Short-Term Modulation of Extracellular Signal-Regulated Kinase 1/2 and Stress-Activated Protein Kinase/c-Jun NH2-Terminal Kinase in Pancreatic Islets by Glucose and Palmitate Possible Involvement of Ceramide

Objectives: The effect of glucose and palmitate on the phosphorylation of proteins associated with cell growth and survival (extracellular signal-regulated kinase 1/2 [ERK1/2] and stress-activated protein kinase/c-Jun NH2-terminal kinase [SAPK/JNK]) and on the expression of immediate early genes was investigated. Methods: Groups of freshly isolated rat pancreatic islets were incubated in 10-mmol/L glucose with palmitate, LY294002, or fumonisin B1 for the measurement of the phosphorylation and the content of ERK1/2, JNK/SAPK, and v-akt murine thymoma viral oncongene (AKT) (serine 473) by immunoblotting. The expressions of the immediate early genes, c-fos and c-jun, were evaluated by reverse transcription-polymerase chain reaction. Results: Glucose at 10 mmol/L induced ERK1/2 and AKT phosphorylations and decreased SAPK/JNK phosphorylation. Palmitate (0.1 mmol/L) abolished the glucose effect on ERK1/2, AKT, and SAPK/JNK phosphorylations. LY294002 caused a similar effect. The inhibitory effect of palmitate on glucose-induced ERK1/2 and AKT phosphorylation changes was not observed in the presence of fumonisin B1. Glucose increased c-fos and decreased c-jun expressions. Palmitate and LY294002 abolished these latter glucose effects. The presence of fumonisin B1 abolished the effect induced by palmitate on c-jun expression. Conclusions: Our results suggest that short-term changes of mitogen-activated protein kinase and AKT signaling pathways and c-fos and c-jun expressions caused by glucose are abolished by palmitate through phosphatidylinositol 3-kinase inhibition via ceramide synthesis.

Deletion of Tumor Necrosis Factor-alpha Receptor 1 (TNFR1) Protects against Diet-induced Obesity by Means of Increased Thermogenesis

In diet-induced obesity, hypothalamic and systemic inflammatory factors trigger intracellular mechanisms that lead to resistance to the main adipostatic hormones, leptin and insulin. Tumor necrosis factor-alpha (TNF-alpha) is one of the main inflammatory factors produced during this process and its mechanistic role as an inducer of leptin and insulin resistance has been widely investigated. Most of TNF-alpha inflammatory signals are delivered by TNF receptor 1 (R1); however, the role played by this receptor in the context of obesity-associated inflammation is not completely known. Here, we show that TNFR1 knock-out (TNFR1 KO) mice are protected from diet-induced obesity due to increased thermogenesis. Under standard rodent chow or a high-fat diet, TNFR1 KO gain significantly less body mass despite increased caloric intake. Visceral adiposity and mean adipocyte diameter are reduced and blood concentrations of insulin and leptin are lower. Protection from hypothalamic leptin resistance is evidenced by increased leptin-induced suppression of food intake and preserved activation of leptin signal transduction through JAK2, STAT3, and FOXO1. Under the high-fat diet, TNFR1 KO mice present a significantly increased expression of the thermogenesis-related neurotransmitter, TRH. Further evidence of increased thermogenesis includes increased O(2) consumption in respirometry measurements, increased expressions of UCP1 and UCP3 in brown adipose tissue and skeletal muscle, respectively, and increased O(2) consumption by isolated skeletal muscle fiber mitochondria. This demonstrates that TNF-alpha signaling through TNFR1 is an important mechanism involved in obesity-associated defective thermogenesis.

