| 1. |
- Andersson, Lotta, et al.
(författare)
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Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line.
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.
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| 2. |
- Fex, Malin, et al.
(författare)
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The pathogenetic role of β-cell mitochondria in type 2 diabetes
- 2018
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Ingår i: Journal of Endocrinology. - 0022-0795. ; 236:3, s. 145-149
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Forskningsöversikt (refereegranskat)abstract
- Mitochondrial metabolism is a major determinant of insulin secretion from pancreatic β-cells. Type 2 diabetes evolves when β-cells fail to release appropriate amounts of insulin in response to glucose. This results in hyperglycemia and metabolic dysregulation. Evidence has recently been mounting that mitochondrial dysfunction plays an important role in these processes. Monogenic dysfunction of mitochondria is a rare condition but causes a type 2 diabetes-like syndrome owing to β-cell failure. Here, we describe novel advances in research on mitochondrial dysfunction in the β-cell in type 2 diabetes, with a focus on human studies. Relevant studies in animal and cell models of the disease are described. Transcriptional and translational regulation in mitochondria are particularly emphasized. The role of metabolic enzymes and pathways and their impact on β-cell function in type 2 diabetes pathophysiology are discussed. The role of genetic variation in mitochondrial function leading to type 2 diabetes is highlighted. We argue that alterations in mitochondria may be a culprit in the pathogenetic processes culminating in type 2 diabetes.
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| 3. |
- Göhring, Isabel, et al.
(författare)
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Chronic high glucose and pyruvate levels differentially affect mitochondrial bioenergetics and fuel-stimulated insulin secretion from clonal INS-1 832/13 cells.
- 2014
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 289:6, s. 3786-3798
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Tidskriftsartikel (refereegranskat)abstract
- Glucotoxicity in pancreatic β-cells is a well-established pathogenetic process in Type 2 Diabetes. It has been suggested that metabolism-derived reactive oxygen species perturb the β-cell transcriptional machi-nery. Less is known about altered mitochondrial function in this condition. We used INS-1 832/13 cells cultured for 48 h in 2.8 mM glucose (low-G), 16.7 mM glucose (high-G) or 2.8 mM glucose plus 13.7 mM pyruvate (high-P) to identify metabolic perturbations. High-G cells showed decreased responsiveness, relative to low-G cells, with respect to mitochondrial membrane hyperpolarization, plasma membrane depolarization and insulin secretion, when stimulated acutely with 16.7 mM glucose or 10 mM pyruvate. In contrast, high-P cells were functionally unimpaired, eliminating chronic provision of saturating mitochondrial substrate as a cause of glucotoxicity. Although cellular insulin content was depleted in high-G cells, relative to low-G and high-P cells, cellular functions were largely recovered following a further 24 h culture in low-G medium. After 2 h at 2.8 mM glucose, high-G cells did not retain increased levels of glycolytic or TCA-cycle intermediates, but nevertheless displayed increased glycolysis, increased respiration and an increased mitochondrial proton leak relative to low-G and high-P cells. This notwithstanding, titration of low-G cells with low protonophore concen-trations, monitoring respiration and insulin secretion in parallel, showed that the perturbed insulin secretion of high-G cells could not be accounted for by increased proton leak. The present study supports the idea that glucose-induced disturbances of stimulus-secretion coupling by extra-mitochondrial metabolism upstream of pyruvate, rather than exhaustion from metabolic overload, underlie glucotoxicity in insulin-producing cells.
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| 4. |
- Koeck, Thomas, et al.
(författare)
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A common variant in TFB1M is associated with reduced insulin secretion and increased future risk of type 2 diabetes.
