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.     

Rapid nitration of adipocyte phosphoenolpyruvate carboxykinase by leptin reduces glyceroneogenesis and induces fatty acid release

Fatty acid (FA) release from white adipose tissue (WAT) is the result of the balance between triglyceride breakdown and FA re-esterification. The latter relies on the induction of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), the key enzyme for glyceroneogenesis. We previously demonstrated that long-term (18 h) leptin treatment of rat epididymal WAT explants reduced glyceroneogenesis through nitric oxide (NO)-induced decrease in PEPCK-C expression. We investigated the effect of a short-term leptin treatment (2 h) on PEPCK-C expression and glyceroneogenesis in relation to NO production. We demonstrate that in WAT explants, leptin-induced NO synthase III (NOS III) phosphorylation was associated with reduced PEPCK-C level and glyceroneogenesis, leading to FA release, while PEPCK-C gene expression remained unaffected. These effects were absent in WAT explants from leptin receptor-deficient Zucker rat. Immunoprecipitation and western blot experiments showed that the leptin-induced decrease in PEPCK-C level was correlated with an increase in PEPCK-C nitration. All these effects were abolished by the NOS inhibitor Nω-nitro-L-arginine methyl ester and mimicked by the NO donor S-nitroso-N-acetyl-DL penicillamine. We propose a mechanism in which leptin activates NOS III and induces NO that nitrates PEPCK-C to reduce its level and glyceroneogenesis, therefore limiting FA re-esterification in WAT.     

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.     

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.     

Glyceroneogenesis is inhibited through HIV protease inhibitor-induced inflammation in human subcutaneous but not visceral adipose tissue

Glyceroneogenesis, a metabolic pathway that participates during lipolysis in the recycling of free fatty acids to triglycerides into adipocytes, contributes to the lipid-buffering function of adipose tissue. We investigated whether glyceroneogenesis could be affected by human immunodeficiency virus (HIV) protease inhibitors (PIs) responsible or not for dyslipidemia in HIV-infected patients. We treated explants obtained from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) depots from lean individuals. We observed that the dyslipidemic PIs nelfinavir, lopinavir and ritonavir, but not the lipid-neutral PI atazanavir, increased lipolysis and decreased glyceroneogenesis, leading to an increased release of fatty acids from SAT but not from VAT. At the same time, dyslipidemic PIs decreased the amount of perilipin and increased interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion in SAT but not in VAT. Parthenolide, an inhibitor of the NFκB pathway, counteracted PI-induced increased inflammation and decreased glyceroneogenesis. IL-6 (100 ng) inhibited the activity of phosphoenolpyruvate carboxykinase, the key enzyme of glyceroneogenesis, in SAT but not in VAT. Our data show that dyslipidemic but not lipid-neutral PIs decreased glyceroneogenesis as a consequence of PI-induced increased inflammation in SAT that could have an affect on adipocytes and/or macrophages. These results add a new link between fat inflammation and increased fatty acids release and suggest a greater sensitivity of SAT than VAT to PI-induced inflammation.     

Molecular docking of Glyceroneogenesis pathway intermediates with Peroxisome Proliferator- Activated Receptor-Alpha (PPAR-α)

Peroxisome proliferator-activated receptor alpha (PPAR-α) belongs to the nuclear receptor superfamily of proteins. It is one of the principle regulators of metabolism and lipid homeostasis whose malfunction leads to complications including obesity and type 2 diabetes. In the adipose tissue, glyceroneogenesis is a unique pathway through which pyruvate is converted into glycerol-3- phosphate (G3P) in a multistep process. Previous findings demonstrated that glyceroneogenesis regulates triacylglycerol synthesis and adipogenesis. This led us to hypothesize that one of the pathway intermediate is physiologically relevant PPAR-α ligand. In the present study using in silico docking, we proved that glycerate, dihydroxy acetone phosphate, glyceraldehyde-3-phosphate, and G3P are key glyceroneogenesis pathway intermediates which bind to PPAR-α. They bind PPAR-α with comparable binding energy and docking score to that of (2s)-2-ethoxy-3-[4-(2-{4-[(methylsulfonyl)oxy]phenyl}ethoxy)phenyl]propanoic acid(AZ-2), a synthetic high affinity ligand of PPAR-α. These intermediates could be studied further as potential physiologically relevant activators of PPAR-α in vitro and in vivo.     

