Leida Maria Botion

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.

Inhibition of Proteasome Activity Blocks the Ability of TNFα to Down-Regulate Gi Proteins and Stimulate Lipolysis

Prolonged treatment of rat adipocytes with TNF alpha increases lipolysis through a mechanism mediated, in part, by down-regulation of inhibitory G proteins (G(i)). Separately, down-regulation of G(i) by prolonged treatment with an A(1)-adenosine receptor agonist, N(6)-phenylisopropyl adenosine (PIA) increases lipolysis. To investigate the role of proteolysis in TNF alpha and PIA-mediated G(i) down-regulation and stimulation of lipolysis, we used the protease inhibitors lactacystin (proteasome inhibitor) and calpeptin (calpain inhibitor). Rat adipocytes were preincubated for 1 h with lactacystin (10 microM) or calpeptin (50 microM), before 30-h treatment with either TNF alpha (50 ng/ml) or PIA (300 nM). We then measured lipolysis (glycerol release), abundance of alpha-subunits of G(i)1 and G(i)2 in plasma membranes (Western blotting) and protease activities (in specific fluorogenic assays). TNF alpha and PIA stimulated lipolysis approximately 2-fold and caused G(i) down-regulation. Although neither lactacystin nor calpeptin affected basal lipolysis, lactacystin completely inhibited both TNF alpha and PIA-stimulated lipolysis (the 50% inhibitory concentration was approximately 2 microM), whereas calpeptin had no effect. Similarly, lactacystin but not calpeptin blocked both PIA and TNF alpha-induced G(i) down-regulation. These findings provide further evidence that the chronic lipolytic effect of TNF alpha and PIA is secondary to G(i) down-regulation and suggest that the mechanism involves proteolytic degradation mediated through the proteasome pathway.

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.

Fenofibrate prevents orotic acid—Induced hepatic steatosis in rats

The experiments performed in this report were designed to investigate the mechanisms involved in the metabolic alterations associated with orotic acid-induced hepatic steatosis and the effect of fenofibrate, a stimulant of peroxisome proliferators-activated receptor alpha (PPARalpha), on these alterations. Male Wistar rats were divided into three experimental groups: 1) fed a balanced diet (C); 2) fed a balanced diet supplemented with 1% orotic acid (OA); 3) fed OA diet containing 100 mg.kg(-1) bw.day(-1) fenofibrate (OA+F), for 9 days. Administration of OA to rats induced significant increase in the hepatic total lipids content, marked microvesicular steatosis and decrease in plasma lipids concentrations compared to control group. Fenofibrate treatment prevented fatty liver induction, caused an additional reduction on plasma lipids concentrations and caused a 40% decrease in the lipogenic rate in adipose tissue. The results also showed a 40% increase in lipoprotein lipase (LPL) activity in adipose tissue from OA treated group and fenofibrate administration induced a 50% decrease in LPL activity. The liver mRNA expression of PPARalpha and ACO (acyl CoA oxidase) were 85% and 68% decreased in OA group when compared to control, respectively. Fenofibrate treatment increased the PPARalpha and ACO expressions whereas the CPT-1 (carnitine palmitoyl transferase-1) expression was not altered. Our results have shown that fenofibrate treatment decreases the hepatic lipid content induced by OA which is mediated by an important increase in fatty acid oxidation consequent to an increase in hepatic mRNA expression of PPARalpha and ACO.

Lack of platelet-activating factor receptor protects mice against diet-induced adipose inflammation and insulin-resistance despite fat pad expansion.

The role of platelet‐activating factor (PAF) on diet‐induced inflammatory and metabolic dysfunction is unknown. The effects of diet‐induced metabolic and inflammatory dysfunction in mice with deletion of the PAF receptor (PAFR−/−) were evaluated in this study.

