Association of serum bilirubin levels with risk of cancer development and total death.

Serum levels of bilirubin, a strong antioxidant, may influence cancer risk. We aimed to assess the association between serum bilirubin levels and cancer risk. Data were retrieved from 10-year electronic medical records at Kyushu University Hospital (Japan) for patients aged 20 to 69 years old. The associations of baseline bilirubin levels with cancer risk (lung, colon, breast, prostate, and cervical) were evaluated using a gradient boosting decision tree (GBDT) model, a machine learning algorithm, and Cox proportional hazard regression model, adjusted for age, smoking, body mass index, and diabetes. The number of study subjects was 29,080. Median follow-up time was 4.7 years. GBDT models illustrated that baseline bilirubin levels were negatively and non-linearly associated with the risk of lung (men), colon, and cervical cancer. In contrast, a U-shaped association was observed for breast and prostate cancer. Cox hazard regression analyses confirmed that baseline bilirubin levels (< 1.2 mg/dL) were negatively associated with lung cancer risk in men (HR = 0.474, 95% CI 0.271-0.828, P = 0.009) and cervical cancer risk (HR = 0.365, 95% CI 0.136-0.977, P = 0.045). Additionally, low bilirubin levels (< 0.6 mg/dL) were associated with total death (HR = 1.744, 95% CI 1.369-2.222, P < 0.001). Serum bilirubin may have a beneficial effect on the risk of some types of cancers.

The dipeptidyl peptidase-4 inhibitor, linagliptin, improves cognitive impairment in streptozotocin-induced diabetic mice by inhibiting oxidative stress and microglial activation.

OBJECTIVE: Accumulating epidemiological studies have demonstrated that diabetes is an important risk factor for dementia. However, the underlying pathological and molecular mechanisms, and effective treatment, have not been fully elucidated. Herein, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin, on diabetes-related cognitive impairment. METHOD: Streptozotocin (STZ)-induced diabetic mice were treated with linagliptin (3 mg/kg/24 h) for 17 weeks. The radial arm water maze test was performed, followed by evaluation of oxidative stress using DNP-MRI and the expression of NAD(P)H oxidase components and proinflammatory cytokines and of microglial activity. RESULTS: Administration of linagliptin did not affect the plasma glucose and body weight of diabetic mice; however, it improved cognitive impairment. Additionally, linagliptin reduced oxidative stress and the mRNA expression of NAD(P)H oxidase component and TNF-α, and the number and body area of microglia, all of which were significantly increased in diabetic mice. CONCLUSIONS: Linagliptin may have a beneficial effect on diabetes-related dementia by inhibiting oxidative stress and microglial activation, independently of glucose-lowering.

Aberrant activation of bone marrow Ly6C high monocytes in diabetic mice contributes to impaired glucose tolerance.

Accumulating evidence indicates that diabetes and obesity are associated with chronic low-grade inflammation and multiple organ failure. Tissue-infiltrated inflammatory M1 macrophages are aberrantly activated in these conditions and contribute to hyperglycemia and insulin resistance. However, it is unclear at which stage these cells become aberrantly activated: as precursor monocytes in the bone marrow or as differentiated macrophages in tissues. We examined the abundance, activation state, and function of bone marrow-derived Ly6Chigh monocytes in mice with diabetes and/or obesity. Ly6Chigh monocytes were FACS-purified from six groups of male mice consisting of type 2 diabetes model db/db mice, streptozotocin (STZ) induced insulin depletion mice, high fat diet (HFD) induced obesity mice and each control mice. Ly6Chigh monocytes were then analyzed for the expression of inflammation markers by qRT-PCR. In addition, bone marrow-derived Ly6Chigh monocytes from db/+ and db/db mice were fluorescently labeled and injected into groups of db/db recipient mice. Cell trafficking to tissues and levels of markers were examined in the recipient mice. The expression of many inflammation-related genes was significantly increased in Ly6Chigh monocytes from db/db mice, compared with the control. Bone marrow-derived Ly6Chigh monocytes isolated from db/db mice, but not from db/+ mice, displayed prominent infiltration into peripheral tissues at 1 week after transfer into db/db mice. The recipients of db/db Ly6Chigh monocytes also exhibited significantly increased serum glucose levels and worsening tolerance compared with mice receiving db/+ Ly6Chigh monocytes. These novel observations suggest that activated Ly6Chigh monocytes may contribute to the glucose intolerance observed in diabetes.

