Akio Kuroda

Probucol preserves pancreatic β-cell function through reduction of oxidative stress in type 2 diabetes

Oxidative stress is induced under diabetic conditions and causes various forms of tissue damage in patients with diabetes. Recently, pancreatic beta-cells have emerged as a putative target of oxidative stress-induced tissue damage and this seems to explain in part the progressive deterioration of beta-cell function in type 2 diabetes. As a step toward clinical trial of antioxidant for type 2 diabetes, we investigated the possible anti-diabetic effects of probucol, an antioxidant widely used as an anti-hyperlipidemic agent, on preservation of beta-cell function in diabetic C57BL/KsJ-db/db mice. Probucol-containing diet was given to mice from 6 to 16 weeks of age. Immunostaining for oxidative stress markers such as 4-hydroxy-2-nonenal (HNE)-modified proteins and heme oxygenase-1 revealed that probucol treatment decreased reactive oxygen species (ROS) in pancreatic islets of diabetic animals. Oxidative stress is known to enhance apoptosis of beta-cells and to suppress insulin biosynthesis, but probucol treatment led to preservation of beta-cell mass and the insulin content. According to intraperitoneal glucose tolerance tests, the probucol treatment preserved glucose-stimulated insulin secretion and improved glucose tolerance at 10 and 16 weeks: insulin, 280+/-82 vs. 914+/-238 pmol/l (120 min, at 16 weeks; P<0.05); glucose, 44.6+/-2.4 vs. 35.2+/-2.6 mmol/l (120 min, at 16 weeks; P<0.05). Thus, our present observations demonstrate the potential usefulness of probucol for treatment of type 2 diabetes.

Important role of the hepatic vagus nerve in glucose uptake and production by the liver.

We examined the role of the hepatic vagus nerve in hepatic and peripheral glucose metabolism. To assess endogenous glucose production (EGP), hepatic uptake of first-pass glucose infused intraportally (HGU), and the metabolic clearance rate of glucose (MCR), rats were subjected to hepatic vagotomy (HV, n = 7) or sham operation (SH, n = 8), after 10 days, they were then subjected to a euglycemic-hyperinsulinemic clamp together with a portal glucose load in the 24-hour fasting state. Metabolic parameters were determined by the dual-tracer method using stable isotopes. During the experiment, [6,6-2H2]glucose was continuously infused into the peripheral vein. To maintain euglycemia (4.5 mmol/L), insulin (54 pmol x kg(-1) x min(-1)) and glucose were infused peripherally after the 90-minute tracer equilibration and 30-minute basal periods, and glucose containing 5% enriched [U-13C]glucose was infused intraportally (50 micromol x kg(-1) x min(-1)) for 120 minutes (clamp period). EGP was significantly higher in HV rats versus SH rats during the basal period (64.3 +/- 7.6 v 43.6 +/- 5.3 micromol x kg(-1) x min(-1), P < .005)) and was comparable to EGP in SH rats during the clamp period (9.3 +/- 21.5 v 1.1 +/- 11.7 micromol x kg(-1) x min(-1)). HGU was reduced in HV rats compared with SH rats during portal glucose infusion (5.9 +/- 2.4 v 10.1 +/- 3.2 micromol x kg(-1) x min(-1)). The MCR in HV rats was significantly higher than in SH rats in the basal period (11.0 +/- 2.0 v 7.9 +/- 0.8 mL x kg(-1) x min(-1), P < .01)) and was comparable to the MCR in SH rats during the clamp period (41.9 +/- 10.0 and 36.6 +/- 5.7 mL x kg(-1) x min(-1)). We conclude that innervation of the hepatic vagus nerve is important for the regulation of hepatic glucose production in the postabsorptive state and HGU in the postprandial state.

Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes

DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of DNA methylation. We previously reported that the mouse insulin-2 gene (Ins2) promoter has three potential methylation (CpG) sites, all of which are unmethylated in insulin-producing cells but methylated in other tissues. In this study we examined Ins2 exon 2 and found a similar tissue-specific methylation pattern. These methylation patterns can differentiate between DNA from insulin-producing beta cells and other tissues. We hypothesized that damaged beta cells release their DNA into circulation at the onset of type 1 diabetes mellitus (T1DM) and sought to develop a quantitative methylation-specific polymerase chain reaction (qMSP) assay for circulating beta cell DNA to monitor the loss of beta cells. Methylation-specific primers were designed to interrogate two or more CpG in the same assay. The cloned mouse Ins2 gene was methylated in vitro and used for development of the qMSP assay. We found the qMSP method to be sensitive and specific to differentiate between insulin-producing cells and other tissues with a detection limit of 10 copies in the presence of non-specific genomic DNA background. We also compared different methods for data analysis and found that the Relative Expression Ratio method is the most robust method since it incorporates both a reference value to normalize day-to-day variability as well as PCR reaction efficiencies to normalize between the methylation-specific and bisulfite-specific components of the calculations. The assay was applied in the streptozotocin-treated diabetic mouse model and detected a significant increase in circulating beta cell DNA before the rise in blood glucose level. These results demonstrate that this qMSP assay can be used for monitoring circulating DNA from insulin-producing cells, which will provide the basis for development of assays to detect beta cell destruction in early T1DM.

Basal Insulin Requirement Is ∼30% of the Total Daily Insulin Dose in Type 1 Diabetic Patients Who Use the Insulin Pump

OBJECTIVE To investigate the basal insulin requirement in total daily insulin dose in Japanese type 1 diabetic patients who use the insulin pump. RESEARCH DESIGN AND METHODS The basal insulin requirement in 35 type 1 diabetic patients without detectable C-peptide using the insulin pump (Paradigm 712) was investigated during 2–3 weeks of hospitalization. The patients were served diabetic diets of 25–30 kcal/kg ideal body weight. Each meal omission was done to confirm stable blood glucose levels within 30 mg/dL variance until the next meal. Target blood glucose level was set at 100 mg/dL before each meal and 150 mg/dL at 2 h after each meal. RESULTS Total daily insulin dose was 31.6 ± 8.5 units, and total basal insulin requirement was 8.7 ± 2.9 units, which was 27.7 ± 6.9% of the total daily dose. CONCLUSIONS Basal insulin requirement is ∼30% of the total daily dose in Japanese type 1 diabetic patients who use the insulin pump.

Relationship Between Carotid Intima-Media Thickness and the Presence and Extent of Coronary Stenosis in Type 2 Diabetic Patients With Carotid Atherosclerosis but Without History of Coronary Artery Disease

OBJECTIVE We examined the relationship between the presence and extent of coronary stenosis and carotid intima-media thickness (CIMT) in type 2 diabetic patients without history of coronary artery disease (CAD) but with carotid atherosclerosis. RESEARCH DESIGN AND METHODS A total of 91 type 2 diabetic patients underwent multi-slice computed tomography coronary angiography. RESULTS Max-IMT in the ≥50% stenosis group by multi-slice computed tomography coronary angiography estimation was significantly greater than the 0–25 and 25–50% stenosis group (2.68 ± 0.77 vs. 1.61 ± 0.49 mm, P < 0.0005, and 2.14 ± 0.81 mm, P < 0.05, respectively), and max-IMT in the 25–50% stenosis group was significantly greater than the 0–25% stenosis group (P < 0.05) after adjustment for age, sex, duration of type 2 diabetes, hypertension, and dyslipidemia. In the analysis for trend through the categories of max-IMT, as max-IMT increased, the percentage of ≥50% stenosis increased and the percentage of 0–25% stenosis decreased. CONCLUSIONS Our data suggest that max-IMT might be closely associated with the extent of coronary stenosis in type 2 diabetic patients without history of CAD but with carotid atherosclerosis.

Subclinical hypothyroidism is independently associated with albuminuria in people with type 2 diabetes.

We examined a possible association between subclinical hypothyroidism and albuminuria in 159 people with type 2 diabetes. Patients with subclinical hypothyroidism had significantly higher levels of urinary albumin-to-creatinine ratio (UACR) than those with euthyroidism. Multivariate logistic regression analyses demonstrated that serum TSH level was an independent risk factor of albuminuria.

Vitamin D deficiency is significantly associated with retinopathy in young Japanese type 1 diabetic patients.

The aim of this study was to examine the possible association of vitamin D deficiency with diabetic retinopathy in 75 young Japanese type 1 diabetic patients. A multivariate regression analysis, duration of diabetes and vitamin D deficiency were independent determinants of diabetic retinopathy.

Improvement of canine islet yield by donor pancreas infusion with a p38MAPK inhibitor.