- 2011
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Ingår i: Cell Metabolism. - : Elsevier BV. - 1550-4131 .- 1932-7420. ; 13:1, s. 80-91
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Tidskriftsartikel (refereegranskat)abstract
- Type 2 diabetes (T2D) evolves when insulin secretion fails. Insulin release from the pancreatic β cell is controlled by mitochondrial metabolism, which translates fluctuations in blood glucose into metabolic coupling signals. We identified a common variant (rs950994) in the human transcription factor B1 mitochondrial (TFB1M) gene associated with reduced insulin secretion, elevated postprandial glucose levels, and future risk of T2D. Because islet TFB1M mRNA levels were lower in carriers of the risk allele and correlated with insulin secretion, we examined mice heterozygous for Tfb1m deficiency. These mice displayed lower expression of TFB1M in islets and impaired mitochondrial function and released less insulin in response to glucose in vivo and in vitro. Reducing TFB1M mRNA and protein in clonal β cells by RNA interference impaired complexes of the mitochondrial oxidative phosphorylation system. Consequently, nutrient-stimulated ATP generation was reduced, leading to perturbed insulin secretion. We conclude that a deficiency in TFB1M and impaired mitochondrial function contribute to the pathogenesis of T2D.
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| 5. |
- Krus, Ulrika, et al.
(författare)
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Pyruvate dehydrogenase kinase 1 controls mitochondrial metabolism and insulin secretion in INS-1 832/13 clonal beta-cells
- 2010
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 429, s. 205-213
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Tidskriftsartikel (refereegranskat)abstract
- Tight coupling between cytosolic and mitochondrial metabolism is key for GSIS (glucose-stimulated insulin secretion). In the present study we examined the regulatory contribution of PDH (pyruvate dehydrogenase) kinase 1, a negative regulator of PDH, to metabolic coupling in 832/13 clonal beta-cells. Knockdown of PDH kinase 1 with siRNA (small interfering RNA) reduced its mRNA (>80 %) and protein level (>40 %) after 72 h. PDH activity, glucose-stimulated cellular oxygen consumption and pyruvate-stimulated mitochondrial oxygen consumption increased 1.7- (P < 0.05), 1.6- (P < 0.05) and 1.6-fold (P < 0.05) respectively. Gas chromatography/MS revealed an altered metabolite profile upon silencing of PDH kinase 1, determined by increased levels of the tricarboxylic acid cycle intermediates malate, fumarate and alpha-ketoglutarate. These metabolic alterations were associated with exaggerated GSIS (5-fold compared with 3.1-fold in control cells; P < 0.01). Insulin secretion, provoked by leucine and dimethylsuccinate, which feed into the tricarboxylic acid cycle bypassing PDH, was unaffected. The oxygen consumption and metabolic data strongly suggest that knockdown of PDH kinase 1 in beta-cells permits increased metabolic flux of glucose-derived carbons into the tricarboxylic acid cycle via PDH. Enhanced insulin secretion is probably caused by increased generation of tricarboxylic acid cycle-derived reducing equivalents for mitochondrial electron transport to generate ATP and/or stimulatory metabolic intermediates. On the basis of these findings, we suggest that PDH kinase 1 is an important regulator of PDH in clonal beta-cells and that PDH kinase 1 and PDH are important for efficient metabolic coupling. Maintaining low PDH kinase I expression/activity, keeping PDH in a dephosphorylated and active state, may be important for beta-cells to achieve the metabolic flux rates necessary for maximal GSIS.
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| 6. |
- Kumar, Parveen, et al.
(författare)
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The transcriptional co-repressor myeloid translocation gene 16 inhibits glycolysis and stimulates mitochondrial respiration.
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7
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Tidskriftsartikel (refereegranskat)abstract
- The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor-containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline-dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4), and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia-stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2) oligomerization domain and the NHR3 protein-protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen-activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti-tumor effect.
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| 7. |
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| 8. |
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| 9. |
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| 10. |
- Malmgren, Siri, et al.
(författare)
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Tight coupling between glucose and mitochondrial metabolism in clonal beta-cells is required for robust insulin secretion.
