacy, Nerchowk, Distt. Mandi, H.P. 175008, India. 3Department of Pharmacognosy, Abhilashi College of Pharmacy, Nerchowk, Distt. Mandi, H.P. 175008, India. 4Department of Pharmaceutical Chemistry, School of Pharmacy, Maharaja Agrasen University, Baddi, Distt. Solan, H.P. 174103, India. *Corresponding Author E-mail: deepikasharma.bhushan@gmail.com

Obesity is abnormal or excessive fats accumulate in adipose tissue. The excess fat accumulates either in waist and trunk or in periphery of the body 1 . Obesity can be explained on the basis of Body mass index (BMI), although another definition is based on waist circumference 2 . According to classification based on BMI, normal healthy body weight is when BMI falls in between 18.5 kg/m 2 -24.9 kg/m 2 , overweight between 25 kg/m 2 -29.9 kg/m 2 and obesity ≥ 30 kg/m 2 . The obesity can be termed as severe when BMI is more than 40 kg/m 2 , shows the maximum changes and is very difficult to treat without surgery [3][4][5] . As obesity is increasing day by day among the population worldwide, the incidence of metabolic disorders is also increasing along with this. Metabolic disorder is connected with a higher risk of a variety of diseases, including cardiovascular disease, insulin resistance, diabetes, hypertension and hyperlipidemia 6 . The increasing BMI can also lead to incidences of several types of cancer 7,8 . The health consequence of obesity increases the possibility of sudden or unexpected death and also leads to severe health conditions that may decreases the overall aspects of life. The use of energy rich, less nutrient foods with large amount of saturated fats and sugar, and also decreased physical activity can increases the obesity rates 9 . Excess fats firstly stored in the adipose tissues and then in non adipose tissue. Adipose tissues are off two types: white adipose tissue and brown adipose tissue. White adipose tissues are the primary location to store the metabolic energy in the form of triglycerides. Whereas, brown adipose tissue which is rich in mitochondria, contains uncoupling protein 1(UCP1), results in the conversion of energy into heat and regulate the energy expenditure. UCP1 is a fatty acid ion, which acts as a H+ symporter, which bypass the electron chemical gradient through the inner mitochondrial membrane with the help of ATP synthase and results in uncoupling of the mitochondrial oxidative phosphorylation and thereby transform the stored energy into heat 10,11 . Studies had shown that the difference in percentage of obesity can be observed between genetically specific subpopulations, for example American Indians and Northern European people living in proportionate environments, which indicate that the obesity may be issues of gene and environment interactions. The mechanism through which the environmental factors and genes interacts basically includes epigenetic factors for example deoxyribonucleic acid (DNA) methylation and histone modifications 12 . Epigenesis includes histone acetylation and also without changing the actual DNA structure, epigenesis changes the structure of chromatin and thus regulates the gene expression 13 . The DNA molecule is wrapped around the histone octamer to form the basic structure of chromatin called nucleosome. Transcriptional co-activators show basic histone acetyltransferase action and acetylate the histone. The acetylated histone thus activates other co-activators and chromatin modifying engines. This result in alteration of the chromatin structure and activate RNA polymerase II, which is then followed by gene transcription 14 .
Histone acetylation is basically maintained through the action of histone acetyltransferases (HATs) 13,15 . Obesity-associated genes may change the encoded amino acid pattern, and also the mass and action of translated proteins, changes may also be related to the eating patterns that affect individual sensitivity to gain weight in specific environment 16 . Histone or lysine deacetylase (HDAC) enzyme mainly act on histone and non histone proteins, which remove acetyl and acyl group from their lysine residue to regulate various transcription, cell death and metabolism 17 . Deacetylation of histone through HDAC results in heterochromatin formation and gene repression 18,19 .
Class I, Class II, Class IV HDAC's members are Zn2+ dependant deacetylases, which catalyzes the removal of acetyl group from lysine residue in histone and other cellular protiens, where as Class III HDAC members are NAD+ dependant deacetylase 13,20 .

