Bromocriptine as a Potential Glucose-lowering Agent for the Treatment of Prolactinoma with Type 2 Diabetes.

A 22-year-old Japanese woman consulted an endocrinologist due to persistent galactorrhea for the past 10 months. She had hyperprolacinemia and had previously been diagnosed with type 2 diabetes mellitus based on her glycohemoglobin level of 11.6%. After two months, she was admitted to our hospital and finally diagnosed with prolactinoma. For the treatment of prolactinoma, bromocriptine 2.5 mg/day was started. After seven days, her post-prandial blood glucose levels, homeostasis model assessment of insulin resistance and plasma C-peptide levels were significantly improved. These results indicate that traditional bromocriptine can be an effective therapeutic alternative in patients with prolactinoma complicated with type 2 diabetes.

A High-Fat/High-Sucrose Diet Induces WNT4 Expression in Mouse Pancreatic ß-cells.

Aims/Introduction: Several lines of evidence suggest that dysregulation of the WNT signaling pathway is involved in the pathogenesis of type 2 diabetes. This study was performed to elucidate the effects of a high-fat/high-sucrose (HF/HS) diet on pancreatic islet functions in relation to modulation of WNT ligand expression in ß-cells. MATERIALS AND METHODS: Mice were fed either standard mouse chow or a HF/HS diet from 8 weeks of age. At 20 weeks of age, intraperitoneal glucose tolerance tests were performed in both groups of mice, followed by euthanasia and isolation of pancreatic islets. WNT-related gene expression in islets and MIN6 cells was measured by quantitative real-time RT-PCR. To explore the direct effects of WNT signals on pancreatic ß-cells, MIN6 cells were exposed to recombinant mouse WNT4 protein (rmWNT4) for 48 h, and glucose-induced insulin secretion was measured. Furthermore, Wnt4 siRNAs were transfected into MIN6 cells, and cell viability and insulin secretion were measured in control and Wnt4 siRNA-transfected MIN6 cells. RESULTS: Mice fed the HF/HS diet were heavier and their plasma glucose and insulin levels were higher compared with mice fed standard chow. Wnt4, Wnt5b, Ror1, and Ror2 expression was upregulated, while Fzd4, Fzd5, Fzd6, Lrp5, and Lrp6 expression was downregulated in the islets of mice fed the HF/HS diet. Wnt4 was the most abundantly expressed WNT ligand in ß-cells, and its expression was increased by the HF/HS diet. Although exposure to recombinant mouse WNT4 protein for 48 h did not alter glucose-induced insulin secretion, it was significantly reduced by knockdown of Wnt4 in MIN6 cells. CONCLUSIONS: We demonstrated that the HF/HS diet-induced increase of WNT4 signaling in ß-cells is involved in augmentation of glucose-induced insulin secretion and impaired ß-cell proliferation. These results strongly indicate an essential role of WNT4 in the regulation of ß-cell functions in mouse pancreatic islets.

Protective effect of 3-hydroxybutyrate against endoplasmic reticulum stress-associated vascular endothelial cell damage induced by low glucose exposure.

AIMS/HYPOTHESIS: The aim of this study was to elucidate the mechanism by which severe hypoglycemia accelerates vascular complications. Furthermore, we assessed the possible protective effect of ketone bodies against the endothelial cell damage caused by glucose deficiency. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured at a glucose level of either 0.56 or 5.6 mmol/L with or without 3-hydroxybutyrate (3-HB) supplementation. Cell viability was assessed with a CCK-8 assay and a lactate dehydrogenase (LDH) release assay. The activity of caspases was measured using fluorogenic substrates. The expression of genes associated with endothelial cell function and endoplasmic reticulum (ER) stress was evaluated by real-time quantitative PCR. Protein levels of ER stress-related molecules were assessed by Western blotting. RESULTS: Culture of HUVECs in low-glucose medium for 24 or 48 h resulted in reduction of cell viability accompanied by activation of caspase-3/7 and caspase-8. The addition of a pan caspase inhibitor attenuated the cell death. After incubation in the low-glucose medium, we found reduced mRNA and protein levels of endothelial nitric oxide synthase. ER stress responses mediated by phosphorylation of protein kinase RNA-like ER kinase (PERK) and cleavage of activating transcription factor 6 (ATF6) were augmented, but X-box binding protein 1 (Xbp1) splicing was reduced. Most of these responses to glucose deficiency were significantly attenuated by supplementation with 3-HB. CONCLUSIONS/INTERPRETATION: These observations showed that exposure to low glucose induces ER stress, caspase activation, endothelial cell dysfunction and cell death. The beneficial effects of 3-HB shown in this study suggest that hypoketonemic severe hypoglycemia induced by insulin injections or insulin secretagogue administration may be more harmful than hyperketonemic severe hypoglycemia.