- 2009
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 284, s. 32395-32404
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Tidskriftsartikel (refereegranskat)abstract
- The biochemical mechanisms underlying glucose-stimulated insulin secretion from pancreatic beta-cells are not completely understood. To identify metabolic disturbances in beta-cells that impair glucose-stimulated insulin secretion, we compared two INS-1-derived clonal beta-cell lines, which are glucose-responsive (832/13) or glucose-unresponsive (832/2). We found that despite a marked impairment of glucose-stimulated insulin secretion, 832/2 cells exhibited a higher rate of glycolysis. Still, no glucose-induced increases in respiratory rate, ATP production or respiratory chain complex I, III and IV activities were seen in the 832/2 cells. Instead, 832/2 cells, which expressed lactate dehydrogenase, released lactate regardless of ambient glucose concentrations. In contrast, the glucose-responsive 832/13 line lacked lactate dehydrogenase and did not produce lactate. Accordingly, in 832/2 cells mRNA expression of genes for glycolytic enzymes were up-regulated, whereas mitochondria-related genes were down-regulated. In human islets, mRNA expression of genes such as lactate dehydrogenase A and hexokinase I correlated positively with long-term glucose homeostasis reflected by HbA1c levels, while that of Slc2a2 (GLUT2) correlated negatively with Hb1Ac. We conclude that tight metabolic regulation enhancing mitochondrial metabolism and restricting glycolysis in 832/13 cells is required for clonal beta-cells to secrete insulin robustly in response to glucose. Moreover, a similar expression pattern of genes controlling glycolytic and mitochondrial metabolism in clonal beta-cells and human islets was observed, suggesting that a similar prioritization of mitochondrial metabolism is required in healthy human beta-cells. The 832 beta-cell lines may be helpful tools to resolve metabolic perturbations occurring in Type 2 Diabetes.
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| 11. |
- Schultz, Julia, et al.
(författare)
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Precise expression of Fis1 is important for glucose responsiveness of beta cells
- 2016
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Ingår i: Journal of Endocrinology. - 0022-0795. ; 230:1, s. 81-91
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial network functionality is vital for glucose-stimulated insulin secretion in pancreatic beta cells. Altered mitochondrial dynamics in pancreatic beta cells are thought to trigger the development of type 2 diabetes mellitus. Fission protein 1 (Fis1) might be a key player in this process. Thus, the aim of this study was to investigate mitochondrial morphology in dependence of beta cell function, after knockdown and overexpression of Fis1. We demonstrate that glucose-unresponsive cells with impaired glucose-stimulated insulin secretion (INS1-832/2) showed decreased mitochondrial dynamics compared with glucose-responsive cells (INS1-832/13). Accordingly, mitochondrial morphology visualised using MitoTracker staining differed between the two cell lines. INS1-832/2 cells formed elongated and clustered mitochondria, whereas INS1-832/13 cells showed a homogenous mitochondrial network. Fis1 overexpression using lentiviral transduction significantly improved glucose-stimulated insulin secretion and mitochondrial network homogeneity in glucose-unresponsive cells. Conversely, Fis1 downregulation by shRNA, both in primary mouse beta cells and glucose-responsive INS1-832/13 cells, caused unresponsiveness and significantly greater numbers of elongated mitochondria. Overexpression of FIS1 in primary mouse beta cells indicated an upper limit at which higher FIS1 expression reduced glucose-stimulated insulin secretion. Thus, FIS1 was overexpressed stepwise up to a high concentration in RINm5F cells using the RheoSwitch system. Moderate FIS1 expression improved glucose-stimulated insulin secretion, whereas high expression resulted in loss of glucose responsiveness and in mitochondrial artificial loop structures and clustering. Our data confirm that FIS1 is a key regulator in pancreatic beta cells, because both glucosestimulated insulin secretion and mitochondrial dynamics were clearly adapted to precise expression levels of this fission protein.
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| 12. |
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| 13. |
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| 14. |
- Sharoyko, Vladimir, et al.
(författare)
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Loss of TFB1M results in mitochondrial dysfunction that leads to impaired insulin secretion and diabetes.