Hepatic denervation impairs the assembly and secretion of VLDL-TAG

VLDL secretion is a regulated process that depends on the availability of lipids, apoB and MTP. Our aim was to investigate the effect of liver denervation upon the secretion of VLDL and the expression of proteins involved in this process. Denervation was achieved by applying a 85% phenol solution onto the portal tract, while control animals were treated with 9% NaCl. VLDL secretion was evaluated by the Tyloxapol method. The hepatic concentration of TAG and cholesterol, and the plasma concentration of TAG, cholesterol, VLDL-TAG, VLDL-cholesterol and HDL-cholesterol were measured, as well as mRNA expression of proteins involved in the process of VLDL assembly. Hepatic acinar distribution of MTP and apoB was evaluated by immunohistochemistry. Denervation increased plasma concentration of cholesterol (125.3 +/- 10.1 vs. 67.1 +/- 4.9 mg dL(-1)) and VLDL-cholesterol (61.6 +/- 5.6 vs. 29.4 +/- 3.3 mg dL(-1)), but HDL-cholesterol was unchanged (45.5 +/- 6.1 vs. 36.9 +/- 3.9 mg dL(-1)). Secretion of VLDL-TAG (47.5 +/- 23.8 vs. 148.5 +/- 27.4 mg dL h(-1)) and mRNA expression of CPT I and apoB were reduced (p < 0.01) in the denervated animals. MTP and apoB acinar distribution was not altered in the denervated animals, but the intensity of the reaction was reduced in relation to controls. Copyright (C) 2008 John Wiley & Sons, Ltd.

Modulation of Bone Morphogenetic Protein-9 Expression and Processing by Insulin, Glucose, and Glucocorticoids: Possible Candidate for Hepatic Insulin-Sensitizing Substance

Bone morphogenetic protein 9 (BMP-9), a member of the TGF-beta superfamily predominantly expressed in nonparenchymal liver cells, has been demonstrated to improve glucose homeostasis in diabetic mice. Along with this therapeutic effect, BMP-9 was proposed as a candidate for the hepatic insulin-sensitizing substance ( HISS). Whether BMP-9 plays a physiological role in glucose homeostasis is still unknown. In the present study, we show that BMP-9 expression and processing is severely reduced in the liver of insulin-resistant rats. BMP-9 expression and processing was directly stimulated by in situ exposition of the liver to the combination of glucose and insulin and oral glucose in overnight fasted rats. Additionally, prolonged fasting ( 72 h) abrogated refeeding-induced BMP-9 expression and processing. Previous exposition to dexamethasone, a known inductor of insulin resistance, reduced BMP-9 processing stimulated by the combination of insulin and glucose. Finally, we show that neutralization of BMP-9 with an anti-BMP-9 antibody induces glucose intolerance and insulin resistance in 12-h fasted rats. Collectively, the present results demonstrate that BMP-9 plays an important role in the control of glucose homeostasis of the normal rat. Additionally, BMP-9 is expressed and processed in an HISS-like fashion, which is impaired in the presence of insulin resistance. BMP-9 regulation according to the feeding status and the presence of diabetogenic factors reinforces the hypothesis that BMP-9 might exert the role of HISS in glucose homeostasis physiology. ( Endocrinology 149: 6326-6335, 2008)

Redox regulation of the mitochondrial K(ATP) channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK(ATP) regulation. mK(ATP) activity was measured by both osmotic swelling and Tl(+) flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK(ATP) in cardioprotection by nitroxyl. Key findings were as follows: (i) mK(ATP) was activated by O(2)(center dot-) and H(2)O(2) but not other peroxides. (ii) mK(ATP) was inhibited by NADPH. (iii) mK(ATP) was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK(ATP)-dependent manner. Overall, these results suggest that the mK(ATP) channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK(ATP) may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection. (C) 2010 Elsevier B.V. All rights reserved.

trans, trans-2,4-decadienal induces mitochondrial dysfunction and oxidative stress

