村尾 直哉

OBJECTIVES: Phosphate (Pi) induces differentiation of arterial smooth muscle cells to the osteoblastic phenotype by inducing the type III Na-dependent Pi transporter Pit-1/solute carrier family member 1. This induction can contribute to arterial calcification, but precisely how Pi stress acts on the vascular wall remains unclear. We investigated the role of extracellular Pi in inducing microstructural changes in the arterial wall. METHODS: Aortae of Pit-1-overexpressing transgenic (TG) rats and their wild-type (WT) littermates were obtained at 8 weeks after birth. The thoracic descending aorta from WT and TG rats was used for the measurement of wall thickness and uniaxial tensile testing. Structural and ultrastructural analyses were performed using light microscopy and transmission electron microscopy. Gene expression of connective tissue components in the aorta was quantified by quantitative real-time polymerase chain reaction. RESULTS: Aortic wall thickness in TG rats was the same as that in WT rats. Uniaxial tensile testing showed that the circumferential breaking stress in TG rats was significantly lower than that in WT rats (p<0.05), although the longitudinal breaking stress, breaking strain, and elastic moduli in both directions in TG rats were unchanged. Transmission electron microscopy analysis of the aorta from TG rats showed damaged formation of elastic fibers in the aortic wall. Fibrillin-1 gene expression levels in the aorta were significantly lower in TG rats than in WT rats (p<0.05). CONCLUSIONS: Pi overload acting via the arterial wall Pit-1 transporter weakens circumferential strength by causing elastic fiber malformation, probably via decreased fibrillin-1 expression.

Hyponatremia is the most common clinical electrolyte disorder. Chronic hyponatremia has been recently reported to be associated with falls, fracture, osteoporosis, neurocognitive impairment, and mental manifestations. In the treatment of chronic hyponatremia, overly rapid correction of hyponatremia can cause osmotic demyelination syndrome (ODS), a central demyelinating disease that is also associated with neurological morbidity and mortality. Using a rat model, we have previously shown that microglia play a critical role in the pathogenesis of ODS. However, the direct effect of rapid correction of hyponatremia on microglia is unknown. Furthermore, the effect of chronic hyponatremia on microglia remains elusive. Using microglial cell lines BV-2 and 6-3, we show here that low extracellular sodium concentrations (36 mmol/L decrease; LS) suppress Nos2 mRNA expression and nitric oxide (NO) production of microglia. On rapid correction of low sodium concentrations, NO production was significantly increased in both cells, suggesting that acute correction of hyponatremia partly directly contributes to increased Nos2 mRNA expression and NO release in ODS pathophysiology. LS also suppressed expression and nuclear translocation of nuclear factor of activated T cells-5 (NFAT5), a transcription factor that regulates the expression of genes involved in osmotic stress. Furthermore, overexpression of NFAT5 significantly increased Nos2 mRNA expression and NO production in BV-2 cells. Expressions of Nos2 and Nfat5 mRNA were also modulated in microglia isolated from cerebral cortex in chronic hyponatremia model mice. These data indicate that LS modulates microglial NO production dependent on NFAT5 and suggest that microglia contribute to hyponatremia-induced neuronal dysfunctions.

