Independent risk factors of rapid glomerular filtration rate decline in patients with type 2 diabetes with preserved kidney function and normoalbuminuria: A multicenter cohort study.

AIMS/INTRODUCTION: Research on the incidence and underlying mechanisms of rapid renal function decline in patients with type 2 diabetes mellitus with preserved renal function and normoalbuminuria is limited. This study aimed to investigate the involvement of hemoglobin level as a risk factor for rapid decliners among patients with type 2 diabetes with preserved renal function and normoalbuminuria. MATERIALS AND METHODS: This was a retrospective observational study of 242 patients with type 2 diabetes with a baseline estimated glomerular filtration rate of ≥60 mL/min/1.73 m2 and normoalbuminuria (<30 mg/gCr), followed up for >1 year. The annual rate of estimated glomerular filtration rate decline during the follow-up period was calculated using least square regression analysis; rapid decliners defined at ≥3.3%/year. Risk factors associated with rapid decliners were identified using a logistic regression analysis of variables previously identified as risk factors of rapid decliners. RESULTS: The median follow-up period was 6.7 years, and 34 patients showed rapid decliners. On multivariate analysis, lower baseline hemoglobin level was a risk factor of rapid decliners (odds ratio 0.69, 95% confidence interval 0.47-0.99; P = 0.045). Furthermore, the baseline hemoglobin levels were correlated positively with iron and ferritin levels, implying that an impaired iron metabolism might cause lower hemoglobin levels in rapid decliners. CONCLUSIONS: In patients with type 2 diabetes with preserved renal function and normoalbuminuria, lower hemoglobin levels were a risk factor for rapid decliners, where disturbed iron metabolism might precede the development of diabetic kidney disease.

[A case of recurrent Clostridium difficile infection with type 2 diabetes mellitus indicating the usefulness of metformin hydrochloride].

A 76-year-old woman undergoing insulin treatment for type 2 diabetes mellitus at our hospital was diagnosed with Clostridium difficile infection (CDI). She was treated with metronidazole, vancomycin hydrochloride, and fidaxomicin. Metformin hydrochloride in combination with fidaxomicin was administered to control the exacerbated symptoms. The diarrhea disappeared two days later, and colonoscopy confirmed the resolution of pseudomembranes eight days later. Till approximately two years after discharge, no recurrence was observed. Here metformin hydrochloride was suggested to be useful for the treatment of recurrent CDI with type 2 diabetes mellitus.

Urinary afamin levels are associated with the progression of diabetic nephropathy.

AIMS: In this study, we applied quantitative proteomic analysis to identify urinary proteins associated with diabetic nephropathy (DN). METHODS: Two-dimensional image-converted analysis of liquid chromatography and mass spectrometry detected the proteins differentially excreted between normoalbuminuric and macroalbuminuric patients with type 2 diabetes mellitus (T2DM) (n = 6 each). Urinary levels of excreted proteins were measured by multiple reaction monitoring (MRM) analysis using an independent sample set (n = 77). Urinary afamin levels were measured by ELISA in T2DM and DN patients enrolled in this cohort study (n = 203). RESULTS: One-hundred-four proteins displayed significant alterations in excretion. Nine of these candidates were validated by MRM analysis. Among them, the levels of afamin, CD44 antigen, and lysosome-associated membrane glycoprotein 2, which have not previously been implicated in DN, were significantly associated with both the urinary albumin to creatinine ratio (ACR) and eGFR. We further measured afamin levels in urine collected from T2DM patients who did not yet have significant kidney disease (ACR < 300 mg/g or eGFR change rate ≤ 3.3%/year). The urinary afamin to creatinine ratio (Afa/Cre) was significantly higher in patients who progressed to a more severe DN stage or had early renal decline than in patients who did not. CONCLUSIONS: Afa/Cre was significantly increased in T2DM patients who subsequently developed DN. Afa/Cre may be useful to predict patients with T2DM at high risk of nephropathy before the development of macroalbuminuria or reduced kidney function, although further validation studies in a larger population are needed.