Bilirubin reduces visceral obesity and insulin resistance by suppression of inflammatory cytokines.

OBJECTIVE: Although previous studies have reported a negative relationship between serum bilirubin concentration and the development of diabetes mellitus (DM), the relationship between bilirubin and insulin resistance has not been thoroughly assessed. This study was designed to determine the relationships between bilirubin, body fat distribution, and adipose tissue inflammation in patients with type 2 DM and the effect of bilirubin in an obese animal model. METHOD: Body fat distribution was measured using an abdominal dual bioelectrical impedance analyzer in patients with type 2 DM. We also measured glycemic control, lipid profile, serum bilirubin concentration and other clinical characteristics, and determined their relationships with body fat distribution. In the animal study, biliverdin (20 mg/kg daily) was orally administered to high-fat diet (HFD)-induced obese (DIO) mice for 2 weeks, after which intraperitoneal insulin tolerance testing was performed. Then, adipocyte area, adipocytokine expression, and macrophage polarization were evaluated in epididymal adipose tissues. RESULTS: In the clinical study, univariate analysis showed that a lower bilirubin concentration was significantly correlated with higher body mass index, waist circumference, triglyceride, uric acid, creatinine, visceral fat area and lower HDL-C. In multivariate analyses, bilirubin concentration significantly correlated with diastolic blood pressure, creatinine, and visceral fat area. However, there was no association between bilirubin concentration and subcutaneous fat area. In the animal study, DIO mice treated with biliverdin had smaller adipocytes than untreated DIO mice and biliverdin improved HFD-induced insulin resistance. Biliverdin treatment reversed the higher gene expression of Cd11c, encoding an M1 macrophage marker, and Tnfa, encoding the proinflammatory cytokine tumor necrosis factor-α, in the adipose tissues of DIO mice. These data suggest biliverdin administration alleviates insulin resistance by ameliorating inflammation and the dysregulation of adipocytokine expression in adipose tissues of DIO mice. CONCLUSIONS: Bilirubin may protect against insulin resistance by ameliorating visceral obesity and adipose tissue inflammation.

Poorly controlled diabetes during pregnancy and lactation activates the Foxo1 pathway and causes glucose intolerance in adult offspring.

Exposure to maternal diabetes during pregnancy results in diabetes in offspring, but its underlying mechanisms are unclear. Here, we investigated the phenotype and molecular defects of the offspring of poorly controlled diabetic female mice generated by streptozotocin (STZ) administration. Offspring was exposed to maternal diabetes during pregnancy and lactation. The body weight of STZ offspring was lower than that of control offspring at birth and in adulthood, and glucose tolerance was impaired in adult STZ offspring. Interestingly, the phenotype was more pronounced in male offspring. We next investigated the morphology of islets and expression of ß cell-related genes, but no significant changes were observed. However, transcriptome analysis of the liver revealed activation of the fork head box protein O1 (Foxo1) pathway in STZ male offspring. Notably, two key gluconeogenesis enzyme genes, glucose 6 phosphatase catalytic subunit (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1), were upregulated. Consistent with this finding, phosphorylation of Foxo1 was decreased in the liver of STZ male offspring. These changes were not obvious in female offspring. The activation of Foxo1 and gluconeogenesis in the liver may have contributed to the impaired glucose tolerance of STZ male offspring.

Serum Bilirubin Concentration is Associated with Left Ventricular Remodeling in Patients with Type 2 Diabetes Mellitus: A Cohort Study.

