High Glucose-induced Changes Research Articles

Empagliflozin and liraglutide ameliorate HFpEF in mice via augmenting the Erbb4 signaling pathway.

Heart failure with preserved ejection fraction (HFpEF) is closely associated with metabolic derangement. Sodium glucose cotransporter-2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) exert anti-HFpEF effects, but the underlying mechanisms remain unclear. In this study, we explored the anti-HFpEF effects of empagliflozin and liraglutide and the underlying molecular mechanisms in a mouse model of HFpEF. This model was established by high-fat diet (HFD) feedingplus Nω-nitro-L-arginine methyl ester (L-NAME) treatment. The mice were treated with empagliflozin (20 mg·kg-1·d-1, i.g.) or liraglutide (0.3 mg·kg-1·d-1, i.p.) or their combination for 4 weeks. At the end of the experimental protocol, cardiac function was measured using ultrasound, then mice were euthanized and heart, liver, and kidney tissues were collected. Nuclei were isolated from frozen mouse ventricular tissue for single-nucleus RNA-sequencing (snRNA-seq). We showed that administration of empagliflozin or liraglutide alone or in combination significantly improved diastolic function, ameliorated cardiomyocyte hypertrophy and cardiac fibrosis, as well as exercise tolerance but no synergism was observed in the combination group. Furthermore, empagliflozin and/or liraglutide lowered body weight, improved glucose metabolism, lowered blood pressure, and improved liver and kidney function. After the withdrawal of empagliflozin or liraglutide for 1 week, these beneficial effects tended to diminish. The snRNA-seq analysis revealed a subcluster of myocytes, in which Erbb4 expression was down-regulated under HFpEF conditions, and restored by empagliflozin or liraglutide. Pseudo-time trajectory analysis and cell-to-cell communication studies confirmed that the Erbb4 pathway was a prominent pathway essential for both drug actions. In the HFpEF mouse model, both empagliflozin and liraglutide reversed Erbb4 down-regulation. In rat h9c2 cells, we showed that palmitic acid- or high glucose-induced changes in PKCα and/or ERK1/2 phosphorylation at least in part through Erbb4. Collectively, the single-cell atlas reveals the anti-HFpEF mechanism of empagliflozin and liraglutide, suggesting that Erbb4 pathway represents a new therapeutic target for HFpEF. Effects and mechanisms of action of empagliflozin and liraglutide in HFpEF mice. HFpEF was induced with a high-fat diet and L-NAME for 15 weeks, and treatment with empagliflozin and liraglutide improved the HFpEF phenotype. Single nucleus RNA sequencing (snRNA-seq) was used to reveal the underlying mechanism of action of empagliflozin and liraglutide.

Curcumin Blocks High Glucose-Induced Podocyte Injury via RIPK3-Dependent Pathway.