Changes in Antidiabetic Drug Prescription and Glycemic Control Trends in Elderly Patients with Type 2 Diabetes Mellitus from 2005-2013: An Analysis of the National Center Diabetes Database (NCDD-03).

Objective To analyze the changes in the pharmacotherapy and glycemic control trends in elderly patients with type 2 diabetes mellitus (T2DM) in Japan. Methods We extracted the data of 7,590 patients (5,396 men and 2,194 women; median year of birth: 1945) with T2DM registered in the National Center Diabetes Database for the years 2005 to 2013, and conducted age-stratified (<65, 65-74, and ≥75 years of age) analyses. Results The hemoglobin A1c (HbA1c) levels declined from 2005 to 2013, and for those who received antihyperglycemic drug prescription, the HbA1c levels were lower in the older age group than in the younger age group. In the ≥75 age group, dipeptidyl peptidase-4 inhibitors (DPP4i) became the most frequently prescribed drug (49.1%) in 2013, and sulfonylureas remained the second-most frequently prescribed drug (37.8%) with decreased prescribed doses. The prescription ratio of oral drugs associated with a risk of hypoglycemia was higher in patients ≥75 years of age than in those <75 years of age (40.5% and 26.4%, respectively in 2013), although it showed a downward trend. The prescription rates of insulin for patients ≥75 years of age increased during the study period. Conclusion The pharmacotherapy trends for elderly patients with T2DM changed dramatically in Japan with the launch of DPP4i in 2009. Glycemic control in a considerable portion of the ≥75 age group in Japan was maintained at the expense of potential hypoglycemia by the frequent, although cautious, use of sulfonylureas, glinides and insulin.

Clinical significance of serum complement factor 3 in patients with type 2 diabetes mellitus.

AIMS: Although serum complement factor 3 (C3) is an acute phase reactant mainly synthesized in the liver, several recent studies have shown high C3 gene expression in adipose tissue (AT). However, the relationship between C3 and AT levels has not been fully clarified in type 2 diabetes mellitus (T2DM) patients. METHODS: A total of 164 T2DM patients (109men and 55 women) participated in this cross-sectional study. A computed tomography scan was performed to measure visceral, subcutaneous, and total AT. The correlation between these factors and C3 levels was examined using Pearson's correlation analysis. A multivariate regression model was used to assess an independent determinant associated with C3 levels after adjusting the explanatory variables (i.e., all ATs [visceral, subcutaneous, and total], and clinical features [sex, age, body mass index, waist circumference, glycated hemoglobin, duration of diabetes, systolic blood pressure, diastolic blood pressure, aspartate aminotransferase levels, alanine aminotransferase levels, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, log(triglyceride levels), estimated glomerular filtration rate, and log(high-sensitivity C-reactive protein levels)]). RESULTS: Serum C3 levels were correlated with visceral, subcutaneous, and total AT among both men (r=0.505, p<0.001; r=0.545, p<0.001; r=0.617, p<0.001, respectively) and women (r=0.396, p=0.003; r=0.517, p<0.001; r=0.548, p<0.001, respectively). In the multivariate regression model, the association between total AT and C3 levels remained significantly positive (ß=0.490, p<0.001). CONCLUSIONS: Serum C3 levels are associated with visceral, subcutaneous, and total AT in T2DM patients. Furthermore, C3 levels seem to be a marker for overall adiposity rather than regional adiposity.

A positive family history of hypertension might be associated with an accelerated onset of type 2 diabetes: Results from the National Center Diabetes Database (NCDD-02).

