Sérgio Henrique Sousa Santos

Mas Deficiency in FVB/N Mice Produces Marked Changes in Lipid and Glycemic Metabolism

OBJECTIVE— Metabolic syndrome is characterized by the variable coexistence of obesity, hyperinsulinemia, insulin resistance, dyslipidemia, and hypertension. It is well known that angiotensin (Ang) II is importantly involved in the metabolic syndrome. However, the role of the vasodilator Ang-(1-7)/Mas axis is not known. The aim of this study was to evaluate the effect of genetic deletion of the G protein–coupled receptor, Mas, in the lipidic and glycemic metabolism in FVB/N mice. RESEARCH DESIGN AND METHODS— Plasma lipid, insulin, and cytokine concentrations were measured in FVB/N Mas-deficient and wild-type mice. A glucose tolerance test was performed by intraperitoneally injecting d-glucose into overnight-fasted mice. An insulin sensitivity test was performed by intraperitoneal injection of insulin. Uptake of 2-deoxy-[3H]glucose by adipocytes was used to determine the rate of glucose transport; adipose tissue GLUT4 was quantified by Western blot. Gene expression of transforming growth factor (TGF)-β, type 1 Ang II receptor, and angiotensinogen (AGT) were measured by real-time PCR. RESULTS— Despite normal body weight, Mas-knockout (Mas-KO) mice presented dyslipidemia, increased levels of insulin and leptin, and an ∼50% increase in abdominal fat mass. In addition, Mas gene–deleted mice presented glucose intolerance and reduced insulin sensitivity as well as a decrease in insulin-stimulated glucose uptake by adipocytes and decreased GLUT4 in adipose tissue. Mas−/− presented increased muscle triglycerides, while liver triglyceride levels were normal. Expression of TGF-β and AGT genes was higher in Mas-KO animals in comparison with controls. CONCLUSIONS— These results show that Mas deficiency in FVB/N mice leads to dramatic changes in glucose and lipid metabolisms, inducing a metabolic syndrome–like state.

Genetic deletion of the angiotensin-(1–7) receptor Mas leads to glomerular hyperfiltration and microalbuminuria

Angiotensin-(1-7), an active fragment of both angiotensins I and II, generally opposes the vascular and proliferative actions of angiotensin II. Here we evaluated effects of the angiotensin-(1-7) receptor Mas on renal physiology and morphology using Mas-knockout mice. Compared to the wild-type animals, Mas knockout mice had significant reductions in urine volume and fractional sodium excretion without any significant change in free-water clearance. A significantly higher inulin clearance and microalbuminuria concomitant with a reduced renal blood flow suggest that glomerular hyperfiltration occurs in the knockout mice. Histological analysis found reduced glomerular tuft diameter and increased expression of collagen IV and fibronectin in the both the mesangium and interstitium, along with increased collagen III in the interstitium. These fibrogenic changes and the renal dysfunction of the knockout mice were associated with an upregulation of angiotensin II AT1 receptor and transforming growth factor-beta mRNA. Our study suggests that Mas acts as a critical regulator of renal fibrogenesis by controlling effects transduced through angiotensin II AT1 receptors in the kidney.

Improved Lipid and Glucose Metabolism in Transgenic Rats With Increased Circulating Angiotensin-(1-7)

Objective—Obesity and diabetes remain among the worlds most pervasive health problems. Although the importance of angiotensin II for metabolic regulation is well documented, the role of the angiotensin-(1-7)/Mas axis in this process is poorly understood. The aim of this study was to evaluate the effect of increased angiotensin-(1-7) plasma levels in lipid and glucose metabolism using transgenic rats that express an angiotensin-(1-7)-releasing fusion protein, TGR(A1-7)3292 (TGR). Methods and Results—The increased angiotensin-(1-7) levels in TGR induced enhanced glucose tolerance, insulin sensitivity, and insulin-stimulated glucose uptake. In addition, TGR presented decreased triglycerides and cholesterol levels, as well as a significant decrease in abdominal fat mass, despite normal food intake. These alterations were accompanied by a marked decrease of angiotensinogen expression and increased Akt in adipose tissue. Furthermore, augmented plasma levels and expression in adipose tissue was observed for adiponectin. Accordingly, angiotensin-(1-7) stimulation increased adiponectin production by primary adipocyte culture, which was blocked by the Mas antagonist A779. Circulating insulin and muscle glycogen content were not altered in TGR. Conclusion—These results show that increased circulating angiotensin-(1-7) levels lead to prominent changes in glucose and lipid metabolism.

