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Autophagy has been reported to play a crucial role in the maintenance of intracellular homeostasis, including in pancreatic beta cells. Rubicon, which interacts with the phosphoinositide 3-kinase (PI3K) complex, through autophagy-related 14 (ATG14), is among the few autophagy regulators that have been reported to inhibit autophagic flux to date and the deletion of Rubicon has been shown to increase autophagic flux. Based on previous results showing a causal relationship between autophagic dysfunction and pancreatic beta-cell impairment, we hypothesized that the deletion of Rubicon in pancreatic beta cells would improve cell integrity and confer protective effects. To test this hypothesis, we first confirmed that Rubicon knockdown (KD) promoted autophagic flux in βTC3 pancreatic beta-cell line. Next, we generated pancreatic beta-cell-specific Rubicon knockout (βKO) mice, by administering tamoxifen to Rubicon flox/flox :MIP-Cre-ERT mice, which showed normal glucose tolerance and insulin secretion under a normal chow diet, despite successful gene recombination. We also attempted to increase insulin resistance by feeding the mice with a high-fat diet for an additional 2 months to find little differences among the parameters evaluated for glucose metabolism. Finally, severe insulin resistance was induced with insulin receptor antagonist treatment, which resulted in comparable glucose homeostasis measurements between Rubicon βKO and control mice. In summary, these results suggest that in pancreatic beta cells, Rubicon plays a limited role in the maintenance of systemic glucose homeostasis.

Pancreatic polypeptide (PP) is a 36-amino acid peptide encoded by the Ppy gene, which is produced by a small population of cells located in the periphery of the islets of Langerhans. Owing to the high amino acid sequence similarity among neuropeptide Y family members, antibodies against PP that are currently available are not convincingly specific to PP. Here we report the development of mouse monoclonal antibodies that specifically bind to PP. We generated Ppy knockout (Ppy-KO) mice in which the Ppy-coding region was replaced by Cre recombinase. The Ppy-KO mice were immunized with mouse PP peptide, and stable hybridoma cell lines producing anti-PP antibodies were isolated. Firstly, positive clones were selected in an enzyme-linked immunosorbent assay for reactivity with PP coupled to bovine serum albumin. During the screening, hybridoma clones producing antibodies that cross-react to the peptide YY (PYY) were excluded. In the second screening, hybridoma clones in which their culture media produce no signal in Ppy-KO islets but detect specific cells in the peripheral region of wild-type islets, were selected. Further studies demonstrated that the selected monoclonal antibody (23-2D3) specifically recognizes PP-producing cells, not only in mouse, but also in human and rat islets. The monoclonal antibodies with high binding specificity for PP developed in this study will be fundamental for future studies towards elucidating the expression profiles and the physiological roles of PP.

Treatment-related quality of life (QOL) is an important aspect of diabetes management. However, no studies have compared the influence of dipeptidyl peptidase-4 inhibitors versus alpha-glucosidase inhibitors on treatment-related QOL. This prespecified sub-analysis of the Linagliptin Study of Effects on Postprandial blood glucose (L-STEP) compared the effects of linagliptin (5 mg once daily) and voglibose (0.2 mg/meal thrice daily) on treatment-related QOL in Japanese patients with type 2 diabetes (T2DM) inadequately controlled with diet and exercise therapy. Among 366 subjects in the original study, 182 in the linagliptin group and 173 in the voglibose group were included in this analysis. The outcome of this study was change in QOL as assessed by the Diabetes Therapy-Related Quality of Life 17 (DTR-QOL17) questionnaire from baseline to week 12. Compared with baseline data, total DTR-QOL17 scores were significantly higher after 12 weeks of linagliptin and voglibose treatment. The change in the total DTR-QOL17 score and the score of one domain, burden on social activities and daily activities, was significantly greater in the linagliptin group than in the voglibose group. In addition, only linagliptin treatment was identified as a factor associated with an increased total DTR-QOL17 score. Linagliptin is superior to voglibose in terms of improving treatment-related QOL in Japanese patients with T2DM.

