Novel time-resolved reporter mouse reveals spatial and transcriptional heterogeneity during alpha cell differentiation.

AIMS/HYPOTHESIS: Glucagon-expressing pancreatic alpha cells have attracted much attention for their plasticity to transdifferentiate into insulin-producing beta cells; however, it remains unclear precisely when, and from where, alpha cells emerge and what regulates alpha cell fate. We therefore explored the spatial and transcriptional heterogeneity of alpha cell differentiation using a novel time-resolved reporter system. METHODS: We established the mouse model, 'Gcg-Timer', in which newly generated alpha cells can be distinguished from more-differentiated cells by their fluorescence. Fluorescence imaging and transcriptome analysis were performed with Gcg-Timer mice during the embryonic and postnatal stages. RESULTS: Fluorescence imaging and flow cytometry demonstrated that green fluorescence-dominant cells were present in Gcg-Timer mice at the embryonic and neonatal stages but not after 1 week of age, suggesting that alpha cell neogenesis occurs during embryogenesis and early neonatal stages under physiological conditions. Transcriptome analysis of Gcg-Timer embryos revealed that the mRNAs related to angiogenesis were enriched in newly generated alpha cells. Histological analysis revealed that some alpha cells arise close to the pancreatic ducts, whereas the others arise away from the ducts and adjacent to the blood vessels. Notably, when the glucagon signal was suppressed by genetic ablation or by chemicals, such as neutralising glucagon antibody, green-dominant cells emerged again in adult mice. CONCLUSIONS/INTERPRETATION: Novel time-resolved analysis with Gcg-Timer reporter mice uncovered spatiotemporal features of alpha cell neogenesis that will enhance our understanding of cellular identity and plasticity within the islets. DATA AVAILABILITY: Raw and processed RNA sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE229090.

Normal pancreatic ß-cell function in mice with RIP-Cre-mediated inactivation of p62/SQSTM1.

Recent studies have suggested that decreased pancreatic ß-cell function and mass are common features of patients with type 2 diabetes mellitus. Pancreatic ß-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 ß 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 ß-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 ß-cell function.

SET7/9 Enzyme Regulates Cytokine-induced Expression of Inducible Nitric-oxide Synthase through Methylation of Lysine 4 at Histone 3 in the Islet ß Cell.

SET7/9 is an enzyme that methylates histone 3 at lysine 4 (H3K4) to maintain euchromatin architecture. Although SET7/9 is enriched in islets and contributes to the transactivation of ß cell-specific genes, including Ins1 and Slc2a, SET7/9 has also been reported to bind the p65 subunit of nuclear factor κB in non-ß cells and modify its transcriptional activity. Given that inflammation is a central component of ß cell dysfunction in Type 1 and Type 2 diabetes, the aim of this study was to elucidate the role of SET7/9 in proinflammatory cytokine signaling in ß cells. To induce inflammation, ßTC3 insulinoma cells were treated with IL-1ß, TNF-α, and IFN-γ. Cytokine treatment led to increased expression of inducible nitric-oxide synthase, which was attenuated by the diminution of SET7/9 using RNA interference. Consistent with previous reports, SET7/9 was co-immunoprecipitated with p65 and underwent cytosolic to nuclear translocation in response to cytokines. ChIP analysis demonstrated augmented H3K4 mono- and dimethylation of the proximal Nos2 promoter with cytokine exposure. SET7/9 was found to occupy this same region, whereas SET7/9 knockdown attenuated cytokine-induced histone methylation of the Nos2 gene. To test this relationship further, islets were isolated from SET7/9-deficient and wild-type mice and treated with IL-1ß, TNF-α, and IFN-γ. Cytokine-induced Nos2 expression was reduced in the islets from SET7/9 knock-out mice. Together, our findings suggest that SET7/9 contributes to Nos2 transcription and proinflammatory cytokine signaling in the pancreatic ß cell through activating histone modifications.

Increased expression of ERp57/GRP58 is protective against pancreatic beta cell death caused by autophagic failure.