Resveratrol attenuates hepatic steatosis in high-fat fed mice by decreasing lipogenesis and inflammation

OBJECTIVE Resveratrol (RSV) is the most studied natural compound that activates sirtuins, which produce beneficial metabolic effects on lipid and glucose metabolism. The aim of the present study was to investigate the role of resveratrol in preventing nonalcoholic fatty liver disease (NAFLD) and expression of liver inflammatory markers in mice treated with a high-fat diet. METHODS AND PROCEDURES Eighteen male mice were divided into three groups and fed for 60 d with a standard diet (ST), high-fat diet (HFD), or high-fat diet plus resveratrol (HFD + RSV, 30 mg/kg/d). Body weight, food intake, and serum total cholesterol, triacylglycerol, insulin, alanine transaminase (ALT), and aspartate aminotransferase (AST) were evaluated. Liver histology was analyzed. Expression of ACC, PPAR-γ, ChREBP, SREBP-1 c, CPT-1, tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), NF-κB, interleukin 1 β (IL-1 β), and SIRT1 were evaluated by quantitative real-time reverse transcriptase PCR (qRT-PCR). RESULTS The major finding of the present study was that RSV reduced body fat, total cholesterol, triacylglycerol, transaminases, and insulin plasma level. These results were accompanied with a significant reduction in TNF-α, IL-6, and NF-κB mRNA expression in the liver. Analyses of liver adipogenesis related genes indicated that ACC, PPAR-γ, and SREBP-1 mRNA expression were significantly suppressed in HFD + RSV mice. In addition, we observed increased expression of SIRT1 in the HFD + RSV group. CONCLUSIONS We observed that treatment with resveratrol improved lipid metabolism, and decreased NAFLD and pro-inflammatory profile in liver of mice with obesity-inducible diets. These data suggest an important clinical application of RSV in preventing liver diseases.

Adipokines, diabetes and atherosclerosis: an inflammatory association.

Cardiovascular diseases can be considered the most important cause of death in diabetic population and diabetes can in turn increase the risk of cardiovascular events. Inflammation process is currently recognized as responsible for the development and maintenance of diverse chronic diseases, including diabetes and atherosclerosis. Considering that adipose tissue is an important source of adipokines, which may present anti and proinflammatory effects, the aim of this review is to explore the role of the main adipokines in the pathophysiology of diabetes and atherosclerosis, highlighting the therapeutic options that could arise from the manipulation of these signaling pathways both in humans and in translational models.

Selective increase of angiotensin(1–7) and its receptor in hearts of spontaneously hypertensive rats subjected to physical training

In the present study we investigated the effects of physical training on plasma and cardiac angiotensin(1–7) [Ang(1–7)] levels. In addition, possible changes in expression of the Ang(1–7) Mas receptor in the heart were also evaluated. Normotensive Wistar rats and spontaneously hypertensive rats (SHR) were subjected to an 8 week period of 5% overload swimming training. Blood pressure was determined by a tail‐cuff system. Heart and left ventricle weights and cardiomyocyte diameter were analysed to evaluate cardiac hypertrophy. Radioimmunoassay was used to measure angiotensin levels. Expression of Mas was determined by semi‐quantitative polymerase chain reaction, immunofluorescence and Western blotting. Physical training induced cardiac hypertrophy in Wistar rats and SHR. A significant decrease of plasma angiotensin II (Ang II) levels in both strains was also observed. Strikingly, trained SHR, but not trained Wistar rats, showed a twofold increase in left ventricular Ang(1–7) levels. No significant changes were observed in plasma Ang(1–7) and left ventricular Ang II concentrations in either strain. Furthermore, Mas mRNA and protein expression in left ventricle were substantially increased in trained SHR. The physical training protocol used did not change blood pressure in either strain. These results suggest that the beneficial effects induced by swimming training in hypertensive rats might include an augmentation of Ang(1–7) and its receptor in the heart.

