Clinical factors associated with bacterial translocation in Japanese patients with type 2 diabetes: A retrospective study.

OBJECTIVE: To explore clinical factors associated with bacterial translocation in Japanese patients with type 2 diabetes mellitus (T2DM). METHODS: The data of 118 patients with T2DM were obtained from two previous clinical studies, and were retrospectively analyzed regarding the clinical parameters associated with bacterial translocation defined as detection of bacteremia and levels of plasma lipopolysaccharide binding protein (LBP), the latter of which is thought to reflect inflammation caused by endotoxemia. RESULTS: LBP level was not significantly different between patients with and without bacteremia. No clinical factors were significantly correlated with the detection of bacteremia. On the other hand, plasma LBP level was significantly correlated with HbA1c (r = 0.312), fasting blood glucose (r = 0.279), fasting C-peptide (r = 0.265), body mass index (r = 0.371), high-density lipoprotein cholesterol (r = -0.241), and inflammatory markers (high-sensitivity C-reactive protein, r = 0.543; and interleukin-6, r = 0.456). Multiple regression analysis identified body mass index, HbA1c, high-sensitivity C-reactive protein, and interleukin-6 as independent determinants of plasma LBP level. CONCLUSION: The plasma LBP level was similar in patients with and without bacteremia. While both bacteremia and LBP are theoretically associated with bacterial translocation, the detection of bacteremia was not associated with LBP level in T2DM.

Everolimus Directly Suppresses Insulin Secretion Independently of Cell Growth Inhibition.

Everolimus, an orally administered mammalian target of rapamycin inhibitor, has been widely used as an immunosuppressant and an anticancer agent. Whereas everolimus can control recurrent hypoglycemia in patients with insulinoma, possibly through tumor regression and/or the direct inhibition of insulin secretion, time-dependent changes in serum insulin levels caused by everolimus still remain unclear. Here we report a clinical case of a patient with metastatic insulinoma, in which frequent monitoring of serum insulin levels demonstrated rapid and substantial changes in insulin secretion levels, a few days after the discontinuation as well as the readministration of everolimus. To further confirm the direct effect of everolimus on ß-cell function, we performed in vitro experiments using mouse insulinoma cells (MIN6) and human induced pluripotent stem cell (hiPSC)-derived insulin-producing cells and found that everolimus significantly suppressed glucose-stimulated insulin secretion in both MIN6 cells and hiPSC-derived insulin-producing cells. Thus, both a patient with metastatic insulinoma and in vitro experiments demonstrated that everolimus directly suppresses insulin secretion, independently of its tumor regression effect.

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.

The impact of PNPLA3 and JAZF1 on hepatocellular carcinoma in non-viral hepatitis patients with type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is an established independent risk factor for hepatocellular carcinoma (HCC). T2DM is associated with non-alcoholic steatohepatitis (NASH), which is a major cause of non-HBV and non-HCV-related HCC; nevertheless, it has been difficult to identify those patients with T2DM who have a high risk of developing HCC. The aim of this study was to identify genetic determinants that predispose T2DM patients to HCC by genotyping T2DM susceptibility loci and PNPLA3. METHODS: We recruited 389 patients with T2DM who satisfied the following three criteria: negative for HBs-Ag and anti-HCV Ab, alcohol intake <60 g/day, and history of T2DM >10 years. These patients were divided into two groups: T2DM patients with HCC (DM-HCC, n = 59) or those without HCC (DM-non-HCC, n = 330). We genotyped 51 single-nucleotide polymorphisms (SNPs) previously reported as T2DM or NASH susceptibility loci (PNPLA3) compared between the DM-HCC and DM-non-HCC groups with regard to allele frequencies at each SNP. RESULTS: The SNP rs738409 located in PNPLA3 was the greatest risk factor associated with HCC. The frequency of the PNPLA3 G allele was significantly higher among DM-HCC individuals than DM-non-HCC individuals (OR 2.53, p = 1.05 × 10(-5)). Among individuals homozygous for the PNPLA3 G allele (n = 115), the frequency of the JAZF1 rs864745 G allele was significantly higher among DM-HCC individuals than DM-non-HCC individuals (OR 3.44, p = 0.0002). CONCLUSIONS: PNPLA3 and JAZF1 were associated with non-HBV and non-HCV-related HCC development among Japanese patients with T2DM.