Autophagy is a tightly regulated self-digestion system. As in other cell types, autophagy plays an essential role in the homeostasis of pancreatic beta cells. However, the mechanisms involved in the deterioration of beta cell function caused by autophagic failure have not yet been fully elucidated. To gain insight into its mechanisms, we compared the protein expression of islets from beta cell-specific Atg7-deficient mice (Atg7(Δß-cell) mice) and their controls (Atg7(f/f) mice). Liquid chromatography/mass spectrometry after 1-dimensional electrophoresis identified the increased expression of ERp57/GRP58 in islets isolated from Atg7(Δß-cell) mice compared with those from Atg7(f/f) mice. The expression level of ERp57 was also elevated in rat insulinoma INS-1 cells by inducible knock-down of the atg7-gene. In Atg7 knock-down INS-1 cells, the suppression of ERp57 expression by siRNA resulted in an increase in the level of cleaved Caspase-3 protein and a decrease in the number of live cells. Furthermore, cell cycle analyses demonstrated that the suppressed expression of ERp57 increased the sub-G1 population. These data reveal that increased expression of ERp57 may contribute to the protection from beta cell death caused by autophagic failure.

Beneficial effects of vildagliptin combined with miglitol on glucose tolerance and islet morphology in diet-controlled db/db mice.

Dipeptidyl peptidase-4 (DPP-4) inhibitors improve glycemic control in patients with type 2 diabetes primarily by increasing plasma active glucagon-like peptide-1 (GLP-1) levels. While various combination therapies based on DPP-4 inhibitors have been proposed for treatment of type 2 diabetes, the effects of combination therapy of DPP-4 inhibitors and alpha-glucosidase inhibitors on ß-cell function are less characterized. We evaluated the effects of long-term treatment with vildagliptin, a DPP-4 inhibitor, on metabolic parameters and ß-cell function, in combination with miglitol, an alpha-glucosidase inhibitor, in diet-controlled db/db mice. In this study, 6-week-old male db/db mice were provided with standard chow twice a day for 6 weeks. Meal tolerance tests and glucose tolerance tests showed that the combination therapy of vildagliptin with miglitol, but not each alone, suppressed postprandial glycemic excursion, enhanced postprandial active GLP-1 levels and prevented deterioration of glucose tolerance in the db/db mice. The combination treatment did not alter ß-cell mass, but resulted in preserved expression of glucose transporter 2, Zinc transporter 8 and MafA and reduced the number of α cells. These results suggest that the combination of vildagliptin and miglitol prevents the development of overt diabetes in diet-controlled pre-diabetic db/db mice by normalizing postprandial glucose and incretin response, and by preserving ß-cell structure and the expression of factors essential for ß-cell function.

Exercise-induced enhancement of insulin sensitivity is associated with accumulation of M2-polarized macrophages in mouse skeletal muscle.

Exercise enhances insulin sensitivity in skeletal muscle, but the underlying mechanism remains obscure. Recent data suggest that alternatively activated M2 macrophages enhance insulin sensitivity in insulin target organs such as adipose tissue and liver. Therefore, the aim of this study was to determine the role of anti-inflammatory M2 macrophages in exercise-induced enhancement of insulin sensitivity in skeletal muscle. C57BL6J mice underwent a single bout of treadmill running (20 m/min, 90 min). Twenty-four hours later, ex vivo insulin-stimulated 2-deoxy glucose uptake was found to be increased in plantaris muscle. This change was associated with increased number of CD163-expressing macrophages (i.e. M2-polarized macrophages) in skeletal muscle. Systemic depletion of macrophages by pretreatment of mice with clodronate-containing liposome abrogated both CD163-positive macrophage accumulation in skeletal muscle as well as the enhancement of insulin sensitivity after exercise, without affecting insulin-induced phosphorylation of Akt and AS160 or exercise-induced GLUT4 expression. These results suggest that accumulation of M2-polarized macrophages is involved in exercise-induced enhancement of insulin sensitivity in mouse skeletal muscle, independently of the phosphorylation of Akt and AS160 and expression of GLUT4.

Inhibition of deoxyhypusine synthase enhances islet {beta} cell function and survival in the setting of endoplasmic reticulum stress and type 2 diabetes.

