A novel mechanism of idiopathic orthostatic hypotension and hypocatecholaminemia due to autoimmunity against aromatic l-Amino acid decarboxylase.

Orthostatic hypotension (OH) is a common condition. Many potential etiologies of OH have been identified, but in clinical practice the underlying cause of OH is often unknown. In the present study, we identified a novel and extraordinary etiology of OH. We describe a first case of acquired severe OH with syncope, and the female patient had extremely low levels of catecholamines and serotonin in plasma, urine and cerebrospinal fluid (CSF). Her clinical and biochemical evidence showed a deficiency of the enzyme aromatic l-amino acid decarboxylase (AADC), which converts l-DOPA to dopamine, and 5-hydroxytryptophan to serotonin, respectively. The consequence of pharmacologic stimulation of catecholaminergic nerves and radionuclide examination revealed her catecholaminergic nerves denervation. Moreover, we found that the patient's serum showed presence of autoantibodies against AADC, and that isolated peripheral blood mononuclear cells (PBMCs) from the patient showed cytokine-induced toxicity against AADC. These observations suggest that her autoimmunity against AADC is highly likely to cause toxicity to adrenal medulla and catecholaminergic nerves which contain AADC, resulting in hypocatecholaminemia and severe OH. Administration of vitamin B6, an essential cofactor of AADC, enhanced her residual AADC activity and drastically improved her symptoms. Our data thus provide a new insight into pathogenesis and pathophysiology of OH.

TDP-43 regulates early-phase insulin secretion via CaV1.2-mediated exocytosis in islets.

TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting both upper and lower motor neurons. Besides motor symptoms, patients with ALS often develop nonneuronal signs including glucose intolerance, but the underlying pathomechanism is still controversial, i.e., whether it is impaired insulin secretion and/or insulin resistance. Here, we showed that ALS subjects reduced early-phase insulin secretion and that the nuclear localization of TDP-43 was lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibited exocytosis by downregulating CaV1.2 calcium channels, thereby reducing early-phase insulin secretion in a cultured ß cell line (MIN6) and ß cell-specific Tardbp knockout mice. Overexpression of CaV1.2 restored early-phase insulin secretion in Tardbp knocked-down MIN6 cells. Our findings suggest that TDP-43 regulates cellular exocytosis mediated by L-type voltage-dependent calcium channels and thus plays an important role in the early phase of insulin secretion by pancreatic islets. Thus, nuclear loss of TDP-43 is implicated in not only the selective loss of motor neurons but also in glucose intolerance due to impaired insulin secretion at an early stage of ALS.

Glucose-dependent insulinotropic polypeptide is required for moderate high-fat diet- but not high-carbohydrate diet-induced weight gain.

Both high-fat (HFD) and high-carbohydrate (ST) diets are known to induce weight gain. Glucose-dependent insulinotropic polypeptide (GIP) is secreted mainly from intestinal K cells upon stimuli by nutrients such as fat and glucose, and it potentiates glucose-induced insulin secretion. GIP is well known to contribute to HFD-induced obesity. In this study, we analyzed the effect of ST feeding on GIP secretion and metabolic parameters to explore the role of GIP in ST-induced weight gain. Both wild-type (WT) and GIP receptor deficient ( GiprKO) mice were fed normal chow (NC), ST, or moderate (m)HFD for 22 wk. Body weight was measured, and then glucose tolerance tests were performed. Insulin secretion from isolated islets also was analyzed. WT mice fed ST or mHFD displayed weight gain concomitant with increased plasma GIP levels compared with WT mice fed NC. WT mice fed mHFD showed improved glucose tolerance due to enhanced insulin secretion during oral glucose tolerance tests compared with WT mice fed NC or ST. GiprKO mice fed mHFD did not display weight gain. On the other hand, GiprKO mice fed ST showed weight gain and did not display obvious glucose intolerance. Glucose-induced insulin secretion was enhanced during intraperitoneal glucose tolerance tests and from isolated islets in both WT and GiprKO mice fed ST compared with those fed NC. In conclusion, enhanced GIP secretion induced by mHFD-feeding contributes to increased insulin secretion and body weight gain, whereas GIP is marginally involved in weight gain induced by ST-feeding.

S100B impairs glycolysis via enhanced poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase in rodent muscle cells.

