Noriyuki Sonoda

β-Arrestin-1 mediates glucagon-like peptide-1 signaling to insulin secretion in cultured pancreatic β cells

Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted from enteroendocrine L cells and potentiates glucose-dependent insulin secretion in pancreatic β cells. Recently the GLP-1 receptor (GLP-1 R) has been a focus for new anti-diabetic therapy with the introduction of GLP-1 analogues and DPP-IV inhibitors, and this has stimulated additional interest in the mechanisms of GLP-1 signaling. Here we identify a mechanism for GLP-1 action, showing that the scaffold protein β-arrestin-1 mediates the effects of GLP-1 to stimulate cAMP production and insulin secretion in β cells. Using a coimmunoprecipitation technique, we also found a physical association between the GLP-1 R and β-arrestin-1 in cultured INS-1 pancreatic β cells. β-Arrestin-1 knockdown broadly attenuated GLP-1 signaling, causing decreased ERK and CREB activation and IRS-2 expression as well as reduced cAMP levels and impaired insulin secretion. However, β-arrestin-1 knockdown did not affect GLP-1 R surface expression and ligand-induced GLP-1 R internalization/desensitization. Taken together, these studies indicate that β-arrestin-1 plays a role in GLP-1 signaling leading to insulin secretion, defining a previously undescribed mechanism for GLP-1 action.

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.

SIRT1 Exerts Anti-Inflammatory Effects and Improves Insulin Sensitivity in Adipocytes

ABSTRACT SIRT1 is a prominent member of a family of NAD+-dependent enzymes and affects a variety of cellular functions ranging from gene silencing, regulation of the cell cycle and apoptosis, to energy homeostasis. In mature adipocytes, SIRT1 triggers lipolysis and loss of fat content. However, the potential effects of SIRT1 on insulin signaling pathways are poorly understood. To assess this, we used RNA interference to knock down SIRT1 in 3T3-L1 adipocytes. SIRT1 depletion inhibited insulin-stimulated glucose uptake and GLUT4 translocation. This was accompanied by increased phosphorylation of JNK and serine phosphorylation of insulin receptor substrate 1 (IRS-1), along with inhibition of insulin signaling steps, such as tyrosine phosphorylation of IRS-1, and phosphorylation of Akt and ERK. In contrast, treatment of cells with specific small molecule SIRT1 activators led to an increase in glucose uptake and insulin signaling as well as a decrease in serine phosphorylation of IRS-1. Moreover, gene expression profiles showed that SIRT1 expression was inversely related to inflammatory gene expression. Finally, we show that treatment of 3T3-L1 adipocytes with a SIRT1 activator attenuated tumor necrosis factor alpha-induced insulin resistance. Taken together, these data indicate that SIRT1 is a positive regulator of insulin signaling at least partially through the anti-inflammatory actions in 3T3-L1 adipocytes.

SIRT1 inhibits inflammatory pathways in macrophages and modulates insulin sensitivity.

Chronic inflammation is an important etiology underlying obesity-related disorders such as insulin resistance and type 2 diabetes, and recent findings indicate that the macrophage can be the initiating cell type responsible for this chronic inflammatory state. The mammalian silent information regulator 2 homolog SIRT1 modulates several physiological processes important for life span, and a potential role of SIRT1 in the regulation of insulin sensitivity has been shown. However, with respect to inflammation, the role of SIRT1 in regulating the proinflammatory pathway within macrophages is poorly understood. Here, we show that knockdown of SIRT1 in the mouse macrophage RAW264.7 cell line and in intraperitoneal macrophages broadly activates the JNK and IKK inflammatory pathways and increases LPS-stimulated TNFalpha secretion. Moreover, gene expression profiles reveal that SIRT1 knockdown leads to an increase in inflammatory gene expression. We also demonstrate that SIRT1 activators inhibit LPS-stimulated inflammatory pathways, as well as secretion of TNFalpha, in a SIRT1-dependent manner in RAW264.7 cells and in primary intraperitoneal macrophages. Treatment of Zucker fatty rats with a SIRT1 activator leads to greatly improved glucose tolerance, reduced hyperinsulinemia, and enhanced systemic insulin sensitivity during glucose clamp studies. These in vivo insulin-sensitizing effects were accompanied by a reduction in tissue inflammation markers and a decrease in the adipose tissue macrophage proinflammatory state, fully consistent with the in vitro effects of SIRT1 in macrophages. In conclusion, these results define a novel role for SIRT1 as an important regulator of macrophage inflammatory responses in the context of insulin resistance and raise the possibility that targeting of SIRT1 might be a useful strategy for treating the inflammatory component of metabolic diseases.

