Acute In Vivo Administration of Compound 21 Stimulates Akt and ERK1/2 Phosphorylation in Mouse Heart and Adipose Tissue.

The angiotensin II type 2 (AT2) receptor has a role in promoting insulin sensitivity. However, the mechanisms underlying the AT2 receptor-induced facilitation of insulin are still not completely understood. Therefore, we investigated whether acute in vivo administration of AT2 receptor agonist compound 21 (C21) could activate insulin signaling molecules in insulin-target tissues. We report that, in male C57BL/6 mice, an acute (5 min, 0.25 mg/kg; i.v.) injection of C21 induces the phosphorylation of Akt and ERK1/2 at activating residues (Ser473 and Thr202/Tyr204, respectively) in both epididymal white adipose tissue (WAT) and heart tissue. In WAT, the extent of phosphorylation (p) of Akt and ERK1/2 induced by C21 was approximately 65% of the level detected after a bolus injection of a dose of insulin known to induce maximal activation of the insulin receptor (IR). In the heart, C21 stimulated p-Akt to a lesser extent than in WAT and stimulated p-ERK1/2 to similar levels to those attained by insulin administration. C21 did not modify p-IR levels in either tissue. We conclude that in vivo injection of the AT2 receptor agonist C21 activates Akt and ERK1/2 through a mechanism that does not involve the IR, indicating the participation of these enzymes in AT2R-mediated signaling.

Chronic administration of the angiotensin type 2 receptor agonist C21 improves insulin sensitivity in C57BL/6 mice.

The renin-angiotensin system modulates insulin action. Angiotensin type 1 receptor exerts a deleterious effect, whereas the angiotensin type 2 receptor (AT2R) appears to have beneficial effects providing protection against insulin resistance and type 2 diabetes. To further explore the role of the AT2R on insulin action and glucose homeostasis, in this study we administered C57Bl/6 mice with the synthetic agonist of the AT2R C21 for 12 weeks (1 mg/kg per day; ip). Vehicle-treated animals were used as control. Metabolic parameters, glucose, and insulin tolerance, in vivo insulin signaling in main insulin-target tissues as well as adipose tissue levels of adiponectin, and TNF-α were assessed. C21-treated animals displayed decreased glycemia together with unaltered insulinemia, increased insulin sensitivity, and increased glucose tolerance compared to nontreated controls. This was accompanied by a significant decrease in adipocytes size in epididymal adipose tissue and significant increases in both adiponectin and UCP-1 expression in this tissue. C21-treated mice showed an increase in both basal Akt and ERK1/2 phosphorylation levels in the liver, and increased insulin-stimulated Akt activation in adipose tissue. This positive modulation of insulin action induced by C21 appeared not to involve the insulin receptor. In C21-treated mice, adipose tissue and skeletal muscle became unresponsive to insulin in terms of ERK1/2 phosphorylation levels. Present data show that chronic pharmacological activation of AT2R with C21 increases insulin sensitivity in mice and indicate that the AT2R has a physiological role in the conservation of insulin action.

Downregulation of the ACE2/Ang-(1-7)/Mas axis in transgenic mice overexpressing GH.

The renin-angiotensin system (RAS) plays a crucial role in the regulation of physiological homeostasis and diseases such as hypertension, coronary artery disease, and chronic renal failure. In this cascade, the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/AT1 receptor axis induces pathological effects, such as vasoconstriction, cell proliferation, and fibrosis, while the ACE2/Ang-(1-7)/Mas receptor axis is protective for end-organ damage. The altered function of the RAS could be a contributing factor to the cardiac and renal alterations induced by GH excess. To further explore this issue, we evaluated the consequences of chronic GH exposure on the in vivo levels of Ang II, Ang-(1-7), ACE, ACE2, and Mas receptor in the heart and the kidney of GH-transgenic mice (bovine GH (bGH) mice). At the age of 7-8 months, female bGH mice displayed increased systolic blood pressure (SBP), a high degree of both cardiac and renal fibrosis, as well as increased levels of markers of tubular and glomerular damage. Angiotensinogen abundance was increased in the liver and the heart of bGH mice, along with a concomitant increase in cardiac Ang II levels. Importantly, the levels of ACE2, Ang-(1-7), and Mas receptor were markedly decreased in both tissues. In addition, Ang-(1-7) administration reduced SBP to control values in GH-transgenic mice, indicating that the ACE2/Ang-(1-7)/Mas axis is involved in GH-mediated hypertension. The data indicate that the altered expression profile of the ACE2/Ang-(1-7)/Mas axis in the heart and the kidney of bGH mice could contribute to the increased incidence of hypertension, cardiovascular, and renal alterations observed in these animals.