Class I HDAC
Class I HDAC has sequel related to the yeast Rpd3 protein and also having a nuclear localization signal (NLS), although it do not contain nuclear export signal. Therefore these HDAC are mainly localized in nucleus only 21 . Thus shows high enzymatic activity towards histone substrate. Structurally Class I HDAC comprises of conserved deacetyalse site plus short amino and carboxy terminal extensions. Comparative to other members HDAC1 and HDACII are almost similar to each other and present along with each other in some complexes such as sin3 (transcriptional regulatory protein), NuRD (nucleosome remodeling deacetyalse), CoREST, and PRC2 (polycomb repressive complex 2). HDAC3 is present in different complexes such as N-CoR-SMRT complex. Also, there is no distinguished complex for HDAC8 22,23 . Studies had shown that HDAC1 is a prominent target in regulating the thermogenic action in brown adipose tissue. Studies had also shown that Class I and Class II HDAC inhibitors show effect on thermogenic gene expressions of brown fat 24 . Studies had shown that the effect of β adrenergic receptor activation on the chromatin state of brown adipocytes. β adrenergic receptor stimulates UCP1 expression and result in acetylation of histone. Studies had investigated that this effect can be seen with down regulation of Class I HDAC specially HDAC3. HDAC3 inhibitor increases the UCP1 expression and cause histone acetylation. Isoproterenol act by down regulating the HDAC3 and increases the UCP1 expression 25 . Li FenFen et al had studied that HDAC1 regulate the H3K27 deacetylation and methylation and due to which HDAC1 negatively regulates the gene expressions of brown adipocytes. Thus by inhibiting the HDAC1 enhances the thermogenic expression of brown adipocytes. Study revealed that by inhibiting the HDAC1 may be the novel target for the treatment of obesity and by enhancing the brown adipocytes thermogenesis and energy expenditure 24 .

Class II HDAC
Class II HDAC contain extra regulatory domain as compared to Class I HDAC, therefore are larger protein than Class I HDACs. Class II HDAC contains nuclear localization signal as well as nuclear export signal and travel between cytoplasm and nucleus which depends on phosphorylation. Class II HDAC again classified in to two more sub category: Class IIa HDAC, which include HDAC4, HDAC5, HDAC7 and HDAC9, Class IIb, which include HDAC6, HDAC10 21,26,27 .
In Class II a, HDAC4 is present in brain and growth plates of skeleton, HDAC 5 and HDAC 9 are present in muscle, brain and heart, and HDAC 7 is found in endothelial cells and thymocyte. Class II a HDAC members contains N-terminal extensions, which also contains the binding site for the transcription factor myocyte enhancer factor 2 (MEF2) and also for the chaperone protein 14-3-3, and make the HDACs signal responsive. Various kinases (calcium/calmodulin-dependent protein kinase (CaMK) and protein kinase D (PKD) phosphorylate the HDACs. These phosphorylated HDAC bind to the 14-3-3 protein, after which the complex travel from the nucleus to the cytoplasm. The phosphorylation of Class IIa HDACs renders a mechanism for connecting extracellular signals with transcription and shows its action in diverse tissues during production and disease 22,28,29 .
Class II b HDAC include only HDAC6, HDAC10. HDAC 6 is the main deacetylase enzyme present in cytoplasm of mammals. HDAC6 is a unique enzyme which basically contain two catalytic/ deacetylase domain and zinc finger motif (HUB) site at the C-terminus. HDAC mainly deacetylases the cytoskeleton proteins for example α-tubulin and contractin and also transmembrane proteins for example the interferon receptor (IFNαR) and chaperones. HDAC10 is closely related to HDAC6. HDAC10 has its catalytic site on its N-terminal (22,30). Studies had shown that HDAC5 is an important component of leptin singal and is found in hypothalamus of brain. Hence HDAC5 play important role in food intake as well as body weight (31). HDAC6 is mainly located in the cytoplasm and deacetylates different cytoplasmic substrates for example tubulin, Hsp90, cortactin, and MFN1. Study had identified that HDAC6 is a key adjuster of mitochondrial thermogenesis, which is related to the lipid metabolism in brown adipose tissue via cAMP-Pka signaling. Inhibition of HDAC6 decreases the cellular cAMP, which is a key adjuster of uncoupling protein1 (UCP1), which in results causes building up of fats in brown adipose tissue (32). Studies had shown that deletion of HDAC9 results in reduction of BMI and modified metabolic homeostasis, by preventing harmful actions of ingested chronic high-fat food on adipogenic differentiation, enhances