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IntroductionGiven an increasing use of dipeptidyl peptidase-4 (DPP-4) inhibitors to treat patients with type 2 diabetes mellitus in the real-world setting, we conducted a prospective observational study (Japan-based Clinical Research Network for Diabetes Registry: J-BRAND Registry) to elucidate the safety and efficacy profile of long-term usage of alogliptin.Research design and methodsWe registered 5969 patients from April 2012 through September 2014, who started receiving alogliptin (group A) or other classes of oral hypoglycemic agents (OHAs; group B), and were followed for 3 years at 239 sites nationwide. Safety was the primary outcome. Symptomatic hypoglycemia, pancreatitis, skin disorders of non-extrinsic origin, severe infections, and cancer were collected as major adverse events (AEs). Efficacy assessment was the secondary outcome and included changes in hemoglobin A1c (HbA1c), fasting blood glucose, fasting insulin and urinary albumin.ResultsOf the registered, 5150 (group A: 3395 and group B: 1755) and 5096 (3358 and 1738) were included for safety and efficacy analysis, respectively. Group A patients mostly (>90%) continued to use alogliptin. In group B, biguanides were the primary agents, while DPP-4 inhibitors were added in up to ~36% of patients. The overall incidence of AEs was similar between the two groups (42.7% vs 42.2%). Kaplan-Meier analysis revealed the incidence of cancer was significantly higher in group A than in group B (7.4% vs 4.8%, p=0.040), while no significant incidence difference was observed in the individual cancer. Multivariate Cox regression analysis revealed that the imbalanced patient distribution (more elderly patients in group A than in group B), but not alogliptin usage per se, contributed to cancer development. The incidence of other major AE categories was with no between-group difference. Between-group difference was not detected, either, in the incidence of microvascular and macrovascular complications. HbA1c and fasting glucose decreased significantly at the 0.5-year visit and nearly plateaued thereafter in both groups.ConclusionsAlogliptin as a representative of DPP-4 inhibitors was safe and durably efficacious when used alone or with other OHAs for patients with type 2 diabetes in the real world setting.

Aims/Introduction To explore the relationships between periodontitis and microvascular complications as well as glycemic control in type 2 diabetes patients. Materials and Methods This multicenter, hospital-based, cross-sectional study included 620 patients with type 2 diabetes. We compared the prevalence and severity of periodontitis between patients with ≥1 microvascular complication and those without microvascular complications. We also compared the prevalence and severity of periodontitis among patients with different degrees of glycemic control. Results After adjusting for confounding factors, multiple logistic regression analysis showed that the severity of periodontitis was significantly associated with the number of microvascular complications (odds ratio 1.3, 95% confidence interval 1.1–1.6), glycated hemoglobin ≥8.0% (64 mmol/mol; odds ratio 1.6; 95% confidence interval 1.1–2.3), and older age (≥50 years; odds ratio 1.7; 95% confidence interval 1.1–2.6). However, the prevalence of periodontitis was not significantly associated with the number of microvascular complications, but was associated with male sex, high glycated hemoglobin (≥8.0% [64 mmol/mol]), older age (≥40 years), longer duration of diabetes (≥15 years) and fewer teeth (≤25). Furthermore, propensity score matching for age, sex, diabetes duration and glycated hemoglobin showed that the incidence of severe periodontitis was significantly higher among patients with microvascular complications than among those without microvascular complications (P < 0.05). Conclusions The number of microvascular complications is a risk factor for more severe periodontitis in patients with type 2 diabetes, whereas poor glycemic control is a risk factor for increased prevalence and severity of periodontitis.

Background Glycated albumin is an intermediate glycaemic control marker for which there are several measurement procedures with entirely different reference intervals. We have developed a reference measurement procedure for the purpose of standardizing glycated albumin measurements. Methods The isotope dilution liquid chromatography/tandem mass spectrometry method was developed as a reference measurement procedure for glycated albumin. The stable isotopes of lysine and fructosyl-lysine, which serve as an internal standard, were added to albumin isolated from serum, followed by hydrogenation. After hydrolysis of albumin with hot hydrochloric acid, the liberated lysine and fructosyl-lysine were measured by liquid chromatography/tandem mass spectrometry, and their concentrations were determined from each isotope ratio. The reference materials (JCCRM611) for determining of glycated albumin were prepared from pooled patient blood samples. Results The isotope dilution–tandem mass spectrometry calibration curve of fructosyl-lysine and lysine showed good linearity (r = 0.999). The inter-assay and intra-assay coefficient of variation values of glycated albumin measurement were 1.2 and 1.4%, respectively. The glycated albumin values of serum in patients with diabetes assessed through the use of this method showed a good relationship with routine measurement procedures (r = 0.997). The relationship of glycated albumin values of the reference material (JCCRM611) between these two methods was the same as the relationship with the patient serum samples. Conclusion The Committee on Diabetes Mellitus Indices of the Japan Society of Clinical Chemistry recommends the isotope dilution liquid chromatography/tandem mass spectrometry method as a reference measurement procedure, and JCCRM611 as a certified reference material for glycated albumin measurement. In addition, we recommend the traceability system for glycated albumin measurement.

The aim of this investigation is to examine the correlation between the HbA1c numbers used by the National Glycohemoglobin Standardization Program (NGSP) and the Japan Diabetes Society (JDS) and to validate the HbA1c cutoff values of 6.5 and 6.1 % set for the diagnosis of diabetes mellitus by the American Diabetes Association (ADA) and the JDS, respectively. NGSP HbA1c values obtained from the monthly monitoring program of the performance of the NGSP Secondary Reference Laboratories (SRLs) were provided by the NGSP. The samples used in the NGSP monitoring program were also assayed to obtain the JDS values at the Laboratory of Reference Material Institute for Clinical Chemistry Standards (ReCCS) using the KO 500 designated comparison method (DCM) calibrated with a reference material, JDS Lot 4, issued by ReCCS. The results were compared, and the correlation between the two data sets was examined by linear regression. The relationship between the NGSP SRLs values and JDS values for the monitoring program samples was as follows: NGSP = 1.029 × JDS + 0.216. For another sample, JDS Lot 4, the correlation was: NGSP = 1.029 × JDS + 0.187. The two equations are consistent with the cutoff points 6.5 and 6.1 %, used by the ADA and the JDS, respectively.