Islet ß cell dysfunction resulting from inflammation, ER stress, and oxidative stress is a key determinant in the progression from insulin resistance to type 2 diabetes mellitus. It was recently shown that the enzyme deoxyhypusine synthase (DHS) promotes early cytokine-induced inflammation in the ß cell. DHS catalyzes the conversion of lysine to hypusine, an amino acid that is unique to the translational elongation factor eIF5A. Here, we sought to determine whether DHS activity contributes to ß cell dysfunction in models of type 2 diabetes in mice and ß cell lines. A 2-week treatment of obese diabetic C57BLKS/J-db/db mice with the DHS inhibitor GC7 resulted in improved glucose tolerance, increased insulin release, and enhanced ß cell mass. Thapsigargin treatment of ß cells in vitro induces a picture of ER stress and apoptosis similar to that seen in db/db mice; in this setting, DHS inhibition led to a block in CHOP (CAAT/enhancer binding protein homologous protein) production despite >30-fold activation of Chop gene transcription. Blockage of CHOP translation resulted in reduction of downstream caspase-3 cleavage and near-complete protection of cells from apoptotic death. DHS inhibition appeared to prevent the cytoplasmic co-localization of eIF5A with the ER, possibly precluding the participation of eIF5A in translational elongation at ER-based ribosomes. We conclude that hypusination by DHS is required for the ongoing production of proteins, particularly CHOP, in response to ER stress in the ß cell.

Liver X receptor agonists augment human islet function through activation of anaplerotic pathways and glycerolipid/free fatty acid cycling.

Recent studies in rodent models suggest that liver X receptors (LXRs) may play an important role in the maintenance of glucose homeostasis and islet function. To date, however, no studies have comprehensively examined the role of LXRs in human islet biology. Human islets were isolated from non-diabetic donors and incubated in the presence or absence of two synthetic LXR agonists, TO-901317 and GW3965, under conditions of low and high glucose. LXR agonist treatment enhanced both basal and stimulated insulin secretion, which corresponded to an increase in the expression of genes involved in anaplerosis and reverse cholesterol transport. Furthermore, enzyme activity of pyruvate carboxylase, a key regulator of pyruvate cycling and anaplerotic flux, was also increased. Whereas LXR agonist treatment up-regulated known downstream targets involved in lipogenesis, we observed no increase in the accumulation of intra-islet triglyceride at the dose of agonist used in our study. Moreover, LXR activation increased expression of the genes encoding hormone-sensitive lipase and adipose triglyceride lipase, two enzymes involved in lipolysis and glycerolipid/free fatty acid cycling. Chronically, insulin gene expression was increased after treatment with TO-901317, and this was accompanied by increased Pdx-1 nuclear protein levels and enhanced Pdx-1 binding to the insulin promoter. In conclusion, our data suggest that LXR agonists have a direct effect on the islet to augment insulin secretion and expression, actions that should be considered either as therapeutic or unintended side effects, as these agents are developed for clinical use.

Postprandial Plasma Glucagon Kinetics in Type 2 Diabetes Mellitus: Comparison of Immunoassay and Mass Spectrometry.

CONTEXT: Accurate glucagon level measurements are necessary for investigation of mechanisms for postprandial hyperglycemia in type 2 diabetes. OBJECTIVE: To evaluate the accuracy of postprandial glucagon level measurements using a sandwich ELISA vs a recently established liquid chromatography-high resolution mass spectrometry (LC-HRMS) method in type 2 diabetes mellitus. DESIGN AND PARTICIPANTS: Twenty patients with type 2 diabetes treated with insulin underwent a meal test before and after administration of the dipeptidyl peptidase-4 inhibitor anagliptin for 4 weeks. Blood samples were taken serially after the meal, and glucagon levels were measured using both ELISA and LC-HRMS. We compared the change from baseline to 4 weeks (Δ0-4W) using the area under the curve for plasma glucagon during the meal test [area under the curve (AUC)0-3h] measured using ELISA and LC-HRMS. RESULTS: ELISA-based glucagon AUC0-3h was higher than LC-HRMS-based AUC0-3h at baseline and 4 weeks. However, differences in Δ0-4W-AUC0-3h measured using ELISA and LC-HRMS were not statistically significant. Additionally, Δ0-4W-AUC0-3h measured using ELISA and LC-HRMS were strongly correlated (r = 0.87, P < 0.001). CONCLUSIONS: Plasma glucagon levels during a meal test in patients with type 2 diabetes measured using ELISA were consistently higher than those measured using LC-HRMS. However, given that the changes in glucagon levels measured using ELISA before and after dipeptidyl peptidase-4 inhibitor therapy were similar to those based on LC-HRMS, this ELISA seems to be useful for evaluating the effect of the drug interventions on postprandial glucagon levels.

