Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H₂O₂‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16^INK4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H₂O₂‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H₂O₂‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H₂O₂ treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.     

Inhibition of P53/miR‐34a improves diabetic endothelial dysfunction via activation of SIRT1

Endothelial dysfunction contributes to diabetic macrovascular complications, resulting in high mortality. Recent findings demonstrate a pathogenic role of P53 in endothelial dysfunction, encouraging the investigation of the effect of P53 inhibition on diabetic endothelial dysfunction. Thus, high glucose (HG)‐treated endothelial cells (ECs) were subjected to pifithrin‐α (PFT‐α)—a specific inhibitor of P53, or P53‐small interfering RNA (siRNA), both of which attenuated the HG‐induced endothelial inflammation and oxidative stress. Moreover, inhibition of P53 by PFT‐α or P53‐siRNA prohibited P53 acetylation, decreased microRNA‐34a (miR‐34a) level, leading to a dramatic increase in sirtuin 1 (SIRT1) protein level. Interestingly, the miR‐34a inhibitor (miR‐34a‐I) and PFT‐α increased SIRT1 protein level and alleviated the HG‐induced endothelial inflammation and oxidative stress to a similar extent; however, these effects of PFT‐α were completely abrogated by the miR‐34a mimic. In addition, SIRT1 inhibition by EX‐527 or Sirt1‐siRNA completely abolished miR‐34a‐I's protection against HG‐induced endothelial inflammation and oxidative stress. Furthermore, in the aortas of streptozotocin‐induced diabetic mice, both PFT‐α and miR‐34a‐I rescued the inflammation, oxidative stress and endothelial dysfunction caused by hyperglycaemia. Hence, the present study has uncovered a P53/miR‐34a/SIRT1 pathway that leads to endothelial dysfunction, suggesting that P53/miR‐34a inhibition could be a viable strategy in the management of diabetic macrovascular diseases.     

UVA influenced the SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in human skin primary fibroblasts

Both SIRT1 and UVA radiation are involved in cellular damage processes such as apoptosis, senescence and ageing. MicroRNAs (miRNAs) have been reported to be closely related to UV radiation, as well as to SIRT1. In this study, we investigated the connections among SIRT1, UVA and miRNA in human skin primary fibroblasts. Our results showed that UVA altered the protein level of SIRT1 in a time point–dependent manner. Using miRNA microarray, bioinformatics analysis, we found that knocking down SIRT1 could cause up‐regulation of miR‐27a‐5p and the latter could down‐regulate SMAD2, and these results were verified by qRT‐PCR or Western blot. Furthermore, UVA radiation (5 J/cm²), knocking down SIRT1 or overexpression of miR‐27a‐5p led to increased expression of MMP1, and decreased expressions of COL1 and BCL2. We also found additive impacts on MMP1, COL1 and BCL2 under the combination of UVA radiation + Sirtinol (SIRT1 inhibitor), or UVA radiation + miR‐27a‐5p mimic. SIRT1 activator resveratrol could reverse damage changes caused by UVA radiation. Besides, absent of SIRT1 or overexpression of miR‐27a‐5p increased cell apoptosis and induced cell arrest in G2/M phase. Taken together, these results demonstrated that UVA could influence a novel SIRT1‐miR‐27a‐5p‐SMAD2‐MMP1/COL1/BCL2 axis in skin primary fibroblasts, and may provide potential therapeutic targets for UVA‐induced skin damage.     