We investigated the efficacy of epalrestat, an aldose reductase inhibitor, for diabetic peripheral neuropathy in Japanese patients with type 2 diabetes.A total of 38 type 2 diabetic patients (22 men and 16 women; mean ± S.E.M. age 63.3 ± 1.0 years; duration of diabetes 9.6 ± 0.8 years) with diabetic neuropathy were newly administered 150 mg/day epalrestat (EP group). Motor nerve conduction velocity (MCV), sensory nerve conduction velocity (SCV), and minimum F-wave latency were evaluated before administration of epalrestat and after 1 and 2 years. Serum N(ɛ)-carboxymethyl lysine (CML) as a parameter of advanced glycation end products (AGEs), lipid peroxide, and soluble vascular cell adhesion molecule (sVCAM)-1 as a parameter of angiopathy were measured before administration and after 1 year. We compared the results with those of 36 duration of diabetes-matched type 2 diabetic patients (mean ± S.E.M. duration of diabetes 8.2 ± 0.7 years) as control (C group).The EP group showed significant suppression of deterioration of MCV (P.01) and minimum F-wave latency (P.01) in the tibial nerve and SCV (P.05) in the sural nerve compared to those in the C group after 2 years. There was a significant difference in change in CML level between groups (-0.18 ± 0.13 mU/ml in the EP group vs. +0.22 ± 0.09 mU/ml in the C group, P.05) after 1 year.Epalrestat suppressed the deterioration of diabetic peripheral neuropathy, especially in the lower extremity. Its effects might be mediated by improvement of the polyol pathway and suppression of production of AGEs.

Conophylline, a vinca alkaloid extracted from the tropical plant Ervatamia microphylla, has been shown to induce the differentiation of insulin-producing beta-cells in cultured cells and in animals. However, its mechanism of action and the molecular target have remained unclear. Therefore, we prepared a fishing probe with conophylline to identify the target protein by using latex nano-beads, which are newly innovated tools for affinity-purification. With these conophylline-linked nano-beads, we found that conophylline directly interacted with ARL6IP. ARL6IP may thus be involved in the mechanism of cellular differentiation of beta-cells, and this probe should be useful to find other target proteins.

Objective: We tested the hypothesis that insulin therapy rather than sulfonylurea (SU) treatment is preferable to reverse or preserve β-cell function among patients with slowly progressive insulin-dependent (type 1) diabetes (SPIDDM) or latent autoimmune diabetes in adults. Methods: This multicenter, randomized, nonblinded clinical study screened 4089 non-insulin-dependent diabetic patients for glutamic acid decarboxylase autoantibodies (GADAb). Sixty GADAb-positive non-insulin-requiring diabetic patients with a 5-yr duration or shorter of diabetes were assigned to either the SU group (n = 30) or the insulin group (n = 30). Serum C-peptide responses to annual oral glucose tolerance tests were followed up for a mean of 57 months. The primary endpoint was an insulin-dependent state defined by the sum of serum C-peptide values during the oral glucose tolerance test (ΣC-peptide) less than 4 ng/ml (1.32 nmol/liter). Results: The progression rate to an insulin-dependent state in the insulin group (three of 30, 10%) was lower than that in the SU group (13 of 30, 43%; P = 0.003, log-rank). Longitudinal analysis demonstrated that ΣC-peptide values were better preserved in the insulin group than in the SU group. Multiple regression analysis demonstrated that insulin treatment, a preserved C-peptide response, and a low GADAb titer at entry were independent factors in preventing progression to an insulin-dependent state. Subgroup analysis suggested that insulin intervention was highly effective for SPIDDM patients with high GADAb titers [≥10 U/ml (180 World Health Organization U/ml)] and preserved β-cell function [ΣC-peptide ≥ 10 ng/ml (3.31 nmol/liter)] at entry. No severe hypoglycemic episodes occurred during the study. Conclusions: Insulin intervention to preserve β-cell function is effective and safe for patients with SPIDDM or latent autoimmune diabetes in adults.

The present study was conducted to establish a method to induce differentiation of bone marrow (MB)-derived mesenchymal cells into insulin-producing cells. When mouse BM-derived mesenchymal cells were cultured for 60 days in medium containing 10% fetal calf serum and 25 mM glucose, they expressed insulin. Addition of activin A and betacellulin (BTC) accelerated differentiation, and immunoreactive insulin was detected 14 days after the treatment. Insulin-containing secretory granules were observed in differentiated cells by electron microscopy. Treatment of BM-derived mesenchymal cells with conophylline (CnP) and BTC-delta4 further accelerated differentiation, and mRNA for insulin was detected 5 to 7 days after the treatment. Mesencymal cells treated with CnP and BTC-delta4 responded to a high concentration of glucose and secreted mature insulin. When these cells were transplanted into streptozotocin-treated mice, they markedly reduced the plasma glucose concentration, and the effect continued for at least 4 weeks. These results indicate an efficacy of the combination of CnP and BTC-delta4 in inducing differentiation of BM-derived mesenchymal cells into insulin-producing cells.

Background Recent genetic studies have shown an association between the T-786C polymorphism in the endothelial NO synthase gene and coronary artery diseases, but any possible association with hypertension has been controversial. Other studies indicate the effect of restricting salt intake differ depending on individual salt-sensitivity, and the mixture of different sensitivity in study subjects may obscure the results. The objective of this study was to investigate the gene–environment interaction between the salt intake and this polymorphism. Methods We genotyped 281 healthy men after excluding 37 men on hypertensive therapy (mean age 44.8 ± 11.9 years) for the mutation, and evaluated their daily salt intake using a validated food frequency questionnaire. Results A quartile classification of salt intake revealed that the blood pressure of subjects with the mutation was significantly higher than that of subjects without the mutation, but only in the 4th quartile (the highest intake group). A multiple logistic regression analysis also showed that the presence of this mutation increased the risk of hypertension only in the 4th quartile (adjusted odds ratio = 6.38, P = .025). Conclusions The presence of this mutation alone does not significantly increase the risk of hypertension. However, high salt intake interacts with the mutation and leads to a significant increase in the risk of hypertension. The T-786C mutation warrants being considered a candidate for further study with the aim of tailor-made hypertension prevention.

