Phosphoenolpyruvate carboxykinase (PEPCK) is expressed at high levels in liver, kidney, and adipose tissue. This enzyme catalyzes the rate-limiting step in hepatic and renal gluconeogenesis and adipose glyceroneogenesis. The regulatory factors important for adipose expression of the PEPCK gene are not well defined. Previous studies with transgenic mice established that the region between bp ؊2086 and ؊888 is required for expression in adipose tissue but not for expression in liver or kidney tissue. We show here that a DNA fragment containing this region can function as an enhancer and direct differentiation-dependent expression of a chloramphenicol acetyltransferase gene from a heterologous promoter in cultured 3T3-F442A preadipocytes and adipocytes. We further demonstrate that the adipocyte-specific transcription factor PPAR␥2, previously identified as a regulator of the adipocyte P2 enhancer, binds in a heterodimeric complex with RXR␣ to the PEPCK 5-flanking region at two sites, termed PCK1 (bp ؊451 to ؊439) and PCK2 (bp ؊999 to ؊987). Forced expression of PPAR␥2 and RXR␣ activates the PEPCK enhancer in non-adipose cells. This activation is potentiated by peroxisome proliferators and fatty acids but not by 9-cis retinoic acid. Mutation of the PPAR␥2 binding site (PCK2) abolishes both the activity of the enhancer in adipocytes and its ability to be activated by PPAR␥2 and RXR␣. These results establish a role for PPAR␥2 in the adipose expression of the PEPCK gene and suggest that this factor functions as a coordinate regulator of multiple adipocyte-specific genes.Phosphoenolpyruvate carboxykinase (PEPCK [EC 4.1.1.32]) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, the rate-limiting step in gluconeogenesis and glyceroneogenesis. The PEPCK gene is expressed at high levels in liver, kidney, and white and brown fat (37). Expression is regulated at the transcriptional level by multiple hormones and second messengers, including insulin, glucocorticoids, retinoic acid, thyroid hormone, and cyclic AMP (10, 21, 33). Hormonal and dietary regulation of PEPCK gene transcription is tissue specific. For example, PEPCK expression in the liver is linked to blood glucose concentration, whereas in the kidney it is primarily regulated by physiologic acid-base status. In the liver and kidney, expression is stimulated by glucocorticoids and cyclic AMP and inhibited by insulin (33). In adipose tissue, however, glucocorticoids are inhibitory (25).The complexity of its transcriptional regulation makes PEPCK an attractive model for the study of hormone-linked and tissue-specific gene expression. The regions of the PEPCK promoter important for the hormonal and dietary regulation of this gene in liver have been analyzed in detail. A complex hormone response region extending from bp Ϫ460 to Ϫ349 from the transcriptional start site contains elements important for response to glucocorticoids, insulin and retinoic acid (14,16,24,26). A thyroid hormone response element (10) and a cyclic AMP response element (31) are located a...

Insulin regulates the synthesis of several proteins in a variety of tissues. Before techniques were available to quantify the amount of specific mRNAs, insulin was thought to regulate the synthesis of proteins by influencing the rate of translation of a fixed amount of mRNA. A very different interpretation is called for by experiments which show that insulin alters the amount of several specific mRNAs, but little is known about the mechanism. Insulin decreases the rate of synthesis of the critical gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) in both liver and H4IIE heptoma cells. We recently showed that insulin acts directly on H4IIE cells to decrease mRNAPEPCK activity without any other hormone intermediaries. This effect is mediated by the insulin receptor and occurs at insulin concentrations which are well within the physiological range range (10(-12)--10(-9) M). Here we extend these studies to show that insulin specifically inhibits transcription of the PEPCK gene. This inhibition results in a rapid decrease in the concentration of nuclear PEPCK transcripts which is followed, in turn, by a proportionate decline in cytoplasmic mRNAPEPCK and synthesis of the protein.

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 ␥, 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 ␥-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.