. Tacrolimus suppresses glucose-induced insulin release from pancreatic islets by reducing glucokinase activity. Am J Physiol Endocrinol Metab 288: E365-E371, 2005. First published October 12, 2004; doi:10.1152/ ajpendo.00390.2004.-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 Ϯ 0.08 vs. tacrolimus 1.75 Ϯ 0.02 ng ⅐ islet Ϫ1 ⅐ 30 min Ϫ1 , P Ͻ 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 Ϯ 0.99 vs. tacrolimus 6.52 Ϯ 0.40 pmol/islet, P Ͻ 0.01; glucose utilization: control 103.8 Ϯ 6.9 vs. tacrolimus 74.4 Ϯ 5.1 pmol ⅐ islet Ϫ1 ⅐ 90 min Ϫ1 , P Ͻ 0.01). However, insulin release from tacrolimus-treated islets was similar to that from control islets in the presence of 16.7 mM ␣-ketoisocaproate, a mitochondrial fuel. Glucokinase activity, which determines glycolytic velocity, was reduced by tacrolimus treatment (control 65.3 Ϯ 3.4 vs. tacrolimus 49.9 Ϯ 2.8 pmol ⅐ islet Ϫ1 ⅐ 60 min Ϫ1 , P Ͻ 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.islet; adenosine 5Ј-triphosphate TACROLIMUS (FK-506) IS AN IMMUNOSUPPRESSANT widely used in human organ transplantation. Immunosuppression by the agent is due to blocking of antigen-stimulated expression of genes, including interleukin-2 in T lymphocytes, which is required for T-cell proliferation (34). Interleukin-2 gene transcription is activated by dephosphorylation and nuclear translocation of a transcriptional cofactor, the nuclear factor of activated T cells (NFAT). Tacrolimus binds specific intracellular proteins, FK-506-binding proteins (FKBPs), and inhibits calcineurin (protein phosphatase-2B), a Ca 2ϩ

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.

Aims/hypothesis Na + /K + -ATPase inhibition by ouabain suppresses ATP production by generating reactive oxygen species (ROS) and impairs glucose-induced insulin secretion from pancreatic islets. To clarify the signal-transducing function of Na + /K + -ATPase in decreasing ATP production by the generation of ROS in pancreatic islets, the involvement of Src was examined. In addition, the significance of Src activation in diabetic islets was examined. Methods Isolated islets from Wistar rats and diabetic GotoKakizaki (GK) rats (a model for diabetes) were used. ROS was measured by 5-(and 6)-chloromethyl-2′,7′-dichlorofluorescein fluorescence using dispersed islet cells. After lysates were immunoprecipitated by anti-Src antibody, immunoblotting was performed. Results Ouabain caused a rapid Tyr 418 phosphorylation, indicating activation of Src in the presence of high glucose. The specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) restored the ouabain-induced decrease in ATP content and the increase in ROS production. Both PP2 and ROS scavenger restored the impaired insulin release and impaired ATP elevation in GK islets, but had no such effect in control islets. PP2 reduced the high glucose-induced increase in ROS generation in GK islet cells but had no effect on that in control islet cells. Moreover, ouabain had no effect on ATP content and ROS production in the presence of high glucose in GK islets. Conclusions/interpretation These results indicate that Src plays a role in the signal-transducing function of Na + /K + -ATPase, in which ROS generation decreases ATP production in control islets. Moreover, ROS generated by Src activation plays an important role in impaired glucoseinduced insulin secretion in GK islets, in which Src is endogenously activated independently of ouabain.

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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.