- 2014
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Ingår i: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 23:21, s. 5733-5749
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Tidskriftsartikel (refereegranskat)abstract
- We have previously identified Transcription Factor B1 Mitochondrial (TFB1M) as a Type 2 Diabetes (T2D) risk gene, using human and mouse genetics. To further understand the function of TFB1M and how it is associated with T2D we created a β-cell specific knockout of Tfb1 m, which gradually developed diabetes. Prior to the onset of diabetes, β-Tfb1 m(-/-) mice exhibited retarded glucose clearance due to impaired insulin secretion. β-Tfb1 m(-/-) islets released less insulin in response to fuels, contained less insulin and secretory granules, and displayed reduced β-cell mass. Moreover, mitochondria in Tfb1 m-deficient β-cells were more abundant with disrupted architecture. TFB1M is known to control mitochondrial protein translation by adenine-dimethylation of 12S ribosomal RNA (rRNA). Here, we found that levels of TFB1M and mitochondrial encoded proteins, mitochondrial 12S rRNA methylation, ATP production and oxygen consumption were reduced in β-Tfb1 m(-/-) islets. Furthermore, levels of reactive oxygen species in response to cellular stress were increased while induction of defense mechanisms was attenuated. We also show increased apoptosis and necrosis as well as infiltration of macrophages and CD4(+)-cells in the islets. Taken together, our findings demonstrate that Tfb1 m-deficiency in β-cells caused mitochondrial dysfunction and subsequently diabetes due to combined loss of β-cell function and mass. These observations reflect pathogenetic processes in human islets: using RNA sequencing, we found that the TFB1M risk variant exhibited a negative gene-dosage effect on islet TFB1M mRNA levels, as well as insulin secretion. Our findings highlight the role of mitochondrial dysfunction in impairments of β-cell function and mass, the hallmarks of T2D.
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| 15. |
- Sharoyko, Vladimir
(författare)
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Mechanism of pure glucose-dependent insulinotropic activity of a novel imidazoline compound BL11282
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- We developed a novel, pure glucose-dependent, insulinotropic imidazoline compound, BL11282, which directly affects the insulin exocytotic machinery and does not block the KATP channel activity. BL11282 does not induce insulin secretion at basal glucose concentrations, whereas it stimulates insulin secretion at an elevated glucose level. Therefore this imidazoline should not provoke hypoglycemic episodes as it has been observed for the sulfonylureas. However, so far, the detailed biochemical and pharmacological mechanisms underlying the insulinotropic effects of BL11282 are not fully established. The overall objective of this study was to investigate signaltransduction pathways involved in the pure glucose-dependent activity of BL11282 on insulin release. Using SUR1(-/-) mice, we unambiguously confirmed the previous notion that the insulinotropic activity of BL11282 is unrelated to its interaction with ATPdependent K+ channels. We have also shown that previously described targets for imidazoline compounds like alpha2-adrenoreceptors, imidazoline I1-receptors and monomeric G-protein Rhes are not involved in the mechanisms of the insulinotropic action of BL11282. To clarify the molecular mechanisms underlying the effects of BL11282 on insulin secretion, we have used an approach involving desensitization of beta-cells to the insulinotropic activity of BL11282 by prolonged incubation with the compound. The data obtained show that overnight pretreatment of pancreatic islets with BL11282 desensitizes the subsequent islet response to this imidazoline. Islets pretreated for the same time with efaroxan, another insulinotropic imidazoline, are unresponsive to subsequent addition of efaroxan but preserve their response to BL11282. The effect of pretreatment with BL11282 is accompanied by an increased insulinotropic response to subsequent high glucose concentration. Desensitization of islet response to BL11282 does not eliminate subsequent islet response to GLP-1, but significantly decreases the fold potentiation of insulin release by this peptide. The latter effect points to the importance of GLP-1 stimulated signal-transduction pathways for the insulinotropic activity of BL11282. Our results support the involvement of the cAMP-GEFII·Rim2 pathway in BL11282 stimulated insulin secretion. Indeed, expression of dominant negative cAMP-GEFII and Rim2 mutant proteins in MIN6 cells lead to a significant reduction in insulin secretion stimulated by the imidazoline. To further investigate this direct mechanism of BL11282 on insulin release, we turned our attention to calcium-independent PLA2 isoform iPLA2beta, which is predominantly expressed in pancreatic islets and plays an important role in insulin secretion in pancreatic islets. Our observations indicate a deficiency in iPLA2beta isoform expression in diabetic Goto-Kakizaki (GK) rat islets compared to Wistar rat islets, this effect being in agreement with an impairment in the glucose-stimulated insulin response in GK rat islets. Pharmacological inhibition of iPLA2 and cytochrome P-450 enzymes completely abolished the insulinotropic effect of BL11282. BL11282 stimulated arachidonic acid release from the islets in the presence of high glucose concentration and this effect was fully blocked by iPLA2 inhibitor, bromoenol lactone. The data suggest that potentiation of glucose-induced insulin release by BL11282, independent of concomitant changes in cytoplasmic free Ca2+ concentration, involves the release of arachidonic acid by iPLA2 and its metabolism to epoxyeicosatrienoic acids through the cytochrome P-450 pathway.