Modulation of the action of insulin by angiotensin-(1-7).

The prevalence of Type 2 diabetes mellitus is predicted to increase dramatically over the coming years and the clinical implications and healthcare costs from this disease are overwhelming. In many cases, this pathological condition is linked to a cluster of metabolic disorders, such as obesity, systemic hypertension and dyslipidaemia, defined as the metabolic syndrome. Insulin resistance has been proposed as the key mediator of all of these features and contributes to the associated high cardiovascular morbidity and mortality. Although the molecular mechanisms behind insulin resistance are not completely understood, a negative cross-talk between AngII (angiotensin II) and the insulin signalling pathway has been the focus of great interest in the last decade. Indeed, substantial evidence has shown that anti-hypertensive drugs that block the RAS (renin-angiotensin system) may also act to prevent diabetes. Despite its long history, new components within the RAS continue to be discovered. Among them, Ang-(1-7) [angiotensin-(1-7)] has gained special attention as a counter-regulatory hormone opposing many of the AngII-related deleterious effects. Specifically, we and others have demonstrated that Ang-(1-7) improves the action of insulin and opposes the negative effect that AngII exerts at this level. In the present review, we provide evidence showing that insulin and Ang-(1-7) share a common intracellular signalling pathway. We also address the molecular mechanisms behind the beneficial effects of Ang-(1-7) on AngII-mediated insulin resistance. Finally, we discuss potential therapeutic approaches leading to modulation of the ACE2 (angiotensin-converting enzyme 2)/Ang-(1-7)/Mas receptor axis as a very attractive strategy in the therapy of the metabolic syndrome and diabetes-associated diseases.

Upregulation of the angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas receptor axis in the heart and the kidney of growth hormone receptor knock-out mice.

OBJECTIVE: Growth hormone (GH) resistance leads to enhanced insulin sensitivity, decreased systolic blood pressure and increased lifespan. The aim of this study was to determine if there is a shift in the balance of the renin-angiotensin system (RAS) towards the ACE2/Ang-(1-7)/Mas receptor axis in the heart and the kidney of a model of GH resistance and retarded aging, the GH receptor knockout (GHR-/-) mouse. DESIGN: RAS components were evaluated in the heart and the kidney of GHR-/- and control mice by immunohistochemistry and Western blotting (n=12 for both groups). RESULTS: The immunostaining of Ang-(1-7) was increased in both the heart and the kidney of GHR-/- mice. These changes were concomitant with an increased immunostaining of the Mas receptor and ACE2 in both tissues. The immunostaining of AT1 receptor was reduced in heart and kidney of GHR-/- mice while that of AT2 receptor was increased in the heart and unaltered in the kidney. Ang II, ACE and angiotensinogen levels remained unaltered in the heart and the kidney of GH resistant mice. These results were confirmed by Western blotting and correlated with a significant increase in the abundance of the endothelial nitric oxide synthase in both tissues. CONCLUSIONS: The shift within the RAS towards an exacerbation of the ACE2/Ang-(1-7)/Mas receptor axis observed in GHR-/- mice could be related to a protective role in cardiac and renal function; and thus, possibly contribute to the decreased incidence of cardiovascular diseases displayed by this animal model of longevity.

The Mas receptor mediates modulation of insulin signaling by angiotensin-(1-7).

