Glucosamine regulates hepatic lipid accumulation by sensing glucose levels or feeding states of normal and excess

Dose-dependent lipid accumulation was induced by glucose in HepG2 cells. GlcN also exerted a promotory effect on lipid accumulation in HepG2 cells under normal glucose conditions (NG, 5 mM) and liver of normal fed zebrafish larvae. High glucose (HG, 25 mM)-induced lipid accumulation was suppressed by l-glutamine-d-fructose 6-phosphate amidotransferase inhibitors. ER stress inhibitors did not suppress HG or GlcN-mediated lipid accumulation. HG and GlcN stimulated protein expression, DNA binding and O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP). Furthermore, both HG and GlcN increased nuclear sterol regulatory element-binding protein-1 (SREBP-1) levels in HepG2 cells. In contrast to its stimulatory effect under NG, GlcN suppressed lipid accumulation in HepG2 cells under HG conditions. Similarly, GlcN suppressed lipid accumulation in livers of overfed zebrafish. In addition, GlcN activity on DNA binding and O-GlcNAcylation of ChREBP was stimulatory under NG and inhibitory under HG conditions. Moreover, GlcN enhanced ChREBP, SREBP-1c, ACC, FAS, L-PK and SCD-1 mRNA expression under NG but inhibited HG-induced upregulation in HepG2 cells. The O-GlcNAc transferase inhibitor, alloxan, reduced lipid accumulation by HG or GlcN while the O-GlcNAcase inhibitor, PUGNAc, enhanced lipid accumulation in HepG2 cells and liver of zebrafish larvae. GlcN-induced lipid accumulation was inhibited by the AMPK activator, AICAR. Phosphorylation of AMPK (p-AMPK) was suppressed by GlcN under NG while increased by GlcN under HG. PUGNAc downregulated p-AMPK while alloxan restored GlcN- or HG-induced p-AMPK inhibition. Our results collectively suggest that GlcN regulates lipogenesis by sensing the glucose or energy states of normal and excess fuel through AMPK modulation.     

Apollon/Bruce is upregulated by Humanin

Hydrogen sulfide (H₂S) protects cardiomyoblasts against high glucose (HG)-induced injury by inhibiting the activation of p38 mitogen-activated protein kinase (MAPK). This study aims to determine whether the leptin–p38 MAPK pathway is involved in HG-induced injury and whether exogenous H₂S prevents the HG-induced insult through inhibition of the leptin–p38 MAPK pathway in H9c2 cells. H9c2 cells were treated with 35 mM glucose (HG) for 24 h to establish a HG-induced cardiomyocyte injury model. Cell viability; mitochondrial membrane potential (ΔΨ _m); apoptosis; reactive oxygen species (ROS) level; and leptin, leptin receptor, and p38 MAPK expression level were measured by the methods indicated. The results showed pretreatment of H9c2 cells with NaHS before exposure to HG led to an increase in cell viability, decrease in apoptotic cells, ROS generation, and a loss of ΔΨ _m. Exposure of H9c2 cells to 35 mM glucose for 24 h significantly upregulated the expression levels of leptin and leptin receptors. The increased expression levels of leptin and leptin receptors were markedly attenuated by pretreatment with 400 μM NaHS. In addition, the HG-induced increase in phosphorylated (p) p38 MAPK expression was ameliorated by pretreatment with 50 ng/ml leptin antagonist. In conclusion, the present study has demonstrated for the first time that the leptin–p38 MAPK pathway contributes to the HG-induced injury in H9c2 cells and that exogenous H₂S protects H9c2 cells against HG-induced injury at least in part by inhibiting the activation of leptin–p38 MAPK pathway.     

