miR-200s contribute to interleukin-6 (IL-6)-induced insulin resistance in hepatocytes.

By influencing the activity of the PI3K/AKT pathway, IL-6 acts as an important regulator of hepatic insulin resistance. miR-200s have been shown to control growth by regulating PI3K, but the role of miR-200s in the development of hepatic insulin resistance remains unclear. The present study showed that elevated serum concentration of IL-6 is associated with decreased levels of miR-200s, impaired activation of the AKT/glycogen synthase kinase (GSK) pathway, and reduced glycogenesis that occurred in the livers of db/db mice. As shown in the murine NCTC 1469 hepatocytes and the primary hepatocytes treated with 10 ng/ml IL-6 for 24 h and in 12-week-old male C57BL/6J mice injected with 16 µg/ml IL-6 by pumps for 7 days, IL-6 administration induced insulin resistance through down-regulation of miR-200s. Moreover, IL-6 treatment inhibited the phosphorylation of AKT and GSK and decreased the glycogenesis. The effects of IL-6 could be diminished by suppression of FOG2 expression. We concluded that IL-6 treatment may impair the activities of the PI3K/AKT/GSK pathway and inhibit the synthesis of glycogen, perhaps via down-regulating miR-200s while augmenting FOG2 expression.

Fibulin-2 deficiency attenuates angiotensin II-induced cardiac hypertrophy by reducing transforming growth factor-ß signalling.

AngII (angiotensin II) is a potent neurohormone responsible for cardiac hypertrophy, in which TGF (transforming growth factor)-ß serves as a principal downstream mediator. We recently found that ablation of fibulin-2 in mice attenuated TGF-ß signalling, protected mice against progressive ventricular dysfunction, and significantly reduced the mortality after experimental MI (myocardial infarction). In the present study, we investigated the role of fibulin-2 in AngII-induced TGF-ß signalling and subsequent cardiac hypertrophy. We performed chronic subcutaneous infusion of AngII in fibulin-2 null (Fbln2-/-), heterozygous (Fbln2+/-) and WT (wild-type) mice by a mini-osmotic pump. After 4 weeks of subpressor dosage of AngII infusion (0.2 µg/kg of body weight per min), WT mice developed significant hypertrophy, whereas the Fbln2-/- showed no response. In WT, AngII treatment significantly up-regulated mRNAs for fibulin-2, ANP (atrial natriuretic peptide), TGF-ß1, Col I (collagen type I), Col III (collagen type III), MMP (matrix metalloproteinase)-2 and MMP-9, and increased the phosphorylation of TGF-ß-downstream signalling markers, Smad2, TAK1 (TGF-ß-activated kinase 1) and p38 MAPK (mitogen-activated protein kinase), which were all unchanged in AngII-treated Fbln2-/- mice. The Fbln2+/- mice consistently displayed AngII-induced effects intermediate between WT and Fbln2-/-. Pressor dosage of AngII (2 mg/kg of body weight per min) induced significant fibrosis in WT but not in Fbln2-/- mice with comparable hypertension and hypertrophy in both groups. Isolated CFs (cardiac fibroblasts) were treated with AngII, in which direct AngII effects and TGF-ß-mediated autocrine effects was observed in WT. The latter effects were totally abolished in Fbln2-/- cells, suggesting that fibulin-2 is essential for AngII-induced TGF-ß activation. In conclusion our data indicate that fibulin-2 is essential for AngII-induced TGF-ß-mediated cardiac hypertrophy via enhanced TGF-ß activation and suggest that fibulin-2 is a potential therapeutic target to inhibit AngII-induced cardiac remodelling.

Loss of fibulin-2 protects against progressive ventricular dysfunction after myocardial infarction.

Remodeling of the cardiac extracellular matrix (ECM) is an integral part of wound healing and ventricular adaptation after myocardial infarction (MI), but the underlying mechanisms remain incompletely understood. Fibulin-2 is an ECM protein upregulated during cardiac development and skin wound healing, yet mice lacking fibulin-2 do not display any identifiable phenotypic abnormalities. To investigate the effects of fibulin-2 deficiency on ECM remodeling after MI, we induced experimental MI by permanent coronary artery ligation in both fibulin-2 null and wild-type mice. Fibulin-2 expression was up-regulated at the infarct border zone of the wild-type mice. Acute myocardial tissue responses after MI, including inflammatory cell infiltration and ECM protein synthesis and deposition in the infarct border zone, were markedly attenuated in the fibulin-2 null mice. However, the fibulin-2 null mice had significantly better survival rate after MI compared to the wild-type mice as a result of less frequent cardiac rupture and preserved left ventricular function. Up-regulation of TGF-ß signaling and ECM remodeling after MI were attenuated in both ischemic and non-ischemic myocardium of the fibulin-2 null mice compared to the wild type counterparts. Increase in TGF-ß signaling in response to angiotensin II was also lessened in cardiac fibroblasts isolated from the fibulin-2 null mice. The studies provide the first evidence that absence of fibulin-2 results in decreased up-regulation of TGF-ß signaling after MI and protects against ventricular dysfunction, suggesting that fibulin-2 may be a potential therapeutic target for attenuating the progression of ventricular remodeling.