S100 calcium-binding protein B (S100B), a multifunctional macromolecule mainly expressed in nerve tissues and adipocytes, has been suggested to contribute to the pathogenesis of obesity. To clarify the role of S100B in insulin action and glucose metabolism in peripheral tissues, we investigated the effect of S100B on glycolysis in myoblast and myotube cells. Rat myoblast L6 cells were treated with recombinant mouse S100B to examine glucose consumption, lactate production, glycogen accumulation, glycolytic metabolites and enzyme activity, insulin signaling, and poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Glycolytic metabolites were investigated by enzyme assays or metabolome analysis, and insulin signaling was assessed by Western blot analysis. Enzyme activity and poly(ADP-ribosyl)ation of GAPDH was evaluated by an enzyme assay and immunoprecipitation followed by dot blot with an anti-poly(ADP-ribose) antibody, respectively. S100B significantly decreased glucose consumption, glucose analog uptake, and lactate production in L6 cells, in either the presence or absence of insulin. In contrast, S100B had no effect on glycogen accumulation and insulin signaling. Metabolome analysis revealed that S100B increased the concentration of glycolytic intermediates upstream of GAPDH. S100B impaired GAPDH activity and increased poly(ADP-ribosyl)ated GAPDH proteins. The effects of S100B on glucose metabolism were mostly canceled by a poly(ADP-ribose) polymerase inhibitor. Similar results were obtained in C2C12 myotube cells. We conclude that S100B as a humoral factor may impair glycolysis in muscle cells independent of insulin action, and the effect may be attributed to the inhibition of GAPDH activity from enhanced poly(ADP-ribosyl)ation of the enzyme.

Endogenous GIP ameliorates impairment of insulin secretion in proglucagon-deficient mice under moderate beta cell damage induced by streptozotocin.

AIMS/HYPOTHESIS: The action of incretin hormones including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) is potentiated in animal models defective in glucagon action. It has been reported that such animal models maintain normoglycaemia under streptozotocin (STZ)-induced beta cell damage. However, the role of GIP in regulation of glucose metabolism under a combination of glucagon deficiency and STZ-induced beta cell damage has not been fully explored. METHODS: In this study, we investigated glucose metabolism in mice deficient in proglucagon-derived peptides (PGDPs)-namely glucagon gene knockout (GcgKO) mice-administered with STZ. Single high-dose STZ (200 mg/kg, hSTZ) or moderate-dose STZ for five consecutive days (50 mg/kg × 5, mSTZ) was administered to GcgKO mice. The contribution of GIP to glucose metabolism in GcgKO mice was also investigated by experiments employing dipeptidyl peptidase IV (DPP4) inhibitor (DPP4i) or Gcg-Gipr double knockout (DKO) mice. RESULTS: GcgKO mice developed severe diabetes by hSTZ administration despite the absence of glucagon. Administration of mSTZ decreased pancreatic insulin content to 18.8 ± 3.4 (%) in GcgKO mice, but ad libitum-fed blood glucose levels did not significantly increase. Glucose-induced insulin secretion was marginally impaired in mSTZ-treated GcgKO mice but was abolished in mSTZ-treated DKO mice. Although GcgKO mice lack GLP-1, treatment with DPP4i potentiated glucose-induced insulin secretion and ameliorated glucose intolerance in mSTZ-treated GcgKO mice, but did not increase beta cell area or significantly reduce apoptotic cells in islets. CONCLUSIONS/INTERPRETATION: These results indicate that GIP has the potential to ameliorate glucose intolerance even under STZ-induced beta cell damage by increasing insulin secretion rather than by promoting beta cell survival.

Fructose induces glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1 and insulin secretion: Role of adenosine triphosphate-sensitive K(+) channels.

Adenosine triphosphate-sensitive K(+) (KATP) channels play an essential role in glucose-induced insulin secretion from pancreatic ß-cells. It was recently reported that the KATP channel is also found in the enteroendocrine K-cells and L-cells that secrete glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), respectively. In the present study, we investigated the involvement of the KATP channel in fructose-induced GIP, GLP-1 and insulin secretion in mice. Fructose stimulated GIP secretion, but pretreatment with diazoxide, a KATP channel activator, did not affect fructose-induced GIP secretion under streptozotocin-induced hyperglycemic conditions. Fructose significantly stimulated insulin secretion in Kir6.2 (+/+) mice, but not in mice lacking KATP channels (Kir6.2 (-/-) ), and fructose stimulated GLP-1 secretion in both Kir6.2 (+/+) mice and Kir6.2 (-/-) mice under the normoglycemic condition. In addition, diazoxide completely blocked fructose-induced insulin secretion in Kir6.2 (+/+) mice and in MIN6-K8 ß-cells. These results show that fructose-induced GIP and GLP-1 secretion is KATP channel-independent and that fructose-induced insulin secretion is KATP channel-dependent.

Effect of hyperglycemia on hepatocellular carcinoma development in diabetes.

