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Aryl Hydrocarbon Receptor Nuclear Translocator/Hypoxia-inducible Factor-1β Plays a Critical Role in Maintaining Glucose-stimulated Anaplerosis and Insulin Release from Pancreatic β-Cells

2010; Elsevier BV; Volume: 286; Issue: 2 Linguagem: Inglês

10.1074/jbc.m110.149062

ISSN

1083-351X

Autores

Renjitha Pillai, Peter Huypens, Mei Huang, Stephanie Schaefer, Tanya Sheinin, Shawn Wettig, Jamie W. Joseph,

Tópico(s)

Adipose Tissue and Metabolism

Resumo

The metabolic pathways that are involved in regulating insulin secretion from pancreatic β-cells are still incompletely understood. One potential regulator of the metabolic phenotype of β-cells is the transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor (HIF)-1β. ARNT/HIF-1β levels are profoundly reduced in islets obtained from type 2 diabetic patients. However, no study to date has investigated key pathways involved in regulating insulin release in β-cells that lack ARNT/HIF-1β. In this study, we confirm that siRNA-mediated knockdown of ARNT/HIF-1β inhibits glucose-stimulated insulin secretion. We next investigated the metabolic consequence of the loss of ARNT/HIF-1β knockdown. We demonstrate that β-cells with reduced ARNT/HIF-1β expression levels exhibit a 31% reduction in glycolytic flux without significant changes in glucose oxidation or the ATP:ADP ratio. Metabolic profiling of β-cells treated with siRNAs against the ARNT/HIF-1β gene revealed that glycolysis, anaplerosis, and glucose-induced fatty acid production were down-regulated, and all are key events involved in glucose-stimulated insulin secretion. In addition, both first and second phase insulin secretion in islets were significantly reduced after ARNT/HIF-1β knockdown. Together, our data suggest an important role for ARNT/HIF-1β in anaplerosis, and it may play a critical role in maintaining normal secretion competence of β-cells. The metabolic pathways that are involved in regulating insulin secretion from pancreatic β-cells are still incompletely understood. One potential regulator of the metabolic phenotype of β-cells is the transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor (HIF)-1β. ARNT/HIF-1β levels are profoundly reduced in islets obtained from type 2 diabetic patients. However, no study to date has investigated key pathways involved in regulating insulin release in β-cells that lack ARNT/HIF-1β. In this study, we confirm that siRNA-mediated knockdown of ARNT/HIF-1β inhibits glucose-stimulated insulin secretion. We next investigated the metabolic consequence of the loss of ARNT/HIF-1β knockdown. We demonstrate that β-cells with reduced ARNT/HIF-1β expression levels exhibit a 31% reduction in glycolytic flux without significant changes in glucose oxidation or the ATP:ADP ratio. Metabolic profiling of β-cells treated with siRNAs against the ARNT/HIF-1β gene revealed that glycolysis, anaplerosis, and glucose-induced fatty acid production were down-regulated, and all are key events involved in glucose-stimulated insulin secretion. In addition, both first and second phase insulin secretion in islets were significantly reduced after ARNT/HIF-1β knockdown. Together, our data suggest an important role for ARNT/HIF-1β in anaplerosis, and it may play a critical role in maintaining normal secretion competence of β-cells. IntroductionThe ability of the pancreatic β-cell to maintain glucose homeostasis critically depends on the existence of a functional glucose sensor that operates within the physiologic range of glucose concentrations (1Matschinsky F.M. Diabetes. 1996; 45: 223-241Crossref PubMed Scopus (0) Google Scholar, 2Matschinsky F.M. Nat. Rev. Drug Discov. 2009; 8: 399-416Crossref PubMed Scopus (328) Google Scholar). 