Retinol binding protein 3 is increased in the retina of patients with diabetes resistant to diabetic retinopathy.

The Joslin Medalist Study characterized people affected with type 1 diabetes for 50 years or longer. More than 35% of these individuals exhibit no to mild diabetic retinopathy (DR), independent of glycemic control, suggesting the presence of endogenous protective factors against DR in a subpopulation of patients. Proteomic analysis of retina and vitreous identified retinol binding protein 3 (RBP3), a retinol transport protein secreted mainly by the photoreceptors, as elevated in Medalist patients protected from advanced DR. Mass spectrometry and protein expression analysis identified an inverse association between vitreous RBP3 concentration and DR severity. Intravitreal injection and photoreceptor-specific overexpression of RBP3 in rodents inhibited the detrimental effects of vascular endothelial growth factor (VEGF). Mechanistically, our results showed that recombinant RBP3 exerted the therapeutic effects by binding and inhibiting VEGF receptor tyrosine phosphorylation. In addition, by binding to glucose transporter 1 (GLUT1) and decreasing glucose uptake, RBP3 blocked the detrimental effects of hyperglycemia in inducing inflammatory cytokines in retinal endothelial and Müller cells. Elevated expression of photoreceptor-secreted RBP3 may have a role in protection against the progression of DR due to hyperglycemia by inhibiting glucose uptake via GLUT1 and decreasing the expression of inflammatory cytokines and VEGF.

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.

Exogenous Insulin Infusion Can Decrease Atherosclerosis in Diabetic Rodents by Improving Lipids, Inflammation, and Endothelial Function.

OBJECTIVE: The objective of this study is to evaluate whether exogenously induced hyperinsulinemia may increase the development of atherosclerosis. APPROACH AND RESULTS: Hyperinsulinemia, induced by exogenous insulin implantation in high-fat fed (60% fat HFD) apolipoprotein E-deficient mice (ApoE-/-) mice, exhibited insulin resistance, hyperglycemia, and hyperinsulinemia. Atherosclerosis was measured by the accumulation of fat, macrophage, and extracellular matrix in the aorta. After 8 weeks on HFD, ApoE-/- mice were subcutaneously implanted with control (sham) or insulin pellet, and phlorizin, a sodium glucose cotransporters inhibitor (1/2)inhibitor, for additional 8 weeks. Intraperitoneal glucose tolerance test showed that plasma glucose levels were lower and insulin and IGF-1 (insulin-like growth factor-1) levels were 5.3- and 3.3-fold higher, respectively, in insulin-implanted compared with sham-treated ApoE-/- mice. Plasma triglyceride, cholesterol, and lipoprotein levels were decreased in mice with insulin implant, in parallel with increased lipoprotein lipase activities. Atherosclerotic plaque by en face and complexity staining showed significant reductions of fat deposits and expressions of vascular adhesion molecule-1, tumor necrosis factor-α, interleukin 6, and macrophages in arterial wall while exhibiting increased activation of pAKT and endothelial nitric oxide synthase (P<0.05) comparing insulin-implanted versus sham HFD ApoE-/- mice. No differences were observed in atherosclerotic plaques between phlorizin-treated and sham HFD ApoE-/- mice, except phlorizin significantly lowered plasma glucose and glycated hemoglobin levels while increased glucosuria. Endothelial function was improved only by insulin treatment through endothelial nitric oxide synthase/nitric oxide activations and reduced proinflammatory (M1) and increased anti-inflammatory (M2) macrophages, which were inhibited by endothelial nitric oxide synthase inhibitor. CONCLUSIONS: Exogenous insulin decreased atherosclerosis by lowering inflammatory cytokines, macrophages, and plasma lipids in HFD-induced hyperlipidemia, insulin resistant and mildly diabetic ApoE-/- mice.

Overexpressing IRS1 in Endothelial Cells Enhances Angioblast Differentiation and Wound Healing in Diabetes and Insulin Resistance.

The effect of enhancing insulin's actions in endothelial cells (ECs) to improve angiogenesis and wound healing was studied in obesity and diabetes. Insulin receptor substrate 1 (IRS1) was overexpressed in ECs using the VE-cadherin promoter to create ECIRS1 TG mice, which elevated pAkt activation and expressions of vascular endothelial growth factor (VEGF), Flk1, and VE-cadherin in ECs and granulation tissues (GTs) of full-thickness wounds. Open wound and epithelialization rates and angiogenesis significantly improved in normal mice and high fat (HF) diet-induced diabetic mice with hyperinsulinemia in ECIRS1 TG versus wild type (WT), but not in insulin-deficient diabetic mice. Increased angioblasts and EC numbers in GT of ECIRS1 mice were due to proliferation in situ rather than uptake. GT in HF-fed diabetic mice exhibited parallel decreases in insulin and VEGF-induced pAkt and EC numbers by >50% without changes in angioblasts versus WT mice, which were improved in ECIRS1 TG mice on normal chow or HF diet. Thus, HF-induced diabetes impaired angiogenesis by inhibiting insulin signaling in GT to decrease the differentiation of angioblasts to EC, which was normalized by enhancing insulin's action targeted to EC, a potential target to improve wound healing in diabetes and obesity.

PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts.

Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients.

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.

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.

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.

[Ruptured true posterior communicating artery fusiform aneurysm: case report].

A 32-year-old female complained of a headache persisting for approximately one week. She was emergently brought to the hospital for sudden consciousness disorder early in the morning. Her neurological state was JCS 200 and WFNS Grade V. CT scan demonstrated a subarachnoid hemorrhage. 3D-CTA and DSA showed a right true posterior communicating artery fusiform aneurysm. We performed open surgery the same day, and found that the aneurysmal wall had thinned markedly. The aneurysm ruptured, and trapping was performed. A perforating artery from the aneurysm was sacrificed, but there was no apparent neurological deficit. The patient was ambulatory when discharged after a VP shunt was implanted, and she has returned to normal daily life. True posterior communicating artery fusiform aneurysm is very rare, and has been reported in detail in only 6 cases to date. A brief clinical review of the literature is presented. Since this type of aneurysm enlarges rapidly in some patients, and the possibility of re-rupture in the acute period is high, prompt trapping after onset is recommended.

Expression of erythropoietin receptor-like molecule in Xenopus laevis and erythrocytopenia upon administration of its recombinant soluble form.

The regulation of hematopoiesis in non-mammalian vertebrates is poorly understood. This is partly because the structures and effects of most hematopoietic regulators have not been identified. As a first step towards studies on the key mechanism of hematopoietic regulation among phyla as well as the diversity of organisms, we have focused on amphibian hematopoiesis. A cDNA sharing the highest degree of homology with mammalian erythropoietin (EPO) receptors, tentatively named xlEPOR, was cloned from a cDNA library of Xenopus laevis immature erythrocytes. The comparative identities of the deduced entire amino acid sequence to mammalian EPO receptors were quite low, although functional domains indispensable for erythropoietic activities were found in the molecule. Northern analysis revealed that xlEPOR were expressed in peripheral blood cells. In the peripheral blood of phenylhydrazine-treated adult Xenopus, immature erythrocytes expressing xlEPOR were identified by in situ hybridization and immunostaining with polyclonal antibodies to xlEPOR. To confirm the biological functions of this molecule, the extracellular domain of xlEPOR (i.e., soluble xlEPOR) was administered to adult Xenopus by consecutive intracardiac injection. The peripheral erythrocyte counts were decreased gradually; meanwhile, immature erythrocytes appeared in the circulation, demonstrating that xlEPOR plays a significant physiological role in erythropoiesis in Xenopus laevis.

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.