Zinc Modulates High Glucose-Induced Apoptosis by Suppressing Oxidative Stress in Renal Tubular Epithelial Cells

Hyperglycemia is a characteristic of diabetic nephropathy, inducing renal tubular cell apoptosis by eliciting oxidative stress and inflammation. Zinc (Zn) is known as an essential trace element in many enzymes and proteins involved in antioxidant defenses, electron transport, and exerting antiapoptotic or cytoprotective effects. In this study, the underlying mechanisms involved in the protective effects of Zn on high glucose-induced cytotoxicity were explored using cultured renal tubular epithelial cells (NRK-52E). The authors discovered that Zn supplementation inhibited high glucose (HG)-induced NRK-52E cell apoptosis by attenuating reactive oxygen species production, inhibiting HG-induced caspase-3 and caspase-9 activation, and inhibiting the release of cytochrome c from mitochondria to the cytosol. Further analysis revealed that Zn supplementation facilitated cell survival through increasing nuclear translocation of NF-E2-related factor 2 (Nrf2), leading to increased regulation of levels of two antioxidant enzymes, hemeoxygenase-1 and glutamate cysteine ligase, which provided an adaptive survival response against the HG-induced oxidative cytotoxicity. Moreover, the Zn-mediated increases in Nrf2 activity were suppressed by the pharmacological inhibition of Akt or extracellular signal-regulated kinase 1/2. Taken together, these findings suggest that Zn antiapoptosis capacity through the activation of Akt and ERK signal pathways leads to Nrf2 activation and, subsequently, Nrf2 target gene induction, thereby protecting the NRK-52E cells from HG-induced apoptosis.     

Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway

Metformin activates both PRKA and SIRT1. Furthermore, autophagy is induced by either the PRKA-MTOR-ULK1 or SIRT1-FOXO signaling pathways. We aimed to elucidate the mechanism by which metformin alleviates hepatosteatosis by examining the molecular interplay between SIRT1, PRKA, and autophagy. ob/ob mice were divided into 3 groups: one with ad libitum feeding of a standard chow diet, one with 300 mg/kg intraperitoneal metformin injections, and one with 3 g/d caloric restriction (CR) for a period of 4 wk. Primary hepatocytes or HepG2 cells were treated with oleic acid (OA) plus high glucose in the absence or presence of metformin. Both CR and metformin significantly improved body weight and glucose homeostasis, along with hepatic steatosis, in ob/ob mice. Furthermore, CR and metformin both upregulated SIRT1 expression and also stimulated autophagy induction and flux in vivo. Metformin also prevented OA with high glucose-induced suppression of both SIRT1 expression and SIRT1-dependent activation of autophagy machinery, thereby alleviating intracellular lipid accumulation in vitro. Interestingly, metformin treatment upregulated SIRT1 expression and activated PRKA even after siRNA-mediated knockdown of PRKAA1/2 and SIRT1, respectively. Taken together, these results suggest that metformin alleviates hepatic steatosis through PRKA-independent, SIRT1-mediated effects on the autophagy machinery.     

High glucose stimulates synthesis of fibronectin via a novel protein kinase C,Rap1b,and B-Raf signaling pathway

The molecular mechanism(s) by which high glucose induces fibronectin expression via G-protein activation in the kidney are largely unknown. This investigation describes the effect of high glucose (HG) on a small GTP-binding protein, Rap1b, expression and activation, and the relevance of protein kinase C (PKC) and Raf pathways in fibronectin synthesis in cultured renal glomerular mesangial cells (MCs). In vivo experiments revealed a dose-dependent increase in Rap1b expression in glomeruli of diabetic rat kidneys. Similarly, in vitro exposure of MCs to HG led to an up-regulation of Rap1b with concomitant increase in fibronectin (FN) mRNA and protein expression. The up-regulation of Rap1b mRNA was mitigated by the PKC inhibitors, calphostin C, and bisindolymaleimide, while also reducing HG- induced FN expression in non-transfected MCs. Overexpression of Rap1b by transfection with pcDNA 3.1/Rap1b in MCs resulted in the stimulation of FN synthesis; however, the PKC inhibitors had no significant effect in reducing FN expression in Rap1b-transfected MCs. Transfection of Rap1b mutants S17N (Ser --> Asn) or T61R (Thr --> Arg) in MCs inhibited the HG-induced increased FN synthesis. B-Raf and Raf-1 expression was investigated to assess whether Rap1b effects are mediated via the Raf pathway. B-Raf, and not Raf-1, expression was increased in MCs transfected with Rap1b. HG also caused activation of Rap1b, which was largely unaffected by anti-platelet-derived growth factor (PDGF) antibodies. HG-induced activation of Rap1b was specific, since Rap2b activation and expression of Rap2a and Rap2b were unaffected by HG. These findings indicate that hyperglycemia and HG cause an activation and up-regulation of Rap1b in renal glomeruli and in cultured MCs, which then stimulates FN synthesis. This effect appears to be PKC-dependent and PDGF-independent, but involves B-Raf, suggesting a novel PKC-Rap1b-B-Raf pathway responsible for HG-induced increased mesangial matrix synthesis, a hallmark of diabetic nephropathy.     

