RNA interference targeting cytosolic NADP(+)-dependent isocitrate dehydrogenase exerts anti-obesity effect in vitro and in vivo

A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome.     

Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism

NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP(+)-dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor gamma were markedly elevated. The hepatic and epididymal fat pad contents of acetyl-CoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.     

Overexpression of glucose-6-phosphate dehydrogenase is associated with lipid dysregulation and insulin resistance in obesity

Glucose-6-phosphate dehydrogenase (G6PD) produces cellular NADPH, which is required for the biosynthesis of fatty acids and cholesterol. Although G6PD is required for lipogenesis, it is poorly understood whether G6PD in adipocytes is involved in energy homeostasis, such as lipid and glucose metabolism. We report here that G6PD plays a role in adipogenesis and that its increase is tightly associated with the dysregulation of lipid metabolism and insulin resistance in obesity. We observed that the enzymatic activity and expression levels of G6PD were significantly elevated in white adipose tissues of obese models, including db/db, ob/ob, and diet-induced obesity mice. In 3T3-L1 cells, G6PD overexpression stimulated the expression of most adipocyte marker genes and elevated the levels of cellular free fatty acids, triglyceride, and FFA release. Consistently, G6PD knockdown via small interfering RNA attenuated adipocyte differentiation with less lipid droplet accumulation. Surprisingly, the expression of certain adipocytokines such as tumor necrosis factor alpha and resistin was increased, whereas that of adiponectin was decreased in G6PD overexpressed adipocytes. In accordance with these results, overexpression of G6PD impaired insulin signaling and suppressed insulin-dependent glucose uptake in adipocytes. Taken together, these data strongly suggest that aberrant increase of G6PD in obese and/or diabetic subjects would alter lipid metabolism and adipocytokine expression, thereby resulting in failure of lipid homeostasis and insulin resistance in adipocytes.     

CCAAT/enhancer-binding protein beta deletion reduces adiposity, hepatic steatosis, and diabetes in Lepr(db/db) mice

CCAAT/enhancer-binding protein beta (C/EBPbeta) plays a key role in initiation of adipogenesis in adipose tissue and gluconeogenesis in liver; however, the role of C/EBPbeta in hepatic lipogenesis remains undefined. Here we show that C/EBPbeta inactivation in Lepr(db/db) mice attenuates obesity, fatty liver, and diabetes. In addition to impaired adipogenesis, livers from C/EBPbeta(-/-) x Lepr(db/db) mice had dramatically decreased triglyceride content and reduced lipogenic enzyme activity. C/EBPbeta deletion in Lepr(db/db) mice down-regulated peroxisome proliferator-activated receptor gamma2 (PPARgamma2) and stearoyl-CoA desaturase-1 and up-regulated PPARalpha independent of SREBP1c. Conversely, C/EBPbeta overexpression in wild-type mice increased PPARgamma2 and stearoyl-CoA desaturase-1 mRNA and hepatic triglyceride content. In FAO cells, overexpression of the liver inhibiting form of C/EBPbeta or C/EBPbeta RNA interference attenuated palmitate-induced triglyceride accumulation and reduced PPARgamma2 and triglyceride levels in the liver in vivo. Leptin and the anti-diabetic drug metformin acutely down-regulated C/EBPbeta expression in hepatocytes, whereas fatty acids up-regulate C/EBPbeta expression. These data provide novel evidence linking C/EBPbeta expression to lipogenesis and energy balance with important implications for the treatment of obesity and fatty liver disease.     

CD38 deficiency suppresses adipogenesis and lipogenesis in adipose tissues through activating Sirt1/PPARγ signaling pathway

It has been recently reported that CD38 was highly expressed in adipose tissues from obese people and CD38‐deficient mice were resistant to high‐fat diet (HFD)‐induced obesity. However, the role of CD38 in the regulation of adipogenesis and lipogenesis is unknown. In this study, to explore the roles of CD38 in adipogenesis and lipogenesis in vivo and in vitro, obesity models were generated with male CD38^?/? and WT mice fed with HFD. The adipocyte differentiations were induced with MEFs from WT and CD38^?/? mice, 3T3‐L1 and C3H10T1/2 cells in vitro. The lipid accumulations and the alternations of CD38 and the genes involved in adipogenesis and lipogenesis were determined with the adipose tissues from the HFD‐fed mice or the MEFs, 3T3‐L1 and C3H10T1/2 cells during induction of adipocyte differentiation. The results showed that CD38^?/? male mice were significantly resistant to HFD‐induced obesity. CD38 expressions in adipocytes were significantly increased in WT mice fed with HFD, and the similar results were obtained from WT MEFs, 3T3‐L1 and C3H10T1/2 during induction of adipocyte differentiation. The expressions of PPARγ, AP2 and C/EBPα were markedly attenuated in adipocytes from HFD‐fed CD38^?/? mice and CD38^?/? MEFs at late stage of adipocyte differentiation. Moreover, the expressions of SREBP1 and FASN were also significantly decreased in CD38^?/? MEFs. Finally, the CD38 deficiency‐mediated activations of Sirt1 signalling were up‐regulated or down‐regulated by resveratrol and nicotinamide, respectively. These results suggest that CD38 deficiency impairs adipogenesis and lipogenesis through activating Sirt1/PPARγ‐FASN signalling pathway during the development of obesity.     

Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells

Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells. [BMB Reports 2014; 47(4): 209-214]     

Dicer has a crucial role in the early stage of adipocyte differentiation, but not in lipid synthesis, in 3T3-L1 cells

Dicer is a rate-limiting enzyme for microRNA (miRNA) synthesis. To determine the effects of Dicer on adipogenesis, we performed stage-specific knockdown of Dicer using adenovirus encoding short-hairpin RNAi against Dicer in 3T3-L1 cells. When cells were infected with the adenovirus before induction of adipocyte differentiation, Dicer RNAi suppressed the gene expression of inducers of adipocyte differentiation such as PPARγ, C/EBPα, and FAS in 3T3-L1 cells during adipocyte differentiation. Concurrently, both adipocyte differentiation and cellular lipid accumulation were cancelled by Dicer RNAi when compared with control RNAi. Meanwhile, we addressed the roles of Dicer in lipid synthesis and accumulation in the final stages of differentiation. When the differentiated cells at day 4 after induction of differentiation were infected with adenovirus Dicer RNAi, cellular lipid accumulation was unchanged. Consistent with this, Dicer RNAi had no effects on the expression of genes related to cellular lipid accumulation, including PPARγ and FAS. Thus, Dicer controls proadipogenic genes such as C/EBPα and PPARγ in the early, but not in the late, stage of adipogenesis via regulation of miRNA synthesis.     

Dissociation between adipose tissue fluxes and lipogenic gene expression in ob/ob mice

Recent evidence has been presented that expression of lipogenic genes is downregulated in adipose tissue of ob/ob mice as well as in human obesity, suggesting a functionally lipoatrophic state. Using (2)H(2)O labeling, we measured three adipose tissue biosynthetic processes concurrently: triglyceride (TG) synthesis, palmitate de novo lipogenesis (DNL), and cell proliferation (adipogenesis). To determine the effect of the ob/ob mutation (leptin deficiency) on these parameters, adipose dynamics were compared in ob/ob, leptin-treated ob/ob, food-restricted ob/ob, and lean control mice. Adipose tissue fluxes for TG synthesis, de novo lipogenesis (DNL), and adipogenesis were dramatically increased in ob/ob mice compared with lean controls. Low-dose leptin treatment (2 microg/day) via miniosmotic pump suppressed all fluxes to control levels or below. Food restriction in ob/ob mice only modestly reduced DNL, with no change in TG synthesis or adipogenesis. Measurement of mRNA levels in age-matched ob/ob mice showed generally normal expression levels for most of the selected lipid anabolic genes, and leptin treatment had, with few exceptions, only modest effects on their expression. We conclude that leptin deficiency per se results in marked elevations in flux through diverse lipid anabolic pathways in adipose tissue (DNL, TG synthesis, and cell proliferation), independent of food intake, but that gene expression fails to reflect these changes in flux.     

Protein tyrosine phosphatase 1B inhibits adipocyte differentiation and mediates TNFα action in obesity

