Background Glucose increases the expression of glycolytic enzymes and other hypoxia-response genes in pancreatic beta-cells. Here, we tested whether this effect results from the activation of Hypoxia-Inducible-factors (HIF) 1 and 2 in a hypoxia-dependent manner. Methodology/Principal Findings Isolated rat islets and insulin-secreting INS-1E cells were stimulated with nutrients at various pO2 values or treated with the HIF activator CoCl2. HIF-target gene mRNA levels and HIF subunit protein levels were measured by real-time RT-PCR, Western Blot and immunohistochemistry. The formation of pimonidazole-protein adducts was used as an indicator of hypoxia. In INS-1E and islet beta-cells, glucose concentration-dependently stimulated formation of pimonidazole-protein adducts, HIF1 and HIF2 nuclear expression and HIF-target gene mRNA levels to a lesser extent than CoCl2 or a four-fold reduction in pO2. Islets also showed signs of HIF activation in diabetic Leprdb/db but not non-diabetic Leprdb/+ mice. In vitro, these glucose effects were reproduced by nutrient secretagogues that bypass glycolysis, and were inhibited by a three-fold increase in pO2 or by inhibitors of Ca2+ influx and insulin secretion. In INS-1E cells, small interfering RNA-mediated knockdown of Hif1α and Hif2α, alone or in combination, indicated that the stimulation of glycolytic enzyme mRNA levels depended on both HIF isoforms while the vasodilating peptide adrenomedullin was a HIF2-specific target gene. Conclusions/Significance Glucose-induced O2 consumption creates an intracellular hypoxia that activates HIF1 and HIF2 in rat beta-cells, and this glucose effect contributes, together with the activation of other transcription factors, to the glucose stimulation of expression of some glycolytic enzymes and other hypoxia response genes.
References
[1]
Semenza GL (2007) Oxygen-dependent regulation of mitochondrial respiration by hypoxia-inducible factor 1. Biochem J 405: 1–9.
[2]
Taylor CT (2008) Mitochondria and cellular oxygen sensing in the HIF pathway. Biochem J 409: 19–26.
Jitrapakdee S, Wutthisathapornchai A, Wallace JC, MacDonald MJ (2010) Regulation of insulin secretion: role of mitochondrial signalling. Diabetologia 53: 1019–1032.
[5]
Henquin JC (2009) Regulation of insulin secretion: a matter of phase control and amplitude modulation. Diabetologia 52: 739–751.
[6]
Hinke SA, Hellemans K, Schuit FC (2004) Plasticity of the β cell insulin secretory competence: preparing the pancreatic β cell for the next meal. J Physiol 558: 369–380.
[7]
Hutton JC, Malaisse WJ (1980) Dynamics of O2 consumption in rat pancreatic islets. Diabetologia 18: 395–405.
[8]
Longo EA, Tornheim K, Deeney JT, Varnum BA, Tillotson D, et al. (1991) Oscillations in cytosolic free Ca2+, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets. J Biol Chem 266: 9314–9319.
[9]
Wang W, Upshaw L, Strong DM, Robertson RP, Reems J (2005) Increased oxygen consumption rates in response to high glucose detected by a novel oxygen biosensor system in non-human primate and human islets. J Endocrinol 185: 445–455.
[10]
Carlsson PO, Jansson L, Ostenson CG, Kallskog O (1997) Islet capillary blood pressure increase mediated by hyperglycemia in NIDDM GK rats. Diabetes 46: 947–952.
[11]
Carlsson PO, Jansson L, Palm F (2002) Unaltered oxygen tension in rat pancreatic islets despite dissociation of insulin release and islet blood flow. Acta Physiol Scand 176: 275–281.
[12]
Jung SK, Kauri LM, Qian WJ, Kennedy RT (2000) Correlated oscillations in glucose consumption, oxygen consumption, and intracellular free Ca(2+) in single islets of Langerhans. J Biol Chem 275: 6642–6650.
[13]
Ortsater H, Liss P, Akerman KE, Bergsten P (2002) Contribution of glycolytic and mitochondrial pathways in glucose-induced changes in islet respiration and insulin secretion. Pflugers Arch 444: 506–512.
[14]
Lau J, Henriksnas J, Svensson J, Carlsson PO (2009) Oxygenation of islets and its role in transplantation. Curr Opin Organ Transplant 14: 688–693.