the adiponectin responses, and via enhancing the beige adipogenesis, this increases the energy expenditure 20 Class III HDAC Class III HDAC is only class of HDAC which comprises of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases, having sequel which is related to the yeast Sir2 protein. Hence, called as Sirtuins (SIRTs). SIRTs act on vast maximum of cellular proteins present in the nucleus, cytoplasm and mitochondria for post-translational alteration by acetylation or ADP-ribosylation. Out of all SIRTs, SIRT1 and SIRT2 travel among the nucleus and cytoplasm. SIRT, through NAD+ hydrolysis, also represents the deacetylation of protein and combine cellular energy and redox elements to produce several signaling and outlast mechanisms that control various transcription factors for example NF-kB, p53andforkheadbox (FOX) proteins. SIRT1 also show its role in tumor initiation. SIRT2 is present in cytoplasm and deacetylase α-tubulin protein. SIRT3, SIRT4, SIRT5 are present in mitochondria. SIRT3 regulate the actions of acetyl coenzyme A synthetase 2 and deacetylating complex I of the respiratory chain involved in ATP synthesis and play a role to maintain the energetic cell homeostasis. SIRT4 shows ADP ribosylase action that deactivates the glutamate dehydrogenase and also hinder the insulin secretion in β cells of pancreas. SIRT5 increases the ammonia detoxification through deacetylation and stimulation of carbamoyl phosphate synthetase 1 to increase the ammonia detoxification. SIRTs 6 and 7 are mainly present in the nucleus where they show a key role in DNA repair and ribosomal RNA transcription, respectively 21,30 . Activation of SIRT1 increases the functions of mitochondrial, increases the expenditure of energy, also increases the sensitivity of insulin and also decreases the inflammation 33 . SIRT1 results in deacylation of PPARγ, which is a master regulator of adipogenesis. SIRT also increases the binding of deacylated PPARγ with (PRDM 16) and promote the conversion of white adipose tissue to beige phenotype. PRDM16 is a co-regulator of PGC1α and increases the UCP1 expressions. Also PRDM16 regulates the adipogenesis of brown adipocytes and also brown adipose tissue functions. Thus SIRT by deacylating PPARγ play a role in obesity 34 . SIRT2, via deacetylation and activation of forkhead box protein O1, promotes the adipogenesis and lipolysis, whereas SIRT4, via deacetylation and inhibition of malonyl-coenzyme A decarboxylase activity, decreases the lipid oxidation. SIRT3 also shows mitochondrial oxidation and fatty acid oxidation and by showing protein deacetylation. SIRT3 increases mitochondrial antioxidative capabilities. SIRT1 and SIRT3 collectively promote the mitochondrial unfolded protein response (UPR), which further act by protecting mitochondria from proteotoxic stress caused by misfolded proteins that increases mitochondrial protein quality. SIRT5 increases the fatty acid oxidation, mitochondrial respiration and also control lysine acylation of mitochondrial proteins. So, above all the SIRT family shows function in regulating mitochondrial function and metabolism 33 .

Class IV HDAC
HDAC11 is the only member of Class IV HDAC. HDAC11 is present in nucleus. Studies had also shown that HDAC11 consists of long chain fatty acid deacetylase. HDAC11 contains a catalytic site in the N-terminal region. HDAC11 is only member of Class IV HDAC and this HDAC11 is found in heart, brain, muscles, kidney and testes. HDAC11 regulates the equilibrium among immune tolerance and immune activation in CD 4+ and T-cells (20)(21)(22). Study had shown that HDAC 11 reduces the brown adipose tissue thermogenic gene expressions and also prevent beiging of white adipose tissue. Study had shown that the HDAC11 associated with bromodomain and extraterminal (BET) family member BRD2, acts as inhibitor of brown adipose tissue differentiation and thermogenic gene expression. The deletion of HDAC11 causes the enhanced brown adipose tissue uncoupling protein 1 expression, enhanced beiging of white adipose tissue, and also increases the thermogenic gene expression in return to β3 adrenergic signal. Therefore the study provides finding to the possibility of the selective HDAC11 inhibitor can develop to enhance the expenditure of energy for obesity treatment and other metabolic disorders 35 . Role and mechanism of HDAC inhibitor in obesity: HDAC inhibitors are considered as powerful therapeutics agents for the treatment of cardiovascular disease, obesity and diabetes 36 . Various studies in previous years evaluated the actions of different types of inhibitors of HDAC in regulating the gene expressions of brown fat 13 .