INTRODUCTION: Previous studies have shown that serum bilirubin concentration is inversely associated with the risk of cardiovascular disease. The relationship between serum bilirubin concentration and left ventricular geometry, however, has not been investigated in patients with diabetes mellitus. METHODS: In this cohort study, 158 asymptomatic patients with type 2 diabetes mellitus without overt heart disease were enrolled. Left ventricular structure and function were assessed using echocardiography. Serum bilirubin concentration, glycemic control, lipid profile, and other clinical characteristics were evaluated, and their association with left ventricular geometry was determined. Patients with New York Heart Association Functional Classification greater than I, left ventricular ejection fraction less than 50%, history of coronary artery disease, severe valvulopathy, chronic atrial fibrillation, or creatinine clearance less than 30 ml/min, and those receiving insulin treatment, were excluded. RESULTS: Univariate analyses showed that relative wall thickness (RWT) was significantly correlated with diastolic blood pressure (P = 0.003), HbA1c (P = 0.024), total cholesterol (P = 0.043), urinary albumin (P = 0.023), and serum bilirubin concentration (P = 0.009). There was no association between left ventricular mass index and serum bilirubin concentration. Multivariate linear regression analysis showed that log RWT was positively correlated with diastolic blood pressure (P = 0.010) and that log RWT was inversely correlated with log bilirubin (P = 0.003). In addition, the patients with bilirubin less than 0.8 mg/dl had a higher prevalence of concentric left ventricular remodeling compared with those with bilirubin 0.8 mg/dl or more. CONCLUSION: Our study shows that the serum bilirubin concentration may be associated with the progression of concentric left ventricular remodeling in patients with type 2 diabetes mellitus.

A novel DPP-4 inhibitor teneligliptin scavenges hydroxyl radicals: In vitro study evaluated by electron spin resonance spectroscopy and in vivo study using DPP-4 deficient rats.

AIMS: Recently various dipeptidyl peptidase-4 (DPP-4) inhibitors have emerged because of their high effectiveness and safety. In spite of their common effect of DPP-4 inhibition, the chemical structures are diverse. Here we show that the structure of teneligliptin, a novel DPP-4 inhibitor, has a scavenging activity on hydroxyl radical (·OH). METHODS: ·OH and superoxide (O2(-)) were detected by electron spin resonance (ESR) spectroscopy. ·OH and O2(-) were generated in vitro by the Fenton reaction and a hypoxanthine-xanthine oxidase system, respectively. The level of free radicals was estimated from the ESR signal intensity. The product via teneligliptin and ·OH reaction was identified by thin layer chromatography and mass spectrometry analysis. In vivo effect was also evaluated using DPP-4 deficient rats with streptozotocin-induced diabetes. RESULTS: ESR spectroscopy analysis showed that teneligliptin did not scavenge O2(-), but scavenged ·OH in a dose dependent manner. Its activity was greater than that of glutathione. The reaction product appeared to have an oxygen-atom added structure to that of teneligliptin, which was identical to the most abundant metabolite of teneligliptin in human plasma. Furthermore, using DPP-4 deficient rat, teneligliptin did not affect plasma glucose levels or body weight, but normalized increased levels of 8-hydroxy-2'-deoxyguanosine in urine, kidney and aorta of diabetic rats, supporting that teneligliptin may have a ·OH scavenging activity in vivo independently of DPP-4 inhibition. CONCLUSIONS: Teneligliptin is not only effective as DPP-4 inhibitor, but may also be beneficial as ·OH scavenger, which may be useful in the prevention of diabetic complications.

Long-term outcomes after total pancreatectomy: special reference to survivors' living conditions and quality of life.

