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• W2006454401 abstract "The mitochondria of pancreatic beta cells are believed to convert insulin secretagogues into products that are translocated to the cytosol where they participate in insulin secretion. We studied the hypothesis that short chain acyl-CoA (SC-CoAs) might be some of these products by discerning the pathways of SC-CoA formation in beta cells. Insulin secretagogues acutely stimulated 1.5–5-fold increases in acetoacetyl-CoA, succinyl-CoA, malonyl-CoA, hydroxymethylglutaryl-CoA (HMG-CoA), and acetyl-CoA in INS-1 832/13 cells as judged from liquid chromatography-tandem mass spectrometry measurements. Studies of 12 relevant enzymes in rat and human pancreatic islets and INS-1 832/13 cells showed the feasibility of at least two redundant pathways, one involving acetoacetate and the other citrate, for the synthesis SC-CoAs from secretagogue carbon in mitochondria and the transfer of their acyl groups to the cytosol where the acyl groups are converted to SC-CoAs. Knockdown of two key cytosolic enzymes in INS-1 832/13 cells with short hairpin RNA supported the proposed scheme. Lowering ATP citrate lyase 88% did not inhibit glucose-induced insulin release indicating citrate is not the only carrier of acyl groups to the cytosol. However, lowering acetoacetyl-CoA synthetase 80% partially inhibited glucose-induced insulin release indicating formation of SC-CoAs from acetoacetate in the cytosol is important for insulin secretion. The results indicate beta cells possess enzyme pathways that can incorporate carbon from glucose into acetyl-CoA, acetoacetyl-CoA, and succinyl-CoA and carbon from leucine into these three SC-CoAs plus HMG-CoA in their mitochondria and enzymes that can form acetyl-CoA, acetoacetyl-CoA, malonyl-CoA, and HMG-CoA in their cytosol. The mitochondria of pancreatic beta cells are believed to convert insulin secretagogues into products that are translocated to the cytosol where they participate in insulin secretion. We studied the hypothesis that short chain acyl-CoA (SC-CoAs) might be some of these products by discerning the pathways of SC-CoA formation in beta cells. Insulin secretagogues acutely stimulated 1.5–5-fold increases in acetoacetyl-CoA, succinyl-CoA, malonyl-CoA, hydroxymethylglutaryl-CoA (HMG-CoA), and acetyl-CoA in INS-1 832/13 cells as judged from liquid chromatography-tandem mass spectrometry measurements. Studies of 12 relevant enzymes in rat and human pancreatic islets and INS-1 832/13 cells showed the feasibility of at least two redundant pathways, one involving acetoacetate and the other citrate, for the synthesis SC-CoAs from secretagogue carbon in mitochondria and the transfer of their acyl groups to the cytosol where the acyl groups are converted to SC-CoAs. Knockdown of two key cytosolic enzymes in INS-1 832/13 cells with short hairpin RNA supported the proposed scheme. Lowering ATP citrate lyase 88% did not inhibit glucose-induced insulin release indicating citrate is not the only carrier of acyl groups to the cytosol. However, lowering acetoacetyl-CoA synthetase 80% partially inhibited glucose-induced insulin release indicating formation of SC-CoAs from acetoacetate in the cytosol is important for insulin secretion. The results indicate beta cells possess enzyme pathways that can incorporate carbon from glucose into acetyl-CoA, acetoacetyl-CoA, and succinyl-CoA and carbon from leucine into these three SC-CoAs plus HMG-CoA in their mitochondria and enzymes that can form acetyl-CoA, acetoacetyl-CoA, malonyl-CoA, and HMG-CoA in their cytosol. Mitochondria play two important roles in insulin secretion. One role is ATP production, which in addition to powering numerous cellular processes triggers insulin exocytosis via its acting on the plasmalemmal ATP-sensitive potassium channel. In addition, it is well established that beta cell mitochondria use carbon from glucose-derived pyruvate to synthesize various metabolic intermediates (anaplerosis) (1Brunengraber H. Roe C.R. J. Inherit. Metab. Dis. 2006; 29: 327-331Crossref PubMed Scopus (152) Google Scholar, 2MacDonald M.J. Fahien L.A. Brown L.J. Hasan N.M. Buss J.D. Kendrick M.A. Am. J. Physiol. 