Lipid peroxidation produces a large number of reactive aldehydes as secondary products. We have previously shown that the reaction of cytochrome c with trans,trans-2, 4-decadienal (DDE), an aldehyde generated as a product of lipid peroxidation in cell membranes, results in the formation of adducts. Mass spectrometry analysis indicated that His-33, Lys-39, Lys-72 and Lys-100 in cytochrome c were modified by DDE. In the present work, we investigated the effect of DDE on isolated rat liver mitochondria. DDE (162 mu M) treatment increases the rate of mitochondrial oxygen consumption. Extensive mitochondrial swelling upon treatment with DDE (900 nM-162 mu M) was observed by light scattering and transmission electron microscopy experiments. DDE-induced loss of inner mitochondrial membrane potentials, monitored by safranin O fluorescence, was also observed. Furthermore, DDE-treated mitochondria showed an increase in lipid peroxidation, as monitored by MDA formation. These results suggest that reactive aldehydes promote mitochondrial dysfunction.

Effect of unsaturated fatty acids on myocardial performance, metabolism and morphology

Diets rich in saturated fatty acids are one of the most important causes of atherosclerosis in men, and have been replaced with diets rich in unsaturated fatty acids (UFA) for the prevention of this disorder. However, the effect of UFA on myocardial performance, metabolism and morphology has not been completely characterized. The objective of the present investigation was to evaluate the effects of a UFA-rich diet on cardiac muscle function, oxidative stress, and morphology. Sixty-day-old male Wistar rats were fed a control (N = 8) or a UFA-rich diet (N = 8) for 60 days. Myocardial performance was studied in isolated papillary muscle by isometric and isotonic contractions under basal conditions after calcium chloride (5.2 mM) and ss-adrenergic stimulation with 1.0 mu M isoproterenol. Fragments of the left ventricle free wall were used to study oxidative stress and were analyzed by light microscopy, and the myocardial ultrastructure was examined in left ventricle papillary muscle. After 60 days the UFA-rich diet did not change myocardial function. However, it caused high lipid hydroperoxide (176 +/- 5 vs 158 +/- 5, P < 0.0005) and low catalase (7 +/- 1 vs 9 +/- 1, P < 0.005) and superoxide-dismutase (18 +/- 2 vs 27 +/- 5, P < 0.005) levels, and discrete morphological changes in UFA-rich diet hearts such as lipid deposits and mitochondrial membrane alterations compared to control rats. These data show that a UFA-rich diet caused myocardial oxidative stress and mild structural alterations, but did not change mechanical function.

Myocardial fibrosis rather than hypertrophy induces diastolic dysfunction in renovascular hypertensive rats

The aim of the present study was to evaluate the-effect of interstitial fibrosis alone or associated with hypertrophy. on diastolic myocardial function in renovascular hypertensive rats. Myocardial function was evaluated in isolated papillary muscle from renovascular hypertensive Wistar rats (RHT, n = 14), renovascular hypertensive rats treated with the angiotensin converting enzyme inhibitor (ACEI) ramipril, 20 mg.kg(-1).day(-1) (RHT RAM, n = 14), and age-matched unoperated and untreated Wistar rats (CONT, n = 12). The ACEI treatment for 3 weeks allowed the regression of myocyte mass and the maintenance of interstitial fibrosis. Myocardial passive stiffness was analyzed by the resting tension - length relationship. The myocardial fibrosis was evaluated by measuring myocardial hydroxyproline (Hyp) concentration and by histological studies of the myocardium stained with hematoxylin and eosin or picrosirius red. Left ventricular weight was significantly higher in RHT (0.97 +/- 0.12 g) compared with CONT (0.66 +/- 0.06 g) and RHT RAM (0.69 +/- 0.14 g). The Hyp levels were 2.9 +/- 0.4, 3.4 +/- 0.3, and 3.8 +/- 0.4 mu g/mg of dry tissue for the CONT, RHT, and RHT RAM, respectively. Perivascular and interstitial fibrosis were observed in RHT and RHT RAM groups. There were lymphomononuclear inflammatory exudate and edema around arteries, involving adjacent myocytes in the RHT group. There was an increased passive stiffness in RHT and RHT RAM groups compared with the CONT group. In conclusion, our results indicate that the Impaired diastolic function in the renovascular hypertensive rats is related to interstitial fibrosis rather than to myocardial hypertrophy.