Recently, we reported that linagliptin had equivalent efficacy to voglibose in reducing postprandial blood glucose levels in drug-naïve patients with type 2 diabetes (L-STEP Study). As a sub-study of the L-STEP Study we examined the effect of linagliptin on postprandial lipids profile. Between October 2012 and April 2014, the study enrolled patients with type 2 diabetes mellitus who had inadequate glycemic control. Patients were randomly assigned to either the linagliptin group (5 mg once daily, n = 85) or the voglibose group (0.2 mg/meal thrice daily, n = 71). Meal tolerance tests were performed at baseline (week 0) and endpoint (week 12). The increments in 4-h postprandial triglyceride, remnant lipoprotein cholesterol (RLP-C), and apolipoprotein B48 (ApoB48) from baseline to endpoint in the linagliptin group were lower (p < 0.001, p = 0.025 and p < 0.001). 4-h postprandial ApoB48 at endpoint was lower in the linagliptin group (p = 0.007), and positive correlation was detected between change of ApoB48 and changes in both triglyceride (r = 0.67, p < 0.001) and RLP-C (r = 0.73, p < 0.001) at 4 h. This study revealed that in drug-naïve Japanese patients with relatively mild type 2 diabetes mellitus, linagliptin improves not only postprandial blood glucose level but also levels of lipids such as TG and RLP-C by reducing the ApoB48 level compared with voglibose.

Recent studies have suggested that decreased pancreatic β-cell function and mass are common features of patients with type 2 diabetes mellitus. Pancreatic β-cell homeostasis is regulated by various types of signaling molecules and stress responses. Sequestosome 1/p62 (SQSTM1, hereafter referred to as p62) is a ubiquitin-binding adaptor protein involved in cell signaling, oxidative stress, and autophagy. Because p62 appears to play an important role in maintaining mitochondrial quality control, it is possible that the loss of p62 in pancreatic β cells contributes to mitochondrial dysfunction, and thus leading to impaired glucose tolerance. In this study we investigated the physiological roles of p62 by inactivating p62 in a β-cell specific manner. We found that firstly, rat insulin-2 promoter-Cre (RIP-Cre)-mediated p62 inactivation did not cause body weight gain, although ubiquitous inactivation of p62 was previously shown to result in severe obesity. Secondly, we found no gross structural disorganization of the islets of p62-deficient mice. Consistent with normal islet morphology, no impairment in glucose tolerance was observed in mice with RIP-Cre-mediated p62 deletion. These results suggest that p62 is dispensable for normal islet organization and β-cell function.

A small number of cells in the adult pancreas are endocrine cells. They are arranged in clusters called islets of Langerhans. The islets make insulin, glucagon, and other endocrine hormones, and release them into the blood circulation. These hormones help control the level of blood glucose. Therefore, a dysfunction of endocrine cells in the pancreas results in impaired glucose homeostasis, or diabetes mellitus. The pancreas is an organ that originates from the evaginations of pancreatic progenitor cells in the epithelium of the foregut endoderm. Pancreas organogenesis and maturation of the islets of Langerhans occurs via a coordinated and complex interplay of transcriptional networks and signaling molecules, which guide a stepwise and repetitive process of the propagation of progenitor cells and their maturation, eventually resulting in a fully functional organ. Increasing our understanding of the extrinsic, as well as intrinsic mechanisms that control these processes should facilitate the efforts to generate surrogate β cells from ES or iPS cells, or to reactivate the function of important cell types within pancreatic islets that are lost in diabetes.

Thyrotoxicosis with diffuse thyroid disease can be caused by Graves' disease (GD) or destructive thyroiditis (DT). Optimal treatment of the underlying condition requires a prompt and accurate method for the diagnosis of thyrotoxicosis. This study evaluated measurement of the mean peak systolic velocity of the superior thyroid artery (STA-PSV) by ultrasonography in detecting thyrotoxicosis in Japanese patients. We recruited 44 patients with untreated GD, 13 with DT, 55 with treated GD, and 49 subjects without thyroid disease. Blood samples were taken to analyze thyroid function and STA-PSV was measured by ultrasonography. The mean STA-PSV was the highest in the untreated GD group, followed by treated GD patients and then those with DT. Receiver operating characteristic curves of the STA-PSV values demonstrated that the area under the curve required discriminating untreated GD from DT was 0.941. The optimal sensitivity and specificity were 83.7% and 92.3%, respectively, using 45 cm/sec as the cutoff value. In conclusion measurement of STA-PSV by ultrasonography is useful for the diagnosis of thyrotoxicosis in Japanese patients.