Background: The Committee on Standardization of Laboratory Testing Related to Diabetes Mellitus of the Japan Diabetes Society (JDS) previously recommended use of the primary calibrator (JDS Lot 1) prepared by the former Committee for Standardization of Glycohemoglobin for standardizing the measurement of haemoglobin A1c (HbA1c). Owing to the depletion of vials of Lot 1 in March 2001, the present committee certified a new reference material, Lot 2, now distributed by the Health Care Technology Foundation (HECTEF). The standardization programme for HbA1c measurement in Japan is currently based on Lot 2, which has values assigned from within Lot 1; the Lot 1 values were consensus values based on assays by laboratories in the Japanese national quality control programme. In this study, for the purpose of international comparison and standardization, Lot 2 was assayed by the JDS reference laboratories, the National Glycoprotein Standardization Program (NGSP) in the USA, and by reference laboratories approved by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Method: The HbA1c values of JDS Lot 2 were transferred from those assigned to Lot 1 using KO500, a high-resolution HPLC method, at three laboratories approved by the JDS committee. Subsequently, vials of JDS Lot 2 were shipped to and assayed by the NGSP in the USA and 10 IFCC reference laboratories. Result: The JDS-assigned HbA1c values (from Lot 1) are 4.04 for Level 1, 5.38 for Level 2, 7.32 for Level 3, 9.88 for Level 4, and 12.63 for Level 5, all expressed as a percentage of total haemoglobin. The values obtained by NGSP and the IFCC laboratories gave the following formulas: NGSP value (%) = JDS value (%) + 0.3%; IFCC value (%) = 1.068 x JDS value (%) -1.741%. Conclusion: Although the values obtained by the IFCC laboratories are significantly lower than the values assigned to Lot 2 by the JDS, the relationship is linear. In addition, standardization of HbA1c based on JDS Lot 2 is currently at a satisfactory level in Japan. As a result, the reassignment of values for Lot 2 to agree with the IFCC values should be relatively easy and will be done after all relevant parties agree to the change.

BACKGROUND: Few studies have investigated the interaction between the Trp64Arg polymorphism of the β3-adrenergic receptor gene (ADRB3) and environmental factors. This study aimed to investigate whether energy intake affects the relationship between this polymorphism and obesity.METHODS: Healthy Japanese men (n=295; age 46.1±11.5 years (mean ±standard deviation); waist circumference 83.9±9.3 cm; body mass index (BMI) 23.3±3.3 kg/m2) recruited in a Japanese chemical industry firm were eligible for analysis. Daily energy intake, protein, fat, and carbohydrate (PFC) ratio and daily physical activity were assessed by self-reported questionnaires. Genotyping for the polymorphism was performed with written informed consent.RESULTS: When the subjects were classified into two groups according to presence of the polymorphism, the groups were not significantly different in waist circumference or BMI. Quartile classification of energy intake, however, demonstrated a significantly larger ratio of obese subjects to non-obese subjects in the group with the polymorphism in the highest 4th quartile alone. Multiple logistic regression analysis also revealed that the presence of the polymorphism increased the risk of obesity significantly in the 4th quartile alone (adjusted odds ratio=3.37, 95% confidence interval=1.12-10.2).CONCLUSION: Presence of the polymorphism alone does not significantly increase the risk of obesity. However, high energy intake interacts with the polymorphism and leads to a significant increase in risk of obesity. The Trp64Arg polymorphism of ADRB3 warrants consideration, along with other polymorphisms involved in the development of obesity, for tailor-made prevention of obesity.J Epidemiol 2005; 15: 203-210.

For reaching near normal glycemic control, multiple daily insulin injections are necessary, although subcutaneous insulin therapy cannot get the physiological profile, results in hypoglycemia, weight gain, peripheral hyperinsulinemia, and may not be accepted for painful injections. Glucagon-like peptide 1(GLP-1) analogs and alternative routes of insulin, especially oral (enteric-gastrointestinal, inhaled) route, are most promising and attractive now. Biotechnology and biochemistry will make it possible to overcome several disadvantages of low absorption, short half-life, low bioavailability, and many clinical trials are now in progress. We will show the review of these drugs and another candidate for the treatment of diabetic complications, protein kinase C inhibitor.

In 1996, we designed a randomized multicenter study to assess the effects of small doses of insulin on beta cell failure in slowly progressive type 1 diabetes (the Tokyo Study). We report here the preliminary results of this study. Glutamic acid decarboxylase 65 antibody (GADA)-positive patients were randomly divided into 2 groups: one group received insulin (Ins group), the other a sulfonylurea (SU group). Fifty-four patients (24 Ins group, 30 SU group) were analyzed at the end of a 4-year period. All patients underwent a 75 g oral-glucose test (O-GTT) every 6-12 months. The insulin-dependent stage was defined based on an integrated value of serum C-peptide levels on O-GTT ( summation operator CPR; sum of CPR at 0, 30, 60, 90, and 120 min) below 4.0 ng/mL. The summation operator CPR in the SU group decreased progressively from 22.0 +/- 10.6 to 11.3 +/- 7.5 ng/mL over the 48-month period (p /= 10 ng/mL), the proportion of SU group subjects who progressed to IDDM was significantly higher than that of the Ins group (7/28, 25% vs. 0/21, 0%, p = 0.015). Among subjects with a high GADA titer (>/=0 U/mL), 9/14 (64.3%) of the SU group, but only 2/16 (12.5%) of the Ins group, developed IDDM (p = 0.0068). As to those with a high GADA titer and a preserved C-peptide response, SU group subjects progressed to IDDM (7/12, 58.3%) more frequently than Ins group subjects (0/14, 0%) (p = 0.0012). In summary, our results suggest that small doses of insulin effectively prevent beta cell failure in slowly progressive type 1 diabetes. We recommend avoiding SU treatment and instead administering insulin to NIDDM patients with high GADA titer.

