Preserving expression of Pdx1 improves ß-cell failure in diabetic mice.

Pdx1, a ß-cell-specific transcription factor, has been shown to play a crucial role in maintaining ß-cell function through transactivation of ß-cell-related genes. In addition, it has been reported that the expression levels of Pdx1 are compromised under diabetic conditions in human and rodent models. We therefore aimed to clarify the possible beneficial role of Pdx1 against ß-cell failure and generated the transgenic mouse that expressed Pdx1 conditionally and specifically in ß cells (ßPdx1) and crossed these mice with Ins2Akita diabetic mice. Whereas Pdx1 mRNA levels were reduced in Ins2Akita mice compared with their non-diabetic littermates, the mRNA levels of Pdx1 were significantly recovered in the islets of ßPdx1; Ins2Akita mice. The ßPdx1; Ins2Akita mice exhibited significantly improved glucose tolerance, compared with control Ins2Akita littermates, accompanied by increased insulin secretion after glucose loading. Furthermore, histological examination demonstrated that ßPdx1; Ins2Akita mice had improved localization of SLC2A2 (GLUT2), and quantitative RT-PCR showed the recovered expression of Mafa and Gck mRNAs in the islets of ßPdx1; Ins2Akita mice. These findings suggest that the sustained expression of Pdx1 improves ß-cell failure in Ins2Akita mice, at least partially through the preserving expression of ß-cell-specific genes as well as improved localization of GLUT2.

Sustained expression of GLP-1 receptor differentially modulates ß-cell functions in diabetic and nondiabetic mice.

Glucagon-like peptide 1 (GLP-1) has been shown to play important roles in maintaining ß-cell functions, such as insulin secretion and proliferation. While expression levels of GLP-1 receptor (Glp1r) are compromised in the islets of diabetic rodents, it remains unclear when and to what degree Glp1r mRNA levels are decreased during the progression of diabetes. In this study, we performed real-time PCR with the islets of db/db diabetic mice at different ages, and found that the expression levels of Glp1r were comparable to those of the islets of nondiabetic db/misty controls at the age of four weeks, and were significantly decreased at the age of eight and 12 weeks. To investigate whether restored expression of Glp1r affects the diabetic phenotypes, we generated the transgenic mouse model Pdx1(PB)-CreER(TM); CAG-CAT-Glp1r (ßGlp1r) that allows for induction of Glp1r expression specifically in ß cells. Whereas the expression of exogenous Glp1r had no measurable effect on glucose tolerance in nondiabetic ßGlp1r;db/misty mice, ßGlp1r;db/db mice exhibited higher glucose and lower insulin levels in blood on glucose challenge test than control db/db littermates. In contrast, four weeks of treatment with exendin-4 improved the glucose profiles and increased serum insulin levels in ßGlp1r;db/db mice, to significantly higher levels than those in control db/db mice. These differential effects of exogenous Glp1r in nondiabetic and diabetic mice suggest that downregulation of Glp1r might be required to slow the progression of ß-cell failure under diabetic conditions.

The usefulness of a cholesterol absorption inhibitor in Japanese type 2 diabetes patients with dyslipidemia.

AIM: Cholesterol absorption has been suggested to be an independent risk factor for cerebral and cardiovascular events. We studied the clinical efficacy of ezetimibe in Japanese patients with type 2 diabetes mellitus complicated by dyslipidemia, in whom increased cholesterol absorption had been reported. SUBJECTS AND METHODS: Ninety-six patients with type 2 diabetes complicated by dyslipidemia received ezetimibe at 10 mg/day for 12 weeks. The lipid profile, a cholesterol synthesis marker (lathosterol), and cholesterol absorption markers (cholestanol, sitosterol, and campesterol) were measured before and after the therapy to evaluate the clinical efficacy of ezetimibe. RESULTS: Serum low-density lipoprotein-cholesterol (LDL-C) levels were positively associated with cholesterol absorption markers but not associated with a cholesterol synthesis marker, suggesting that serum LDL-C levels are more strongly related to cholesterol absorption than synthesis. During the 12-week ezetimibe treatment period, cholesterol absorption markers significantly decreased, and serum lipid profiles, including LDL-C levels, significantly improved. The LDL-C-lowering rate was greater in those patients who had been receiving statin therapy and were newly started on ezetimibe additionally than in the ezetimibe monotherapy group (-31.4% vs. -18.4%; P<0.001). CONCLUSIONS: It is suggested that ezetimibe improves the lipid profile in Japanese type 2 diabetes patients with dyslipidemia through the substantial reduction of cholesterol absorption.