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Cell. 2005; 122: 337-349Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar).In this study, our aim was to obtain a metabolic footprint of β-cells with low ARNT/HIF-1β levels and identify the metabolic pathways that are affected by the transcription factor. We demonstrate that impairment in GSIS become eminent when ARNT/HIF-1β is silenced in our INS-1-derived 832/13 cells. Our novel findings that ARNT/HIF-1β plays a role in regulating biphasic insulin secretion and anaplerosis as well as other key metabolic pathways suggest that the mechanism of ARNT/HIF-1β-regulated insulin release appears to be independent of ATP production and likely involves the altered KATP-independent pathway of insulin release.DISCUSSIONExpression profiling of type 2 diabetic human islets showed that ARNT/HIF-1β was reduced by 90% (35Gunton J.E. Kulkarni R.N. Yim S. Okada T. Hawthorne W.J. Tseng Y.H. Roberson R.S. Ricordi C. O'Connell P.J. Gonzalez F.J. Kahn C.R. Cell. 2005; 122: 337-349Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). It was also demonstrated that ARNT/HIF-1β may control the expression levels of other candidate genes for type 2 diabetes, such as HNF4α, the insulin receptor, IRS-2, and Akt2, all of which play an important role in glucose homeostasis (44Vaxillaire M. Froguel P. Endocr. Rev. 2008; 29: 254-264Crossref PubMed Scopus (117) Google Scholar, 45Stoffel M. Duncan S.A. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 13209-13214Crossref PubMed Scopus (332) Google Scholar, 46Withers D.J. Gutierrez J.S. Towery H. Burks D.J. Ren J.M. Previs S. Zhang Y. Bernal D. Pons S. Shulman G.I. Bonner-Weir S. White M.F. Nature. 1998; 391: 900-904Crossref PubMed Scopus (1325) Google Scholar, 47Kubota N. Tobe K. Terauchi Y. Eto K. Yamauchi T. Suzuki R. Tsubamoto Y. Komeda K. Nakano R. Miki H. Satoh S. Sekihara H. Sciacchitano S. Lesniak M. Aizawa S. Nagai R. Kimura S. Akanuma Y. Taylor S.I. Kadowaki T. Diabetes. 2000; 49: 1880-1889Crossref PubMed Scopus (423) Google Scholar). In this study, we assessed the metabolic profile of pancreatic β-cells with low ARNT/HIF-1β expression levels to provide more insight into the metabolic pathways that are affected by ARNT/HIF-1β knockdown and to identify the underlying mechanisms that impair GSIS under these conditions.Based on the available literature, we hypothesized that ARNT/HIF-1β would be required for maintaining glycolytic flux and normal GSIS. Although we confirmed that GSIS was dramatically reduced, we found that glucose utilization was only decreased by 31%, and glucose oxidation and glucose-stimulated ATP production were not significantly altered. To gain more insight into the metabolic consequence of the loss of ARNT/HIF-1β, we employed a metabolomics approach to assess a wide range of cellular metabolites. As expected, glycolytic intermediates were significantly reduced in siARNT1-treated cells; however, the most dramatic reductions were seen for TCA intermediates suggesting that anaplerosis is an important target of ARNT/HIF-1β. These studies also demonstrate that the role of ARNT/HIF-1β in regulating insulin release in response to glucose is independent of altered glucose oxidation and ATP production and likely involves altered anaplerosis.Anaplerosis has been suggested to play an important role in insulin secretion. In β-cells, when glucose is abundant, the mitochondrial entry of pyruvate into the TCA cycle occurs via PDH and PC in almost equal proportions, giving rise to the existence of two separate pyruvate pools, with one feeding acetyl-CoA into the TCA cycle for oxidation (PDH pathway) and the other leading to a net accumulation of TCA intermediates (PC pathway) (21Lu 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 (219) Google Scholar, 48MacDonald M.J. Arch. Biochem. Biophys. 1993; 305: 205-214Crossref PubMed Scopus (79) Google Scholar, 49MacDonald M.J. Metabolism. 1993; 42: 1229-1231Abstract Full Text PDF PubMed Scopus (47) Google Scholar, 50Khan A. Ling Z.C. Landau B.R. J. Biol. Chem. 1996; 271: 2539-2542Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 51Schuit F. De Vos A. Farfari S. Moens K. Pipeleers D. Brun T. Prentki M. J. Biol. Chem. 1997; 272: 18572-18579Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). In the PDH pathway, the increase in mitochondrial glucose metabolism and acetyl-CoA will promote the transfer of reducing equivalents into the respiratory chain, resulting in mitochondrial membrane hyperpolarization and increased ATP production. The net accumulation of TCA intermediates in the PC pathway can play an important role in other proposed pathways involved in regulating insulin release, including pyruvate cycling, GTP, glutamate, α-ketoglutarate, and LC-CoA hypothesis (11Jensen M.V. Joseph J.W. Ronnebaum S.M. Burgess S.C. Sherry A.D. Newgard C.B. Am. J. Physiol. Endocrinol. Metab. 