2005; 288: E1-E15Crossref PubMed Scopus (202) Google Scholar). About one-half of the pyruvate derived from glucose, the most potent insulin secretagogue, is carboxylated by pyruvate carboxylase to oxaloacetate (2MacDonald M.J. Fahien L.A. Brown L.J. Hasan N.M. Buss J.D. Kendrick M.A. Am. J. Physiol. 2005; 288: E1-E15Crossref PubMed Scopus (202) Google Scholar, 3MacDonald M.J. Arch. Biochem. Biophys. 1993; 300: 201-205Crossref PubMed Scopus (43) Google Scholar, 4MacDonald M.J. Arch. Biochem. Biophys. 1993; 305: 205-214Crossref PubMed Scopus (79) Google Scholar, 5MacDonald M.J. Metabolism. 1993; 42: 1229-1231Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 6MacDonald M.J. J. Biol. Chem. 1995; 270: 20051-20058Abstract Full Text Full Text PDF PubMed Google Scholar, 7Khan A. Ling Z.C. Landau B.R. J. Biol. Chem. 1996; 271: 2539-2542Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Although most of this oxaloacetate is used for anaplerosis and many studies have suggested that anaplerosis is important for insulin secretion (8Farfari S. Schulz V. Corkey B. Prentki M. Diabetes. 2000; 49: 718-726Crossref PubMed Scopus (224) Google Scholar, 9Flamez D. Berger V. Kruhoffer M. Orntoft T. Pipeleers D. Schuit F.C. Diabetes. 2002; 51: 2018-2024Crossref PubMed Scopus (95) Google Scholar, 10Lu D. Mulder H. Zhao P. Burgess S.C. Jensen M.V. Kamzolova S. Newgard C.B. Sherry A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2708-2713Crossref PubMed Scopus (221) Google Scholar, 11Cline G.W. Lepine R.L. Papas K.K. Kibbey R.G. Shulman G.I. J. Biol. Chem. 2004; 279: 44370-44375Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 12Gunawardana S.C. Liu Y.J. MacDonald M.J. Straub S.G. Sharp G.W. Am. J. Physiol. 2004; 287: E828-E833Crossref PubMed Scopus (12) Google Scholar), the purpose of anaplerosis in the beta cell is unclear. It seems clear, however, that if the carboxylation of pyruvate increases the levels of citric acid cycle intermediates inside the mitochondria, this would alter mitochondrial function because many citric acid cycle intermediates inhibit or activate citric acid cycle enzymes (as described in biochemistry texts and briefly reviewed in Refs. 2MacDonald M.J. Fahien L.A. Brown L.J. Hasan N.M. Buss J.D. Kendrick M.A. Am. J. Physiol. 2005; 288: E1-E15Crossref PubMed Scopus (202) Google Scholar and 13MacDonald M.J. Fahien L.A. Buss J.D. Hasan N.M. Fallon M.J. Kendrick M.A. J. Biol. Chem. 2003; 278: 51894-51900Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Therefore, this indicates that these metabolites must be transferred to the extramitochondrial space where they might have signaling or supporting roles in insulin secretion. To explain part of the role of anaplerosis in insulin secretion, we recently proposed the “succinate mechanism” (14Fahien L.A. MacDonald M.J. Diabetes. 2002; 51: 2669-2676Crossref PubMed Scopus (45) Google Scholar). This hypothesis was based on our own data and data from the literature and speculated that succinate metabolism supplies both NADPH and short chain acyl-CoA precursors of mevalonic acid that have signaling or supporting roles in insulin secretion (2MacDonald M.J. Fahien L.A. Brown L.J. Hasan N.M. Buss J.D. Kendrick M.A. Am. J. Physiol. 2005; 288: E1-E15Crossref PubMed Scopus (202) Google Scholar, 14Fahien L.A. MacDonald M.J. Diabetes. 