Yoo Jeong Lee

Reactive Oxygen Species Facilitate Adipocyte Differentiation by Accelerating Mitotic Clonal Expansion

Growth-arrested 3T3-L1 preadipocytes rapidly express CCAAT/enhancer-binding protein-β (C/EBPβ) upon hormonal induction of differentiation. However, the DNA binding activity of C/EBPβ is not activated until the cells synchronously reenter S phase during the mitotic clonal expansion (MCE) phase of differentiation. In this period, C/EBPβ is sequentially phosphorylated by MAPK and glycogen synthase kinase-3β, inducing C/EBPβ DNA binding activity and transcription of its target genes. Because the DNA binding activity of C/EBPβ is further enhanced by oxidation in vitro, we investigated how redox state affects C/EBPβ DNA binding and MCE during adipogenesis. When 3T3-L1 cells were treated with H2O2 and hormonal stimuli, differentiation was accelerated with increased expression of peroxisome proliferator-activated receptor γ. Interestingly, cell cycle progression (S to G2/M phase) was markedly enhanced by H2O2, whereas antioxidants caused an S phase arrest during the MCE. H2O2 treatment resulted in the early appearance of a punctate pattern observed by immunofluorescent staining of C/EBPβ, which is a hallmark for C/EBPβ binding to regulatory elements, whereas a short antioxidant treatment rapidly dispersed the centromeric localization of C/EBPβ. Consistently, reactive oxygen species production was increased during 3T3-L1 differentiation. Our results indicate that redox-induced C/EBPβ DNA binding activity, along with the dual phosphorylation of C/EBPβ, is required for the MCE and terminal differentiation of adipocytes.

Nuclear receptor PPARγ-regulated monoacylglycerol O-acyltransferase 1 (MGAT1) expression is responsible for the lipid accumulation in diet-induced hepatic steatosis.

Recently, hepatic peroxisome proliferator-activated receptor (PPAR)γ has been implicated in hepatic lipid accumulation. We found that the C3H mouse strain does not express PPARγ in the liver and, when subject to a high-fat diet, is resistant to hepatic steatosis, compared with C57BL/6 (B6) mice. Adenoviral PPARγ2 injection into B6 and C3H mice caused hepatic steatosis, and microarray analysis demonstrated that hepatic PPARγ2 expression is associated with genes involved in fatty acid transport and the triglyceride synthesis pathway. In particular, hepatic PPARγ2 expression significantly increased the expression of monoacylglycerol O-acyltransferase 1 (MGAT1). Promoter analysis by luciferase assay and electrophoretic mobility shift assay as well as chromatin immunoprecipitation assay revealed that PPARγ2 directly regulates the MGAT1 promoter activity. The MGAT1 overexpression in cultured hepatocytes enhanced triglyceride synthesis without an increase of PPARγ expression. Importantly, knockdown of MGAT1 in the liver significantly reduced hepatic steatosis in 12-wk-old high-fat–fed mice as well as ob/ob mice, accompanied by weight loss and improved glucose tolerance. These results suggest that the MGAT1 pathway induced by hepatic PPARγ is critically important in the development of hepatic steatosis during diet-induced obesity.

Transcription factor Snail is a novel regulator of adipocyte differentiation via inhibiting the expression of peroxisome proliferator-activated receptor γ

Snail belongs to the superfamily of zinc-finger transcription factors and plays a crucial role in processes regulating cell fate, such as the formation of mesoderm and initiation of epithelial–mesenchymal transition. We have previously discovered that Snail modulates adiponectin expression in 3T3-L1 cells during adipogenesis. In the present study, we elucidated the functional role of Snail in adipocyte differentiation and its underlying molecular mechanism. Snail expression was dramatically decreased during adipogenesis in 3T3-L1 cells. Overexpression of Snail blocked adipocyte differentiation by suppressing the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer-binding protein alpha, while knockdown of Snail expression stimulated adipogenesis in 3T3-L1 cells. Chromatin immunoprecipitation assay and luciferase assay showed that Snail inhibits the transcriptional activity of the PPARγ gene by directly binding to the E-box motifs in the PPARγ promoter. Wnt10b induced phosphorylation of glycogen synthase kinase 3 beta (GSK3β), leading to inhibition of adipogenesis in 3T3-L1 cells in accordance with increased expression of Snail, whereas adipogenic capacity was restored in Snail siRNA-transfected preadipocytes. LiCl (a GSK3β inhibitor)-treated cells also showed increased expression of Snail, with a reduced adipogenic potential. Snail-overexpressing 3T3-F442A cells did not differentiate into mature adipocytes in immunodeficient nude mice. Taken together, Snail is a novel regulator of adipocyte differentiation, which acts by direct suppression of PPARγ expression. Our data also indicate that the expression of Snail is mediated by the Wnt–GSK3β signaling pathway.

