Partha Chakrabarti

Mammalian Target of Rapamycin Complex 1 Suppresses Lipolysis, Stimulates Lipogenesis, and Promotes Fat Storage

OBJECTIVE In metazoans, target of rapamycin complex 1 (TORC1) plays the key role in nutrient- and hormone-dependent control of metabolism. However, the role of TORC1 in regulation of triglyceride storage and metabolism remains largely unknown. RESEARCH DESIGN AND METHODS In this study, we analyzed the effect of activation and inhibition of the mammalian TORC1 (mTORC1) signaling pathway on the expression of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), lipolysis, lipogenesis, and lipid storage in different mammalian cells. RESULTS Activation of mTORC1 signaling in 3T3-L1 adipocytes by ectopic expression of Rheb inhibits expression of ATGL and HSL at the level of transcription, suppresses lipolysis, increases de novo lipogenesis, and promotes intracellular accumulation of triglycerides. Inhibition of mTORC1 signaling by rapamycin or by knockdown of raptor stimulates lipolysis primarily via activation of ATGL expression. Analogous results have been obtained in C2C12 myoblasts and mouse embryonic fibroblasts with genetic ablation of tuberous sclerosis 2 (TSC2) gene. Overexpression of ATGL in these cells antagonized the lipogenic effect of TSC2 knockout. CONCLUSIONS Our findings demonstrate that mTORC1 promotes fat storage in mammalian cells by suppression of lipolysis and stimulation of de novo lipogenesis.

FoxO1 Controls Insulin-dependent Adipose Triglyceride Lipase (ATGL) Expression and Lipolysis in Adipocytes

FoxO1 represents a central regulator of metabolism in several cell types. Although FoxO1 is abundant in adipocytes, its biological functions in these cells remain largely unknown. We show here that the promotor region of the rate-limiting lipolytic enzyme, adipose triglyceride lipase (ATGL), has two FoxO1-binding sites, and co-transfection with wild type and unphosphorylated FoxO1 mutant activates the expression of luciferase driven by the ATGL promotor. In 3T3-L1 adipocytes, insulin controls nucleo-cytoplasmic shuttling of FoxO1 and regulates its interaction with endogenous ATGL promotors. Knockdown of FoxO1 in 3T3-L1 adipocytes decreases the expression of ATGL and attenuates basal and isoproterenol-stimulated lipolysis. Infection of mouse embryonic fibroblasts with FoxO1-encoding lentivirus increases ATGL expression and renders it sensitive to regulation by insulin. Thus, FoxO1 may play an important role in the regulation of lipolysis in adipocytes by controlling the expression of ATGL.

SIRT1 controls lipolysis in adipocytes via FOXO1-mediated expression of ATGL

Recent studies have established SIRT1 as an important regulator of lipid metabolism, although the mechanism of its action at the molecular level has not been revealed. Here, we show that knockdown of SIRT1 with the help of small hairpin RNA decreases basal and isoproterenol-stimulated lipolysis in cultured adipocytes. This effect is attributed, at least in part, to the suppression of the rate-limiting lipolytic enzyme, adipose triglyceride lipase (ATGL), at the level of transcription. Mechanistically, SIRT1 controls acetylation status and functional activity of FoxO1 that directly binds to the ATGL promoter and regulates ATGL gene transcription. We have also found that depletion of SIRT1 decreases AMP-dependent protein kinase (AMPK) activity in adipocytes. To determine the input of AMPK in regulation of lipolysis, we have established a stable adipose cell line that expresses a dominant-negative α1 catalytic subunit of AMPK under the control of the inducible TET-OFF lentiviral expression vector. Reduction of AMPK activity does not have a significant effect on the rates of lipolysis in this cell model. We conclude, therefore, that SIRT1 controls ATGL transcription primarily by deacetylating FoxO1.

Insulin Inhibits Lipolysis in Adipocytes via the Evolutionarily Conserved mTORC1-Egr1-ATGL-Mediated Pathway

ABSTRACT One of the basic functions of insulin in the body is to inhibit lipolysis in adipocytes. Recently, we have found that insulin inhibits lipolysis and promotes triglyceride storage by decreasing transcription of adipose triglyceride lipase via the mTORC1-mediated pathway (P. Chakrabarti et al., Diabetes 59:775–781, 2010), although the mechanism of this effect remained unknown. Here, we used a genetic screen in Saccharomyces cerevisiae in order to identify a transcription factor that mediates the effect of Tor1 on the expression of the ATGL ortholog in yeast. This factor, Msn4p, has homologues in mammalian cells that form a family of early growth response transcription factors. One member of the family, Egr1, is induced by insulin and nutrients and directly inhibits activity of the ATGL promoter in vitro and expression of ATGL in cultured adipocytes. Feeding animals a high-fat diet increases the activity of mTORC1 and the expression of Egr1 while decreasing ATGL levels in epididymal fat. We suggest that the evolutionarily conserved mTORC1-Egr1-ATGL regulatory pathway represents an important component of the antilipolytic effect of insulin in the mammalian organism.