2002; 51: 2669-2676Crossref PubMed Scopus (45) Google Scholar). Although succinate metabolism was instrumental in suggesting the original hypothesis, succinate has become less central to the hypothesis as it has evolved, in part, into a scheme involving the mitochondrial synthesis of multiple short chain acyl-CoAs from the carbon derived from all metabolized insulin secretagogues. Another hypothesis of insulin secretion that has been discussed for 2 decades involves the mitochondrial export of citrate as a carrier of acetyl groups to the cytosol where they are converted to malonyl-CoA, which has a special role in insulin secretion via its inhibition of mitochondrial fatty acid oxidation. This is believed to increase cytosolic long chain acyl-CoA molecules, which might perform signaling roles in insulin secretion (for a review see Ref. 15Corkey B.E. Deeney J.T. Yaney G.C. Tornheim K. Prentki M. J. Nutr. 2000; 130: S299-S304Crossref PubMed Google Scholar). Previous reports have described the possible involvement of NADPH in insulin secretion (6MacDonald M.J. J. Biol. Chem. 1995; 270: 20051-20058Abstract Full Text Full Text PDF PubMed Google Scholar, 8Farfari S. Schulz V. Corkey B. Prentki M. Diabetes. 2000; 49: 718-726Crossref PubMed Scopus (224) Google Scholar, 10Lu D. Mulder H. Zhao P. Burgess S.C. Jensen M.V. Kamzolova S. Newgard C.B. Sherry A.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2708-2713Crossref PubMed Scopus (221) Google Scholar, 16Ivarsson R. Quintens R. Dejonghe S. Tsukamoto K. in't Veld P. Renstrom E. Schuit F.C. Diabetes. 2005; 54: 2132-2142Crossref PubMed Scopus (211) Google Scholar, 17Ronnebaum S.M. Ilkayeva O. Burgess S.C. Joseph J.W. Lu D. Stevens R.D. Becker T.C. Sherry A.D. Newgard C.B. Jensen M.V. J. Biol. Chem. 2006; 281: 30593-30602Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). In this study we examined the possibility that multiple short chain acyl-CoAs, not just mevalonic acid or malonyl-CoA, might be some of the many products of anaplerosis in the beta cell. We first showed that acetyl-CoA, acetoacetyl-CoA, HMG-CoA, 2The abbreviations used are: HMG-CoA, hydroxymethylglutaryl-CoA; KIC, α-ketoisocaproate; shRNA, short hairpin RNA; RT, reverse transcription. succinyl-CoA, and malonyl-CoA are increased to various extents in INS-1 cells stimulated by many different insulin secretagogues as judged from liquid chromatography-tandem mass spectrometry measurements. We then considered the pathways by which multiple short chain acyl-CoAs might be synthesized from insulin secretagogues in mitochondria and their acyl groups transferred to the cytosol where they could be re-activated with coenzyme A and have roles in insulin secretion. Acyl-CoA molecules themselves cannot be transported across the inner mitochondrial membrane and, therefore, after they are synthesized in mitochondria, the coenzyme A moiety is removed, and their acyl groups are transported as carboxylate ions out of the mitochondria to the extramitochondrial space. Outside the mitochondria they are reconverted to their CoA derivatives. The pathways of the original “succinate mechanism” scheme (14Fahien L.A. MacDonald M.J. Diabetes. 2002; 51: 2669-2676Crossref PubMed Scopus (45) Google Scholar) were proposed on the assumption of pathways known to be present in non-beta cells without verification of the presence of the enzymes in the beta cell that might synthesize and metabolize short chain acyl-CoAs or, if present, their intracellular location (mitochondrial versus extramitochondrial). Thus, the scheme did not fully address the pathways by which short chain acyl-CoAs could be synthesized in mitochondria and re-formed in the cytosol. The malonyl-CoA hypothesis seemed to consider citrate as the only