Pivotal Role of TNF-α in the Development and Progression of Nonalcoholic Fatty Liver Disease in a Murine Model.

Previously, we have shown that the adipocyte-specific nuclear form of sterol regulatory element-binding protein-1c (nSREBP-1c) transgenic mice spontaneously developed hepatic lesions that are similar to those of human nonalcoholic steatohepatitis (NASH) with a concomitant elevation of plasma TNF-α. In this study, we analyzed the role of TNF-α in the progression of nonalcoholic fatty liver disease (NAFLD). We established a Tnf knockout nSREBP-1c transgenic mouse line. Glucose tolerance and liver histology were examined at the age of 20 weeks. The gene expression and protein levels were assessed by quantitative RT-PCR and Western blot, respectively. The Tnf knockout improved glucose tolerance and significantly reduced the prevalence of hepatic steatosis (20% vs. 100%, p<0.0001) and fibrosis (15% vs. 65%, p=0.0057). The expressions of Acaca, Scd1, Mcp1, Tgfb1, Col1a1, and Timp1 were increased in the liver from the original nSREBP-1c transgenic mice. However, gene upregulation was reduced in the livers from the Tnf(-/-) nSREBP-1c transgenic mice. Furthermore, the hepatic levels of TIMP1 protein were increased in the original nSREBP-1c transgenic mice but not in Tnf(-/-) nSREBP-1c transgenic mice. To assess the direct effect of TNF-α on the expression of the genes, we cultured primary hepatocytes in the presence of TNF-α and found that TNF-α increased the expression of Mcp1, Tgfb1, and Timp1 in hepatocytes. These observations indicate that TNF-α plays a pivotal role in the development of NAFLD and progression to NASH through upregulating key molecules associated with lipid metabolism, inflammatory cytokines, and fibrosis in the liver.

Hyperoxia reduces insulin release and induces mitochondrial dysfunction with possible implications for hyperoxic treatment of neonates.

We previously showed that hyperoxia in vitro negatively affects beta cells of the rat. Here, we tested for possible clinical significance as well as mitochondrial interactions by hyperoxia, using human islets (function and viability), INS-1 832/13 cells (mitochondrial metabolism), and mouse neonates (effects in vivo). Lastly, we assessed relevant parameters in a cohort of individuals born preterm and then exposed to hyperoxia. Human islets and INS-1 832/13 cells were exposed to 24 h of hyperoxia (90-92% oxygen). Mouse neonates were subjected to 5 days of continuous hyperoxia. Individuals born preterm were evaluated in terms of glucose homeostasis and beta cell function by HbA1c and the HOMA2 formula. In human islets, hyperoxia significantly reduced glucose-stimulated insulin secretion by 42.2 ± 5.3% and viability assessed by MTT by 22.5 ± 5.4%. Hyperoxia down-regulated mitochondrial complex II by 21 ± 5% and upregulated complex III by 26 ± 10.1% and complex IV by 37 ± 10.6%. Partly similar effects on mitochondrial complexes were found in hyperoxia-exposed INS-1 832/13 cells. Exposure to hyperoxia swiftly reduced oxygen consumption in these cells and increased mitochondrial uncoupling. Hyperoxia transiently but significantly reduced insulin release in mouse neonates. Individuals born preterm displayed higher HbA1c versus controls, as well as insulin resistance. Thus, hyperoxia exerts negative effects in vitro on human beta cells and results indicate inhibitory effects on insulin secretion in vivo in mouse neonates. Negative effects may be lessened by the demonstrated swift and profound mitochondrial adaptability. Our findings open the possibility that hyperoxia could negatively affect beta cells of preterm human neonates.