ACE2‐EPC‐EXs protect ageing ECs against hypoxia/reoxygenation‐induced injury through the miR‐18a/Nox2/ROS pathway

Oxidative stress is one of the mechanisms of ageing‐associated vascular dysfunction. Angiotensin‐converting enzyme 2 (ACE2) and microRNA (miR)‐18a have shown to be down‐regulated in ageing cells. Our previous study has shown that ACE2‐primed endothelial progenitor cells (ACE2‐EPCs) have protective effects on endothelial cells (ECs), which might be due to their released exosomes (EXs). Here, we aimed to investigate whether ACE2‐EPC‐EXs could attenuate hypoxia/reoxygenation (H/R)‐induced injury in ageing ECs through their carried miR‐18a. Young and angiotensin II‐induced ageing ECs were subjected to H/R and co‐cultured with vehicle (medium), EPC‐EXs, ACE2‐EPCs‐EXs, ACE2‐EPCs‐EXs + DX600 or ACE2‐EPCs‐EXs with miR‐18a deficiency (ACE2‐EPCs‐EXs^{anti‐miR‐18a}). Results showed (1) ageing ECs displayed increased senescence, apoptosis and ROS production, but decreased ACE2 and miR‐18a expressions and tube formation ability; (2) under H/R condition, ageing ECs showed higher rate of apoptosis, ROS overproduction and nitric oxide reduction, up‐regulation of Nox2, down‐regulation of ACE2, miR‐18a and eNOS, and compromised tube formation ability; (3) compared with EPC‐EXs, ACE2‐EPC‐EXs had better efficiencies on protecting ECs from H/R‐induced changes; (4) The protective effects were less seen in ACE2‐EPCs‐EXs + DX600 and ACE2‐EPCs‐EXs^{anti‐miR‐18a} groups. These data suggest that ACE‐EPCs‐EXs have better protective effects on H/R injury in ageing ECs which could be through their carried miR‐18a and subsequently down‐regulating the Nox2/ROS pathway.     

A miR‐34a‐SIRT6 axis in the squamous cell differentiation network

Squamous cell carcinomas (SCCs) are highly heterogeneous tumours, resulting from deranged expression of genes involved in squamous cell differentiation. Here we report that microRNA‐34a (miR‐34a) functions as a novel node in the squamous cell differentiation network, with SIRT6 as a critical target. miR‐34a expression increases with keratinocyte differentiation, while it is suppressed in skin and oral SCCs, SCC cell lines, and aberrantly differentiating primary human keratinocytes (HKCs). Expression of this miRNA is restored in SCC cells, in parallel with differentiation, by reversion of genomic DNA methylation or wild‐type p53 expression. In normal HKCs, the pro‐differentiation effects of increased p53 activity or UVB exposure are miR‐34a‐dependent, and increased miR‐34a levels are sufficient to induce differentiation of these cells both in vitro and in vivo. SIRT6, a sirtuin family member not previously connected with miR‐34a function, is a direct target of this miRNA in HKCs, and SIRT6 down‐modulation is sufficient to reproduce the miR‐34a pro‐differentiation effects. The findings are of likely biological significance, as SIRT6 is oppositely expressed to miR‐34a in normal keratinocytes and keratinocyte‐derived tumours.     

Sodium hydrosulphide restores tumour necrosis factor‐α‐induced mitochondrial dysfunction and metabolic dysregulation in HL‐1 cells

Tumour necrosis factor (TNF)‐α induces cardiac metabolic disorder and mitochondrial dysfunction. Hydrogen sulphide (H₂S) contains anti‐inflammatory and biological effects in cardiomyocytes. This study investigated whether H₂S modulates TNF‐α‐dysregulated mitochondrial function and metabolism in cardiomyocytes. HL‐1 cells were incubated with TNF‐α (25 ng/mL) with or without sodium hydrosulphide (NaHS, 0.1 mmol/L) for 24 hours. Cardiac peroxisome proliferator‐activated receptor (PPAR) isoforms, pro‐inflammatory cytokines, receptor for advanced glycation end products (RAGE) and fatty acid metabolism were evaluated through Western blotting. The mitochondrial oxygen consumption rate and adenosine triphosphate (ATP) production were investigated using Seahorse XF24 extracellular flux analyzer and bioluminescence assay. Fluorescence intensity using 2′, 7′‐dichlorodihydrofluorescein diacetate was used to evaluate mitochondrial oxidative stress. NaHS attenuated the impaired basal and maximal respiration, ATP production and ATP synthesis and enhanced mitochondrial oxidative stress in TNF‐α‐treated HL‐1 cells. TNF‐α‐treated HL‐1 cells exhibited lower expression of PPAR‐α, PPAR‐δ, phosphorylated 5′ adenosine monophosphate‐activated protein kinase‐α2, phosphorylated acetyl CoA carboxylase, carnitine palmitoyltransferase‐1, PPAR‐γ coactivator 1‐α and diacylglycerol acyltransferase 1 protein, but higher expression of PPAR‐γ, interleukin‐6 and RAGE protein than control or combined NaHS and TNF‐α‐treated HL‐1 cells. NaHS modulates the effects of TNF‐α on mitochondria and the cardiometabolic system, suggesting its therapeutic potential for inflammation‐induced cardiac dysfunction.     