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| 16. |
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| 17. |
- Spégel, Peter, et al.
(författare)
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Metabolomic analyses reveal profound differences in glycolytic and tricarboxylic acid cycle metabolism in glucose-responsive and -unresponsive clonal beta-cell lines
- 2011
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Ingår i: Biochemical Journal. - 0264-6021. ; 435, s. 277-284
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Tidskriftsartikel (refereegranskat)abstract
- Insulin secretion from pancreatic beta-cells is controlled by complex metabolic and energetic changes provoked by exposure to metabolic fuels. Perturbations in these processes lead to impaired insulin secretion, the ultimate cause of T2D (Type 2 diabetes). To increase our understanding of stimulus secretion coupling and metabolic processes potentially involved in the pathogenesis of T2D, a comprehensive investigation of the metabolic response in the glucose-responsive INS-1 832/13 and glucose-unresponsive INS-1 832/2 beta-cell lines was performed. For this metabolomics analysis, we used GC/MS (gas chromatography/mass spectrometry) combined with multivariate statistics. We found that perturbed secretion in the 832/2 line was characterized by disturbed coupling of glycolytic and TCA (tricarboxylic acid)-cycle metabolism. The importance of this metabolic coupling was reinforced by our observation that insulin secretion partially could be reinstated by stimulation of the cells with mitochondrial fuels which bypass glycolytic metabolism. Furthermore, metabolic and functional profiling of additional beta-cell lines (INS-1, INS-1 832/1) confirmed the important role of coupled glycolytic and TCA-cycle metabolism in stimulus-secretion coupling. Dependence of the unresponsive clones on glycolytic metabolism was paralleled by increased stabilization of HIF-1 alpha (hypoxia-inducible factor 1 alpha). The relevance of a similar perturbation for human T2D was suggested by increased expression of HIF-1 alpha target genes in islets from T2D patients.
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| 18. |
- Spégel, Peter, et al.
(författare)
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Time-resolved metabolomics analysis of beta-cells implicates the pentose phosphate pathway in the control of insulin release
- 2013
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Ingår i: Biochemical Journal. - 0264-6021. ; 450, s. 595-605
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Tidskriftsartikel (refereegranskat)abstract
- Insulin secretion is coupled with changes in beta-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP(+), NADPH and insulin secretion were measured within 15 mm of stimulation of clonal INS-1 832/13 beta-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the beta-cell metabolic response. This was fulfilled by the NADPH/NADP(+) ratio, which was elevated (8-fold; P < 0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P < 0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 beta-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in beta-cell stimulus-secretion coupling.
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| 19. |
- Spégel, Peter, et al.
(författare)
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Unique and Shared Metabolic Regulation in Clonal β-cells and Primary Islets Derived from Rat Revealed by Metabolomics Analysis.