The multi-level action of fatty acids on adiponectin production by fat cells.

Current epidemics of diabetes mellitus is largely caused by wide spread obesity. The best-established connection between obesity and insulin resistance is the elevated and/or dysregulated levels of circulating free fatty acids that cause and aggravate insulin resistance, type 2 diabetes, cardiovascular disease and other hazardous metabolic conditions. Here, we investigated the effect of a major dietary saturated fatty acid, palmitate, on the insulin-sensitizing adipokine adiponectin produced by cultured adipocytes. We have found that palmitate rapidly inhibits transcription of the adiponectin gene and the release of adiponectin from adipocytes. Adiponectin gene expression is controlled primarily by PPARγ and C/EBPα. Using mouse embryonic fibroblasts from C/EBPα-null mice, we have determined that the latter transcription factor may not solely mediate the inhibitory effect of palmitate on adiponectin transcription leaving PPARγ as a likely target of palmitate. In agreement with this model, palmitate increases phosphorylation of PPARγ on Ser273, and substitution of PPARγ for the unphosphorylated mutant Ser273Ala blocks the effect of palmitate on adiponectin transcription. The inhibitory effect of palmitate on adiponectin gene expression requires its intracellular metabolism via the acyl-CoA synthetase 1-mediated pathway. In addition, we found that palmitate stimulates degradation of intracellular adiponectin by lysosomes, and the lysosomal inhibitor, chloroquine, suppressed the effect of palmitate on adiponectin release from adipocytes. We present evidence suggesting that the intracellular sorting receptor, sortilin, plays an important role in targeting of adiponectin to lysosomes. Thus, palmitate not only decreases adiponectin expression at the level of transcription but may also stimulate lysosomal degradation of newly synthesized adiponectin.

Adipose triglyceride lipase: a new target in the regulation of lipolysis by insulin.

In adipose tissue, the primary physiological function of insulin is the suppression of lipolysis, the hydrolysis of stored fat. Mechanistically, insulin suppresses lipolysis both in transcriptional and post-transcriptional levels. Insulin signaling acutely inhibits beta-adrenergic signaling by decreasing intracellular cyclic AMP levels and the rate of lipolysis. Insulin also suppresses lipolysis by down-regulating the expression of the rate-limiting lipolytic enzyme, adipose triglyceride lipase or ATGL. In insulin resistance and type 2 diabetes, insulin mediated attenuation of lipolysis is impaired leading to an increased rate of lipolysis and increased release of free fatty acids (FFA) in the circulation. This is one of the potential mechanisms behind the development of hyperlipidemia and subsequent metabolic abnormalities in type 2 diabetes. In this article, we focus on the recent findings that highlight distinct molecular mechanisms by which insulin action is mediated and possible implications of the deregulation of these pathways in the pathophysiological context.

Arsenic-induced promoter hypomethylation and over-expression of ERCC2 reduces DNA repair capacity in humans by non-disjunction of the ERCC2–Cdk7 complex

Arsenic in drinking water is of critical concern in West Bengal, India, as it results in several physiological symptoms including dermatological lesions and cancers. Impairment of the DNA repair mechanism has been associated with arsenic-induced genetic damage as well as with several cancers. ERCC2 (Excision Repair Cross-Complementing rodent repair, complementation group 2), mediates DNA-repair by interacting with Cdk-activating kinase (CAK) complex, which helps in DNA proof-reading during transcription. Arsenic metabolism alters epigenetic regulation; we tried to elucidate the regulation of ERCC2 in arsenic-exposed humans. Water, urine, nails, hair and blood samples from one hundred and fifty seven exposed and eighty eight unexposed individuals were collected. Dose dependent validation was done in vitro using HepG2 and HEK-293. Arsenic content in the biological samples was higher in the exposed individuals compared with the content in unexposed individuals (p < 0.001). Bisulfite-modified methylation specific PCR showed a significant (p < 0.0001) hypomethylation of the ERCC2 promoter in the arsenic-exposed individuals. Densitometric analysis of immunoblots showed a nearly two-fold increase in expression of ERCC2 in exposed individuals, but there was an enhanced genotoxic insult as measured by micronuclei frequency. Immuno-precipitation and western blotting revealed an increased (p < 0.001) association of Cdk7 with ERCC2 in highly arsenic exposed individuals. The decrease in CAK activity was determined by observing the intensity of Ser(392) phosphorylation in p53, in vitro, which decreased with an increase in arsenic dose. Thus we infer that arsenic biotransformation leads to promoter hypomethylation of ERCC2, which in turn inhibits the normal functioning of the CAK-complex, thus affecting DNA-repair; this effect was highest among the arsenic exposed individuals with dermatological lesions.