Repetitive hypoglycemia increases circulating adrenaline level with resultant worsening of intimal thickening after vascular injury in male Goto-Kakizaki rat carotid artery.

Hypoglycemia associated with diabetes management is a potential risk for cardiovascular diseases. However, the effect of hypoglycemic episodes including a surge of sympathetic activity on the progression of neointima formation after vascular injury remains largely unknown. In this study, insulin was injected intraperitoneally into nonobese diabetic Goto-Kakizaki (GK) rats, once every 3 days for 4 weeks after balloon injury of carotid artery to induce hypoglycemia. Then, we evaluated balloon injury-induced neointima formation. Insulin treatment enhanced neointima formation and increased the number of proliferating cell nuclear antigen (PCNA)-positive cells in the carotid artery. Injection of glucose with insulin prevented hypoglycemia and abrogated intimal thickening. Also, bunazosin, an α1 adrenergic receptor antagonist, prevented intimal thickening and accumulation of PCNA-positive cells induced by insulin treatment despite the presence of concomitant hypoglycemia and high adrenaline levels. Incubation of cultured smooth muscle cells with adrenaline resulted in a significant increase in their proliferation and G0/G1 to S phase progression, which was associated with activation of extracellular signal-regulated kinase, enhanced expression of cell cycle regulatory molecules such as cyclin D1, and cyclin E, and phosphorylation of retinoblastoma protein. These adrenaline-induced effects were abrogated by bunazosin. Our data indicated that increased adrenaline induced by repetitive hypoglycemia promotes intimal thickening and smooth muscle cell proliferation after endothelial denudation in GK rats.

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.

The diabetes-susceptible gene SLC30A8/ZnT8 regulates hepatic insulin clearance.

Recent genome-wide association studies demonstrated that common variants of solute carrier family 30 member 8 gene (SLC30A8) increase susceptibility to type 2 diabetes. SLC30A8 encodes zinc transporter-8 (ZnT8), which delivers zinc ion from the cytoplasm into insulin granules. Although it is well known that insulin granules contain high amounts of zinc, the physiological role of secreted zinc remains elusive. In this study, we generated mice with ß cell-specific Slc30a8 deficiency (ZnT8KO mice) and demonstrated an unexpected functional linkage between Slc30a8 deletion and hepatic insulin clearance. The ZnT8KO mice had low peripheral blood insulin levels, despite insulin hypersecretion from pancreatic ß cells. We also demonstrated that a substantial amount of the hypersecreted insulin was degraded during its first passage through the liver. Consistent with these findings, ZnT8KO mice and human individuals carrying rs13266634, a major risk allele of SLC30A8, exhibited increased insulin clearance, as assessed by c-peptide/insulin ratio. Furthermore, we demonstrated that zinc secreted in concert with insulin suppressed hepatic insulin clearance by inhibiting clathrin-dependent insulin endocytosis. Our results indicate that SLC30A8 regulates hepatic insulin clearance and that genetic dysregulation of this system may play a role in the pathogenesis of type 2 diabetes.

Anagliptin, a DPP-4 inhibitor, suppresses proliferation of vascular smooth muscles and monocyte inflammatory reaction and attenuates atherosclerosis in male apo E-deficient mice.

