Toyoshi Inoguchi

Protein Kinase C–Dependent Increase in Reactive Oxygen Species (ROS) Production in Vascular Tissues of Diabetes: Role of Vascular NAD(P)H Oxidase

Hyperglycemia seems to be an important causative factor in the development of micro- and macrovascular complications in patients with diabetes. Several hypotheses have been proposed to explain the adverse effects of hyperglycemia on vascular cells. Both protein kinase C (PKC) activation and oxidative stress theories have increasingly received attention in recent years. This article shows a PKC-dependent increase in oxidative stress in diabetic vascular tissues. High glucose level stimulated reactive oxygen species (ROS) production via a PKC-dependent activation of NAD(P)H oxidase in cultured aortic endothelial cells, smooth muscle cells, and renal mesangial cells. In addition, expression of NAD(P)H oxidase components were shown to be upregulated in vascular tissues and kidney from animal models of diabetes. Furthermore, several agents that were expected to block the mechanism of a PKC-dependent activation of NAD(P)H oxidase clearly inhibited the increased oxidative stress in diabetic animals, as assessed by in vivo electron spin resonance method. Taken together, these findings strongly suggest that the PKC-dependent activation of NAD(P)H oxidase may be an essential mechanism responsible for increased oxidative stress in diabetes.

Increased expression of NAD(P)H oxidase subunits, NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment.

Aim/hypothesisAn increased production of reactive oxygen species (ROS) could contribute to the development of diabetic nephropathy. NAD(P)H oxidase might be an important source of ROS production in kidney as reported in blood vessels. In this study, we show the increased expression of essential subunits of NAD(P)H oxidase, NOX4 and p22phox, in the kidney of diabetic rats.MethodsThe levels of mRNA of both NOX4 and p22phox were evaluated in kidney from streptozotocin-induced diabetic rats and age-matched control rats at 4 and 8 weeks after onset of diabetes by Northern blot analysis. The localization and expression levels of these components and 8-hydroxy-deoxyguanosine (8-OHdG), which is a marker of ROS-induced DNA damage, were also evaluated by immunostaining.ResultsThe levels of both NOX4 and p22phox mRNA were increased in the kidney of diabetic rats as compared with control rats. Immunostaining analysis showed that the expression levels of NOX4 and p22phox were clearly increased in both distal tubular cells and glomeruli from diabetic rats. Both the localization and the expression levels of these components were in parallel with those of 8-OHdG. Interventive insulin treatment for 2 weeks completely restored the increased levels of these components in the diabetic kidney to control levels in parallel with those of 8-OHdG.Conclusions/interpretationThis study provides evidence that NAD(P)H oxidase subunits, NOX4 and p22phox, were increased in the kidney of diabetic rats. Thus, NAD(P)H-dependent overproduction of ROS could cause renal tissue damage in diabetes. This might contribute to the development of diabetic nephropathy.

Antithrombin III stimulates prostacyclin production by cultured aortic endothelial cells

Prostacyclin (PGI2) is a potent vasodilator and an inhibitor of platelet aggregation. We found that antithrombin III (AT III), an anticoagulant present in circulating blood, stimulated PGI2 production by cultured bovine aortic endothelial cells in a dose- and time-dependent manner. The stimulation of PGI2 production by AT III was observed at physiological concentrations and was inhibited by the addition of anti-AT III antiserum and heparin. These results suggest that AT III may stimulate PGI2 production by binding to heparin-like molecules on the endothelial cell membrane.

Biochemical and molecular mechanisms in the development of diabetic vascular complications

Hyperglycemia is the major causal factor in the development of diabetic vascular complications. The mechanism by which hyperglycemia causes the complications is not clear; however, it is very likely that hyperglycemia is mediating its adverse effects through multiple mechanisms. We have summarized some of these mechanisms in this review, with particular attention to the effect of hyperglycemia on the activation of diacylglycerol (DAG)-protein kinase C (PKC) pathway. We have reviewed existing information regarding various vascular tissues that show increased DAG and PKC levels. In addition, the mechanism by which hyperglycemia increases DAG as well as the cellular physiological consequences on the activation of PKC have been reviewed.

NAD(P)H Oxidase Activation: A Potential Target Mechanism for Diabetic Vascular Complications, Progressive β-Cell Dysfunction and Metabolic Syndrome

Both protein kinase C (PKC) activation and increased oxidative stress have been paid attention to as important causative factors for diabetic vascular complications. In this article, we show a PKC-dependent increase in oxidative stress in vascular tissues of diabetes and insulin resistant state. High glucose level and free fatty acids stimulate de novo diacylglycerol (DAG)-PKC pathway and subsequently stimulate reactive oxygen species (ROS) production through a PKC-dependent activation of NAD(P)H oxidase. Increasing evidence has also shown that NAD(P)H oxidase components are upregulated in micro- and macro- vascular tissues of animal models and patients of diabetes and obesity. It is also noted that increased intrinsic angiotensin II production may amplify such a PKC-dependent activation of NAD(P)H oxidase in diabetic vascular tissues. These mechanisms may play an important role in the diabetic vascular complications and the accelerated atherosclerosis associated with diabetes and obesity. In addition, recent reports have shown that NAD(P)H oxidases exist in pancreatic beta-cells and adipocytes, and this oxidase-generated ROS production may play an important role in both the progressive beta-cell dysfunction and the dysregulated adipocytokine production and subsequent obesity-induced metabolic syndrome. These results suggest that an NAD(P)H oxidase activation may be a useful therapeutic target for preventing diabetic vascular complications, progressive beta-cell dysfunction and metabolic syndrome.