Oral Angiotensin-(1-7) prevented obesity and hepatic inflammation by inhibition of resistin/TLR4/MAPK/NF-κB in rats fed with high-fat diet.

Obesity is characterized by a pro-inflammatory state commonly associated with type 2 diabetes and fat-liver disease. In the last few years, different studies pointed out the role of Angiotensin (Ang)-(1-7) in the metabolic regulation. The aim of the present study was to evaluate the effect of oral-administration of Ang-(1-7) in metabolism and inflammatory state of high-fat feed rats. Twenty-four male Sprague Dawley rats were randomized into three groups: High Fat Diet (HFD); Standard Diet (ST); High Fat Diet+Angiotensin-(1-7) [HFD+Ang-(1-7)]. Glycemic profile was evaluated by glucose tolerance and insulin sensitivity tests, plasmatic glucose and insulin. Cholesterol, HDL and triglycerides analyses presented lipidic profile. RT-PCR evaluated mRNA expression to ACE, ACE2, resistin, TLR4, IL-6, TNF-α and NF-κB genes. The main results showed that oral Ang-(1-7) decreased body weight and abdominal fat-mass. In addition, HFD+Ang-(1-7) treated rats presented enhanced glucose tolerance, insulin-sensitivity and decreased plasma-insulin levels, as well as a significant decrease in circulating lipid levels. These alterations were accompanied by a marked decreased expression of resistin, TLR4, ACE and increased ACE2 expression in liver. Furthermore, Ang-(1-7) decreases phosphorylation of MAPK and increases NF-κB expression. These alterations diminished expression of interleukin-6 and TNF-α, ameliorate inflammatory state in liver. In summary, the present study showed that oral-treatment with Ang-(1-7) in high-fat feed rats improved metabolism down-regulating resistin/TLR4/NF-κB-pathway.

Oral Formulation of Angiotensin-(1–7) Improves Lipid Metabolism and Prevents High-Fat Diet–Induced Hepatic Steatosis and Inflammation in Mice

Angiotensin (Ang)-(1–7) has been described as an important tool on treating and preventing metabolic disorders. In this study, we aimed to evaluate the effect of an oral formulation of Ang-(1–7) included in hydroxypropyl&bgr;-cyclodextrin (HP&bgr;CD/Ang-[1–7]) on hepatic function, steatosis, and on liver inflammatory markers expression in mice treated with a high-fat diet. Male FVB/N mice were divided into 4 groups and fed for 60 days, with each group receiving 1 of the following diets: standard diet+HP&bgr;CD, standard diet+Ang-(1–7)/HP&bgr;CD, high-fat diet+HP&bgr;CD, or high-fat diet+Ang-[1–7]/HP&bgr;CD. Body weight, food intake, and blood parameters, such as total cholesterol, triglyceride, alaninetransaminases, and aspartate transaminases, were evaluated. Immunohistochemical analyses were performed for inflammatory markers tumor necrosis factor-&agr; and interleukin-6. Expression of angiotensin converting enzyme, angiotensin-converting enzyme-2, interleukin-1&bgr;, tumor necrosis factor-&agr;, interleukin-6, transforming growth factor-&bgr;, acetyl-CoA carboxylase, carbohydrate-responsive element–binding protein, peroxisome proliferator–activated receptor-&ggr;, and sterol regulatory element–binding proteins-1c was evaluated by quantitative real-time polymerase chain reaction. The major findings of our study included reduced liver fat mass and weight, decreased plasma total cholesterol, triglyceride, and alaninetransaminase enzyme levels in the oral Ang-(1–7)–treated groups compared with the control groups. These results were accompanied by a significant reduction in tumor necrosis factor-&agr; and interleukin-6 mRNA expression in the liver. Analyses of liver adipogenesis-related genes by quantitative real-time polymerase chain reaction showed that acetyl-CoA carboxylase, peroxisome proliferator–activated receptor-&ggr;, and sterol regulatory element–binding proteins-1c mRNA expression were significantly suppressed. In conclusion, we observed that treatment with Ang-(1–7) improved metabolism and decreased proinflammatory profile and fat deposition in liver of mice.