Angiotensin (Ang)-(1-7) stimulates proteins belonging to the insulin signaling pathway and ameliorates the Ang II negative effects at this level. However, up to date, receptors involved and mechanisms behind these observations remain unknown. Accordingly, in the present study, we explored the in vivo effects of antagonism of the Ang-(1-7) specific Mas receptor on insulin signal transduction in rat insulin-target tissues. We evaluated the acute modulation of insulin-stimulated phosphorylation of Akt, GSK-3ß (Glycogen synthase kinase-3ß) and AS160 (Akt substrate of 160kDa) by Ang-(1-7) and/or Ang II in the presence and absence of the selective Mas receptor antagonist A-779 in insulin-target tissues of normal rats. Also using A-779, we determined whether the Mas receptor mediates the improvement of insulin sensitivity exerted by chronic Ang-(1-7) treatment in fructose-fed rats (FFR), a model of insulin resistance, dyslipidemia and mild hypertension. The two major findings of the present work are as follows; 1) Ang-(1-7) attenuates acute Ang II-mediated inhibition of insulin signaling components in normal rats via a Mas receptor-dependent mechanism; and 2). The Mas receptor appears to be involved in beneficial effects of Ang-(1-7) on the phosphorylation of crucial insulin signaling mediators (Akt, GSK-3ß and AS160), in liver, skeletal muscle and adipose tissue of FFR. These results shed light into the mechanism by which Ang-(1-7) exerts its positive physiological modulation of insulin actions in classical metabolic tissues and reinforces the central role of Akt in these effects.

Angiotensin-(1-7) attenuates diabetic nephropathy in Zucker diabetic fatty rats.

Angiotensin (ANG)-(1-7) is known to attenuate diabetic nephropathy; however, its role in the modulation of renal inflammation and oxidative stress in type 2 diabetes is poorly understood. Thus in the present study we evaluated the renal effects of a chronic ANG-(1-7) treatment in Zucker diabetic fatty rats (ZDF), an animal model of type 2 diabetes and nephropathy. Sixteen-week-old male ZDF and their respective controls [lean Zucker rats (LZR)] were used for this study. The protocol involved three groups: 1) LZR + saline, 2) ZDF + saline, and 3) ZDF + ANG-(1-7). For 2 wk, animals were implanted with subcutaneous osmotic pumps that delivered either saline or ANG-(1-7) (100 ng·kg(-1)·min(-1)) (n = 4). Renal fibrosis and tissue parameters of oxidative stress were determined. Also, renal levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), ED-1, hypoxia-inducible factor-1α (HIF-1α), and neutrophil gelatinase-associated lipocalin (NGAL) were determined by immunohistochemistry and immunoblotting. ANG-(1-7) induced a reduction in triglyceridemia, proteinuria, and systolic blood pressure (SBP) together with a restoration of creatinine clearance in ZDF. Additionally, ANG-(1-7) reduced renal fibrosis, decreased thiobarbituric acid-reactive substances, and restored the activity of both renal superoxide dismutase and catalase in ZDF. This attenuation of renal oxidative stress proceeded with decreased renal immunostaining of IL-6, TNF-α, ED-1, HIF-1α, and NGAL to values similar to those displayed by LZR. Angiotensin-converting enzyme type 2 (ACE2) and ANG II levels remained unchanged after treatment with ANG-(1-7). Chronic ANG-(1-7) treatment exerts a renoprotective effect in ZDF associated with a reduction of SBP, oxidative stress, and inflammatory markers. Thus ANG-(1-7) emerges as a novel target for treatment of diabetic nephropathy.

Novel roles of galectin-1 in hepatocellular carcinoma cell adhesion, polarization, and in vivo tumor growth.