carrier of short chain acyl groups from mitochondria to the cytosol (15Corkey B.E. Deeney J.T. Yaney G.C. Tornheim K. Prentki M. J. Nutr. 2000; 130: S299-S304Crossref PubMed Google Scholar). To discern the feasibility of the various possible relevant pathways, we determined the presence or absence, as well as the intracellular locations, of short chain acyl-CoA-synthesizing enzymes in rat and human pancreatic islets and in INS-1 832/13 cells with measurements of their enzyme activities, immunoblot analysis of their protein levels, and/or RT-PCR analysis of their cognate mRNAs. The results showed that beta cells possess enzymes that can form all of the short chain acyl-CoAs studied except malonyl-CoA in their mitochondria and all of the CoAs except succinyl-CoA in their extramitochondrial space. Proof that citrate is not the only carrier of acyl groups from mitochondria to the cytosol was obtained by lowering ATP citrate lyase activity with shRNA and showing that this did not inhibit glucose-induced insulin release. Evidence that acetoacetate is a carrier of acyl groups from mitochondria to the cytosol in the beta cell was obtained by lowering acetoacetyl-CoA synthetase activity with shRNA and observing that this partially inhibited insulin release. Thus, we believe the data suggest that multiple short chain acyl-CoAs formed by anaplerosis could play roles in signaling or supporting insulin secretion and that there are at least two pathways, one involving acetoacetate and the other citrate, for translocation of acyl groups from mitochondria to the cytosol in beta cells. Materials—β-[3-14C]Hydroxybutyrate was from American Radiolabeled Chemicals, Inc. Succinic acid monomethyl ester and all other chemicals were from Sigma. Auxiliary enzymes used in analysis of enzyme activity were from Roche Diagnostics. An antibody against rat acetoacetyl-CoA synthetase was a generous gift of Dr. Tetsuya Fukui (18Ito M. Fukui T. Saito T. Tomita K. Biochim. Biophys. Acta. 1986; 876: 280-287Crossref PubMed Scopus (15) Google Scholar). Rabbit antibodies against the β subunits of rat succinyl-CoA synthetase were from David O. Lambeth (19Lambeth D.O. Tews K.N. Adkins S. Frohlich D. Milavetz B.I. J. Biol. Chem. 2004; 279: 36621-36624Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). A rabbit polyclonal antiserum against the rat peroxisomal acetyl-CoA acetyltransferase (Acaa1a) 3When the same isoforms of both a human and a rat enzyme are discussed together, the abbreviation for the human enzyme (upper case) is used. When either human or rat enzymes are individually mentioned, the convention of using the upper case for the human enzyme and the lower case for rat enzyme is used. was from Paul Van Veldhoven (20Antonenkov V.D. Van Veldhoven P.P. Waelkens E. Mannaerts G.P. J. Biol. Chem. 1997; 272: 26023-26031Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). An antibody to human HMG-CoA lyase was from Grant Mitchell (21Ashmarina L.I. Rusnak N. Miziorko H.M. Mitchell G.A. J. Biol. Chem. 1994; 269: 31929-31932Abstract Full Text PDF PubMed Google Scholar). A rabbit polyclonal antibody to human HMG-CoA reductase was from Upstate Cell Signaling Solutions (Lake Placid, NY). A rabbit polyclonal antibody to human cytosolic acetyl-CoA acetyltransferase (ACAT2) was from Abcam. Human pancreatic islets were from the Islet Isolation Core at Washington University School of Medicine, St. Louis. INS-1 832/13 cells were from Chris Newgard (22Hohmeier H.E. Mulder H. Chen G. Henkel-Rieger R. Prentki M. Newgard C.B. Diabetes. 