Adiponectin: an indispensable molecule in rosiglitazone cardioprotection following myocardial infarction.

RATIONALE: Patients treated with peroxisome proliferator-activated receptor (PPAR)-gamma agonist manifest favorable metabolic profiles associated with increased plasma adiponectin (APN). However, whether increased APN production as a result of PPAR-gamma agonist treatment is an epiphenomenon or is causatively related to the cardioprotective actions of PPAR-gamma remains unknown. OBJECTIVE: To determine the role of APN in rosiglitazone (RSG) cardioprotection against ischemic heart injury. METHODS AND RESULTS: Adult male wild-type (WT) and APN knockdown/knockout (APN(+ or -) and APN(- or -)) mice were treated with vehicle or RSG (20 mg/kg per day), and subjected to coronary artery ligation 3 days after beginning treatment. In WT mice, RSG (7 days) significantly increased adipocyte APN expression, elevated plasma APN levels (2.6-fold), reduced infarct size (17% reduction), decreased apoptosis (0.23 + or - 0.02% versus 0.47 + or - 0.04% TUNEL-positive in remote nonischemic area), attenuated oxidative stress (48.5% reduction), and improved cardiac function (P<0.01). RSG-induced APN production and cardioprotection were significantly blunted (P<0.05 versus WT) in APN(+ or -), and completely lost in APN(- or -) (P>0.05 versus vehicle-treated APN(- or -) mice). Moreover, treatment with RSG for up to 14 days significantly improved the postischemic survival rate of WT mice (P<0.05 versus vehicle group) but not APN knockdown/knockout mice. CONCLUSIONS: The cardioprotective effects of PPAR-gamma agonists are critically dependent on its APN stimulatory action, suggesting that under pathological conditions where APN expression is impaired (such as advanced type 2 diabetes), the harmful cardiovascular effects of PPAR-gamma agonists may outweigh its cardioprotective benefits.

Nitrative thioredoxin inactivation as a cause of enhanced myocardial ischemia/reperfusion injury in the aging heart.

Several recent studies have demonstrated that thioredoxin (Trx) is an important antiapoptotic/cytoprotective molecule. The present study was designed to determine whether Trx activity is altered in the aging heart in a way that may contribute to increased susceptibility to myocardial ischemia/reperfusion (MI/R). Compared to young animals, MI/R-induced cardiomyocyte apoptosis and infarct size were increased in aging animals (p<0.01). Trx activity was decreased in the aging heart before MI/R, and this difference was further amplified after MI/R. Trx expression was moderately increased and Trx nitration, a posttranslational modification that inhibits Trx activity, was increased in the aging heart. Moreover, Trx-aptosis-regulating kinase-1 (Trx-ASK1) complex formation was reduced and activity of p38 mitogen-activated protein kinase (MAPK) was increased. Treatment with FP15 (a peroxynitrite decomposition catalyst) reduced Trx nitration, increased Trx activity, restored Trx-ASK1 interaction, reduced P38 MAPK activity, attenuated caspase 3 activation, and reduced infarct size in aging animals (p<0.01). Our results demonstrated that Trx activity is decreased in the aging heart by posttranslational nitrative modification. Interventions that restore Trx activity in the aging heart may be novel therapies to attenuate MI/R injury in aging patients.

[Effects of insulin on cardiac function and coronary circulation in acute myocardial ischemia and reperfusion experiment with dogs].

