A TCR-like antibody against a proinsulin-containing fusion peptide ameliorates type 1 diabetes in NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by destruction of insulin-producing ß cells. The response of autoreactive T cells to ß cell antigens plays a central role in the development of T1D. Recently, fusion peptides composed by insulin C-peptide fragments and other proteins were reported as ß cell target antigens for diabetogenic CD4+ T cells in non-obese diabetic (NOD) mice. In this study, we generated a T cell-receptor (TCR)-like monoclonal antibody (mAb) against a fusion peptide bound to major histocompatibility complex (MHC) class II component to elucidate the function of the fusion peptides in T1D. In addition, we developed a novel NFAT-GFP TCR reporter system to evaluate the TCR-like mAb. The NFAT-GFP reporter T cells expressing the diabetogenic TCR were specifically activated by the fusion peptide presented on the MHC class II molecules. By using the NFAT-GFP reporter T cells, we showed that the TCR-like mAb blocks the diabetogenic T cell response against the fusion peptide presented on the MHC class II molecules. Furthermore, the development of T1D was ameliorated when pre-diabetic NOD mice were treated with this mAb. These findings suggest that NFAT-GFP reporter T cells are useful to assess the function of specific TCR and the recognition of fusion peptides by T cells is crucial for the pathogenesis of T1D.

Modeling Congenital Hyperinsulinism with ABCC8-Deficient Human Embryonic Stem Cells Generated by CRISPR/Cas9.

Congenital hyperinsulinism (CHI) is a rare genetic disorder characterized by excess insulin secretion, which results in hypoglycemia. Mutation of sulfonylurea receptor 1 (SUR1), encoded by the ABCC8 gene, is the main cause of CHI. Here, we captured the phenotype of excess insulin secretion through pancreatic differentiation of ABCC8-deficient stem cells generated by the CRISPR/Cas9 system. ABCC8-deficient insulin-producing cells secreted higher insulin than their wild-type counterparts, and the excess insulin secretion was rescued by nifedipine, octreotide and nicorandil. Further, we tested the role of SUR1 in response to different potassium levels and found that dysfunction of SUR1 decreased the insulin secretion rate in low and high potassium environments. Hence, pancreatic differentiation of ABCC8-deficient cells recapitulated the CHI disease phenotype in vitro, which represents an attractive model to further elucidate the function of SUR1 and to develop and screen for novel therapeutic drugs.

Gallic acid ameliorates hyperglycemia and improves hepatic carbohydrate metabolism in rats fed a high-fructose diet.

Herein, we investigated the hypoglycemic effect of plant gallic acid (GA) on glucose uptake in an insulin-resistant cell culture model and on hepatic carbohydrate metabolism in rats with a high-fructose diet (HFD)-induced diabetes. Our hypothesis is that GA ameliorates hyperglycemia via alleviating hepatic insulin resistance by suppressing hepatic inflammation and improves abnormal hepatic carbohydrate metabolism by suppressing hepatic gluconeogenesis and enhancing the hepatic glycogenesis and glycolysis pathways in HFD-induced diabetic rats. Gallic acid increased glucose uptake activity by 19.2% at a concentration of 6.25 µg/mL in insulin-resistant FL83B mouse hepatocytes. In HFD-induced diabetic rats, GA significantly alleviated hyperglycemia, reduced the values of the area under the curve for glucose in an oral glucose tolerance test, and reduced the scores of the homeostasis model assessment of insulin resistance index. The levels of serum C-peptide and fructosamine and cardiovascular risk index scores were also significantly decreased in HFD rats treated with GA. Moreover, GA up-regulated the expression of hepatic insulin signal transduction-related proteins, including insulin receptor, insulin receptor substrate 1, phosphatidylinositol-3 kinase, Akt/protein kinase B, and glucose transporter 2, in HFD rats. Gallic acid also down-regulated the expression of hepatic gluconeogenesis-related proteins, such as fructose-1,6-bisphosphatase, and up-regulated expression of hepatic glycogen synthase and glycolysis-related proteins, including hexokinase, phosphofructokinase, and aldolase, in HFD rats. Our findings indicate that GA has potential as a health food ingredient to prevent diabetes mellitus.

Inhibition of cortisol biosynthesis decreases circulating leptin levels in obese humans.

