A large-scale functional analysis of genes expressed differentially in insulin secreting MIN6 sublines with high versus mildly reduced glucose-responsiveness.

Molecular mechanisms of glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells are not fully understood. GSIS deteriorations are believed to underlie the pathogenesis of type 2 diabetes mellitus. By comparing transcript levels of 3 insulin secreting MIN6 cell sublines with strong glucose-responsiveness and 3 with mildly reduced responsiveness, we identified 630 differentially expressed genes. Using our recently developed system based on recombinase-mediated cassette exchange, we conducted large-scale generation of stable clones overexpressing such genes in the doxycycline-regulated manner. We found that overexpressions of 18, out of 83, genes altered GSIS. Sox11 ((sex determining region Y)-box 11) was selected to confirm its roles in regulating insulin secretion, and the gene was subjected to shRNA-mediated suppression. While Sox11 overexpression decreased GSIS, its suppression increased GSIS, confirming the role of Sox11 as a negative regulator of insulin secretion. Furthermore, metabolic experiments using radiolabelled glucose showed Sox11 to participate in regulating glucose metabolism. Our data suggested that overexpression screening is a feasible option for systemic functional testing to identify important genes in GSIS.

Glucagon Response to Glucose Challenge in Patients with Idiopathic Postprandial Syndrome.

BACKGROUND: Postprandial syndrome is characterized by hunger, weakness, and anxiety neurosis after meals. Although abnormal glucagon response is a suggested mechanism, inaccuracies in conventional glucagon measurement methods have prevented precise analysis. Recently, a more reliable dual-antibody sandwich enzyme-linked immunosorbent assay for glucagon was developed. METHODS: We conducted a 75-g oral glucose tolerance test (OGTT) extending to 4 hours in 14 patients with idiopathic postprandial syndrome. In addition to blood glucose and insulin, glucagon concentration was measured with the novel method and analyzed retrospectively. RESULTS: Median (lower quartile, upper quartile) age and body-mass index were 40 years (30, 49) and 24.9 (23.1, 26.2), respectively. The OGTT revealed that one patient had a diabetic pattern, and two were glucose intolerant. Fasting insulin was 7.6 µU/mL (6.8, 8.8) and reached 73.7 µU/mL (54.3, 82.6) at 30 min. Insulin remained elevated until 180 min. Fasting glucagon was 21.1 pg/mL (16.1, 33.8), reached a nadir of 6.9 (3.5, 10.3) at 60 min, one-third the baseline level, and remained suppressed until 180 min. We observed two types of glucagon dynamics: a lower fasting glucagon with further suppression and a normal or higher fasting glucagon with a subsequent large decrease. CONCLUSIONS: These data suggest that glucagon suppression is greater in patients with idiopathic postprandial syndrome than in previously studied healthy subjects. The present data will contribute to our understanding and future research of this syndrome.

Using recombinase-mediated cassette exchange to engineer MIN6 insulin-secreting cells based on a newly identified safe harbor locus.

AIMS/INTRODUCTION: Recent studies have identified genomic and transcript level changes along with alterations in insulin secretion in patients with diabetes and in rodent models of diabetes. It is important to establish an efficient system for testing functional consequences of these changes. We aimed to generate such a system using insulin-secreting MIN6 cells. MATERIALS AND METHODS: MIN6 cells were first engineered to have a tetracycline-regulated expression system. Then, we used the recombination-mediated cassette exchange strategy to explore the silencing-resistant site in the genome and generated a master cell line based on this site. RESULTS: We identified a site 10.5 kbps upstream from the Zxdb gene as a locus that allows homogenous transgene expression from a tetracycline responsible promoter. Placing the Flip/Frt-based platform on this locus using CRISPR/Cas9 technology generated modified MIN6 cells applicable to achieving cassette exchange on the genome. Using this cell line, we generated MIN6 subclones with over- or underexpression of glucokinase. By analyzing a mixed population of these cells, we obtained an initial estimate of effects on insulin secretion within 6 weeks. Furthermore, we generated six MIN6 cell sublines simultaneously harboring genes of inducible overexpression with unknown functions in insulin secretion, and found that Cited4 and Arhgef3 overexpressions increased and decreased insulin secretion, respectively. CONCLUSIONS: We engineered MIN6 cells, which can serve as a powerful tool for testing genetic alterations associated with diabetes, and studied the molecular mechanisms of insulin secretion.

A rare case of fulminant type 1 diabetes mellitus accompanied by both acute pancreatitis and myocarditis - case report.

BACKGROUND: Fulminant type 1 diabetes mellitus (FT1D) is a newly established subtype of type 1 diabetes. Its etiology has not been fully elucidated. Several cases with FT1D have exhibited pancreatitis or myocarditis. CASE PRESENTATION: We report a 31-year-old Japanese woman who showed upper abdominal pain and was admitted to a local hospital. She was initially diagnosed with acute pancreatitis based on serum amylase elevation and swelling of the pancreas on computed tomography. Four days after admission, she developed diabetic ketoacidosis and was transferred to our hospital. Her symptoms and laboratory findings met the FT1D criteria. On the 3rd hospital day, electrocardiography (ECG) showed ST-segment elevation, and serum cardiac enzymes were markedly elevated. Because she exhibited late gadolinium enhancement in the apical wall on contrast-enhanced cardiac magnetic resonance imaging, she was diagnosed as acute myocarditis. Abnormal ECG findings and elevations of biomarkers associated with myocarditis showed improvement on the next day. CONCLUSIONS: This is the first case of FT1D accompanied by both pancreatitis and myocarditis and suggests that the pathophysiology of FT1D is related to the common etiology of acute pancreatitis and myocarditis.

Addition of sitagliptin or metformin to insulin monotherapy improves blood glucose control via different effects on insulin and glucagon secretion in hyperglycemic Japanese patients with type 2 diabetes.

This study aimed to explore the effects of the dipeptidyl peptidase-4 inhibitor sitagliptin and the biguanide metformin on the secretion of insulin and glucagon, as well as incretin levels, in Japanese subjects with type 2 diabetes mellitus poorly controlled with insulin monotherapy. This was a single-center, randomized, open-label, parallel group study, enrolling 25 subjects. Eleven patients (hemoglobin A1c [HbA1c] 8.40 ± 0.96%) and 10 patients (8.10 ± 0.54%) on insulin monotherapy completed 12-week treatment with sitagliptin (50 mg) and metformin (750 mg), respectively. Before and after treatment, each subject underwent a meal tolerance test. The plasma glucose, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), C-peptide, and glucagon responses to a meal challenge were measured. HbA1c reductions were similar in patients treated with sitagliptin (0.76 ± 0.18%) and metformin (0.77 ± 0.17%). In the sitagliptin group, glucose excursion during a meal tolerance test was reduced and accompanied by elevations in active GLP-1 and active GIP concentrations. C-peptide levels were unaltered despite reduced glucose responses, while glucagon responses were significantly suppressed (-7.93 ± 1.95% of baseline). In the metformin group, glucose excursion and incretin responses were unaltered. C-peptide levels were slightly increased but glucagon responses were unchanged. Our data indicate that sitagliptin and metformin exert different effects on islet hormone secretion in Japanese type 2 diabetic patients on insulin monotherapy. A glucagon suppressing effect of sitagliptin could be one of the factors improving blood glucose control in patients inadequately controlled with insulin therapy.