A Role for ATP-Citrate Lyase, Malic Enzyme, and Pyruvate/Citrate Cycling in Glucose-induced Insulin Secretion

Claudiane Guay,S.R Murthy Madiraju,Alexandre Aumais,Érik Joly,Marc Prentki

doi:10.1074/jbc.m707294200

Claudiane Guay, S.R Murthy Madiraju + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m707294200

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Abstract

In pancreatic beta-cells, metabolic coupling factors generated during glucose metabolism and pyruvate cycling through anaplerosis/cataplerosis processes contribute to the regulation of insulin secretion. Pyruvate/citrate cycling across the mitochondrial membrane leads to the production of malonyl-CoA and NADPH, two candidate coupling factors. To examine the implication of pyruvate/citrate cycling in glucose-induced insulin secretion (GIIS), different steps of the cycle were inhibited in INS 832/13 cells by pharmacological inhibitors and/or RNA interference (RNAi) technology: mitochondrial citrate export, ATP-citrate lyase (ACL), and cytosolic malic enzyme (ME1). The inhibitors of the di- and tri-carboxylate carriers, n-butylmalonate and 1,2,3-benzenetricarboxylate, respectively, reduced GIIS, indicating the importance of transmitochondrial transport of tri- and dicarboxylates in the action of glucose. To directly test the role of ACL and ME1 in GIIS, small hairpin RNA (shRNA) were used to selectively decrease ACL or ME1 expression in transfected INS 832/13 cells. shRNA-ACL reduced ACL protein levels by 67%, and this was accompanied by a reduction in GIIS. The amplification/K(ATP)-independent pathway of GIIS was affected by RNAi knockdown of ACL. The ACL inhibitor radicicol also curtailed GIIS. shRNA-ME1 reduced ME1 activity by 62% and decreased GIIS. RNAi suppression of either ACL or ME1 did not affect glucose oxidation. However, because ACL is required for malonyl-CoA formation, inhibition of ACL expression by shRNA-ACL decreased glucose incorporation into palmitate and increased fatty acid oxidation in INS 832/13 cells. Taken together, the results underscore the importance of pyruvate/citrate cycling in pancreatic beta-cell metabolic signaling and the regulation of GIIS.

Highlights

Is clear that an increase in the cytosolic ATP to ADP ratio plays a critical role in this process via the closure of ATP-dependent potassium (KATP)3 channels, leading to the opening of voltagegated Ca2ϩ channels [1,2,3]
Inhibition of Mitochondrial Citrate Export Alters glucose-induced insulin secretion (GIIS)—We have previously reported that an elevation in glucose concentration causes a rise in citrate in both mitochondrial and cytosolic compartments in the ␤-cell [27], suggesting that during glucose stimulation, citrate formed by the tricarboxylic acid cycle is exported from the mitochondria into the cytosol
These results suggest that export of citrate from mitochondria is needed for GIIS

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—INS 832/13 cells [33] (passages 51– 64) were grown in monolayer cultures in RPMI 1640 complete medium at 11.1 mmol/liter glucose supplemented with 10% (w/v) fetal bovine serum, 10 mmol/liter HEPES, 2 mmol/liter L-glutamine, 1 mmol/liter sodium pyruvate and 50 ␮mol/liter ␤-mercaptoethanol at 37 °C in a humidified atmosphere (5% CO2, 95% air). The cell pellets were washed once with cold PBS, resuspended in 20 mmol/liter Tris-HCl lysis buffer containing 150 mmol/liter NaCl, 1 mmol/ liter EDTA, 1 mmol/liter EGTA, 1% (v/v) Triton X-100, 0.1% SDS, supplemented with protease inhibitor mix, incubated on ice for 45 min, and centrifuged 10 000 ϫ g for 10 min at 4 °C. The cells were incubated for 45 min in KRBH containing 0.5% BSA, 1 mmol/ liter glucose, and 0.1 ␮Ci/ml of [U-14C]glucose (Amersham Biosciences) or 10 mmol/liter glucose and 0.2 ␮Ci/ml of [U-14C]glucose. For fatty acid oxidation experiments, the cells were incubated for 45 min in KRBH containing 0.5% BSA, 1, 5, or 10 mmol/liter glucose in the presence of 1 mmol/liter of carnitine plus 0.2 mmol/liter of palmitate, and 0.1 ␮Ci/ml of [1-14C]palmitate (PerkinElmer Life Sciences). The statistical analyses were performed using the InStat program (GraphPad Software, San Diego, CA)

RESULTS

G 5 G 10 G KCl 1 G 5 G 10 G

G 10 G

DISCUSSION