UNLABELLED: Galectin-1 (Gal-1), a widely expressed ß-galactoside-binding protein, exerts pleiotropic biological functions. Gal-1 is up-regulated in hepatocarcinoma cells, although its role in liver pathophysiology remains uncertain. We investigated the effects of Gal-1 on HepG2 hepatocellular carcinoma (HCC) cell adhesion and polarization. Soluble and immobilized recombinant Gal-1 (rGal-1) promoted HepG2 cell adhesion to uncoated plates and also increased adhesion to laminin. Antibody-mediated blockade experiments revealed the involvement of different integrins as critical mediators of these biological effects. In addition, exposure to rGal-1 markedly accelerated the development of apical bile canaliculi as shown by TRITC-phalloidin labeling and immunostaining for multidrug resistance associated-protein 2 (MRP2). Notably, rGal-1 did not interfere with multidrug resistance protein 1/P-glycoprotein or MRP2 apical localization, neither with transfer nor secretion of 5-chloromethylfluorescein diacetate through MRP2. Stimulation of cell adhesion and polarization by rGal-1 was abrogated in the presence of thiodigalactoside, a galectin-specific sugar, suggesting the involvement of protein-carbohydrate interactions in these effects. Additionally, Gal-1 effects were abrogated in the presence of wortmmanin, PD98059 or H89, suggesting involvement of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase and cyclic adenosine monophosphate-dependent protein kinase signaling pathways in these functions. Finally, expression levels of this endogenous lectin correlated with HCC cell adhesion and polarization and up-regulation of Gal-1-favored growth of hepatocarcinoma in vivo. CONCLUSION: Our results provide the first evidence of a role of Gal-1 in modulating HCC cell adhesion, polarization, and in vivo tumor growth, with critical implications in liver pathophysiology.

Angiotensin-(1-7) reduces proteinuria and diminishes structural damage in renal tissue of stroke-prone spontaneously hypertensive rats.

Angiotensin (ANG)-(1-7) constitutes an important functional end-product of the renin-angiotensin-aldosterone system that acts to balance the physiological actions of ANG II. In the kidney, ANG-(1-7) exerts beneficial effects by inhibiting growth-promoting pathways and reducing proteinuria. We examined whether a 2-wk treatment with a daily dose of ANG-(1-7) (0.6 mg·kg(-1)·day(-1)) exerts renoprotective effects in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). Body weight, glycemia, triglyceridemia, cholesterolemia, as well as plasma levels of Na+ and K+ were determined both at the beginning and at the end of the treatment. Also, the weekly evolution of arterial blood pressure, proteinuria, and creatinine clearance was evaluated. Renal fibrosis was determined by Masson's trichrome staining. Interleukin (IL)-6, tumor necrosis factor (TNF)-α, and nuclear factor-κB (NF-κB) levels were determined by immunohistochemistry and confirmed by Western blotting analysis. The levels of glomerular nephrin were assessed by immunofluorescence. Chronic administration of ANG-(1-7) normalized arterial pressure, reduced glycemia and triglyceridemia, improved proteinuria, and ameliorated structural alterations in the kidney of SHRSP as shown by a restoration of glomerular nephrin levels as detected by immunofluorescence. These results were accompanied with a decrease in both the immunostaining and abundance of IL-6, TNF-α, and NF-κB. In this context, the current study provides strong evidence for a protective role of ANG-(1-7) in the kidney.

Angiotensin-(1-7) stimulates the phosphorylation of Akt in rat extracardiac tissues in vivo via receptor Mas.

The in vivo effect of angiotensin (ANG)-(1-7) on the activation of insulin signaling transduction in rat extracardiac tissues is unknown. Thus, in the present study, we evaluated the ability of ANG-(1-7) to stimulate the phosphorylation of Akt, a main mediator of insulin action in rat extracardiac tissues (adipose tissue, liver and skeletal muscle). We proved that ANG-(1-7) induces the phosphorylation of Akt at both threonine 308 and serine 473 in all tissues analyzed. Selective antagonism of the Mas receptor with A779 blocked the ANG-(1-7)-induced Akt phosphorylation in extracardiac tissues. Reinforcing this evidence, we determined that ANG-(1-7) induces the in vivo activation of the downstream target of Akt, glycogen synthase kinase-3beta in liver and skeletal muscle. Moreover, in every tissue analyzed, the presence of the Mas receptor was detected by immunohistochemical analysis. Based on the current results, we postulate that ANG-(1-7) could be a positive physiological contributor to the actions of insulin in extracardiac tissues. Therefore, our findings extend the possibilities for new approaches in the study of ANG-(1-7)/Mas receptor axis and show the therapeutic potential of ANG-(1-7) in the treatment of metabolic disorders such as insulin resistance as well as other disorders associated with diminished Akt activity.

