Impaired metabolism-secretion coupling in pancreatic beta-cells: role of determinants of mitochondrial ATP production.

Glucose-induced insulin secretion from beta-cells is often impaired in diabetic condition and by exposure to diabetogenic pharmacological agents. In pancreatic beta-cells, intracellular glucose metabolism regulates exocytosis of insulin granules, according to metabolism-secretion coupling in which glucose-induced mitochondrial ATP production plays an essential role. Impaired glucose-induced insulin secretion often results from impaired glucose-induced ATP elevation in beta-cells. Mitochondrial ATP production is driven by the proton-motive force including mitochondrial membrane potential (DeltaPsi(m)) generated by the electron transport chain. These electrons are derived from reducing equivalents, generated in the Krebs cycle and transferred from cytosol by the shuttles. Here, roles of the determinants of mitochondrial ATP production in impaired glucose-induced insulin secretion are discussed. Cytosolic alkalization, H(+) leak in the inner membrane by uncoupler (e.g. free fatty acid exposure), decrease in the supply of electron donors including NADH and FADH(2) to the respiratory chain, and endogenous mitochondrial ROS (e.g. Na(+)/K(+)-ATPase inhibition) all reduce hyperpolarlization of DeltaPsi(m) and ATP production, causing decresed glucose-induced insulin release. The decrease in the supply of NADH and FADH(2) to the respiratory chain derives from impairments in glucose metabolism including glycolysis (e.g. MODY2 and exposure to NO) and the shuttles (e.g. diabetic state and exposure to ketone body).

Diphenylhydantoin suppresses glucose-induced insulin release by decreasing cytoplasmic H+ concentration in pancreatic islets.

Diphenylhydantoin (DPH), which is clinically used in the treatment of epilepsy, inhibits glucose-induced insulin release from pancreatic islets by a mechanism that remains unknown. In the present study, DPH is shown to suppress glucose-induced insulin release concentration-dependently. In dynamic experiments, 20 microm DPH suppressed 16.7 mm glucose-induced biphasic insulin release. DPH also suppressed insulin release in the presence of 16.7 mm glucose, 200 microm diazoxide, and 30 mm K+ without affecting the intracellular Ca2+ concentration. DPH suppressed ATP content and mitochondrial membrane hyperpolarization in the presence of 16.7 mm glucose without affecting glucose utilization, glucose oxidation, and reduced nicotinamide adenine dinucleotide phosphate fluorescence. DPH increased cytoplasmic pH in the presence of high glucose, but the increase was abolished under Na+ -deprived conditions and HCO3- -deprived conditions, suggesting that Na+ and HCO3- transport across the plasma membrane are involved in the increase in cytoplasmic pH by DPH. Alkalization by adding NH4+ to the extracellular medium also suppressed insulin release, ATP content, and mitochondrial membrane hyperpolarization. Because ATP production from the mitochondrial fraction in the presence of substrates was decreased by increased pH in the medium, DPH suppresses mitochondrial ATP production by reducing the H+ gradient across mitochondrial membrane. Using permeabilized islets, the increase in pH was shown to decrease Ca2+ efficacy at a clamped concentration of ATP in the exocytotic system. Taken together, DPH inhibits glucose-induced insulin secretion not only by inhibiting mitochondrial ATP production, but also by reducing Ca2+ efficacy in the exocytotic system through its alkalizing effect on cytoplasm.

A Novel homozygous missense mutation of melanocortin-4 receptor (MC4R) in a Japanese woman with severe obesity.

The melanocortin-4 receptor (MC4R) is a member of the seven membrane-spanning G protein-coupled receptor superfamily and signals through the activation of adenylyl cyclase. The MC4R mutations are the most common known monogenic cause of human obesity. However, no such mutations have been found in Japanese obese subjects. Here we report a novel homozygous missense mutation of MC4R (G98R) in a nondiabetic Japanese woman with severe early-onset obesity, which is located in its second transmembrane domain. Her birth weight was 3,360 g, and she gained weight progressively from 10 months of age. At 40 years of age, her weight reached 160 kg and a BMI of 62 kg/m(2). Her parents, who are heterozygous for the mutation, have BMIs of 26 and 27 kg/m(2). In vitro transient transfection assays revealed no discernable agonist ligand binding and cAMP production in HEK293 cells expressing the mutant receptor, indicating a severe loss-of-function mutation. This study represents the first demonstration of a pathogenic mutation of MC4R in Japan and will provide further insight into the pathophysiologic role of the hypothalamic melanocortin system in human obesity.