OBJECTIVE: To study the effect of insulin on cardiac functional recovery, coronary blood flow (CBF), coronary arterial function and coronary vascular endothelial cell apoptosis following acute myocardial ischemia/reperfusion (MI/R). METHODS: In adult dogs, the left anterior descending coronary artery (LAD) was partially occluded (80% reduction in its blood flow) for 50 min and reperfused for 4 h. Vehicle (0.9% NaCl), GIK (glucose: 250 gxL(-1), insulin: 60 UxL(-1), potassium: 80 mmolxL(-1)), or GK (glucose: 250 gxL(-1), potassium: 80 mmolxL(-1)) were intravenously infused (2 mlxkg(-1)xh(-1)) 5 min before reperfusion, and CBF and left ventricular pressure were monitored. At the end of 4 h reperfusion period, coronary arteries were isolated, and the coronary vascular dysfunction, nitric oxide (NO) production and endothelial apoptosis were determined. RESULTS: During reperfusion, compared with the vehicle, GIK increased CBFLAD (19.2 ml/min +/- 2.2 ml/min) vs (14.6 ml/min +/- 1.8 ml/min) of vehicle at the end of reperfusion, P < 0.05, improved recovery of LVSP and +/- LVdP/dtmax. In vivo ischemia/reperfusion caused significant coronary vascular endothelial dysfunction as evidenced by reduced endothelium dependent vasorelaxation, decreased total NO production, and endothelial cell apoptosis as determined by TUNEL staining. Reperfusion with GIK, but not GK, markedly improved the endothelium-dependent vasorelaxation (80.3% +/- 3.8%) vs. vehicle (28.1% +/- 2.3%, P < 0.01) of coronary artery in response to ACh. GIK significantly increased total NO production (17.19 micromol/L +/- 2.18 micromol/L) versus vehicle (4.74 micromol/L +/- 2.01 micromol/L, P < 0.01) and inhibited apoptosis in coronary arterial endothelial cell (12% +/- 4%) vs vehicle (45% +/- 7%, P < 0.01). GK failed to show any significant vasculoprotection against MI/R-induced coronary vascular injury. CONCLUSION: These results demonstrate that insulin exerts cardioprotective effect by increasing CBF, reducing coronary artery injury and improving cardiac functional recovery during reperfusion, which may be partly attributable to the coronary vasculoprotective effect of insulin. The insulin-induced, NO-mediated anti-endothelial apoptotic effect may play a critical role in the insulin-induced coronary artery protective effect in MI/R.

Protein phosphatase 4 (PP4) functions as a critical regulator in tumor necrosis factor (TNF)-α-induced hepatic insulin resistance.

Protein phosphatase 4 (PP4) was shown to participate in multiple cellular processes, including DNA damage response, cell cycle and embryo development. Recent studies demonstrated a looming role of PP4 in glucose metabolism. However, whether PP4 is involved in hepatic insulin resistance remains poorly understood. The objective of this study was to estimate the role of PP4 in tumor necrosis factor (TNF)-α-induced hepatic insulin resistance. db/db mice and TNF-α-treated C57BL/6J mice were used as hepatic insulin resistance animal models. In vitro models were established in both HepG2 cells and primary hepatocytes by TNF-α treatment. We found that increased expression and activity of PP4 occurred in the livers of db/db mice and TNF-α-induced hepatic insulin resistance both in vitro and in vivo. Actually, PP4 silencing and suppression of PP4 activity ameliorated TNF-α-induced hepatic insulin resistance, whereas over-expression of PP4 caused insulin resistance. We then further investigated the prodiabetic mechanism of PP4 in TNF-α-induced insulin resistance. We found that PP4 formed a complex with IRS-1 to promote phosphorylation of IRS-1 on serine 307 via JNK activation and reduce the expression of IRS-1. Thus, PP4 is an important regulator in inflammatory related insulin resistance.

Apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ.

Apelin, a novel adipokine, is the specific endogenous ligand of G protein-coupled receptor APJ. Consistent with its putative role as an adipokine, apelin has been linked to states of insulin resistance. However, the function of apelin in hepatic insulin resistance, a vital part of insulin resistance, and its underlying mechanisms still remains unclear. Here we define the impacts of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes. Our studies indicate that apelin reversed TNF-α-induced reduction of glycogen synthesis in HepG2 cells, mouse primary hepatocytes and liver tissues of C57BL/6J mice by improving JNK-IRS1-AKT-GSK pathway. Moreover, Western blot revealed that APJ, but not apelin, expressed in the hepatocytes and liver tissues of mice. We found that F13A, a competitive antagonist for G protein-coupled receptor APJ, suppressed the effects of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes, suggesting APJ is involved in the function of apelin. In conclusion, we show novel evidence suggesting that apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ. Apelin appears as a beneficial adipokine with anti-insulin resistance properties, and thus as a promising therapeutic target in metabolic disorders.

Tbx20 functions as an important regulator of estrogen-mediated cardiomyocyte protection during oxidative stress.

