NR4A1 counteracts JNK activation incurred by ER stress or ROS in pancreatic ß-cells for protection.

Sustained hyperglycaemia and hyperlipidaemia incur endoplasmic reticulum stress (ER stress) and reactive oxygen species (ROS) overproduction in pancreatic ß-cells. ER stress or ROS causes c-Jun N-terminal kinase (JNK) activation, and the activated JNK triggers apoptosis in different cells. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an inducible multi-stress response factor. The aim of this study was to explore the role of NR4A1 in counteracting JNK activation induced by ER stress or ROS and the related mechanism. qPCR, Western blotting, dual-luciferase reporter and ChIP assays were applied to detect gene expression or regulation by NR4A1. Immunofluorescence was used to detect a specific protein expression in ß-cells. Our data showed that NR4A1 reduced the phosphorylated JNK (p-JNK) in MIN6 cells encountering ER stress or ROS and reduced MKK4 protein in a proteasome-dependent manner. We found that NR4A1 increased the expression of cbl-b (an E3 ligase); knocking down cbl-b expression increased MKK4 and p-JNK levels under ER stress or ROS conditions. We elucidated that NR4A1 enhanced the transactivation of cbl-b promoter by physical association. We further confirmed that cbl-b expression in ß-cells was reduced in NR4A1-knockout mice compared with WT mice. NR4A1 down-regulates JNK activation by ER stress or ROS in ß-cells via enhancing cbl-b expression.

NR4A1 retards adipocyte differentiation or maturation via enhancing GATA2 and p53 expression.

Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor with diverse functions. It has been reported that NR4A1, as a transcriptional activator, is implicated in glucose and lipid metabolism. The aim of this study was to investigate the regulatory role of NR4A1 in adipogenesis and explore the underlying mechanisms. Quantitative real-time PCR and Western blotting were used to analyse the expression of genes involved in synthesis and mobilization of fats in vivo and in vitro. Dual-luciferase reporter assay was conducted to study the regulatory mechanisms of NR4A1. Our data from in vivo study confirmed that NR4A1 knockout (KO) mice fed with high-fat diet were more prone to obesity, and gene expression levels of PPARγ and FAS were increased in KO mice compared to controls; our data from in vitro study showed that NR4A1 overexpression in 3T3-L1 pre-adipocytes inhibited adipogenesis. Moreover, NR4A1 enhanced GATA binding protein 2 (GATA2) expression, which in turn inhibited peroxisome proliferator-activated receptor γ (PPARγ); NR4A1 inhibited sterol regulatory element binding transcription factor 1 (SREBP1) and its downstream gene fatty acid synthase (FAS) by up-regulating p53. NR4A1 inhibits the differentiation and lipid accumulation of adipocytes by enhancing the expression of GATA2 and p53.

GLP-1/GLP-1R Signaling in Regulation of Adipocyte Differentiation and Lipogenesis.

BACKGROUND/AIMS: The aim of this study was to determine the direct role of liraglutide (LG) in adipogenesis and lipid metabolism. METHODS: Lipid accumulation was evaluated by oil red O staining, quantitative real-time PCR (qPCR) was performed to determine glucagon-like peptide 1 receptor (GLP-1R), fatty acid synthase (FASN) and adipose triglyceride lipase (ATGL) expression in 3T3-L1 preadipocytes, differentiated adipocytes and in adipose tissues from mice. The effects of LG on 3T3-L1 adipogenesis and lipid metabolism were analyzed with qPCR, Western Blotting, oil red O staining, immunohistochemistry (IHC) and immunofluorescence (IF). All measurements were performed at least three times. RESULTS: LG increased the expression of differentiation marker genes and lipid accumulation during preadipocyte differentiation. In differentiated adipocytes, LG decreased FASN expression, and simultaneously led to CREB phosphorylation and ERK1/2 activation which were abolished by a GLP-1R antagonist, exendin (9-39). LG induced-FASN down-regulation was partially reversed by PKA and ERK1/2 inhibitors. Consistent with above in vitro findings, LG treatment significantly reduced FASN expression in visceral adipose tissues of ob/ob mice, and reduced body weight gain. CONCLUSION: LG promotes preadipocytes differentiation, and inhibits FASN expression in adipocytes. LG induced down-regulation of FASN is at least partially mediated by PKA and MAPK signaling pathways.

Hydrogen sulfide improves diabetic wound healing in ob/ob mice via attenuating inflammation.

