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Aims/introduction: Several research groups have reported methods for quantifying pancreatic beta cell (β-cell) injury by measuring β-cell-specific CpG unmethylation of the insulin gene in circulation using digital droplet PCR or next-generation sequencing. However, these methods have certain disadvantages, such as the need to consider the background signal owing to the small number of target CpG sites and the need for unique equipment., Materials and Methods: We established a novel method for detecting four CpG unmethylations of the insulin gene using two-step amplification refractory mutation system PCR. We applied it to type 1 diabetes (T1D) patients with a wide range of disease durations and to healthy adults., Results: The assay showed high linearity and could detect a single copy of unmethylated insulin DNA in experiments using methylated and unmethylated plasmid DNA. The unmethylated insulin DNA level in the type 1 diabetes group, whose β-cell mass was considerably reduced, was similar to that of healthy adults. An inverse correlation was observed between copy number and disease duration in patients with unmethylated insulin DNA-positive type 1 diabetes., Conclusions: We developed a novel method for detecting unmethylated insulin DNA in circulation that can be performed using a conventional real-time PCR system. This method would be useful for analyzing dynamic profiles of β-cells in human disease such as type 1 diabetes., (© 2022 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

The incidence of type 2 diabetes increases with age. The age-dependent decline in functional β-cell mass contributes to the increased risk of onset of diabetes, reflecting the central role of pancreatic β-cells in glucose homeostasis. Indeed, the replication rate of human and rodent β-cells is known to decline sharply with age, and such a characteristic of β-cells might explain the increased onset of type 2 diabetes in the older population. The molecular mechanism involved in the age-dependent decline of β-cell proliferation has been extensively studied, mainly using rodents and in vitro culture systems, but its molecular basis is still largely unknown. A mechanism by which glucagon-like peptide-1 receptor activation induces human β-cell proliferation only within a restricted time window was recently suggested in a study in which human islets were grafted into immunodeficient mice. The authors found that the mitogenic effects of exendin-4 require calcineurin/nuclear factor of activated T-cells signaling, and that only in juvenile islets, exendin-4 induced the expression of nuclear factor of activated T-cells signaling components, as well as downstream target genes that facilitate β-cell proliferation. These findings provide a mechanistic explanation as to why glucagon-like peptide 1 exerts mitogenic effects only in juvenile human β-cells., (© 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Aims/introduction: Dipeptidyl peptidase-4 inhibitors and glinides are effective in reducing postprandial hyperglycemia. However, little information is available on the comparative effects of the two drugs on the levels of postprandial glucose. The aim of the present study was to compare the effects of sitagliptin and nateglinide on meal tolerance tests in drug-naïve patients with type 2 diabetes mellitus., Materials and Methods: The study participants were 19 patients with type 2 diabetes mellitus, which was inadequately controlled by diet and exercise. An open-label, prospective, cross-over trial was carried out to compare the effects of single-dose sitagliptin and nateglinide on the postprandial glucose level and its related hormones during meal tests., Results: The change in area under the curve (AUC) of glucose from 0 to 180 min (AUC0-180 min) during the meal test by nateglinide was similar to that by sitagliptin. As expected, the change in active glucagon like peptide-1 was significantly higher after a single-dose of sitagliptin than nateglinide. Then, insulin secretion relative to glucose elevation (ISG) (ΔISG0-180 min: ΔAUC0-180 min insulin/AUC0-180 min glucose) was significantly enhanced by nateglinide compared with sitagliptin. Conversely, glucagon level (ΔAUC0-180 min glucagon) was increased by administration of nateglinide, whereas the glucagon level was reduced by administration of sitagliptin., Conclusions: The effects of sitagliptin on postprandial glucose levels were similar to those of nateglinide in drug-naïve type 2 diabetes patients. However, the induced changes in insulin, active glucagon-like peptide-1 and glucagon during meal loading suggest that reduction of postprandial hyperglycemia was achieved by the unique effect of each drug.