Compared with other cancers, diabetes mellitus is more closely associated with hepatocellular carcinoma (HCC). However, whether hyperglycemia is associated with hepatic carcinogenesis remains uncertain. In this study, we investigate the effect of hyperglycemia on HCC development. Mice pretreated with 7,12-dimethylbenz (a) anthracene were divided into three feeding groups: normal diet (Control), high-starch diet (Starch), and high-fat diet (HFD) groups. In addition, an STZ group containing mice that were fed a normal diet and injected with streptozotosin to induce hyperglycemia was included. The STZ group demonstrated severe hyperglycemia, whereas the Starch group demonstrated mild hyperglycemia and insulin resistance. The HFD group demonstrated mild hyperglycemia and severe insulin resistance. Multiple HCC were macroscopically and histologically observed only in the HFD group. Hepatic steatosis was observed in the Starch and HFD groups, but levels of inflammatory cytokines, interleukin (IL)-6, tumor necrosis factor-α, and IL-1ß, were elevated only in the HFD group. The composition of gut microbiota was similar between the Control and STZ groups. A significantly higher number of Clostridium cluster XI was detected in the feces of the HFD group than that of all other groups; it was not detectable in the Starch group. These data suggested that hyperglycemia had no effect on hepatic carcinogenesis. Different incidences of HCC between the Starch and HFD groups may be attributable to degree of insulin resistance, but diet-induced changes in gut microbiota including Clostridium cluster XI may have influenced hepatic carcinogenesis. In conclusion, in addition to the normalization of blood glucose levels, diabetics may need to control insulin resistance and diet contents to prevent HCC development.

Long-term pancreatic beta cell exposure to high levels of glucose but not palmitate induces DNA methylation within the insulin gene promoter and represses transcriptional activity.

Recent studies have implicated epigenetics in the pathophysiology of diabetes. Furthermore, DNA methylation, which irreversibly deactivates gene transcription, of the insulin promoter, particularly the cAMP response element, is increased in diabetes patients. However, the underlying mechanism remains unclear. We aimed to investigate insulin promoter DNA methylation in an over-nutrition state. INS-1 cells, the rat pancreatic beta cell line, were cultured under normal-culture-glucose (11.2 mmol/l) or experimental-high-glucose (22.4 mmol/l) conditions for 14 days, with or without 0.4 mmol/l palmitate. DNA methylation of the rat insulin 1 gene (Ins1) promoter was investigated using bisulfite sequencing and pyrosequencing analysis. Experimental-high-glucose conditions significantly suppressed insulin mRNA and increased DNA methylation at all five CpG sites within the Ins1 promoter, including the cAMP response element, in a time-dependent and glucose concentration-dependent manner. DNA methylation under experimental-high-glucose conditions was unique to the Ins1 promoter; however, palmitate did not affect DNA methylation. Artificial methylation of Ins1 promoter significantly suppressed promoter-driven luciferase activity, and a DNA methylation inhibitor significantly improved insulin mRNA suppression by experimental-high-glucose conditions. Experimental-high-glucose conditions significantly increased DNA methyltransferase activity and decreased ten-eleven-translocation methylcytosine dioxygenase activity. Oxidative stress and endoplasmic reticulum stress did not affect DNA methylation of the Ins1 promoter. High glucose but not palmitate increased ectopic triacylglycerol accumulation parallel to DNA methylation. Metformin upregulated insulin gene expression and suppressed DNA methylation and ectopic triacylglycerol accumulation. Finally, DNA methylation of the Ins1 promoter increased in isolated islets from Zucker diabetic fatty rats. This study helps to clarify the effect of an over-nutrition state on DNA methylation of the Ins1 promoter in pancreatic beta cells. It provides new insights into the irreversible pathophysiology of diabetes.

KATP channel as well as SGLT1 participates in GIP secretion in the diabetic state.

Glucose-dependent insulinotropic polypeptide (GIP), a gut hormone secreted from intestinal K-cells, potentiates insulin secretion. Both K-cells and pancreatic ß-cells are glucose-responsive and equipped with a similar glucose-sensing apparatus that includes glucokinase and an ATP-sensitive K(+) (KATP) channel comprising KIR6.2 and sulfonylurea receptor 1. In absorptive epithelial cells and enteroendocrine cells, sodium glucose co-transporter 1 (SGLT1) is also known to play an important role in glucose absorption and glucose-induced incretin secretion. However, the glucose-sensing mechanism in K-cells is not fully understood. In this study, we examined the involvement of SGLT1 (SLC5A1) and the KATP channels in glucose sensing in GIP secretion in both normal and streptozotocin-induced diabetic mice. Glimepiride, a sulfonylurea, did not induce GIP secretion and pretreatment with diazoxide, a KATP channel activator, did not affect glucose-induced GIP secretion in the normal state. In mice lacking KATP channels (Kir6.2(-/-) mice), glucose-induced GIP secretion was enhanced compared with control (Kir6.2(+) (/) (+)) mice, but was completely blocked by the SGLT1 inhibitor phlorizin. In Kir6.2(-/-) mice, intestinal glucose absorption through SGLT1 was enhanced compared with that in Kir6.2(+) (/) (+) mice. On the other hand, glucose-induced GIP secretion was enhanced in the diabetic state in Kir6.2(+) (/) (+) mice. This GIP secretion was partially blocked by phlorizin, but was completely blocked by pretreatment with diazoxide in addition to phlorizin administration. These results demonstrate that glucose-induced GIP secretion depends primarily on SGLT1 in the normal state, whereas the KATP channel as well as SGLT1 is involved in GIP secretion in the diabetic state in vivo.