BACKGROUND: As a transcription factor mainly expressed in cardiovascular system, T-box 20 (Tbx20) plays an important role in embryonic cardiovascular system development and adult heart function. Here, we determined the mechanism by which Tbx20 regulates cardiomyocyte apoptosis and cardiomyopathies. METHODS: We analyzed Tbx20 expression levels and apoptosis rates in normal and heart failure human autopsy heart samples. Female C57BL/6 mice were ovariectomized and treated with 17ß-estradiol to determine Tbx20 expression levels. ROS production, TUNEL, DNA laddering, qRT-PCR, Western blot, immunohistochemistry and ChIP analyses were performed on male C57BL/6 transverse aortic constriction-induced heart failure samples and on neonatal rat ventricular myocytes that were treated with H2O2 to investigate the role of Tbx20 in estrogen-mediated heart protection. RESULTS: Tbx20 expression was down regulated during heart failure, accompanied by elevated cardiomyocyte apoptotic levels in humans and mice. H2O2 led to a concurrent decrease in Tbx20 expression and increase in apoptosis in cultured neonatal rat cardiomyocytes. Tbx20 overexpression reduced H2O2-induced cardiomyocyte apoptosis and was associated with a profound inhibition of p38MAPK, Bax and caspase3 and the activation of Bcl-2. Estrogen was able to protect cardiomyocytes from H2O2-induced apoptosis by upregulating Tbx20 expression in a concentration-dependent manner. Tbx20 silencing increased oxidative stress-induced apoptosis in H9c2 cells. Moreover, Tbx20 directly regulated Esrra expression to enhance the heart-protective effect of estrogen. CONCLUSIONS: These results indicate that Tbx20 functions as an important regulator of estrogen-mediated cardiomyocyte protection during oxidative stress, suggesting that estorgen-Tbx20-ERR-α may represent a crucial regulatory cascade and a potential therapeutic target for heart failure.

APOBEC3G upregulation by alpha interferon restricts human immunodeficiency virus type 1 infection in human peripheral plasmacytoid dendritic cells.

APOBEC3G (A3G), a member of cytidine deaminase family, has potent anti-human immunodeficiency virus type 1 (HIV-1) activity. It has been demonstrated that alpha interferon (IFN-alpha) can significantly enhance the expression of A3G in human primary resting CD4(+) T-cells, macrophages and primary hepatocytes, subsequently decreasing their viral susceptibility. Plasmacytoid dendritic cells (pDCs) are key effectors in innate host immunity, mediating adaptive immune responses and stimulating IFN-alpha production in reaction to various stimuli. In this report, we demonstrate that IFN-alpha, either exogenously added to- or endogenously secreted by pDCs, can enhance the expression of A3G and its family members such as A3A, A3C and A3F. We have also shown that IFN-alpha can inhibit HIV-1 expression in pDCs. This inhibitory effect could be countered by addition of an A3G-specific short interfering RNA, indicating that IFN-alpha-induced A3G plays a key role in mediating pDCs response to HIV-1. Given the central role played by pDCs in orchestrating the IFN-alpha/A3G intercellular network and intracellular signal pathway, our data indicate that pDCs themselves are also protected by an IFN-alpha/A3G-mediated innate immunity barrier from HIV-1 infection.

Physiologically tolerable insulin reduces myocardial injury and improves cardiac functional recovery in myocardial ischemic/reperfused dogs.

This study was designed to examine whether physiologically tolerable insulin, which maintains lower blood glucose, can protect the myocardium against ischemia/reperfusion (I/R) injury in a preclinical large animal model. Adult dogs were subjected to 50 minutes of myocardial ischemia (80% reduction in coronary blood flow) followed by 4 hours of reperfusion and treated with vehicle, glucose-insulin-potassium (GIK; glucose, 250 g/L; insulin, 60 U/L; potassium, 80 mmol/L), GK, or low-dose insulin (30 U/L) 10 minutes before reperfusion. Treatment with GIK exerted significant cardioprotective effects as evidenced by improved cardiac function, improved coronary blood flow, reduced infarct size, and myocardial apoptosis. In contrast, treatment with GK increased blood glucose level and aggravated myocardial I/R injury. It is interesting that treatment with insulin alone at the dose that reduced blood glucose to a clinically tolerable level exerted significant cardioprotective effects that were comparable to that seen in the GIK-treated group. This low-dose insulin had no effect on coronary blood flow after reperfusion but markedly reduced coronary reactive hyperemia and switched myocardial substrate uptake from fat to carbohydrate. Our results suggest that lower glucose supply to the ischemic myocardium at early reperfusion may create a "metabolic postconditioning" and thus reduce myocardial ischemia/reperfusion injury after prolonged reperfusion.