BACKGROUND: The proposed mechanisms of impaired wound healing in diabetes involve sustained inflammation, excess oxidative stress and compromised agiogenesis. Hydrogen sulfide (H2S) has been reported to have multiple biological activities. We aim to investigate the role of H2S in impaired wound healing in ob/ob mice and explore the possible mechanisms involved. PROCEDURES: Full-thickness skin dorsal wounds were created on ob/ob mice and C57BL/6 mice. Cystathionine-γ-lyase (CSE) expression and H2S production were determined in granulation tissues of the wounds. Effects of NaHS on wound healing were evaluated. Inflammation and angiogenesis in granulation tissues of the wounds were examined. RESULTS: CSE expression, and H2S content were significantly reduced in granulation tissues of wounds in ob/ob mice compared with control mice. NaHS treatment significantly improved wound healing in ob/ob mice, which was associated with reduced neutrophil and macrophage infiltration, decreased production of tumor necrosis factor (TNF)-α, interleukin (IL)-6. NaHS treatment decreased metalloproteinase (MMP)-9, whereas increased collagen deposition and vascular-like structures in granulation tissues of wounds in ob/ob mice. CONCLUSION: CSE down-regulation may play a role in the pathogenesis of diabetic impaired wound healing. Exogenous H2S could be a potential agent to improve diabetic impaired wound healing by attenuating inflammation and increasing angiogenesis.

The stress-response molecule NR4A1 resists ROS-induced pancreatic ß-cells apoptosis via WT1.

Pancreatic ß-cells often face endoplasmic reticulum stress and/or ROS-associated oxidative stress under adverse conditions. Our previous work has verified that NR4A1 protects pancreatic ß-cells from ER-stress induced apoptosis. However, It remains unknown whether NR4A1 is able to protect pancreatic ß-cells against ROS-associated oxidative stress. In the present study, our data showed that NR4A1 protein expression rapidly increased in MIN6 cells upon H2O2 treatment, and overexpression of NR4A1 in MIN6 cells conferred resistance to cell apoptosis induced by H2O2. These results were further substantiated in isolated islets from mice infected with an adenovirus overexpressing NR4A1. 8-hydroxy-2'-deoxyguanosine (8-OHdG) was used as a biomarker for oxidative stress or a marker for ROS damage. We found that the 8-OHdG level in the islets from NR4A1 knockout mice fed with high-fat diet was much higher than that in the islets from parental control mice; and higher apoptotic rate was observed in the islets from NR4A1 KO mice compared to control mice. Further investigation of underlying mechanisms of NR4A1's protective effects showed that NR4A1 overexpression in MIN6 cells reduced Caspase 3 activation caused by H2O2, and increased expression of WT1 and SOD1. There is a putative NR4A1 binding site (-1118bp to -1111bp) in WT1 promoter; our data demonstrated that NR4A1 protein physically associates with the WT1 promoter, and enhanced WT1 promoter transactivation and knockdown of WT1 in MIN6 cells induced apoptosis. These findings suggest that NR4A1 protects pancreatic ß-cells against H2O2 mediated apoptosis by up-regulating WT1 expression.

Cytochrome P450 (CYP) epoxygenases as potential targets in the management of impaired diabetic wound healing.

Epoxyeicosatrienoic acids (EETs) are the epoxidation products of arachidonic acid catalyzed by cytochrome P450 (CYP) epoxygenases, which possess multiple biological activities. In the present study, we aimed to explore the role and effects of CYP epoxygenases/EETs in wound healing in ob/ob mice. Full-thickness skin dorsal wounds were made on ob/ob mice and C57BL/6 control mice. The mRNA and protein expression of CYP epoxygenases were determined in granulation tissues of wounds. Effects of EETs on wound healing were evaluated. Inflammation and angiogenesis in wounds were also observed. Compared with C57BL/6 mice, the mRNA and protein expression of CYP2C65 and CYP2J6 in the granulation tissues in ob/ob mice were significantly reduced. 11,12-EET treatment significantly improved wound healing in ob/ob mice, whereas 14,15-EEZE, an EET antagonist, showed the opposite effect. 11,12-EET treatment decreased neutrophil and macrophage infiltration to the wound sites, resulting in reduced production of inflammatory cytokines, decreased MMP-9 expression, and increased collagen accumulation in the granulation tissues of ob/ob mice. In addition, 11,12-EET increased angiogenesis in the granulation tissues of wounds in ob/ob mice. These findings indicate that reduced expression of CYP epoxygenases may contribute to impaired diabetic wound healing, and exogenous EETs may improve diabetic wound healing by modulating inflammation and angiogenesis.

The Effect of Growth Hormone on Lipid Accumulation or Maturation in Adipocytes.