Short- and long-term effect of sitagliptin after near normalization of glycemic control with insulin in poorly controlled Japanese type 2 diabetic patients.

AIMS/INTRODUCTION: The aim of the present study was to examine the short- and long-term effect of sitagliptin on glucose tolerance after near normalization of glycemic control with insulin in poorly controlled type 2 diabetic patients. MATERIALS AND METHODS: We consecutively enrolled a total of 30 type 2 diabetic patients whose glycated hemoglobin levels (National Glycohemoglobin Standardization Program) were ≥7.4%, stopped all oral antidiabetic drugs and started insulin therapy. When fasting plasma glucose levels became <140 mg/dL, we carried out the first oral glucose tolerance test (OGTT). After 1-week sitagliptin treatment (50 mg/day), the second OGTT was carried out. Furthermore, we evaluated the long-term efficacy of sitagliptin on glucose tolerance after near normalization of glycemic control with insulin. RESULTS: After 1-week sitagliptin treatment, the area under the curve of insulin was markedly increased, and the area under the curve of glucagon and glucose was markedly decreased. Duration of diabetes and insulin secretory capacity were correlated with the effect of sitagliptin. Furthermore, interestingly, near normalization of glycemic control with insulin therapy for 1-2 weeks brought out the long-term effectiveness of sitagliptin on glucose tolerance for 24 weeks, which was not observed with other antidiabetic drugs. CONCLUSIONS: These findings suggest that near normalization of glycemic control with insulin improves the clinical response to sitagliptin in poorly controlled type 2 diabetic patients.

Chronological analysis with fluorescent timer reveals unique features of newly generated ß-cells.

Although numerous studies have uncovered the molecular mechanisms regulating pancreas development, it remains to be clarified how ß-cells arise from progenitors and how recently specified ß-cells are different from preexisting ß-cells. To address these questions, we developed a mouse model in which the insulin 1 promoter drives DsRed-E5 Timer fluorescence that shifts its spectrum over time. In transgenic embryos, green fluorescent ß-cells were readily detected by FACS and could be distinguished from mature ß-cells only until postnatal day 0, suggesting that ß-cell neogenesis occurs exclusively during embryogenesis. Transcriptome analysis with green fluorescent cells sorted by FACS demonstrated that newly differentiated ß-cells highly expressed progenitor markers, such as Sox9, Neurog3, and Pax4, showing the progenitor-like features of newborn ß-cells. Flow cytometric analysis of cell cycle dynamics showed that green fluorescent cells were mostly quiescent, and differentiated ß-cells were mitotically active. Thus, the precise temporal resolution of this model enables us to dissect the unique features of newly specified insulin-producing cells, which could enhance our understanding of ß-cell neogenesis for future cell therapy.

A novel function of Onecut1 protein as a negative regulator of MafA gene expression.