Dipeptyl peptidase-4 (DPP-4) inhibitors modulate the progression of atherosclerosis. To gain insights into their mechanism of action, 9-wk-old male apolipoprotein E (apoE)-deficient mice were fed a DPP-4 inhibitor, anagliptin-containing diet. The effects of anagliptin were investigated in, a monocyte cell line, human THP-1 cells, and rat smooth muscle cells (SMCs). Treatment with anagliptin for 16 wk significantly reduced accumulation of monocytes and macrophages in the vascular wall, SMC content in plaque areas, and oil red O-stained area around the aortic valve without affecting glucose tolerance or body weight. Serum DPP-4 concentrations were significantly higher in apoE-deficient mice than control mice, and the levels increased with aging, suggesting the involvement of DPP-4 in the progression of atherosclerosis. Indeed, soluble DPP-4 augmented cultured SMC proliferation, and anagliptin suppressed the proliferation by inhibiting ERK phosphorylation. In THP-1 cells, anagliptin reduced lipopolysaccharide-induced TNF-α production with inhibiting ERK phosphorylation and nuclear translocation of nuclear factor-κB. Quantitative analysis also showed that anagliptin reduced the area of atherosclerotic lesion in apoE-deficient mice. These results indicated that the anti-atherosclerotic effect of anagliptin is mediated, at least in part, through its direct inhibition of SMC proliferation and inflammatory reaction of monocytes.

Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces intimal thickening after vascular injury.

Glucagon-like peptide-1 is a hormone secreted by L cells of the small intestine and stimulates glucose-dependent insulin response. Glucagon-like peptide-1 receptor agonists such as exendin-4 are currently used in type 2 diabetes, and considered to have beneficial effects on the cardiovascular system. To further elucidate the effect of glucagon-like peptide-1 receptor agonists on cardiovascular diseases, we investigated the effects of exendin-4 on intimal thickening after endothelial injury. Under continuous infusion of exendin-4 at 24 nmol/kg/day, C57BL/6 mice were subjected to endothelial denudation injury of the femoral artery. Treatment of mice with exendin-4 reduced neointimal formation at 4weeks after arterial injury without altering body weight or various metabolic parameters. In addition, in vitro studies of isolated murine, rat and human aortic vascular smooth muscle cells showed the expression of GLP-1 receptor. The addition of 10nM exendin-4 to cultured smooth muscle cells significantly reduced their proliferation induced by platelet-derived growth factor. Our results suggested that exendin-4 reduced intimal thickening after vascular injury at least in part by the suppression of platelet-derived growth factor-induced smooth muscle cells proliferation.

Efficacy and safety of modified Yale insulin infusion protocol in Japanese diabetic patients after open-heart surgery.

To our knowledge, there is currently no insulin infusion protocol for critically ill patients especially designed for Asian diabetics although many such protocols are used in Western countries. In this study, we modified the Yale insulin infusion protocol taking into consideration the characteristics of Japanese diabetics and hospital environment. We tested the modified protocol in 40 type 2 diabetic patients after elective open-heart surgery (MY group) comparing with 35 type 2 diabetic patients under empirical blood glucose control (EC group). Analyses of 1656 blood glucose measurements during insulin infusion revealed that percentage of samples that showed achievement of target blood glucose level (80-140 mg/dl) was higher under MY (78+/-15%, n=870) than EC (57+/-23%, n=786, p<0.0001). On the other hand, the percentage of samples in which blood glucose was less than 60 mg/dl was comparable in the two groups (MY: 0.5+/-5.9 per thousand, EC: 5.1+/-18.5 per thousand). None of the patients with hypoglycemia showed significant clinical adverse effects. In conclusion, our modified Yale insulin infusion protocol is effective and safe for tight blood glucose control in Japanese diabetic patients after open-heart surgery.

Wnt5b partially inhibits canonical Wnt/beta-catenin signaling pathway and promotes adipogenesis in 3T3-L1 preadipocytes.