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of systemic glucose and insulin homeostasis; however, its exact role in adipocytes is poorly understood. This study was to elucidate the role of PTP1B in adipocyte differentiation and its implication in obesity. During differentiation of 3T3-L1 white preadipocytes, PTP1B decreased progressively with adipocyte maturation. Lentivirus-mediated PTP1B overexpression in preadipocytes delayed adipocyte differentiation, shown as lack of mature adipocytes, low level of lipid accumulation, and down-regulation of main markers (PPARγ2, SREBP-1c, FAS and LPL). In contrast, lentivirus-mediated PTP1B knockdown accelerated adipocyte differentiation, demonstrated as full of mature adipocytes, high level of lipid accumulation, and up-regulation of main markers. Dominant-negative inhibition on endogenous PTP1B by lentivirus-mediated overexpression of PTP1B double mutant in Tyr-46 and Asp-181 residues (LV-D/A-Y/F) also stimulated adipogenesis, more efficient than PTP1B knockdown. Diet-induced obesity mice exhibited an up-regulation of PTP1B and TNFα accompanied by a down-regulation of PPARγ2 in white adipose tissue. TNFα recombinant protein impeded PTP1B reduction and inhibited adipocyte differentiation in vitro; this inhibitory effect was prevented by LV-D/A-Y/F. Moreover, PTP1B inhibitor treatment improved adipogenesis and suppressed TNFα in adipose tissue of obese mice. All together, PTP1B negatively regulates adipocyte development and may mediate TNFα action to impair adipocyte differentiation in obesity. Our study provides novel evidence for the importance of PTP1B in obesity and for the potential application of PTP1B inhibitors.     

HRASLS3 is a PPARgamma-selective target gene that promotes adipocyte differentiation

The prevalence of obesity and its associated metabolic diseases worldwide has focused attention on understanding the mechanisms underlying adipogenesis. The nuclear receptor PPARgamma has emerged as a central regulator of adipose tissue function and formation. Despite the identification of numerous PPARgamma targets involved in a range of processes, from lipid droplet formation to adipokine secretion, information is still lacking on targets downstream of PPARgamma that directly affect fat cell differentiation. Here we identify HRASLS3 as a novel PPARgamma regulated gene with a role in adipogenesis. HRASLS3 expression increases during the differentiation of preadipocyte cell lines and is highly expressed in white and brown adipose tissue in mice. HRASLS3 expression is induced by PPARgamma ligands in preadipocyte cell lines as well in adipose tissue in vivo. We demonstrate that the HRASLS3 promoter contains a functional PPAR response element and is a direct target for regulation by PPARgamma/RXR heterodimers. Finally, we show that overexpression of HRASLS3 augments PPARgamma-driven lipid accumulation and adipogenesis, whereas siRNA-mediated knockdown of HRASLS3 expression decreases differentiation. Together, these results identify HRASLS3 as one of the downstream effectors of PPARgamma action in adipogenesis.     

Upregulation of cytosolic NADP+-dependent isocitrate dehydrogenase by hyperglycemia protects renal cells against oxidative stress

Hyperglycemia-induced oxidative stress is widely recognized as a key mediator in the pathogenesis of diabetic nephropathy, a complication of diabetes. We found that both expression and enzymatic activity of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) were upregulated in the renal cortexes of diabetic rats and mice. Similarly, IDPc was induced in murine renal proximal tubular OK cells by high hyperglycemia, while it was abrogated by co-treatment with the antioxidant N-Acetyl-Cysteine (NAC). In OK cells, increased expression of IDPc by stable transfection prevented hyperglycemia-mediated reactive oxygen species (ROS) production, subsequent cellular oxidative stress and extracellular matrix accumulation, whereas these processes were all stimulated by decreased IDPc expression. In addition, production of NADPH and GSH in the cytosol was positively correlated with the expression level of IDPc in OK cells. These results together indicate that upregulation of IDPc in response to hyperglycemia might play an essential role in preventing the progression of diabetic nephropathy, which is accompanied by ROS-induced cellular damage and fibrosis, by providing NADPH, the reducing equivalent needed for recycling reduced glutathione and low molecular weight antioxidant thiol proteins.     

Sulforaphane attenuates obesity by inhibiting adipogenesis and activating the AMPK pathway in obese mice

Obesity is associated with metabolic disorders. Sulforaphane, an isothiocyanate, inhibits adipogenesis and the occurrence of cardiovascular disease. In this study, we investigated whether sulforaphane could prevent high-fat diet (HFD)-induced obesity in C57BL/6N mice. Mice were fed a normal diet (ND), HFD or HFD plus 0.1% sulforaphane (SFN) for 6 weeks. Food efficiency ratios and body weight were lower in HFD-SFN-fed mice than in HFD-fed mice. SFN attenuated HFD-induced visceral adiposity, adipocyte hypertrophy and fat accumulation in the liver. Serum total cholesterol and leptin, and liver triglyceride levels were lower in HFD-SFN-fed mice than in HFD-fed mice. SFN decreased the expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and leptin in the adipose tissue of HFD-SFN mice and increased adiponectin expression. Phosphorylation of AMP-activated protein kinase α (AMPKα) and acetyl-CoA carboxylase in the adipose tissue of HFD-SFN-fed mice was elevated, and HMG-CoA reductase expression was decreased compared with HFD-fed mice. Thus, these results suggest that SFN may induce antiobesity activity by inhibiting adipogenesis through down-regulation of PPARγ and C/EBPα and by suppressing lipogenesis through activation of the AMPK pathway.     