Type 2 diabetes, which is characterized by a combination of decreased insulin secretion and decreased insulin sensitivity, can be delayed or prevented by healthy lifestyle behaviors. Therefore, it is important that the population in general understands their personal risk at an early age to reduce their chances of ever developing the disease. A family history of hypertension is known to be associated with insulin resistance, but the effect of a family history of hypertension on the onset of type 2 diabetes has not well been examined. We performed a retrospective study examining patient age at the time of the diagnosis of type 2 diabetes by analyzing a dataset of 1,299 patients (1,021 men and 278 women) who had been diagnosed as having type 2 diabetes during a health checkup. The mean ± standard deviation of the patient age at the time of the diagnosis of diabetes was 49.1 ± 10.4 years for patients with a family history of hypertension and 51.8 ± 11.4 years for patients without a family history of hypertension (p < 0.001). A multivariate linear regression analysis showed a significant association between a family history of hypertension and a younger age at the time of the diagnosis of type 2 diabetes, independent of a family history of diabetes mellitus and a male sex, suggesting that a positive family history of hypertension might be associated with the accelerated onset of type 2 diabetes.

Proteomic analysis of serum biomarkers for prediabetes using the Long-Evans Agouti rat, a spontaneous animal model of type 2 diabetes mellitus.

AIMS/INTRODUCTION: To identify candidate serum molecules associated with the progression of type 2 diabetes mellitus, differential serum proteomic analysis was carried out on a spontaneous animal model of type 2 diabetes mellitus without obesity, the Long-Evans Agouti (LEA) rat. MATERIALS AND METHODS: We carried out quantitative proteomic analysis using serum samples from 8- and 16-week-old LEA and control Brown Norway (BN) rats (n = 4/group). Differentially expressed proteins were validated by multiple reaction monitoring analysis using the sera collected from 8-, 16-, and 24-week-old LEA (n = 4/each group) and BN rats (n = 5/each group). Among the validated proteins, we also examined the possible relevance of the human homolog of serine protease inhibitor A3 (SERPINA3) to type 2 diabetes mellitus. RESULTS: The use of 2-D fluorescence difference gel electrophoresis analysis and the following liquid chromatography-multiple reaction monitoring analysis showed that the serum levels of five proteins were differentially changed between LEA rats and BN rats at all three time-points examined. Among the five proteins, SERPINA3N was increased significantly in the sera of LEA rats compared with age-matched BN rats. The serum level of SERPINA3 was also found to be significantly higher in type 2 diabetes mellitus patients than in healthy control participants. Furthermore, glycated hemoglobin, fasting insulin and estimated glomerular filtration rate were independently associated with the SERPINA3 levels. CONCLUSIONS: These findings suggest a possible role for SERPINA3 in the development of the early stages of type 2 diabetes mellitus, although further replication studies and functional investigations regarding their role are required.

Efficacy and safety of ipragliflozin in Japanese patients with type 2 diabetes receiving conventional therapy: clinical implication of the importance of exercise habits during treatment with ipragliflozin.

AIMS: To evaluate the efficacy and safety of ipragliflozin in Japanese patients with type 2 diabetes mellitus and inadequate glycemic control and investigate the impact of maintaining exercise habits during treatment. MATERIALS AND METHODS: A total of 20 patients were enrolled. Patients aged 20-70 years with type 2 diabetes mellitus were administered 50 mg of ipragliflozin once daily for 12 weeks. The primary endpoint was the change in glycated hemoglobin levels from baseline to week 12. Key secondary endpoints included changes in total body weight, body composition, serum lipid levels, and diabetes therapy-related quality of life from baseline to week 12. Adverse events were recorded throughout the study. RESULTS: The patients' glycated hemoglobin levels were 0.69% lower at week 12 versus baseline (adjusted mean difference from baseline; P < 0.01, n = 18). Mean total body weight, body composition, and serum lipid levels also improved significantly from baseline. Of note, stratification analysis by physical activity level revealed slight but significant reductions in skeletal muscle mass and muscle mass, but not body fat mass, in the minimal exercise group compared to the data for the moderate exercise group. One of the subdomain structures in diabetes therapy-related quality of life questionnaire, "Anxiety and dissatisfaction with treatment," was significantly improved. Although no major hypoglycemic episodes occurred, two adverse events were reported. CONCLUSIONS: Daily treatment with ipragliflozin was associated with marked improvements in glycemic control and body composition without major side effects, and this improvement was affected by exercise habits. This study was registered with UMIN CTR (no. UMIN000014388).