Thyrotoxicosis with diffuse thyroid disease can be caused by Graves' disease (GD) or destructive thyroiditis (DT). Optimal treatment of the underlying condition requires a prompt and accurate method for the diagnosis of thyrotoxicosis. This study evaluated measurement of the mean peak systolic velocity of the superior thyroid artery (STA-PSV) by ultrasonography in detecting thyrotoxicosis in Japanese patients. We recruited 44 patients with untreated GD, 13 with DT, 55 with treated GD, and 49 subjects without thyroid disease. Blood samples were taken to analyze thyroid function and STA-PSV was measured by ultrasonography. The mean STA-PSV was the highest in the untreated GD group, followed by treated GD patients and then those with DT. Receiver operating characteristic curves of the STA-PSV values demonstrated that the area under the curve required discriminating untreated GD from DT was 0.941. The optimal sensitivity and specificity were 83.7% and 92.3%, respectively, using 45 cm/sec as the cutoff value. In conclusion measurement of STA-PSV by ultrasonography is useful for the diagnosis of thyrotoxicosis in Japanese patients.

The increased flux of polyol pathway induced by hyperglycemia is implicated in the pathogenesis of various complications associated with diabetic, which results in increased oxidative stress. Because oxidative stress causes tissue damage in patients with diabetes, searching for an effective strategy to reduce oxidative stress in clinical setting is important in order to prevent diabetic complications. The aim of this study was to evaluate the effects of aldose reductase inhibition on oxidative stress in patients with type 2 diabetes mellitus. The subjects of this study were 21 patients with type 2 diabetes. We compared the levels of various oxidative stress markers and antioxidants including plasma thiobarbituric acid-reactive substances, malondialdehyde-modified low-density lipoprotein, vitamin E, beta-carotene and lipid hydroperoxides in erythrocytes at baseline with those measured after a 3-month course of epalrestat (150 mg/day), an aldose reductase inhibitor. While administration of epalrestat did not result in significant changes in plasma thiobarbituric acid-reactive substances, malondialdehyde-modified low-density lipoprotein, vitamin E, or beta-carotene, it significantly reduced lipid hydroperoxides in erythrocytes. Given the importance of measuring lipid hydroperoxides in erythrocytes as an index of oxidative stress, these results highlight the potential usefulness of epalrestat in reducing oxidative stress in type 2 diabetes mellitus.

Neurogenin 3 (Ngn3) is a transcription factor that regulates an initial step of differentiation from uncommitted pancreatic progenitors into endocrine cells. Additional transcription factors are required for complete differentiation into mature pancreatic beta cells. In this study, we established an in vitro model system of beta-cell differentiation by adenovirus-mediated expression of several transcription factors in AR42J-B13 cells, a pancreatic progenitor-like cell line derived from exocrine pancreas. Exogenous expression of Ngn3 in AR42J-B13 cells induced expression of Nkx2.2, Pax4, and Pax6, which are all essential for beta-cell differentiation in mouse embryos. However, Ngn3 did not induce more downstream regulators of beta-cell differentiation, Nkx6.1 and Maf A. Coexpression of Nkx6.1 and Ngn3 induced endogenous expression of the insulin 2 gene, while coexpression of Maf A and Ngn3 induced both insulin 1 and 2 genes in AR42J-B13 cells. Our data demonstrated that Ngn3 expressed together with Nkx6.1 or MafA induces AR42J-B13 cells to differentiate into insulin-producing cells, supporting the use of these cells as a model system for studying beta-cell differentiation in vitro.

To compare the effects of postprandial hypertriglyceridemia and postprandial hyperglycemia on monocyte adhesion to endothelial cells, we investigated the effects of twice-daily standard diet (5% fat) and high-fat diet (30% fat) for 3 weeks on monocyte adhesion to endothelial cells and the expression of adhesion molecules in the aortic artery in non-obese type 2 diabetic Goto-Kakizaki rats. Fasting glucose, insulin, non-esterified fatty acid (NEFA), HbA1c, and body weight were comparable between the two diet groups. Postprandial glucose and insulin were higher in the standard diet group, while postprandial NEFA and triglyceride were higher in the high fat diet group, compared with the other group. The number of monocyte adherent to endothelial cells was higher in the high-fat diet group than the standard diet group. Consumption of high-fat diet resulted in overexpression of heme oxygenase-1, intercellular adhesion molecule-1 (ICAM-1), and connecting segment-1 fibronectin on the arterial wall, compared with standard diet. Thus, our data demonstrated that short-term intermittent high-fat diet prevented postprandial hyperglycemia in a model of type 2 diabetes without a significant increase in body weight. However, the resulting postprandial hypertriglyceridemia induces more monocyte adhesion to endothelial cells than postprandial hyperglycemia. This increased monocyte adhesion is associated with the increased aortic expression of adhesion molecules such as ICAM-1, and connecting segment-1 fibronectin.

