Biomedical subjects

P Maechler

Publications and source records attributed to P Maechler.

At least 19 recordsLinked to original sources

PubMed2002

Mitochondria as the conductor of metabolic signals for insulin exocytosis in pancreatic beta-cells.

Mitochondrial metabolism is crucial for the coupling of glucose recognition to the exocytosis of the insulin granules. This is illustrated by in vitro and in vivo observations discussed in the present review. Mitochondria generate ATP, which is the main coupling messenger in insulin secretion. However, the subsequent Ca2+ signal in the cytosol is necessary but not sufficient for full development of sustained insulin secretion. Hence, mitochondria generate ATP and other coupling factors serving as fuel sensors for the control of the exocytotic process. Numerous studies have sought to identify the factors that mediate the amplifying pathway over the Ca2+ signal in glucose-stimulated insulin secretion. Predominantly, these factors are nucleotides (GTP, ATP, cAMP, NADPH), although metabolites have also been proposed, such as long-chain acyl-CoA derivatives and glutamate. Hence, the classical neurotransmitter glutamate receives a novel role, that of an intracellular messenger or co-factor in insulin secretion. This scenario further highlights the importance of glutamate dehydrogenase, a mitochondrial enzyme well recognized to play a key role in the control of insulin secretion. Therefore, additional putative messengers of mitochondrial origin are likely to participate in insulin secretion.

Animals↗

PubMed2002

Mitochondria respond to Ca2+ already in the submicromolar range: correlation with redox state.

Rapid formation of high-Ca2+ perimitochondrial cytoplasmic microdomains has been shown to evoke mitochondrial Ca2+ signal and activate mitochondrial dehydrogenases, however, the significance of submicromolar cytoplasmic Ca2+ concentrations in the control of mitochondrial metabolism has not been sufficiently elucidated. Here we studied the mitochondrial response to application of Ca2+ at buffered concentrations in permeabilized rat adrenal glomerulosa cells, in an insulin-producing cell line (INS-1/EK-3) and in an osteosarcoma cell line (143BmA-13). Mitochondrial Ca2+ concentration was measured with the fluorescent dye rhod-2 and, using an in situ calibration method, with the mitochondrially targeted luminescent protein mt-aequorin. In both endocrine cell types, mitochondrial Ca2+ concentration increased in response to elevated cytoplasmic Ca2+ concentration (between 60 and 740 nM) and an increase in mitochondrial Ca2+ concentration could be revealed already at a cytoplasmic Ca2+ concentration step from 60-140 nM. Similar responses were observed in the osteosarcoma cell line, although a clearcut response was first observed at 280 nM extramitochondrial Ca2+ only. As examined in glomerulosa cells, graded increases in cytoplasmic Ca2+ concentration were associated with graded increases in the reduction of mitochondrial pyridine nucleotides, consistent with Ca2+-dependent activation of mitochondrial dehydrogenases. Our data indicate that in addition to the recognized role of high-Ca2+ cytoplasmic microdomains, also small Ca2+ signals may influence mitochondrial metabolism.

Aequorin↗

PubMed2001

Mitochondrial function in normal and diabetic beta-cells.

The aetiology of type 2, or non-insulin-dependent, diabetes mellitus has been characterized in only a limited number of cases. Among these, mitochondrial diabetes, a rare subform of the disease, is the consequence of pancreatic beta-cell dysfunction caused by mutations in mitochondrial DNA, which is distinct from the nuclear genome. The impact of such mutations on beta-cell function reflects the importance of mitochondria in the control of insulin secretion. The beta-cell mitochondria serve as fuel sensors, generating factors that couple nutrient metabolism to the exocytosis of insulin-containing vesicles. The latter process requires an increase in cytosolic Ca2+, which depends on ATP synthesized by the mitochondria. This organelle also generates other factors, of which glutamate has been proposed as a potential intracellular messenger.

Animals↗

PubMed2001

GAD65-mediated glutamate decarboxylation reduces glucose-stimulated insulin secretion in pancreatic beta cells.

