Control of NAD+ homeostasis by autophagic flux modulates mitochondrial and cardiac function.

Impaired autophagy is known to cause mitochondrial dysfunction and heart failure, in part due to altered mitophagy and protein quality control. However, whether additional mechanisms are involved in the development of mitochondrial dysfunction and heart failure in the setting of deficient autophagic flux remains poorly explored. Here, we show that impaired autophagic flux reduces nicotinamide adenine dinucleotide (NAD+) availability in cardiomyocytes. NAD+ deficiency upon autophagic impairment is attributable to the induction of nicotinamide N-methyltransferase (NNMT), which methylates the NAD+ precursor nicotinamide (NAM) to generate N-methyl-nicotinamide (MeNAM). The administration of nicotinamide mononucleotide (NMN) or inhibition of NNMT activity in autophagy-deficient hearts and cardiomyocytes restores NAD+ levels and ameliorates cardiac and mitochondrial dysfunction. Mechanistically, autophagic inhibition causes the accumulation of SQSTM1, which activates NF-κB signaling and promotes NNMT transcription. In summary, we describe a novel mechanism illustrating how autophagic flux maintains mitochondrial and cardiac function by mediating SQSTM1-NF-κB-NNMT signaling and controlling the cellular levels of NAD+.

Late-in-life treadmill training rejuvenates autophagy, protein aggregate clearance, and function in mouse hearts.

Protein quality control mechanisms decline during the process of cardiac aging. This enables the accumulation of protein aggregates and damaged organelles that contribute to age-associated cardiac dysfunction. Macroautophagy is the process by which post-mitotic cells such as cardiomyocytes clear defective proteins and organelles. We hypothesized that late-in-life exercise training improves autophagy, protein aggregate clearance, and function that is otherwise dysregulated in hearts from old vs. adult mice. As expected, 24-month-old male C57BL/6J mice (old) exhibited repressed autophagosome formation and protein aggregate accumulation in the heart, systolic and diastolic dysfunction, and reduced exercise capacity vs. 8-month-old (adult) mice (all p < 0.05). To investigate the influence of late-in-life exercise training, additional cohorts of 21-month-old mice did (old-ETR) or did not (old-SED) complete a 3-month progressive resistance treadmill running program. Body composition, exercise capacity, and soleus muscle citrate synthase activity improved in old-ETR vs. old-SED mice at 24 months (all p < 0.05). Importantly, protein expression of autophagy markers indicate trafficking of the autophagosome to the lysosome increased, protein aggregate clearance improved, and overall function was enhanced (all p < 0.05) in hearts from old-ETR vs. old-SED mice. These data provide the first evidence that a physiological intervention initiated late-in-life improves autophagic flux, protein aggregate clearance, and contractile performance in mouse hearts.

Insulin receptor substrates differentially exacerbate insulin-mediated left ventricular remodeling.

Pressure overload (PO) cardiac hypertrophy and heart failure are associated with generalized insulin resistance and hyperinsulinemia, which may exacerbate left ventricular (LV) remodeling. While PO activates insulin receptor tyrosine kinase activity that is transduced by insulin receptor substrate 1 (IRS1), the present study tested the hypothesis that IRS1 and IRS2 have divergent effects on PO-induced LV remodeling. We therefore subjected mice with cardiomyocyte-restricted deficiency of IRS1 (CIRS1KO) or IRS2 (CIRS2KO) to PO induced by transverse aortic constriction (TAC). In WT mice, TAC-induced LV hypertrophy was associated with hyperactivation of IRS1 and Akt1, but not IRS2 and Akt2. CIRS1KO hearts were resistant to cardiac hypertrophy and heart failure in concert with attenuated Akt1 activation. In contrast, CIRS2KO hearts following TAC developed more severe LV dysfunction than WT controls, and this was prevented by haploinsufficiency of Akt1. Failing human hearts exhibited isoform-specific IRS1 and Akt1 activation, while IRS2 and Akt2 activation were unchanged. Kinomic profiling identified IRS1 as a potential regulator of cardioprotective protein kinase G-mediated signaling. In addition, gene expression profiling revealed that IRS1 signaling may promote a proinflammatory response following PO. Together, these data identify IRS1 and Akt1 as critical signaling nodes that mediate LV remodeling in both mice and humans.