Haemoglobin variants may cause significant differences in haemoglobin A1c as measured by high-performance liquid chromatography and enzymatic methods in diabetic patients: a cross-sectional study.

Background We aimed to determine whether the discrepancy between haemoglobin A1c values determined by high-performance liquid chromatography and enzymatic haemoglobin A1c measurements in diabetic patients was clinically relevant. Methods We randomly recruited 1421 outpatients undergoing diabetic treatment and follow-up who underwent at least three haemoglobin A1c measurements between April 2014 and March 2015 at our clinic. In 6369 samples, haemoglobin A1c was simultaneously measured by HA-8160 and MetaboLead (enzymatic assay), and the values were compared. Results haemoglobin A1c measurements by high-performance liquid chromatography and enzymatic assay were strongly correlated (correlation coefficient: 0.9828, linear approximation curve y = 0.9986x - 0.2507). Mean haemoglobin A1c (6.8 ± 1.0%) measured by high-performance liquid chromatography was significantly higher than that measured by enzymatic assay (6.5 ± 1.0%, P < 0.0001). During the sample processing, four (0.3%) subjects presented consistently lower haemoglobin A1c values (<0.7%) by high-performance liquid chromatography than those from enzymatic assay. Of these, three had Hb Toranomon [ß112 (G14) Cys→Trp]. The fourth had Hb Ube-2 [α68 (E17) Asn→Asp]. One other subject presented consistently higher haemoglobin A1c values (>1%) by high-performance liquid chromatography than those from enzymatic assay and was diagnosed with a -77 (T > C) mutation in the δ-globin gene. These unrelated asymptomatic subjects had normal erythrocyte profiles, without anaemia. Conclusions We showed that haemoglobin A1c values measured by high-performance liquid chromatography were significantly higher than those measured by enzymatic assay in diabetic subjects. However, when an oversized deviation (>0.7%) between glycaemic control status and haemoglobin A1c is apparent, clinicians should check the methods used to measure haemoglobin A1c and consider the possible presence of a haemoglobin variant.

Cross-sectional and Longitudinal Analyses of Factors Contributing to the Progressive Loss of the ß-cell Function in Type 2 Diabetes.

OBJECTIVE: Type 2 diabetes is a progressive disease characterized by insulin resistance and insulin secretory dysfunction. In this study, we assessed the factors contributing to an insulin secretory defect in type 2 diabetes patients. METHODS: The subjects consisted of 382 patients with type 2 diabetes, aged 57±13 years. We estimated the ß-cell function using 6-min post-glucagon increments in C-peptide (ΔCPR). RESULTS: A significant inverse correlation was observed between the time since the diagnosis of diabetes and ΔCPR. A simple liner regression analysis showed that ΔCPR decreases at a rate of 0.056 ng/mL/year. According to a multiple regression model, body mass index (BMI) and log (triglyceride) were positively correlated with ΔCPR. Time since the diagnosis of diabetes, diabetes in 1st degree relatives, the presence of diabetic retinopathy, and HbA1c were inversely correlated with ΔCPR. In 50 patients who underwent the glucagon stimulation test twice, the ΔCPR decreased from 2.27±1.47 to 1.72±1.08 ng/mL over a period of 6.5±0.9 years. A multiple regression analysis revealed the BMI and fasting plasma glucose level to be significant contributing factors to the decline in ΔCPR. CONCLUSION: The duration of diabetes, a low BMI, genetic factors, and the presence of microangiopathy may be associated with ß-cell dysfunction in diabetic patients. The observations in this study suggest that obese subjects showed a rapid decline in the ß-cell function despite an initial high CPR response. Environmental factors causing insulin resistance and glucotoxicity may therefore be involved in progressive ß-cell failure.