Estresse crônico melhora a função miocárdica sem alterar a atividade do canal-L para Ca+2 em ratos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ventricular Remodeling Induced by Tissue Vitamin A Deficiency in Rats

Background/Aims: Experimental studies suggest that vitamin A plays a role in regulating cardiac structure and function. We tested the hypothesis that cardiac vitamin A deficiency is associated with adverse myocardial remodeling in young adult rats. Methods: Two groups of young female rats, control (C - n = 29) and tissue vitamin A deficient (RVA - n = 31), were subjected to transthoracic echocardiography exam, isolated rat heart study and biochemical study. Results: The RVA rats showed a reduced total vitamin A concentration in both the liver and heart [vitamin A in heart, mu mol/kg (C = 0.95 +/- 0.44 and RVA = 0.24 +/- 0.16, p = 0.01)] with the same serum retinol levels (C = 0.73 +/- 0.29 mu mol/L e RVA = 0.62 +/- 0.17 mu mol/L, p = 0.34). The RVA rats showed higher left ventricular diameters and reduced systolic function. The RVA rats also demonstrated increased lipid hydroperoxide/total antioxidant capacity ratio and cardiac levels of IFN-gamma and TNF-alpha but not of metalloproteinase (MMP)-2 and -9 activity. on the other hand, the RVA rats had decreased levels of beta-hydroxyacylcoenzyme A dehydrogenase and lactate dehydrogenase. Conclusions: Tissue vitamin A deficiency stimulated cardiac remodeling and ventricular dysfunction. Additionally, the data support the involvement of oxidative stress, energy metabolism, and cytokine production in this remodeling process. Copyright (C) 2010 S. Karger AG, Basel

Diabetes evolution in rats after neonatal treatment with alloxan

Physical exercises have been recommended in the prevention of non-insulin dependent diabetes mellitus (NIDDM), but the mechanisms involved in this intervention are not yet fully understood. Experimental models offer the opportunity for the study of this matter. The present study was designed to analyze the diabetes evolution in rats submitted to neonatal treatment with alloxan with the objective of verifying the suitability of the model to future studies with exercises. For this, newly born rats (6 days old) received intraperitoneal alloxan (A = 200 mg/kg of body weight). Rats injected with vehicle (citrate buffer) were used as controls (C). The fasting blood glucose level (mg/dL) was higher in the alloxan group at the day 28 (C=47.25 +/- 5.08; A=54.51 +/- 7.03) but not at the 60 day of age (C=69.18 +/- 8.31; A=66.81 +/- 6.08). The alloxan group presented higher blood glucose level during glucose tolerance test (GTT) (mg/dL. 120 min) in relation to the control group both at day 28 (C=16908.9 +/- 1078.8; A=21737,7 +/- 1106.4) and at day 60 (C=11463.45 +/- 655.30; A=15282.21 +/- 1221.84). Insulinaemia during GTT (ng/mL.120 min) was lower at day 28 (C=158.67 +/- 33.34; A=123.90 +/- 19.80), but presented no difference at day 60 (C=118.83 +/- 26.02; A=97.8 +/- 10.88). At day 60, the glycogen concentration in the soleus muscle (mg/100mg) was lower in the alloxan group (0.3 +/- 0.13) in relation to the control group (0.5 +/- 0.07). No difference was observed between groups in relation to (mu mol/g.h): Glucose Uptake (C = 5.8 +/- 0.63; A = 5.2 +/- 0.73); Glucose Oxidation (C= 4.3 +/- 1.13; A= 3.9 +/- 0.44); Glycogen Synthesis (C= 0.8 +/- 0.18; A= 0.7 +/- 0.18) and Lactate Production (C= 3.8 +/- 0.8; A= 3.8 0.7) by the isolated soleus muscle. The glucose-stimulated insulin secretion (16.7mM) by the isolated islets (ng/5 islets. h) of the alloxan group was lower (14.3 +/- 4.7) than the control group (32.0 +/- 7.9). Thus, we may conclude that this neonatal diabetes induction model gathers interesting characteristics and may be useful for further studies on the role of the exercise in the diabetes mellitus appearance.