Identification of a Paracrine Signaling Mechanism Linking CD34high Progenitors to the Regulation of Visceral Fat Expansion and Remodeling.

Accumulation of visceral (VIS) is a predictor of metabolic disorders and insulin resistance. This is due in part to the limited capacity of VIS fat to buffer lipids allowing them to deposit in insulin-sensitive tissues. Mechanisms underlying selective hypertrophic growth and tissue remodeling properties of VIS fat are not well understood. We identified subsets of adipose progenitors (APs) unique to VIS fat with differential Cd34 expression and adipogenic capacity. VIS low (Cd34 low) APs are adipogenic, whereas VIS high (Cd34 high) APs are not. Furthermore, VIS high APs inhibit adipogenic differentiation of SUB and VIS low APs in vitro through the secretion of soluble inhibitory factor(s). The number of VIS high APs increased with adipose tissue expansion, and their abundance in vivo caused hypertrophic growth, fibrosis, inflammation, and metabolic dysfunction. This study unveils the presence of APs unique to VIS fat involved in the paracrine regulation of adipogenesis and tissue remodeling.

Suppression of Cardiac Autophagy by Hyperinsulinemia in Insulin Receptor-Deficient Hearts Is Mediated by Insulin-Like Growth Factor Receptor Signaling.

Aims: Autophagy is a catabolic process required for the maintenance of cardiac health. Insulin and insulin-like growth factor 1 (IGF-1) are potent inhibitors of autophagy and as such, one would predict that autophagy will be increased in the insulin-resistant/diabetic heart. However, autophagy is rather decreased in the hearts of diabetic/insulin-resistant mice. The aim of this study is to determine the contribution of IGF-1 receptor signaling to autophagy suppression in insulin receptor (IR)-deficient hearts. Results: Absence of IRs in the heart was associated with reduced autophagic flux, and further inhibition of autophagosome clearance reduced survival, impaired contractile function, and enhanced myocyte loss. Contrary to the in vivo setting, isolated cardiomyocytes from IR-deficient hearts exhibited unrestrained autophagy in the absence of insulin, whereas addition of insulin was able to suppress autophagy. To investigate the mechanisms involved in the maintenance of the responsiveness to insulin in IR-deficient hearts, we generated mice lacking both IRs and one copy of the IGF-1 receptor (IGF-1R) in cardiac cells and showed that these mice had increased autophagy. Innovation and Conclusion: This study unveils a new mechanism by which IR-deficient hearts can still respond to insulin to suppress autophagy, in part, through activation of IGF-1R signaling. This is a highly significant observation because it is the first to show that systemic hyperinsulinemia can suppress autophagy in IR-deficient hearts through IGF-1R signaling.

Autophagy Ablation in Adipocytes Induces Insulin Resistance and Reveals Roles for Lipid Peroxide and Nrf2 Signaling in Adipose-Liver Crosstalk.

Autophagy is a homeostatic cellular process involved in the degradation of long-lived or damaged cellular components. The role of autophagy in adipogenesis is well recognized, but its role in mature adipocyte function is largely unknown. We show that the autophagy proteins Atg3 and Atg16L1 are required for proper mitochondrial function in mature adipocytes. In contrast to previous studies, we found that post-developmental ablation of autophagy causes peripheral insulin resistance independently of diet or adiposity. Finally, lack of adipocyte autophagy reveals cross talk between fat and liver, mediated by lipid peroxide-induced Nrf2 signaling. Our data reveal a role for autophagy in preventing lipid peroxide formation and its transfer in insulin-sensitive peripheral tissues.

Activation of IGF-1 receptors and Akt signaling by systemic hyperinsulinemia contributes to cardiac hypertrophy but does not regulate cardiac autophagy in obese diabetic mice.