Age-related changes in the diurnal variation of ketogenesis in patients with type 2 diabetes and relevance to hypoglycemic medications.

To assess the significance of ketogenesis in the management of diabetes mellitus, we analyzed the factors associated with the diurnal variation of the plasma ketone body levels. The subjects consisted of 220 patients with type 2 diabetes, aged 60 ± 15 years, without advanced complications. They ate a standardized, low-fat meal at 8:00, 12:00, and 18:00. The plasma levels of 3-hydroxybutyrate (3HB) and free fatty acid (FFA) were increased before breakfast and before dinner. The plasma glucose concentration was almost the same at any blood sampling time point among age quartiles. However, the 3HB levels were significantly decreased with age, which was most obvious before dinner. The FFA levels also decreased with age, but the decline was mild. A multiple regression analysis with stepwise selection revealed that age was an independent, negative contributor and that the pre-breakfast FFA concentration was an independent, positive contributor to the pre-breakfast 3HB levels. Regarding the pre-dinner 3HB levels, in addition to age and the pre-dinner FFA concentration, the uses of sulfonylurea and dipeptidyl peptidase-4 inhibitors were independent negative contributors. The metabolism of ketone bodies is an alternative energy source for the brain under conditions of starvation. While excessive ketogenesis leads to critical ketoacidosis, inadequate ketone body production could be associated with a propensity to develop neurohypoglycemia in elderly patients treated with insulin secretagogues. Because age-related changes in ketogenesis were the most significant before dinner, attention should be paid not only to fasting but also to the pre-dinner levels of 3HB.

Low serum level of high-sensitivity C-reactive protein in a Japanese patient with maturity-onset diabetes of the young type 3 (MODY3).

High-sensitivity C-reactive protein (hs-CRP) levels in European populations are lower in patients with maturity-onset diabetes of the young type 3 (MODY3) than in those with type 2 diabetes. hs-CRP levels have been suggested to be useful for discriminating MODY3 from type 2 diabetes. As hs-CRP levels are influenced by various factors including race and body mass index, it is worthwhile to examine whether hs-CRP can serve as a biomarker for MODY3 in Japanese. Here we describe the case of a Japanese MODY3 patient with a nonsense mutation in the HNF1A gene. Two measurements showed consistently lower hs-CRP levels (<0.05 and 0.09 mg/L) than in Japanese patients with type 1 and type 2 diabetes. Hepatic expression of Crp messenger ribonucleic acid was significantly decreased in Hnf1a knockout mice. The hs-CRP level might be a useful biomarker for MODY3 in both Japanese and European populations.

Prevalence and clinical characteristics of unremembered nocturnal eating in diabetic subjects: Kurume sleep trouble in obesity and metabolic disorders (KUSTOMED) study.

Nighttime food intake is associated with weight gain and higher HbA1c levels. We experienced night eaters who have no memory of their nocturnal eating in the morning. In this study, the curious night eating behavior was designated as "unremembered nocturnal eating syndrome (UNES)". We screened 1,169 patients with diabetes for sleep quality and abnormal eating behavior at night using the Pittsburgh Sleep Quality Index questionnaire with an additional question regarding UNES. When abnormal nocturnal eating behavior was noted, detailed clinical information was extracted from interviews with the patients. We identified 9 patients who experienced UNES. They had a higher BMI compared with subjects who reported no such episodes. Among them, 6 patients who consumed food at night without memory 2-5 times per month or more had significantly higher HbA1c levels. Continuous glucose monitoring in a patient with type 1 diabetes revealed an abrupt elevation of glucose levels from midnight when some foods were consumed. Eight of the 9 patients were taking benzodiazepine and/or non-benzodiazepine hypnotic agents when they experienced the episodes. The prevalence of UNES was 0.8% in all subjects and 4% in those taking hypnotic drugs. The ratio of hypnotic drug use in subjects with UNES was significantly higher than for individuals without UNES (89% vs. 17%, p<0.0001). Although UNES seems to be etiologically heterogeneous, hypnotics-induced parasomnia and/or anterograde amnesia may be associated with the behavior. UNES is not rare in diabetic patients on hypnotic medicine and may be a hidden cause of unexpected morning hyperglycemia.