BACKGROUND: Although recent studies have confirmed the safety of total pancreatectomy (TP), appropriate selection of patients for TP has not been well documented. Because patients require lifelong medical treatment and self-management of pancreatic insufficiency after TP, indications for TP should be determined carefully according not only to disease factors but also to the social background of patients. We aimed to clarify long-term outcomes after TP, including the living conditions and quality of life (QoL), of surviving patients. METHODS: Medical records of 44 consecutive patients who underwent TP between 1990 and 2013 were reviewed retrospectively; 25 survivors completed cross-sectional clinical surveys and responded to a questionnaire about QoL using Short Form 36v2. RESULTS: Prevalence of morbidity and mortality after TP was 32 and 5 %, respectively. Postoperative complications occurred more frequently in elderly patients than in young patients (48 vs. 14 %; P = 0.02); however, there was no significant difference in mortality, postoperative hospital stay, or survival. Twenty-four of 25 survivors (96 %) could manage pancreatogenic diabetes by themselves, and the median level of glycosylated hemoglobin was 7.4 %. Although one-third of patients after TP complained of diarrhea and the QoL scores of patients with diarrhea were lower than those of patients without diarrhea, QoL scores after TP were virtually comparable with those of the national population, even in elderly patients. CONCLUSIONS: TP can be performed safely, even in elderly patients. QoL after TP seems to be acceptable if patients are capable of self-management.

Brachial-ankle pulse wave velocity predicts all-cause mortality and cardiovascular events in patients with diabetes: the Kyushu Prevention Study of Atherosclerosis.

OBJECTIVE: Whether brachial-ankle pulse wave velocity (baPWV), a noninvasive marker for arterial stiffness, is a useful predictive maker for cardiovascular events in subjects with diabetes is not established. In the present cohort study, we evaluated the benefit of baPWV for the prediction of cardiovascular morbidity and mortality in subjects with diabetes. RESEARCH DESIGN AND METHODS: A total of 4,272 outpatients with diabetes were enrolled in the Kyushu Prevention Study of Atherosclerosis. Of these, 3,628 subjects, excluding those with an ankle-brachial index of <0.9, were prospectively followed for 3.2 ± 2.2 years. The baPWV at baseline was classified by recursive partitioning (RP) for each end point. We plotted the Kaplan-Meier curves for high- and low-baPWV groups, which were designated based on the cutoff points, and calculated Cox proportional hazards models. RESULTS: The elevation of baPWV quartiles was significantly correlated to the incidence of coronary artery events, cerebrovascular events, and all-cause mortality. RP revealed baPWVs of 14 and 24 m/s as statistically adequate cutoff points for cardiovascular events and mortality, respectively. High-baPWV classes showed significantly low event-free ratios in Kaplan-Meier curves for all end points and remained independent risks for all-cause mortality and cerebrovascular events, but not for coronary artery events after adjustments for age, sex, BMI, hypertension, hyperlipidemia, smoking, and hemoglobin A1c by Cox proportional hazards models. CONCLUSIONS: This large-scale cohort study provided evidence that high baPWV is a useful independent predictor of mortality and cardiovascular morbidity in subjects with diabetes.

Exenatide improves hepatic steatosis by enhancing lipid use in adipose tissue in nondiabetic rats.

AIM: To investigate the metabolic changes in skeletal muscle and/or adipose tissue in glucagon-like peptide-1-induced improvement of nonalcoholic fatty liver disease (NAFLD). METHODS: Male Wistar rats were fed either a control diet (control group) or a high-fat diet (HFD). After 4 wk, the HFD-fed rats were subdivided into two groups; one group was injected with exenatide [HFD-Ex(+) group] and the other with saline [HFD-Ex(-) group] every day for 12 wk. The control group received saline and were fed a control diet. Changes in weight gain, energy intake, and oxygen consumption were analyzed. Glucose tolerance tests were performed after 8 wk of treatment. Histological assessments were performed in liver and adipose tissue. RNA expression levels of lipid metabolism related genes were evaluated in liver, skeletal muscle, and adipose tissue. RESULTS: Exenatide attenuated weight gain [HFD-Ex(-) vs HFD-Ex(+)] and reduced energy intake, which was accompanied by an increase in oxygen consumption and a decrease in the respiratory exchange ratio [HFD-Ex(-) vs HFD-Ex(+)]. However, exenatide did not affect glucose tolerance. Exenatide reduced lipid content in the liver and adipose tissue. Exenatide did not affect the expression of lipid metabolism-related genes in the liver or skeletal muscle. In adipose tissue, exenatide significantly upregulated lipolytic genes, including hormone-sensitive lipase, carnitine palmitoyltransferase-1, long-chain acyl-CoA dehydrogenase, and acyl-CoA oxidase 1 [HFD-Ex(-) vs HFD-Ex(+)]. Exenatide also upregulated catalase and superoxide dismutase 2 [HFD-Ex(-) vs HFD-Ex(+)]. CONCLUSION: In addition to reducing appetite, enhanced lipid use by exenatide in adipose tissue may reduce hepatic lipid content in NAFLD, most likely by decreasing lipid influx into the liver.