2000; 494: 24-30Google Scholar). Short Chain Acyl-CoAs—INS-1 832/13 cells were maintained as monolayers on 150-mm diameter plates in INS-1 medium (RPMI 1640 tissue culture medium (the glucose concentration in this medium is 11.1 mm) supplemented with 10% fetal bovine serum, 1 mm pyruvate, 50 μm β-mercaptoethanol, and 10 mm Hepes buffer (23Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar)) and penicillin (100 units/ml) and streptomycin (100 μg/ml). Twenty four hours before an experiment was to be performed, this medium was replaced with fresh medium modified to contain 5 mm glucose. On the day of the experiment plates were washed twice with 10 ml of phosphate-buffered saline and once with 10 ml of Krebs-Ringer bicarbonate solution modified to contain 15 mm sodium bicarbonate and 15 mm sodium Hepes buffer, pH 7.3. Secretagogues were added to the plates in 10 ml of the modified Krebs-Ringer solution, and after 30 min at 37 °C, this solution was quickly withdrawn and replaced with 2 ml of 1% trifluoroacetic acid in 50% methanol. The extracts were then prepared for analysis and analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry exactly as described previously (24MacDonald M.J. J. Biol. Chem. 2007; 282: 6043-6052Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Subcellular Fractionation—Subcellular fractionation of pancreatic islets was as described previously (6MacDonald M.J. J. Biol. Chem. 1995; 270: 20051-20058Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, islets were homogenized in 220 mm mannitol, 70 mm sucrose, 5 mm potassium Hepes buffer, pH 7.5 (MSH). The homogenate was centrifuged at 600 × g for 10 min to precipitate the nuclei and cell debris fraction, and the resulting supernatant fraction was centrifuged at 5,500 × g for 10 min to precipitate the mitochondrial fraction. The resulting supernatant fraction or a 20,000 × g for 20 min supernatant fraction was the cytosol. In some instances, the 5,500 × g supernatant fractions were centrifuged at 20,000 or 50,000 × g for 20 min to obtain a pellet enriched in microsomes and peroxisomes. Liver, kidney, and heart subcellular fractions were prepared as described previously (25MacDonald M.J. Mol. Cell. Biochem. 2004; 258: 201-210Crossref PubMed Scopus (6) Google Scholar). INS-1 cells were fractionated with reagents from the mitochondrial isolation kit (catalog number 8974, Pierce). About 100 μl of loosely packed INS-1 832/13 cells were homogenized in 800 μl of mitochondria isolation reagent A containing a protease inhibitor EDTA-free mixture (Pierce). After vortexing and a 2-min incubation on ice, 10 μl of reagent B was added. The mixture sat on ice for 5 min and was then homogenized with 30 strokes up and down in a Potter-Elvehjem homogenizer. Reagent C (800 μl) containing Halt protease inhibitor was added, and the mixture was centrifuged at 700 × g for 10 min. The resulting supernatant fraction was centrifuged at 12,000 × g for 10 min to give the mitochondrial pellet that was resuspended in MSH solution and freeze-thawed three times before use. The resulting supernatant fraction (cytosol) and mitochondrial fraction were used for enzyme assays and immunoblot analysis. In some instances, the resulting supernatant fraction was centrifuged at higher speeds to obtain a pellet enriched in microsomes and peroxisomes as described for islets. Enzyme Assays—Succinyl-CoA:3-ketoacid CoA transferase was measured in the presence of 50 mm sodium acetoacetate, 0.2 mm succinyl-CoA, 5 mm MgCl2, 5 mm iodoacetamide in 50 mm Tris chloride buffer, pH 8.0 (26Williamson D.H. Bates M.W. Page M.A. Krebs H.A. Biochem. J. 1971; 121: 41-47Crossref PubMed Scopus (277) Google Scholar), at 30 °C. The rate of acetoacetyl-CoA formation was followed by measuring the increase in absorbance at 310 nm. Acetyl-CoA acetyltransferase was measured in the presence of 25 μm acetoacetyl-CoA, 60 μm coenzyme A, 5 mm MgCl2 in 50 mm Tris chloride buffer, pH 7.6, at 30 °C. The disappearance of acetoacetyl-CoA was followed spectrophotometrically by monitoring the decrease in absorbance at 303 nm (27Middleton B. Biochem. J. 1974; 139: 109-121Crossref PubMed Scopus (60) Google Scholar). Acetoacetyl-CoA synthetase was measured with slight modifications of a radiochemical assay (28Bergstrom J.D. Edmond J. Methods Enzymol. 