Reduced serum level of leukocyte cell-derived chemotaxin 2 is associated with the presence of diabetic retinopathy.

BACKGROUND: Vascular endothelial growth factor (VEGF) signaling is an important pathway in the development of diabetic retinopathy (DR). A recent report showed that leukocyte cell-derived chemotaxin 2 (LECT2) suppresses the VEGF signaling in endothelial cells. However, the clinical relevance of LECT2 in DR is unknown. This study aimed to investigate serum LECT2 levels and the presence of DR. METHODS: The study included 230 people with type 2 diabetes mellitus (DM), 95 with DR and 135 without DR. Serum LECT2 levels were measured using an enzyme-linked immunosorbent assay. Data were evaluated using Spearman's rank correlation, univariate and multivariate logistic regression. RESULTS: Serum LECT2 levels were significantly lower in participants with DM having DR than in those not having DR (35.6±14.9ng/ml vs. 44.5±17.6ng/ml, P<0.001). Spearman's rank correlation analysis revealed a significant association between serum LECT2 levels and the presence of DR (P<0.001). Multiple regression analysis revealed that serum LECT2 levels were independently related to DR (P<0.001). CONCLUSIONS: These findings indicated that serum LECT2 level is negatively associated with the presence of DR and suggest that low circulating LECT2 level is a risk factor for DR.

Quantitative assessment of visual estimation of the infrared indocyanine green imaging of lymph nodes retrieved at sentinel node navigation surgery for gastric cancer.

BACKGROUND: Although the infrared indocyanine green (ICG) imaging is an effective method to identify sentinel lymph nodes (SLNs) of gastric cancer, its objectivity has not been verified. METHODS: We studied 563 lymph nodes under infrared light observation from the ICG-positive lymphatic basins of 36 patients who underwent SLN-navigated gastrectomy for clinically node-negative gastric cancer. First, the rate of SLN detection, the number of SLNs and sensitivities were compared between ordinary light observation and infrared light observation. Second, 563 lymph nodes were grouped into ICG-positive and -negative under infrared light observation. The intensities of the region of interest for each lymph node defined as the lymph node on which digital imaging was performed using an imaging-software, and the region of reference defined as its surrounding background, were compared and quantified. RESULTS: In the comparison of ordinary light observation with infrared light observation, the SLN identification rates were 28/36 (78 %) vs. 36/36 (100 %), the mean ± SD (minimum to maximum) number of SLNs was 3.4 ± 3.7 (0-16) vs. 9.2 ± 5.9 (2-25), and the sensitivities were 1/5 (20 %) vs. 5/5 (100 %). The ICG-positive group contained 358 lymph nodes with an intensity of 0.323 ± 1.56 (mean ± SD), and the ICG-negative group contained 205 lymph nodes with an intensity of 0.639 ± 1.93 (mean ± SD), demonstrating a significant difference between these two groups (P < 0.0001). CONCLUSIONS: The significant difference in the intensity as measured by an imaging-software between ICG-positive and ICG-negative lymph nodes would erase the concern about the objectivity of the infrared ICG method for SLN-navigated surgery for early gastric cancer.

Charting weight four times daily as an effective behavioural approach to obesity in patients with type 2 diabetes.

Although many obese people successfully lose weight by dieting and/or behavioural therapy, most of them subsequently regain the lost weight. Thus, new therapeutic strategies are required to maintain weight loss. We report a woman with type 2 diabetes and moderate obesity who succeeded in achieving good glycaemic control and long-term weight loss with weaning from insulin therapy, while charting her weight four times daily. This charting method might be useful for long-term maintenance of weight reduction in obese diabetic patients. Obese patients can monitor their irregular weight fluctuations produced by overeating and correct both their food intake and their lifestyle. Further studies, including randomized control trials, will be needed to confirm the efficacy of this approach in patients with type 2 diabetes.