[15]
Sweet IR, Gilbert M (2006) Contribution of calcium influx in mediating glucose-stimulated oxygen consumption in pancreatic islets. Diabetes 55: 3509–3519.
[16]
Sato Y, Endo H, Okuyama H, Takeda T, Iwahashi H, et al. (2011) Cellular hypoxia of pancreatic β-cells due to high levels of oxygen consumption for insulin secretion in vitro. J Biol Chem 286: 12524–12532.
[17]
Hosokawa H, Hosokawa YA, Leahy JL (1995) Upregulated hexokinase activity in isolated islets from diabetic 90% pancreatectomized rats. Diabetes 44: 1328–1333.
[18]
Jonas JC, Sharma A, Hasenkamp W, Ilkova H, Patane G, et al. (1999) Chronic hyperglycemia triggers loss of pancreatic β cell differentiation in an animal model of diabetes. J Biol Chem 274: 14112–14121.
[19]
Laybutt DR, Sharma A, Sgroi DC, Gaudet J, Bonner-Weir S, et al. (2002) Genetic regulation of metabolic pathways in β-cells disrupted by hyperglycemia. J Biol Chem 277: 10912–10921.
[20]
Li X, Zhang L, Meshinchi S, Dias-Leme C, Raffin D, et al. (2006) Islet microvasculature in islet hyperplasia and failure in a model of type 2 diabetes. Diabetes 55: 2965–2973.
[21]
Bensellam M, Van Lommel L, Overbergh L, Schuit FC, Jonas JC (2009) Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations. Diabetologia 52: 463–476.
[22]
Zehetner J, Danzer C, Collins S, Eckhardt K, Gerber PA, et al. (2008) pVHL is a regulator of glucose metabolism and insulin secretion in pancreatic β cells. Genes Dev 22: 3135–3146.
[23]
Buchwald P (2009) FEM-based oxygen consumption and cell viability models for avascular pancreatic islets. Theor Biol Med Model 6: Article 5. DOI 10.1186/1742-4682-6-5.
[24]
Arteel GE, Thurman RG, Raleigh JA (1998) Reductive metabolism of the hypoxia marker pimonidazole is regulated by oxygen tension independent of the pyridine nucleotide redox state. Eur J Biochem 253: 743–750.
[25]
Trube G, Rorsman P, Ohno-Shosaku T (1986) Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic β-cells. Pflugers Arch 407: 493–499.
[26]
Kjorholt C, Akerfeldt MC, Biden TJ, Laybutt DR (2005) Chronic hyperglycemia, independent of plasma lipid levels, is sufficient for the loss of β-cell differentiation and secretory function in the db/db mouse model of diabetes. Diabetes 54: 2755–2763.
[27]
Jung SR, Reed BJ, Sweet IR (2009) A highly energetic process couples calcium influx through L-type calcium channels to insulin secretion in pancreatic beta-cells. Am J Physiol Endocrinol Metab 297: E717–E727.
[28]
Pipeleers D, Kiekens R, Ling Z, Wilikens A, Schuit F (1994) Physiologic relevance of heterogeneity in the pancreatic beta-cell population. Diabetologia 37: Suppl 2S57–S64.
[29]
Moritz W, Meier F, Stroka DM, Giuliani M, Kugelmeier P, et al. (2002) Apoptosis in hypoxic human pancreatic islets correlates with HIF-1α expression. FASEB J 16: 745–747.
[30]
Cantley J, Selman C, Shukla D, Abramov AY, Forstreuter F, et al. (2009) Deletion of the von Hippel-Lindau gene in pancreatic β cells impairs glucose homeostasis in mice. J Clin Invest 119: 125–135.
[31]
Heinis M, Simon MT, Ilc K, Mazure NM, Pouyssegur J, et al. (2010) Oxygen tension regulates pancreatic β-cell differentiation through hypoxia-inducible factor 1α. Diabetes 59: 662–669.
[32]
Roche E, Assimacopoulos-Jeannet F, Witters LA, Perruchoud B, Yaney G, et al. (1997) Induction by glucose of genes coding for glycolytic enzymes in a pancreatic β-cell line (INS-1). J Biol Chem 272: 3091–3098.