1985; 110: 3-9Crossref PubMed Google Scholar). The complete assay mixture contained final concentrations of 0.5 mm β-[3-14C]hydroxybutyrate (specific radioactivity, 3 mCi/mmol), 1 mm coenzyme A, 1.5 mm ATP, 3 mm NAD, 10 mm MgCl2, 100 mm KCl, and 0.05 units/ml of β-hydroxybutyrate dehydrogenase from Rhodobacter sphaeroides in Tris chloride buffer, pH 7.5. The mixture was maintained at 37 °C for 10 min to generate [3-14C]acetoacetate before pancreatic islet cytosol or INS-1 cytosol was added to bring the final volume to 50 μl. After 20 more min the enzyme reaction was stopped by the addition of 12 μl of acetic acid to the reaction mixture, and part of or the entire volume was spotted on Whatman SG81 chromatography paper. Chromatography was run in a mixture of ethyl ether:formic acid (7:1) for 60–90 min. Acetoacetyl-CoA remains at the origin and acetoacetate and 3-hydroxybutyrate migrate with the solvent front. Liquid scintillation spectrometry was used to measure radioactivity in squares of paper cut from the origin to estimate synthetase activity. Radioactivity in blanks containing cytosol and reaction mixtures minus ATP was subtracted from the radioactivity in samples containing the complete reaction mixture plus cytosol to calculate activity attributable to the synthetase. The activity with ATP present in the enzyme assay mixture was 17–26-fold higher than with no ATP present or when a blank with no cellular extract was present in the reaction mixture when INS-1 cytosol was used and 5-fold higher when rat islet cytosol was used. ATP citrate lyase activity was measured in a reaction mixture containing 5 mm citrate, 0.3 mm coenzyme A, 3 mm ATP, 0.15 mm NADH, 10 mm MgCl2, 10 mm dithiothreitol, and 6 units/ml of malate dehydrogenase from pig heart mitochondria in Tris chloride buffer, pH 8.5, at 37 °C (29Ki S.W. Ishigami K. Kitahara T. Kasahar K. Yoshida M. Horinouchi S. J. Biol. Chem. 2000; 275: 39231-39236Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The disappearance of NADH, which corresponded to oxaloacetate formation, was monitored spectrophotometrically at 340 nm. Glutamate dehydrogenase activity was measured in the presence of 10 mm α-ketoglutarate, 50 mm NH4Cl, 100 μm NADH, 2 mm ADP, 100 μm EDTA, 1 mm KCN, 0.1% Triton X-100, and 50 mm imidazole buffer, pH 7.0 at 30 °C (30MacDonald M.J. McKenzie D.I. Kaysen J.H. Walker T.M. Moran S.M. Fahein L.A. Towle H.C. J. Biol. Chem. 1991; 266: 1335-1340Abstract Full Text PDF PubMed Google Scholar). NADH disappearance was monitored spectrophotometrically at 340 nm. In each spectrophotometric assay, the background rate measured in the absence of one substrate was subtracted from the rate in the presence of the complete reaction mixture to give the rate attributable to the enzyme. Usually for positive controls, enzyme activities were measured in non-islet tissues known to contain the enzyme of interest (data not shown). RT-PCR—Total RNA was isolated from cultured INS-1 (832/13) cells and from freshly isolated rat islets and liver using TRIzol reagent (Invitrogen) and further purified on an RNeasy Mini column (Qiagen). cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Invitrogen) and oligo(dT) primers (Promega, Madison, WI). PCR was performed with the primers shown in supplemental Table 1. cDNAs from tissues known to contain specific transcripts were run as positive controls (data not shown). Immunoblotting—For immunoblotting of