BACKGROUND: Adipogenesis of adipocytes includes two stages: initiation and maturation. Growth hormone (GH) secretion is decreased in obese subjects and GH levels are inversely correlated with abdominal fat mass. The effects of growth hormone (GH) on lipids accumulation or maturation of adipocytes remains elusive. METHODS: In the present study, effect of GH on lipid accumulation in vitro and in vivo was examined. cDNA microarray, quantitative real time-PCR (qPCR) and western blotting was used to analyze the expression of genes related to adipocyte lipid accumulation or degradation in pre- or mature 3T3-F442A adipocytes treated with GH and in epididymal adipose tissue of C57BL/6 mice administrated with GH. Level of adiponectin in supernatants of cultured F442A adipocytes was determined by enzyme-linked immune-sorbent assay. RESULTS: We found that in 3T3-F442A especially 6 days post initiation of adipogenesis, GH intervention resulted in decreased expression of adipocyte maturation regulators (C/EBPα, PPARγ) and prominent genes related to lipid synthesis such as FAS and FABP, while the expression of UCP1 was markedly enhanced. cDNA microarray analysis and qPCR showed that the expression of SOCS2 and Adipor2 was increased under GH-treatment in mature 3T3-F442A adipocytes. GH treatment increased the mRNA expression of adiponectin and UCP1 in mature adipocytes. The above results were confirmed by in vivo study. CONCLUSIONS: GH potentially negatively modulates the maturation and accumulation of lipid in adipocytes.

Signal transducer and activator of transcription 5B (STAT5B) modulates adipocyte differentiation via MOF.

The role and mechanism of signal transducer and activator of transcription 5B (STAT5B) in adipogenesis remain unclear. In this study, our data showed that Males absent on the first (MOF) protein expression was increased during 3 T3-L1 preadipocytes differentiation accompanied with STAT5B expression increasing. Over-expression STAT5B enhanced MOF promoter trans-activation in HeLa cells. Mutagenesis assay and ChIP analysis exhibited that STAT5B was able to bind MOF promoter. Knocking-down STAT5B in 3 T3-L1 preadipocytes led to decreased expression of MOF, but resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and fatty acid-binding protein 4 (Fabp4), which were important factors or enzymes for adipogenesis. We also found that knocking-down MOF in 3 T3-L1 preadipocytes resulted in increased expression of PPARγ, C/EBPα and Fabp4, which was in the same trend as STAT5B knocking-down. Over-expression MOF resulted in reduced promoter trans-activation activity of C/EBPα. These results suggest that STAT5B and MOF work as negative regulators in adipogenesis, and STAT5B modulates preadipocytes differentiation partially by regulating MOF expression.

Effects of hydrogen sulfide on high glucose-induced glomerular podocyte injury in mice.

The aim of this study was to assess the effects of hydrogen sulfide on high glucose-induced mouse podocyte (MPC) injury and the underlying mechanisms. Mouse podocytes were randomly divided into 4 groups, including high glucose (HG), normal glucose (NG), normal glucose + DL-propargylglycine (PPG), and high glucose + NaHS (HG + NaHS) groups for treatment. Then, ZO-2, nephrin, ß-catenin, and cystathionine γ-lyase (CSE) protein expression levels were determined by western blot. We found that high glucose significantly reduced nephrin, ZO-2, and CSE expression levels (P<0.05), and overtly elevated ß-catenin amounts (P<0.05), in a time-dependent manner. Likewise, PPG at different concentrations in normal glucose resulted in significantly lower CSE, ZO-2, and nephrin levels (P<0.05), and increased ß-catenin amounts (P<0.05). Interestingly, significantly increased ZO-2 and nephrin levels, and overtly reduced ß-catenin amounts were observed in the HG + NaHS group compared with HG treated cells (P<0.01). Compared with NG treated cells, decreased ZO-2 and nephrin levels and higher ß-catenin amounts were obtained in the HG + NaHS group. In conclusion,CSE downregulation contributes to hyperglycemia induced podocyte injury, which is alleviated by exogenous H2S possibly through ZO-2 upregulation and the subsequent suppression of Wnt/ß-catenin pathway.

The Orphan Nuclear Receptor NR4A1 Protects Pancreatic ß-Cells from Endoplasmic Reticulum (ER) Stress-mediated Apoptosis.

The role of NR4A1 in apoptosis is controversial. Pancreatic ß-cells often face endoplasmic reticulum (ER) stress under adverse conditions such as high free fatty acid (FFA) concentrations and sustained hyperglycemia. Severe ER stress results in ß-cell apoptosis. The aim of this study was to analyze the role of NR4A1 in ER stress-mediated ß-cell apoptosis and to characterize the related mechanisms. We confirmed that upon treatment with the ER stress inducers thapsigargin (TG) or palmitic acid (PA), the mRNA and protein levels of NR4A1 rapidly increased in both MIN6 cells and mouse islets. NR4A1 overexpression in MIN6 cells conferred resistance to cell loss induced by TG or PA, as assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and TUNEL assays indicated that NR4A1 overexpression also protected against ER stress-induced apoptosis. This conclusion was further confirmed by experiments exploiting siRNA to knockdown NR4A1 expression in MIN6 cells or exploiting NR4A1 knock-out mice. NR4A1 overexpression in MIN6 cells reduced C/EBP homologous protein (CHOP) expression and Caspase3 activation induced by TG or PA. NR4A1 overexpression in MIN6 cells or mouse islets resulted in Survivin up-regulation. A critical regulatory element was identified in Survivin promoter (-1872 bp to -1866 bp) with a putative NR4A1 binding site; ChIP assays demonstrated that NR4A1 physically associates with the Survivin promoter. In conclusion, NR4A1 protects pancreatic ß-cells against ER stress-mediated apoptosis by up-regulating Survivin expression and down-regulating CHOP expression, which we termed as "positive and negative regulation."

