Abstract
Pyruvate carboxylase (PC) plays a crucial role in various metabolic pathways, including gluconeogenesis, lipogenesis, and glucose-induced insulin secretion. Here we showed for the first time that the PC gene is transcriptionally regulated by peroxisome proliferator-activated receptor-gamma (PPARgamma) in vitro and in vivo in white and brown adipose tissue. PC mRNA and protein are markedly increased during differentiation of 3T3-L1 cells and HIB-1B, in parallel with the expression of the adipogenic transcription factors, CCAAT-enhancer binding protein alpha, PPARgamma1, and PPARgamma2. Tumor necrosis factor-alpha, a cytokine that blocks differentiation of 3T3-L1 cells, suppressed PC expression. Co-transfection studies in 3T3-L1 preadipocytes or HEK293T cells with a 2.3-kb promoter fragment of mouse PC gene linked to a luciferase reporter construct and with plasmids overexpressing retinoid X receptor alpha/PPARgamma1 or retinoid X receptor alpha/PPARgamma2 showed a 6-8-fold increase above the basal promoter activity. Furthermore, treatment of these transfected cells with the PPARgamma agonist doubled the promoter activity. Mutation of the putative PPAR-response element-(-386/-374) of this 2.3-kb PC promoter fragment abolished the PPARgamma response. Gel shift and chromatin immunoprecipitation assays demonstrated that endogenous PPARgamma binds to this functional PPAR-response element of the PC promoter. Mice with targeted disruption of the PPARgamma2 gene displayed approximately 50-60% reduction of PC mRNA and protein in white adipose tissue. Similarly, in brown adipose tissue of PPARgamma2-deficient mice subjected to cold exposure, PC mRNA was 40% lower than that of wild type mice. Impaired in vitro differentiation of white adipocytes of PPARgamma2 knock-out mice was also associated with a marked reduction of PC mRNA. Our findings identified PC as a PPARgamma-regulated gene and suggested a role for PPARgamma regulating intermediary metabolism.
Highlights
Pyruvate carboxylase (PC) plays a crucial role in various metabolic pathways, including gluconeogenesis, lipogenesis, and glucose-induced insulin secretion
PC Is Expressed Concomitant with Adipogenic Transcription Factors during 3T3-L1 and HIB-1B Differentiation—It has been reported previously that PC activity and mRNA are induced during the conversion of 3T3-L1 preadipocyte to mature adipocyte by hormonal induction (1, 13, 15), suggesting PC may play a lipogenic role during terminal differentiation
De novo fatty acid synthesis in differentiating adipocytes starts with the reaction catalyzed by acetyl-CoA carboxylase, which converts acetyl-CoA to malonyl-CoA, a building block of long chain fatty acids
Summary
C/EBP␣, CCAAT-enhancer binding protein ␣; PC, pyruvate carboxylase; PPAR␥, peroxisome proliferator activated receptor-␥; TNF␣, tumor necrosis factor-␣; RXR␣, retinoid X receptor-␣; PPRE, PPAR-response element; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; mPC, murine PC; E2F, elongation factor 2F. Decarboxylated to yield pyruvate and NADPH, with the latter being necessary for de novo fatty acid synthesis. Pyruvate carboxylation was shown to be necessary in hamster brown adipose tissue for maximal oxygen consumption in norepinephrine-stimulated respiration, even when drainage of the citric acid cycle for amino acid synthesis is eliminated, suggesting that the provision of oxaloacetate promotes the oxidation of acetyl-CoA from fatty acid degradation (18, 19). PPAR␥2 null mice (20) show a marked reduction of PC protein and PC mRNA both in white and brown adipose tissues
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