The effects of diosgenin in the Regulation of renal proximal tubular fibrosis

Fibrosis is the important pathway for end-stage renal failure. Glucose has been demonstrated to be the most important fibrogenesis-inducing agent according to previous studies. Despite diosgenin has been demonstrated to be anti-inflammatory, the possible role in fibrosis regulation of diosgenin remain to be investigated. In this study, renal proximal tubular epithelial cells (designated as HK-2) were treated with high concentration of glucose (HG, 27.5 mM) to determine whether diosgenin (0.1, 1 and 10 μM) has the effects to regulate renal cellular fibrosis. We found that 10 μM of diosgenin exert optimal inhibitory effects on high glucose-induced fibronectin expression in HK-2 cells. In addition, diosgenin markedly inhibited HG-induced increase in α-smooth muscle actin (α-SMA) and HG-induced decrease in E-cadherin. In addition, diosgenin antagonizes high glucose-induced epithelial-to-mesenchymal transition (EMT) signals partly by enhancing the catabolism of Snail in renal cells. Collectively, these data suggest that diosgenin has the potential to inhibit high glucose-induced renal tubular fibrosis possibly through EMT pathway.     

高糖诱导的大鼠原代心肌细胞损伤对程序性坏死的影响及其机制*

目的: 探讨程序性坏死在高糖诱导的大鼠原代心肌细胞损伤中的变化及可能机制。方法: 原代大鼠心肌细胞随机分为4组(n=9):正常对照组(Control,5.5 mmol/L葡萄糖培养心肌细胞48 h)、高糖组(HG,30 mmol/L葡萄糖培养心肌细胞48 h)、HG+Nec-1(30 mmol/L葡萄糖+100 μmol/L程序性坏死关键蛋白RIP1抑制剂Nec-1共同培养心肌细胞48 h)组、高渗组(HPG,5.5 mmol/L葡萄糖+24.5 mmol/L甘露醇共同培养心肌细胞48 h)。MTT法检测各组心肌细胞活力,DHE荧光染色检测细胞氧化应激水平,ELISA法检测心肌细胞TNF-α、IL-6及IL-1β水平,Real-time PCR和Western blot分别检测各组程序性坏死关键蛋白RIP1、RIP3、MLKL mRNA和蛋白水平的表达情况。结果: 与Control组相比,HG组心肌细胞活力明显降低(P＜0.01),氧化应激水平明显增高(P＜0.01),TNF-α、IL-6及IL-1β水平升高明显(P＜0.01),RIP1、RIP3、MLKL mRNA及蛋白水平表达均明显升高(P＜0.05);与HG组相比,HG+Nec-1组心肌细胞活力明显升高(P＜0.01),氧化应激水平明显下降(P＜0.01),TNF-α、IL-6及 IL-1β水平明显降低(P＜0.01), RIP1、RIP3、MLKL mRNA及蛋白水平表达均下降(P＜0.05)。结论: 高糖诱导的原代大鼠心肌细胞损伤可引起程序性坏死的发生;抑制程序性坏死可减轻细胞损伤的机制,可能与抑制氧化应激、减轻炎症反应有关。     

