Proposed involvement of adipocyte glyceroneogenesis and phosphoenolpyruvate carboxykinase in the metabolic syndrome

Elevated concentration of plasma non-esterified fatty acids (NEFA) is now recognized as a key factor in the onset of insulin-resistance and type 2 diabetes mellitus. During fasting, circulating NEFAs arise from white adipose tissue (WAT) as a consequence of lipolysis from stored triacylglycerols. However, a significant part of these FAs (30-70%) is re-esterified within the adipocyte, so that a recycling occurs and net FA output is much less than < true > lipolysis. Indeed, a balance between two antagonistic processes, lipolysis and FA re-esterification, controls the rate of net FA release from WAT. During fasting, re-esterification requires glyceroneogenesis defined as the de novo synthesis of glycerol-3-P from pyruvate, lactate or certain amino acids. The key enzyme in this process is the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C; EC 4.1.1.32). Recent advance has stressed the role of glyceroneogenesis and of PEPCK-C in FA release from WAT. Results indicate that glyceroneogenesis is indeed important to lipid homeostasis and that a disregulation in this pathway may have profound pathophysiological effects. The present review focuses on the regulation of glyceroneogenesis and of PEPCK-C gene expression and activity by FAs, retinoic acids, glucocorticoids and the hypolipidemic class of drugs, thiazolidinediones.     

PCK1 and PCK2 as candidate diabetes and obesity genes

The PCK1 gene (Pck1 in rodents) encodes the cytosolic isozyme of phosphoenolpyruvate carboxykinase (PEPCK-C), which is well-known for its function as a gluconeogenic enzyme in the liver and kidney. Mouse studies involving whole body and tissue-specific Pck1 knockouts as well as tissue-specific over-expression of PEPCK-C have resulted in type 2 diabetes as well as several surprising phenotypes including obesity, lipodystrophy, fatty liver, and death. These phenotypes arise from perturbations not only in gluconeogenesis but in two additional metabolic functions of PEPCK-C: (1) cataplerosis which maintains metabolic flux through the Krebs cycle by removing excess oxaloacetate, and (2) glyceroneogenesis which produces glycerol-3-phosphate as a precursor for fatty acid esterification into triglycerides. PEPCK-C catalyzes the conversion of oxaloacetate + GTP to phosphoenolpyruvate + GDP + CO₂. It is in part the tissue-specificity of this simple reaction that results in the variety of phenotypes listed above. Briefly: (1) A 7-fold over-expression of PEPCK-C in the livers of mice causes excessive glucose production. (2) Mice with a whole-body knockout of Pck1 die within 2–3 days of birth, not from hypoglycemia, but probably because the Krebs cycle slows to approximately 10% of normal in the absence of cataplerosis. (3) Mice with a liver-specific knockout have an inability to remove oxaloacetate from the Krebs cycle, which leads to a fatty liver following a fast. (4) An adipose-specific knockout of Pck1 results in a fraction of the mice developing lipodystrophy due to lost glyceroneogenesis and a consequent decrease in fatty acid re-esterification. (5) Finally, disregulated over-expression of PEPCK-C in adipose tissue increases fatty acid re-esterification leading to obesity. These varied experimental phenotypes in mice have led us to postulate that abnormal production of PEPCK isozymes encoded by two PEPCK genes, PCK1 and PCK2, in humans could have similar consequences (Beale, E. G. et al. (2004). Trends in Endocrinology and Metabolism, 15, 129–135). The purpose of this review is to further explore these possibilities.     

The gene encoding the Acyl-CoA-binding protein is activated by peroxisome proliferator-activated receptor gamma through an intronic response element functionally conserved between humans and rodents

The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular protein that specifically binds acyl-CoA esters with high affinity and is structurally and functionally conserved from yeast to mammals. In vitro studies indicate that ACBP may regulate the availability of acyl-CoA esters for various metabolic and regulatory purposes. The protein is particularly abundant in cells with a high level of lipogenesis and de novo fatty acid synthesis and is significantly induced during adipocyte differentiation. However, the molecular mechanisms underlying the regulation of ACBP expression in mammalian cells have remained largely unknown. Here we report that ACBP is a novel peroxisome proliferator-activated receptor (PPAR)gamma target gene. The rat ACBP gene is directly activated by PPARgamma/retinoid X receptor alpha (RXRalpha) and PPARalpha/RXRalpha, but not by PPARdelta/RXRalpha, through a PPAR-response element in intron 1, which is functionally conserved in the human ACBP gene. The intronic PPAR-response element (PPRE) mediates induction by endogenous PPARgamma in murine adipocytes and confers responsiveness to the PPARgamma-selective ligand BRL49653. Finally, we have used chromatin immunoprecipitation to demonstrate that the intronic PPRE efficiently binds PPARgamma/RXR in its natural chromatin context in adipocytes. Thus, the PPRE in intron 1 of the ACBP gene is a bona fide PPARgamma-response element.     