Aims/introduction: Recent data have shown that ectopic fat accumulation in the liver worsens hepatic glucose metabolism, suggesting that fatty liver in patients with type 2 diabetes is a therapeutic target. Glucagon-like peptide (GLP)-1 improves fatty liver, but the effect of dipeptidyl peptidase-4 inhibitor on fatty liver is still unclear. The present pilot study determined the effects of 12-week treatment with sitagliptin, a dipeptidyl peptidase-4 inhibitor, on liver fat content in type 2 diabetes with fatty liver. We also evaluated intramyocellular lipid (IMCL) and glucose kinetics during oral glucose tolerance test (OGTT) before and after the treatment., Materials and Methods: The study participants were seven type 2 diabetes patients with fatty liver who were studied at baseline and 12 weeks after sitagliptin treatment. Intrahepatic lipid (IHL) and IMCL were assessed by (1)H magnetic resonance spectroscopy. Glucose kinetics was assessed during double-tracer OGTT (U-[(13)C]-glucose orally and 6,6-[(2)H2]-glucose intravenously)., Results: Sitagliptin significantly reduced glycated hemoglobin (from 7.1 ± 0.2 to 6.5 ± 0.3%, P < 0.005), but had no effects on IHL and IMCL. The glucose level diminished, whereas GLP-1 concentration increased during OGTT at the end of treatment. These changes were not accompanied by significant changes in insulin or glucagon levels. However, long-term sitagliptin treatment partially decreased the rate of appearance of oral glucose during OGTT, but did not affect endogenous glucose production or the rate of disappearance., Conclusions: It was found that 12-week sitagliptin treatment improved glycated hemoglobin and glucose excursion during OGTT in type 2 diabetes with fatty liver, independent of changes in lipid accumulation in the liver. This trial was registered with the Japan Clinical Trials Registry (UMIN-CTR000005666).

Aims/introduction: To investigate the efficacy and safety of vildagliptin, a potent dipeptidyl peptidase-4 inhibitor, as add-on to nateglinide, compared with switching to vildagliptin in Japanese type 2 diabetes patients poorly controlled with nateglinide., Materials and Methods: A total of 40 patients inadequately controlled with nateglinide were randomized to the switching group (n = 20, switching from nateglinide to vildagliptin) or combination group (n = 20, nateglinide plus vildagliptin). A meal tolerance test was carried out at weeks 0 and 24., Results: The mean changes in glycated hemoglobin from baseline to week 24 were -1.2 ± 0.3% and -0.3 ± 0.5% in patients of the combination and switching groups, respectively, and the difference between the groups was statistically significant (P < 0.001). The mean changes in area under the curve of glucose from 0 to 180 min (AUC0-180 min) from baseline to week 24 was -361 ± 271.3 mmol·min/L in patients of the combination group compared with 141 ± 241.9 mmol·min/L in those of the switching group (P < 0.001). The incidence of hypoglycemic events was low (three in the combination group), and none of the patients developed severe hypoglycemia. Although the addition of vildagliptin to nateglinide did not significantly increase insulin secretion relative to glucose elevation (ISG) after meal load (ISG0-180 min: AUC0-180 min insulin / AUC0-180 min glucose) in comparison with that in baseline, the mean ISG0-30 min 24 weeks after addition of vildagliptin to nateglinide was significantly higher than that at baseline. In contrast, switching from nateglinide to vildagliptin reduced the mean ISG0-180 min, relative to baseline., Conclusions: The combination therapy of vildagliptin and nateglinide is effective and safe in Japanese type 2 diabetes, and the improved glycemic control is as a result of augmentation of nateglinide-induced early phase insulin secretion. This trial was registered with UMIN (no. ID000004010).

Aims/introduction: 'Morningness' and 'eveningness' represent the sleep-wake patterns of the circadian rhythm might also affect glycemic control in patients with type 2 diabetes. The aim of this study was to examine the relationship between the morningness-eveningness trait and metabolic parameters., Materials and Methods: The study participants comprised 101 Japanese male workers with type 2 diabetes treated in an outpatient clinic. Blood samples were obtained, and a morningness-eveningness questionnaire (MEQ), where a high score represents morningness; and the Pittsburg Sleep Quality Index (PSQI), where the higher the score the worse the sleep quality, were carried out., Results: MEQ correlated positively with age, and high-density lipoprotein cholesterol (HDL-C), and negatively with glycated hemoglobin (HbA1c) and PSQI. Multivariate regression analysis showed that MEQ was significantly associated with HbA1c and HDL-C. In addition, we classified the study patients into three groups: 'morning type', 'neither type' and 'evening type' according to the sum of the MEQ score, and analyzed the difference between morning type (n = 32) and evening type (n = 11). We found that HbA1c, low-density lipoprotein cholesterol and PSQI of the morning type group were significantly lower than those of the evening type group., Conclusions: The present study suggests that 'eveningness' type male Japanese workers with type 2 diabetes suffer inadequate glycemic control.