To elucidate the functional roles of Wnt5b in adipogenesis, we characterized gene expression profiles in Wnt5b overexpressing 3T3-L1 cells using microarray analysis. Of the approximately 20,000 genes screened, we found that 85 genes were up-regulated and 211 genes were down-regulated in 3T3-L1 cells overexpressing Wnt5b. Among the genes regulated by Wnt5b, the expressions of insulin like growth factor-1 (IGF-1), vascular endothelial growth factor-C (VEGF-C), and WNT1 inducible signaling pathway protein 1 (WISP-1), which were known to be up-regulated by Wnt1/beta-catenin signaling, were decreased in the Wnt5b overexpressing cells. This result was subsequently confirmed by real-time quantitative RT-PCR (IGF-1; 0.74+/-0.08 and 0.56+/-0.08, WISP-1; 0.71+/-0.03 and 0.56+/-0.08, and VEGF-C; 0.67+/-0.01 and 0.80+/-0.07, mean+/-SEM, compared with the control at zero and two days after induction of differentiation, respectively). We also found that Wnt5b overexpression in 3T3-L1 preadipocytes was able to partially prevent the inhibitory effect of Wnt3a on adipogenesis. Furthermore, the overexpression of Wnt5b was able to inhibit Wnt3a-induced activation of the canonical Wnt/beta-catenin pathway as evidenced by the reduced translocation of beta-catenin into the nucleus. These findings indicate that Wnt5b may promote adipogenesis in 3T3-L1 cells, at least in part, by antagonizing the canonical Wnt/beta-catenin pathway.

PKCepsilon induces interleukin-6 expression through the MAPK pathway in 3T3-L1 adipocytes.

Recent reports have suggested that PKCepsilon contributes to systemic insulin resistance, and is involved in the pathogenesis of type 2 diabetes, however, the exact mechanism is still unknown. To elucidate the possible involvement of PKCepsilon in the pathogenesis of type 2 diabetes, we examined the role of PKCepsilon in differentiated adipocytes using mouse 3T3-L1 adipocytes. We found that the over-expression of PKCepsilon resulted in the increase of IL-6 expression in differentiated adipocytes. This PKCepsilon-induced IL-6 expression could be completely inhibited by U0126, an inhibitor of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase. We also demonstrated that PKCepsilon increased the transcriptional activity of Est-like transcription factor (Elk-1) as well as the DNA-binding activity of activator protein-1 (AP-1) in differentiated 3T3-L1 adipocytes. These results suggest that PKCepsilon is able to increase IL-6 expression via the ERK-AP-1 pathway in differentiated adipocytes, and that PKCepsilon is involved in systemic insulin resistance by regulating plasma IL-6 concentrations.

Association of the gene encoding wingless-type mammary tumor virus integration-site family member 5B (WNT5B) with type 2 diabetes.

Recent reports have suggested that WNT signaling is an important regulator for adipogenesis or insulin secretion and might be involved in the pathogenesis of type 2 diabetes. To investigate possible roles of the WNT genes in conferring susceptibility to type 2 diabetes, we examined the association of the genes that encode members of the WNT family with type 2 diabetes in the Japanese population. First, 40 single-nucleotide polymorphism (SNP) loci within 11 WNT genes were analyzed in 188 subjects with type 2 diabetes (case-1) and 564 controls (control-1). Among them, six SNP loci exhibited a significant difference (P<.05) in the allele and/or genotype distributions between case and control subjects. These SNP loci were further analyzed in another set of case (case-2; n=733) and control (control-2; n=375) subjects to confirm their statistical significance. As a result, one SNP locus in the WNT5B gene was strongly associated with type 2 diabetes ( chi 2=15.6; P=.00008; odds ratio=1.74; 95% confidence interval 1.32-2.29). Expression of the WNT5B gene was detectable in several tissues, including adipose, pancreas, and liver. Subsequent in vitro experiments identified the fact that expression of the Wnt5b gene was increased at an early phase of adipocyte differentiation in mouse 3T3-L1 cells. Furthermore, overexpression of the Wnt5b gene in preadipocytes resulted in the promotion of adipogenesis and the enhancement of adipocytokine-gene expression. These results indicate that the WNT5B gene may contribute to conferring susceptibility to type 2 diabetes and may be involved in the pathogenesis of this disease through the regulation of adipocyte function.