Abstract
It has been proposed that de novo synthesis of long-chain acyl-CoA (LC-CoA) is a signal for glucose-stimulated insulin secretion (GSIS). Key enzymes involved in synthesis of fatty acids from glucose include ATP-citrate lyase (CL) and fatty acid synthase (FAS). An inhibitor of CL, hydroxycitrate (HC), has been reported to inhibit insulin secretion in some laboratories but not in others. Here we show that high concentrations of NaCl created during preparation of HC by standard methods explain the inhibition of GSIS, and that removal of the excess NaCl prevents the effect. To further investigate the role of CL, two small interfering RNA adenoviruses (Ad-siCL2 and Ad-siCL3) were generated. Ad-siCL3 reduced CL mRNA levels by 92 +/- 6% and CL protein levels by 75 +/- 4% but did not affect GSIS in 832/13 cells compared with cells treated with a control adenovirus (Ad-siControl). Similar results were obtained with Ad-siCL2. Ad-siCL3-treated cells also exhibited a 52 +/- 7% reduction in cytosolic oxaloacetate, an 83 +/- 4% reduction in malonyl-CoA levels, and inhibition of [U-(14)C]glucose incorporation into lipid by 43 +/- 4%, all expected metabolic out-comes of CL suppression. Similarly, treatment of 832/13 cells with a recombinant adenovirus specific to FAS (Ad-siFAS) reduced FAS mRNA levels by 81 +/- 2% in 832/13 cells, resulting in a 59 +/- 4% decrease in [U-(14)C]glucose incorporation into lipid, without affecting GSIS. Finally, treatment of primary rat islets with Ad-siCL3 or Ad-siFAS reduced CL and FAS mRNA levels by 65 +/- 4% and 52 +/- 3%, respectively, but had no effect on GSIS relative to Ad-siControl-treated islets. These findings demonstrate that a normal rate of flux of glucose carbons through CL and FAS is not required for GSIS in insulinoma cell lines or rat islets.
Highlights
Understanding the nature of all of the glucose-derived signals for insulin secretion will be required to understand the functional failure of the -cell in diabetes and for the development of new drugs for correcting this problem
Treatment of primary rat islets with Ad-siCL3 or Ad-siFAS reduced citrate lyase (CL) and fatty acid synthase (FAS) mRNA levels by 65 ؎ 4% and 52 ؎ 3%, respectively, but had no effect on glucose-stimulated insulin secretion (GSIS) relative to Ad-siControl-treated islets. These findings demonstrate that a normal rate of flux of glucose carbons through CL and FAS is not required for GSIS in insulinoma cell lines or rat islets
The currently accepted pathway for GSIS involves an elevation in the ATP:ADP ratio, closure of ATP-sensitive Kϩ channels (KATP channels), elevation of cytosolic Ca2ϩ levels, and activation of insulin granule exocytosis
Summary
Cell Lines—The INS-1-derived cell line 832/13, which exhibits robust GSIS, was used in these studies [30]. Protein was precipitated from the cytosolic fraction by addition of 0.25 M perchloric acid at 4 °C for 20 min, followed by neutralization with 0.94 M KOH. Supernatant or oxaloacetate standard in perchloric acid (0.2– 2.0 pmol) was added to 200 l of reaction buffer (75 mM K2HPO4, pH 7.4, 80 nM [acetyl-3H]acetyl-CoA, 50 g/ml citrate synthase) at room temperature for 60 min. 832/13 cells (5 ϫ 106) were washed 2 times in phosphate-buffered saline, 0.02% EDTA and extracted with 6% perchloric acid followed by neutralization with 1.67 M KHCO3. Assays were performed in 0.2 ml of 0.2 M potassium phosphate buffer (pH 7.2) containing 0.2 mM EDTA, 2.5 mM dithiothreitol, 1 mg/ml fatty acid-free albumin, 0.25 mM NADPH, 0.68 M [3H]acetyl-CoA (0.5 Ci/nmol), and either 150 l of HClO4-soluble 832/13 cell extract or 0, 1, 100, 400, or 1000 pmol of malonyl-CoA standard.
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