Proliferative diabetic retinopathy is a predictor of coronary artery disease in Japanese patients with type 2 diabetes.

A retrospective cohort study was performed to investigate the relationship between diabetic retinopathy and coronary artery disease in 371 Japanese adult patients with type 2 diabetes. We found that proliferative retinopathy was significantly associated with an increased risk of coronary artery disease, even after adjustment for classical coronary risk factors.

Establishment and characterization of HER2-positive cell line derived from pleural effusion of human breast scirrhous carcinoma.

We have established a cell line derived from a pleural effusion of breast scirrhous carcinoma. This cell line presented immunohistochemically negative for estrogen receptor and slightly positive for progesterone receptor, and positive for c-erbB/HER2/neu. However the original tumor was found to be positive for estrogen receptor and progesterone receptor and slightly positive for c-erbB/HER2/neu expression. Enzyme and electrochemiluminescence immunoassays of tumor markers in the conditioned medium by the cell line revealed they secrete CA15-3, NCC-ST-439 and HER2 protein. We believe the cell line will contribute to the therapeutic study of malignant breast cancer.

Establishment and characterization of a cell line (IGSK-3) secreting human chorionic gonadotropin, adrenocorticotropic hormone and parathyroid hormone-related protein derived from primary poorly differentiated adenocarcinoma of the stomach.

We recently established human chorionic gonadotropin-, adrenocorticotropic hormone- and parathyroid hormone-related protein-secreting cell line derived from primary poorly differentiated adenocarcinoma of the stomach. The cell line was designated as IGSK-3. Inverted-phase contrast microscopy revealed that the IGSK-3 cells consist of two morphological subtypes. One type has visible nucleoli and clear nuclei, but nucleoli and nuclear membrane of the other type are invisible. The population-doubling time was about 43 h. An analysis of conditioned medium by IGSK-3 cells cultured for 4 days revealed the IGSK-3 cells secrete human chorionic gonadotropin-beta (0.5 ng/mL), adrenocorticotropic hormone (5.5 pg/mL), parathyroid hormone-related protein (3.4 pmol/mL) and epidermal growth factor (14.2 pg/mL). Histopathological diagnosis of the graft of IGSK-3 cells revealed that IGSK-3 cells built a poorly differentiated adenocarcinoma which resembled the original tumor. In addition, the IGSK-3 cell line was immunocytochemically positive for human chorionic gonadotropin-beta and epidermal growth factor receptor, and negative for vascular endothelial growth factor.

Establishment and characterization of a cell line (BTIC) including HER-2-positive cells derived from pleural effusion of recurrent breast invasive ductal carcinoma, scirrhous type.

Human epidermal growth factor receptor-2 (HER-2) overexpression in breast cancer occurs in 20% to 30% of patients with breast cancer. Trastuzumab (Herceptin) targets HER-2 tyrosine kinase receptors expressed on tumor cells and mediates anti-proliferative effects against HER-2-positive tumor cells. Adjuvant chemotherapy with trastuzumab has improved the prognosis of patients with HER-2 positive high-grade breast cancer. However, patients often experience appearance and proliferation of recurrent tumor cells after trastuzumab treatment. In this study, we report the successful establishment and characterization of a cell line (BTIC) derived from a patient with recurrent breast cancer after adjuvant chemotherapy with trastuzumab. Characteristics of the BTIC cell line were investigated by phase contrast or electron microscopic observations, chromosome analysis, xenotransplantation, immunohistochemistry and radioimmunoassay for tumor markers. We confirmed that the BTIC cell line grown as multilayered culture in culture dishes, has a poorly developed endoplasmic reticulum in the cytoplasm and some desmosomes. The population doubling time was approximately 44 hr. A graft in nude mouse after xenotransplantation was diagnosed as scirrhous carcinoma. Immunohistochemistry on cultured BTIC cells revealed that the BTIC cells were negative for estrogen receptor and progesterone receptor, and 30% positive for HER-2. Radioimmunoassay indicated secretion of HER-2 protein, NCC-ST-439 and CA15-3.

Hypoglycemia and hyperglycemia due to insulin antibodies against therapeutic human insulin: treatment with double filtration plasmapheresis and prednisolone.

Two diabetic patients, who had been treated with human insulin, suffered from fasting hypoglycemia and postprandial hyperglycemia. The insulin-binding capacities of their sera were high, and a large amount of total insulin and prolonged presence of free insulin in the sera were shown. Scatchard analysis of these insulin antibodies revealed that high-affinity insulin antibodies had larger capacity and stronger affinity compared with commonly insulin-treated patients. Treatment with double filtration plasmapheresis and subsequent administration of prednisolone in the second patient reduced such antibodies and resulted in recovery of glycemic control by insulin. Hypoglycemia and hyperglycemia could be incurred when insulin antibodies with strong affinity and high capacity in high-affinity sites arise. This condition can be treated with double filtration plasmapheresis and subsequent administration of prednisolone.

Role of uncoupling protein-2 up-regulation and triglyceride accumulation in impaired glucose-stimulated insulin secretion in a beta-cell lipotoxicity model overexpressing sterol regulatory element-binding protein-1c.

Triglyceride (TG) accumulation in pancreatic beta-cells is thought to be associated with impaired insulin secretory response to glucose (lipotoxicity). To better understand the mechanism of the impaired insulin secretory response to glucose in beta-cell lipotoxicity, we overexpressed a constitutively active form of the sterol regulatory element-binding protein- 1c (SREBP-1c), a master transcriptional factor of lipogenesis, in INS-1 cells with an adenoviral vector. This treatment was associated with strong activation of transcription of the genes involved in fatty acid biosynthesis, increased cellular TG content, severely blunted glucose-stimulated insulin secretion, and enhanced expression of the uncoupling protein-2 (UCP-2), which supposedly dissipates the mitochondrial electrochemical potential. To decrease the up-regulated UCP-2 expression, small interfering RNA for UCP-2 was used. Introduction of the small interfering RNA increased the ATP/ADP ratio and partially rescued the glucose-stimulated insulin secretion in the cells overexpressing SREBP-1c, but did not affect the cellular TG content. Next, the effect of the AMP-activated protein kinase (AMPK) agonist, 5-amino-4-imidazolecarboxamide riboside, was examined in the lipotoxicity model. Exposure of the cells with lipotoxicity to 5-amino-4-imidazolecarboxamide riboside increased free fatty acid oxidation, partially reversed the TG accumulation, phosphorylated AMPK and acetyl-coenzyme A carboxylase, and improved the impaired glucose-stimulated insulin secretion. These results suggest that UCP-2 down-regulation and AMPK activation could be candidate targets for releasing beta-cells from lipotoxicity.

A kindred of familial acromegaly without evidence for linkage to MEN-1 locus.

Familial acromegaly (FA) is a rare inherited disease characterized by clustering of somatotrophic adenomas and acromegaly within a family without other manifestations of multiple endocrine neoplasia-type 1 (MEN-1). The genetic basis of this pituitary-specific phenotype is largely unknown, and its relationship to the MEN-1 locus on chromosome 11q13 also remains unclear. To test the hypothesis that FA results from a germline mutation of the MEN-1 locus, we performed a linkage analysis in a Japanese family with 2 members showing manifestations of acromegaly due to somatotroph adenomas. We also examined the adenoma of one patient for loss of heterozygosity (LOH) at 11q13 locus and for the presence of mutations of codon 201 and 227 in the gene for Gsalpha. Our results provided no evidence that either germline alterations of the MEN-1 locus, LOH at 11q13, or somatic mutation of Gsalpha plays a causative role in the development of somatotroph adenomas in our FA family. Together with the previous reports, these results suggest that there are at least two distinct subgroups of FA: one that results from a mutation in MEN-1 locus and the other whose causative gene is located outside the 11q13 locus.