Sitagliptin‐mediated preservation of endothelial progenitor cell function via augmenting autophagy enhances ischaemic angiogenesis in diabetes

Recently, the dipeptidyl peptidase‐4 (DPP‐4) inhibitor sitagliptin, a major anti‐hyperglycaemic agent, has received substantial attention as a therapeutic target for cardiovascular diseases via enhancing the number of circulating endothelial progenitor cells (EPCs). However, the direct effects of sitagliptin on EPC function remain elusive. In this study, we evaluated the proangiogenic effects of sitagliptin on a diabetic hind limb ischaemia (HLI) model in vivo and on EPC culture in vitro. Treatment of db/db mice with sitagliptin (Januvia) after HLI surgery efficiently enhanced ischaemic angiogenesis and blood perfusion, which was accompanied by significant increases in circulating EPC numbers. EPCs derived from the bone marrow of normal mice were treated with high glucose to mimic diabetic hyperglycaemia. We found that high glucose treatment induced EPC apoptosis and tube formation impairment, which were significantly prevented by sitagliptin pretreatment. A mechanistic study found that high glucose treatment of EPCs induced dramatic increases in oxidative stress and apoptosis; pretreatment of EPCs with sitagliptin significantly attenuated high glucose‐induced apoptosis, tube formation impairment and oxidative stress. Furthermore, we found that sitagliptin restored the basal autophagy of EPCs that was impaired by high glucose via activating the AMP‐activated protein kinase/unc‐51‐like autophagy activating kinase 1 signalling pathway, although an autophagy inhibitor abolished the protective effects of sitagliptin on EPCs. Altogether, the results indicate that sitagliptin‐induced preservation of EPC angiogenic function results in an improvement of diabetic ischaemia angiogenesis and blood perfusion, which are most likely mediated by sitagliptin‐induced prevention of EPC apoptosis via augmenting autophagy.     

MPK‐1/ERK is required for the full activity of resveratrol in extended lifespan and reproduction

Resveratrol (RSV) extends the lifespan of various organisms through activation of sirtuin. However, whether RSV‐mediated longevity is entirely dependent upon sirtuin is still controversial. Thus, understanding additional mechanisms concerning the genetic requirements for the biological activity of RSV needs to be clarified to utilize the beneficial effects of RSV. In this study using Caenorhabditis elegans as a model system, we found that MPK‐1 (an ERK homolog) signaling is necessarily required for RSV‐mediated longevity of sir‐2.1/sirtuin mutants as well as for wild‐type worms. We demonstrated that MPK‐1 contributes to RSV‐mediated longevity through nuclear accumulation of SKN‐1 in a SIR‐2.1/DAF‐16 pathway‐independent manner. The positive effect of RSV in regulating lifespan was completely abolished by RNA interference against mpk‐1 in the sir‐2.1 and daf‐16 mutants, strongly indicating that the MPK‐1/SKN‐1 pathway is involved in RSV‐mediated longevity, independently of SIR‐2.1/DAF‐16. We additionally found that RSV protected worms from oxidative stress via MPK‐1. In addition to organismal aging, RSV prevented the age‐associated loss of mitotic germ cells, brood size, and reproductive span through MPK‐1 in C. elegans germline. Therefore, our findings not only provide new mechanistic insight into the controversial effects of RSV on organismal longevity, but additionally have important implications in utilizing RSV to improve the outcome of aging‐related diseases.     