The transcription factor MafA is a key regulator of insulin gene expression and maturation of islet ß cells. Despite its importance, the regulatory mechanism of MafA gene expression is still unclear. To identify the transcriptional regulators of MafA, we examined various transcription factors, which are potentially involved in ß cell differentiation. An adenovirus-mediated overexpression study clearly demonstrated that Onecut1 suppresses the promoter activity of MafA through the Foxa2-binding cis-element on the MafA enhancer region (named area A). However, ChIP analysis showed that Foxa2 but not Onecut1 could directly bind to area A. Furthermore, overexpression of Onecut1 inhibited the binding of Foxa2 onto area A upon ChIP analysis. Importantly, insertion of a mutation in the Foxa2-binding site of area A significantly decreased the promoter activity of MafA. These findings suggest that Onecut1 suppresses MafA gene expression through the Foxa2-binding site. In the mouse pancreas, MafA expression was first detected at the latest stage of ß cell differentiation and was scarcely observed in Onecut1-positive cells during pancreas development. In addition, Onecut1 expression was significantly increased in the islets of diabetic db/db mice, whereas MafA expression was markedly decreased. The improved glucose levels of db/db mice with insulin injections significantly reduced Onecut1 expression and rescued the reduction of MafA expression. These in vivo experiments also suggest that Onecut1 is a negative regulator of MafA gene expression. This study implicates the novel role of Onecut1 in the control of normal ß cell differentiation and its involvement in ß cell dysfunction under diabetic conditions by suppressing MafA gene expression.

Circulating soluble RAGE as a predictive biomarker of cardiovascular event risk in patients with type 2 diabetes.

It is still controversial whether circulating soluble form of receptor for AGE (sRAGE) is associated with atherosclerosis in diabetic patients. In this study, we enrolled 276 Japanese type 2 diabetic subjects without history of cardiovascular disease (CVD), assessed their baseline clinical and biochemical data including serum sRAGE levels, and prospectively evaluated the association between these parameters and CVD events. The median follow-up period was 5.6 years and there were 25 new CVD events. The tertile analysis showed that the risk for CVD events was higher as serum sRAGE levels were increased (p for trend = 0.046). A multivariate Cox proportional hazards regression analysis revealed that serum sRAGE levels were independently associated with CVD (HR per 1SD = 1.59, 95% CI 1.04-2.45, p = 0.034), even after adjusting for conventional coronary risk factors. In summary, elevated sRAGE levels were associated with the increased risk of CVD in Japanese type 2 diabetic subjects.

Usefulness of a novel system for measuring glucose area under the curve while screening for glucose intolerance in outpatients.

AIMS/INTRODUCTION: To realize the effectiveness of a novel system for measuring glucose area under the curve (AUC) using minimally invasive interstitial fluid extraction technology (MIET), outpatients undergoing oral glucose tolerance tests (OGTT) were investigated for the efficacy of screening for glucose intolerance using this system. MATERIALS AND METHODS: Fifty outpatients scheduled to undergo a 75-g OGTT for medical reasons were recruited to the study. An area of skin on the forearm was pretreated with microneedle arrays before the application of hydrogels for interstitial fluid extraction. Plasma glucose (PG) levels were measured every 30 min for 2 h to calculate reference (actual) AUC. The AUC was predicted by MIET on the basis of glucose extracted by the hydrogel using sodium ion levels as the internal standard. RESULTS: Good correlation between MIET-predicted and reference AUCs obtained using PG levels was confirmed for a wide AUC range. By introducing a threshold level for AUC to separate glucose intolerance with peak glucose ≥180 mg/dL from normal glucose tolerance, the system was demonstrated to provide better screening accuracy compared with conventional methods that use HbA1c and fasting PG levels. The results of a questionnaire-based survey administered to the subjects suggested that this system was readily accepted by the majority as a painless monitoring method. CONCLUSIONS: The findings suggest that our glucose AUC measurement system using MIET would be useful for screening of glucose intolerance. In the future, this system may prove to be a useful aid as a screen for glucose intolerance before performing an OGTT for diagnosis.

Ultrasonic tissue characterization of carotid plaque improves the prediction of cardiovascular events in diabetic patients: a pilot study.

