Fatty acid recycling in adipocytes: a role for glyceroneogenesis and phosphoenolpyruvate carboxykinase.

FA (fatty acid) recycling in adipose tissue appears to be an important pathway for regulating FA release into the blood during fasting. Re-esterification requires G3P (glycerol 3-phosphate), which cannot be synthesized from glucose because glycolysis is much reduced under such circumstances. In addition, G3P can scarcely originate from glycerol since glycerol kinase has a very low activity in white adipose tissue. It was shown about 35 years ago that a metabolic pathway named glyceroneogenesis, which allows G3P synthesis from non-carbohydrate precursors like pyruvate, lactate or amino acids, is activated during fasting. The major enzyme in this pathway was shown to be PEPCK-C [cytosolic phosphoenolpyruvate carboxykinase (GTP); EC 4.1.1.32]. The present review analyses the mechanisms by which a series of hormones and nutrients affect PEPCK-C gene transcription and glyceroneogenesis and describes evidence for dysregulation of this pathway in type 2 diabetes.

A single element in the phosphoenolpyruvate carboxykinase gene mediates thiazolidinedione action specifically in adipocytes.

Phosphoenolpyruvate carboxykinase (PEPCK) is the key enzyme in glyceroneogenesis, an important metabolic pathway that functions to restrain the release of non-esterified fatty acids (NEFAs) from adipocytes. The antidiabetic drugs known as thiazolidinediones (TZDs) are thought to achieve some of their benefits by lowering elevated plasma NEFAs. Moreover, peroxisome proliferator activated receptor gamma (PPARgamma) mediates the antidiabetic effects of TZDs, though many TZD responses appear to occur via PPARgamma-independent pathways. PPARgamma is required for adipocyte PEPCK expression, hence PEPCK could be a major target gene for the antidiabetic actions of TZDs. Here we used tissue culture and transfection assays to confirm that the TZD, rosiglitazone, stimulates PEPCK gene transcription specifically in adipocytes. We made the novel observation that this effect was by far the most rapid and robust among several other genes expressed in adipocytes. Adipocytes were transfected with a PEPCK/chloramphenicol acetyltransferase chimeric gene, in which either of the two previously discovered PPARgamma/retinoid X receptor alpha response elements, PCK2 and gAF1/PCK1, had been inactivated by mutagenesis. We demonstrate that PCK2 alone is a bona fide thiazolidinedione response element. We show also that the regulation of PEPCK by PPARs is cell-specific and isotype-specific since rosiglitazone induces PEPCK gene expression selectively in adipocytes, and PPARalpha- and PPARbeta-specific activators are inefficient. Hence, TZDs could lower plasma NEFAs via PPARgamma and PEPCK by enhancing adipocyte glyceroneogenesis.

Peroxisome proliferator-activated receptor gamma and chicken ovalbumin upstream promoter transcription factor II negatively regulate the phosphoenolpyruvate carboxykinase promoter via a common element.

A heterodimer of peroxisome proliferator-activated receptor gamma (PPARgamma) and retinoid X receptor (RXR) is required for adipocyte differentiation. The gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is a PPARgamma/RXR target gene in adipose tissue. Of the two PPARgamma response elements, gAF1/PCK1 and PCK2, only PCK2 is required for PEPCK expression and responsiveness to the PPARgamma agonist, rosiglitazone, in adipose tissue even though both elements bind PPARgamma/RXR in vitro. In contrast, gAF1/PCK1 is essential for glucocorticoid inhibition of PPARgamma-induced PEPCK gene expression in adipocytes. We report that chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is the predominant nuclear receptor bound to gAF1/PCK1 in preadipocytes. COUP-TFII declines during adipogenesis in reciprocal fashion to PPARgamma. In transiently transfected fibroblasts COUP-TFII acts at gAF1/PCK1 to inhibit PPARgamma/RXR activation via PCK2. In contrast COUP-TFs are transcriptional activators of PEPCK in hepatocytes. PPARgamma/RXR occupies gAF1/PCK1 in adipocytes, and mutation of gAF1/PCK1 enhances PEPCK promoter transactivation by PPARgamma/RXR in fibroblasts, suggesting that this element is also a negative PPARgamma response element. These results indicate that gAF1/PCK1 is a pleiotropic element through which COUP-TFII inhibits premature PEPCK expression, and perhaps adipogenesis in general, and PPARgamma/RXR uses this same element in adipocytes to participate in PEPCK modulation by glucocorticoids.

Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in adipocyte lipolysis. Short-term hormonal regulation of HSL activity is well characterized, whereas little is known about the control of HSL gene expression. We have measured HSL mRNA content of 3T3-F442A and BFC-1 adipocytes in response to the cAMP analogue 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and to the phorbol ester phorbol 12-myristate 13-acetate (PMA) by Northern blot, using a specific mouse cDNA fragment. Treatment of the cells for 12 or 6 h with, respectively, 0.5 mM 8-CPT-cAMP or 1 microM PMA produced a maximal decrease of about 60% in HSL mRNA. These effects were unaffected by the protein-synthesis inhibitor anisomycin, suggesting that cAMP and PMA actions were direct. The reduction in HSL mRNA was accompanied by a reduction in HSL total activity. The intracellular routes that cAMP and PMA follow for inducing such an effect seemed clearly independent. (i) After desensitization of the protein kinase C regulation pathway by a 24 h treatment of the cells with 1 microM PMA, PMA action was abolished whereas cAMP was still fully active. (ii) Treatment with saturating concentrations of both agents produced an additive effect. (iii) The synthetic glucocorticoid dexamethasone had no proper effect on HSL gene expression but potentiated cAMP action without affecting PMA action. cAMP inhibitory action on HSL is unexpected. Indeed, the second messenger of catecholamines is the main activator of HSL by phosphorylation. We envision that a long-term cAMP treatment of adipocytes induces a counter-regulatory process that reduces HSL content and, ultimately, limits fatty acid depletion from stored triacylglycerols.