HDAC inhibitors represent the different types of chemotherapeutic agents that interrupt the functions of HDAC through indirect stimulation of histone acetylation, HDAC inhibitors also modify the gene expression. HDAC inhibitors can be obtained by natural as well as synthetic sources 30 . Structurally HDAC inhibitors consists of zinc binding site, surface binding site and a link that connect these two binding sites along with the hydrophobic catalytic site channels. HDAC inhibitors mainly bind on the zinc ion on the catalytic site of enzyme and inhibit the activity of HDAC 37,38 .  (30,37). Hydroxamates: Hydroxamates are the most commonly used HDAC inhibitors and have very high binding affinity with the zinc ion. The first HDAC inhibitors approved by the FDA was Suberoylanilide hydroxamic acid (SAHA), which shows its inhibitory action as zing binding group and thus inhibit the deacylation by HDAC. Trichostatin A (TSA) is a naturally derived compound which shows the highest HDAC inhibition activity.
Hydroxamates have ability to inhibit all types of HDAC as compred to other HDAC inhibitors. Various advantages of hydraxamtes are: higher zinc binding ability, good solubility, can be easily synthesized. As these hydroxamtes can bind to other zinc dependant enzymes such as aminopeptidases, metalloproteinases and carbonic anhydrase, which lead produce undesirable effects 37,38 .
R e c e n t s t u d i e s h a d s h o w n t h a t hydroxamtes for example trichostatin A (TSA) inhibit the gene expressions of uncoupling protein 1, peroxisome proliferator activated resecptor γ, PR domain-containing protein 16 (prdm-16). TSA also act through the stimulation of Pgc1α expressions in brown adipose tissue 1 brown adipocyte. TSA act dose dependently to show the action. Suberoylanilide hydroxamic acid (SAHA) also act dose dependently, and act the same way as TSA. TSA and SAHA show their inhibitory action on both Class I HDAC and Class II HDAC. Study represent that TSA and SAHA show their maximum inhibitory action at dose 500 ηM and 5ηM respectively 13,39 . Aliphatic acids: Aliphatic acids or mainly carboxylic acid derivative have less zinc ion binding affinity as compared to hydroxamtes or benzamide. Aliphatic acid with short chain fatty acids, such as valproic acid, butyric acid/ sodium butyrate, phenylbutyric acid/ phenylbutyrate shows HDAC inhibitory action. These aliphatic acids HDAC inhibitor inhibits mainly Class I HDAC and Class IIa HDAC 37,38 .
Valproic acid is widely used as anti epileptic drug. But most recently it can be used as HDAC inhibitor. Valproic acid, which is a short chain fatty acid, also shows HDAC inhibition activity. However it was previously suggested that valproic acid induces the weight gain. But, recently study had shown that Valproic acid suppresses adipogenesis. Valproic acid and other HDAC inhibitors are mainly involved in the improvement of growth and differentiation of adipocytes by activating the adenosine monophosphate activated protein kinase, which is a controller mechanism of cellular metabolism. Study had shown that valproic acid acts via inhibiting fatty acid synthase (FAS) enzyme. Fatty acid synthase plays a major role in lipogenesis in mammals and also catalyses the long chain fatty acid synthesis from acetyl co-enzyme A and malonyl co-enzyme A. FAS activity is regulated by the various hormones and nutrients as well. Enhanced FAS gene expressions in adipose tissue results in accumulation of visceral fat and also changes the insulin sensitivity in humans. Therefore FAS is the major target for the obesity and valproic acid causes the down regulation of the fatty acid synthase enzyme and thus reduces the lipid accumulation in human adipose tissue. Valproic acid also reduces the Pparg and Cebpa gene expression and regulate the expressions of lipolytic genes such as Atgl, Hsl and Mgl, as well as the adipogenic genes such as Fabp4 (fatty acid binding protein 4), Lpl (lipoprotein lipase) and Cd36, a transporter of long-chain fatty acids. Study also showed that valproic acid also inhibit USF-1 gene, as polymorphism of USF-1 genes is associated with the hyperlipidemia and obesity 40,41 . Benzamides: Benzamides such as MS-275 also known as Entinostat, inhibits some of Class I HDAC. The amino group present in benzamide form chelate with zinc ion and therefore show its inhibitory action 37,38 .