Angiotensin-(1-7) improves cardiac remodeling and inhibits growth-promoting pathways in the heart of fructose-fed rats.

The present study examined whether chronic treatment with angiotensin (ANG)-(1-7) reduces cardiac remodeling and inhibits growth-promoting signaling pathways in the heart of fructose-fed rats (FFR), an animal model of insulin resistance. Sprague-Dawley rats were fed either normal rat chow (control) or the same diet plus 10% fructose in drinking water. For the last 2 wk of a 6-wk period of the corresponding diet, control and FFR were implanted with osmotic pumps that delivered ANG-(1-7) (100 ng.kg(-1).min(-1)). A subgroup of each group of animals (control or FFR) underwent a sham surgery. We determined heart weight, myocyte diameter, interstitial fibrosis, and perivascular collagen type III deposition as well as the phosphorylation degree of ERK1/2, JNK1/2, and p38MAPK. FFR showed a mild hypertension that was significantly reduced after ANG-(1-7) treatment. Also, FFR displayed higher ANG II circulating and local levels in the heart that remained unaltered after chronic ANG-(1-7) infusion. An increased heart-to-body weight ratio, myocyte diameter, as well as left ventricular fibrosis and perivascular collagen type III deposition were detected in the heart of FFR. Interestingly, significant improvements in these cardiac alterations were obtained after ANG-(1-7) treatment. Finally, FFR that received ANG-(1-7) chronically displayed significantly lower phosphorylation levels of ERK1/2, JNK1/2, and p38MAPK. The beneficial effects obtained by ANG-(1-7) were associated with normal values of Src-homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity in the heart. In conclusion, chronic ANG-(1-7) treatment ameliorated cardiac hypertrophy and fibrosis and attenuated the growth-promoting pathways in the heart. These findings show an important protective role of ANG-(1-7) in the heart of insulin-resistant rats.

Ames dwarf (Prop1(df)/Prop1(df)) mice display increased sensitivity of the major GH-signaling pathways in liver and skeletal muscle.

CONTEXT: Growth hormone (GH) is an anabolic hormone that regulates growth and metabolism. Ames dwarf mice are natural mutants for Prop1, with impaired development of anterior pituitary and undetectable levels of circulating GH, prolactin and TSH. They constitute an endocrine model of life-long GH-deficiency. The main signaling cascades activated by GH binding to its receptor are the JAK2/STATs, PI-3K/Akt and the MAPK Erk1/2 pathways. OBJECTIVES: We have previously reported that GH-induced STAT5 activation was higher in Ames dwarf mice liver compared to non-dwarf controls. The aim of this study was to evaluate the principal components of the main GH-signaling pathways under GH-deficiency in liver and skeletal muscle, another GH-target tissue. METHODS: Ames dwarf mice and their non-dwarf siblings were assessed. Animals were injected i.p. with GH or saline 15min before tissue removal. Protein content and phosphorylation of signaling mediators were determined by immunoblotting of tissue solubilizates. RESULTS: GH was able to induce STAT5 and STAT3 tyrosine phosphorylation in both liver and muscle, but the response was higher for Ames dwarf mice than for non-dwarf controls. When Erk1/2 activation was assessed in liver, only dwarf mice showed GH-induced phosphorylation, while in muscle no response to the hormone was found in either genotype. GH-induced Akt phosphorylation at Ser473 in liver was only detected in dwarf mice. In skeletal muscle, both normal and dwarf mice responded to a GH stimulus, although dwarf mice presented higher GH activation levels. The phosphorylation of GSK-3, a substrate of Akt, increased upon hormone stimulation only in dwarf mice in both tissues. In contrast, no differences in the phosphorylation of mTOR, another substrate of Akt, were observed after GH stimulus, either in normal or dwarf mice in liver, while we were unable to determine mTOR in muscle. Protein content of GH-receptor and of the signaling mediators studied did not vary between normal and dwarf animals in the assessed tissues. CONCLUSION: These results show that several components of the main GH-signaling pathways exhibit enhanced sensitivity to the hormone in liver and muscle of Ames dwarf mice.