Deacetylation of LAMP1 drives lipophagy-dependent generation of free fatty acids by Abrus agglutinin to promote senescence in prostate cancer

Therapy-induced senescence in cancer cells is an irreversible antiproliferative state, which inhibits tumor growth and is therefore a potent anti-neoplastic mechanism. In this study, low doses of Abrus agglutinin (AGG)-induced senescence through autophagy in prostate carcinoma cells (PC3) and inhibited proliferation. The inhibition of autophagy with 3-methyl adenine reversed AGG-induced senescence, thus confirming that AGG-triggered senescence required autophagy. AGG treatment also led to lipophagy-mediated accumulation of free fatty acids (FFAs), with a concomitant decrease in the number of lipid droplets. Lalistat, a lysosomal acid lipase inhibitor, abrogated AGG-induced lipophagy and senescence in PC3 cells, indicating that lipophagy is essential for AGG-induced senescence. The accumulation of FFAs increased reactive oxygen species generation, a known facilitator of senescence, which was also reduced in the presence of lalistat. Furthermore, AGG upregulated silent mating type information regulator 2 homolog 1 (SIRT1), while the presence of sirtinol reduced autophagy flux and the senescent phenotype in the AGG-treated cells. Mechanistically, AGG-induced cytoplasmic SIRT1 deacetylated a Lys residue on the cytoplasmic domain of lysosome-associated membrane protein 1 (LAMP1), an autolysosomal protein, resulting in lipophagy and senescence. Taken together, our findings demonstrate a novel SIRT1/LAMP1/lipophagy axis mediating AGG-induced senescence in prostate cancer cells.     

Rosiglitazone attenuates NF-κB-mediated Nox4 upregulation in hyperglycemia-activated endothelial cells

Vascular complications, a major cause of morbidity and mortality in diabetic patients, are related to hyperglycemia-induced oxidative stress. Previously, we reported that rosiglitazone (RSG) attenuated vascular expression and activity of NADPH oxidases in diabetic mice. The mechanisms underlying these effects remain to be elucidated. We hypothesized that RSG acts directly on endothelial cells to modulate vascular responses in diabetes. To test this hypothesis, human aortic endothelial cells (HAECs) were exposed to normal glucose (NG; 5.6 mmol/l) or high glucose (HG; 30 mmol/l) concentrations. Select HAEC monolayers were treated with RSG, caffeic acid phenethyl ester (CAPE), diphenyleneiodonium (DPI), small interfering (si)RNA (to NF-κB/p65 or Nox4), or Tempol. HG increased the expression and activity of the NADPH oxidase catalytic subunit Nox4 but not Nox1 or Nox2. RSG attenuated HG-induced NF-κB/p65 phosphorylation, nuclear translocation, and binding to the Nox4 promoter. Inhibiting NF-κB with CAPE or siNF-κB/p65 also reduced HG-induced Nox4 expression and activity. HG-induced H(2)O(2) production was attenuated by siRNA-mediated knockdown of Nox4, and HG-induced HAEC monocyte adhesion was attenuated by treatment with RSG, DPI, CAPE, or Tempol. These results indicate that HG exposure stimulates HAEC NF-κB activation, Nox4 expression, and H(2)O(2) production and that RSG attenuates HG-induced oxidative stress and subsequent monocyte-endothelial interactions by attenuating NF-κB/p65 activation and Nox4 expression. This study provides novel insights into mechanisms by which the thiazolidinedione peroxisome proliferator-activated receptor-γ ligand RSG favorably modulates endothelial responses in the diabetic vasculature.     