There is little information on direct comparison between metformin and glucagon-like peptide-1 (GLP-1) receptor agonists in the Asian population. This study examined the efficacy and safety of liraglutide monotherapy compared with metformin monotherapy in overweight/obese Japanese patients with type 2 diabetes (T2DM). The study was a 24-week, open-labeled, randomized controlled study. Overweight or obese patients with T2DM aged 20-75 years with suboptimal glycemic control were randomized to liraglutide or metformin monotherapy. The primary endpoint was change in HbA1c at week 24. Secondary endpoints included changes in daily glycemic profile, body weight, incidence of hypoglycemia and other adverse events. The study, which was originally planned to enroll 50 subjects in each group, was ended with insufficient recruitment. A total of 46 subjects completed the study, and analysis was conducted in this cohort. Reduction in HbA1c at week 24 was comparable between the metformin (n = 24) and liraglutide (n = 22) groups (-0.95 ± 0.80% vs. -0.80 ± 0.88%, p = 0.77), while the liraglutide group reached maximal reduction more rapidly than did the metformin group. There was no significant difference in weight gain or incidence of hypoglycemia between the groups. Diarrhea was more frequent in the metformin group, while constipation was more frequent in the liraglutide group. There was no significant difference in treatment satisfaction between the groups. In conclusion, liraglutide and metformin monotherapy showed similar reduction in HbA1c during 24 weeks, with no difference in weight gain or incidence of hypoglycemia in overweight or obese Japanese patients with T2DM.

BACKGROUND A major objective of the IFCC Task Force on Implementation of HbA1c Standardization is to develop a model to define quality targets for glycated hemoglobin (Hb A1c). METHODS Two generic models, biological variation and sigma-metrics, are investigated. We selected variables in the models for Hb A1c and used data of external quality assurance/proficiency testing programs to evaluate the suitability of the models to set and evaluate quality targets within and between laboratories. RESULTS In the biological variation model, 48% of individual laboratories and none of the 26 instrument groups met the minimum performance criterion. In the sigma-metrics model, with a total allowable error (TAE) set at 5 mmol/mol (0.46% NGSP), 77% of the individual laboratories and 12 of 26 instrument groups met the 2σ criterion. CONCLUSIONS The biological variation and sigma-metrics models were demonstrated to be suitable for setting and evaluating quality targets within and between laboratories. The sigma-metrics model is more flexible, as both the TAE and the risk of failure can be adjusted to the situation—for example, requirements related to diagnosis/monitoring or international authorities. With the aim of reaching (inter)national consensus on advice regarding quality targets for Hb A1c, the Task Force suggests the sigma-metrics model as the model of choice, with default values of 5 mmol/mol (0.46%) for TAE and risk levels of 2σ and 4σ for routine laboratories and laboratories performing clinical trials, respectively. These goals should serve as a starting point for discussion with international stakeholders in the field of diabetes.

In 1999, the Japan Diabetes Society (JDS) launched the previous version of the diagnostic criteria of diabetes mellitus, in which JDS took initiative in adopting glycated hemoglobin (HbA1c) as an adjunct to the diagnosis of diabetes. In contrast, in 2009 the International Expert Committee composed of the members of the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) manifested the recommendation regarding the use of HbA1c in diagnosing diabetes mellitus as an alternative to glucose measurements based on the updated evidence showing that HbA1c has several advantages as a marker of chronic hyperglycemia2–4. The JDS extensively evaluated the usefulness and feasibility of more extended use of HbA1c in the diagnosis of diabetes based on Japanese epidemiological data, and then the ‘Report of the Committee on the Classification and Diagnostic Criteria of Diabetes Mellitus’ was published in the Journal of Diabetes Investigation5 and Diabetology International6. The new diagnostic criterion in Japan came into effect on 1 July 2010. According to the new version of the criteria, HbA1c (JDS) ≥6.1% is now considered to indicate a diabetic type, but the previous diagnosis criteria of high plasma glucose (PG) levels to diagnose diabetes mellitus also need to be confirmed. Those are as follows: (i) FPG ≥126 mg/dL (7.0 mmol/L); (ii) 2‐h PG ≥200 mg/dL (11.1 mmol/L) during an oral glucose tolerance test; or (iii) casual PG ≥200 mg/dL (11.1 mmol/L). If both PG criteria and HbA1c in patients have met the diabetic type, those patients are immediately diagnosed to have diabetes mellitus5,6.

Objective. This clinical trial was designed to investigate whether goshajinkigan reduces the onset of diabetic complications or not.Materials and Methods. A total of 332 type 2 diabetic mellitus patients were registered from 9 clinical centers from March 2000 to August 2007. Patients were randomly assigned to take goshajinkigan extract powder, 2.5 grams for 3 times a day or no kampo therapy, additionally to the regular treatment. The primary endpoints were the onset of macrovascular diseases or progression of nephropathy or retinopathy. Statistical analysis was performed by the intention-to-treat method.Results. After 5 years of observation, 116 patients were submitted to analysis. Among them, no macrovascular events were observed in both groups. Although 43 participants had upstaging of retinopathy or nephropathy in total, there was no significant difference between goshajinkigan group and control group. Deterioration of ankle reflex was suppressed in goshajinkigan group. Also glycated hemoglobin, and fasting plasma glucose were decreased in the goshajinkigan group.Conclusion. Although the power of analysis was too low to demonstrate any effects of goshajinkigan on the progression of macrovascular diseases, retinopathy or nephropathy, goshajinkigan may be beneficial for diabetic neuropathy and glycemic control.

It has been proposed that cytokine responses of memory CD4+ cells change from a T-helper 2 (Th2)-to a T-helper 1 (Th1)-dominant response as the disease progresses in non-obese diabetic (NOD) mice. However, the regulation of Th1/Th2 balance in spontaneous diabetes development in this model is not well understood. In this study, higher glutamic acid decarboxylase 65 (GAD65)-specific IL-10 production was observed at 10-12 weeks in NOD mice, and a marked increase of Th1-type response (IFN-gamma production) upon polyclonal (anti-CD3 antibody) stimulation was observed just before diabetes development along with a decline of GAD65-specific IL-10 production. Moreover, there was a clear negative correlation between IL-10 level upon GAD65 stimulation and log(IFN-gamma) level upon anti-CD3 antibody stimulation (r=-0.999, p

We investigated the efficacy of additional administration of 400 mg troglitazone (+T), which became available as a treatment for type 2 diabetes following the demonstration of its ability to reduce insulin resistance, in combination with diet (D + T) or sulfonylurea (S + T) therapy. Body fat area as determined by computed tomographic (CT) scanning at the umbilical level, as well as several clinical and biochemical parameters of glycemic control and lipid metabolism, were compared before and after 3 months of additional treatment with troglitazone. The body mass index (BMI) tended to increase in both groups (22.7 +/- 0.6 v 23.2 +/- 0.6 kg/m2 in D + T, nonsignificant [NS]; 22.2 +/- 0.5 v 22.3 +/- 0.5 kg/m2 in S + T, NS), while it tended to decrease in the control group (only diet therapy, 23.6 +/- 0.6 v 23.1 +/- 0.8 kg/m2, NS). Mean blood pressure ([BP] 96 +/- 3 v 89 +/- 4 mm Hg, P.05) decreased significantly in the D + T group. Changes in the glycemic and lipid profile and leptin did not reach statistical significance. The D + T group showed a significant decline in immunoreactive insulin ([IRI] 12.4 +/- 1.2 v 8.0 +/- 1.0 microU/mL, P.05), reflecting markedly reduced insulin resistance, as well as a significant increase in plasma insulin-like growth factor-1 ([IGF-1] 175.7 +/- 14.2 v 189.8 +/- 12.6 ng/mL, P.05). A slight weight gain was associated with a tendency for subcutaneous fat to increase, while visceral fat decreased in both troglitazone-treated groups. The decrease in the visceral to subcutaneous fat ratio (V/S ratio) was statistically significant in the D + T group (1.09 +/- 0.11 v 0.94 +/- 0.09, P.05), while the V/S ratio in the control group did not change. A notable finding of this study is the difference in the response to troglitazone between subcutaneous and visceral adipose tissue. It is suggested that troglitazone may exert beneficial effects by reducing visceral fat.

We previously demonstrated that intramuscular plasmid injection serves as a useful method of long-term systemic delivery of cytokines. In the present study, we assess intramuscular DNA injection as a means of systemically delivering interleukin 10 (IL-10), a cytokine with immunosuppressive properties, and preventing the progression of autoimmune diabetes in the nonobese diabetic (NOD) mouse, an excellent model for human insulin-dependent diabetes mellitus (IDDM). We injected IL-10 expression plasmid (pCAGGS-IL10) or a control pCAGGS plasmid into the muscles of NOD mice twice at 3 and 5 weeks of age. IL-10 was detectable by ELISA in the sera of mice injected with pCAGGS-IL10 for more than 2 weeks after the injection. Although the severity of insulitis at 13 weeks of age was not improved by the intramuscular injection of pCAGGS-IL10, the incidence of diabetes was markedly reduced in NOD mice injected with pCAGGS-IL10 as compared with those injected with pCAGGS or as compared with nontreated NOD mice. These results show that the progression of autoimmune diseases in mice can effectively be suppressed by intramuscular DNA injection, and suggest that this method is potentially applicable to the treatment of human autoimmune diseases.

It has been demonstrated that intramuscularly injected expression plasmid DNA is taken up by myofibers and subsequently expresses exogenous genes. In the present study, we assessed intramuscular DNA injection as a means of systemically delivering interleukin-5 (IL-5). We constructed an IL-5 expression plasmid, pCAGGS-IL-5, containing murine IL-5 cDNA under the control of the CAG promoter. The soleus muscle of mice was pretreated with bupivacaine. Two days later, mice were injected with pCAGGS-IL-5 or a control pCAGGS plasmid DNA at the same site. At 2 weeks after DNA injection, eosinophils had increased from 2-3% to 8-29% of peripheral white blood cells in 9 of 10 mice injected with pCAGGS-IL-5, while eosinophils never exceeded 3% in control mice injected with pCAGGS. IL-5 mRNA was present in the muscle area injected with pCAGGS-IL-5. IL-5 was also detectable by ELISA in the sera of most mice injected with pCAGGS-IL-5, but in none of the control mice. These results demonstrate that intramuscular plasmid injection serves as a useful method of systemically delivering cytokines by combining the strong CAG promoter and bupivacaine pretreatment.

Diagnostic criteria in diabetes mellitus has been revised after 11 years. In addition to previous diagnostic criteria, HbAlc was added to the new criteria as a biomarker of chronic diabetes. Although NGSP6.5 has become a marker of diabetes, the equation of HbA1c (NGSP) = 1.02 x HbA1c (JDS) + 0.25% was reported by analysis of global standardization. According to this equation, HbA1c (NGSP) 6.5% corresponded to HbA1c (JDS) 6.1%. In this symposium, we presented the viewpoint of the Japanese Society of Diabetes Mellitus for standardization of HbAlc and the issue of using NGSP instead of JDS.

Autoantibodies against heat shock protein (hsp) 60 have been reported to be detected in sera of non-obese diabetic mice, in an experimental model of IDDM. However, there are only a few studies which have examined IDDM patients for antibodies against mammalian hsp60. We produced murine hsp60 derived from pancreatic beta cells which has high homology to human hsp60 and examined antibodies against the hsp60 in IDDM patients using an enzyme-linked immunosorbent assay. We extended the analysis to patients with other immune-mediated diseases and non-insulin-dependent diabetes mellitus (NIDDM). Positive sera for hsp60 antibody were more frequently detected in 13 out of 84 IDDM (15.5%) and 5 out of 25 rheumatoid arthritis patients (20%), when compared to healthy subjects (1/85; 1.2%, P0.001 and P0.01, respectively). The levels of hsp60 antibodies of IDDM (0.218 +/- 0.227) and rheumatoid arthritis patients (0.259 +/- 0.191) were significantly higher than those of healthy subjects (0.076 +/- 0.131, P0.001, P0.01, respectively). Patients with slowly progressive IDDM (n = 26), autoimmune thyroid disease (n = 42), or NIDDM (n = 40) had levels of hsp60 antibodies similar to those in healthy subjects. We found no relationship between the levels of hsp60 antibodies and islet cell antibodies (ICA) or antibodies to glutamic acid decarboxylase (GAD65) in IDDM patients. In conclusion, hsp60 antibodies were detected in Japanese IDDM as well as in rheumatoid arthritis patients. Although the positivity was low, the detection of hsp60 antibodies may be helpful for diagnosis of IDDM especially in GAD65 Ab- or JCA-negative Japanese patients.

Recent studies indicate that experimentally induced hyperinsulinemia may reduce serum dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulfate (DHEA-S). Serum DHEA and DHEA-S decrease in diabetic patients, but the mechanism by which hyperglycemia decreases DHEA and DHEA-S is unknown. In this study, we investigated the effect of hyperglycemia on DHEA and DHEA-S in impaired glucose tolerance (IGT) by means of the 75g-oral glucose tolerance test (OGTT). We selected 30 male IGT patients receiving diet therapy only, whose insulinogenic Index was under 0.3. Oral glucose challenge significantly reduced DHEA (P = 0.0001) and DHEA-S (P < 0.05) at 60 and 120 min after OGTT. Setting the value of DHEA and DHEA-S at time zero as 100%, we calculated the DHEA and DHEA-S values at 60 and 120 min after OGTT as %DHEA(-S) 60 min and %DHEA(-S) 120 min, respectively. DHEA and DHEA-S at time zero showed no correlation with BMI, HbA1c, the sum of insulin values (sigma IRI) or the area under the curve of plasma glucose (AUC). We found decreases in %DHEA 60 min (r = -0.411, P < 0.05), %DHEA-S 60 min (r = -0.508, P < 0.01) and %DHEA-S 120 min (r = -0.393, P < 0.05) as AUC increased, but sigma IRI showed no correlation with %DHEA(-S) 60 min or %DHEA(-S)120 min. We conclude that the depression of DHEA and DHEA-S after OGTT is attributable to hyperglycemia in male Japanese IGT with low insulin response.

Microorganisms and plants produce bioactive metabolites that are potentially useful in the treatment of disease. We have designed and synthesized DHMEQ as a specific inhibitor of NF-κB based on the structure of epoxyquinomicin. It directly binds to NF-κB components to inhibit DNA-binding and was shown to be endowed with inhibiting activity in various inflammatory and cancer models in experimental animals. It was also effective to improve the success of islet transplantation especially when administered to donor mice. We have also isolated from the leaves of Ervatamia microphylla conophylline, a compound that induces differentiation of beta cells from the precursor cells and was recently found to suppress islet fibrosis in diabetes model rats.

Excessive nitric oxide (NO) plays a pivotal role in the progression of β-cell apoptosis in type 1 diabetes mellitus. We used mouse insulinoma Min6 cells as a model of β cells in this research. We found that (−)-DHMEQ, an NF-κB inhibitor, rescued β cells from NO-induced apoptosis, and then studied the mechanism of apoptosis inhibition. (−)-DHMEQ activated Nrf2 and induced transcription of Nrf2-target genes following the increase of antioxidant response element (ARE) reporter activity. Similarly, tert-butyl hydroquinone (tBHQ), a known activator of Nrf2, inhibited NO-induced cell death along with the transcriptional activation of ARE. RNAi-mediated knockdown of Nrf2 lowered the cytoprotective effect of (−)-DHMEQ against NO, suggesting that (−)-DHMEQ inhibited NO-induced cell death via Nrf2 activation. Furthermore, overexpression of Nrf2 rendered cells to be more resistant to NO, indicating that Nrf2 activation provides critical defense function against NO in Min6 cells. Taken together, we conclude that (−)-DHMEQ may be a useful therapeutic agent for type 1 diabetes mellitus in the onset of disease by protecting β cells from apoptosis.

Background & Aims Conophylline (CnP) is a vinca alkaloid purified from a tropical plant and inhibits activation of pancreatic stellate cells. We investigated the effect of CnP on hepatic stellate cells (HSC) in vitro. We also examined whether CnP attenuates hepatic fibrosis in vivo. Method We examined the effect of CnP on the expression of α-smooth muscle actin (α-SMA) and collagen-1, DNA synthesis and apoptosis in rat HSC and Lx-2 cells. We also examined the effect of CnP on hepatic fibrosis induced by thioacetamide (TAA). Results In rat HSC and Lx-2 cells, CnP reduced the expression of α-SMA and collagen-1. CnP inhibited DNA synthesis induced by serum. CnP also promoted activation of caspase-3 and induced apoptosis as assessed by DNA ladder formation and TUNEL assay. In contrast, CnP did not induce apoptosis in AML12 cells. We then examined the effect of CnP on TAA-induced cirrhosis. In TAA-treated rats, the surface of the liver was irregular and multiple nodules were observed. Histologically, formation of pseudolobules surrounded by massive fibrous tissues was observed. When CnP was administered together with TAA, the surface of the liver was smooth and liver fibrosis was markedly inhibited. Collagen content was significantly reduced in CnP-treated liver. Conclusion Conophylline suppresses HSC and induces apoptosis in vitro. CnP also attenuates formation of the liver fibrosis induced by TAA in vivo.

We retrospectively analyzed the courses of 37 non-insulin dependent diabetics (hemodialyzed:HD group) with end-stage renal disease (ESRD), to identify factors predisposing to renal failure. The factors analyzed were: diabetic (non-proliferative and proliferative) retinopathy, family histories of diabetes and hypertension, smoking, dyslipidemia, first examination proteinuria and non-compliance. These factors were statistically compared in 37 NIDDM without renal failure (non-HD group). There were no significant differences in age or duration of diabetes between the two groups. Significant differences (P < 0.001) were, however, recognized in diabetic proliferative retinopathy and hypertension between the two groups. Hypertension was present in 35/36 (97.2%) HD patients and in 21/36 (58.3%) non-HD patients. A family history of hypertension was recognized in 16/37 HD (43.2%) and in 7/33 (21.2%) non-HD (P < 0.05). Differences were recognized in HDL-cholesterol, LDL-cholesterol and TG levels (38.2 +/- 12.5 mg/dl and 56.7 +/- 18.5 mg/dl, 140.4 +/- 57.1 mg/dl and 115.6 +/- 33.6 mg/dl, 169.9 +/- 89.4 mg/dl and 115.7 +/- 75.1 mg/dl, in HD and non-HD, respectively, P < 0.05). First visit proteinuria was found in all HD patients, and in 6/34 (17.6%) non-HD. The difference in previous treatment refusal, for 7 or more years, was significant with 23/36 (58.9%) HD patients and only 1/25 (4.0%) non-HD patients (P < 0.001) having a history of prolonged non-compliance with diabetic treatment. Diabetic retinopathy, non-proliferative and proliferative, hypertension and a family history of hypertension, elevated triglyceride and LDL-cholesterol, low HDL-cholesterol, first visit proteinuria, and prolonged non-compliance correlated with progression to ESRD. We advocate expanding diabetic education to include prevention of complications such as diabetic nephropathy.

A murine diabetes mellitus induced with a new diabetogenic variant (DK-27) which we isolated from the M variant of the encephalomyocarditis (EMC) virus was characterized. Male DBA/2 mice (9.5 weeks old) were infected with various infectious doses of DK-27 intraperitoneally. Blood glucose and insulin levels were examined in association with the viral replication. Pancreatic pathology and hormone contents and stable hemoglobin A1c (St-A1c) levels were also examined on the final day of observation (35 days of post-infection). In infected mice, blood glucose levels rapidly elevated at 72 hr, slightly decreased between 7 and 10 days and finally became sustained hyperglycemia. On the other hand, blood insulin levels elevated at 48 hr, promptly decreased, and subsequently became sustained hypoinsulinemia. Viral replication in pancreases reached the highest titers at 48 hr and rapidly disappeared with all infectious doses used. Pancreatic insulin contents in infected mice were not detectable, and glucagon contents were not affected. In pathological examination, atrophy of islets and marked diminution of B-cells were observed, and A-cells occupied the major part of an infected islet. St-A1c levels reflected lasting hyperglycemia. These findings show that DK-27 causes insulin-dependent diabetes mellitus by the specific and direct destruction of pancreatic B-cells in susceptible mice. Such a diabetic model mouse will be useful for therapeutic studies.

Macrophages from recent onset non-obese diabetic (NOD) mice showed cytotoxicity against the NOD mouse derived beta-cell line, MIN6N-9a. In this report, we examined whether nitric oxide is associated with beta-cell destruction. Peritoneal exudate cells (PEC), obtained from cyclophosphamide treated NOD mice showed higher cytotoxicity against MIN6N-9a compared to PECs from saline injected NOD mice (P

Chronic inflammation is involved in the pathogenesis of cardiovascular diseases (CVD). Several prospective studies have indicated that an elevated high sensitive C-reactive protein (hs-CRP) level is a risk factor for CVD. These results were also confirmed by prospective studies in Japan both for primary and secondary prevention. A randomized control study using statins also revealed that lower levels of both LDL cholesterol and hs-CRP were independently related to the incidence of CVD. Recent meta-analysis revealed that hs-CRP was a risk factor not only for CVD but for other diseases including cancers. It revealed that the absolute value of hs-CRP varied among the study populations. The mechanism of how hs-CRP is associated with the pathogenesis of CVD is not fully understood. Generally, inflammation in the vascular wall and the release of inflammatory cytokines from macrophages was considered to the main mechanism, but infection with such as chlamydia or Helicobacter pylori, and periodontal disease have been postulated as the causes of systemic inflammation. Recently, visceral fat accumulation and its cross-interaction with inflammatory cells have been proposed as the cause of systemic inflammation as "innate inflammation". Our original cross sectional studies also showed the correlations of hs-CRP with BMI and triglyceride. Although there is no specific therapy for the reduction of hs-CRP, we have to consider hs-CRP as a risk factor for CVD which complements other classical risk factors.

To elucidate the roles of macrophages in the pathogenesis of NOD murine diabetes, peritoneal macrophages from NOD mice were injected into young NOD mice. We used 12 to 20 week-old NOD mice of both sexes as donors, and sex-matched 2-week-old NOD mice as recipients. Cyclophosphamide (CY), 200 mg/kg, was intraperitoneally injected into the donors. Two weeks later, peritoneal exudate cells (PEC) were collected from the diabetic donors. Macrophagerich fractions (MRF) were collected by adherence. Then PEC(5–8 × 10 6 ) or MRF(3–7 × 10 6 ) were transferred, intraperitoneally, to the recipients. Two weeks later, some of the recipients were killed in order to perform immunofluorescent analysis of splenocytes and to assess pancreatic histology. Mac 1 positive splenocytes were increased in PEC- and in MRF-injected recipient mice. Insulitis was seen in PEC- and MRF-injected mice, but not in controls. Some of the recipients were injected with CY, 200 mg/kg, intraperitoneally, at two weeks post cell transfer. Two weeks after CY injection, the animals were examined for the presence of diabetes. The incidences of diabetes were 67% in PEC-injected mice, 40% in the MRF-injected group, and 3% in the controls. These results suggest that peritoneal macrophages accelerate the disease process in NOD mice.

A long-term investigation of bone mineral metabolism in a newly developed strain, the WBN/Kob rat, which spontaneously develops diabetes, possibly due in part to hemosiderin deposition, was conducted. WBN/Kob rats used in this study developed diabetes after 9 months of age. Bone mass peaked at 6 months or 8 months of age, and femoral breaking strength was maximal at 8 months of age, declining rapidly after the development of diabetes. In contrast, both the bone mass and the mechanical strength increased up to 14 months of age in controls. The serum osteocalcin (BGP) levels were lower at 4 months of age and serum 1.25(OH)2D levels were significantly lower throughout the study in WBN/Kob rats than in controls. These results suggest that abnormal bone and mineral metabolism is present in WBN/Kob rats before the onset of diabetes, and that bone strength and BMD decrease simultaneously with the development of diabetes. This strain can serve as a useful model, not only of hemosiderosis and diabetes, but also of osteopenia.

Transgenic expression of the MHC (major histocompatibility complex) class II I-Ak molecule was previously shown to effectively reduce the incidence of insulitis in non-obese diabetic (NOD) mice at the age of 20 weeks. We have further characterized the expression and function of the I-Ak molecule and examined its effects on the incidence of diabetes in NOD mice. The newly expressed I-Ak molecule was recognized as an alloantigen by the T lymphocytes of normal NOD mice as shown by mixed lymphocyte reaction (MLR). The levels of endogenous I-Ag7 expression on peripheral blood lymphocytes were not affected by the transgene expression. Transgenic NOD mice were completely resistant to spontaneous diabetes, but the treatment by cyclophosphamide, which effectively induces diabetes in normal NOD mice, caused diabetes, although at a much lower incidence than that of normal NOD mice. On the basis of these findings, we discuss the role of I-Ak in the prevention of diabetes in NOD mice.

