Érica Guilhen Mario

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.

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.

Angiotensin-(1-7) Mas-receptor deficiency decreases peroxisome proliferator-activated receptor gamma expression in adipocytes

The renin-angiotensin system is an important link between metabolic syndrome and cardiovascular diseases. Besides angiotensin II, other angiotensin peptides such as angiotensin-(1-7), have important biological activities. It has been demonstrated that angiotensin-(1-7), acting through the G protein-coupled receptor encoded by the Mas protooncogene have important actions on the cardiovascular system. However, the role of angiotensin-(1-7)-Mas axis in lipidic profile is not well established. In the present study, the adipocyte metabolism was investigated in wild type and FVB/N Mas-deficient male mice. The gene expression of peroxisome proliferator-activated receptor gamma, acetyl-CoA carboxylase and the amount of fatty acid synthase protein were reduced in the Mas-knockout mice. Serum nonesterified fatty acids of Mas-knockout showed a 50% increase in relation to wild type group. Basal and isoproterenol-stimulated lipolysis was similar between the groups, however, a significant decrease of the glycerol release (lipolytic index) in response to insulin was observed in wild type animals, while no effect of the insulin action was observed in a Mas-knockout group. The data suggest that the lack of angiotensin-(1-7) action through Mas receptor alters the response of adipocytes to insulin action. These effects might be related to decreased expression of PPARγ.

Increased expression of oxidative enzymes in adipose tissue following PPARα-activation.

OBJECTIVE Evaluate the effect of fenofibrate treatment on the expression of PPARα and oxidative enzymes in adipose tissue. MATERIALS/METHODS Wistar male rats were fed a balanced diet supplemented with 100mg.Kg-1 bw.day-1 fenofibrate (Sigma) during nine days. Plasma glucose, free fatty acids (FFA) leptin and insulin were determined. PPARα, ACO and CPT-1 mRNA expression and amount of PPARα and PPARγ protein were assessed in epididymal adipose tissue. Oral glucose tolerance test was evaluated into overnight fasted rats. Glucose uptake was measured in adipocytes isolated from epididymal fat pads in the presence or absence of insulin (25ng/mL). RESULTS Fenofibrate treatment increased PPARα and PPARγ protein abundance in adipose tissue. In addition to it well- known effect on oxidative enzymes in liver, fenofibrate treatment also induces a high expression of Acyl CoA Oxidase (ACO) and Carnitine palmitoyltransferase 1 (CPT-1) in adipose tissue. Furthermore, we have shown that the fenofibrate treatment improves the glucose tolerance and enhance the glucose uptake by adipocytes. CONCLUSION Altogether, the data suggest that fenofibrate have a direct effect in adipose tissue contributing to the low adiposity and improvement of glucose homeostasis.

Long-term effects of angiotensin-(1–7) on lipid metabolism in the adipose tissue and liver

HIGHLIGHTSThe overexpression of Ang‐(1–7) inhibits adipose tissue lipogenesis and LPL activity.Increased Ang‐(1–7) plasma level regulates the adipose tissue LPL activity independently of the PPAR&ggr; expression.Increased Ang‐(1–7) plasma level has a stimulatory effect on cytosolic lipase activity and inhibits FAS and FATP expression in the liver, suggesting a decrease in de novo fatty acid synthesis and fatty acid uptake.The data clearly show that Ang‐(1–7) overexpression regulates lipid metabolism in the adipose tissue and liver. ABSTRACT The angiotensin (Ang) converting enzyme 2/Ang‐(1–7)/Mas axis has been described to have a beneficial role on metabolic disorders. In the present study, the use of a transgenic rat model that chronically overexpresses Ang‐(1–7) enabled us to investigate the chronic effects of this peptide on lipid accumulation in the liver and adipose tissue. The transgenic group showed a marked tendency toward increased expression of peroxisome proliferator‐activated receptor‐&ggr; (PPAR&ggr;) and decreased lipoprotein lipase (LPL) expression and activity in epididymal adipose tissue. We also showed that Mas receptor‐knockout mice had decreased PPAR&ggr; expression in adipose tissue, accompanied by an increase in LPL activity. These results confirm the regulation of adipose tissue LPL activity by Ang‐(1–7) and suggest that this occurs independent of PPAR&ggr; expression. The reduced adiposity index of transgenic rats, due to the effect of Ang‐(1–7), was accompanied by a decrease in lipogenesis. These findings suggest a direct effect of Ang‐(1–7) on lipogenesis, independent of the stimulatory effect of insulin. Furthermore, the decreased concentration of triacylglycerol in the liver of transgenic rats may result from increased activity of cytosolic lipases and decreased fatty acid uptake from the adipose tissue, determined from fatty acid‐binding protein expression, and hepatic de novo fatty acid synthesis, evaluated by fatty acid synthase expression. The data clearly show that Ang‐(1–7) regulates lipid metabolism in the adipose tissue and liver.

Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies

Mas receptor (MasR) is a G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang (1–7) protective axis of renin–angiotensin system. Because the role of this receptor is not definitively clarified, determination of MasR tissue distribution and expression levels constitutes a critical knowledge to fully understanding its function. Commercially available antibodies have been widely employed for MasR protein localization and quantification, but they have not been adequately validated. In this study, we carried on an exhaustive evaluation of four commercial MasR antibodies, following previously established criteria. Western Blotting (WB) and immunohistochemistry studies starting from hearts and kidneys from wild type (WT) mice revealed that antibodies raised against different MasR domains yielded different patterns of reactivity. Furthermore, staining patterns appeared identical in samples from MasR knockout (MasR-KO) mice. We verified by polymerase chain reaction analysis that the MasR-KO mice used were truly deficient in this receptor as MAS transcripts were undetectable in either heart or kidney from this animal model. In addition, we evaluated the ability of the antibodies to detect the human c-myc-tagged MasR overexpressed in human embryonic kidney cells. Three antibodies were capable of detecting the MasR either by WB or by immunofluorescence, reproducing the patterns obtained with an anti c-myc antibody. In conclusion, although three of the selected antibodies were able to detect MasR protein at high expression levels observed in a transfected cell line, they failed to detect this receptor in mice tissues at physiological expression levels. As a consequence, validated antibodies that can recognize and detect the MasR at physiological levels are still lacking.

Carbohydrate-enriched diet impairs cardiac performance by decreasing the utilization of fatty acid and glucose

Aims: We hypothesized that a high-carbohydrate diet affects the cardiac performance by interfering in the metabolic steps involved in energy transfer in this organ. To verify this, we investigated the myocardial utilization of different substrates and contractile function in rats fed a high-carbohydrate diet, under normal flow and ischemia. Methods and Results: Male Wistar rats were fed over 9 days with standard (39.5% carbohydrate, 8% fiber) or high-carbohydrate diet (58% carbohydrate) and, afterwards, their cardiac function was examined using isolated heart preparations. The high-carbohydrate diet decreased the activity of the lipoprotein lipase, utilization of fatty acids, expression of the gene of peroxisome proliferator-activated receptor α and its target enzymes. In addition, decreased GLUT4 mass, glucose uptake, glycogen content and glycolytic intermediates were also observed. High-carbohydrate hearts displayed weaker activation of the glycolytic pathway during ischemia, according to minor production of lactate, in relation to control hearts. The functional impairment caused by high-carbohydrate diet shown by the decrease in the ventricular systolic strength, +dT/dt and −dT/dt was, at least in part, due to the low availability of adenosine triphosphate (ATP). Conclusion: Our data suggest that a high-carbohydrate diet can damage myocardial contractile function by decreasing the cardiac utilization of glucose and fatty acids and, consequently, the ATP pool.