2008; 295: E1287-E1297Crossref PubMed Scopus (189) Google Scholar, 24Meredith M. Rabaglia M.E. Metz S.A. J. Clin. Invest. 1995; 96: 811-821Crossref PubMed Scopus (47) Google Scholar, 25Corkey B.E. Glennon M.C. Chen K.S. Deeney J.T. Matschinsky F.M. Prentki M. J. Biol. Chem. 1989; 264: 21608-21612Abstract Full Text PDF PubMed Google Scholar, 26Prentki M. Vischer S. Glennon M.C. Regazzi R. Deeney J.T. Corkey B.E. J. Biol. Chem. 1992; 267: 5802-5810Abstract Full Text PDF PubMed Google Scholar, 27Newgard C.B. Lu D. Jensen M.V. Schissler J. Boucher A. Burgess S. Sherry A.D. Diabetes. 2002; 51: S389-S393Crossref PubMed Google Scholar, 28Broca C. Brennan L. Petit P. Newsholme P. Maechler P. FEBS Lett. 2003; 545: 167-172Crossref PubMed Scopus (46) Google Scholar, 29Ivarsson R. Quintens R. Dejonghe S. Tsukamoto K. in 't Veld P. Renström E. Schuit F.C. Diabetes. 2005; 54: 2132-2142Crossref PubMed Scopus (209) Google Scholar, 30Joseph J.W. Jensen M.V. Ilkayeva O. Palmieri F. Alárcon C. Rhodes C.J. Newgard C.B. J. Biol. Chem. 2006; 281: 35624-35632Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 31Kibbey R.G. Pongratz R.L. Romanelli A.J. Wollheim C.B. Cline G.W. Shulman G.I. Cell Metab. 2007; 5: 253-264Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Our results support the idea that ARNT/HIF-1β plays a role in regulating anaplerosis as both anaplerotic related genes (PC, DIC, and OGC) and TCA intermediates are both reduced in response to lower ARNT/HIF-1β expression levels. We also found that anaplerosis was lower at low glucose, and this was associated with a nonsignificant reduction in basal insulin secretion in siARNT1- and siARNT2-treated cells. This suggests that anaplerosis may play a role in basal insulin secretion as well as high glucose-stimulated insulin secretion.Pyruvate cycling has been proposed to be a novel pathway involved in regulating insulin secretion by numerous groups (11Jensen M.V. Joseph J.W. Ronnebaum S.M. Burgess S.C. Sherry A.D. Newgard C.B. Am. J. Physiol. Endocrinol. Metab. 2008; 295: E1287-E1297Crossref PubMed Scopus (189) Google Scholar, 30Joseph J.W. Jensen M.V. Ilkayeva O. Palmieri F. Alárcon C. Rhodes C.J. Newgard C.B. J. Biol. Chem. 2006; 281: 35624-35632Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 37Joseph J.W. Odegaard M.L. Ronnebaum S.M. Burgess S.C. Muehlbauer J. Sherry A.D. Newgard C.B. J. Biol. 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Han J. Long Y.S. Lock J. Weir G.C. Epstein P.N. Liu Y.Q. Diabetologia. 2008; 51: 2281-2289Crossref PubMed Scopus (26) Google Scholar, 55Pongratz R.L. Kibbey R.G. Shulman G.I. Cline G.W. J. Biol. Chem. 2007; 282: 200-207Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 56Guay C. Madiraju S.R. Aumais A. Joly E. Prentki M. J. Biol. Chem. 2007; 282: 35657-35665Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Increased anaplerotic input is critical for proper functioning of all pyruvate cycling pathways. Pyruvate cycling has been suggested to generate two potentially important signaling molecules for insulin release, α-ketoglutarate and NADPH. NADPH can be produced via one of three pyruvate cycling pathways, the pyruvate/malate pathway, the pyruvate/citrate pathway, or the pyruvate/isocitrate pathway. A key NADPH-producing enzyme for both the pyruvate malate and pyruvate citrate pathways is the MEc (11Jensen M.V. Joseph J.W. Ronnebaum S.M. Burgess S.C. Sherry A.D. Newgard C.B. Am. J. Physiol. Endocrinol. Metab. 2008; 295: E1287-E1297Crossref PubMed Scopus (189) Google Scholar, 52Ronnebaum S.M. Jensen M.V. Hohmeier H.E. Burgess S.C. Zhou Y.P. Qian S. MacNeil D. Howard A. Thornberry N. Ilkayeva O. Lu D. Sherry A.D. Newgard C.B. J. Biol. Chem. 2008; 283: 28909-28917Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 53Brown L.J. Longacre M.J. Hasan N.M. Kendrick M.A. Stoker S.W. Macdonald M.J. J. Biol. Chem. 2009; 284: 35359-35367Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The key enzyme for the pyruvate isocitrate pathway is the cytosolic isocitrate dehydrogenase (38Ronnebaum 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 (180) Google Scholar). Although strong evidence for a role of the pyruvate-isocitrate pathways exists (11Jensen M.V. Joseph J.W. Ronnebaum S.M. Burgess S.C. Sherry A.D. Newgard C.B. Am. J. Physiol. Endocrinol. Metab. 