Fat mass and obesity-associated protein regulates lipogenesis via m6A modification in fatty acid synthase mRNA

As the first identified N⁶-methyladenosine (m⁶A) demethylase, fat mass and obesity-associated (FTO) protein is associated with fatty acid synthase (FASN) and lipid accumulation. However, little is known about the regulatory role of FTO in the expression of FASN and de novo lipogenesis through m⁶A modification. In this study, we used FTO small interfering RNA to explore the effects of FTO knockdown on hepatic lipogenesis and its underlying epigenetic mechanism in HepG2 cells. We found that knockdown of FTO increased m⁶A levels in total RNA and enhanced the expression of YTH domain family member 2 which serves as the m⁶A-binding protein. The de novo lipogenic enzymes and intracellular lipid content were significantly decreased under FTO knockdown. Mechanistically, knockdown of FTO dramatically enhanced m⁶A levels in FASN messenger RNA (mRNA), leading to the reduced expression of FASN mRNA through m⁶A-mediated mRNA decay. The protein expressions of FASN along with acetyl CoA carboxylase and ATP-citrate lyase were further decreased, which inhibited de novo lipogenesis, thereby resulting in the deficiency of lipid accumulation in HepG2 cells and the induction of cellular apoptosis. The results reveal that FTO regulates hepatic lipogenesis via FTO-dependent m⁶A demethylation in FASN mRNA and indicate the critical role of FTO-mediated lipid metabolism in the survival of HepG2 cells. This study provides novel insights into a unique RNA epigenetic mechanism by which FTO mediates hepatic lipid accumulation through m⁶A modification and indicates that FTO could be a potential target for obesity-related diseases and cancer.     

Adaptive failure to high-fat diet characterizes steatohepatitis in Alms1 mutant mice

The biochemical differences between simple steatosis, a benign liver disease, and non-alcoholic steatohepatitis, which leads to cirrhosis, are unclear. Fat aussie is an obese mouse strain with a truncating mutation (foz) in the Alms1 gene. Chow-fed female foz/foz mice develop obesity, diabetes, and simple steatosis. We fed foz/foz and wildtype mice a high-fat diet. Foz/foz mice developed serum ALT elevation and severe steatohepatitis with hepatocyte ballooning, inflammation, and fibrosis; wildtype mice showed simple steatosis. Biochemical pathways favoring hepatocellular lipid accumulation (fatty acid uptake; lipogenesis) and lipid disposal (fatty acid beta-oxidation; triglyceride egress) were both induced by high-fat feeding in wildtype but not foz/foz mice. The resulting extremely high hepatic triglyceride levels were associated with induction of mitochondrial uncoupling protein-2 and adipocyte-specific fatty acid binding protein-2, but not cytochrome P4502e1 or lipid peroxidation. In this model of metabolic syndrome, transition of steatosis to steatohepatitis was associated with hypoadiponectinemia, a mediator of hepatic fatty acid disposal pathways.     

The chemical chaperone 4-phenylbutyrate inhibits adipogenesis by modulating the unfolded protein response

Recent studies have shown a link between obesity and endoplasmic reticulum (ER) stress. Perturbations in ER homeostasis cause ER stress and activation of the unfolded protein response (UPR). Adipocyte differentiation contributes to weight gain, and we have shown that markers of ER stress/UPR activation, including GRP78, phospho-eIF2α, and spliced XBP1, are upregulated during adipogenesis. Given these findings, the objective of this study was to determine whether attenuation of UPR activation by the chemical chaperone 4-phenylbutyrate (4-PBA) inhibits adipogenesis. Exposure of 3T3-L1 preadipocytes to 4-PBA in the presence of differentiation media decreased expression of ER stress markers. Concomitant with the suppression of UPR activation, 4-PBA resulted in attenuation of adipogenesis as measured by lipid accumulation and adiponectin secretion. Consistent with these in vitro findings, female C57BL/6 mice fed a high-fat diet supplemented with 4-PBA showed a significant reduction in weight gain and had reduced fat pad mass, as compared with the high-fat diet alone group. Furthermore, 4-PBA supplementation decreased GRP78 expression in the adipose tissue and lowered plasma triglyceride, glucose, leptin, and adiponectin levels without altering food intake. Taken together, these results suggest that UPR activation contributes to adipogenesis and that blocking its activation with 4-PBA prevents adipocyte differentiation and weight gain in mice.