Previous studies reported that both cilnidipine and azelnidipine have a renoprotective effect compared with amlodipine. The aim of this study was to compare the effects of cilnidipine and azelnidipine on blood pressure, heart rate and albuminuria. An open-label prospective crossover trial was carried out. We recruited 19 type 2 diabetics treated with amlodipine (5 mg/day) at least for 12 weeks. At study entry, amlodipine was changed to cilnidipine (10 mg/day) or azelnidipine (16 mg/day) and each administered for 16 weeks. Then, the drugs were switched and the treatment was continued for another 16 weeks. Despite no differences in 24-h blood pressure and heart rate between cilnidipine and azelnidipine, treatment with cilnidipine resulted in a greater reduction in urinary albumin:creatinine ratio than azelnidipine. Our results suggested that cilnidipine is more efficient in reducing albuminuria than azelnidipine independent of its blood pressure lowering effect in type 2 diabetic patients with hypertension. This trial was registered with UMIN (no. 000007201).

The level of intramyocellular lipids (IMCL) is a physiological marker of skeletal muscle function. (1)H-magnetic resonance spectroscopy (MRS) is an established method to measure IMCL contents in vivo. However, all of the MR systems do not always contain measurement instruments for (1)H-MRS, thus in a clinical setting, alternative methods for estimation of IMCL content are needed. Here, we investigated the association between T1 and T2 relaxation times, determined by MR imaging, and IMCL measured by (1)H-MRS in the soleus and tibialis anterior muscles of 15 healthy male subjects. Intriguingly, in the soleus muscle, but not in the tibialis anterior muscle, T2 relaxation time correlated significantly with IMCL (r = 0.65, P < 0.05). The result suggests the possibility that T2 relaxation time of the soleus muscle can be used to estimate IMCL in a clinical setting. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00108.x, 2011).

Unlabelled: Aims/Introduction:  Accumulation of intramyocellular lipid (IMCL) is associated with insulin resistance. However, the factors affecting the change in IMCL remain to be elucidated. The aim of the present study was to determine the factors that influence the change in IMCL level after high-fat loading., Materials and Methods:   The study subjects were 37 non-obese men. Each subject consumed a high-fat diet for 3 days after a normal-fat diet for 3 days. After each diet program, IMCL levels in the tibialis anterior (TA-IMCL) and soleus (SOL-IMCL) were measured by proton magnetic resonance spectroscopy. Glucose infusion rate (GIR) was evaluated by euglycemic hyperinsulinemic clamp as an index of peripheral insulin sensitivity., Results:   The high-fat diet significantly increased TA-IMCL and SOL-IMCL by ∼30 and ∼20%, respectively (P < 0.05), whereas it did not significantly alter GIR. The increase in SOL-IMCL, but not in TA-IMCL, negatively correlated with serum high molecular weight (HMW)-adiponectin (r = -0.36, P < 0.05) and HMW-/total-adiponectin ratio (r = -0.46, P < 0.05). Although high-fat diet-related changes in SOL-IMCL showed high inter-individual variations, in subjects doing exercise, changes in SOL-IMCL (r = 0.55, P < 0.05) and TA-IMCL (r = 0.61, P < 0.05) positively correlated with daily physical activity level. In contrast, in sedentary subjects, changes in SOL-IMCL (r = -0.50, P < 0.01) and TA-IMCL (r = -0.48, P < 0.05) negatively correlated with daily physical activity., Conclusions:   HMW-adiponectin and daily physical activity are determinants of IMCL accumulation by a high-fat diet. Intriguingly, the effect of daily physical activity on the change in IMCL depends on the level of regular exercise. (J Diabetes Invest,doi: 10.1111/j.2040-1124.2010.00091.x, 2011).

Unlabelled: Aims/Introduction:  2-Methoxyestradiol (2ME) is an estradiol metabolite with little estrogenic activity. Previous data identified its anti-carcinogenic properties and possible cardiovascular benefits. However, its effect on diabetes mellitus has not been fully elucidated. The aim of the present study was to determine the effects of 2ME on glucose metabolism in the diabetic state., Materials and Methods:   To evaluate the effects of 2ME, pellets of two different doses of the drug were implanted into female db/db mice at the age of 5 weeks. Intraperitoneal glucose tolerance test and insulin tolerance test were carried out at the age of 8 weeks. The pancreas was harvested for morphological analysis and β-cell function at the age of 9 weeks., Results:   2ME improved random blood glucose levels and glucose tolerance with increases in insulin levels during an intraperitoneal glucose tolerance test. Insulin sensitivity judged by an insulin tolerance test was comparable in the low- and high-dose 2ME groups and the control group. Although glucose-stimulated insulin secretion in isolated islets was comparable among the three groups, β-cell mass in 2ME-treated groups was higher than the control group. In the 2ME-treated groups, the number of Ki67-positive cells in islets was higher, whereas the number of cleaved caspase-3-positive cells was comparable with the control., Conclusions:   2ME ameliorates glucose tolerance by promoting the proliferation of β-cell mass in db/db mice. Our data suggests its potential clinical usefulness as a disease-modifying drug for type 2 diabetes mellitus. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00087.x, 2011).