TSH/TSHR Signaling Suppresses Fatty Acid Synthase (FASN) Expression in Adipocytes.

TSH/TSHR signaling plays a role in the regulation of lipid metabolism in adipocytes. However, the precise mechanisms are not known. In the present study, we determined the effect of TSH on fatty acid synthase (FASN) expression, and explored the underlying mechanisms. In vitro, TSH reduced FASN expression in both mRNA and protein levels in mature adipocytes and was accompanied by protein kinase A (PKA) activation, cAMP-response element binding protein (CREB) phosphorylation, as well as extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2 -terminal kinase (JNK) activation. TSH-induced downregulation of FASN was partially abolished by inhibition of PKA and ERK, but not JNK. TSHR and FASN expression in visceral tissue was significantly increased in C57BL/6 mice with diet-induced obesity compared with control animals, whereas thyroid TSHR expression was normal. These findings suggest that activation of TSHR directly inhibits FASN expression in mature adipocytes, possibly mediated by PKA and ERK. In obese animals, this function of TSHR seems to be counteracted. The precise mechanisms need further investigation.

Involvement of CSE/ H2S in high glucose induced aberrant secretion of adipokines in 3T3-L1 adipocytes.

BACKGROUND: Deregulated secretion of adipokines contributes to subclinical systemic inflammation associated with type 2 diabetes mellitus (T2DM). However, the mechanisms underlying are not fully understood. Hydrogen sulfide (H2S), as an endogenous gasotransmitter, possesses an anti-inflammation activity. The aim of this study was to examine the possible involvement of H2S in high glucose induced adipokine secretion in 3T3-L1 adipocytes. METHODS: The expression of cystathionine-gamma-lyase (CSE), the H2S-forming enzyme, was evaluated by Western-blotting and real-time PCR. The secretion of TNF-α, MCP-1 and adiponectin was determined by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA), respectively. Lentiviral empty vector and vector expressing mouse CSE were used for in vitro transduction. RESULTS: High glucose (HG) significantly decreased CSE expression at both protein and mRNA levels in mature 3T3-L1 adipocytes. In parallel, HG significantly increased secretion of MCP-1 while decreasing secretion of adiponectin, but had no effect on secretion of TNF-α. HG induced changes in MCP-1 and adiponectin secretion were partly attenuated by forced expression of CSE or sodium hydrosulfide (NaHS), a source of exogenous H2S. CONCLUSION: High glucose induces aberrant secretion of adipokines in mature 3T3-L1 adipocytes, favoring inflammation. The mechanism is partly related to inhibition of CSE/ H2S system.

Hydrogen sulfide suppresses high glucose-induced expression of intercellular adhesion molecule-1 in endothelial cells.

Hydrogen sulfide (H2S) is a newly identified endogenous gasotransmitter that has been implicated in the pathophysiology of several biologic systems. However, the role of H2S in the pathogenesis of diabetic vascular injury remains unclear. The aims of this study were to determine the effect of H2S on the high glucose (HG)-induced expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells and to explore the possible underlying mechanisms. Human umbilical vein endothelial cells were exposed either to a normal concentration of D-glucose (5.5 mmol/L) or to HG (16.7 mmol/L) in the absence or presence of NaHS for the indicated periods. The ICAM-1 protein and messenger RNA (mRNA) levels were analyzed by Western blotting and real-time reverse transcriptase-polymerase chain reaction, respectively. Exposure to HG for 48 or 72 hours significantly increased ICAM-1 expression at both the protein and mRNA levels, and these increases correlated with increases in both the production of intracellular reactive oxygen species and the activation of nuclear factor-κB. Pretreatment with NaHS inhibited HG-induced ICAM-1 expression at both the protein and mRNA levels and resulted in a reduction in the intracellular reactive oxygen species level and the suppression of nuclear factor-κB activity. NaHS also inhibited tumor necrosis factor-α-induced ICAM-1 protein expression, which was similar to the effect of antioxidant N-acetyl-L-cysteine. These findings indicate that H2S might protect against HG-induced vascular damage by down-regulating ICAM-1 expression in endothelial cells.