Combined expression of transcription factors induces AR42J-B13 cells to differentiate into insulin-producing cells.

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.

Enhanced Expression of the Key Mitosis Regulator Cyclin B1 Is Mediated by PDZ-Binding Kinase in Islets of Pregnant Mice.

The proliferation of pancreatic ß cells is enhanced to enable an increase in ß-cell mass and to compensate for insulin resistance during pregnancy. To elucidate the mechanisms involved, we previously investigated islets from pregnant and nonpregnant mice by gene expression profiling and found that the expression of postsynaptic density-95/Discs large/zonula occludens-1 (PDZ)-binding kinase (Pbk), a member of the mitogen-activated protein kinase kinase family, is increased in pregnant mouse islets compared with control mouse islets. Among the pregnancy hormones, treatment with estradiol upregulated Pbk expression. Inhibition of Pbk expression using a small interfering RNA for Pbk reduced bromodeoxyuridine incorporation in mouse insulinoma 6 cells, which was accompanied by a decreased expression of Ccnb1, a regulatory gene involved in mitosis. Ccnb1 expression was augmented in mouse islets during pregnancy. The forced expression of Pbk using an adenovirus system in isolated mouse islets increased Ccnb1 expression, and the Pbk inhibitor HI-TOPK-032 suppressed Ccnb1 expression in islets isolated from pregnant mice. Our results suggest that Pbk contributes to the expansion of islets during pregnancy and that Ccnb1 may assist Pbk in its role in ß-cell proliferation.

Activin A-induced expression of PAX4 in AR42J-B13 cells involves the increase in transactivation of E47/E12.

Pax4 is a paired-homeodomain containing transcriptional factor that controls the differentiation of pancreatic beta cells. The aim of this study was to investigate the mechanism of PAX4 expression by activin A. By reporter gene analysis using AR42J-B13 cells, in which treatment with activin A induced PAX4 mRNA expression, we identified that a short sequence located approximately 1930 bp upstream of the transcriptional start site is essential for activin A induced PAX4 promoter activation. This region contains an E box and binding sites for hepatocyte nuclear factor (HNF)-1alpha. Mutation introduced in each binding site markedly reduced activin A responsiveness. It has been reported that HNF-1alpha synergizes with basic helix-loop-helix (bHLH) proteins in activating the PAX4 promoter, and we demonstrated that activin A strongly enhanced the functional activity of E47/E12 without the increase in its binding ability. In addition, suppression of E47/E12 expression in AR42J-B13 cells using siRNA oligonucleotides results in the significant decrease in the intrinsic activin A-induced PAX4 expression. Our results suggest that activin A enhances PAX4 expression by enhanced transactivation of E47/E12 proteins and might result in a cumulative transactivation of the promoter.

Eicosapentaenoic acid reduces the progression of carotid intima-media thickness in patients with type 2 diabetes.

To investigate the effect of highly purified eicosapentaenoic acid (EPA) on the progression of diabetic macroangiopathy, we performed an open-label randomized prospective trial. A total of 81 Japanese type 2 diabetes were randomly assigned to the EPA (1800 mg/day) treated group or the control group. Carotid intima-media thickness (IMT) and brachial-ankle pulse wave velocity (baPWV) were evaluated before and after treatment in both groups. Sixty patients (EPA group, n=30; control group, n=30) completed this study. During the study period of 2.1+/-0.2 years, the mean IMT and max IMT of the EPA treated group showed a significant annual decrease compared with that of the control group (mean IMT, -0.029+/-0.112 mm versus 0.016+/-0.109 mm, respectively, P=0.029; max IMT, -0.084+/-0.113 mm versus -0.005+/-0.108 mm, respectively, P=0.0008). The baPWV was also improved significantly in the EPA treated group compared with the control group (-22.1+/-127.9 cm/s versus 62.3+/-223 cm/s, respectively, P=0.021). Multiple regression analysis showed that the administration of EPA was a significant and independent factor associated with an annual improvement of mean IMT (R2=0.067). In summary, this is the first demonstration that administration of purified EPA improves the carotid IMT and the baPWV in patients with type 2 diabetes.