Signs and symptoms of hypernatremia largely indicate central nervous system dysfunction. Acute hypernatremia can cause demyelinating lesions similar to that observed in osmotic demyelination syndrome (ODS). We have previously demonstrated that microglia accumulate in ODS lesions and minocycline protects against ODS by inhibiting microglial activation. However, the direct effect of rapid rise in the sodium concentrations on microglia is largely unknown. In addition, the effect of chronic hypernatremia on microglia also remains elusive. Here, we investigated the effects of acute (6 or 24 h) and chronic (the extracellular sodium concentration was increased gradually for at least 7 days) high sodium concentrations on microglia using the microglial cell line, BV-2. We found that both acute and chronic high sodium concentrations increase NOS2 expression and nitric oxide (NO) production. We also demonstrated that the expression of nuclear factor of activated T-cells-5 (NFAT5) is increased by high sodium concentrations. Furthermore, NFAT5 knockdown suppressed NOS2 expression and NO production. We also demonstrated that high sodium concentrations decreased intracellular Ca2+ concentration and an inhibitor of Na+/Ca2+ exchanger, NCX, suppressed a decrease in intracellular Ca2+ concentrations and NOS2 expression and NO production induced by high sodium concentrations. Furthermore, minocycline inhibited NOS2 expression and NO production induced by high sodium concentrations. These in vitro data suggest that microglial activity in response to high sodium concentrations is regulated by NFAT5 and Ca2+ efflux through NCX and is suppressed by minocycline.

(1) Background: Proglucagon-derived peptides (PDGPs) including glucagon (Gcg), GLP-1, and GLP-2 regulate lipid metabolism in the liver, adipocytes, and intestine. However, the mechanism by which PGDPs participate in alterations in lipid metabolism induced by high-fat diet (HFD) feeding has not been elucidated. (2) Methods: Mice deficient in PGDP (GCGKO) and control mice were fed HFD for 7 days and analyzed, and differences in lipid metabolism in the liver, adipose tissue, and duodenum were investigated. (3) Results: GCGKO mice under HFD showed lower expression levels of the genes involved in free fatty acid (FFA) oxidation such as Hsl, Atgl, Cpt1a, Acox1 (p < 0.05), and Pparα (p = 0.05) mRNA in the liver than in control mice, and both FFA and triglycerides content in liver and adipose tissue weight were lower in the GCGKO mice. On the other hand, phosphorylation of hormone-sensitive lipase (HSL) in white adipose tissue did not differ between the two groups. GCGKO mice under HFD exhibited lower expression levels of Pparα and Cd36 mRNA in the duodenum as well as increased fecal cholesterol contents compared to HFD-controls. (4) Conclusions: GCGKO mice fed HFD exhibit a lesser increase in hepatic FFA and triglyceride contents and adipose tissue weight, despite reduced β-oxidation in the liver, than in control mice. Thus, the absence of PGDP prevents dietary-induced fatty liver development due to decreased lipid uptake in the intestinal tract.

OBJECTIVE: Insulin secretion is regulated by ATP-sensitive potassium (KATP) channels in pancreatic beta-cells. Peroxisome proliferator-activated receptors (PPAR) α ligands are clinically used to treat dyslipidemia. A PPARα ligand, fenofibrate, and PPARγ ligands troglitazone and 15-deoxy-∆12,14-prostaglandin J2 are known to close KATP channels and induce insulin secretion. The recently developed PPARα ligand, pemafibrate, became a new entry for treating dyslipidemia. Because pemafibrate is reported to improve glucose intolerance in mice treated with a high fat diet and a novel selective PPARα modulator, it may affect KATP channels or insulin secretion. RESULTS: The effect of fenofibrate (100 µM) and pemafibrate (100 µM) on insulin secretion from MIN6 cells was measured by using batch incubation for 10 and 60 min in low (2 mM) and high (10 mM) glucose conditions. The application of fenofibrate for 10 min significantly increased insulin secretion in low glucose conditions. Pemafibrate failed to increase insulin secretion in all of the conditions experimented in this study. The KATP channel activity was measured by using whole-cell patch clamp technique. Although fenofibrate (100 µM) reduced the KATP channel current, the same concentration of pemafibrate had no effect. Both fenofibrate and pemafibrate had no effect on insulin mRNA expression.