Dexras1 mediates glucocorticoid-associated adipogenesis and diet-induced obesity

Significance Glucocorticoids are well known to play a major role in obesity, but underlying mechanisms have been obscure. We demonstrate that the small G protein Dexras1, first identified based on its dramatic induction by glucocorticoids, mediates adipogenic differentiation of preadipocytes, as well as diet-induced obesity in intact rodents. Thus, the adipogenesis of preadipocytes is abolished by Dexras1 deletion and selectively induced by Dexras1 expression. Relevance to intact animals is evident from our experiments wherein diet-induced obesity is prevented in mice with knockout of Dexras1. Thus, pharmacotherapy involving Dexras1 may afford a promising approach to the therapy of obesity. Adipogenesis, the conversion of precursor cells into adipocytes, is associated with obesity and is mediated by glucocorticoids acting via hitherto poorly characterized mechanisms. Dexras1 is a small G protein of the Ras family discovered on the basis of its marked induction by the synthetic glucocorticoid dexamethasone. We show that Dexras1 mediates adipogenesis and diet-induced obesity. Adipogenic differentiation of 3T3-L1 cells is abolished with Dexras1 depletion, whereas overexpression of Dexras1 elicits adipogenesis. Adipogenesis is markedly reduced in mouse embryonic fibroblasts from Dexras1-deleted mice, whereas adiposity and diet-induced weight gain are diminished in the mutant mice.

Krüppel-like factor KLF8 plays a critical role in adipocyte differentiation.

KLF8 (Krüppel-like factor 8) is a zinc-finger transcription factor known to play an essential role in the regulation of the cell cycle, apoptosis, and differentiation. However, its physiological roles and functions in adipogenesis remain unclear. In the present study, we show that KLF8 acts as a key regulator controlling adipocyte differentiation. In 3T3-L1 preadipocytes, we found that KLF8 expression was induced during differentiation, which was followed by expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Adipocyte differentiation was significantly attenuated by the addition of siRNA against KLF8, whereas overexpression of KLF8 resulted in enhanced differentiation. Furthermore, luciferase reporter assays demonstrated that overexpression of KLF8 induced PPARγ2 and C/EBPα promoter activity, suggesting that KLF8 is an upstream regulator of PPARγ and C/EBPα. The KLF8 binding sites were localized by site mutation analysis to −191 region in C/EBPα promoter and −303 region in PPARγ promoter, respectively. Taken together, these data reveal that KLF8 is a key component of the transcription factor network that controls terminal differentiation during adipogenesis.

Chronic alcohol consumption potentiates the development of diabetes through pancreatic β-cell dysfunction

Chronic ethanol consumption is well established as a major risk factor for type-2 diabetes (T2D), which is evidenced by impaired glucose metabolism and insulin resistance. However, the relationships between alcohol consumption and the development of T2D remain controversial. In particular, the direct effects of ethanol consumption on proliferation of pancreatic β-cell and the exact mechanisms associated with ethanol-mediated β-cell dysfunction and apoptosis remain elusive. Although alcoholism and alcohol consumption are prevalent and represent crucial public health problems worldwide, many people believe that low-to-moderate ethanol consumption may protect against T2D and cardiovascular diseases. However, the J- or U-shaped curves obtained from cross-sectional and large prospective studies have not fully explained the relationship between alcohol consumption and T2D. This review provides evidence for the harmful effects of chronic ethanol consumption on the progressive development of T2D, particularly with respect to pancreatic β-cell mass and function in association with insulin synthesis and secretion. This review also discusses a conceptual framework for how ethanol-produced peroxynitrite contributes to pancreatic β-cell dysfunction and metabolic syndrome.

Chronic Ethanol Consumption Inhibits Glucokinase Transcriptional Activity by Atf3 and Triggers Metabolic Syndrome in Vivo