Mitochondrial metabolism plays a pivotal role in the pancreatic beta cell by generating signals that couple glucose sensing to insulin secretion. We have demonstrated previously that mitochondrially derived glutamate participates directly in the stimulation of insulin exocytosis. The aim of the present study was to impose altered cellular glutamate levels by overexpression of glutamate decarboxylase (GAD) to repress elevation of cytosolic glutamate. INS-1E cells infected with a recombinant adenovirus vector encoding GAD65 showed efficient overexpression of the GAD protein with a parallel increase in enzyme activity. In control cells glutamate levels were slightly increased by 7.5 mm glucose (1.4-fold) compared with the effect at 15 mm (2.3-fold) versus basal 2.5 mm glucose. Upon GAD overexpression, glutamate concentrations were no longer elevated by 15 mm glucose as compared with controls (-40%). Insulin secretion was stimulated in control cells by glucose at 7.5 mm (2.5-fold) and more efficiently at 15 mm (5.2-fold). INS-1E cells overexpressing GAD exhibited impaired insulin secretion on stimulation with 15 mm glucose (-37%). The secretory response to 30 mm KCl, used to raise cytosolic Ca(2+) levels, was unaffected. Similar results were obtained in perifused rat pancreatic islets following adenovirus transduction. This GAD65-mediated glutamate decarboxylation correlating with impaired glucose-induced insulin secretion is compatible with a role for glutamate as a glucose-derived factor participating in insulin exocytosis.

Adenosine Triphosphate↗

PubMed2001

Pdx1 level defines pancreatic gene expression pattern and cell lineage differentiation.

The absence of Pdx1 and the expression of brain-4 distinguish alpha-cells from other pancreatic endocrine cell lineages. To define the transcription factor responsible for pancreatic cell differentiation, we employed the reverse tetracycline-dependent transactivator system in INS-I cell-derived subclones INSralphabeta and INSrbeta to achieve tightly controlled and conditional expression of wild type Pdx1 or its dominant-negative mutant, as well as brain-4. INSralphabeta cells express not only insulin but also glucagon and brain-4, while INSrbeta cells express only insulin. Overexpression of Pdx1 eliminated glucagon mRNA and protein in INSralphabeta cells and promoted the expression of beta-cell-specific genes in INSrbeta cells. Induction of dominant-negative Pdx1 in INSralphabeta cells resulted in differentiation of insulin-producing beta-cells into glucagon-containing alpha-cells without altering brain4 expression. Loss of Pdx1 function alone in INSrbeta cells, which do not express endogenous brain-4 and glucagon, was also sufficient to abolish the expression of genes restricted to beta-cells and to cause alpha-cell differentiation. In contrast, induction of brain-4 in INSrbeta cells initiated detectable expression of glucagon but did not affect beta-cell-specific gene expression. In conclusion, Pdx1 confers the expression of pancreatic beta-cell-specific genes, such as genes encoding insulin, islet amyloid polypeptide, Glut2, and Nkx6.1. Pdx1 defines pancreatic cell lineage differentiation. Loss of Pdx1 function rather than expression of brain4 is a prerequisite for alpha-cell differentiation.

Animals↗

PubMed2000

Hepatocyte nuclear factor 4alpha regulates the expression of pancreatic beta -cell genes implicated in glucose metabolism and nutrient-induced insulin secretion.

Mutations in the HNF4alpha gene are associated with the subtype 1 of maturity-onset diabetes of the young (MODY1), which is characterized by impaired insulin secretory response to glucose in pancreatic beta-cells. Hepatocyte nuclear factor 4alpha (HNF4alpha) is a transcription factor critical for liver development and hepatocyte-specific gene expression. However, the role of HNF4alpha in the regulation of pancreatic beta-cell gene expression and its correlation with metabolism secretion coupling have not been previously investigated. The tetracycline-inducible system was employed to achieve tightly controlled expression of both wild type (WT) and dominant-negative mutant (DN) of HNF4alpha in INS-1 cells. The induction of WT-HNF4alpha resulted in a left shift in glucose-stimulated insulin secretion, whereas DN-HNF4alpha selectively impaired nutrient-stimulated insulin release. Induction of DN-HNF4alpha also caused defective mitochondrial function substantiated by reduced [(14)C]pyruvate oxidation, attenuated substrate-evoked mitochondrial membrane hyperpolarization, and blunted nutrient-generated cellular ATP production. Quantitative evaluation of HNF4alpha-regulated pancreatic beta-cell gene expression revealed altered mRNA levels of insulin, glucose transporter-2, L-pyruvate kinase, aldolase B, 2-oxoglutarate dehydrogenase E1 subunit, and mitochondrial uncoupling protein-2. The patterns of HNF4alpha-regulated gene expression are strikingly similar to that of its downstream transcription factor HNF1alpha. Indeed, HNF4alpha changed the HNF1alpha mRNA levels and HNF1alpha promoter luciferase activity through altered HNF4alpha binding. These results demonstrate the importance of HNF4alpha in beta-cell metabolism-secretion coupling.