Rosiglitazone controls fatty acid cycling in human adipose tissue by means of glyceroneogenesis and glycerol phosphorylation

Control of fatty acid homeostasis is crucial to prevent insulin resistance. During fasting, the plasma fatty acid level depends on triglyceride lipolysis and fatty acid re-esterification within fat cells. In rodents, Rosiglitazone controls fatty acid homeostasis by stimulating two pathways in the adipocytes, glyceroneogenesis and glycerol phosphorylation, that provide the glycerol 3-phosphate necessary for fatty acid re-esterification. Here, we analyzed the functionality of both pathways for controlling fatty acid release in subcutaneous adipose tissue samples from lean and overweight women before and after Rosiglitazone ex vivo treatment. In controls, pyruvate, used as a substrate of glyceroneogenesis, could contribute to the re-esterification of up to 65% of the fatty acids released after basal lipolysis, whereas glycerol phosphorylation accounted for only 14 +/- 9%. However, the efficiency of glyceroneogenesis diminished as body mass index (BMI) of women increased. After Rosiglitazone treatment, increase of either pyruvate- or glycerol-dependent fatty acid re-esterification was strictly correlated to that of phosphoenolpyruvate carboxykinase and glycerol kinase, the key enzymes of each pathway, but depended on BMI of the women. Whereas the Rosiglitazone responsiveness of glyceroneogenesis was rather constant according to the BMI of the women, glycerol phosphorylation was mostly enhanced in lean women (BMI < 27). Overall, these data indicate that, whereas glyceroneogenesis is more utilized than glycerol phosphorylation for fatty acid re-esterification in human subcutaneous adipose tissue in the physiological situation, both are solicited in response to Rosiglitazone but with lower efficiency when BMI is increased.     

PEPCK-C reexpression in the liver counters neonatal hypoglycemia in <Emphasis Type="Italic">Pck1</Emphasis><Superscript><Emphasis Type="Italic">del/del</Emphasis></Superscript> mice,unmasking role in non-gluconeogenic tissues

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism.     

Immunocytochemical localization of glucose 6-phosphatase and cytosolic phosphoenolpyruvate carboxykinase in gluconeogenic tissues reveals unsuspected metabolic zonation

Immunohistochemical analysis was used to define the precise cell-specific localization of Glucose-6-phosphatase (Glc6Pase) and cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) in the digestive system (liver, small intestine and pancreas) and the kidney. Co-expression of Glc6Pase and PEPCK-C was shown to take place in hepatocytes, in proximal tubules of the cortex kidney and at the top of the villi of the small intestine suggesting that these tissues are all able to perform complete gluconeogenesis. On the other hand, intrahepatic bile ducts, collecting tubes of the nephron and the urinary epithelium in the calices of the kidney, as well as the crypts of the small intestine, express Glc6Pase without significant levels of PEPCK-C. In such cases, the function of Glc6Pase could be related to the transepithelial transport of glucose characteristic of these tissues, rather than to the neoformation of glucose. Lastly, PEPCK-C expression in the absence of Glc6Pase was noted in both the exocrine pancreas and the endocrine islets of Langerhans. Possible roles of PEPCK-C in exocrine pancreas might be the provision of gluconeogenic intermediates for further conversion into glucose in the liver, whereas PEPCK-C would be instrumental in pyruvate cycling, which has been suggested to play a regulatory role in insulin secretion by the β-cells of the islets. An erratum to this article can be found at