Autophagy plays an important role in the maintenance of normal heart function. However, the role of autophagy in the inulin resistant and diabetic heart is not well understood. Furthermore, the upstream signaling and the downstream targets involved in cardiac autophagy regulation during obesity and type 2 diabetes mellitus (T2DM) are not fully elucidated. The aim of this study was to measure autophagic flux and to dissect the upstream and downstream signaling involved in cardiac autophagy regulation in the hearts of obese T2DM mice. Our study demonstrated that cardiac autophagic flux is suppressed in the heart of obese diabetic (ob/ob) mice due to impaired autophagosome formation. We showed that suppression of autophagy was due to sustained activation of mTOR as we could restore cardiac autophagy by inhibiting mTOR. Moreover, the novel finding of this study is that while IGF-1 receptor-mediated Akt activation contributes to cardiac hypertrophy, it is not involved in mTOR activation and autophagy suppression in obesity and T2DM. In contrast, inhibition of ERK signaling abolished mTOR activation and restored autophagy in the heart of obese diabetic (ob/ob) mice. The study identifies mechanisms regulating cardiac autophagy in obesity and T2DM that are mediated by ERK/mTOR but are distinct from Akt. The findings are of significant importance as they demonstrate for the first time the contribution of IGF-1 receptors (IGF-1R) and Akt signaling in cardiac hypertrophy but not in cardiac autophagy regulation in obesity and T2DM.

Endothelial Cell Autophagy Maintains Shear Stress-Induced Nitric Oxide Generation via Glycolysis-Dependent Purinergic Signaling to Endothelial Nitric Oxide Synthase.

OBJECTIVE: Impaired endothelial cell (EC) autophagy compromises shear stress-induced nitric oxide (NO) generation. We determined the responsible mechanism. APPROACH AND RESULTS: On autophagy compromise in bovine aortic ECs exposed to shear stress, a decrease in glucose uptake and EC glycolysis attenuated ATP production. We hypothesized that decreased glycolysis-dependent purinergic signaling via P2Y1 (P2Y purinoceptor 1) receptors, secondary to impaired autophagy in ECs, prevents shear-induced phosphorylation of eNOS (endothelial nitric oxide synthase) at its positive regulatory site S1117 (p-eNOSS1177) and NO generation. Maneuvers that restore glucose transport and glycolysis (eg, overexpression of GLUT1 [glucose transporter 1]) or purinergic signaling (eg, addition of exogenous ADP) rescue shear-induced p-eNOSS1177 and NO production in ECs with impaired autophagy. Conversely, inhibiting glucose transport via GLUT1 small interfering RNA, blocking purinergic signaling via ectonucleotidase-mediated ATP/ADP degradation (eg, apyrase), or inhibiting P2Y1 receptors using pharmacological (eg, MRS2179 [2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetrasodium salt]) or genetic (eg, P2Y1-receptor small interfering RNA) procedures inhibit shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Supporting a central role for PKCδT505 (protein kinase C delta T505) in relaying the autophagy-dependent purinergic-mediated signal to eNOS, we find that (1) shear stress-induced activating phosphorylation of PKCδT505 is negated by inhibiting autophagy, (2) shear-induced p-eNOSS1177 and NO generation are restored in autophagy-impaired ECs via pharmacological (eg, bryostatin) or genetic (eg, constitutively active PKCδ) activation of PKCδT505, and (3) pharmacological (eg, rottlerin) and genetic (eg, PKCδ small interfering RNA) PKCδ inhibition prevents shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Key nodes of dysregulation in this pathway on autophagy compromise were revealed in human arterial ECs. CONCLUSIONS: Targeted reactivation of purinergic signaling and PKCδ has strategic potential to restore compromised NO generation in pathologies associated with suppressed EC autophagy.

OPA1 deficiency promotes secretion of FGF21 from muscle that prevents obesity and insulin resistance.

Mitochondrial dynamics is a conserved process by which mitochondria undergo repeated cycles of fusion and fission, leading to exchange of mitochondrial genetic content, ions, metabolites, and proteins. Here, we examine the role of the mitochondrial fusion protein optic atrophy 1 (OPA1) in differentiated skeletal muscle by reducing OPA1 gene expression in an inducible manner. OPA1 deficiency in young mice results in non-lethal progressive mitochondrial dysfunction and loss of muscle mass. Mutant mice are resistant to age- and diet-induced weight gain and insulin resistance, by mechanisms that involve activation of ER stress and secretion of fibroblast growth factor 21 (FGF21) from skeletal muscle, resulting in increased metabolic rates and improved whole-body insulin sensitivity. OPA1-elicited mitochondrial dysfunction activates an integrated stress response that locally induces muscle atrophy, but via secretion of FGF21 acts distally to modulate whole-body metabolism.