Adenosine Triphosphate↗

PubMed2000

Mitochondrial signals in glucose-stimulated insulin secretion in the beta cell.

Glucose-induced insulin secretion is determined by signals generated in the mitochondria. The elevation of ATP is necessary for the membrane-dependent increase in cytosolic Ca2+, the main trigger of insulin exocytosis. Beta cells depleted of mitochondrial DNA fail to respond to glucose while still secreting insulin in response to membrane depolarisation. This cell model resembles the situation of defective insulin secretion in patients with mitochondrial diabetes. On the other hand, infants with activating mutations in the mitochondrial enzyme glutamate dehydrogenase are characterised by hyperinsulinism and hypoglycaemia. We have recently proposed that glutamate, generated by this enzyme, participates in insulin secretion as a glucose-derived metabolic messenger. In this model, glutamate acts downstream of the mitochondria by sensitising the exocytotic process to Ca2+. The evidence in favour of such a role for glutamate is discussed in the present review.

Animals↗

PubMed2000

Mitochondrial calcium oscillations in C2C12 myotubes.

Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) was monitored in C2C12 skeletal muscle cells stably expressing the Ca(2+)-sensitive photoprotein aequorin targeted to mitochondria. In myotubes, KCl-induced depolarization caused a peak of 3.03 +/- 0.14 micrometer [Ca(2+)](m) followed by an oscillatory second phase (5.1 +/- 0.1 per min). Chelation of extracellular Ca(2+) or blockade of the voltage-operated Ca(2+) channel attenuated both phases of the KCl response. The inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, cyclopiazonic acid, reduced the amplitude of the KCl-induced [Ca(2+)](m) peak and prevented the oscillations, suggesting that these were generated intracellularly. No such [Ca(2+)](m) oscillations occurred with the nicotinic agonist carbachol, cyclopiazonic acid alone, or the purinergic agonist ATP. In contrast, caffeine produced an oscillatory behavior, indicating a role of ryanodine receptors as mediators of the oscillations. The [Ca(2+)](m) response was desensitized when cells were exposed to two consecutive challenges with KCl separated by a 5-min wash, whereas a second pulse of carbachol potentiated [Ca(2+)](m), indicating differences in intracellular Ca(2+) redistribution. Cross-desensitization between KCl and carbachol and cross-potentiation between carbachol and KCl were observed. These results suggest that close contacts between mitochondria and sarcoplasmic reticulum exist permitting Ca(2+) exchanges during KCl depolarization. These newly demonstrated dynamic changes in [Ca(2+)](m) in stimulated skeletal muscle cells might contribute to the understanding of physiological and pathological processes in muscular disorders.

Animals↗

PubMed2000

Molecular targets of a human HNF1 alpha mutation responsible for pancreatic beta-cell dysfunction.