Podocyte loss is well known to play a critical role in the early progression of diabetic nephropathy. A growing number of studies are paying attention to necroptosis, a programmed form of cell necrosis as a mechanism of podocyte loss. Although necroptosis is a recently established concept, the significance of receptor interacting serine/threonine kinase 3 (RIPK3), a gene that encodes for the homonymous enzyme RIPK3 responsible for the progression of necroptosis, is well studied. Curcumin, a natural hydrophobic polyphenol compound responsible for the yellow color of Curcuma longa, has drawn attention due to its antioxidant and anti-inflammatory effects on cells prone to necroptosis. Nonetheless, effects of curcumin on high glucose-induced podocyte necroptosis have not been reported yet. Therefore, this study investigated RIPK3 expression in high glucose-treated podocytes to identify the involvement of necroptosis via the RIPK3 pathway and the effects of curcumin treatment on RIPK3-dependent podocytopathy in a hyperglycemic environment. The study discovered that increased reactive oxygen species (ROS) in renal podocytes induced by high glucose was improved after curcumin treatment. Curcumin treatment also significantly restored the upregulated levels of VEGF, TGF-β, and CCL2 mRNAs and the downregulated level of nephrin mRNA in cultured podocytes exposed to a high glucose environment. High glucose-induced changes in protein expression of TGF-β, nephrin, and CCL2 were considerably reverted to their original levels after curcumin treatment. Increased expression of RIPK3 in high glucose-stimulated podocytes was alleviated by curcumin treatment as well as N-acetyl cysteine (NAC, an antioxidant) or GSK′872 (a RIPK3 inhibitor). Consistent with this, the increased necroptosis-associated molecules, such as RIPK3, pRIPK3, and pMLKL, were also restored by curcumin in high glucose-treated mesangial cells. DCF-DA assay confirmed that such a result was attributed to the reduction of RIPK3 through the antioxidant effect of curcumin. Further observations of DCF-DA-sensitive intracellular ROS in NAC-treated and GSK′872-treated podocyte groups showed a reciprocal regulatory relationship between ROS and RIPK3. The treatment of curcumin and GSK′872 in podocytes incubated with high glucose protected from excessive intracellular superoxide anion production. Taken together, these results indicate that curcumin treatment can protect against high glucose-induced podocyte injuries by suppressing the abnormal expression of ROS and RIPK3. Thus, curcumin might be a potential therapeutic agent for diabetic nephropathy as an inhibitor of RIPK3.

Wogonoside alleviates high glucose-induced dysfunction of retinal microvascular endothelial cells and diabetic retinopathy in rats by up-regulating SIRT1

To investigate the effects of wogonoside on high glucose-induced dysfunction of human retinal microvascular endothelial cells (hRMECs) and streptozotocin (STZ)-induced diabetic retinopathy in rats and explore the underlying molecular mechanism. HRMECs in routine culture were treated with 25 mmol/L mannitol or exposed to high glucose (30 mmol/L glucose) and treatment with 10, 20, 30, 40 μmol/L wogonoside. CCK-8 assay and Transwell assay were used to examine cell proliferation and migration, and the changes in tube formation and monolayer cell membrane permeability were tested. ROS, NO and GSH-ST kits were used to evaluate oxidative stress levels in the cells. The expressions of IL-1β and IL-6 in the cells were examined with qRT-PCR and ELISA, and the protein expressions of VEGF, HIF-1α and SIRT1 were detected using Western blotting. We also tested the effect of wogonoside on retinal injury and expressions of HIF-1α, ROS, VEGF, TNF-α, IL-1β, IL-6 and SIRT1 proteins in rat models of STZ-induced diabetic retinopathy. High glucose exposure caused abnormal proliferation and migration, promoted angiogenesis, increased membrane permeability (P < 0.05), and induced inflammation and oxidative stress in hRMECs (P < 0.05). Wogonoside treatment concentration-dependently inhibited high glucose-induced changes in hRMECs. High glucose exposure significantly lowered the expression of SIRT1 in hRMECs, which was partially reversed by wogonoside (30 μmol/L) treatment; interference of SIRT1 obviously attenuated the inhibitory effects of wogonoside against high glucose-induced changes in proliferation, migration, angiogenesis, membrane permeability, inflammation and oxidative stress in hRMECs. In rat models of STZ-induced diabetic retinopathy, wogonoside effectively suppressed retinal thickening (P < 0.05), alleviated STZ-induced retinal injury, and increased the expression of SIRT1 in the retinal tissues (P < 0.001). Wogonoside alleviates retinal damage caused by diabetic retinopathy by up-regulating SIRT1 expression.

Melatonin Attenuates High Glucose-Induced Changes in Beta Amyloid Precursor Protein Processing in Human Neuroblastoma Cells.