Class I membrane glycoproteins encoded by major histocompatibility complex (MHC) genes are not expressed during early stages of development. This regulation is thought to play an important role in maternal tolerance of the fetal allograft. To elucidate the significance of developmental regulation of MHC class I genes, we produced transgenic mice expressing transgenes encoding the class I Ld heavy chain or the light chain, β2-microglobulin, in the developing mouse embryo. These transgenes were driven by the chiken β-actin promoter, which is very active in the developing mouse embryo. The heavy chain and light chain transgenic mice were mated, and the resultant double transgenic offspring expressed Ld antigen in all the tissues examined by immunostaining and Northern blot analysis. The Ld antigen was also detected by immunostaining in the placenta of the double transgenic fetus. The double transgenic fetus was not rejected by the immunocompetent nontransgenic mother. These results suggest that expression of MHC class I antigens in embryos is not sufficient to provoke a maternal immune response.

Glutamic acid decarboxylase antibodies (GAD65Ab) are common in new onset Caucasian insulin-dependent diabetic (IDDM) patients but it is unclear if this marker is also prevalent in patients of other ethnic backgrounds. We determined antibodies against human recombinant GAD in Japanese diabetic patients using a radioimmunoassay with competition between in vitro translated 35S-GAD65 and non-labelled recombinant human GAD65 (rhGAD65). GAD67 antibodies (GAD67Ab) were similarly analyzed but without antigen competition. In 73 Japanese diabetic patients, GAD65Ab were found in 11/16 (69%) of patients with short-duration (less than 5 yrs) IDDM, 6/23 (26%) with long-duration (5 or more yrs) IDDM and 10/20 (50%) with slowly progressive diabetes. High GAD65Ab levels were associated with concomitant autoimmune diseases (p = 0.021). GAD67Ab were found in 4/16 (25%) of patients with short-duration IDDM, 3/23 (13%) with long-duration IDDM and 2/20 (10%) with slowly progressive diabetes. In 14 non-insulin dependent diabetic (NIDDM) patients, GAD65Ab and GAD67Ab were not found (0/14) and 1/50 (2%) healthy controls were positive in either assay. Among the GAD67Ab-positive samples, 8/9 (88%) were also high level GAD65Ab positive, 7/9 (77%) were displaced by an excess of rhGAD65 and the antibody levels correlated (r2 = 0.573; p = 0.003). Our data are consistent with a strong association of GAD65Ab also in Japanese IDDM, and suggest that, when present, GAD67Ab are frequently directed to epitope(s) common to GAD65 and GAD67.

Activin A is a differentiation factor for β-cells and is effective to promote β-cell neogenesis. Activin A is also an autocrine activator of pancreatic stellate cells, which play a critical role in fibrogenesis of the pancreas. Conophylline (CnP) is a natural compound, which reproduces the effect of activin on β-cell differentiation and promotes β-cell neogenesis when administered in vivo. However, its effect on stellate cells is not known. We therefore investigated the effect of CnP on stellate cells both in vitro and in vivo. Unlike activin A, CnP inhibited activation of cultured stellate cells and reduced the production of collagen. We then analyzed the involvement of stellate cells in islet fibrosis in Goto-Kakizaki (GK) rats, a model of type 2 diabetes mellitus. In pancreatic sections obtained from 6-wk-old GK rats, CD68-positive macrophages and glial fibrillary acidic protein- and α-smooth muscle actin-positive stellate cells infiltrated into islets. Later, the number of macrophages was increased, and the α-smooth muscle actin staining of stellate cells became stronger, indicating the involvement of stellate cells in islet fibrosis in GK rats. When CnP was administered orally for 4 wk, starting from 6 wk of age, invasion of stellate cells and macrophages was markedly reduced and islet fibrosis was significantly improved. The insulin content was twice as high in CnP-treated rats. These results indicate that CnP exerts antifibrotic actions both in vitro and in vivo and improves islet fibrosis in Goto-Kakizaki rats.

The cytotoxicity of macrophages from non-obese diabetic (NOD) mice against murine mastocytoma (P-815), and murine beta-cell lines having the NOD gene background (MIN6N-9a), were examined. Peritoneal exudate cells from 20-week-old mice showed higher cytotoxicity, measured as inhibition of thymidine uptake into P-815, than those from 12-week-old mice (p

We have investigated whether antibodies to heat shock protein (hsp) 65 are present in sera from patients with insulin-dependent diabetes mellitus by using Mycobacterium leprae hsp65. Fifty-two sera from patients with IDDM, 36 from patients with unclassified insulin-treated diabetes mellitus and 41 from normal healthy controls were examined by ELISA assay. Seventeen (32.7%) out of 52 IDDM sera and 10 (27.8%) out of unclassified insulin-treated diabetic sera were positive for anti- Mycobacterium (anti- M. leprae ) hsp65 antibodies while none of the healthy control sera were positive. Based on western blot analysis, 12 of the 17 IDDM sera and 1 of 2 sera from the unclassified insulin-treated diabetics were positive for anti- M. leprae hsp65 antibodies while all normal control sera were negative. These results support the idea that hsp65 may play a role in the pathogenesis of IDDM. Future studies are necessary to elucidate the role of hsp65 in the pathogenesis of IDDM.

A 21-yr-old female with hyperthyroidism is described. Though her serum-free T3 was 17.8 pmol/L and free T4 was 60.2 pmol/L, TSH was as high as 10.7 mU/L. TRH stimulated an increase in TSH from 10.7-91.7 mU/L. T3 administration in gradually increasing doses of 100, 200, and 400 mg/day resulted in gradual reduction in serum TSH. Cranial computed tomography and magnetic resonance imaging revealed a microadenoma of the pituitary gland. Histology of the surgical specimen showed a TSH-producing adenoma with TSH cell cluster islets and decreased numbers of TSH cells in the nonneoplastic pituitary. Cultured cells from the adenoma secreted TSH spontaneously and in response to TRH. This TRH-stimulated TSH secretion was suppressed by T3 in a dose-dependent manner. One year postoperatively, neither residual tumor nor recurrence were seen by computed tomography and magnetic resonance imaging. However TSH, as well as free T3 or T4, was still high and overresponsive to TRH.