Studies had shown that benzamide MS-275 acts dose dependently and stimulate the thermogenic gene expressions of brown fat, which include uncoupling protein 1, peroxisome proliferator activated receptor γ, fatty acid elongase 3 (Elovl3) and peroxisome proliferator activated receptor gamma coactivator 1 alpha (Pgc1α). Studies showed that the treatment of HIB-1b cell line with benzamide MS-275 significantly increases the basal and isoproterenol stimulated gene expressions of brown fat including uncoupling protein 1, peroxisome proliferator activated receptor gamma coactivator 1 alpha, peroxisome proliferator activated receptor gamma coactivator 1β, acyl-CoA oxidase1 (ACOX 1), cell death inducing DFFA like effector A (CIDEA), and peroxisome proliferator activated receptor and study also show that MS-275 do not any effect on COX-1 expressions. The study also showed that along with the stimulating brown adipocyte thermogenic regulations, this MS-275 treatment also regulate enhanced uncoupling protein 1 messenger RNA expressions in 3T3-L1 cell line of white adipocytes. Ucp1 is a mitochondrial uncoupling agent in brown adipose tissues and Pgc1α play as a master controller of brown fat thermogenesis 13,39,42 .
MS-275 enhances the oxidative metabolism in adipose tissue and also in muscle tissues, thus increases the energy expenditure, which can lead to the reduction in body weight. Study had shown that MS-275 inhibit action of both HDAC1 and HDAC3. Study also showed that animal reduces 10% of body weight via treatment of MS-275 43 . Tetrapeptide/Depsipeptide: Apicidin, romidepsin, trapoxin B are the example of tetrapeptide/ depsipetide. Apicidin is a cyclic tetrapeptide which is isolated from the fungus Fusarium Spp. Apicidin is known as selective inhibitor of HDAC class 1. Apicidin bind to HDAC class 1 and interfere with the deaetylation of protein 44,45 .
Romidepsin is another tetrapeptide/ depsipeptide which inhibits HDAC1 and HDAC2 and is widely used as anti cancer drug. Recent study had shown that romidepsin, via the stimulation of adipocytes linked with transcriptional factors which is required for the adipocytes differentiation for example proliferator-activated gamma receptor (PPARγ2) and other genes related to adipogenesis including AD1, POQ, apetala 2 (AP2), phosphoenolpyruvate carboxykinase 1 (PCK1), lipoprotein lipase (LPL), lipase E (LIPE), induces the adipocytic differentiation. Expression of these genes can lead to obesity and diabetes 46,47 .

Sirtuin inhibitor (SIRTi):
Nicotinamide is the most widely used SIRT inhibitor, which inhibits the deacetylation of class III HDAC. Nicotinamide is form of vitamin B3 or amide form of niacin, which play important role in production of nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), which also takes part in biological processes such as energy production, redox reactions and biological rhythm. Nicotinamide deacetylases the SIRT1 and results in modification of various target protein actions and help the cell to survive in various stress conditions. Study had shown that circulating nicotinamide in form of nicotinamide phosphoribosyltransferase is related to obesity, insulin resistance, lipid profile and inflammation. Study had shown that there is increased expression of adipose tissue towards nicotinamide phosphoribosyl transferase, which results in induction of obesity (48)(49)(50). Study had shown that the nicotinamide can be considered as novel marker of systemic inflammation which is associated with fat depot, in the elderly population 50 .
Another drug which is used as SIRT inhibitor is EX-527. Study had shown that EX-527 inhibits the SIRT1 and thus reduces the SIRT-1 phosphorylation and PRDM-16 deacetylation 34 .

ConCluSIon
The study explains the different classes of Histone deacetylase as Class I, Class II, Class III and Class IV and their mechanism by which they play role in obesity. Class I, Class II and Class IV HDAC are Zn+ dependant deacetylase and by binding on the Zn+ they increases the incidence of obesity, whereas Class III HDAC, alternatively known as SIRT, is NAD+ dependant and reduces the incidence of obesity. In this study, different HDAC inhibitors were also explained. Out of which HDAC inhibitor hydroxmates, was the first approved HDAC inhibitor, aliphatic acids and benzamides act by binding on Zn+ and increases the gene expression required for acylation of histone protein and thus by increasing the gene expression these may lead to beiging or browning of white adipose tissue as well as energy expenditure. Other class of HDAC inhibitor which includes SIRT inhibitor or specifically Class III HDAC inhibitor shows opposite effect than other classes of HDAC inhibitor. SIRT inhibitor interferes with NAD+ synthesis. Nicotinamide is example of SIRT inhibitor which increases the lipid profile as well as fat depot and thus induces obesity and inflammation, whereas Resveratrol, a polyphenolic compound, act by stimulating SIRT and show pharmacological properties in cardiovascular disease, obesity, aging and also in diabetes mellitus.

Conflict of Interest
There are no conflicts of interest.

Funding Source
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.