- 2015
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Ingår i: Endocrinology. - : The Endocrine Society. - 0013-7227 .- 1945-7170. ; 156:6, s. 1995-2005
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Tidskriftsartikel (refereegranskat)abstract
- As models for β-cell metabolism, rat islets are, to some extent, a, heterogeneous cell-population stressed by the islet isolation procedure, while rat-derived clonal β-cells exhibit a tumor-like phenotype. To describe to what extent either of these models reflect normal cellular metabolism, we compared metabolite profiles and gene expression in rat islets and the INS-1 832/13 line, a widely used clonal β-cell model. We found that insulin secretion and metabolic regulation provoked by glucose were qualitatively similar in these β-cell models. However, rat islets exhibited a more pronounced glucose-provoked increase of glutamate, glycerol-3-phosphate, succinate and lactate levels while INS-1 832/13 cells showed a higher glucose-elicited increase in glucose-6-phosphate, alanine, isocitrate, and α-ketoglutarate levels. Glucose induced a decrease in levels of γ-aminobutyrate (GABA) and aspartate in rat islets and INS-1 832/13 cells, respectively. Genes with cellular functions related to proliferation and the cell cycle were more highly expressed in the INS-1 832/13 cells. Most metabolic pathways that were differentially expressed included GABA metabolism, in line with altered glucose responsiveness of GABA. Also, lactate dehydrogenase A, which is normally expressed at low levels in mature β-cells, was more abundant in rat islets than in INS-1 832/13 cells, confirming the finding of elevated glucose-provoked lactate production in the rat islets. Overall, our results suggest that metabolism in rat islets and INS-1 832/13 cells is qualitatively similar, albeit with quantitative differences. Differences may be accounted for by cellular heterogeneity of islets and proliferation of the INS-1 832/13 cells.
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| 20. |
- Stamenkovic, Jelena, et al.
(författare)
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Inhibition of the malate-aspartate shuttle in mouse pancreatic islets abolishes glucagon secretion without affecting insulin secretion
- 2015
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Ingår i: Biochemical Journal. - 0264-6021. ; 468, s. 49-63
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Tidskriftsartikel (refereegranskat)abstract
- Altered secretion of insulin as well as glucagon has been implicated in the pathogenesis of Type 2 diabetes (T2D), but the mechanisms controlling glucagon secretion from alpha-cells largely remain unresolved. Therefore, we studied the regulation of glucagon secretion from alpha TC1-6 (alpha TC1 clone 6) cells and compared it with insulin release from INS-1 832/13 cells. We found that INS-1 832/13 and alpha TC1-6 cells respectively secreted insulin and glucagon concentration-dependently in response to glucose. In contrast, tight coupling of glycolytic and mitochondrial metabolism was observed only in INS-1 832/13 cells. Although glycolytic metabolism was similar in the two cell lines, TCA (tricarboxylic acid) cycle metabolism, respiration and ATP levels were less glucose-responsive in alpha TC1-6 cells. Inhibition of the malate-aspartate shuttle, using phenyl succinate (PhS), abolished glucose-provoked ATP production and hormone secretion from alpha TC1-6 but not INS-1 832/13 cells. Blocking the malate-aspartate shuttle increased levels of glycerol 3-phosphate only in INS-1 832/13 cells. Accordingly, relative expression of constituents in the glycerol phosphate shuttle compared with malate-aspartate shuttle was lower in alpha TC1-6 cells. Our data suggest that the glycerol phosphate shuttle augments the malate-aspartate shuttle in INS-1 832/13 but not alpha TC1-6 cells. These results were confirmed in mouse islets, where PhS abrogated secretion of glucagon but not insulin. Furthermore, expression of the rate-limiting enzyme of the glycerol phosphate shuttle was higher in sorted primary beta-than in alpha-cells. Thus, suppressed glycerol phosphate shuttle activity in the alpha-cell may prevent a high rate of glycolysis and consequently glucagon secretion in response to glucose. Accordingly, pyruvate-and lactate-elicited glucagon secretion remains unaffected since their signalling is independent of mitochondrial shuttles.
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| 21. |
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