The reverse tetracycline-dependent transactivator system was employed in insulinoma INS-1 cells to achieve controlled inducible expression of hepatocyte nuclear factor-1 alpha (HNF1 alpha)-P291fsinsC, the most common mutation associated with subtype 3 of maturity-onset diabetes of the young (MODY3). Nuclear localized HNF1 alpha-P291fsinsC protein exerts its dominant-negative effects by competing with endogenous HNF1 alpha for the cognate DNA-binding site. HNF1 alpha controls multiple genes implicated in pancreatic beta-cell function and notably in metabolism- secretion coupling. In addition to reduced expression of the genes encoding insulin, glucose transporter-2, L-pyruvate kinase, aldolase B and 3-hydroxy-3-methylglutaryl coenzyme A reductase, induction of HNF1 alpha-P291fsinsC also significantly inhibits expression of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [(14)C]pyruvate oxidation were also reduced. In contrast, the mRNA and protein levels of mitochondrial uncoupling protein-2 were dramatically increased by HNF1 alpha-P291fsinsC induction. As predicted from this altered gene expression profile, HNF1 alpha-P291fsinsC also inhibits insulin secretory responses to glucose and leucine, correlated with impaired nutrient-evoked mitochondrial ATP production and mitochondrial membrane hyperpolarization. These unprecedented results suggest the molecular mechanism of HNF1 alpha-P291fsinsC causing beta-cell dysfunction.

Adenosine Triphosphate↗

PubMed2000

Modulation of glutamate generation in mitochondria affects hormone secretion in INS-1E beta cells.

The mitochondria play a pivotal role in regulating glucose-induced insulin secretion in the pancreatic beta cell. We have recently demonstrated that glutamate derived from mitochondria participates directly in the stimulation of insulin exocytosis. In the present study, mitochondria isolated from the beta cell line INS-1E generated glutamate when incubated with the tricarboxylic acid cycle intermediate succinate. The generation of glutamate correlated with stimulated mitochondrial activity monitored as oxygen consumption and was inhibited by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Glutamate is formed by the mitochondrial enzyme glutamate dehydrogenase from alpha-ketoglutarate. Transient overexpression of glutamate dehydrogenase in INS-1E cells resulted in potentiation of glucose-stimulated hormone secretion without affecting basal release. These results further point to glutamate as an intracellular messenger playing a key role in the control of insulin exocytosis.

Animals↗

PubMed1999

Mitochondrial glutamate acts as a messenger in glucose-induced insulin exocytosis.

The hormone insulin is stored in secretory granules and released from the pancreatic beta-cells by exocytosis. In the consensus model of glucose-stimulated insulin secretion, ATP is generated by mitochondrial metabolism, promoting closure of ATP-sensitive potassium (KATP) channels, which depolarizes the plasma membrane. Subsequently, opening of voltage-sensitive Ca2+ channels increases the cytosolic Ca2+ concentration ([Ca2+]c) which constitutes the main trigger initiating insulin exocytosis. Nevertheless, the Ca2+ signal alone is not sufficient for sustained secretion. Furthermore, glucose elicits a secretory response under conditions of clamped, elevated [Ca2+]c. A mitochondrial messenger must therefore exist which is distinct from ATP. We have now identified this as glutamate. We show that glucose generates glutamate from beta-cell mitochondria. A membrane-permeant glutamate analogue sensitizes the glucose-evoked secretory response, acting downstream of mitochondrial metabolism. In permeabilized cells, under conditions of fixed [Ca2+]c, added glutamate directly stimulates insulin exocytosis, independently of mitochondrial function. Glutamate uptake by the secretory granules is likely to be involved, as inhibitors of vesicular glutamate transport suppress the glutamate-evoked exocytosis. These results demonstrate that glutamate acts as an intracellular messenger that couples glucose metabolism to insulin secretion.

Adenosine Triphosphate↗

PubMed1999

Hydrogen peroxide alters mitochondrial activation and insulin secretion in pancreatic beta cells.