2008; 295: E1287-E1297Crossref PubMed Scopus (189) Google Scholar, 30Joseph J.W. Jensen M.V. Ilkayeva O. Palmieri F. Alárcon C. Rhodes C.J. Newgard C.B. J. Biol. Chem. 2006; 281: 35624-35632Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 37Joseph J.W. Odegaard M.L. Ronnebaum S.M. Burgess S.C. Muehlbauer J. Sherry A.D. Newgard C.B. J. Biol. Chem. 2007; 282: 31592-31600Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 38Ronnebaum 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 (180) Google Scholar, 40Jensen M.V. Joseph J.W. Ilkayeva O. Burgess S. Lu D. Ronnebaum S.M. Odegaard M. Becker T.C. Sherry A.D. Newgard C.B. J. Biol. Chem. 2006; 281: 22342-22351Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 52Ronnebaum S.M. Jensen M.V. Hohmeier H.E. Burgess S.C. Zhou Y.P. Qian S. MacNeil D. Howard A. Thornberry N. Ilkayeva O. Lu D. Sherry A.D. Newgard C.B. J. Biol. Chem. 2008; 283: 28909-28917Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 53Brown L.J. Longacre M.J. Hasan N.M. Kendrick M.A. Stoker S.W. Macdonald M.J. J. Biol. Chem. 2009; 284: 35359-35367Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), others have shown a role for the other two other pyruvate cycling pathways (54Xu J. Han J. Long Y.S. Lock J. Weir G.C. Epstein P.N. Liu Y.Q. Diabetologia. 2008; 51: 2281-2289Crossref PubMed Scopus (26) Google Scholar, 55Pongratz R.L. Kibbey R.G. Shulman G.I. Cline G.W. J. Biol. Chem. 2007; 282: 200-207Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 56Guay C. Madiraju S.R. Aumais A. Joly E. Prentki M. J. Biol. Chem. 2007; 282: 35657-35665Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Which pyruvate cycling pathway(s) that is critical for insulin release is unknown. Our results support a role for ARNT/HIF-1β in pyruvate cycling as important genes in the pathway are down-regulated (PC and MEc).Gunton et al. (35Gunton J.E. Kulkarni R.N. Yim S. Okada T. Hawthorne W.J. Tseng Y.H. Roberson R.S. Ricordi C. O'Connell P.J. Gonzalez F.J. Kahn C.R. Cell. 2005; 122: 337-349Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar) showed that the suppression or complete lack of ARNT/HIF-1β leads to abnormalities in insulin release in ARNT/HIF-1β-silenced Min6 cells and in primary islets obtained from β-cell-specific ARNT knock-out mice. Here, we confirm their findings as siRNA-mediated knockdown of ARNT/HIF-1β in INS-1-derived 832/13 cells also reduced GSIS by 60% when compared with control cells. We also found that both first and second phase insulin secretion was down-regulated in perifused pseudoislets. To identify the underlying mechanisms that cause this loss in glucose responsiveness, we initially measured glucose utilization and glucose oxidation. At 2 mm glucose, ARNT/HIF-1β knockdown did not significantly affect glucose utilization or glucose oxidation, which is in agreement with the observation that pyruvate levels did not deviate from control under these conditions. However, when exposed to 16.7 mm glucose, the glycolytic flux was clearly suppressed by ∼31% in siARNT1-treated cells, and this reduction in glycolysis was not accompanied by a drop in the glucose oxidation. These findings are in agreement with an earlier report showing that the expression levels of several glycolytic enzymes are lowered upon ARNT/HIF-1β knockdown (35Gunton J.E. Kulkarni R.N. Yim S. Okada T. Hawthorne W.J. Tseng Y.H. Roberson R.S. Ricordi C. O'Connell P.J. Gonzalez F.J. Kahn C.R. Cell. 2005; 122: 337-349Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Our data showing a lack of an effect on the oxidative entry of pyruvate into the TCA cycle is surprising considering that glycolytic flux was markedly reduced in ARNT/HIF-1β silenced β-cells.Others have established that either suppression or