Background: Chronic ethanol consumption induces pancreatic β-cell dysfunction and metabolic syndrome. Results: Ethanol-induced Atf3 inhibits glucokinase transcriptional activity through direct binding or Atf3/Pdx-1/Hdac1 axis on glucokinase promoter. Conclusion: ATf3 fosters β-cell dysfunction via Gck down-regulation and triggers T2D, which is ameliorated by in vivo Atf3 silencing. Significance: The presented data uncover a new role for Atf3 as a potential therapeutic target in treating type 2 diabetes. Chronic ethanol consumption induces pancreatic β-cell dysfunction through glucokinase (Gck) nitration and down-regulation, leading to impaired glucose tolerance and insulin resistance, but the underlying mechanism remains largely unknown. Here, we demonstrate that Gck gene expression and promoter activity in pancreatic β-cells were suppressed by chronic ethanol exposure in vivo and in vitro, whereas expression of activating transcription factor 3 (Atf3) and its binding to the putative Atf/Creb site (from −287 to −158 bp) on the Gck promoter were up-regulated. Furthermore, in vitro ethanol-induced Atf3 inhibited the positive effect of Pdx-1 on Gck transcriptional regulation, enhanced recruitment of Hdac1/2 and histone H3 deacetylation, and subsequently augmented the interaction of Hdac1/Pdx-1 on the Gck promoter, which were diminished by Atf3 siRNA. In vivo Atf3-silencing reversed ethanol-mediated Gck down-regulation and β-cell dysfunction, followed by the amelioration of impaired glucose tolerance and insulin resistance. Together, we identified that ethanol-induced Atf3 fosters β-cell dysfunction via Gck down-regulation and that its loss ameliorates metabolic syndrome and could be a potential therapeutic target in treating type 2 diabetes. The Atf3 gene is associated with the induction of type 2 diabetes and alcohol consumption-induced metabolic impairment and thus may be the major negative regulator for glucose homeostasis.

Peroxisome Proliferator-Activated Receptor Gamma/Signal Transducers and Activators of Transcription 5A Pathway Plays a Key Factor in Adipogenesis of Human Bone Marrow-Derived Stromal Cells and 3T3-L1 Preadipocytes

Adipogenesis is largely dependent on the signal transducers and activators of transcription (STAT) pathway. However, the molecular mechanism of the STAT pathway in the adipogenesis of human bone marrow-derived stromal cells (hBMSCs) remains not well understood. The purpose of this research was to characterize the transcriptional regulation involved in expression of STAT5A and STAT5B during adipogenesis in hBMSCs and 3T3-L1 cells. The expression of STAT5A and STAT5B increases with the onset of adipogenesis in hBMSCs and 3T3-L1 cells. The PPAR response elements regulatory element of STAT5A exists at a promoter region ranging from -346 to -101, and the CCAAT/enhancer-binding protein (C/EBP) regulatory element is located at -196 to -118 of the STAT5B promoter. C/EBPβ and C/EBPα bound to the STAT5B promoter region, whereas peroxisome proliferator-activated receptor γ (PPARγ) bound to STAT5A. RNA interference of STAT5A completely blocked differentiation, whereas the inhibition of STAT5B only partially blocked differentiation. We propose that C/EBPα, C/EBPβ, and PPARγ control adipogenesis by regulating STAT5B and STAT5A and that STAT5A is necessary, whereas STAT5B plays a supplementary role during adipogenesis. Further, the regulation of PPARγ-STAT5 by C/EBPβ signaling seems to be the crucial adipogenesis pathway-initiating cascade of the various adipogenic genes.

Suppression of PPARγ-mediated monoacylglycerol O-acyltransferase 1 expression ameliorates alcoholic hepatic steatosis.

Alcohol consumption is one of the major causes of hepatic steatosis, fibrosis, cirrhosis, and superimposed hepatocellular carcinoma. Ethanol metabolism alters the NAD+/NADH ratio, thereby suppressing the activity of sirtuin family proteins, which may affect lipid metabolism in liver cells. However, it is not clear how long-term ingestion of ethanol eventually causes lipid accumulation in liver. Here, we demonstrate that chronic ethanol ingestion activates peroxisome proliferator-activated receptor γ (PPARγ) and its target gene, monoacylglycerol O-acyltransferase 1 (MGAT1). During ethanol metabolism, a low NAD+/NADH ratio repressed NAD-dependent deacetylase sirtuin 1 (SIRT1) activity, concomitantly resulting in increased acetylated PPARγ with high transcriptional activity. Accordingly, SIRT1 transgenic mice exhibited a low level of acetylated PPARγ and were protected from hepatic steatosis driven by alcohol or PPARγ2 overexpression, suggesting that ethanol metabolism causes lipid accumulation through activation of PPARγ through acetylation. Among the genes induced by PPARγ upon alcohol consumption, MGAT1 has been shown to be involved in triglyceride synthesis. Thus, we tested the effect of MGAT1 knockdown in mice following ethanol consumption, and found a significant reduction in alcohol-induced hepatic lipid accumulation. These results suggest that MGAT1 may afford a promising approach to the treatment of fatty liver disease.

Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation.

Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator of mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans.