A Pyruvate Cycling Pathway Involving Cytosolic NADP-dependent Isocitrate Dehydrogenase Regulates Glucose-stimulated Insulin Secretion

Abstract

Glucose-stimulated insulin secretion (GSIS) from pancreatic islet ␤-cells is central to control of mammalian fuel homeostasis.Glucose metabolism mediates GSIS in part via ATP-regulated K ؉ (K ATP ) channels, but multiple lines of evidence suggest participation of other signals. Here we investigated the role of cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) in control of GSIS in ␤-cells. Delivery of small interfering RNAs specific for ICDc caused impairment of GSIS in two independent robustly glucose-responsive rat insulinoma (INS-1-derived) cell lines and in primary rat islets. Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity and in NADPH levels, a predicted by-product of pyruvate cycling pathways, as well as the total cellular NADP(H) content. Metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused increases in lactate production in both INS-1-derived cell lines and primary islets, consistent with the attenuation of pyruvate cycling, with no significant changes in other intermediates. Based on these studies, we propose that a pyruvate cycling pathway involving ICDc plays an important role in control of GSIS.Glucose metabolism in pancreatic islet ␤-cells generates signals for acute stimulation of insulin secretion, and it is widely accepted that an increase in the ATP:ADP ratio brought about by glucose flux through glycolysis and the tricarboxylic acid cycle is central to this process. The rise in the ATP:ADP ratio results in closure of ATP-regulated K ϩ (K ATP ) 2 channels, plasma membrane depolarization, activation of voltage-dependent Ca 2ϩ channels, and subsequent influx of Ca 2ϩ to stimulate insulin release (1, 2). This K ATP channel-dependent pathway has been suggested to be especially important for the acute first phase of glucose-stimulated insulin secretion (GSIS). In the second and more prolonged phase of GSIS, glucose-derived factors in addition to ATP have been implicated (3, 4), including glutamate (5), malonyl-CoA/cytosolic long chain acyl-CoA esters (6 -9), and transport of NADH reducing equivalents into the mitochondria via hydrogen shuttles (10), but data arguing against a role for some of these events have also been presented (11-13).Pyruvate carboxylase (PC), which catalyzes the conversion of pyruvate to oxaloacetate, is highly active in ␤-cells and accounts for ϳ40 -50% of pyruvate entry into mitochondrial metabolism at stimulatory glucose concentrations (14 -17). The high PC activity in ␤-cells is remarkable in light of the absence of gluconeogenesis (18) and relatively low lipogenic activity (19) in these cells. Furthermore, it has been estimated that only 25% of the glucose-derived carbons entering the tricarboxylic acid cycle via PC are channeled into protein synthesis (17). These findings suggest that PC-catalyzed entry of metabolites into mitochondrial metabolic pathways (anaplerosis) may play other roles in ␤-cell function.Previously, we examined glucose flux in a set of INS-derived ce...

The Mitochondrial Citrate/Isocitrate Carrier Plays a Regulatory Role in Glucose-stimulated Insulin Secretion

“…Citrate emanating from mitochondrial metabolism can also be cleaved by ATP-citrate lyase to produce malonyl-CoA and LC-CoA (19). We and others have previously established that anaplerotic metabolism of pyruvate and pyruvate cycling flux are closely correlated with the capacity for glucose-stimulated insulin secretion in ␤-cells (9,13,18,20,21). Also supporting a key role for anaplerosis is the earlier finding that ϳ40 -50% of pyruvate that enters mitochondrial pathways at high glucose does so via pyruvate carboxylase, the anaplerotic entry point (22)(23)(24).…”

“…RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). CIC mRNA levels were detected by real time PCR as previously described (13,18), using prevalidated CIC and 18 S RNA-specific fluorescent probes obtained from Applied Biosystems (Foster City, CA).…”

“…NADPH can be produced via one of three pyruvate cycling pathways, the pyruvate/malate pathway, the pyruvate/citrate pathway, or the pyruvate/isocitrate pathway, via cytosolic NADP ϩ -dependent isoforms of malic enzyme (used in the pyruvate/malate and pyruvate/citrate pathways) or cytosolic, NADP ϩ -dependent isocitrate dehydrogenase (ICDc) (used in the pyruvate/isocitrate cycle) (18,19). Citrate emanating from mitochondrial metabolism can also be cleaved by ATP-citrate lyase to produce malonyl-CoA and LC-CoA (19).…”

“…Moreover, recent reports indicate that mouse islets lack malic enzyme activity (25) and that siRNA-mediated suppression of cytosolic NADP-dependent ICDc activity strongly impairs GSIS, pyruvate cycling, and NADPH production (18), suggesting that CIC could play a particularly important role in regulation of insulin secretion. In the current study, we have tested this hypothesis and demonstrate that inhibition of CIC with the specific substrate analogue 1,2,3-benzenetricarboxylate (BTC) or by siRNA-mediated suppression of its expression results in potent inhibition of GSIS, whereas CIC overexpression stimulates GSIS.…”

Leptin therapy in insulin-deficient type I diabetes

“…Metabolic Profiling Studies. Acylcarnitines, fatty acyl CoAs, and amino acids were analyzed in liver homogenates by tandem MS/MS, and organic acids were analyzed by gas GC/MS as described previously (33)(34)(35)(36)(37)(38). All MS analyses employed stable-isotope dilution with internal standards from Isotec, Cambridge Isotope Laboratories, and CDN Isotopes.…”