complete loss of ARNT/HIF-1β in β-cells does not significantly affect the expression levels of the glycolytic pacemaker, glucokinase, or the glucose transporters GLUT1 and GLUT2 (35Gunton J.E. Kulkarni R.N. Yim S. Okada T. Hawthorne W.J. Tseng Y.H. Roberson R.S. Ricordi C. O'Connell P.J. Gonzalez F.J. Kahn C.R. Cell. 2005; 122: 337-349Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar); however, our results suggest that there may be an effect on GLUT2 and GK. Although in our experiments GLUT2 and GK expressions are lower, the metabolomics results support the idea that overall glucose entry and glucokinase activity are not affected by ARNT/HIF-1β knockdown as glucose 6-phosphate levels were similar in siARNT1-treated cells exposed to the high glucose concentration. Therefore, it seems more likely that steps downstream of glucokinase have attained rate-limiting properties for glycolysis in β-cells where ARNT/HIF-1β function is impaired. One possible explanation is that low expression levels of other glycolytic enzymes, such as glucose-6-phosphate isomerase, phosphofructokinase, and aldolase, become limiting for the substrate flow-through glycolysis (35Gunton J.E. Kulkarni R.N. Yim S. Okada T. Hawthorne W.J. Tseng Y.H. Roberson R.S. Ricordi C. O'Connell P.J. Gonzalez F.J. Kahn C.R. Cell. 2005; 122: 337-349Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). In agreement with this notion, we observed that the dihydroxyacetone phosphate and pyruvate levels were significantly lower in siARNT1-treated cells than in siControl-treated cells exposed to 16.7 mm glucose. The 31% reduction in glycolytic flux in siARNT1-treated cells was not reflected in a lack of an increase in glucose oxidation and the ATP:ADP ratio in response to glucose stimulation.The novel finding that reducing ARNT/HIF-1β levels leads to a profound reduction in PC, DIC, and OGC expression levels and a reduction in TCA metabolites, even though glucose oxidation and ATP production were unaltered, is an unexpected result. These collective changes in metabolite levels demonstrate that the oxidative entry of pyruvate is preserved when siARNT1-treated cells are placed under a stimulatory glucose regime and such is likely to occur at the expense of the anaplerotic input into the TCA cycle.Several arguments can be brought forward to support the idea that both anaplerotic input and pyruvate cycling activity are negatively affected by ARNT/HIF-1β knockdown. First, the reduction in glycolytic flux by ∼31% is disproportionate to the 65% lower pyruvate levels detected in ARNT/HIF-1β-silenced 832/13 cells suggesting that anaplerotic and pyruvate cycling pathways are sacrificed at the expenses of maintaining mitochondrial oxidation.Second, the glycolytic end product pyruvate and the TCA intermediates malate, citrate, and isocitrate, which collectively play a flux-determining role in the pyruvate cycling activity via the pyruvate-malate, pyruvate-citrate, and pyruvate-isocitrate shuttle, were significantly reduced by 50–70%. We also found that a key pyruvate cycling gene MEc was significantly reduced. As such, these pyruvate cycling metabolites control the amount of cytosolic NADPH being produced at stimulatory glucose concentrations, and this becomes particularly interesting from the perspective that NADPH has been proposed to act as a metabolic coupling factor (11Jensen M.V. Joseph J.W. Ronnebaum S.M. Burgess S.C. Sherry A.D. Newgard C.B. Am. J. Physiol. Endocrinol. Metab. 2008; 295: E1287-E1297Crossref PubMed Scopus (189) Google Scholar, 29Ivarsson R. Quintens R. Dejonghe S. Tsukamoto K. in 't Veld P. Renström E. Schuit F.C. Diabetes. 