The effects of a transient exposure to hydrogen peroxide (10 min at 200 microM H(2)O(2)) on pancreatic beta cell signal transduction and insulin secretion have been evaluated. In rat islets, insulin secretion evoked by glucose (16.7 mM) or by the mitochondrial substrate methyl succinate (5 mM) was markedly blunted following exposure to H(2)O(2). In contrast, the secretory response induced by plasma membrane depolarization (20 mM KCl) was not significantly affected. Similar results were obtained in insulinoma INS-1 cells using glucose (12.8 mM) as secretagogue. After H(2)O(2) treatment, glucose no longer depolarized the membrane potential (DeltaPsi) of INS-1 cells or increased cytosolic Ca(2+). Both DeltaPsi and Ca(2+) responses were still observed with 30 mM KCl despite an elevated baseline of cytosolic Ca(2+) appearing approximately 10 min after exposure to H(2)O(2). The mitochondrial DeltaPsi of INS-1 cells was depolarized by H(2)O(2) abolishing the hyperpolarizing action of glucose. These DeltaPsi changes correlated with altered mitochondrial morphology; the latter was not preserved by the overexpression of the antiapoptotic protein Bcl-2. Mitochondrial Ca(2+) was increased following exposure to H(2)O(2) up to the micromolar range. No further augmentation occurred after glucose addition, which normally raises this parameter. Nevertheless, KCl was still efficient in enhancing mitochondrial Ca(2+). Cytosolic ATP was markedly reduced by H(2)O(2) treatment, probably explaining the decreased endoplasmic reticulum Ca(2+). Taken together, these data point to the mitochondria as primary targets for H(2)O(2) damage, which will eventually interrupt the transduction of signals normally coupling glucose metabolism to insulin secretion.

Animals↗

PubMed1999

Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells. Studies in aequorin-expressing intact and permeabilized ins-1 cells.

The precise regulation of the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) is important for protein processing and signal transduction. In the pancreatic beta-cell, dysregulation of [Ca2+]er may cause impaired insulin secretion. The Ca2+-sensitive photoprotein aequorin mutated to lower its Ca2+ affinity was stably expressed in the endoplasmic reticulum (ER) of rat insulinoma INS-1 cells. The steady state [Ca2+]er was 267 +/- 9 microM. Both the Ca2+-ATPase inhibitor cyclopiazonic acid and 4-chloro-m-cresol, an activator of ryanodine receptors, caused an almost complete emptying of ER Ca2+. The inositol 1,4,5-trisphosphate generating agonists, carbachol, and ATP, reduced [Ca2+]er by 20-25%. Insulin secretagogues that raise cytosolic [Ca2+] by membrane depolarization increased [Ca2+]er in the potency order K+ >> glucose > leucine, paralleling their actions in the cytosolic compartment. Glucose, which augmented [Ca2+]er by about 25%, potentiated the Ca2+-mobilizing effect of carbachol, explaining the corresponding observation in cytosolic [Ca2+]. The filling of ER Ca2+ by glucose is not directly mediated by ATP production as shown by the continuous monitoring of cytosolic ATP in luciferase expressing cells. Both glucose and K+ increase [Ca2+]er, but only the former generated whereas the latter consumed ATP. Nonetheless, drastic lowering of cellular ATP with a mitochondrial uncoupler resulted in a marked decrease in [Ca2+]er, emphasizing the requirement for mitochondrially derived ATP above a critical threshold concentration. Using alpha-toxin permeabilized cells in the presence of ATP, glucose 6-phosphate did not change [Ca2+]er, invalidating the hypothesis that glucose acts through this metabolite. Therefore, insulin secretagogues that primarily stimulate Ca2+ influx, elevate [Ca2+]er to ensure beta-cell homeostasis.

Adenosine Triphosphate↗

PubMed1999

Free radical modulation of insulin release in INS-1 cells exposed to alloxan.

Generation of free radicals is thought to mediate the cytotoxic action of alloxan on the pancreatic beta-cell. In this investigation, the early effects of alloxan on cell function were studied. When INS-1D insulinoma cells were exposed to alloxan (1 mM) for 45 min followed by a 3-hr recovery period, the drug increased basal insulin release while abolishing the effect of glucose in static incubations. This was associated with impaired stimulation of cellular metabolism by glucose and reduced viability, both monitored colorimetrically with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). These alterations were largely counteracted by the antioxidant butylated hydroxyanisol (BHA). Similar changes occurred when glucose was added directly after 5 min of alloxan treatment, whereas KCl-induced secretion was only partially inhibited. In perifusion, alloxan caused transient insulin secretion to 50% of the rates obtained with glucose 30 min later. Under these conditions, epinephrine abolished the stimulation due to both agents. Membrane potential and cytosolic calcium concentrations ([Ca2+]i) were recorded to clarify the action of alloxan. Alloxan-induced insulin release correlated with depolarization of INS-1D cells and a rise in [Ca2+]i. Alloxan did not augment [Ca2+]i in the presence of BHA or the absence of extracellular calcium. Nickel chloride blocked the effect of alloxan on [Ca2+]i, whereas verapamil was ineffective. This suggests that alloxan promotes Ca2+ influx through channels distinct from L-type channels, perhaps through non-selective cation channels. Thus, alloxan causes changes in INS-1D cells prevented by antioxidant treatment, suggesting that free radicals may modulate the ionic permeability leading to functional activation.