Diabetes mellitus (DM), one of metabolic diseases, has been suggested as a risk factor for Alzheimer's disease (AD). However, how the metabolic pathway activates amyloid precursor protein (APP) processing enzymes then contributes to the increase of amyloid-beta (Aβ) production, is not clearly understood. In the present study, we aimed to examine the protective effect of melatonin against hyperglycemia-induced alterations in the amyloidogenic pathway. High concentration of glucose was used to induce hyperglycemia in human neuroblastoma SH-SY5Y cells. We found that 30mM glucose affected the expression of insulin receptors and glucose transporters, which indicated the disruption of glucose sensing. High glucose induced the activation of the phosphorylated protein kinase B (pAkt)/GSK-3β signaling pathway and a significant increase in the expression of β-site beta APP cleaving enzyme (BACE1), presenilin1 (PS1) and Aβ42. Pretreatment with melatonin significantly reversed these parameters. We also showed that these effects are similar to those effects in the presence of the GSK-3β blocker, N-(4-methoxybenyl)-N'-(5-nitro-1,3-thiazol-2-yl) urea (ARA) in glucose-treated hyperglycemic cells. These suggested that melatonin exerted an inhibitory effect on the activation of APP-cleaving enzymes via the GSK-3β signaling pathway. Pretreatment with luzindole, a melatonin receptor MT1 antagonist, significantly prevented the effect of melatonin on the glucose-induced increase level of APP processing enzymes. This suggested that melatonin attenuated the toxic effect on hyperglycemia involving the amyloidogenic pathway partially mediated via melatonin receptor. Taken together the present results suggested that melatonin has a beneficial role in preventing Aβ generation in a cellular model of hyperglycemia-induced DM.

An untargeted metabolomic strategy based on liquid chromatography-mass spectrometry to study high glucose-induced changes in HK-2 cells

Diabetes mellitus is a major health concern nowadays. It is estimated that 40% of diabetics are affected by diabetic nephropathy, one of the complications derived from high glucose blood levels which can lead to chronic loss of kidney function. It is now clear that the renal proximal tubule plays a critical role in the progression of diabetic nephropathy but research focused on studying the molecular mechanisms involved is still needed. The aim of this work was to develop a liquid chromatography-mass spectrometry platform to carry out, for the first time, the untargeted metabolomic analysis of high glucose-induced changes in cultured human proximal tubular HK-2 cells. In order to find the metabolites which were affected by high glucose and to expand the metabolite coverage, intra- and extracellular fluid from HK-2 cells exposed to high glucose (25 mM), normal glucose (5.5 mM) or osmotic control (5.5 mM glucose +19.5 mM mannitol) were analyzed by two complementary chromatographic modes: hydrophilic interaction and reversed-phase liquid chromatography. Non-supervised principal components analysis showed a good separation among the three groups of samples. Statistically significant variables were chosen for further metabolite identification. Different metabolic pathways were affected mainly those derived from amino acidic, polyol, and nitrogenous bases metabolism.

Protective effects of Schisandrin on high glucose-induced changes of RhoA and eNOS activity in human umbilical vein endothelial cells

Schisandrin, derived from the Chinese medicinal herb Schisandra chinensis, has been found to confer protective effects on circulation systems. But the underlying molecular mechanisms remain unclear. The aim of this study was to investigate the effects of a high level of glucose on RhoA and eNOS activity in human umbilical vein endothelial cells(HUVECs) and how Schisandrin plays a role in mediating these effects. To find the optimal treatment time, HUVECs were cultured at a high glucose concentration (30 mM) for different lengths of time (0, 12, 24, and 48 h). Subsequently, the cells were randomized into five groups: a normal group, a high glucose group, and three high glucose groups that were given different doses (5, 10, and 20 μM) of Schisandrin. The cells were pretreated with Schisandrin for 24 h before stimulation with high glucose. The morphology of HUVECs in the various groups was assessed under a light microscope. Immunocytochemical staining was used to detect the level of p-MYPT1 expression. The levels of RhoA activity were determined using the RhoA Activation Assay Biochem Kit. The levels of eNOS activity were examined using a nitrate reduction test. The results showed that in the high glucose group, the activity of RhoA was increased and the activity of eNOS was reduced, thus decreasing the secretion of NO. However, after pretreatment with Schisandrin (10, 20 μM), the activity of RhoA was inhibited and the activity of eNOS increased, which led to an increase in NO production compared with the high glucose group. There was no evident difference between the 5 μM Schisandrin group and the high glucose group. Taken together, these findings indicate that Schisandrin can improve the function of endothelial cells by lowering the activity of RhoA/Rho kinase and raising both the activity of eNOS and the production of NO.