Adipocyte-Specific Deletion of Manganese Superoxide Dismutase Protects From Diet-Induced Obesity Through Increased Mitochondrial Uncoupling and Biogenesis.

Obesity and insulin resistance are associated with oxidative stress (OS). The causal role of adipose OS in the pathogenesis of these conditions is unknown. To address this issue, we generated mice with an adipocyte-selective deletion of manganese superoxide dismutase (MnSOD). When fed a high-fat diet (HFD), the AdSod2 knockout (KO) mice exhibited less adiposity, reduced adipocyte hypertrophy, and decreased circulating leptin. The resistance to diet-induced adiposity was the result of an increased metabolic rate and energy expenditure. Furthermore, palmitate oxidation was elevated in the white adipose tissue (WAT) and brown adipose tissue of AdSod2 KO mice fed an HFD, and the expression of key fatty acid oxidation genes was increased. To gain mechanistic insight into the increased fat oxidation in HFD-fed AdSod2 KO mice, we quantified the mitochondrial function and mitochondrial content in WAT and found that MnSOD deletion increased mitochondrial oxygen consumption and induced mitochondrial biogenesis. This effect was preserved in cultured adipocytes from AdSod2 KO mice in vitro. As expected from the enhanced fat oxidation, circulating levels of free fatty acids were reduced in the HFD-fed AdSod2 KO mice. Finally, HFD-fed AdSod2 KO mice were protected from hepatic steatosis, adipose tissue inflammation, and glucose and insulin intolerance. Taken together, these results demonstrate that MnSOD deletion in adipocytes triggered an adaptive stress response that activated mitochondrial biogenesis and enhanced mitochondrial fatty acid oxidation, thereby preventing diet-induced obesity and insulin resistance.

Pulmonary and hepatic injury after sub-chronic exposure to sublethal doses of microcystin-LR.

We had previously shown that microcystin-LR (MCLR) could induce lung and liver inflammation after acute exposure. The biological outcomes following prolonged exposure to MCLR, although more frequent, are still poorly understood. Thus, we aimed to verify whether repeated doses of MCLR could damage lung and liver and evaluate the dose-dependence of the results. Male Swiss mice received 10 intraperitoneal injections (i.p.) of distilled water (60 µL, CTRL) or different doses of MCLR (5 µg/kg, TOX5), 10 µg/kg (TOX10), 15 µg/kg (TOX15) and 20 µg/kg (TOX20) every other day. On the tenth injection respiratory mechanics (lung resistive and viscoelastic/inhomogeneous pressures, static elastance, and viscoelastic component of elastance) was measured. Lungs and liver were prepared for histology (morphometry and cellularity) and inflammatory mediators (KC and MIP-2) determination. All mechanical parameters and alveolar collapse were significantly higher in TOX5, 10, 15 and 20 than CTRL, but did not differ among them. Lung inflammatory cell content increased dose-dependently in all TOX groups in relation to CTRL, being TOX20 the largest. The production of KC was increased in lung and liver homogenates. MIP-2 increased in the liver of all TOX groups, but in lung homogenates it was significantly higher only in TOX20 group. All TOX mice livers showed steatosis, necrosis, inflammatory foci and a high degree of binucleated hepatocytes. In conclusion, sub-chronic exposure to MCLR damaged lung and liver in all doses, with a more important lung inflammation in TOX20 group.

Grape skin extract-derived polyphenols modify programming-induced renal endowment in prenatal protein-restricted male mouse offspring.