Long-term treatment with an angiotensin II receptor blocker decreases adipocyte size and improves insulin signaling in obese Zucker rats.

BACKGROUND: Angiotensin II (Ang II) has been shown to contribute to the pathogenesis of hypertension and insulin resistance. In addition, administration of selective Ang II type-1 receptor blockers has been shown to improve insulin sensitivity. However, only a few studies have addressed the molecular mechanisms involved in this association. OBJECTIVE AND DESIGN: The current study was undertaken to determine whether an Ang II receptor blocker (irbesartan) is effective in improving insulin resistance in adipose tissue from obese Zucker rats, a model of metabolic syndrome. METHODS: Ten-week-old male obese Zucker rats (fa/fa) were treated daily with either vehicle or 50 mg/kg irbesartan for 6 months, and their age-matched lean (+/?) (lean Zucker rats) was used as a control. We determined systolic blood pressure (SBP), together with plasma levels of insulin, triglycerides, cholesterol and glucose. In addition, we evaluated insulin signaling through the insulin receptor/insulin receptor substrate-1/phosphatidylinositol 3 kinase/Akt/glucose transporter 4 pathway as well as the inflammatory status of adipose tissue. RESULTS: Obese Zucker rats displayed hyperglycemia, hypertriglyceridemia, hyperinsulinemia and hypercholesterolemia and increased SBP together with decreased activation of insulin signaling through the insulin receptor/insulin receptor substrate-1/phosphatidylinositol 3 kinase/Akt pathway in adipose tissue as well as increased adipocytes size, macrophage infiltration and augmented levels of inflammatory mediators such tumor necrosis factor-alpha, monocyte chemoattractant protein-1 and Ang II. Chronic irbesartan treatment resulted in an improvement of all alterations. CONCLUSION: The present study provides substantial information that demonstrates that long-term selective Ang II blockade ameliorates insulin resistance in adipose tissue from a model of metabolic syndrome via a mechanism that could involve the modulation of insulin signaling.

Chronic infusion of angiotensin-(1-7) improves insulin resistance and hypertension induced by a high-fructose diet in rats.

The current study was undertaken to determine whether Ang-(1-7) is effective in improving metabolic parameters in fructose-fed rats (FFR), a model of metabolic syndrome. Six-week-old male Sprague-Dawley rats were fed either normal rat chow (control) or the same diet plus 10% fructose in drinking water. For the last 2 wk of a 6-wk period of either diet, control and FFR were implanted with subcutaneous osmotic pumps that delivered Ang-(1-7) (100 ng.kg(-1).min(-1)). A subgroup of each group of animals (control or FFR) underwent a sham surgery. We measured systolic blood pressure (SBP) together with plasma levels of insulin, triglycerides, and glucose. A glucose tolerance test (GTT) was performed, with plasma insulin levels determined before and 15 and 120 min after glucose administration. In addition, we evaluated insulin signaling through the IR/IRS-1/PI3K/Akt pathway as well as the phosphorylation levels of IRS-1 at inhibitory site Ser(307) in skeletal muscle and adipose tissue. FFR displayed hypertriglyceridemia, hyperinsulinemia, increased SBP, and an exaggerated release of insulin during a GTT, together with decreased activation of insulin signaling through the IR/IRS-1/PI3K/Akt pathway in skeletal muscle, liver, and adipose tissue, as well as increased levels of IRS-1 phospho-Ser(307) in skeletal muscle and adipose tissue, alterations that correlated with increased activation of the kinases mTOR and JNK. Chronic Ang-(1-7) treatment resulted in normalization of all alterations. These results show that Ang-(1-7) ameliorates insulin resistance in a model of metabolic syndrome via a mechanism that could involve the modulation of insulin signaling.

ACE inhibition and AT1 receptor blockade prevent fatty liver and fibrosis in obese Zucker rats.