Modulation by adiponectin of circadian clock rhythmicity in model mice for metabolic syndrome.

To assess the effect of adiponectin on the circadian rhythm disturbances associated with metabolic syndrome, we generated a KK/Ta mouse line expressing the human adiponectin transgene in the liver. Locomotor activity of control C57BL/6 mice was highest during the beginning of the dark period and low during the light period. Under constant darkness, the length of locomotor activity rhythm of control mice was slightly shorter than 24 h. In KK/Ta mice the peak of locomotor activity was blunted and significant activity was observed during the light period. Furthermore, KK/Ta mice showed shorter average period length of free-running locomotor activity rhythm when compared with control mice. However, the transgenic expression of adiponectin in the liver significantly altered the circadian rhythm of locomotor activity and the length of free-running rhythm of KK/Ta mice towards those of C57BL/6 mice. In the liver and skeletal muscles from control mice, mRNA levels of Arntl and Cry1 were increased during the dark period, whereas those of Dbp, Cry2, Per1 and Per2 were elevated during the light period. KK/Ta mice exhibited phase advances in circadian rhythms of Arntl, Dbp, Cry2 and Per2 in both tissues. The phase shifts of the circadian clock gene expression in the liver were attenuated in adiponectin-transgenic mice. These results suggest that adiponectin is a peripheral regulator of the circadian clocks in the brain and peripheral organs, and may be a novel target for the treatment of obesity-associated disorders of circadian rhythms.

Identification of hepatocyte growth factor activator (Hgfac) gene as a target of HNF1α in mouse ß-cells.

HNF1α is a transcription factor that is expressed in pancreatic ß-cells and mutations of the HNF1α gene cause a form of monogenic diabetes. To understand the role of HNF1α in pancreatic ß-cells, we established the MIN6 ß-cell line that stably expressed HNF1α-specific shRNA. Expression of the gene encoding hepatocyte growth factor (HGF) activator (Hgfac), a serine protease that efficiently activates HGF, was decreased in HNF1α KD-MIN6 cells. Down-regulation of Hgfac expression was also found in the islets of HNF1α (+/-) mice. Reporter gene analysis and the chromatin immunoprecipitation assay indicated that HNF1α directly regulates the expression of Hgfac in ß-cells. It has been reported that HGF has an important influence on ß-cell mass and ß-cell function. Thus, HNF1α might regulate ß-cell mass or function at least partly by modulating Hgfac expression.

Long-term efficacy of sitagliptin for the treatment of type 2 diabetic patients in Japan.

We studied the efficacy of sitagliptin in type 2 diabetic patients of our outpatient clinics. Since December in 2009, 164 patients have been treated by sitagliptin for their management of diabetes. HbA1c decreased by 0.8% in all patients without any change in mean body weight after 3 months. However, actually HbA1c did not decrease in 30 patients, and more than half of patients showed weight gain to some extent. Patients were classified according to the reduction of HbA1c and analyzed based on this category. Baseline characters such as age, gender, duration of diabetes, BMI, concomitantly used oral hypoglycemic agents and the score for life-style assessment were not related to glucose-lowering effect of sitagliptin. Ninety eight patients whose HbA1c had decreased after 3 months were further followed-up for another 3 months. Among them 45 patients showed some relapsing of HbA1c after 6 months, and they were compared with 53 patients without relapsing. More cases had been switched from α-glucosidase inhibitor (α-GI) and the score for life-style assessment was lower in relapsing patients compared to those in patients without relapsing. In conclusion, clinicians should keep the fact in mind that the individual variation of glucose-lowering effects and the possibility of relapsing exist during sitagliptin treatment, and that concern about life-style is still a quite important issue to prevent weight gain and the relapsing of blood glucose control.