PURPOSE: Protein-restricted diet during pregnancy is related to oxidative stress and, as a consequence, damage to nephrogenesis. We investigated the effects of vinifera grape skin extract (ACH09)-derived polyphenols on preserving renal morphology of maternal protein-restricted 1-day-old offspring. METHODS: Female C57/Bl-6 mice were fed two different isocaloric diets: control diet (19.3 % protein) and low-protein diet (6 % protein) with access to water or to the extract dissolved in drinking water (19.3 % protein plus ACH09 200 mg kg(-1) day(-1) and 6 % protein plus ACH09 200 mg kg(-1) day(-1)) throughout gestation. Renal morphology-glomerular number N[glom]; renal maturity-vascular glomeruli and avascular glomeruli ratio (v-N[glom]/a-N[glom]); medullar and cortical volumes, as well as mean glomerular volume, were analyzed in male offspring. Hepatic superoxide dismutase and catalase (CAT) activities were evaluated, and renal lipid peroxidation levels were measured. RESULTS: Maternal protein restriction affected birth weight and naso-anal length in low-protein offspring compared to control and ACH09 restored both parameters. Protein restriction increased lipid peroxidation in kidney and liver and reduced CAT activity in low-protein group compared to control. Supplementation with ACH09 reduced the kidney oxidative damage and restored the antioxidant activity of CAT. ACH09 prevented glomerular loss and renal immaturity in the offspring. CONCLUSION: The treatment of low-protein-fed dams during pregnancy with ACH09 provides protection from early-life deleterious renal morphological changes. The protective effect of ACH09 may involve antioxidant action and vasodilator effect of the extract.

Antioxidant treatment normalizes mitochondrial energetics and myocardial insulin sensitivity independently of changes in systemic metabolic homeostasis in a mouse model of the metabolic syndrome.

Cardiac dysfunction in obesity is associated with mitochondrial dysfunction, oxidative stress and altered insulin sensitivity. Whether oxidative stress directly contributes to myocardial insulin resistance remains to be determined. This study tested the hypothesis that ROS scavenging will improve mitochondrial function and insulin sensitivity in the hearts of rodent models with varying degrees of insulin resistance and hyperglycemia. The catalytic antioxidant MnTBAP was administered to the uncoupling protein-diphtheria toxin A (UCP-DTA) mouse model of insulin resistance (IR) and obesity, at early and late time points in the evolution of IR, and to db/db mice with severe obesity and type-two diabetes. Mitochondrial function was measured in saponin-permeabilized cardiac fibers. Aconitase activity and hydrogen peroxide emission were measured in isolated mitochondria. Insulin-stimulated glucose oxidation, glycolysis and fatty acid oxidation rates were measured in isolated working hearts, and 2-deoxyglucose uptake was measured in isolated cardiomyocytes. Four weeks of MnTBAP attenuated glucose intolerance in 13-week-old UCP-DTA mice but was without effect in 24-week-old UCP-DTA mice and in db/db mice. Despite the absence of improvement in the systemic metabolic milieu, MnTBAP reversed cardiac mitochondrial oxidative stress and improved mitochondrial bioenergetics by increasing ATP generation and reducing mitochondrial uncoupling in all models. MnTBAP also improved myocardial insulin mediated glucose metabolism in 13 and 24-week-old UCP-DTA mice. Pharmacological ROS scavenging improves myocardial energy metabolism and insulin responsiveness in obesity and type 2 diabetes via direct effects that might be independent of changes in systemic metabolism.

Treatment with a SOD mimetic reduces visceral adiposity, adipocyte death, and adipose tissue inflammation in high fat-fed mice.

OBJECTIVE: Obesity is associated with enhanced reactive oxygen species (ROS) accumulation in adipose tissue. However, a causal role for ROS in adipose tissue expansion after high fat feeding is not established. The aim of this study is to investigate the effect of the cell permeable superoxide dismutase mimetic and peroxynitrite scavenger Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) on adipose tissue expansion and remodeling in response to high fat diet (HFD) in mice. DESIGN AND METHODS: Male C57BL/6j mice were fed normal chow or high fat diet (HFD) and treated with saline or MnTBAP for 5 weeks. The effects of MnTBAP on body weights, whole body energy expenditure, adipose tissue morphology, and gene expression were determined. RESULTS: MnTBAP attenuated weight gain and adiposity through a reduction in adipocyte hypertrophy, adipogenesis, and fatty acid uptake in epididymal (eWAT) but not in inguinal (iWAT) white adipose tissue. Furthermore, MnTBAP reduced adipocyte death and inflammation in eWAT and diminished circulating levels of free fatty acids and leptin. Despite these improvements, the development of systemic insulin resistance and diabetes after HFD was not prevented with MnTBAP treatment. CONCLUSIONS: Taken together, these data suggest a causal role for ROS in the development of diet-induced visceral adiposity but not in the development of insulin resistance and type 2 diabetes.