OBJECTIVE: Non-alcoholic steatohepatitis (NASH), which is a common liver disease in industrialized countries, is associated with obesity, hypertension, and type-2 diabetes (metabolic syndrome). Since angiotensin II (ANG II) has been suggested to play an important role in liver inflammation and fibrosis, the purpose of this study was to investigate whether therapy against renin-angiotensin system (RAS) may provide some beneficial effect in liver of an animal model of metabolic syndrome. METHODS AND PROCEDURES: For 6 months, obese Zucker rats (OZRs) were treated as follows: OZR-group, OZR + Perindopril (P) group, OZR + Irbesartan (IRB) group, OZR + Amlodipine (AML) group, and lean Zucker rats (LZRs) group as a control. Livers were evaluated by immunohistochemistry techniques using corresponding antibodies. RESULTS: All treated groups showed a similar reduction in blood pressure compared to untreated OZR. Therapy either with IRB or P improves insulin sensitivity and reduces hepatic enzyme level with respect to untreated OZR. Conversely, AML failed to modify both parameters. Untreated OZR displayed higher hepatic ANG II levels and steatosis together with a marked increase in tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-beta1) level compared to LZR. Following RAS inhibition either by P or IRB, a significant reduction (P < 0.01) in the immunostaining of TNF-alpha, IL-6 and TGF-beta1 compared to untreated OZR was observed. DISCUSSION: These results indicate that ANG II expression is increased in the liver of these animals with steatohepatitis. Furthermore, RAS control by either angiotensin-converting enzyme inhibition or AT1 receptor blockade seems to provide a beneficial modulation concerning the inflammatory response to liver injury in this model. Consequently, blockade of RAS could be a new approach to prevent or to treat patients with NASH.

Angiotensin-(1 7) stimulates the phosphorylation of JAK2, IRS-1 and Akt in rat heart in vivo: role of the AT1 and Mas receptors.

Angiotensin (ANG) II exerts a negative modulation on insulin signal transduction that might be involved in the pathogenesis of hypertension and insulin resistance. ANG-(1-7), an endogenous heptapeptide hormone formed by cleavage of ANG I and ANG II, counteracts many actions of ANG II. In the current study, we have explored the role of ANG-(1-7) in the signaling crosstalk that exists between ANG II and insulin. We demonstrated that ANG-(1-7) stimulates the phosphorylation of Janus kinase 2 (JAK2) and insulin receptor substrate (IRS)-1 in rat heart in vivo. This stimulating effect was blocked by administration of the selective ANG type 1 (AT(1)) receptor blocker losartan. In contrast to ANG II, ANG-(1-7) stimulated cardiac Akt phosphorylation, and this stimulation was blunted in presence of the receptor Mas antagonist A-779 or the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. The specific JAK2 inhibitor AG-490 blocked ANG-(1-7)-induced JAK2 and IRS-1 phosphorylation but had no effect on ANG-(1-7)-induced phosphorylation of Akt, indicating that activation of cardiac Akt by ANG-(1-7) appears not to involve the recruitment of JAK2 but proceeds through the receptor Mas and involves PI3K. Acute in vivo insulin-induced cardiac Akt phosphorylation was inhibited by ANG II. Interestingly, coadministration of insulin with an equimolar mixture of ANG II and ANG-(1-7) reverted this inhibitory effect. On the basis of our present results, we postulate that ANG-(1-7) could be a positive physiological contributor to the actions of insulin in heart and that the balance between ANG II and ANG-(1-7) could be relevant for the association among insulin resistance, hypertension, and cardiovascular disease.

Behaviour of the anti-oxidant defence system and heme oxygenase-1 protein expression in fructose-hypertensive rats.

1. Addition of fructose to a rat diet for various periods of time leads to hypertension, hyperinsulinaemia and dyslipidaemia and provides a model for testing oxidative stress parameters in the animals. 2. In the present study, oxidative stress generation, the soluble and enzymatic defence system and heme oxygenase-1 (HO-1) protein expression were investigated in the heart, liver and kidney of rats fed fructose for a period of 1 or 8 months. 3. Compared with the control group, fructose-hypertensive rats showed increased in lipid peroxidation only in the heart after both 1 and 8 months of fructose treatment. Changes in the behaviour of the soluble and enzymatic defence system and HO-1 protein expression were different depending on the organ. Increased or unaltered activities of anti-oxidant enzymes were found in the liver and kidney, respectively. Induction of HO-1 prevented the generation of oxidative stress in the liver, where the activity of anti-oxidant defence enzymes was not reduced. Increased expression of HO-1 protein was not able to prevent the generation of oxidative stress in the heart, where fructose treatment diminished the activity of anti-oxidant enzymes. 4. The results of the present study demonstrate that upregulation of HO-1 may prevent the generation of oxidative stress only when the anti-oxidant defence system is still operative.