CONTEXT: Glucocorticoids increase both appetite and leptin secretion; the hyperleptinemic effect might be a counterregulatory response to the orexigenic effect of glucocorticoids. However, the effect of glucocorticoid inhibition on leptin production has not been reported. OBJECTIVE: We tested the hypothesis that if glucocorticoid-induced hyperleptinemia plays a physiological role, then inhibition of endogenous cortisol biosynthesis should decrease leptin secretion. DESIGN: A randomized, placebo-controlled, cross-over study design was used. SETTING: The study was carried out at a General Clinical Research Center. PARTICIPANTS: Eight obese subjects (four men, four women; mean age, 30.4 +/- 1.56 yr; mean body mass index, 42.0 +/- 1.33 kg/m2) participated in the study. INTERVENTION: The subjects were treated with metyrapone (750 mg every 4 h) or placebo for 24 h during two overnight admissions, 2 wk apart. Blood sampling for measurement of cortisol, leptin glucose, insulin, and C-peptide was performed hourly for 6 h and every 2 h for 24 h. MAIN OUTCOME MEASURE: The change in plasma leptin from baseline during metyrapone vs. placebo treatment was measured. RESULTS: Metyrapone treatment was associated with a significant decrease in plasma cortisol level; the cortisol nadir was 4.84 +/- 1.22 microg/dl during placebo and 2.80 +/- 0.65 microg/dl during metyrapone treatment (P = 0.009). Compared with placebo, metyrapone treatment was associated with a significant reduction in circulating leptin levels and marked attenuation of the nocturnal rise in plasma leptin (+28.45 +/- 11.12% vs. +55.51 +/- 5.42%; P = 0.01). CONCLUSIONS: We conclude that metyrapone-induced inhibition of cortisol biosynthesis results in hypoleptinemia, which indicates that glucocorticoids may play an important role in the physiological regulation of leptin.

A hyperinsulinaemic, sequentially eu- and hypoglycaemic clamp test to characterize autonomous insulin secretion in patients with insulinoma.

To better characterize autonomous insulin secretory behaviour in insulinoma patients and to establish diagnostic criteria with high accuracy, hyper-insulinaemic, sequentially eu- and hypoglycaemic clamp tests were performed in insulinoma patients and control subjects. Ten patients with insulinoma (benign in nine, histologically proven in nine) and 10 patients with suspected episodes of hypoglycaemia, in whom thorough clinical evaluation excluded an insulinoma, were examined. Five insulinoma patients were restudied after successful extirpation of the tumour. Suppression of C-peptide during low-dose [2 pmol kg-1 min-1 (20 mU kg-1 h-1) for 90 min, plasma insulin approximately 120 pmol L-1 (20 mUL-1)] and high-dose [8 pmol kg-1 h-1 (80 mU kg-1 h-1) for 90 min, plasma insulin approximately 450 pmol L-1 (75 mU L-1)] insulin infusion under euglycaemic conditions [plasma glucose 4.4-5.0 mmol L-1 (80-90 mg dL-1)] and during high-dose insulin infusion under hypoglycaemic conditions [glucose 2-2.2 mmol L-1 (40-45 mg dL-1)] was evaluated by radioimmunoassay (RIA). Euglycaemic hyper-insulinaemia suppressed C-peptide in control subjects (P < 0.0001), whereas in insulinoma patients apparently irregular changes in C-peptide concentrations (with spontaneous or paradoxical increments, P = 0.0006 vs. controls) were observed. The combination of hyper-insulinaemia and controlled hypoglycaemia led to a nearly complete suppression of C-peptide in normal subjects (from basal, 0.76 +/- 0.08-0.06 +/- 0.01 nmol L-1; maximum observed value 0.10 nmol L-1), which was more pronounced than at the point of discontinuation of prolonged fasting (> 48 h; 0.26 +/- 0.16 nmol L-1; P = 0.005). In insulinoma patients, C-peptide remained elevated under all conditions (P = 0.51 vs. prolonged fasting). All these findings were reversible after successful surgical removal of the insulinoma. Insulinoma patients could be identified as abnormal by (a) non-suppression of C-peptide even under hyperinsulinaemic/hypoglycaemic conditions (10 out of 10 patients) and (b) irregular increments in C-peptide under conditions that led to at least partial suppression in all normal subjects (9 out of 10 patients) and/or by an apparent shift to the left of insulin secretion relative to glucose concentrations (7 out of 10 patients). Controlled exposure to hyperinsulinaemic/hypoglycaemic conditions can help to characterize autonomous secretion in insulinoma patients and may be used as a diagnostic procedure when conventional methods yield equivocal results.