The influence of 5-lipoxygenase on cigarette smoke-induced emphysema in mice.

BACKGROUND: Pulmonary emphysema is characterized by the loss of lung architecture. Our hypothesis is that the inhibition of 5-lipoxygenase (5-LO) production may be an important strategy to reduce inflammation, oxidative stress, and metalloproteinases in lung tissue resulting from cigarette smoke (CS)-induced emphysema. METHODS: 5-LO knockout (129S2-Alox5(tm1Fun)/J) and wild-type (WT) mice (129S2/SvPas) were exposed to CS for 60days. Mice exposed to ambient air were used as Controls. Oxidative, inflammatory, and proteolytic markers were analyzed. RESULTS: The alveolar diameter was decreased in CS 5-LO(-/-) mice when compared with the WT CS group. The CS exposure resulted in less pronounced pulmonary inflammation in the CS 5-LO(-/-) group. The CS 5-LO(-/-) group showed leukotriene B4 values comparable to those of the Control group. The expression of MMP-9 was decreased in the CS 5-LO(-/-) group when compared with the CS WT group. The expression of superoxide dismutase, catalase, and glutathione peroxidase were decreased in the CS 5-LO(-/-) group when compared with the Control group. The protein expression of nuclear factor (erythroid-derived 2)-like 2 was reduced in the CS 5-LO(-/-) group when compared to the CS WT group. CONCLUSION: In conclusion, we show for the first time that 5-LO deficiency protects 129S2 mice against emphysema caused by CS. We suggest that the main mechanism of pathogenesis in this model involves the imbalance between proteases and antiproteases, particularly the association between MMP-9 and TIMP-1. General significance This study demonstrates the influence of 5-LO mediated oxidative stress, inflammation, and proteolytic markers in CS exposed mice.

Antioxidant action of propolis on mouse lungs exposed to short-term cigarette smoke.

Propolis is a natural product with antioxidant properties. In this study, we tested the efficacy of propolis against acute lung inflammation (ALI) caused by cigarette smoke (CS). C57BL6 male mice were exposed to CS and treated with propolis (200mg/kg orally, CS+P) or only with propolis (P). A Control group treated with propolis was sham-smoked (Control+P). We collected the lungs for histological and biochemical analyses. We observed an increase in alveolar macrophages and neutrophils in the CS group compared with the Control+P. These counts reduced in the CS+P group compared to the CS group. The treatment with propolis normalized all biochemical parameters in the CS+P group compared with the CS group, including nitrite, myeloperoxidase level, antioxidant enzyme activities (superoxide dismutase, catalase and glutathione peroxidase), reduced glutathione/oxidized glutathione ratio and malondialdehyde. Additionally, TNF-α expression reduced in the CS+P group when compared with the CS group. These data imply a potential antioxidant and anti-inflammatory role for propolis with regard to ALI caused by CS in mice.

Insulin receptor substrate signaling suppresses neonatal autophagy in the heart.

The induction of autophagy in the mammalian heart during the perinatal period is an essential adaptation required to survive early neonatal starvation; however, the mechanisms that mediate autophagy suppression once feeding is established are not known. Insulin signaling in the heart is transduced via insulin and IGF-1 receptors (IGF-1Rs). We disrupted insulin and IGF-1R signaling by generating mice with combined cardiomyocyte-specific deletion of Irs1 and Irs2. Here we show that loss of IRS signaling prevented the physiological suppression of autophagy that normally parallels the postnatal increase in circulating insulin. This resulted in unrestrained autophagy in cardiomyocytes, which contributed to myocyte loss, heart failure, and premature death. This process was ameliorated either by activation of mTOR with aa supplementation or by genetic suppression of autophagic activation. Loss of IRS1 and IRS2 signaling also increased apoptosis and precipitated mitochondrial dysfunction, which were not reduced when autophagic flux was normalized. Together, these data indicate that in addition to prosurvival signaling, insulin action in early life mediates the physiological postnatal suppression of autophagy, thereby linking nutrient sensing to postnatal cardiac development.