Irbesartan restores the in-vivo insulin signaling pathway leading to Akt activation in obese Zucker rats.

BACKGROUND: Angiotensin II (AII) has been shown to contribute to the pathogenesis of hypertension and insulin resistance. In addition, the administration of selective AII type 1 receptor blockers has been shown to improve insulin sensitivity. However, only a few studies have addressed the molecular mechanisms involved in this association. Furthermore, in a previous study we illustrated that obese Zucker rats (OZR) present increased serine 994 (Ser994) phosphorylation of hepatic insulin receptor, and this event seems to be implicated in the regulation of the intrinsic IRK in this model of insulin resistance. OBJECTIVE AND DESIGN: We examined the effects of chronic treatment with irbesartan (50 mg/kg a day for 6 months) on the hepatic insulin signaling system of OZR. METHODS: The extent of phosphorylation of several components of the insulin signaling system was assessed by immunoprecipitation, followed by immunoblotting with phosphospecific antibodies. In addition, liver AII levels and fat deposits were determined by immunohistochemistry and Oil red O, respectively. RESULTS: OZR displayed a marked attenuation in the in-vivo phosphorylation of several components of the insulin signaling pathways in the liver, together with significantly higher hepatic AII levels and hepatic steatosis when compared with lean Zucker rats. We found that in the livers of OZR long-term administration of irbesartan is associated with: (i) increased insulin-stimulated insulin receptor tyrosine phosphorylation; (ii) decreased insulin receptor Ser994 phosphorylation; (iii) augmented insulin receptor substrate (IRS) 1 and 2 abundance and tyrosine phosphorylation; (iv) augmented association between IRS and the p85 regulatory subunit of phosphatidylinositol 3-kinase; (v) increased insulin-induced Akt phosphorylation; and (vi) decreased hepatic steatosis. CONCLUSION: The present study provides substantial information that demonstrates that long-term selective AII blockade by irbesartan improves insulin signaling and is associated with decreased insulin receptor Ser994 phosphorylation in the liver of a representative animal model of the human metabolic syndrome.

Use of a purified Trypanosoma cruzi antigen and CpG oligodeoxynucleotides for immunoprotection against a lethal challenge with trypomastigotes.

The crucial role played by Ag163B6/cruzipain, the major cystein proteinase of Trypanosoma cruzi, in the process of parasite internalization into mammalian cells and IgG hydrolysis, signals this antigen as a potential target for raising a protective immune response against Chagas' disease. On the other hand, synthetic oligodeoxynucleotides containing CpG-motifs (CpG-ODN) are capable of driving immunity toward a Th1 bias. Considering the importance of Th1 mechanisms in resistance against this intracellular parasite, we analyzed the ability of Ag163B6/cruzipain plus CpG-ODN to induce immunoprotection against a lethal challenge with trypomastigotes. Mice were immunized with Ag163B6+CpG-ODN showing high specific antibody titers, mostly IgG2a. Spleen cells from these mice strongly proliferated and presented significant increase of IL-2 and IFN-gamma concentrations in their supernatant upon antigen stimulation. Trypomastigote challenge rendered elevated parasitemia and mortality in all control groups, meanwhile Ag163B6+CpG-ODN mice displayed the lowest level of blood parasites and 100% survival to acute infection. Besides, we demonstrated that other parasite antigens introduced into mice when challenged, and consequently never seen before by the immune system, also elicited a Th1 immune response. Taken together, these results plus others provide the basis for the design of a multicomponent anti-T. cruzi vaccine which may ultimately be used not only to protect humans at risk of infection, but also may alleviate or prevent the pathogenic responses characteristic of chronic Chagas' disease by reducing or perhaps eliminating tissue parasites from infected patients.