Role of ATP Production and Uncoupling Protein-2 in the Insulin Secretory Defect Induced by Chronic Exposure to High Glucose or Free Fatty Acids and Effects of Peroxisome Proliferator–Activated Receptor- Inhibition

In rat pancreatic islets chronically exposed to high glucose or high free fatty acid (FFA) levels, glucoseinduced insulin release and mitochondrial glucose oxidation are impaired. These abnormalities are associated with high basal ATP levels but a decreased glucoseinduced ATP production ( of increment over baseline 0.7 0.5 or 0.5 0.3 pmol/islet in islets exposed to glucose or FFA vs. 12.0 0.6 in control islets, n 3; P < 0.01) and, as a consequence, with an altered ATP/ADP ratio. To investigate further the mechanism of the impaired ATP formation, we measured in rat pancreatic islets glucose-stimulated pyruvate dehydrogenase (PDH) activity, a key enzyme for pyruvate metabolism and for the subsequent glucose oxidation through the Krebs cycle, and also the uncoupling protein-2 (UCP-2) content by Western blot. In islets exposed to high glucose or FFA, glucose-stimulated PDH activity was impaired and UCP-2 was overexpressed. Because UCP-2 expression is modulated by a peroxisome proliferator– activated receptor (PPAR)-dependent pathway, we measured PPARcontents by Western blot and the effects of a PPARantagonist. PPARlevels were overexpressed in islets cultured with high FFA levels but unaffected in islets exposed to high glucose. In islets exposed to high FFA concentration, a PPARantagonist was able to prevent UCP-2 overexpression and to restore insulin secretion and the ATP/ADP ratio. These data indicate that in rat pancreatic islets chronically exposed to high glucose or FFA, glucose-induced impairment of insulin secretion is associated with (and might be due to) altered mitochondrial function, which results in impaired glucose oxidation, overexpression of the UCP-2 protein, and a consequent decrease of ATP production. This alteration in FFA cultured islets is mediated by the PPARpathway. Diabetes 51: 2749–2756, 2002 Patients with type 2 diabetes are characterized by a progressive decline of insulin secretion that becomes more severe with the increasing duration of the disease (1–4). In these patients, the mechanisms that cause the progressive -cell failure are currently under investigation: the altered insulin secretory pattern depends, at least in part, on the negative influence of chronic high glucose (5–8) and/or high free fatty acid (FFA) (9–12) plasma concentrations (glucoor lipotoxicity). These metabolites are believed to affect pancreatic -cell function by chronic -cell stimulation and consequent “desensitization” to glucose. However, the molecular mechanisms of glucose desensitization induced by hyperglycemia or hypernefemia are still unclear (13,14). Rat pancreatic islets that are chronically exposed to high glucose or FFAs have an impaired glucose oxidation (15). Because glucose oxidation generates ATP and the rise of ATP and ATP/ADP ratio plays a central role in glucose-induced insulin release by causing K -ATP channel closure, membrane depolarization, increased calcium influx, and insulin granule exocytosis, we first measured ATP and ADP levels in islets that were chronically exposed to high glucose or FFAs. In addition to mitochondrial glucose oxidation, ATP synthesis and ATP/ADP ratio are regulated by uncoupling protein-2 (UCP-2) expression. UCP-2 is a member of a family of proteins that are located in the mitochondrial inner membrane and that act as proton channels to uncouple mitochondrial oxidative phosphorylation. By this mechanism, energy is wasted through heat, and cellular ATP synthesis is decreased. UCP-2 overexpression in rat pancreatic islets has already been shown to inhibit glucose-stimulated insulin secretion by decreasing ATP formation (16,17). Therefore, to investigate the role of two mechanisms involved in ATP synthesis in pancreatic islets that are chronically exposed to high glucose and FFAs, we studied the activity of pyruvate dehydrogenase (PDH), a key enzyme for pyruvate metabolism, and the subsequent glucose oxidation through the Krebs cycle and UCP-2 levels. To study further the mechanisms implicated in the transcriptional regulation of the UCP-2 gene in pancreatic From the Institute of Internal Medicine, Endocrinology and Metabolism, “Signorelli” Diabetes Center, University of Catania, Ospedale Garibaldi and Ospedale Cannizzaro, Catania, Italy. Address correspondence and reprint requests to Francesco Purrello, Unità Operativa di Medicina Interna, Ospedale Cannizzaro, Via Messina 829, 95126 Catania, Italy. E-mail: [email protected]. Received for publication 21 February 2002 and accepted in revised form 3 May 2002. BADGE, bisphenol A diglycidyl ether; FFA, free fatty acid; KRHB, Krebs Ringer HEPES buffer; PDH, pyruvate dehydrogenase; PPAR, peroxisome proliferator–activated receptor; UCP-2, uncoupling protein-2.