Insulin suppresses ischemic preconditioning-mediated cardioprotection through Akt-dependent mechanisms.

It is believed that the diabetic myocardium is refractory to cardioprotection by ischemic preconditioning (IPC) mainly because of impaired insulin signaling to phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB or Akt). However, human as well as animal studies have clearly showed that the hearts of type 2 diabetic humans and animals may exhibit increased signaling through PI3K-Akt but yet are resistant to cardioprotection by IPC or ischemic post-conditioning. Therefore, this study was designed to determine whether activation of insulin signaling prior to IPC is detrimental for cardioprotection and to assess the role of insulin receptors (IRs) and Akt in mediating this effect. Wild-type (WT) hearts, hearts lacking IRs or hearts expressing an active form of Akt (myrAkt1) were perfused ex vivo using a Langendorff preparation and were subjected to IPC (3cycles of 5min ischemia followed by 5min reflow before 30min no flow ischemia and then by 45min reperfusion) in the presence or absence of 1nmol/L insulin. Interestingly, whereas insulin was protective against I/R (30min no flow ischemia and 45min reperfusion), it completely abolished cardioprotection by IPC in WT hearts but not in mice lacking insulin receptors (IRs) in cardiomyocytes (CIRKO) or in all cardiac cells (TIRKO). The suppression of IPC-mediated cardioprotection was mediated through downstream signaling to Akt and Gsk3ß. In addition, transgenic induction of Akt in the heart was sufficient to abrogate IPC even when insulin was absent, further confirming the involvement of Akt in insulin's suppression of cardioprotection by IPC. These data provide evidence that excessive insulin signaling to Akt is detrimental for cardioprotection by IPC and could explain the failure of the diabetic myocardium to precondition.

Critical role for CCR2 and HMGB1 in induction of experimental endotoxic shock.

Our aim was to investigate CCR2 and HMGB1 involvement in a murine model of endotoxic shock. We used C57BL/6 CCR2 knockout (KO) mice and wild-type (WT) littermates to establish an optimal dose of LPS. CCR2 KO mice survived more frequently than WT mice after 80, 40 and 20 mg/kg of LPS i.p. Inflammation and redox markers were high in WT mice than in CCR2 KO mice. HMGB1 expression was reduced in CCR2 KO mice in parallel to ERK 1/2 activation. Therefore, we used glycyrrhizic acid (50 mg/kg), an HMGB1 inhibitor in WT mice injected with LPS, and mortality was fully abolished. Thus, drugs targeting CCR2 and HMGB1 could represent future resources for sepsis treatment.

Effects of exercise on monocrotaline-induced changes in right heart function and pulmonary artery remodeling in rats.

Pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT) is an experimental protocol of right heart failure. We analyzed the role of exercise training on the right ventricle structure and function, pulmonary artery remodeling, and GSK-3ß expression. Rats were divided among the following groups: sedentary control (SC), sedentary monocrotaline (SM), trained control (TC), and trained monocrotaline (TM). Rats underwent exercise training for a period of 5 weeks, with 3 weeks post-MCT injection. Rats in the SM and TM groups presented with an increase in right ventricle hypertrophy indexes and lung congestion. The right ventricular end diastolic pressure (RVEDP), right ventricular systolic pressure (RVSP), and its minimum and maximal pressure derivates were increased in the SM and TM groups. The right ventricle interstitial volume pulmonary artery thickness and p-GSK-3ß/GSK-3ß were increased in the MCT groups as compared with the control groups. The TM group had a reduction in interstitial volume, p-GSK-3ß/GSK-3ß ratio, pulmonary artery thickness, RVEDP, and an increase in intramyocardial vessels volume as compared with the SM group. The overall results have shown that the exercise protocol used promoted positive changes in right ventricle and pulmonary artery remodeling. These observations also suggest that structural remodeling may be influenced by signaling proteins, such as GSK-3ß.