Mthfr deficiency induces endothelial progenitor cell senescence via uncoupling of eNOS and downregulation of SIRT1

Hyperhomocysteinemia (HHcy) has been shown to induce endothelial dysfunction in part as a result of enhanced oxidative stress. Function and survival of endothelial progenitor cells (EPCs, defined as sca1(+) c-kit(+) flk-1(+) bone marrow-derived cells), which significantly contribute to neovascularization and endothelial regeneration, depend on controlled production of reactive oxygen species (ROS). Mice heterozygous for the gene deletion of methylenetetrahydrofolate reductase (Mthfr(+/-)) have a 1.5- to 2-fold elevation in plasma homocysteine. This mild HHcy significantly reduced the number of circulating EPCs as well as their differentiation. Mthfr deficiency was also associated with increased ROS production and reduced nitric oxide (NO) generation in Mthfr(+/-) EPCs. Treatment of EPCs with sepiapterin, a precursor of tetrahydrobiopterin (BH(4)), a cofactor of endothelial nitric oxide synthase (eNOS), significantly reduced ROS and improved NO production. mRNA and protein expression of eNOS and the relative amount of eNOS dimer compared with monomer were decreased by Mthfr deficiency. Impaired differentiation of EPCs induced by Mthfr deficiency correlated with increased senescence, decreased telomere length, and reduced expression of SIRT1. Addition of sepiapterin maintained cell senescence and SIRT1 expression at levels comparable to the wild type. Taken together, these results demonstrate that Mthfr deficiency impairs EPC formation and increases EPC senescence by eNOS uncoupling and downregulation of SIRT1.     

Kruppel-like Factor 4 Is a Novel Mediator of Kallistatin in Inhibiting Endothelial Inflammation via Increased Endothelial Nitric-oxide Synthase Expression

Kallistatin is a plasma protein that exhibits pleiotropic effects in vasodilation, anti-angiogenesis, and anti-inflammation. To isolate a kallistatin-binding protein that mediates the vascular actions of kallistatin, we screened and identified a positive clone from a human heart cDNA expression library by using an alkaline phosphatase-kallistatin fusion protein binding assay. Sequence analysis revealed that kallistatin-binding protein is human Kruppel-like factor 4 (KLF4). KLF4 was localized on the plasma membrane of HEK-293 cells and endothelial cells overexpressing KLF4. KLF4 and kallistatin complex formation was identified in endothelial cells by immunoprecipitation followed by immunoblotting. We showed that kallistatin inhibits tumor necrosis factor-α-induced NF-κB activation, as well as vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression in endothelial cells, whereas knockdown of KLF4 by small interfering RNA oligonucleotide abolished the effect of kallistatin. Kallistatin increased endothelial nitric-oxide synthase (eNOS) expression and nitric oxide levels, and these effects were also blocked by KLF4 small interfering RNA oligonucleotide. Moreover, inhibition of eNOS by RNA interference or by NOS inhibitor abolished the blocking effect of kallistatin on vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression. In summary, we identified KLF4 as a kallistatin-binding protein, which has a novel role in mediating the anti-inflammatory actions of kallistatin via increasing eNOS expression in endothelial cells. This study provides a new target for modulating endothelial function in vascular disease.     