PCK1 expression is correlated with the plasma glucose level in the duck

Phosphoenolpyruvate carboxykinase 1 (soluble) (PCK1) is a key gene in gluconeogenesis and glyceroneogenesis. Although its functions have been extensively studied in mice, bats and humans, little is known in ducks. Here, PCK1 functions were studied using a duck domestication model and a 48‐h fasting experiment. We found PCK1 expression significantly decreased in two breeds of domestic ducks (Jinyun Pockmark ducks and Cherry Valley ducks) as compared with wild ducks (Anas platyrhynchos). Simultaneously, plasma levels of glucose, triglycerides and free fatty acid in domestic ducks were lower than in wild ducks. When compared with fed ducks, the plasma triglyceride level was observed to be significantly decreased, while the glucose and free fatty acid levels remained constant in 48‐h fasting ducks. The expression analysis of gluconeogenic genes revealed that fructose‐1,6‐bisphosphatase genes (FBP1 and FBP2) and the glucose‐6‐phosphatase gene (G6PC2) were not changed, whereas PCK1 was significantly upregulated. In addition, the reported regulators of PCK1, including forkhead box A2 (FOXA2) gene and orphan nuclear receptor NR4A family genes (NR4A1, NR4A2 and NR4A3), exhibited similar expression levels between 48‐h fasting ducks and fed ducks, suggesting that PCK1 is not regulated by these genes in the duck under fasting conditions. In conclusion, PCK1 expression may affect plasma levels of glucose, triglycerides and free fatty acid during the duck domestication process. This work demonstrates for the first time in duck that PCK1 is a key gene in maintaining plasma glucose homeostasis during fasting and that the upregulated expression of PCK1 may be responsible for constant plasma free fatty acid level by the glyceroneogenesis process.     

Thiazolidinediones block fatty acid release by inducing glyceroneogenesis in fat cells

Thiazolidinediones are used to treat type 2 diabetes mellitus because they decrease plasma glucose, insulin, triglyceride, and fatty acid levels. Thiazolidinediones are agonists for peroxisome proliferator-activated receptor gamma, a nuclear receptor that is highly expressed in fat tissue. We identify glyceroneogenesis as a target of thiazolidinediones in cultured adipocytes and fat tissues of Wistar rats. The activation of glyceroneogenesis by thiazolidinediones occurs mainly in visceral fat, the same fat depot that is specifically implicated in the progression of obesity to type 2 diabetes. The increase in glyceroneogenesis is a result of the induction of its key enzyme, phosphoenolpyruvate carboxykinase, whose gene expression is peroxisome proliferator-activated receptor gamma-dependent in adipocytes. The main role of this metabolic pathway is to allow the re-esterification of fatty acids via a futile cycle in adipocytes, thus lowering fatty acid release into the plasma. The importance of such a fatty acid re-esterification process in the control of lipid homeostasis is highlighted by the existence of a second thiazolidinedione-induced pathway involving glycerol kinase. We show that glyceroneogenesis accounts for at least 75% of the whole thiazolidinedione effect. Because elevated plasma fatty acids promote insulin resistance, these results suggest that the glyceroneogenesis-dependent fatty acid-lowering effect of thiazolidinediones could be an essential aspect of the antidiabetic action of these drugs.     

Analysis of the transcriptional regulation of the FABP2 promoter haplotypes by PPARgamma/RXRalpha and Oct-1

Variants of the human intestinal fatty acid binding protein 2 gene (FABP2) are associated with traits of the metabolic syndrome. Relevant FABP2 promoter polymorphisms c.-80_-79insT, c.-136_-132delAGTAG, c.-168_-166delAAGinsT, c.-260G>A, c.-471G>A, and c.-778G>T result in two haplotypes A and B. Activation of haplotypes by rosiglitazone stimulated PPARgamma/RXRalpha leads to 2-fold higher activity of haplotype B than A. As shown by chimeric FABP2 promoter constructs, the higher responsiveness of FABP2 haplotype B is mainly but not solely determined by polymorphism c.-471G>A. As shown by EMSA and promoter-reporter assays, Oct-1 interacts with the -471 region of FABP2 promoters, induces the activities of both FABP2 promoter haplotypes and abolishes the different activities of haplotypes induced by rosiglitazone activated PPARgamma/RXRalpha. In conclusion, our findings suggest a functional role of PPARgamma/RXRalpha and Oct-1 in the regulation of the FABP2 gene.