MicroRNA-145 alleviates high glucose-induced proliferation and migration of vascular smooth muscle cells through targeting ROCK1

The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are important steps in atherosclerosis. The present study aimed to investigate whether the high glucose-induced changes of VSCMs are mediated by miR-145 and the potential molecular mechanism involved. We found that loss of miR-145 accompanied with increased proliferation and migration was observed in cultured human VSMCs exposed to high glucose. Exogenous overexpression of miR-145 effectively suppressed the high glucose-induced excessive proliferation and migration of VSMCs. Furthermore, we identified ROCK1 as a downstream target gene product of miR-145, and ROCK1 partially rescues the effects of miR-145 on high glucose-induced VSMC proliferation and migration. Taken together, our results suggested a protective role of miR-145 in high glucose-treated VSMCs by suppressing ROCK1, which might provide a therapeutic target for diabetic atherosclerosis.

High glucose-induced changes in hyaloid-retinal vessels during early ocular development of zebrafish: a short-term animal model of diabetic retinopathy.

Although a variety of animal models have been used to test drug candidates and examine the pathogenesis of diabetic retinopathy, time-saving and inexpensive models are still needed to evaluate the increasing number of therapeutic approaches. We developed a model for diabetic retinopathy using the early stage of transgenic zebrafish (flk:EGFP) by treating embryos with 130 mM glucose, from 3-6 days post fertilisation (high-glucose model). On day 6, lenses from zebrafish larvae were isolated and treated with 3% trypsin, and changes in hyaloid-retinal vessels were analysed using fluorescent stereomicroscopy. In addition, expression of tight junction proteins (such as zonula occludens-1), effects of hyperosmolar solutions and of hypoxia, and Vegf expression were assessed by RT -PCR. NO production was assessed with a fluorescent substrate. Effects of inhibitors of the VEGF receptor, NO synthesis and a VEGF antibody (ranibizumab) were also measured. In this high-glucose model, dilation of hyaloid-retinal vessels, on day 6, was accompanied by morphological lesions with disruption of tight junction proteins, overproduction of Vegf mRNA and increased NO production. Treatment of this high-glucose model with an inhibitor of VEGF receptor tyrosine kinase or an inhibitor of NO synthase or ranibizumab decreased dilation of hyaloid-retinal vessels. These findings suggest that short-term exposure of zebrafish larvae to high-glucose conditions could be used for screening and drug discovery for diabetic retinopathy and particularly for disorders of retinal vessels related to disruption of tight junction proteins and excessive VEGF and NO production.

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

ObjectiveMetformin 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. MethodsProduction 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. ResultsHigh 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. ConclusionsMetformin 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.

P16 Tadalafil stimulates hydrogen sulfide-AMPK pathway to inhibit high glucose stimulation of protein synthesis in mouse podocytes