Caveolin‐1 deficiency induces premature senescence with mitochondrial dysfunction

Paradoxical observations have been made regarding the role of caveolin‐1 (Cav‐1) during cellular senescence. For example, caveolin‐1 deficiency prevents reactive oxygen species‐induced cellular senescence despite mitochondrial dysfunction, which leads to senescence. To resolve this paradox, we re‐addressed the role of caveolin‐1 in cellular senescence in human diploid fibroblasts, A549, HCT116, and Cav‐1^?/? mouse embryonic fibroblasts. Cav‐1 deficiency (knockout or knockdown) induced cellular senescence via a p53‐p21‐dependent pathway, downregulating the expression level of the cardiolipin biosynthesis enzymes and then reducing the content of cardiolipin, a critical lipid for mitochondrial respiration. Our results showed that Cav‐1 deficiency decreased mitochondrial respiration, reduced the activity of oxidative phosphorylation complex I (CI), inactivated SIRT1, and decreased the NAD⁺/NADH ratio. From these results, we concluded that Cav‐1 deficiency induces premature senescence via mitochondrial dysfunction and silent information regulator 2 homologue 1 (SIRT1) inactivation.     

TNF‐α promotes survival and migration of MSCs under oxidative stress via NF‐κB pathway to attenuate intimal hyperplasia in vein grafts

The oxidative stress caused by endothelial injury is involved in intimal hyperplasia (IH) in vein grafts. Mesenchymal stem cells (MSCs) can home to injured intima and promote endothelial repair. However, MSC apoptosis is increased accompanied by decreased functional activity under oxidative stress. Thus, we investigate whether tumour necrosis factor‐α (TNF‐α) can promote the survival and activity of MSCs under oxidative stress to reduce IH more effectively, and establish what role the NF‐κB pathway plays in this. In this study, we preconditioned MSCs with TNF‐α (^{TNF‐α‐PC}MSCs) for 24 hrs and measured the activation of the IKK/NF‐κB pathway. EdU and transwell assays were performed to assess proliferation and migration of ^{TNF‐α‐PC}MSCs. Apoptosis and migration of ^{TNF‐α‐PC}MSCs were evaluated in conditions of oxidative stress by analysis of the expression of Bcl‐2 and CXCR4 proteins. ^{TNF‐α‐PC}MSCs were transplanted into a vein graft model, so that cell homing could be tracked, and endothelial apoptosis and IH of vein grafts were measured. The results demonstrated that TNF‐α promotes proliferation and migration of MSCs. Furthermore, survival and migration of ^{TNF‐α‐PC}MSCs under oxidative stress were both enhanced. A greater number of MSCs migrated to the intima of vein grafts after preconditioning with TNF‐α, and the formation of neointima was significantly reduced. These effects could be partially abolished by IKK XII (NF‐κB inhibitor). All these results indicate that preconditioning with TNF‐α can promote survival and migration of MSCs under oxidative stress via the NF‐κB pathway and thus attenuate IH of vein grafts.     

MicroRNA‐216a induces endothelial senescence and inflammation via Smad3/IκBα pathway

Vascular endothelial senescence contributes to atherosclerosis and coronary artery disease (CAD), but the mechanisms are yet to be clarified. We identified that microRNA‐216a (miR‐216a) significantly increased in senescent endothelial cells. The replicative senescence model of human umbilical vein endothelial cells (HUVECs) was established to explore the role of miR‐216a in endothelial ageing and dysfunction. Luciferase assay indicated that Smad3 was a direct target of miR‐216a. Stable expression of miR‐216a induced a premature senescence‐like phenotype in HUVECs with an impairment in proliferation and migration and led to an increased adhesion to monocytes by inhibiting Smad3 expression and thereafter modulating the degradation of NF‐κB inhibitor alpha (IκBα) and activation of adhesion molecules. Conversely, inhibition of endogenous miR‐216a in senescent HUVECs rescued Smad3 and IκBα expression and inhibited monocytes attachment. Plasma miR‐216a was significantly higher in old CAD patients (>50 years) and associated with increased 31% risk for CAD (odds ratio 1.31, 95% confidence interval 1.03‐1.66; P = .03) compared with the matched healthy controls (>50 years). Taken together, our data suggested that miR‐216a promotes endothelial senescence and inflammation as an endogenous inhibitor of Smad3/IκBα pathway, which might serve as a novel target for ageing‐related atherosclerotic diseases.     