HMG-CoA synthase, rat islets and INS-1 cells, or human islets, were washed twice in phosphate-buffered saline and lysed with M-PER mammalian protein extraction reagent (Pierce) containing protease inhibitors. Rat liver and heart tissue were homogenized, and protein was extracted using T-PER tissue protein extraction reagent (Pierce) containing protease inhibitors. Whole rat islets were either homogenized in MSH and boiled in sample buffer or boiled directly in sample buffer (1% SDS, 5% glycerol, 65 mm Tris chloride, pH 6.8, 0.01% bromphenol blue, and either 100 mm dithiothreitol or 2.5% β-mercaptoethanol). Proteins were separated by electrophoresis on 7.5% SDS-polyacrylamide gels and electrotransfered to nitrocellulose membranes. The membranes were blocked with a mixture of 5% nonfat milk and 1% bovine serum albumin and 0.05% Tween 20 in Tris-buffered saline (TBST) and incubated overnight at 4 °C with primary antiserum diluted 1:500 to 1:2000 in blocking buffer. Primary antisera were raised in rabbits immunized with internal peptides of the rat cytosolic HMG-CoA synthase (HIPSPAKKVPRLPAT) and the rat mitochondrial HMG-CoA synthase (CSTIPPAPLAKTDT) conjugated to keyhole limpet hemocyanin (the amino-terminal cysteine was added to the native sequence to facilitate conjugation). The membranes were washed and treated with the secondary antibody labeled with goat anti-rabbit horseradish peroxidase diluted 1:15,000 in blocking buffer, and proteins were visualized with the Supersignal West Pico chemiluminescent substrate kit (Pierce). Luminescence was captured on a Chemi Doc XRS Gel documentation system (Bio-Rad) or on KSR-B Luc Ultra Autorad x-ray film (ISC Bioexpress). To demonstrate equal loading of proteins per lane, the membranes were then washed in TBST and incubated in Restore Western blot stripping buffer (Pierce) for 30 min at 42 °C and re-probed with anti-β-actin antibody (Sigma). Immunoblotting to detect other proteins was done similarly using dilutions of antibody recommended by the supplier. For each enzyme as appropriate, proteins were also measured in subcellular fractions from kidney, liver, or heart as positive or negative controls (data not always shown). Silencing of ATP Citrate Lyase and Acetoacetyl-CoA Synthetase Genes—Five different 65-bp inserts containing 19-bp sequences targeted against the ATP citrate lyase gene were cloned into the BamHI and HindIII sites of the pRNA-U6.1/Hygro plasmid downstream of the U6 promoter (Genescript) and transfected into INS-1 832/13 cells with Lipofectamine 2000 reagent (Invitrogen). Hygromycin-resistant cells were selected and maintained in INS-1 tissue culture medium containing 150 μg/ml of hygromycin. The target sequence that provided the largest decrease in enzyme activity and enzyme protein was CAACAGACCTATGACTACG (nucleotides 1012–1030 of GenBank™ accession number NM_016987). Three 64–69-bp inserts containing 19–22-bp sequences targeted against the acetoacetyl synthetase gene were cloned into the BamHI and HindIII sites of plasmid pSilencer 2.1-U6 Hygro (Ambion) downstream of the U6 promoter and transfected into the INS-1 cells and selected with hygromycin. The target sequence that provided the largest decrease in enzyme activity and insulin release was ACAGCATGTTTCTGGATGA (nucleotides 872–891 of GenBank™ accession number NM_023104). Sequences that gave less knockdown of enzyme activity and insulin release were GGAATTACGATGACTTATA and GGAAGGCTTACTTCTCCAAATA. As an additional control, cells were also transfected with a vector containing a nontargeting shRNA sequence. Data Analysis—Statistical significance was confirmed with Student's t test. Short Chain Acyl-CoA Measurements—The first phase of insulin release normally begins within 3 min of an increase in glucose or other insulin secretagogues that come in contact with the beta cell. The second phase of insulin release, in which the largest amount of insulin is released, is caused by metabolism of insulin secretagogues (31Henquin J.C. Ravier M.A. Nenquin M. Jonas J.C. Gilon P. Eur. J. Clin. Investig. 