2005; 54: 2132-2142Crossref PubMed Scopus (209) Google Scholar, 30Joseph J.W. Jensen M.V. Ilkayeva O. Palmieri F. Alárcon C. Rhodes C.J. Newgard C.B. J. Biol. Chem. 2006; 281: 35624-35632Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 38Ronnebaum 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 (180) Google Scholar).A third argument to support the idea that ARNT/HIF-1β knockdown negatively affects the glucose-induced anaplerotic input into the TCA cycle follows from the observation that there was a reduction in the expression of both FAS and CPT1α, and the glucose-induced rise in fatty acid production is virtually absent in siARNT1-treated cells. These observations suggest that the mitochondrial export of citrate via the citrate/isocitrate carrier (CIC), which provides an essential source of glucose carbon for lipogenesis (30Joseph J.W. Jensen M.V. Ilkayeva O. Palmieri F. Alárcon C. Rhodes C.J. Newgard C.B. J. Biol. Chem. 2006; 281: 35624-35632Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), is severely diminished in ARNT/HIF-1β silenced β-cells. The abrogated glucose-stimulated fatty acid production in siARNT1-treated cells is relevant as the glucose-stimulated production of malonyl-CoA and LC-CoA has been suggested to serve as a metabolic coupling factor in GSIS (25Corkey B.E. Glennon M.C. Chen K.S. Deeney J.T. Matschinsky F.M. Prentki M. J. Biol. Chem. 1989; 264: 21608-21612Abstract Full Text PDF PubMed Google Scholar, 26Prentki M. Vischer S. Glennon M.C. Regazzi R. Deeney J.T. Corkey B.E. J. Biol. Chem. 1992; 267: 5802-5810Abstract Full Text PDF PubMed Google Scholar). The importance of mitochondrial export of substrate via CIC has been established as pharmacologic inhibition of the mitochondrial carrier protein in 832/13 cells, and isolated rat islets cause profound impairments in GSIS (30Joseph J.W. Jensen M.V. Ilkayeva O. Palmieri F. Alárcon C. Rhodes C.J. Newgard C.B. J. Biol. Chem. 2006; 281: 35624-35632Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 56Guay C. Madiraju S.R. Aumais A. Joly E. Prentki M. J. Biol. Chem. 2007; 282: 35657-35665Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Our metabolic data also show that β-cells with low ARNT/HIF-1β levels have lost the ability to generate l-glutamate when glucose levels are raised from basal to stimulatory levels. This may represent another facet in the stimulus-secretion coupling that is affected by a profound reduction in ARNT/HIF-1β as l-glutamate has been proposed to act as a metabolic signal in the KATP channel-independent pathway that controls GSIS (42Maechler P. Wollheim C.B. Nature. 1999; 402: 685-689Crossref PubMed Scopus (436) Google Scholar).Our data support the concept that reducing ARNT/HIF-1β expression inhibits insulin secretion by lowering glucose-stimulated anaplerosis without affecting glucose oxidation and ATP production. ARNT/HIF-1β may lower insulin secretion by decreasing a novel anaplerotic dependent pathway involved in insulin secretion called pyruvate cycling. However, the link between ARNT/HIF-1β and pyruvate cycling has not yet been established.In summary, we performed an extensive metabolic survey of β-cells with reduced ARNT/HIF-1β levels to identify the metabolic pathways that are affected by the transcription factor. We provide proof that ARNT/HIF-1β plays an important role in β-cell glucose metabolism as siRNA-mediated knockdown of the transcription factor causes a significant reduction in glycolytic flux, biphasic insulin secretion, and TCA substrate levels. Considering that glucose oxidation or glucose-induced ATP production was not significantly affected by ARNT/HIF-1β knockdown, the observed shift in the metabolic profile seems to be consistent with a marked reduction in anaplerosis. The idea that low levels of ARNT/HIF-1β negatively affects the anaplerotic input into the TCA cycle is furthermore supported by the observation that glucose-induced fatty acid and glutamate production are completely absent in siARNT1-treated 832/13 cells. Taken together, we provide evidence that ARNT/HIF-1β is absolutely required to keep pancreatic β-cells in a glucose-responsive state by ensuring sufficient substrate flow through the KATP-independent pathways that regulate GSIS (Fig. 8). IntroductionThe ability of the pancreatic β-cell to maintain glucose homeostasis critically depends on the existence of a functional glucose sensor that operates within the physiologic range of glucose concentrations (1Matschinsky F.M. Diabetes. 1996; 45: 223-241Crossref PubMed Scopus (0) Google Scholar, 2Matschinsky F.M. Nat. Rev. Drug Discov. 2009; 8: 399-416Crossref PubMed Scopus (328) Google Scholar). 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