CTLA-4Ig immunotherapy of obesity-induced insulin resistance by manipulation of macrophage polarization in adipose tissues.

It has been established that obesity alters the metabolic and endocrine function of adipose tissue and, together with accumulation of adipose tissue macrophages, contributes to insulin resistance. Although numerous studies have reported that shifting the polarization of macrophages from M1 to M2 can alleviate adipose tissue inflammation, manipulation of macrophage polarization has not been considered as a specific therapy. Here, we determined whether cytotoxic T-lymphocyte-associated antigen-4IgG1 (CTLA-4Ig) can ameliorate insulin resistance by induction of macrophages from proinflammatory M1 to anti-inflammatory M2 polarization in the adipose tissues of high fat diet-induced insulin-resistant mice. CTLA4-Ig treatment prevented insulin resistance by changing gene expression to M2 polarization, which increased the levels of arginase 1. Furthermore, flow cytometric analysis confirmed the alteration of polarization from CD11c (M1)- to CD206 (M2)-positive cells. Concomitantly, CTLA-4Ig treatment resulted in weight reductions of epididymal and subcutaneous adipose tissues, which may be closely related to overexpression of apoptosis inhibitors in macrophages. Moreover, proinflammatory cytokine and chemokine levels decreased significantly. In contrast, CCAAT enhancer binding protein α, peroxisome proliferator-activated receptor γ, and adiponectin expression increased significantly in subcutaneous adipose tissue. This novel mechanism of CTLA-4lg immunotherapy may lead to an ideal anti-obesity/inflammation/insulin resistance agent.

Phycocyanin and phycocyanobilin from Spirulina platensis protect against diabetic nephropathy by inhibiting oxidative stress.

We and other investigators have reported that bilirubin and its precursor biliverdin may have beneficial effects on diabetic vascular complications, including nephropathy, via its antioxidant effects. Here, we investigated whether phycocyanin derived from Spirulina platensis, a blue-green algae, and its chromophore phycocyanobilin, which has a chemical structure similar to that of biliverdin, protect against oxidative stress and renal dysfunction in db/db mice, a rodent model for Type 2 diabetes. Oral administration of phycocyanin (300 mg/kg) for 10 wk protected against albuminuria and renal mesangial expansion in db/db mice, and normalized tumor growth factor-ß and fibronectin expression. Phycocyanin also normalized urinary and renal oxidative stress markers and the expression of NAD(P)H oxidase components. Similar antioxidant effects were observed following oral administration of phycocyanobilin (15 mg/kg) for 2 wk. Phycocyanobilin, bilirubin, and biliverdin also inhibited NADPH dependent superoxide production in cultured renal mesangial cells. In conclusion, oral administration of phycocyanin and phycocyanobilin may offer a novel and feasible therapeutic approach for preventing diabetic nephropathy.

GLP-1 analog liraglutide protects against oxidative stress and albuminuria in streptozotocin-induced diabetic rats via protein kinase A-mediated inhibition of renal NAD(P)H oxidases.