AIMS/INTRODUCTION: Glucagon is secreted from pancreatic α-cells and plays an important role in amino acid metabolism in liver. Various animal models deficient in glucagon action show hyper-amino acidemia and α-cell hyperplasia, indicating that glucagon contributes to feedback regulation between the liver and the α-cells. In addition, both insulin and various amino acids, including branched-chain amino acids and alanine, participate in protein synthesis in skeletal muscle. However, the effect of hyperaminoacidemia on skeletal muscle has not been investigated. In the present study, we examined the effect of blockade of glucagon action on skeletal muscle using mice deficient in proglucagon-derived peptides (GCGKO mice). MATERIALS AND METHODS: Muscles isolated from GCGKO and control mice were analyzed for their morphology, gene expression and metabolites. RESULTS: GCGKO mice showed muscle fiber hypertrophy, and a decreased ratio of type IIA and an increased ratio of type IIB fibers in the tibialis anterior. The expression levels of myosin heavy chain (Myh) 7, 2, 1 and myoglobin messenger ribonucleic acid were significantly lower in GCGKO mice than those in control mice in the tibialis anterior. GCGKO mice showed a significantly higher concentration of arginine, asparagine, serine and threonine in the quadriceps femoris muscles, and also alanine, aspartic acid, cysteine, glutamine, glycine and lysine, as well as four amino acids in gastrocnemius muscles. CONCLUSIONS: These results show that hyperaminoacidemia induced by blockade of glucagon action in mice increases skeletal muscle weight and stimulates slow-to-fast transition in type II fibers of skeletal muscle, mimicking the phenotype of a high-protein diet.

AIMS/INTRODUCTION: Glutamine is the most abundant amino acid in the circulation. In this study, we investigated cell signaling in the amplification of insulin secretion by glutamine. MATERIALS AND METHODS: Clonal pancreatic β-cells MIN6-K8, wild-type B6 mouse islets, glutamate dehydrogenase (GDH) knockout clonal β-cells (Glud1KOβCL), and glutamate-oxaloacetate transaminase 1 (GOT1) knockout clonal β-cells (Got1KOβCL) were studied. Insulin secretion from these cells and islets was examined under various conditions, and intracellular glutamine metabolism was assessed by metabolic flux analysis. Intracellular Ca2+ concentration ([Ca2+ ]i ) was also measured. RESULTS: Glutamine dose-dependently amplified insulin secretion in the presence of high glucose in both MIN6-K8 cells and Glud1KOβCL. Inhibition of glutaminases, the enzymes that convert glutamine to glutamate, dramatically reduced the glutamine-amplifying effect on insulin secretion. A substantial amount of glutamate was produced from glutamine through direct conversion by glutaminases. Glutamine also increased [Ca2+ ]i at high glucose, which was abolished by inhibition of glutaminases. Glutamic acid dimethylester (dm-Glu), a membrane permeable glutamate precursor that is converted to glutamate in cells, increased [Ca2+ ]i as well as induced insulin secretion at high glucose. These effects of glutamine and dm-Glu were dependent on calcium influx. Glutamine also induced insulin secretion in clonal β-cells MIN6-m14, which otherwise exhibit no insulin secretory response to glucose. CONCLUSIONS: Glutamate converted from glutamine is an essential mediator that enhances calcium signaling in the glutamine-amplifying effect on insulin secretion. Our data also suggest that glutamine exerts a permissive effect on glucose-induced insulin secretion.

AIMS/INTRODUCTION: Pancreatic islets are heterogenous. To clarify the relationship between islet heterogeneity and incretin action in the islets, we studied gene expression and metabolic profiles of non-large and enlarged islets of the Zucker fatty diabetes mellitus rat, an obese diabetes model, as well as incretin-induced insulin secretion (IIIS) in these islets. MATERIALS AND METHODS: Pancreatic islets of control (fa/+) and fatty (fa/fa) rats at 8 and 12 weeks-of-age were isolated. The islets of fa/fa rats at 12 weeks-of-age were separated into non-large islets (≤200 μm in diameter) and enlarged islets (>300 μm in diameter). Morphological analyses, insulin secretion experiments, transcriptome analysis, metabolome analysis and oxygen consumption analysis were carried out on these islets. RESULTS: The number of enlarged islets was increased with age in fatty rats, and IIIS was significantly reduced in the enlarged islets. Markers for β-cell differentiation were markedly decreased in the enlarged islets, but those for cell proliferation were increased. Glycolysis was enhanced in the enlarged islets, whereas the tricarboxylic acid cycle was suppressed. The oxygen consumption rate under glucose stimulation was reduced in the enlarged islets. Production of glutamate, a key signal for IIIS, was decreased in the enlarged islets. CONCLUSIONS: The enlarged islets of Zucker fatty diabetes mellitus rats, which are defective for IIIS, show tumor cell-like metabolic features, including a dedifferentiated state, accelerated aerobic glycolysis and impaired mitochondrial function. The age-dependent increase in such islets could contribute to the pathophysiology of obese diabetes.