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.

Myosin 5a Is an Insulin-Stimulated Akt2 (Protein Kinase Bβ) Substrate Modulating GLUT4 Vesicle Translocation

ABSTRACT Phosphatidylinositol 3-kinase activation of Akt signaling is critical to insulin-stimulated glucose transport and GLUT4 translocation. However, the downstream signaling events following Akt activation which mediate glucose transport stimulation remain relatively unknown. Here we identify an Akt consensus phosphorylation motif in the actin-based motor protein myosin 5a and show that insulin stimulation leads to phosphorylation of myosin 5a at serine 1650. This Akt-mediated phosphorylation event enhances the ability of myosin 5a to interact with the actin cytoskeleton. Small interfering RNA-induced inhibition of myosin 5a and expression of dominant-negative myosin 5a attenuate insulin-stimulated glucose transport and GLUT4 translocation. Furthermore, knockdown of Akt2 or expression of dominant-negative Akt (DN-Akt) abolished insulin-stimulated phosphorylation of myosin 5a, inhibited myosin 5a binding to actin, and blocked insulin-stimulated glucose transport. Taken together, these data indicate that myosin 5a is a newly identified direct substrate of Akt2 and, upon insulin stimulation, phosphorylated myosin 5a facilitates anterograde movement of GLUT4 vesicles along actin to the cell surface.

FOXO1 Transrepresses Peroxisome Proliferator-activated Receptor γ Transactivation, Coordinating an Insulin-induced Feed-forward Response in Adipocytes

The transcriptional factor FoxO1 plays an important role in metabolic homeostasis. Herein we identify a novel transrepressional function that converts FoxO1 from an activator of transcription to a promoter-specific repressor of peroxisome proliferator-activated receptor γ (PPARγ) target genes that regulate adipocyte biology. FoxO1 transrepresses PPARγ via direct protein-protein interactions; it is recruited to PPAR response elements (PPRE) on PPARγ target genes by PPARγ bound to PPRE and interferes with promoter DNA occupancy of the receptor. The FoxO1 transrepressional function, which is independent and dissectible from the transactivational effects, does not require a functional FoxO1 DNA binding domain, but dose require an evolutionally conserved 31 amino acids LXXLL-containing domain. Insulin induces FoxO1 phosphorylation and nuclear exportation, which prevents FoxO1-PPARγ interactions and rescues transrepression. Adipocytes from insulin resistant mice show reduced phosphorylation and increased nuclear accumulation of FoxO1, which is coupled to lowered expression of endogenous PPARγ target genes. Thus the innate FoxO1 transrepression function enables insulin to augment PPARγ activity, which in turn leads to insulin sensitization, and this feed-forward cycle represents positive reinforcing connections between insulin and PPARγ signaling.

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.

Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells

OBJECTIVE Metformin and glucagon like peptide-1 (GLP-1) prevent diabetic cardiovascular complications and atherosclerosis. However, the direct effects on hyperglycemia-induced oxidative stress in endothelial cells are not fully understood. Thus, we aimed to evaluate the effects of metformin and a GLP-1 analog, liraglutide on high glucose-induced oxidative stress. METHODS Production of reactive oxygen species (ROS), activation of protein kinase C (PKC) and NAD(P)H oxidase, and changes in signaling molecules in response to high glucose exposure were evaluated in human aortic endothelial cells with and without treatment of metformin and liraglutide, alone or in combination. PKC-NAD(P)H oxidase pathway was assessed by translocation of GFP-fused PKCβ2 isoform and GFP-fused p47phox, a regulatory subunit of NAD(P)H oxidase, in addition to endogenous PKC phosphorylation and NAD(P)H oxidase activity. RESULTS High glucose-induced ROS overproduction was blunted by metformin or liraglutide treatment, with a further decrease by a combination of these drugs. Exposure to high glucose caused PKCβ2 translocation and a time-dependent phosphorylation of endogenous PKC but failed to induce its translocation and phosphorylation in the cells treated with metformin and liraglutide. Furthermore, both drugs inhibited p47phox translocation and NAD(P)H oxidase activation, and prevented the high glucose-induced changes in intracellulalr diacylglycerol (DAG) level and phosphorylation of AMP-activated protein kinase (AMPK). A combination of these drugs further enhanced all of these effects. CONCLUSIONS Metformin and liraglutide ameliorate high glucose-induced oxidative stress by inhibiting PKC-NAD(P)H oxidase pathway. A combination of these two drugs provides augmented protective effects, suggesting the clinical usefulness in prevention of diabetic vascular complications.

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.