Alloxan↗

PubMed1999

Functional and morphological abnormalities of mitochondria harbouring the tRNA(Leu)(UUR) mutation in mitochondrial DNA derived from patients with maternally inherited diabetes and deafness (MIDD) and progressive kidney disease.

AIMS/HYPOTHESIS: An A to G transition at nucleotide position 3243 in the mitochondrial tRNA Leu(UUR) gene has been identified in patients with maternally inherited diabetes and deafness, as well as in patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes, chronic progressive external ophpthalmoplegia, cardiomyopathy and progressive kidney disease. Variations in the mitochondrial DNA haplotype as well as differences in the degree and distribution of heteroplasmy in a certain tissue are factors that may contribute to the variety in phenotypical expression of the 3243 tRNA(Leu)(UUR) mutation. We have done morphological and functional experiments on mitochondria carrying the 3243 mutation derived from patients with either maternally inherited diabetes and deafness or progressive kidney disease to prove the pathogenicity of the 3243 mutation and to examine whether the mtDNA haplotype modulates the pathobiochemistry of this mutation. METHODS: We constructed clonal cell lines that contain predominantly mutated or exclusively wild-type mtDNA with a distinct mtDNA haplotype by the methodology of mitochondria-mediated transformation. Cells lacking mitochondrial DNA (rho(o)) were used as recipients and donor mitochondria were derived from fibroblasts of a patient with either maternally inherited diabetes and deafness or progressive kidney disease. The fibroblasts from these clinically distinct patients carry different mitochondrial DNA haplotypes with the 3243 mutation in heteroplasmic form. RESULTS: Heteroplasmy in the clonal cybrid cells ranged from 0 to 100%, reflecting the heterogeneity of the mitochondrial donor cell. Cybrid cells containing predominantly mutant mitochondrial DNA showed lactic acidosis, poor respiration and marked defects in mitochondrial morphology and respiratory chain complex I and IV activities. No differences were observed in the extent of the mitochondrial dysfunction between the mutant cells derived from the two donors. CONCLUSION/INTERPRETATION: These results provide evidence for a pathogenic effect of the tRNA(Leu)(UUR) mutation in maternally inherited diabetes and deafness and progressive kidney disease, and show no evidence of a contribution of the mitochondrial DNA haplotype as a modulating the biochemical expression of the mutation.

Cell Line↗

PubMed1999

Reactive oxygen metabolites increase mitochondrial calcium in endothelial cells: implication of the Ca2+/Na+ exchanger.