ATP enhances exocytosis of insulin secretory granules in pancreatic islets under Ca2+-depleted condition.

Glucose and other nutrients have been shown to stimulate insulin release from pancreatic islets under Ca2+-depleted condition when protein kinase A (PKA) and protein kinase C (PKC) are activated simultaneously. We investigated the role of metabolic nucleotide signals including ATP, ADP, and GTP in exocytosis of insulin secretory granules under Ca2+-depleted condition using electrically permeabilized rat islets. ATP under PKC activation augmented insulin release concentration-dependently by 100 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in Ca2+-depleted condition, while ADP could not suppress ATP-dependent insulin release in this condition. Neither GTP nor activated PKA in the absence of PKC activation increased insulin release under Ca2+-depleted condition in the presence of ATP, but both enhanced insulin secretion in the presence of ATP when PKC was activated. In conclusion, activated PKC and the presence of ATP both are required in the insulin secretory process under Ca2+-depleted condition. While PKA activation and GTP cannot substitute for PKC activation and ATP, respectively, under Ca2+-depleted condition, they enhance ATP-dependent insulin secretion when PKC is activated.

Missense mutation of TRPS1 in a family of tricho-rhino-phalangeal syndrome type III.

We report a new Japanese family with tricho-rhino-phalangeal syndrome type III (TRPS III) who have a missense mutation (Arg908Gln) of theTRPS1 gene (TRPS1) in affected individuals of the family. This study supports the notion that TRPS III results from missense mutations in exon 6 of TRPS1.

Case of type 1 diabetes mellitus following interferon ß-1a treatment for multiple sclerosis.

A 57-year-old woman who had been treated with interferon ß-1a (IFNß-1a) for multiple sclerosis was diagnosed with diabetic ketosis. Her fasting serum C-peptide (F-CPR) was 1.9 ng/mL and her daily urinary C-peptide (U-CPR) was 24.1 µg/day. Her anti-glutamic acid decarboxylase (GAD) antibody was 3.5 U/mL. Seven months later, she was hospitalized with body weight loss and a high level of hemoglobin A1c [11.1% (JDS)]. Her F-CPR and U-CPR were very low (0.1 ng/mL and 8.35 µg/day, respectively), and anti-GAD antibody became distinctly positive (12.4 U/mL). She had HLA-DRB1*04:05, A24, and B54. For these reasons, IFNß-1a administration was considered a possible cause of type 1 diabetes mellitus in this case.

Rapamycin impairs metabolism-secretion coupling in rat pancreatic islets by suppressing carbohydrate metabolism.

Rapamycin, an immunosuppressant used in human transplantation, impairs beta-cell function, but the mechanism is unclear. Chronic (24 h) exposure to rapamycin concentration dependently suppressed 16.7 mM glucose-induced insulin release from islets (1.65+/-0.06, 30 nM rapamycin versus 2.35+/-0.11 ng/islet per 30 min, control, n=30, P<0.01) without affecting insulin and DNA contents. Rapamycin also decreased alpha-ketoisocaproate-induced insulin release, suggesting reduced mitochondrial carbohydrate metabolism. ATP content in the presence of 16.7 mM glucose was significantly reduced in rapamycin-treated islets (13.42+/-0.47, rapamycin versus 16.04+/-0.46 pmol/islet, control, n=30, P<0.01). Glucose oxidation, which indicates the velocity of metabolism in the Krebs cycle, was decreased by rapamycin in the presence of 16.7 mM glucose (30.1+/-2.7, rapamycin versus 42.2+/-3.3 pmol/islet per 90 min, control, n=9, P<0.01). Immunoblotting revealed that the expression of complex I, III, IV, and V was not affected by rapamycin. Mitochondrial ATP production indicated that the respiratory chain downstream of complex II was not affected, but that carbohydrate metabolism in the Krebs cycle was reduced by rapamycin. Analysis of enzymes in the Krebs cycle revealed that activity of alpha-ketoglutarate dehydrogenase (KGDH), which catalyzes one of the slowest reactions in the Krebs cycle, was reduced by rapamycin (10.08+/-0.82, rapamycin versus 13.82+/-0.84 nmol/mg mitochondrial protein per min, control, n=5, P<0.01). Considered together, these findings indicate that rapamycin suppresses high glucose-induced insulin secretion from pancreatic islets by reducing mitochondrial ATP production through suppression of carbohydrate metabolism in the Krebs cycle, together with reduced KGDH activity.

