The transcription factor SREBP1c (sterol-regulatory-element-binding protein 1c) is highly expressed in adipose tissue and plays a central role in several aspects of adipocyte development including the induction of PPARγ (peroxisome-proliferator-activated receptor γ), the generation of an endogenous PPARγ ligand and the expression of several genes critical for lipid biosynthesis. Despite its significance, the regulation of SREBP1c expression during adipogenesis is not well characterized. We have noted that in several models of adipogenesis, SREBP1c expression closely mimics that of known C/EBPβ (CCAAT/enhancer-binding protein β) targets. Inhibition of C/EBP activity during adipogenesis by expressing either the dominant-negative C/EBPβ LIP (liver-enriched inhibitory protein) isoform, the co-repressor ETO (eight-twenty one/MTG8) or using siRNAs (small interfering RNAs) targeting either C/EBPβ or C/EBPδ significantly impaired early SREBP1c induction. Furthermore, ChIP (chromatin immunoprecipitation) assays identified specific sequences in the SREBP1c promoter to which C/EBPβ and C/EBPδ bind in intact cells, demonstrating that these factors may directly regulate SREBP1c expression. Using cells in which C/EBPα expression is inhibited using shRNA (short hairpin RNA) and ChIP assays we show that C/EBPα replaces C/EBPβ and C/EBPδ as a regulator of SREBP1c expression in maturing adipocytes. These results provide novel insight into the induction of SREBP1c expression during adipogenesis. Moreover, the findings of the present study identify an important additional mechanism via which the C/EBP transcription factors may control a network of gene expression regulating adipogenesis, lipogenesis and insulin sensitivity.
Microsomal triglyceride transfer protein (MTP) is rate limiting for the assembly and secretion of apolipoprotein B-containing lipoproteins. Elevated hepatic MTP mRNA level, presumably as a result of impaired insulin signaling, has been implicated in the pathophysiology of dyslipidemia associated with insulin resistance/type 2 diabetes. In this study, we showed that insulin decreases MTP mRNA level mainly through transcriptional regulation in HepG2 cells. We further characterized the corresponding signal transduction pathway, using chemical inhibitors and constitutively active and dominant negative forms of regulatory enzymes. We demonstrated that insulin inhibits MTP gene transcription through MAPK erk cascade but not through the PI 3-kinase pathway. Activation of ras through farnesylation is not a prerequisite for the inhibition. Diabetes is not only a disease characterized by elevated blood glucose but also a serious vascular disease with poor prognosis (1). Individuals with type 2 diabetes demonstrate an increased risk of cardiovascular abnormalities, which are closely associated with an increased level of plasma apolipoprotein B (apoB)-containing lipoproteins, i.e., VLDLs and LDLs (2,3). Metabolic labeling studies indicate that elevated plasma levels of lipoproteins in patients with diabetes are caused, at least in part, by an increased hepatic output of apoB-containing lipoproteins (1,4,5). A better understanding of how hepatic lipoprotein production is regulated in patients with diabetes could potentially lead to the development of more effective strategic therapeutics to alleviate diabetic symptoms.Recent advances have established the fundamental role of the microsomal triglyceride transfer protein (MTP) in the assembly of apoB-containing lipoproteins. MTP is an important enabler for the secretion of VLDLs by the liver, chylomicrons by the intestine (reviewed in ref. 6), and even LDLs by the heart (7). Functionally, MTP catalyzes the loading of lipids to the nascent apoB in the endoplasmic reticulum (ER). This stabilizes the newly synthesized apoB (8,9) and facilitates further processing, leading to its secretion. Reduction of MTP activity in animals by inhibitors (10) or gene knockouts (11-13) effectively lowers the plasma lipoprotein level, whereas enforced expression of hepatic MTP in mice (14) increases the plasma level of apoB-containing lipoproteins.Emerging evidence has indicated that the pathophysiology of dyslipidemia observed under insulin resistance/type 2 diabetes is associated with an increased hepatic MTP mRNA level (15)(16)(17)(18). This is presumably due to an impaired insulin-regulatory system as insulin is a negative regulator of the MTP gene (19,20). A recent study demonstrated that improving insulin sensitivity is associated with the normalization of the hepatic MTP expression and the reduction of VLDL secretion in insulin-resistant hamsters (19). Therefore, knowledge regarding how insulin regulates hepatic MTP gene transcription (21) would provide important insights toward the unders...
Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triacylglycerol (TG) synthesis. Despite the existence of an alternative acyltransferase (DGAT1), mice lacking DGAT2 have a severe deficiency of TG in adipose tissue, indicating a nonredundant role for this enzyme in adipocyte TG synthesis. We have studied the regulation of DGAT2 expression during adipogenesis. In both isolated murine preadipocytes and 3T3-L1 cells the temporal pattern of DGAT2 expression closely mimicked that of genes whose expression is regulated by CAAT/enhancerbinding protein  (C/EBP). Inhibition of C/EBP expression in differentiating preadipocytes reduced DGAT2 expression, and electrophoretic mobility shift assay and chromatin immunoprecipitation experiments identified a promoter element in the DGAT2 gene that is likely to mediate this effect. The importance of C/EBP in adipocyte expression of DGAT2 was confirmed by the finding of reduced DGAT2 expression in the adipose tissue of C/EBP-null animals. However, DGAT2 expression is maintained at high levels during the later stages of adipogenesis, when C/EBP levels decline. We show that, at these later stages of differentiation, C/EBP␣ is capable of substituting for C/EBP at the same promoter element. These observations provide novel insight into the transcriptional regulation of DGAT2 expression. Moreover, they further refine the complex and serial roles of the C/EBP family of transcription factors in inducing and maintaining the metabolic properties of mature adipocytes.The increased adipose tissue mass of obesity results from a combination of increased lipid storage in existing adipocytes and the generation of new adipocytes from precursors residing within the adipose tissue (1). The induction of genes responsible for the formation of triacylglycerol (TG) 3 within developing or pre-existing adipocytes is therefore likely to make an important contribution to the enlargement of adipose mass. In contrast, pathologically decreased lipid accumulation or impaired adipogenesis in lipodystrophic subjects has deleterious metabolic consequences superficially like those seen in obesity, including insulin resistance and dyslipidemia, with attendant increases in cardiovascular disease. Thus good metabolic control is likely to require the body to restrain adipose tissue mass while still maintaining the capacity to respond accurately to substrate availability. In this way, when necessary, lipids can be partitioned appropriately into adipose tissue and away from other insulin-sensitive tissues where they may have detrimental effects. That mutations of AGPAT2, a key enzyme in TG synthesis, can cause near total lipodystrophy demonstrates the importance of this pathway in such diseases of adipose development and function (2, 3). Rational therapeutic strategies for both obesity and lipodystrophy will require a detailed knowledge of the regulatory pathways required for the formation of an appropriate mass of metabolically active adipocytes, capable of tightly controlling lipid synthesis...
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