In endothelial cells, a bolus of hydrogen peroxide (H2O2) or oxygen metabolites generated by hypoxanthine-xanthine oxidase (HX-XO) increased the mitochondrial calcium concentration [Ca2+]m. Both agents caused a biphasic increase in [Ca2+]m which was preceded by a rise in cytosolic free calcium concentration [Ca2+]c (18 and 6 seconds for H2O2 and HX-XO, respectively). The peak and plateau elevations of [Ca2+] were consistently higher in the mitochondrial matrix than in the cytosol. In Ca2+-free/EGTA medium, the plateau phase of elevated [Ca2+] evoked by H2O2 due to capacitative Ca2+ influx was abolished in the cytosol, but was maintained in the mitochondria. In contrast to H2O2 and HX-XO, ATP which binds the P2Y purinoceptors induced an increase in [Ca2+]m that was similar to that of [Ca2+]c. When cells were first stimulated with inositol 1,4, 5-trisphosphate-generating agonists or the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA), subsequent addition of H2O2 did not affect [Ca2+]c, but still caused an elevation of [Ca2+]m. Moreover, the specific inhibitor of the mitochondrial Ca2+/Na+ exchanger, 7-chloro-3,5-dihydro-5-phenyl-1H-4.1-benzothiazepine-2-on (CGP37157), did not potentiate the effects of H2O2 and HX-XO on [Ca2+]m, while causing a marked increase in the peak [Ca2+]m and a significant attenuation of the rate of [Ca2+]m efflux upon addition of histamine or CPA. In permeabilized cells, H2O2 mimicked the effects of CGP37157 causing an increase in the basal level of matrix free Ca2+ and decreased efflux. Dissipation of the electrochemical proton gradient by carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), and blocade of the Ca2+ uptake by ruthenium red prevented [Ca2+]m increases evoked by H2O2. These results demonstrate that the H2O2-induced elevation in [Ca2+]m results from a transfer of Ca2+ secondary to increased [Ca2+]c, and an inhibition of the Ca2+/Na+ electroneutral exchanger of the mitochondria.

Adenosine Triphosphate↗

PubMed1998

Dominant-negative suppression of HNF-1alpha function results in defective insulin gene transcription and impaired metabolism-secretion coupling in a pancreatic beta-cell line.

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) have been linked to subtype 3 of maturity-onset diabetes of the young (MODY3), which is characterized by a primary defect in insulin secretion. The role of HNF-1alpha in the regulation of pancreatic beta-cell function was investigated. Gene manipulation allowed graded overexpression of HNF-1alpha and controlled dominant-negative suppression of HNF-1alpha function in insulinoma INS-1 cells. We show that HNF-1alpha is essential for insulin gene transcription, as demonstrated by a pronounced decrease in insulin mRNA expression and in insulin promoter activity under dominant-negative conditions. The expression of genes involved in glucose transport and metabolism including glucose transporter-2 and L-type pyruvate kinase is also regulated by HNF-1alpha. Loss of HNF-1alpha function leads to severe defects in insulin secretory responses to glucose and leucine, resulting from impaired glucose utilization and mitochondrial oxidation. The nutrient-evoked ATP production and subsequent changes in plasma membrane potential and intracellular Ca2+ were diminished by suppression of HNF-1alpha function. These results suggest that HNF-1alpha function is essential for maintaining insulin storage and nutrient-evoked release. The defective mitochondrial oxidation of metabolic substrates causes impaired insulin secretion, indicating a molecular basis for the diabetic phenotype of MODY3 patients.

Adenosine Triphosphate↗

PubMed1998

Desensitization of mitochondrial Ca2+ and insulin secretion responses in the beta cell.

The role of mitochondria in the desensitization of insulin secretion was investigated. In rat pancreatic beta cells, both insulin secretion and mitochondrial [Ca2+] increases were desensitized following two challenges with the mitochondrial substrate methyl succinate. In the beta cell line INS-1, similar results were observed when a 5-min interval separated two 5-min pulses. In contrast, ATP generation monitored in luciferase-expressing INS-1 cells was stimulated to the same extent during both exposures to methyl succinate. Succinate, like alpha-glycerophosphate, activates the electron transport chain at complex II. As a consequence, the mitochondrial membrane hyperpolarizes, promoting ATP synthesis and Ca2+ influx into the mitochondria through the uniporter. The mitochondrial desensitization was further studied in permeabilized INS-1 cells. Increasing extramitochondrial [Ca2+] from 100 to 500 nM enhanced succinate oxidation 4-fold. At 500 nM Ca2+, 1 mM succinate caused a blunted mitochondrial [Ca2+] increase upon the second, compared with the first, stimulation. These effects were mimicked by alpha-glycerophosphate, and there was cross-desensitization between the two compounds. Succinate hyperpolarized the mitochondrial membrane during both the first and second applications. This suggests that the uniporter itself, rather than the respiratory chain, is desensitized. These results emphasize the key role of the mitochondria not only in the stimulation of insulin secretion, but also in its desensitization.

Adenosine Triphosphate↗