Bilirubin and biliverdin protect rodents against diabetic nephropathy by downregulating NAD(P)H oxidase

We recently found a markedly lower prevalence of vascular complications, including kidney disease, in diabetic patients with Gilbert syndrome, a congenital form of hyperbilirubinemia, suggesting a beneficial effect of bilirubin (BIL) on diabetic nephropathy. To directly examine this, we determined whether hereditary hyperbilirubinemic Gunn j/j rats and biliverdin (BVD)-treated diabetic db/db mice were resistant to the development of renal disease. Both rodent models had less albuminuria and complete protection against the progression of mesangial expansion accompanied by normalization of transforming growth factor-β1 and fibronectin expression. Simultaneously, there was normalization of urinary and renal oxidative stress markers, and the expression of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase subunits in the kidney. In cultured vascular endothelial and mesangial cells, BIL and BVD significantly inhibited NADPH-dependent superoxide production, and both high glucose- and angiotensin II-induced production of reactive oxygen species. Collectively, our findings suggest that BIL and BVD may protect against diabetic nephropathy and may lead to novel antioxidant therapies for diabetic nephropathy.

Confirmation of Superoxide Generation via Xanthine Oxidase in Streptozotocin-induced Diabetic Mice

Reactive oxygen species (ROS) may play key roles in vascular inflammation and atherogenesis in patients with diabetes. In this study, xanthine oxidase (XO) system was examined as a potential source of superoxide in mice with streptozotocin (STZ)-induced experimental diabetes. Plasma XO activity increased 3-fold in diabetic mice (50±33 μU/ml) 2 weeks after the onset of diabetes, as compared with non-diabetic control mice (15±6 μU/ml). In vivo superoxide generation in diabetic mice was evaluated by an in vivo electron spin resonance (ESR)/spin probe method. Superoxide generation was significantly enhanced in diabetic mice, and the enhancement was restored by the administration of superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), which was reported to scavenge superoxide. Pretreatment of diabetic mice with XO inhibitors, allopurinol and its active metabolite oxipurinol, normalized the increased superoxide generation. In addition, there was a correlation (r=0.78) between the level of plasma XO activity and the relative degree of superoxide generation in diabetic and non-diabetic mice. Hence, the results of this study strongly suggest that superoxide should be generated through the increased XO seen in the diabetic model mice, which may be involved in the pathogenesis of diabetic vascular complications.

The lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets

CONTEXT The molecular mechanisms by which triglycerides in lipid droplets (LDs) are synthesized, stored, and degraded need to be elucidated. OBJECTIVE The objectives were to report siblings with neutral lipid storage disease with myopathy (NLSDM) with a novel mutation of adipose triglyceride lipase (ATGL) and determine whether the C-terminal part of ATGL containing the hydrophobic region plays a role in the interaction with LDs. DESIGN AND PATIENTS Skin fibroblasts and peripheral blood leukocytes were obtained from NLSDM patients. In vitro experiments were performed with fibroblasts and COS7 cells. MAIN OUTCOME MEASURES Transfection studies were used to assess the effects of various recombinant ATGL proteins on lipase activities and lipid contents. Fluorescence microscopy were used for determination of intracellular distribution of ATGL proteins. RESULTS The direct sequence of ATGL cDNA reveals that a patient is a homozygote for the 4-bp deletion, leading to a premature stop codon and causes the lack of the C terminus of the protein including the hydrophobic domain. Overexpressed control ATGL in NLSDM fibroblasts was found around the rims of LDs and caused significantly reduced cellular lipid accumulation. In contrast, NLSDM ATGL was homogeneously located in the cytoplasm despite the presence of LDs and had almost no effect on LD degradation despite its similar lipase activity. A series of C-terminal truncated ATGLs without the intact hydrophobic domain failed to localize around and degrade LDs. CONCLUSIONS These findings indicate that the domain including the hydrophobic region of ATGL was essential for association with LDs.

High glucose stimulates hepatic stellate cells to proliferate and to produce collagen through free radical production and activation of mitogen-activated protein kinase.

Abstract: Background: Nonalcoholic steatohepatitis is a clinicopathologic condition that may progress to liver fibrosis. Hyperglycemia is supposed to be one of the factors inducing hepatic fibrogenesis, but the mechanism has not been fully clarified. Oxidative stress is increasingly found in patients with diabetes/hyperglycemia in which conditions reactive oxygen species (ROS) are produced.

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-β 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.