[33]
Ma Z, Portwood N, Brodin D, Grill V, Bjorklund A (2007) Effects of diazoxide on gene expression in rat pancreatic islets are largely linked to elevated glucose and potentially serve to enhance β-cell sensitivity. Diabetes 56: 1095–1106.
[34]
da Silva Xavier G, Leclerc I, Salt IP, Doiron B, Hardie DG, et al. (2000) Role of AMP-activated protein kinase in the regulation by glucose of islet beta cell gene expression. Proc Natl Acad Sci U S A 97: 4023–4028.
[35]
Wang H, Wollheim CB (2002) ChREBP rather than USF2 regulates glucose stimulation of endogenous L-pyruvate kinase expression in insulin-secreting cells. J Biol Chem 277: 32746–32752.
[36]
Collier JJ, Zhang PL, Pedersen KB, Burke SJ, Haycock JW, et al. (2007) c-Myc and ChREBP regulate glucose-mediated expression of the L-type pyruvate kinase gene in INS-1-derived 832/13 cells. Am J Physiol Endocrinol Metab 293: E48–E56.
[37]
Diraison F, Ravier MA, Richards SK, Smith RM, Shimano H, et al. (2008) SREBP1 is required for the induction by glucose of pancreatic β-cell genes involved in glucose sensing. J Lipid Res 49: 814–822.
[38]
Hu CJ, Wang LY, Chodosh LA, Keith B, Simon MC (2003) Differential roles of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α in hypoxic gene regulation. Mol Cell Biol 23: 9361–9374.
[39]
Collier JJ, Doan TT, Daniels MC, Schurr JR, Kolls JK, et al. (2003) c-Myc is required for the glucose-mediated induction of metabolic enzyme genes. J Biol Chem 278: 6588–6595.
[40]
Matschinsky FM (1996) A lesson in metabolic regulation inspired by the glucokinase glucose sensor paradigm. Diabetes 45: 223–241.
[41]
Quintens R, Hendrickx N, Lemaire K, Schuit F (2008) Why expression of some genes is disallowed in beta-cells. Biochem Soc Trans 36: 300–305.
[42]
Gunton JE, Kulkarni RN, Yim S, Okada T, Hawthorne WJ, et al. (2005) Loss of ARNT/HIF1β mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes. Cell 122: 337–349.
[43]
Cheng K, Ho K, Stokes R, Scott C, Lau SM, et al. (2010) Hypoxia-inducible factor-1α regulates β cell function in mouse and human islets. J Clin Invest 120: 2171–2183.
[44]
Khaldi MZ, Guiot Y, Gilon P, Henquin JC, Jonas JC (2004) Increased glucose sensitivity of both triggering and amplifying pathways of insulin secretion in rat islets cultured for one week in high glucose. Am J Physiol Endocrinol Metab 287: E207–E217.
[45]
Olsson R, Carlsson PO (2011) A low-oxygenated subpopulation of pancreatic islets constitutes a functional reserve of endocrine cells. Diabetes 60: 2068–2075.
[46]
Ishihara H, Wang H, Drewes LR, Wollheim CB (1999) Overexpression of monocarboxylate transporter and lactate dehydrogenase alters insulin secretory responses to pyruvate and lactate in β cells. J Clin Invest 104: 1621–1629.
[47]
Sekine N, Cirulli V, Regazzi R, Brown LJ, Gine E, et al. (1994) Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic β-cells. Potential role in nutrient sensing. J Biol Chem 269: 4895–4902.
[48]
Puri S, Cano DA, Hebrok M (2009) A role for von Hippel-Lindau protein in pancreatic β-cell function. Diabetes 58: 433–441.
[49]
Richard DE, Berra E, Gothie E, Roux D, Pouyssegur J (1999) p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1α (HIF-1α) and enhance the transcriptional activity of HIF-1. J Biol Chem 274: 32631–32637.
[50]
Elouil H, Bensellam M, Guiot Y, Vander Mierde D, Pascal SM, et al. (2007) Acute nutrient regulation of the unfolded protein response and integrated stress response in cultured rat pancreatic islets. Diabetologia 50: 1442–1452.
[51]
Benita Y, Kikuchi H, Smith AD, Zhang MQ, Chung DC, et al. (2009) An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia. Nucleic Acids Res 37: 4587–4602.