Lack of evident atherosclerosis despite multiple risk factors in glycogen storage disease type 1a with hyperadiponectinemia.

We report a 60-year-old Japanese patient with glycogen storage disease type 1a (GSD1a) who was thoroughly evaluated for risk factors of atherosclerosis. As often observed in patients with GSD1a, this patient has multiple risk factors for atherosclerosis including hyperlipidemia, hypertension, glucose intolerance with insulin resistance, and chronic kidney disease. However, she lacked clinically evident atherosclerosis as generally observed in GSD1a patients. Unexpectedly, this patient had marked hyperadiponectinemia (27.6 microg/mL; reference range, 4.1-18.9 microg/mL) with increase in the ratio of high-molecular weight to total adiponectin. Although the reason for the hyperadiponectinemia was not clear, at least it seemed to protect against enhanced atherosclerogenesis otherwise promoted by a battery of risk factors. Although further studies are needed, hyperadiponectinemia in addition to hypoinsulinemia might explain at least in part the lack of evident atherosclerosis in patients with GSD1a.

Relationships between lifestyle patterns and cardio-renal-metabolic parameters in patients with type 2 diabetes mellitus: A cross-sectional study.

INTRODUCTION: While individuals tend to show accumulation of certain lifestyle patterns, the effect of such patterns in real daily life on cardio-renal-metabolic parameters remains largely unknown. This study aimed to assess clustering of lifestyle patterns and investigate the relationships between such patterns and cardio-renal-metabolic parameters. PARTICIPANTS AND METHODS: The study participants were 726 Japanese type 2 diabetes mellitus (T2DM) outpatients free of history of cardiovascular diseases. The relationship between lifestyle patterns and cardio-renal-metabolic parameters was investigated by linear and logistic regression analyses. RESULTS: Factor analysis identified three lifestyle patterns. Subjects characterized by evening type, poor sleep quality and depressive status (type 1 pattern) had high levels of HbA1c, alanine aminotransferase and albuminuria. Subjects characterized by high consumption of food, alcohol and cigarettes (type 2 pattern) had high levels of γ-glutamyl transpeptidase, triglycerides, HDL-cholesterol, blood pressure, and brachial-ankle pulse wave velocity. Subjects characterized by high physical activity (type 3 pattern) had low uric acid and mild elevation of alanine aminotransferase and aspartate aminotransferase. In multivariate regression analysis adjusted by age, gender and BMI, type 1 pattern was associated with higher HbA1c levels, systolic BP and brachial-ankle pulse wave velocity. Type 2 pattern was associated with higher HDL-cholesterol levels, triglycerides, aspartate aminotransferase, ɤ- glutamyl transpeptidase levels, and diastolic BP. CONCLUSIONS: The study identified three lifestyle patterns that were associated with distinct cardio-metabolic-renal parameters in T2DM patients. TRIAL REGISTRATION: UMIN000010932.

Efficacy and Safety of Vildagliptin as an Add-On Therapy in Inadequately Controlled Type 2 Diabetes Patients Treated With Basal Insulin.