Switch to anaerobic glucose metabolism with NADH accumulation in the beta-cell model of mitochondrial diabetes. Characteristics of betaHC9 cells deficient in mitochondrial DNA transcription.

To elucidate the mechanism underlying diabetes caused by mitochondrial gene mutations, we created a model by applying 0.4 microg/ml ethidium bromide (EtBr) to the murine pancreatic beta cell line betaHC9; in this model, transcription of mitochondrial DNA, but not that of nuclear DNA, was suppressed in association with impairment of glucose-stimulated insulin release (Hayakawa, T., Noda, M., Yasuda, K., Yorifuji, H., Taniguchi, S., Miwa, I., Sakura, H., Terauchi, Y., Hayashi, J.-I., Sharp, G. W. G., Kanazawa, Y., Akanuma, Y., Yazaki, Y., and Kadowaki, T. (1998) J. Biol. Chem. 273, 20300-20307). To elucidate fully the metabolism-secretion coupling in these cells, we measured glucose oxidation, utilization, and lactate production. We also evaluated NADH autofluorescence in betaHC9 cells using two-photon excitation laser microscopy. In addition, we recorded the membrane potential and determined the ATP and ADP contents of the cells. The results indicated 22.2 mm glucose oxidation to be severely decreased by EtBr treatment compared with control cells (by 63% on day 4 and by 78% on day 6; both p < 0.01). By contrast, glucose utilization was only marginally decreased. Lactate production under 22.2 mm glucose was increased by 2.9- and 3.5-fold by EtBr treatment on days 4 and 6, respectively (both p < 0.01). Cellular NADH at 2.8 mm glucose was increased by 35 and 43% by EtBr on days 4 and 6 (both p < 0.01). These data suggest that reduced expression of the mitochondrial electron transport system causes NADH accumulation in beta cells, thereby halting the tricarboxylic acid cycle on one hand, and on the other hand facilitating anaerobic glucose metabolism. Glucose-induced insulin secretion was lost rapidly along with the EtBr treatment with concomitant losses of membrane potential depolarization and the [Ca(2+)](i) increase, whereas glibenclamide-induced changes persisted. This is the first report to demonstrate the connection between metabolic alteration of electron transport system and that of tricarboxylic acid cycle and its impact on insulin secretion.

Phosphatidylinositol 3-kinase suppresses glucose-stimulated insulin secretion by affecting post-cytosolic [Ca(2+)] elevation signals.

The role of phosphatidylinositol (PI) 3-kinase in the regulation of pancreatic beta-cell function was investigated. PI 3-kinase activity in p85 alpha regulatory subunit-deficient (p85 alpha(-/-)) islets was decreased to approximately 20% of that in wild-type controls. Insulin content and mass of rough endoplasmic reticula were decreased in beta-cells from p85 alpha(-/-) mice with increased insulin sensitivity. However, p85 alpha(-/-) beta-cells exhibited a marked increase in the insulin secretory response to higher concentrations of glucose. When PI 3-kinase in wild-type islets was suppressed by wortmannin or LY294002, the secretion was also substantially potentiated. Wortmannin's potentiating effect was not due to augmentation in glucose metabolism or cytosolic [Ca(2+)] elevation. Results of p85 alpha(-/-) islets and wortmannin-treated wild-type islets stimulated with diazoxide and KCl showed that inhibition of PI 3-kinase activity exerted its effect on secretion, at least in part, distal to a cytosolic [Ca(2+)] elevation. These results suggest that PI 3-kinase activity normally plays a crucial role in the suppression of glucose-stimulated insulin secretion.