OBJECTIVE: The aim of this study is to evaluate whether noninvasive ultrasonic tissue characterization of carotid plaque using integrated backscatter (IBS) analysis can be a predictor of future cardiovascular events (CVE) in asymptomatic type 2 diabetic patients. RESEARCH DESIGN AND METHODS: We prospectively evaluated the association between Calibrated-IBS value, an ultrasonic marker for tissue characteristics of carotid plaque, and CVE in 85 asymptomatic type 2 diabetic patients with carotid plaque. RESULTS: The median follow-up period was 7.9 years, and there were 20 new CVE. The risk of CVE was significantly higher in the subjects with low Calibrated-IBS values (<-17.1 dB; n = 42) as compared with those with high values (≥-17.1 dB; n = 43) (P = 0.004, log-rank test). Cox proportional hazards regression analysis revealed that both Calibrated-IBS value (hazard ratio [HR] 0.802 [95% CI 0.710-0.906]; P < 0.0001) and plaque thickness (1.938 [1.170-3.213]; P = 0.010) were independently associated with CVE, even after adjustment for the 10-year risk for a general cardiovascular disease estimated by Framingham risk scoring (FRS). Time-dependent receiver operating characteristic curve analysis for CVE at 10 years after the baseline examinations revealed that area under the curve for Calibrated-IBS was 0.76 (0.60-0.90) and substantially higher than those for plaque thickness (0.60 [0.45-0.79]) and FRS (0.60 [0.40-0.78]). These analyses also revealed that the addition of both plaque thickness and Calibrated-IBS value to conventional risk factors significantly improved the event prediction. CONCLUSIONS: Calibrated-IBS value could improve the risk prediction of CVE in asymptomatic type 2 diabetic patients with carotid plaque.

Large adrenal ganglioneuroma.

We herein report the case of a 41-year-old male patient with an incidentally identified large adrenal ganglioneuroma (GN). His endocrine examinations were normal except for one episode of elevated urinary dopamine and noradrenaline levels. Abdominal computed tomography (CT) and magnetic resonance imaging (MRI) showed a large solid tumor with calcifications and a slightly lobular edge in the right adrenal gland. We performed open tumor excision and diagnosed it as adrenal ganglioneuroma. Adrenal GN is a rare benign tumor, and its hormonal activity and imaging characteristics are occasionally very similar to those of other adrenal tumors. Therefore, it needs careful evaluation by endocrine examinations and multiple imaging procedures to rule out other types of tumors.

Association of coronary artery stenosis with carotid atherosclerosis in asymptomatic type 2 diabetic patients.

AIMS: Carotid atherosclerosis assessed by ultrasound is an established surrogate marker for systemic atherosclerosis. This study aimed to evaluate the association between coronary artery stenosis assessed by coronary computed tomography angiography (CCTA) and intima-media thickness (IMT) in asymptomatic type 2 diabetic patients. METHODS: In a cross-sectional study, carotid IMT was measured by ultrasound and CCTA in 169 asymptomatic type 2 diabetic patients. RESULTS: Among 169 patients, 77 (46%) had coronary artery stenosis on CCTA. Multivariate logistic regression analysis revealed four independent predictors of coronary artery stenosis: diabetes duration (OR 4.07, 95%CI 2.34-7.12; p< 0.001), gender (OR 1.66, 95% CI 1.05-2.61; p=0.029), dyslipidemia (OR 2.07, 95%CI 1.34-3.18; p=0.001), and max-IMT (OR 2.71, 95%CI 1.70-4.33; p< 0.001). By ROC curve analyses, the area under the curve (AUC) for max-IMT was 0.73 (95%CI 0.66-0.81; p< 0.001) and mean-IMT was 0.64 (95%CI 0.56-0.73; p=0.001). The cut-off level for the greatest sensitivity and specificity for max-IMT was 1.55 mm (sensitivity 0.90, specificity 0.46), and mean-IMT was 1.05 mm (sensitivity 0.55, specificity 0.72). CONCLUSION: Carotid IMT was associated with coronary artery stenosis assessed by CCTA in asymptomatic type 2 diabetic patients. Measurement of both mean- and max-carotid IMT is useful for selecting asymptomatic type 2 diabetic patients who should undergo CCTA screening.