Kidney injury is one of the most serious complications of diabetes. Current therapies only slow down but do not stop its progression to end stage kidney failure; thus, there is a need for novel approaches. Accumulation of extracellular matrix proteins, e.g., laminin, fibronectin, contributes to the loss of functioning parenchyma and progressive loss of kidney function in diabetes. We have recently reported that H2S inhibits high glucose-induced increase in synthesis of proteins including matrix proteins (H.J. Lee et al., J. Biol. Chem. 287 (2012) 4451). We wish to identify H2S generating agents that can be used in humans to treat diabetic kidney disease. We hypothesized that tadalafil, a H2S donor and phosphodiesterase 5 (PDE5) inhibitor, inhibits high glucose stimulation of synthesis of proteins including ECM proteins by generating H2S in mouse kidney podocytes. High glucose (30 mM), but not equimolar mannitol, significantly promoted global protein synthesis, cellular hypertrophy and expression of laminin-γ1 and fibronectin. These events were inhibited by 10 μM tadalafil. Because mRNA translation is rate-limiting for protein synthesis, we examined tadalafil effect on events in translation. Tadalafil abrogated high glucose-induced mTORC1 activation as indicated by phosphorylation of p70S6 kinase and 4E-BP1; both these events are important for the initiation phase of translation. Tadalafil reversed high glucose-induced changes in the phosphorylation of eEF2 and eEF2 kinase, which regulate the elongation phase of translation. Upstream regulators of mTORC1 were investigated. Tadalafil stimulated activating phosphorylation of AMPK in a dose dependent manner, peaking at 10 μM. Pre-incubation with Compound C, an AMPK inhibitor, completely abolished tadalafil effect on high glucose stimulation of events in mRNA translation as well as global protein and laminin synthesis. Tadalafil-induced AMPK phosphorylation was abolished by dl -propargylglycine (PAG), an inhibitor of cystathionine γ lyase (CSE), a H2S generating enzyme. Tadalafil rapidly increased CSE protein expression in 1 h which was inhibited by cycloheximide but not actinomycin-D, suggesting regulation at the level of mRNA translation. In an enzymatic assay, PAG abrogated tadalafil stimulation of generation of H2S. PAG completely abolished tadalafil effect on high glucose stimulation of events in mRNA translation as well as global protein and laminin synthesis. Tadalafil is a PDE5 inhibitor that increases cGMP content. ODQ, an inhibitor of guanylyl cyclase that generates cGMP, abolished tadalafil induction of AMPK phosphorylation. ODQ abrogated tadalafil inhibition of high glucose-induced increase in laminin expression. Renal cortical content of cystathionine β synthase (CBS) and CSE, H2S generating enzymes, was significantly reduced in the kidney in mice with type 1 or type 2 diabetes. Our data show that tadalafil stimulates H2S-AMPK axis to inhibit mTORC1 and mRNA translation leading to amelioration of high glucose-induced global and matrix protein increment in renal podocytes. It is likely that tadalafil stimulation of H2S is cGMP-dependent in the podocyte. Future work is needed to define whether tadalafil can ameliorate kidney injury in animal models of diabetes.

Angiotensin-(1–7) attenuates high glucose-induced proximal tubular epithelial-to-mesenchymal transition via inhibiting ERK1/2 and p38 phosphorylation

AimsThe kidney is an important target for both Angiotensin II and angiotensin-(1–7) [Ang-(1–7)] in the renin–angiotensin system. However, the renal function of Ang-(1–7) remains unclear. This study is aimed at investigating the effect of Ang-(1–7) on high glucose-induced epithelial to mesenchymal transition (EMT) in cultured renal epithelial cells. Main methodsCultured renal epithelial (NRK-52E) cell line was used in the experiment. Fluorescence immunocytochemistry was performed to observe α-smooth muscle actin (α-SMA). Real-time PCR and Western blot were used to determine mRNA and protein levels. Enzyme-linked immunosorbent assay was used to measure the concentration of transforming growth factor-β1 (TGF-β1) in the culture media. Key findingsHigh glucose-induced decreased in both angiotensin-converting enzyme-related carboxypeptidase (ACE2) and Mas mRNA levels. Meanwhile, high glucose induced increases in α-SMA and vimentin, decreases in E-cadherin, elevations in TGF-β1 and fibronectin secretions. Ang-(1–7) partially reversed high glucose-induced changes in α-SMA, vimentin, E-cadherin, TGF-β1 and fibronectin. High glucose stimulated ERK, p38 and JNK phosphorylation and Ang-(1–7) reversed the changes in ERK and p38 but not JNK phosphorylation. SignificanceInhibition and insufficiency in ACE2-Ang-(1–7)–Mas axis under high glucose condition participate EMT. Supplementation of Ang-(1–7) attenuates high glucose-induced EMT. ERK and p38 intracellular signaling pathways, not JNK, mediate the effect of Ang-(1–7) on EMT.