Background. The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury of donor organs. The islet isolation process, from pancreas procurement through islet collection, may activate p38MAPK leading to cytokine release and islet damage. This damage may be prevented by treating pancreata with a p38MAPK inhibitor (p38IH) before cold preservation. Methods. Pancreata removed from Beagle dogs were infused with University of Wisconsin solution containing the p38IH, SB203580, and Pefabloc (n=6) or vehicle (dimethyl sulfoxide and Pefabloc) alone (n=7), through the pancreatic duct and preserved using the two-layer method. After 20 to 22 hr, islets were isolated and 3000 IEQ/kg were autotransplanted into the corresponding dog to monitor glucose metabolism. Results. p38IH-treated pancreata yielded significantly more islets than control pancreata (IEQ/g: 2134±297 vs. 1477±145 IEQ/g or 65,012±9385 vs. 45,700±5103 IEQ/pancreas; P<0.05). Apoptotic &bgr;-cell percentages assessed by laser scanning cytometry were lower in p38IH-treated than the controls (44%±9.4% vs. 61.6%±4.8%, P<0.05). Tumor necrosis factor-&agr; expression assessed by real-time reverse transcription polymerase chain reaction was significantly lower in the p38IH-treated group than controls. All dogs (3000 IEQ/kg) transplanted with p38IH-treated islets (n=5) became euglycemic versus four of five dogs that received untreated islets. Plasma C-peptide levels after glucagon challenge were higher in animals receiving p38IH-treated islets (n=5) versus untreated islets (n=4) (0.40±0.78 vs. 0.21±0.05 ng/mL, P<0.05). Conclusions. Infusion of pancreata with University of Wisconsin solution containing p38IH through the duct before preservation suppresses cytokine release, prevents &bgr;-cell apoptosis, and improves islet yield significantly with no adverse effect on islet function after transplantation. p38IH treatment of human pancreata may improve islet yield for use in clinical transplantation.

Long-Term Pancreatic Beta Cell Exposure to High Levels of Glucose but Not Palmitate Induces DNA Methylation within the Insulin Gene Promoter and Represses Transcriptional Activity

Recent studies have implicated epigenetics in the pathophysiology of diabetes. Furthermore, DNA methylation, which irreversibly deactivates gene transcription, of the insulin promoter, particularly the cAMP response element, is increased in diabetes patients. However, the underlying mechanism remains unclear. We aimed to investigate insulin promoter DNA methylation in an over-nutrition state. INS-1 cells, the rat pancreatic beta cell line, were cultured under normal-culture-glucose (11.2 mmol/l) or experimental-high-glucose (22.4 mmol/l) conditions for 14 days, with or without 0.4 mmol/l palmitate. DNA methylation of the rat insulin 1 gene (Ins1) promoter was investigated using bisulfite sequencing and pyrosequencing analysis. Experimental-high-glucose conditions significantly suppressed insulin mRNA and increased DNA methylation at all five CpG sites within the Ins1 promoter, including the cAMP response element, in a time-dependent and glucose concentration-dependent manner. DNA methylation under experimental-high-glucose conditions was unique to the Ins1 promoter; however, palmitate did not affect DNA methylation. Artificial methylation of Ins1 promoter significantly suppressed promoter-driven luciferase activity, and a DNA methylation inhibitor significantly improved insulin mRNA suppression by experimental-high-glucose conditions. Experimental-high-glucose conditions significantly increased DNA methyltransferase activity and decreased ten-eleven-translocation methylcytosine dioxygenase activity. Oxidative stress and endoplasmic reticulum stress did not affect DNA methylation of the Ins1 promoter. High glucose but not palmitate increased ectopic triacylglycerol accumulation parallel to DNA methylation. Metformin upregulated insulin gene expression and suppressed DNA methylation and ectopic triacylglycerol accumulation. Finally, DNA methylation of the Ins1 promoter increased in isolated islets from Zucker diabetic fatty rats. This study helps to clarify the effect of an over-nutrition state on DNA methylation of the Ins1 promoter in pancreatic beta cells. It provides new insights into the irreversible pathophysiology of diabetes.

YKL-40, a new biomarker of endothelial dysfunction, is independently associated with albuminuria in type 2 diabetic patients

The aim of this study was to examine the possible association between YKL-40, a new biomarker of endothelial dysfunction, and albuminuria in 180 type 2 diabetic patients. Multivariate logistic regression analyses demonstrated that serum YKL-40 level was a determinant of albuminuria independently of conventional risk factors.