Lipophagy deficiency exacerbates ectopic lipid accumulation and tubular cells injury in diabetic nephropathy

Autophagy-mediated lipotoxicity plays a critical role in the progression of diabetic nephropathy (DN), but the precise mechanism is not fully understood. Whether lipophagy, a selective type of autophagy participates in renal ectopic lipid deposition (ELD) and lipotoxicity in the kidney of DN is unknown. Here, decreased lipophagy, increased ELD and lipotoxcity were observed in tubular cells of patients with DN, which were accompanied with reduced expression of AdipoR1 and p-AMPK. Similar results were found in db/db mice, these changes were reversed by AdipoRon, an adiponectin receptor activator that promotes autophagy. Additionally, a significantly decreased level of lipophagy was observed in HK-2 cells, a human proximal tubular cell line treated with high glucose, which was consistent with increased lipid deposition, apoptosis and fibrosis, while were partially alleviated by AdipoRon. However, these effects were abolished by pretreatment with ULK1 inhibitor SBI-0206965, autophagy inhibitor chloroquine and enhanced by AMPK activator AICAR. These data suggested by the first time that autophagy-mediated lipophagy deficiency plays a critical role in the ELD and lipid-related renal injury of DN.Subject terms: Chronic kidney disease, Diabetes complications     

SIRT3 inhibited the formation of calcium oxalate-induced kidney stones through regulating NRF2/HO-1 signaling pathway

Oxidative stress is important for the calcium oxalate (CaOx)-induced kidney stone formation. Sirtuin 3 (SIRT3) plays an essential role in the amelioration of oxidative damages. This study aims to explore the effect of SIRT3 on the formation of CaOx-induced kidney stones and the underlying mechanism. SIRT3 expression in renal tissues was detected by immunohistochemistry. Apoptosis in renal tissues was examined by TUNEL staining. Crystal-cell adherence and cell apoptosis in HK-2 cells were assessed by analyzing Ca²⁺ concentration and by the flow cytometry analysis, respectively. Protein expression of SIRT3, nuclear factor erythroid 2-related factor (NRF2), heme oxygenase-1 (HO-1), and Bax in renal tissues or HK-2 cells was examined by Western blot analysis. Renal pathological changes and the adhesion of CaOx crystals in the kidneys were examined by hematoxylin-eosin and von Kossa staining, respectively. Human kidneys with stones showed enhanced renal apoptosis, downregulated SIRT3 expression, and upregulated NRF2/HO-1 expression, compared with the controls. Furthermore, SIRT3 overexpression inhibited the CaOx-induced promotion of crystal-cell adherence and cell apoptosis in human proximal tubular cell line HK-2 cells, which was reversed by the NRF2 knockdown. Moreover, our in vivo assay further confirmed that SIRT3 overexpression alleviated the glyoxylate administration-induced renal damage, renal apoptosis, and crystals deposition in the kidneys from the stone model mice, which was also associated with its activation of the NRF2/HO-1 pathway. Our findings support the notion that overexpression of SIRT3 may inhibit the formation of CaOx-induced kidney stones, at least in part, through regulating the NRF2/HO-1 signaling pathway.     

High glucose enhances MMP-2 production in adventitial fibroblasts via Akt1-dependent NF-kappaB pathway

To understand the role of adventitial fibroblasts (AF) in diabetic vascular diseases, the importance of high glucose (HG, 25mM) on matrix metalloproteinase-2 (MMP-2) production in AF was determined. HG enhanced mRNA, protein and gelatinolytic activity of MMP-2. The enhanced MMP-2 activity was significantly attenuated not only by a PI3K inhibitor but also by an Akt inhibitor. These HG-induced MMP-2 responses were markedly reduced in Akt1-deficient (1KO) cells. The diminished HG-induced MMP-2 responses were completely restored by re-expression of Akt1. Both the reporter activity and electrophoretic mobility shift assay for activator protein-1 and nuclear factor-kappa B (NF-kappaB) were enhanced by HG, but NF-kappaB were not increased in 1KO cells. Furthermore, HG-induced MMP-2 responses were markedly suppressed by NF-kappaB decoy oligodeoxynucleotides. Based on these results, it is suggested that HG augments MMP-2 production via PI3K/Akt1/NF-kappaB pathway.