Nrf2 and NF-&amp;#954;B Modulation by Sulforaphane Counteracts Multiple Manifestations of Diabetic Neuropathy in Rats and High Glucose-Induced Changes

High glucose driven reactive oxygen intermediates production and inflammatory damage are recognized contributors of nerve dysfunction and subsequent damage in diabetic neuropathy. Sulforaphane, a known chemotherapeutic agent holds a promise for diabetic neuropathy because of its dual antioxidant and anti-inflammatory activities. The present study investigated the effect of sulforaphane in streptozotocin (STZ) induced diabetic neuropathy in rats. For in vitro experiments neuro2a cells were incubated with sulforaphane in the presence of normal (5.5 mM) and high glucose (30 mM). For in vivo studies, sulforaphane (0.5 and 1 mg/kg) was administered six weeks post diabetes induction for two weeks. Motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and pain behavior were improved and malondialdehyde (MDA) level was reduced by sulforaphane. Antioxidant effect of sulforaphane is derived from nuclear erythroid 2-related factor 2 (Nrf2) activation as demonstrated by increased expression of Nrf2 and downstream targets hemeoxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1) in neuro2a cells and sciatic nerve of diabetic animals. Nuclear factor-kappa B (NF-κB) inhibition seemed to be responsible for antiinflammatory activity of sulforaphane as there was reduction in NF-κB expression and IκB kinase (IKK) phosphorylation along with abrogation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and tumor necrosis factor-α (TNF-α) and interleukine-6 (IL-6) levels. Here in this study we provide an evidence that sulforaphane is effective in reversing the various deficits in experimental diabetic neuropathy. This study supports the defensive role of Nrf2 in neurons under conditions of oxidative stress and also suggests that the NF-κB pathway is an important modulator of inflammatory damage in diabetic neuropathy.

Effect of resveratrol derivative BTM-0512 on high glucose-induced dysfunction of endothelial cells: role of SIRT1

Hyperglycemia impairs the function of endothelial cells. Sirtuin 1 (SIRT1) is involved in regulating the function of endothelial cells. Resveratrol, a polyphenol found in many plant species, exerts protective effects on endothelial cells through activation of SIRT1. The aims of this work were to explore whether BTM-0512, a novel derivative of resveratrol, is able to exert beneficial effects on high glucose-induced dysfunction of endothelial cells through regulation of SIRT1. We found that high glucose significantly impaired the function of endothelial cells as shown by reduced tube formation, cell migration, and cell adhesion concomitantly with downregulation of mRNA expression of SIRT1 and vascular endothelial growth factor as well as increased tumor necrosis factor-α release and reactive oxygen species production. These effects of high glucose were inhibited by pretreatment with BTM-0512. The beneficial effects of BTM-0512 on high glucose-induced cell dysfunction were abolished by splitomicin, a specific inhibitor of SIRT1. The regulatory effects of BTM-0512 on high glucose-induced changes in vascular endothelial growth factor mRNA expression and tumor necrosis factor-α release were also abolished by splitomicin. The results suggest that BTM-0512 exerts beneficial effects on high glucose-induced endothelial cell dysfunction through regulation of the SIRT1 - reactive oxygen species - vascular endothelial growth factor - tumor necrosis factor-α pathway.

Oxidative Stress and Diabetic Kidney Disease

The number of people with diabetic kidney disease continues to increase worldwide despite current treatments. Of the pathophysiologic mechanisms that have been identified in the development and progression of diabetic nephropathy, oxidative stress (more accurately described as increased levels of reactive oxygen species; ROS) is of major importance. The increase in ROS is due to both increased production and to decreased and/or inadequate antioxidant function. To date, human clinical trials with antioxidants have not been shown to be effective. This is likely due, at least in part, to the lack of specificity of current agents. Recent research has determined both major sources of high glucose-induced cellular ROS production as well as high glucose-induced changes in antioxidant function. Treatments targeted at one or more of the specific diabetes-induced alterations in the regulation of ROS levels will likely lead to effective treatments that prevent the development and progression of diabetic kidney disease.

Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells

High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1–AMPK axis.

NO/peroxynitrite dynamics of high glucose-exposed HUVECs: Chemiluminescent measurement and computational model

Pathogenesis of many of diabetes-related vascular complications is associated with endothelial cell (EC) dysfunction, which is reduced bioavailability of EC-released nitric oxide (NO). Interaction dynamics of NO, superoxide (O 2 −) and peroxynitrite (ONOO −) are dependent on both their productions and consumptions through various pathways. Quantitative knowledge of these interaction dynamics in high glucose-induced EC dysfunction remains poorly understood. We developed an integrated experimental and computational approach to gain a quantitative understanding of the interactions of NO, O 2 − and ONOO − in high glucose-exposed ECs. End-products, nitrite and nitrate, were measured using a chemiluminescence analyzer. A computational biochemical reaction network model was developed to predict the effect of high glucose on ECs NO, O 2 − and ONOO −. ECs NO and O 2 − productions increased in high glucose as evidenced by increased total NOx concentration, primarily increasing nitrate concentration. The model predicted an increase in O 2 − and ONOO − concentrations and a decrease in NO concentration in high glucose conditions. Administration of superoxide dismutase (SOD) decreased O 2 − concentration and increased NO concentration, thus SOD improved high glucose-induced changes in these interactions. An important finding of this study was that the NO bioavailability decreased in high glucose conditions even though NO production of EC increased. The integrated approach provides a framework to predict NO, O 2 − and ONOO − concentrations and productions that are difficult to measure in one experiment and will be useful in further EC dysfunction studies.

Fluvastatin inhibits activation of JAK and STAT proteins in diabetic rat glomeruli and mesangial cells under high glucose conditions

The aim of the present study was to further elucidate the mechanism of the protective role of fluvastatin on diabetic nephropathy. Streptozotocin-induced diabetic rats were treated daily with fluvastatin (4 mg/kg body weight) by gavage. The animals were killed 4 weeks later and urine and blood samples were collected. The kidney tissues were removed and subjected to the following experiments. Rat glomerular mesangial cells (GMC) were cultured under normal glucose (5.5 mmol/L), high glucose (HG, 30 mmol/L), HG+AG490 (10 micromol/L), or HG with fluvastatin (1 micromol/L). Glomeruli or the GMC lysate was immunoprecipitated and/or immunoblotted with antibodies against Janus kinase 2 (JAK2), SH2-domain containing tyrosine phosphatase-1 (SHP-1), phosphospecific SHP-2, and signal transducer and activators of transcription (STAT), respectively. Transforming growth factor-beta (TGF-beta1) mRNA was measured by RT-PCR. The protein synthesis of TGF-beta1 and fibronectin in the culture medium of GMC was detected by ELISA. The phosphorylation levels of JAK2, STAT1, STAT3, and SHP-2 increased significantly, and SHP-1 phosphorylation was reduced in glomeruli of diabetic rats. Treatment with fluvastatin reduced phosphorylation levels of JAK2, STAT1, STAT3, and SHP-2 in glomeruli of diabetic rats, but it had no effect on the dephosphorylation of SHP-1. The exposure of GMC to 30 mmol/L glucose caused the activation of JAK2, STAT1, STAT3, and SHP-2. It upregulated TGF-beta1 expression and increased protein synthesis of fibronectin. These high glucose-induced changes were suppressed by fluvastatin, as well as AG490, a JAK2 inhibitor. The regulation of the phosphorylation of JAK/STAT by fluvastatin may be responsible for its renal protective effects on diabetic nephropathy.