Angiotensin II‐induced redox‐sensitive SGLT1 and 2 expression promotes high glucose‐induced endothelial cell senescence

High glucose (HG)‐induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co‐transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG‐induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence‐associated beta‐galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT‐PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2‐NBD‐glucose. HG increased ECs senescence markers and oxidative stress, down‐regulated eNOS expression and NO formation, and induced the expression of VCAM‐1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX‐4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H₂O₂ in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG‐induced ECs senescence and SGLT1 and 2 expression. Thus, HG‐induced ECs ageing is driven by the local angiotensin system via the redox‐sensitive up‐regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.     

Crosstalk between AMPK activation and angiotensin II‐induced hypertrophy in cardiomyocytes: the role of mitochondria

AMP‐kinase (AMPK) activation reduces cardiac hypertrophy, although underlying molecular mechanisms remain unclear. In this study, we elucidated the anti‐hypertrophic action of metformin, specifically, the role of the AMPK/eNOS/p53 pathway. H9c2 rat cardiomyocytes were treated with angiotensin II (AngII) for 24 hrs in the presence or absence of metformin (AMPK agonist), losartan [AngII type 1 receptor (AT1R) blocker], Nω‐nitro‐L‐arginine methyl ester (L‐NAME, pan‐NOS inhibitor), splitomicin (SIRT1 inhibitor) or pifithrin‐α (p53 inhibitor). Results showed that treatment with metformin significantly attenuated AngII‐induced cell hypertrophy and death. Metformin attenuated AngII‐induced activation (cleavage) of caspase 3, Bcl‐2 down‐regulation and p53 up‐regulation. It also reduced AngII‐induced AT1R up‐regulation by 30% (P < 0.05) and enhanced AMPK phosphorylation by 99% (P < 0.01) and P‐eNOS levels by 3.3‐fold (P < 0.01). Likewise, losartan reduced AT1R up‐regulation and enhanced AMPK phosphorylation by 54% (P < 0.05). The AMPK inhibitor, compound C, prevented AT1R down‐regulation, indicating that metformin mediated its effects via AMPK activation. Beneficial effects of metformin and losartan converged on mitochondria that demonstrated high membrane potential (Δψ_m) and low permeability transition pore opening. Thus, this study demonstrates that the anti‐hypertrophic effects of metformin are associated with AMPK‐induced AT1R down‐regulation and prevention of mitochondrial dysfunction through the SIRT1/eNOS/p53 pathway.     

Endogenous CSE/H2S system mediates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes

Tumour necrosis factor‐α (TNF‐ α)is a major contributor to the pathogenesis of insulin resistance associated with obesity and type 2 diabetes. It has been found that endogenous hydrogen sulfide (H₂S) contributes to the pathogenesis of diabetes. We have hypothesized that TNF‐α‐induced insulin resistance is involved in endogenous H₂S generation. The aim of the present study is to investigate the role of endogenous H₂S in TNF‐α‐induced insulin resistance by studying 3T3‐L1 adipocytes. We found that treatment of 3T3‐L1 adipocytes with TNF‐α leads to deficiency in insulin‐stimulated glucose consumption and uptake and increase in endogenous H₂S generation. We show that cystathionine γ‐lyase (CSE) is catalysed in 3T3‐L1 adipocytes to generate H₂S and that CSE expression and activity are upregulated by TNF‐α treatment. Inhibited CSE by its potent inhibitors significantly attenuates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes, whereas H₂S treatment of 3T3‐L1 adipocytes impairs insulin‐stimulated glucose consumption and uptake. These data indicate that endogenous CSE/H₂S system contributes to TNF‐α‐caused insulin resistance in 3T3‐L1 adipocytes. Our findings suggest that modulation of CSE/H₂S system is a potential therapeutic avenue for insulin resistance. Copyright © 2012 John Wiley & Sons, Ltd.