β-Cell-β-cell interactions are required for normal regulation of insulin secretion. We previously found that formation of spheroid clusters (called K20-SC) from MIN6-K20 clonal β-cells lacking incretin-induced insulin secretion (IIIS) under monolayer culture (called K20-MC) drastically induced incretin responsiveness. Here we investigated the mechanism by which an incretin-unresponsive state transforms to an incretin-responsive state using K20-SC as a model. Glutamate production by glucose through the malate-aspartate shuttle and cAMP signaling, both of which are critical for IIIS, were enhanced in K20-SC. SC formed from β-cells deficient for aspartate aminotransferase 1, a critical enzyme in the malate-aspartate shuttle, exhibited reduced IIIS. Expression of the sodium-coupled neutral amino acid transporter 5 (SNAT5), which is involved in glutamine transport, was downregulated in K20-SC and pancreatic islets of normal mice but was upregulated in K20-MC and islets of rodent models of obesity and diabetes, both of which exhibit impaired IIIS. Inhibition of SNAT5 significantly increased cellular glutamate content and improved IIIS in islets of these models and in K20-MC. These results suggest that suppression of SNAT5 activity, which results in increased glutamate production, and enhancement of cAMP signaling endows incretin-unresponsive β-cells with incretin responsiveness.

Incretins (GLP-1 and GIP) potentiate insulin secretion through cAMP signaling in pancreatic β-cells in a glucose-dependent manner. We recently proposed a mechanistic model of incretin-induced insulin secretion (IIIS) that requires two critical processes: 1) generation of cytosolic glutamate through the malate-aspartate (MA) shuttle in glucose metabolism and 2) glutamate transport into insulin granules by cAMP signaling to promote insulin granule exocytosis. To directly prove the model, we have established and characterized CRISPR/Cas9-engineered clonal mouse β-cell lines deficient for the genes critical in these two processes: aspartate aminotransferase 1 (AST1, gene symbol Got1), a key enzyme in the MA shuttle, which generates cytosolic glutamate, and the vesicular glutamate transporters (VGLUT1, VGLUT2, and VGLUT3, gene symbol Slc17a7, Slc17a6, and Slc17a8, respectively), which participate in glutamate transport into secretory vesicles. Got1 knockout (KO) β-cell lines were defective in cytosolic glutamate production from glucose and showed impaired IIIS. Unexpectedly, different from the previous finding that global Slc17a7 KO mice exhibited impaired IIIS from pancreatic islets, β-cell specific Slc17a7 KO mice showed no significant impairment in IIIS, as assessed by pancreas perfusion experiment. Single Slc17a7 KO β-cell lines also retained IIIS, probably due to compensatory upregulation of Slc17a6. Interestingly, triple KO of Slc17a7, Slc17a6, and Slc17a8 diminished IIIS, which was rescued by exogenously introduced wild-type Slc17a7 or Slc17a6 genes. The present study provides direct evidence for the essential roles of AST1 and VGLUTs in β-cell glutamate signaling for IIIS and also shows the usefulness of the CRISPR/Cas9 system for studying β-cells by simultaneous disruption of multiple genes.