Oral Administration of an Angiotensin-Converting Enzyme 2 Activator Ameliorates Diabetes-Induced Cardiac Dysfunction

We evaluated the hypothesis that activation of endogenous angiotensin-converting enzyme (ACE) 2 would improve cardiac dysfunction induced by diabetes. Ten days after diabetes induction (streptozotocin, 50 mg/kg, i.v.), male Wistar rats were treated with the ACE2 activator 1-[[2-(dimethylamino)ethyl]amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl)sulfonyl]oxy]-9H-xanthen-9-one (XNT, 1 mg/kg/day, gavage) or saline (control) for 30 days. Echocardiography was performed to analyze the cardiac function and kinetic fluorogenic assays were used to determine cardiac ACE and ACE2 activities. Cardiac ACE2, ACE, Mas receptor, AT(1) receptor, AT(2) receptor and collagen types I and III mRNA and ACE2, ACE, Mas, AT(1) receptor, AT(2) receptor, ERK1/2, Akt, AMPK-α and AMPK-β(1) protein were measured by qRT-PCR and western blotting techniques, respectively. Histological sections of hearts were analyzed to evaluate the presence of hypertrophy and fibrosis. Diabetic animals presented hyperglycemia and diastolic dysfunction along with cardiac hypertrophy and fibrosis. XNT treatment prevented further increase in glycemia and improved the cardiac function, as well as the hypertrophy and fibrosis. These effects were associated with increases in cardiac ACE2/ACE ratios (activity: ~26%; mRNA: ~113%; and protein: ~188%) and with a decrease in AT(1) receptor expression. Additionally, XNT inhibited ERK1/2 phosphorylation and prevented changes in AMPK-α and AMPK-β(1) expressions. XNT treatment did not induce any significant change in AT(2) receptor and Akt expression. These results indicate that activation of intrinsic cardiac ACE2 by oral XNT treatment protects the heart against diabetes-induced dysfunction through mechanisms involving ACE, ACE2, ERK1/2, AMPK-α and AMPK-β(1) modulations.

Brain Activation of SIRT1: Role in Neuropathology

Sirtuins (SIRTs) are a family of regulatory proteins of genetic information with a high degree of conservation among species. The SIRTs are heavily involved in several physiological functions including control of gene expression, metabolism, and aging. SIRT1 has been the most studied sirtuin and plays important role in the prevention and progression of neurodegenerative diseases acting in different pathways of proteins involved in brain function. SIRT1 activation regulates important genes that also exert neuroprotective actions such as p53, nuclear factor kappa B, peroxisome proliferator-activated receptor-gamma (PPARγ), PPARγ coactivator-1α, liver X receptor, and forkhead box O. It is well established in literature that growing population aging, oxidative stress, inflammation, and genetic factors are important conditions to development of neurodegenerative disorders. However, the exact pathophysiological mechanisms leading to these diseases remain obscure. The sirtuins show strong potential to become valuable predictive and prognostic markers for diseases and as therapeutic targets for the treatment of a variety of neurodegenerative disorders. In this context, the aim of the current review is to present an actual view of the potential role of SIRT1 in modulating the interaction between target genes and neurodegenerative diseases on the brain.