Accumulating evidence has implicated that GLP-1 may have a beneficial effect on cardiovascular and renal diseases but the mechanism is not fully understood. Here we show that GLP-1 analog, liraglutide, inhibits oxidative stress and albuminuria in streptozotocin (STZ)-induced type 1 diabetes mellitus rats, via a protein kinase A (PKA)-mediated inhibition of renal NAD(P)H oxidases. Diabetic rats were randomly treated with subcutaneous injections of liraglutide (0.3 mg/kg/12 h) for 4 weeks. Oxidative stress markers (urinary 8-hydroxy-2'-deoxyguanosine and renal dihydroethidium staining), expression of renal NAD(P)H oxidase components, transforming growth factor-ß (TGF-ß), fibronectin and urinary albumin excretion were measured. In vitro effect of liraglutide was evaluated using cultured renal mesangial cells. Administration of liraglutide did not affect plasma glucose levels or body weights in STZ diabetic rats, but normalized oxidative stress markers, expression of NAD(P)H oxidase components, TGF-ß, fibronectin in renal tissues and urinary albumin excretion, all of which were significantly increased in diabetic rats. In addition, in cultured renal mesangial cells, incubation with liraglutide for 48 h inhibited NAD(P)H-dependent superoxide production evaluated by lucigenin chemiluminescence in a dose-dependent manner. This effect was reversed by both PKA inhibitor H89 and adenylate cyclase inhibitor SQ22536, but not by Epac2 inhibition via its small interfering RNA. Liraglutide may have a direct beneficial effect on oxidative stress and diabetic nephropathy via a PKA-mediated inhibition of renal NAD(P)H oxidase, independently of a glucose-lowering effect.

Reduced expression of adipose triglyceride lipase enhances tumor necrosis factor alpha-induced intercellular adhesion molecule-1 expression in human aortic endothelial cells via protein kinase C-dependent activation of nuclear factor-kappaB.

We examined the effects of adipose triglyceride lipase (ATGL) on the initiation of atherosclerosis. ATGL was recently identified as a rate-limiting triglyceride (TG) lipase. Mutations in the human ATGL gene are associated with neutral lipid storage disease with myopathy, a rare genetic disease characterized by excessive accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, shows massive TG accumulation in both coronary atherosclerotic lesions and the myocardium. Recent reports show that myocardial triglyceride content is significantly higher in patients with prediabetes or diabetes and that ATGL expression is decreased in the obese insulin-resistant state. Therefore, we investigated the effect of decreased ATGL activity on the development of atherosclerosis using human aortic endothelial cells. We found that ATGL knockdown enhanced monocyte adhesion via increased expression of TNFα-induced intercellular adhesion molecule-1 (ICAM-1). Next, we determined the pathways (MAPK, PKC, or NFκB) involved in ICAM-1 up-regulation induced by ATGL knockdown. Both phosphorylation of PKC and degradation of IκBα were increased in ATGL knockdown human aortic endothelial cells. In addition, intracellular diacylglycerol levels and free fatty acid uptake via CD36 were significantly increased in these cells. Inhibition of the PKC pathway using calphostin C and GF109203X suppressed TNFα-induced ICAM-1 expression. In conclusion, we showed that ATGL knockdown increased monocyte adhesion to the endothelium through enhanced TNFα-induced ICAM-1 expression via activation of NFκB and PKC. These results suggest that reduced ATGL expression may influence the atherogenic process in neutral lipid storage diseases and in the insulin-resistant state.

Pycnogenol, an extract from French maritime pine, suppresses Toll-like receptor 4-mediated expression of adipose differentiation-related protein in macrophages.

Adipose differentiation-related protein (ADRP) is highly expressed in macrophages and human atherosclerotic lesions. We demonstrated that Toll-like receptor (TLR) 4-mediated signals, which are involved in atherosclerosis formation, enhanced the expression of ADRP in macrophages. Lipopolysaccharide (LPS) enhanced the ADRP expression in RAW264.7 cells or peritoneal macrophages from wild-type mice, but not in macrophages from TLR4-deficient mice. Actinomycin D almost completely abolished the LPS effect, whereas cycloheximide decreased the expression at 12 h, indicating that the LPS-induced ADRP expression was stimulated at the transcriptional level and was also mediated by new protein synthesis. LPS enhanced the ADRP promoter activity, in part, by stimulating activator protein (AP)-1 binding to the Ets/AP-1 element. In addition, preceding the increase of the ADRP mRNA, LPS induced the expression of interleukin (IL)-6, IL-1alpha, and interferon-beta mRNAs, all of which stimulated the ADRP expression. Antibodies against these cytokines or inhibitors of c-Jun NH(2)-terminal kinase and nuclear factor (NF)-kappaB suppressed the ADRP mRNA level. Thus TLR4 signals stimulate the ADRP expression both in direct and indirect manners. Pycnogenol (PYC), an extract of French maritime pine, suppressed the expression of ADRP and the above-mentioned cytokines. PYC suppressed the ADRP promoter activity and enhancer activity of AP-1 and NF-kappaB, whereas it did not affect the LPS-induced DNA binding of these factors. In conclusion, TLR4-mediated signals stimulate the ADRP expression in macrophages while PYC antagonizes this process. PYC, a widely used dietary supplement, might be useful for prevention of atherosclerosis.

Differential effect of sulfonylureas on production of reactive oxygen species and apoptosis in cultured pancreatic beta-cell line, MIN6.

Sulfonylureas are considered to cause beta-cell apoptosis. However, it is unclear how this occurs and whether there is a difference in such effects among various sulfonylureas. Here, we examined the effects of various sulfonylureas and a short-acting insulin secretagogue, nateglinide, on oxidative stress and apoptosis using the beta-cell line MIN6. After cultured MIN6 cells were exposed to various concentrations of sulfonylureas (glibenclamide, glimepiride, and gliclazide) or nateglinide, intracellular production of reactive oxygen species (ROS) was evaluated by staining with 2',7'-dichlorofluorescein diacetate. The effect of these agents on apoptosis was also evaluated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling technique. Exposure of beta-cells to glibenclamide, glimepiride, and nateglinide significantly increased intracellular ROS production in a concentration-dependent manner (0.1-10 micromol/L). These effects were completely blocked by nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase inhibitors (diphenylene iodonium or apocynin) or a protein kinase C inhibitor (calphostin C). After exposure to these agents for 48 hours, the numbers of apoptotic cells were also significantly increased. These effects were significantly blocked by apocynin and antioxidant N-acetyl-L-cysteine. In contrast, exposure to any concentrations of gliclazide did not affect either intracellular ROS production or the numbers of apoptotic cells. Sulfonylureas (glibenclamide and glimepiride, but not gliclazide) and nateglinide stimulated ROS production via protein kinase C-dependent activation of NAD(P)H oxidase and consequently caused beta-cell apoptosis in vitro. Because of the lack of such adverse effects, gliclazide may have a benefit in the preservation of functional beta-cell mass.

Therapeutic angiogenesis by ex vivo expanded erythroid progenitor cells.

Recent reports have demonstrated that erythroid progenitor cells contain and secrete various angiogenic cytokines. Here, the impact of erythroid colony-forming cell (ECFC) implantation on therapeutic angiogenesis was investigated in murine models of hindlimb ischemia. During the in vitro differentiation, vascular endothelial growth factor (VEGF) secretion by ECFCs was observed from day 3 (burst-forming unit erythroid cells) to day 10 (erythroblasts). ECFCs from day 5 to day 7 (colony-forming unit erythroid cells) showed the highest VEGF productivity, and day 6 ECFCs were used for the experiments. ECFCs contained larger amounts of VEGF and fibroblast growth factor-2 (FGF-2) than peripheral blood mononuclear cells (PBMNCs). In tubule formation assays with human umbilical vein endothelial cells, ECFCs stimulated 1.5-fold more capillary growth than PBMNCs, and this effect was suppressed by antibodies against VEGF and FGF-2. Using an immunodeficient hindlimb ischemia model and laser-Doppler imaging, we evaluated the limb salvage rate and blood perfusion after intramuscular implantation of ECFCs. ECFC implantation increased both the salvage rate (38% vs. 0%, P < 0.05) and the blood perfusion (82.8% vs. 65.6%, P < 0.01). In addition, ECFCs implantation also significantly increased capillaries with recruitment of vascular smooth muscle cells and the capillary density was 1.6-fold higher than in the control group. Continuous production of human VEGF from ECFCs in the skeletal muscle was confirmed at least 7 days after the implantation. Implantation of ECFCs promoted angiogenesis in ischemic limbs by supplying angiogenic cytokines (VEGF and FGF-2), suggesting a possible novel strategy for therapeutic angiogenesis.