A 66-year-old woman with type C hepatitis had been treated for hepatocellular carcinoma (HCC) with transcatheter arterial embolization and radiofrequency ablation. Liver function worsened gradually to decompensated liver cirrhosis. She had recurrence of HCC and was later admitted to Juntendo University Hospital for living-donor liver transplantation. Although blood glucose was high, she had never been diagnosed with diabetes mellitus. No diabetes-related complications were detected at that time. We started treatment with multiple insulin injections. There is a unique time called the anhepatic phase during liver transplantation during which the liver does not exist in the body. Recent reports show that it is not necessary to administer glucose for patients with normal glucose tolerance during the anhepatic phase since plasma glucose could be maintained at normoglycemia to hyperglycemia (100-150 mg/dl). In our patient, plasma glucose concentration was rather high during the anhepatic phase without glucose administration. We analyzed the levels of blood glucose, insulin and various other hormones during the anhepatic phase. This could be the first report on glucose homeostasis during the anhepatic phase in a diabetic patient.

To assess and compare the efficacy and safety of insulin glargine as intensive replacement of basal insulin in Japanese patients with type 1 (n = 72) and type 2 (n = 46) diabetes, we switched their intensive insulin regimen from NPH plus regular or rapid-acting insulin to glargine plus bolus insulin, which included regular and rapid-acting insulin, and recorded changes in glycemic control and frequency of hypoglycemia for 18 months. The dose titration of basal and bolus insulin was based on home self-monitored blood glucose measurements and monthly HbA(1C). Mean HbA(1C) level was improved significantly at 3 months after switching to glargine plus bolus insulin regimen and these effects continued for 18 months in both type 1 and type 2 diabetes patients (HbA(1C) level: type 1: baseline 8.9 +/- 2.6%, 18 months 7.8 +/- 1.5% (p<0.05), type 2: baseline 8.2 +/- 2.6%, 18 months 7.7 +/- 1.5%. Body weight was slightly but significantly increased at 18 months only in type 2 diabetes. Total daily bolus insulin doses were not changed but basal insulin could be increased significantly after switching regimens in both types diabetes compared with baseline. The frequency of mild to moderate hypoglycemia (self-assisted episodes, blood glucose <70 mg/dl) was marginally lower with glargine but not significantly. Self-monitored fasting blood glucose level was significantly improved after switching in type 2 diabetes. Patients with the worst HbA(1C) level at baseline exhibited more than 10% improvement in HbA(1C) level after switching both type 1 and type 2 diabetes. The HbA(1C) levels of the effectively treated patients were comparable to those of ineffectively treated ones at 6 months and the same improvement was seen at 18 months. Our results suggested that insulin glargine is more effective than NPH insulin as intensive replacement of basal insulin, particularly in those Japanese patients with difficult glycemic control with NPH insulin, equally in both type 1 and type 2 diabetes.

2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol with no affinity for estrogen receptors. It inhibits cell proliferation, thus is a potentially useful drug to block the progression of atherosclerosis. As a first step to examining the anti-atherosclerotic effects of 2-ME, we investigated monocyte adhesion to aortic endothelial cells, which is considered a prerequisite to atherosclerosis in vivo. Eight-week-old Sprague-Dawley rats were ovariectomized then treated by slow-release pellets with placebo, 17-beta-estradiol (5 microg/day), low-dose 2-ME (10 microg/day), or high-dose 2-ME (100 microg/day). After 6 weeks, en face analysis showed an increased number of monocytes adhering to endothelial cells of the thoracic aorta in ovariectomized rats compared with sham-operated controls. This increase was predominantly inhibited by treatment with 17beta-estradiol, and low-dose or high-dose 2-ME. The observed effects were unrelated to changes in serum lipids, blood glucose, or blood pressure. Our data suggested that 2-ME mediates the anti-atherosclerotic actions of estradiol at least in part by preventing monocyte adhesion to the aortic endothelium.