Hancornia speciosa Gomes (Apocynaceae) as a potential anti-diabetic drug

ETHNOPHARMACOLOGICAL RELEVANCE The leaves of Hancornia speciosa Gomes are traditionally used to treat diabetes in Brazil. The aim of the study is to evaluate the potential anti-diabetic effect of Hancornia speciosa extract and derived fractions. MATERIALS AND METHODS The ethanolic extract from Hancornia speciosa leaves and chromatographic fractions thereof were evaluated on α-glucosidase assay, on hyperglycemic effect and glucose uptake. The chemical composition of the extract and its most active fraction was investigated by ESI-LC-MS. RESULTS The ethanolic extract and derived fractions inhibited α-glucosidase in vitro. However, only the crude extract and the dichloromethane fraction inhibited the hyperglycemic effect induced by starch or glucose. Both the extract and dichloromethane fraction were also able to increase glucose uptake in adipocytes. Bornesitol, quinic acid, and chorogenic acid were identified in the extract, along with flavonoid glycosides, whereas the dichloromethane fraction is majorly composed by esters of lupeol and/or α/β-amirin. CONCLUSIONS Hancornia speciosa has a potential anti-diabetic effect through a mechanism dependent on inhibition of α-glucosidase and increase on glucose uptake. These results give support to the use on traditional medicine of this medicinal plant.

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.

Improvement of the energy supply and contractile function in normal and ischemic rat hearts by dietary orotic acid

AIMS As cardiac performance is closely related to its energy supply, our study investigated the effect of the orotic acid cardioprotective agent on the pathways of energy supply, in both conditions of normal flow and ischemia. MAIN METHODS Male Wistar rats were fed during nine days with a balanced diet only or supplemented with 1% orotic acid. KEY FINDINGS Dietary administration of orotic acid increased the cardiac utilization of fatty acids, activity of the lipoprotein lipase, expression of the gene of peroxisome proliferator-activated receptor α and its target enzymes. In addition, orotic acid increased the myocardial uptake and incorporation of glucose, glycogen content and level of GLUT4, concentration of glycolytic metabolites and lactate production in both experimental conditions, baseline and after regional ischemia. SIGNIFICANCE Thus, in orotic acid hearts there was a simultaneous stimulus of fatty acid oxidation and glycolytic pathway, reflected in increased energetic content even in pre-ischemia. The analysis of the cardiac contractility index showed a positive inotropic effect of orotic acid due, at least in part, to the increased availability of energy. The result allows us to suggest that the metabolic changes induced by orotic acid result in appreciable alterations on myocardial contractile function.

Changes in peripheral energy metabolism during audiogenic seizures in rats.

Plasma glucose and lactate, hepatic glycogen and epididymal adipose tissue lipogenesis and lipolysis were studied in Wistar audiogenic rats (WARs), a genetic model of epilepsy, under three experimental conditions, i.e., before, 3 min after and 10 min after seizures induced by intense sound exposure. Plasma glucose increased 3 min after the seizure onset and rose to a peak after 10 min. Hepatic glycogen decreased significantly in susceptible audiogenic rats compared to nonepileptic controls, even before sound stimulation. A marked ( approximately 10-fold) rise was observed in plasma lactate levels 3 and 10 min after the seizures compared to the response of the control group. Lipogenic activity showed a marked decrease even after stimulation with 25 ng/ml insulin. Based on these results, WARs showed reduced isoproterenol-stimulated lipolysis compared to control animals, whereas basal levels only differed significantly at 10 min after seizure activity. In conclusion, it can be inferred from these results that (a) the increase in plasma glucose after stimulation might result from sequential interaction of autonomic activation at seizure onset; (b) excessive muscular activity was at least partially responsible for the steady rise in plasma lactate concentrations; (c) audiogenic seizures, which increase adrenergic activity, induce desensitization of the beta-adrenergic lipolytic pathway in epididymal adipose tissue; (d) genetic selection for audiogenic seizure susceptibility results in pronounced alterations at multiple levels of metabolic regulation.