BACKGROUND: The aim of this study was to investigate the efficacy and safety of vildagliptin as an add-on therapy for patients with type 2 diabetes mellitus inadequately controlled with basal insulin. METHODS: Twenty-four patients treated with basal insulin and oral anti-diabetes drugs were randomly allocated into two groups: the control group (did not receive any add-on drugs) and vildagliptin group (received vildagliptin 100 mg/day for 6 months). The primary outcome was changes in hemoglobin A1c (HbA1c) from baseline to end of study. RESULTS: Treatment with vildagliptin significantly reduced HbA1c from 8.1±0.7% at baseline to 7.1±0.7% (P < 0.01), while there was no significant change of HbA1c in the control group. Vildagliptin group showed significant reduction of HbA1c compared with control group (-1.0±0.3% vs. 0.2±0.8%, P < 0.01). In addition, vildagliptin group showed a significant increase in 1,5-anhydroglucitol compared with the control group (4.5 ± 3.4 vs. 0.5 ± 4.1 µg/mL, P < 0.05). Mild hypoglycemia was reported in one patient of the vildagliptin group and two patients of the control group. CONCLUSION: Vildagliptin improved glycemic control without increasing hypoglycemia in Japanese type 2 diabetes inadequately controlled with basal insulin treatment and other oral anti-diabetes drugs. This study was registered with UMIN (University Hospital Medical Information Network ID#000010849).

One year follow-up after a randomized controlled trial of a 130 g/day low-carbohydrate diet in patients with type 2 diabetes mellitus and poor glycemic control.

BACKGROUND & AIMS: Recently, we conducted a prospective randomized controlled trial (RCT) showing that a 6-month 130g/day low-carbohydrate diet (LCD) reduced HbA1c and BMI more than a calorie restricted diet (CRD). [1] To assess whether the benefits of the LCD persisted after the intensive intervention, we compared HbA1c and BMI between the LCD and CRD groups at 1 year after the end of the 6-month RCT. METHODS: Following the end of the 6-month RCT, patients were allowed to manage their own diets with periodic outpatient visits. One year later, we analyzed clinical and nutrition data. RESULTS: Of the 66 participants in the original study, 27 in the CRD group and 22 in the LCD group completed this trial. One year after the end of the original RCT, the carbohydrate intake was comparable between the groups (215 [189-243]/day in the CRD group and 214 (176-262) g/day in the LCD group). Compared with the baseline data, HbA1c and BMI were decreased in both groups (CRD: HbA1c -0.4 [-0.9 to 0.3] % and BMI -0.63 [-1.20 to 0.18] kg/m2; LCD: HbA1c -0.35 [-1.0 to 0.35] % and BMI -0.77 [-1.15 to -0.12] kg/m2). There were no significant differences in HbA1c and BMI between the groups. CONCLUSIONS: One year after the diet therapy intervention, the beneficial effect of the LCD on reduction of HbA1c and BMI did not persist in comparison with CRD. However, combining the data of both groups, significant improvements in HbA1c and BMI from baseline were observed. Although the superiority of the LCD disappeared 1 year after the intensive intervention, these data suggest that well-constructed nutrition therapy programs, both CRD and LCD, were equally effective in improving HbA1c for at least 1 year. TRIAL REGISTRATION: University Hospital Medical Information Network (UMIN) ID000010663.

Safety and efficacy of metformin up-titration in Japanese patients with type 2 diabetes mellitus treated with vildagliptin and low-dose metformin.

BACKGROUND: This study investigated the safety and efficacy of metformin up-titration in Japanese patients with type 2 diabetes mellitus treated with vildagliptin (100 mg/day) and low-dose metformin (500 or 750 mg/day). RESEARCH DESIGN AND METHODS: Fifty patients were randomly allocated to the control group (maintaining the initial low-dose of metformin) and the dose increase group (up-titrating of metformin to 1,500-2,250 mg/day) for 24 weeks. The primary outcome was change in HbA1c from baseline to 24 weeks. RESULTS: Among the 25 patients allocated to the dose increase group, four patients were not able to complete the study protocol because of gastrointestinal symptoms. HbA1c in the dose increase group was significantly but modestly lower than in the control group (change in HbA1c: 0.22 ± 0.57 vs. -0.15 ± 0.58%, group comparison, P < 0.05). The dose increase group did not gain weight during the study period, and no hypoglycemic events were reported in both groups. The rate of gastrointestinal symptoms in the dose increase group was profoundly higher than in the control group (32 vs. 0%, P < 0.01). CONCLUSIONS: In Japanese patients with type 2 diabetes treated with vildagliptin and low-dose metformin, metformin up-titration significantly but modestly improved glycemic control without hypoglycemia and weight gain.