Unlabelled: Aims/Introduction:  The present study was designed to determine the effects of pioglitazone on glycemic control and atherosclerosis in patients with poorly controlled type 2 diabetes on insulin therapy., Materials and Methods:   The study was a prospective, randomized controlled trial involving 48 patients with inadequately controlled type 2 diabetes treated with insulin. We assigned patients to oral pioglitazone titrated from 15-30 mg (n = 22) or no pioglitazone (n = 26), to be taken in addition to their glucose-lowering drugs and other medications. Daily insulin doses and numbers were recorded during the study period., Results:   The adjusted mean glycosylated hemoglobin (HbA1c) values decreased significantly by 1.13 ± 1.50% and 0.55 ± 0.76% in the pioglitazone and control groups, respectively. Significant decrease of HbA1c level was observed in the pioglitazone group compared with the control group (P < 0.05). The insulin dose lowered by 0.04 ± 0.10 units/kg/day in the pioglitazone group and increased by 0.03 ± 0.10 units/kg/day in the control group (P < 0.05). The number of insulin injections decreased by 0.1 ± 0.6 times/day in the pioglitazone group and increased by 0.2 ± 0.4 times/day in the control group (P < 0.05). The carotid intima-media thickness estimated by B-mode echography was carried out in both groups and decreased significantly at the end-point only in the pioglitazone group, relative to the baseline., Conclusions:   These findings show that pioglitazone is useful in improving glycemic control and preventing the progression of atherosclerosis in poorly-controlled type 2 diabetics on insulin therapy. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00064.x, 2010).

Unlabelled: Aim/Introduction:  Preservation of β-cell mass is crucial for maintaining long-term glucose homeostasis. Therapies based on incretin and its mimetics are expected to achieve this goal through various biological functions, particularly the restoration of β-cell mass. Here we tested the effects of gastrin and exendin-4 in type 2 diabetic animals., Materials and Methods:   The effects of exendin-4 and gastrin on β-cell function and mass were examined in 8-week-old db/db mice. INS-1 beta cells and AR42J cells were used to determine the molecular mechanism underlying the effects of the two agents. Immunohistochemistry, western blotting and RT-PCR assays were used to assess the biological effects of the two agents., Results:   Two weeks of combination administration of exendin-4 plus gastrin resulted in a significant improvement of glucose tolerance associated with a marked preservation of β-cell mass in db/db mice. Immunohistochemical analysis showed that such treatment resulted in the appearance of numerous irregularly-shaped small islets and single insulin-positive cells. While gastrin had little biological effect on INS-1 β-cells consistent with low expression of its intrinsic receptor on these cells, it caused differentiation of AR42J cells into insulin-producing cells. Co-stimulation with exendin-4 significantly enhanced gastrin-induced endocrine differentiation of AR42J precursor cells. These findings were further supported by enhanced expression of key genes involved in β-cell differentiation and maturation, such as neurogenin3 (Ngn3) and MafA., Conclusions:   These results suggest that combination treatment of db/db mice with exendin-4 and gastrin preserves β-cell mass by stimulating β-cell growth and differentiation. (J Diabetes Invest, doi: 10.1111/j.2040-1124.00044.x, 2010).

Unlabelled: Aims/Introduction:  Basal-bolus intensive insulin therapy has been believed to achieve best the glycemic control, but is also complicated as a result of the number of injections required and the type of insulin. This study compared the effect of thrice-daily lispro 50/50 (prandial premixed therapy [PPT]) with thrice daily lispro given in combination with sulfonylureas (prandial bolus therapy with sulfonylurea [PBTS]) as initial insulin therapy for type 2 diabetes., Materials and Methods:   This 24-week, observational, parallel trial comprised a 12-week screening period and a 24-week intervention period for 31 diabetes patients who were poorly controlled with submaximal sulfonylurea. At the start of the intervention period, we commenced thrice-daily insulin injections and divided the 31 patients into either lispro 50/50 with discontinuation of sulfonylurea (PPT, n = 15) or lispro added to sulfonylurea (PBTS, n = 16). The same dose-adjustment algorithm was used for analyzing both groups; HbA1c, plasma glucose, insulin daily dose, bodyweight and number of hypoglycemic episodes were evaluated., Results:   At the end of the study, HbA1c was significantly improved in both groups (P < 0.00001), but no difference was apparent between the groups. The daily doses of PPT were more than those of PBTS, albeit the difference was statistically insignificant (P = 0.051). There were significantly fewer hypoglycemic episodes encountered with PPT than with PBTS., Conclusions:   Thrice-daily injections of lispro 50/50 provide an effective and safe regimen as initial insulin therapy for type 2 diabetes. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00025.x, 2010).