Efeitos do treinamento de corrida em diferentes intensidades sobre a capacidade aeróbia e produção de lactato pelo músculo de ratos Wistar

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metabolic syndrome markers in wistar rats of different ages

In recent decades, metabolic syndrome has become a public health problem throughout the world. Longitudinal studies in humans have several limitations due to the invasive nature of certain analyses and the size and randomness of the study populations. Thus, animal models that are able to mimic human physiological responses could aid in investigating metabolic disease. Thus, the present study was designed to analyze metabolic syndrome markers in albino Wistar rats (Rattus norvegicus) of different ages. The following parameters were assessed at two (young), four (adult), six (adult), and twelve (mature) months of age: glucose tolerance (glucose tolerance test); insulin sensitivity (insulin tolerance test); fasting serum glucose, triglycerides, total cholesterol, HDL cholestero, and LDL cholesterol concentrations; glucose uptake in isolated soleus muscle; and total lipid concentration in subcutaneous, mesenteric, and retroperitoneal adipose tissue. We found that aging triggered signs of metabolic syndrome in Wistar rats. For example, mature rats showed a significant increase in body weight that was associated. In addition, mature rats showed an increase in the serum concentration of triglycerides, total cholesterol, and LDL cholesterol, which is characteristic of dyslipidemia. There was also an increase in serum glucose compared with the younger groups of animals. Therefore, aging Wistar rats appear to be an interesting model to study the changes related to metabolic syndrome.

Iron chelating-mediated antioxidant activity of Plectranthus barbatus extract on mitochondria

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Myocyte necrosis is the basis for fibrosis inrenovascular hypertensive rats

The pathogenesis of fibrosis and the functional features of pressure overload myocardial hypertrophy are still controversial. The objectives of the present study were to evaluate the function and morphology of the hypertrophied myocardium in renovascular hypertensive (RHT) rats. Male Wistar rats were sacrificed at week 4 (RHT4) and 8 (RHT8) after unilateral renal ischemia (Goldblatt II hypertension model). Normotensive rats were used as controls. Myocardial function was analyzed in isolated papillary muscle preparations, morphological features were defined by light microscopy, and myocardial hydroxyproline concentration (HOP) was determined by spectrophotometry. Renal artery clipping resulted in elevated systolic arterial pressure (RHT4: 178 ± 19 mmHg and RHT8: 194 ± 24 mmHg, P<0.05 vs control: 123 ± 7 mmHg). Myocardial hypertrophy was observed in both renovascular hypertensive groups. The myocardial HOP concentration was increased in the RHT8 group (control: 2.93 ± 0.38 µg/mg; RHT4: 3.02 ± 0.40 µg/mg; RHT8: 3.44 ± 0.45 µg/mg of dry tissue, P<0.05 vs control and RHT4 groups). The morphological study demonstrated myocyte necrosis, vascular damage and cellular inflammatory response throughout the experimental period. The increased cellularity was more intense in the adventitia of the arterioles. As a consequence of myocyte necrosis, there was an early, local, conjunctive stroma collapse with disarray and thickening of the argyrophilic interstitial fibers, followed by scarring. The functional data showed an increased passive myocardial stiffness in the RHT4 group. We conclude that renovascular hypertension induces myocyte and arteriole necrosis. Reparative fibrosis occurred as a consequence of the inflammatory response to necrosis. The mechanical behavior of the isolated papillary muscle was normal, except for an early increased myocardial passive stiffness