2003; 33: 742-750Crossref PubMed Scopus (155) Google Scholar) and lasts for several hours or until the stimulus stops. We stimulated INS-1 832/13 cells with various insulin secretagogues, as well as nonsecretagogues as negative controls, and selected 30 min as a time point to measure short chain acyl-CoAs in beta cells when the cells would likely be showing changes in metabolite levels related to a stimulated state. Relative insulin release caused by the various secretagogues and nonsecretagogue controls is listed in the legend of Fig. 2. In INS-1 832/13 cells used for these studies numerous concomitant measurements of insulin release showed that glucose, pyruvate, and leucine plus glutamine were the most potent stimulants of insulin release from these cells, closely followed by 2 mm α-ketoisocaproate plus 10 mm monomethyl succinate (24MacDonald M.J. J. Biol. Chem. 2007; 282: 6043-6052Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 4M. J. MacDonald, unpublished data. Leucine provides a moderate stimulus of insulin release in these cells. 4M. J. MacDonald, unpublished data. Incubation of INS-1 832/13 cells for 30 min under conditions that stimulate insulin release caused large relative increases in several short chain acyl-CoAs. The unstimulated concentration of acetoacetyl-CoA was the lowest of any of the CoAs measured, and the relative increases in acetoacetyl-CoA were the largest (2–5-fold). Succinyl-CoA, HMG-CoA, and malonyl-CoA were present at intermediate levels. Succinyl-CoA and malonyl-CoA were increased 2–3-fold. HMG-CoA was increased 30–60% and acetyl-CoA, which was present at the highest level of the CoAs studied, was increased 55–80%. Pyruvate, which is as potent an insulin secretagogue as glucose in INS-1 832/13 cells (24MacDonald M.J. J. Biol. Chem. 2007; 282: 6043-6052Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 32Ishihara H. Wang H. Drewes L.R. Wollheim C.B. J. Clin. Investig. 1999; 104: 1621-1629Crossref PubMed Scopus (162) Google Scholar, 33Antinozzi P.A. Ishihara H. Newgard C.B. Wollheim C.B. J. Biol. Chem. 2002; 277: 11746-11765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), also caused large increases in the acyl-CoA levels (Fig. 2). In fresh rat pancreatic islets α-ketoisocaproate at a 10 mm concentration is as potent an insulin stimulant as glucose, but α-ketoisocaproate alone does not stimulate insulin in INS-1 832/13 cells (24MacDonald M.J. J. Biol. Chem. 2007; 282: 6043-6052Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). α-Ketoisocaproate alone at either 2 or 10 mm also caused large increases in the short chain CoAs. Methyl succinate alone and leucine alone each stimulate about one-third the amount of insulin release of glucose in fresh rat pancreatic islets (24MacDonald M.J. J. Biol. Chem. 2007; 282: 6043-6052Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 34Fahien L.A. MacDonald M.J. Kmiotek E.H. Mertz R.J. Fahien C.M. J. Biol. Chem. 1988; 263: 13610-13614Abstract Full Text PDF PubMed Google Scholar, 35MacDonald M.J." @default.
• W2006454401 created "2016-06-24" @default.
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• W2006454401 date "2007-10-01" @default.
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• W2006454401 title "Feasibility of Pathways for Transfer of Acyl Groups from Mitochondria to the Cytosol to Form Short Chain Acyl-CoAs in the Pancreatic Beta Cell" @default.
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