Chronic exposure to beta-hydroxybutyrate inhibits glucose-induced insulin release from pancreatic islets by decreasing NADH contents.

To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca2+ and metabolism, and Ca2+ efficacy of the exocytotic system in rat pancreatic islets. Fifteen-hour exposure to 5 mM d-beta-hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca2+-independent and ATP-independent component of insulin release, which was suppressed by the GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca2+ concentration ([Ca2+](i)) was not affected 10 min after exposure to high glucose. Additionally, [Ca2+](i) efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP-to-ADP ratio in the HB-cultured islets in the presence of high glucose were lower, whereas glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB, and that the decrease in ATP production is due to decreased NADH content in the mitochondrial matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.

Tacrolimus suppresses glucose-induced insulin release from pancreatic islets by reducing glucokinase activity.

Tacrolimus is widely used for immunosuppressant therapy, including various organ transplantations. One of its main side effects is hyperglycemia due to reduced insulin secretion, but the mechanism remains unknown. We have investigated the metabolic effects of tacrolimus on insulin secretion at a concentration that does not influence insulin content. Twenty-four-hour exposure to 3 nM tacrolimus reduced high glucose (16.7 mM)-induced insulin secretion (control 2.14 +/- 0.08 vs. tacrolimus 1.75 +/- 0.02 ng.islet(-1).30 min(-1), P < 0.01) without affecting insulin content. In dynamic experiments, insulin secretion and NAD(P)H fluorescence during a 20-min period after 10 min of high-glucose exposure were reduced in tacrolimus-treated islets. ATP content and glucose utilization of tacrolimus-treated islets in the presence of 16.7 mM glucose were less than in control (ATP content: control 9.69 +/- 0.99 vs. tacrolimus 6.52 +/- 0.40 pmol/islet, P < 0.01; glucose utilization: control 103.8 +/- 6.9 vs. tacrolimus 74.4 +/- 5.1 pmol.islet(-1).90 min(-1), P < 0.01). However, insulin release from tacrolimus-treated islets was similar to that from control islets in the presence of 16.7 mM alpha-ketoisocaproate, a mitochondrial fuel. Glucokinase activity, which determines glycolytic velocity, was reduced by tacrolimus treatment (control 65.3 +/- 3.4 vs. tacrolimus 49.9 +/- 2.8 pmol.islet(-1).60 min(-1), P < 0.01), whereas hexokinase activity was not affected. These results indicate that glucose-stimulated insulin release is decreased by chronic exposure to tacrolimus due to reduced ATP production and glycolysis derived from reduced glucokinase activity.

A case of TSH receptor antibody-positive hyperthyroidism with functioning metastases of thyroid carcinoma.

The presence of TSH receptor antibody (TRAb) is rarely responsible for hyperthyroidism due to metastatic lesions of thyroid carcinoma. A 70-year-old woman was incidentally found to be thyrotoxic around the time that external irradiation was performed for multiple bone metastases 9 years after subtotal thyroidectomy for follicular carcinoma. Hyperthyroidism persisted after oral administration of thiamazole. Relevant laboratory data were as follows: FT4 9.6 ng/L, FT3 7.3 ng/L, TSH <0.19 mU/L, TBII 70, TSAb 735, and Tg 32,000 microg/L. 131I-total body scan showed 131I accumulation in the occipital bone, cervical vertebra, thoracic vertebra, ilium, and residual thyroid gland. Since the ilium uptake (11.6) was markedly higher compared to the residual thyroid gland uptake (0.14), four subsequent 131I therapies were performed. The patient became hypothyroid, and TBII became negative. TSAb became negative after the first 131I-therapy but has increased again to 204 at present. Tg was 1,962 microg/L despite high TSH levels. 131I accumulation in the residual thyroid, cervical vertebra, and thoracic vertebra disappeared. Also 131I accumulation in the ilium has gradually decreased, but the image in the occipital bone has become markedly distinctive. This is a rare case characterized by TRAb-positive hyperthyroidism, by T3-predominant thyrotoxicosis, and by stronger accumulation of 131I in the metastatic tumor than in the residual thyroid gland. Thus, the response to TRAb and 131I-therapy is different among metastatic thyroid tissues.