Adipose Recruitment and Activation of Plasmacytoid Dendritic Cells Fuel Metaflammation

In obese individuals, visceral adipose tissue (VAT) is the seat of chronic low-grade inflammation (metaflammation), but the mechanistic link between increased adiposity and metaflammation largely remains unclear. In obese individuals, deregulation of a specific adipokine, chemerin, contributes to innate initiation of metaflammation by recruiting circulating plasmacytoid dendritic cells (pDCs) into VAT through chemokine-like receptor 1 (CMKLR1). Adipose tissue–derived high-mobility group B1 (HMGB1) protein activates Toll-like receptor 9 (TLR9) in the adipose-recruited pDCs by transporting extracellular DNA through receptor for advanced glycation end products (RAGE) and induces production of type I interferons (IFNs). Type I IFNs in turn help in proinflammatory polarization of adipose-resident macrophages. IFN signature gene expression in VAT correlates with both adipose tissue and systemic insulin resistance (IR) in obese individuals, which is represented by ADIPO-IR and HOMA2-IR, respectively, and defines two subgroups with different susceptibility to IR. Thus, this study reveals a pathway that drives adipose tissue inflammation and consequent IR in obesity.

4E-BPs Control Fat Storage by Regulating the Expression of Egr1 and ATGL

Background: Insulin suppresses lipolysis in adipocytes by inducing Egr1 that inhibits expression of ATGL. Results: Stimulation of mTORC1 or genetic ablation of 4E-BP1/2 increase expression of Egr1 regardless of its mRNA levels. Conclusion: Regulation of Egr1 by insulin takes place predominantly at the level of translation via mTORC1/4E-BP. Significance: Translational control of the Egr1 expression explains the role of mTORC1 in regulation of lipolysis. Early growth response transcription factor Egr1 controls multiple aspects of cell physiology and metabolism. In particular, Egr1 suppresses lipolysis and promotes fat accumulation in adipocytes by inhibiting the expression of adipose triglyceride lipase. According to current dogma, regulation of the Egr1 expression takes place primarily at the level of transcription. Correspondingly, treatment of cultured adipocytes with insulin stimulates expression of Egr1 mRNA and protein. Unexpectedly, the MEK inhibitor PD98059 completely blocks insulin-stimulated increase in the Egr1 mRNA but has only a moderate effect on the Egr1 protein. At the same time, mTORC1 inhibitors rapamycin and PP242 suppress expression of the Egr1 protein and have an opposite effect on the Egr1 mRNA. Mouse embryonic fibroblasts with genetic ablations of TSC2 or 4E-BP1/2 express less Egr1 mRNA but more Egr1 protein than wild type controls. 35S-labeling has confirmed that translation of the Egr1 mRNA is much more effective in 4E-BP1/2-null cells than in control. A selective agonist of the CB1 receptors, ACEA, up-regulates Egr1 mRNA, but does not activate mTORC1 and does not increase Egr1 protein in adipocytes. These data suggest that although insulin activates both the Erk and the mTORC1 signaling pathways in adipocytes, regulation of the Egr1 expression takes place predominantly via the mTORC1/4E-BP-mediated axis. In confirmation of this model, we show that 4E-BP1/2-null MEFs express less ATGL and accumulate more fat than control cells, while knock down of Egr1 in 4E-BP1/2-null MEFs increases ATGL expression and decreases fat storage.

mTORC2 controls cancer cell survival by modulating gluconeogenesis

For rapid tumor growth, cancer cells often reprogram the cellular metabolic processes to obtain enhanced anabolic precursors and energy. The molecular changes of such metabolic rewiring are far from established. Here we explored the role of mTOR (mechanistic target of rapamycin), which serves as a key regulator of cell growth, proliferation and survival, in the metabolic reprograming of cancer cells. When we inhibited mTOR in human hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) cells, using pharmacologic inhibitors or by RNA interference, we noticed shuttle of the glycolytic flux to gluconeogenesis pathway along with reduction in cellular proliferation and survival. Augmentation of gluconeogenesis was mechanistically linked to upregulation of the key gluconeogenic enzymes PCK1 and G6PC expressions, enhanced lactate dehydrogenase activity and glucose-derived lipogenesis without causing any attenuation in mitochondrial function. Interestingly, concomitant knocking down of PCK1 and not G6PC along with mTOR pathway could overcome the inhibition of cancer cell proliferation and survival. These observations were validated by identifying distinctive diminution of PCK1 and G6PC expressions in human HCC and RCC transcriptome data. Significant correlation between mTOR-dependent upregulation of PCK1 and cell death in different cancer cell lines further emphasizes the physiological relevance of this pathway. We reveal for the first time that inhibition of mTORC2 and consequent redistribution of glycolytic flux can have a prosurvival role in HCC and RCC cancer cells only in the presence of downregulation of gluconeogenesis pathway genes, thus identifying novel pivots of cancer cell metabolic rewiring and targets for therapy.