Expression of adipose differentiation-related protein (ADRP) is conjointly regulated by PU.1 and AP-1 in macrophages.

ADRP is associated with intracellular lipid droplets. We demonstrate the regulatory mechanism for ADRP expression in RAW264.7 macrophages. The ADRP mRNA expression was stimulated by PMA, and synergistically enhanced in association with its protein level in the presence of lipids. A proteasome inhibitor protected the protein from degradation under the lipid-free conditions. One of the possible sites of the PMA action was proved to be an Ets/AP-1 element in the promoter, since mutations of this site reduced the PMA-induced promoter activity, and ligation of this element led to a significant increase in the PMA-responsiveness of homologous or heterologous promoters. Mutations of this site diminished the synergistic effect on the promoter activity induced by PMA and oleic acid, suggesting a possible interaction between this site and the downstream PPARdelta site. EMSA revealed that PU.1 and AP-1 conjointly bound to this site. The juxtaposition of the two sequences was requisite for full activity, since spacer sequences between them decreased the PMA-induced activity. PI3 kinase inhibitor was found to reduce the PMA-induced mRNA expression and promoter activity in parallel with PU.1/AP-1 complex formation on EMSA. From these results, we concluded that the Ets/AP-1 site is an important cis-acting element that regulates the ADRP gene expression in macrophages.

Erythropoietin attenuated high glucose-induced apoptosis in cultured human aortic endothelial cells.

High glucose-induced apoptosis in vascular endothelial cells may contribute to the acceleration of atherosclerosis associated with diabetes. Here, we show that erythropoietin attenuates high glucose-induced apoptosis in cultured human aortic endothelial cells (HAECs). Exposure of HAECs to high glucose level for 72h significantly increased the number of apoptotic cells compared with normal glucose level, as evaluated by TUNEL assay. Simultaneous addition of erythropoietin (100 U/ml) significantly attenuated high glucose-induced apoptosis. In parallel, exposure to high glucose level induced caspase-3 activation and erythropoietin also prevented it. Erythropoietin stimulated Akt phosphorylation in a dose-dependent manner (1-100 U/ml). PI3 kinase inhibitor, wortmannin or LY294002 eliminated erythropoietin's inhibitory effect on caspase-3 activity. In conclusion, erythropoietin may attenuate high glucose-induced endothelial cell apoptosis via PI-3 kinase pathway. Replacing therapy with erythropoietin is often used for correction of renal anemia, but may have potential in preventing atherosclerosis in diabetic patients with end-stage renal failure.

Increased expression of NAD(P)H oxidase in islets of animal models of Type 2 diabetes and its improvement by an AT1 receptor antagonist.

This study was undertaken to reveal the role of NAD(P)H oxidase in increased oxidative stress in islets of Type 2 diabetes. Immunostaining analysis showed that staining intensities of NAD(P)H oxidase components, gp91phox and p22phox, significantly increased in islets of animal models of Type 2 diabetes, OLETF rats (60 weeks of age) and db/db mice (14 weeks of age), compared with age-matched controls, respectively, correlating with increased levels of oxidative stress marker, 8-hydroxy-deoxyguanosine or 4-hydroxy-2-nonenal modified protein. In db/db mice, oral administration of angiotensin II Type 1 receptor antagonist valsartan (5 mg/kg) for 4 weeks significantly attenuated the increased expression of gp91phox and p22phox together with inhibition of oxidative stress and partially restored decreased insulin contents in islets. Angiotensin II-related increased expression of NAD(P)H oxidase may play an important role in increased oxidative stress in islets of Type 2 diabetes. This mechanism may be a novel therapeutic target for preventing beta-cell damage.