Anti-hypertensive agents with antioxidative effects are potentially useful for diabetic patients with hypertension to prevent the onset and progression of their complication. While dihydropyridine-type calcium antagonists are among the frequently used anti-hypertensive drugs, azelnidipine, a novel calcium antagonist, has been reported to have a unique anti-oxidative effect in vitro and in animals. In this study, we measured lipid hydroperoxides in human sample using diphenyl-1-pyrenylphosphine for the first time, and used the value of lipid hydroperoxides as an index of oxidative stress. Then, we compared the antioxidative properties of azelnidipine and amlodipine, a frequently used calcium antagonist in hypertensive diabetic patients. Administration of vitamin C and E for 8 weeks significantly reduced lipid hydroperoxides in erythrocyte membrane in normal subjects. In hypertensive diabetic patients, azelnidipine treatment for 12 weeks induced a more significant fall in erythrocyte lipid hydroperoxide level than amlodipine, though blood pressure during each treatment was comparable. Our data confirm the usefulness of lipid hydroperoxides in erythrocyte membrane as a marker of oxidative stress in vivo, and indicate that azelnidipine has a unique antioxidative property in human.

In this study, we described the effectiveness of metformin on Japanese type 2 diabetes patients receiving sulfonylureas and the clinical characteristics of the patients whose glycemic control were significantly improved with metformin administration. Our results showed that the reduction of glycohemoglobin (HbA1C), serum concentration of total cholesterol, and diastolic blood pressure was statistically significant through the administration of metformin. The clinical characteristics of the patients who responded to metformin therapy exhibited lower systolic blood pressure in addition to higher HbA1C value just before administration of metformin when compared with DeltaHbA1C (HbA1C 6 months after administration of metformin--HbA1C before administration of metformin). Moreover, effectiveness of metformin was weakened, in comparison with non-hypertensive patients, even though the blood pressure of hypertensive patients was reduced to normal range by medication with antihypertensive drugs. But average reduction of HbA1C level of hypertensive patients without antihypertensive medications was smaller than those of patients with high blood pressure with such medication. These results suggested that high blood pressure and hypertension phenotype itself were suppressive factors of metformin but antihypertensive therapy itself enhanced the effectiveness of metformin regardless of the improvement of blood pressure.

We have previously reported the therapeutic efficacy of mitiglinide combined with once daily insulin glargine (mitiglinide regimen) after switching from multiple daily insulin regimen of aspart insulin and glargine (intensive insulin regimen) in inpatients with type 2 diabetes mellitus in two consecutive days. In the present study, we followed up 9 of the 15 responsive patients with these novel regimens for 6 months after discharge. The data collected from these patients were compared to those of 15 randomly chosen patients who had well matched background and received intensive insulin regimen during hospitalization which was continued after discharge. The average HbA1c level of these 9 patients with mitiglinide regimen at 6 months was 6.7 +/- 0.8% and was comparable to that of the patients with intensive insulin regimen (HbA1c = 7.0 +/- 1.0%). In conclusion, mitiglinide and insulin glargine combination therapy maintained fair glycemic control for as long as 6 months in subpopulation of Japanese patients with type 2 diabetes.

We saw a 57-yr-old female non-insulin dependent diabetes mellitus (NIDDM) patient with antiinsulin antibody, in whom insulin-like growth factor-I (IGF-I) was shown to be effective as a blood glucose-lowering agent. Due to the presence of the antibody, the patient suffered from postprandial hyperglycemia and frequent hypoglycemia, which were uncontrollable even by multiple insulin injection therapy. In contrast to insulin injection (0.24 IU/kg body wt) which showed a delayed glucose-lowering effect, subcutaneous injection of recombinant human IGF-I (0.05-0.1 mg/kg body wt) caused a quick fall in plasma glucose levels. Prolongation of the glucose-lowering effect of "rapid-acting" insulin frequently caused subsequent hypoglycemia in this patient, but the effect of IGF-I seemed to have disappeared within the first three hours.