Participation of the liver gluconeogenesis in the glibenclamide-induced hypoglycaemia in rats

We previously demonstrated an increased liver gluconeogenesis (LG) during insulin-induced hypoglycaemia. Thus, an expected effect of sulphonylureas induced hypoglycaemia (SIH) could be the activation of LG. However, sulphonylureas infused directly in to the liver inhibits LG. Considering these opposite effects we investigated herein LG in rats submitted to SIH. For this purpose, 24 h fasted rats that received glibenclamide (10 mg kg(-1)) were used (SIH group). Control group received oral saline. Glycaemia at 30, 60, 90, 120 and 150 min after oral administration of glibenclamide were evaluated. Since the lowest glycaemia was obtained 120 min after glibenclamide administration, this time was chosen to investigate LG in situ perfused livers. The gluconeogenesis from precursors that enters in this metabolic pathway before the mitochondrial step, i.e. L-alanine (5 mM), L-lactate (2 mM), pyruvate (5 mM) and L-glutamine were decreased (p < 0.05). However, the gluconeogenic activity using glycerol (2 mM), which enters in the gluconeogenesis after the mitochondrial step was maintained. Taken together, the results suggest that the inhibition of LG promoted by SIH overcome the activation of this metabolic pathway promoted by IIH and could be attributed, at least in part, to its effect on mitochondrial function. Copyright (C) 2011 John Wiley & Sons, Ltd.

Multicenter Analysis of Glucocerebrosidase Mutations in Parkinson`s Disease

Background Recent studies indicate an increased frequency of mutations in the gene encoding glucocerebrosidase (GBA), a deficiency of which causes Gaucher`s disease, among patients with Parkinson`s disease. We aimed to ascertain the frequency of GBA mutations in an ethnically diverse group of patients with Parkinson`s disease. Methods Sixteen centers participated in our international, collaborative study: five from the Americas, six from Europe, two from Israel, and three from Asia. Each center genotyped a standard DNA panel to permit comparison of the genotyping results across centers. Genotypes and phenotypic data from a total of 5691 patients with Parkinson`s disease (780 Ashkenazi Jews) and 4898 controls (387 Ashkenazi Jews) were analyzed, with multivariate logistic-regression models and the Mantel-Haenszel procedure used to estimate odds ratios across centers. Results All 16 centers could detect two GBA mutations, L444P and N370S. Among Ashkenazi Jewish subjects, either mutation was found in 15% of patients and 3% of controls, and among non-Ashkenazi Jewish subjects, either mutation was found in 3% of patients and less than 1% of controls. GBA was fully sequenced for 1883 non-Ashkenazi Jewish patients, and mutations were identified in 7%, showing that limited mutation screening can miss half the mutant alleles. The odds ratio for any GBA mutation in patients versus controls was 5.43 across centers. As compared with patients who did not carry a GBA mutation, those with a GBA mutation presented earlier with the disease, were more likely to have affected relatives, and were more likely to have atypical clinical manifestations. Conclusions Data collected from 16 centers demonstrate that there is a strong association between GBA mutations and Parkinson`s disease.

A comparative study on the effects of the benzodiazepine midazolam and the dopamine agents, apomorphine and sulpiride, on rat behavior in the two-way avoidance test

In recent years. studies in behavioral pharmacology have shown the involvement of dopaminergic mechanisms in avoidance behavior as assessed by the two-way active avoidance test (CAR). Changes in dopaminergic transmission also occur in response to particularly threatening challenges. However, studies on the effects of benzodiazepine (BZD) drugs ill this test are still unclear. Given the interplay of dopamine and other neurotransmitters in the neurobiology of anxiety and schizophrenia the aim of this work was to evaluate the effects of systemic administration of midazolam, the dopaminergic agonist apomorphine, and the D(2) receptor antagonist sulpiride using the CAR, a test that shows good sensitivity to typical neuroleptic drugs. Whereas midazolam did not alter the avoidance response. apomorphine increased and sulpiride reduced them in this test. Escape was not affected by any drug treatments. Heightened avoidance was not associated with the increased motor activity caused by apomorphine. In contrast with the benzodiazepine midazolam, activation of post-synaptic D(2) receptors with apomorphine facilitates, whereas the D(2) receptor antagonism with sulpiride inhibited the acquisition of the avoidance behavior. Together, these results bring additional evidence for a role of D(2) mechanisms in the acquisition of the active avoidance. (C) 2009 Elsevier Inc. All rights reserved.

Can physical exercise during gestation attenuate the effects of a maternal perinatal low-protein diet on oxygen consumption in rats?

A protocol of physical exercise, based on maximal oxygen uptake ((V) over dot(O2max)), for female rats before and during pregnancy was developed to evaluate the impact of a low-protein diet on oxygen consumption during gestation and growth rate of the offspring. Virgin female Wistar rats were divided into four groups as follows: untrained (NT, n = 5); trained (T, n = 5); untrained with low-protein diet (NT+LP, n = 5); and trained with low-protein diet (T+LP, n = 5). Trained rats were submitted to a protocol of moderate physical training on a treadmill over a period of 4 weeks (5 days week(-1) and 60 min day(-1), at 65% of (V) over dot(O2max)). At confirmation of pregnancy, the intensity and duration of the exercise was reduced. Low-protein groups received an 8% casein diet, and their peers received a 17% casein diet. The birthweight and growth rate of the pups up to the 90th day were recorded. Oxygen consumption ((V) over dot(O2)), CO(2) production and respiratory exchange ratio (RER) were determined using an indirect open-circuit calorimeter. Exercise training increased. (V) over dot(O2max) by about 20% when compared with the initial values (45.6 +/- 1.0 ml kg(-1) min(-1)). During gestation, all groups showed a progressive reduction in the resting (V) over dot(O2) values. Dams in the NT+LP group showed lower values of resting (V) over dot(O2) than those in the NT group. The growth rate of pups from low-protein-fed mothers was around 50% lower than that of their respective controls. The T group showed an increase in body weight from the 60th day onwards, while the NT+LP group presented a reduced body weight from weaning onwards. In conclusion, physical training attenuated the impact of the low- protein

Palmitate Activates Insulin Signaling Pathway in Pancreatic Rat Islets

Objective: To investigate the action of palmitate on insulin receptor (IR) signaling pathway in rat pancreatic islets. The following proteins were studied: IR substrate-1 and -2 (IRS1 and IRS2), phosphatidylinositol 3-kinase, extracellular signal-regulated protein kinase-1 and -2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3). Methods: Immunoblotting and immunoprecipitation assays were used to evaluate the phosphorylation states of IRS1 and IRS2 (tyrosine [Tyr]), ERK1/2 (threonine 202 [Thr202]/Tyr204), and STAT3 (serine [Ser727]). Results: The exposure of rat pancreatic islets to 0.1-mmol/L palmitate for up to 30 minutes produced a significant increase of Tyr phosphorylation in IRS2 but not in IRS1. The association of phosphatidylinositol 3-kinase with IRS2 was also upregulated by palmitate. Exposure to 5.6-mmol/L glucose caused a gradual decrease in ERK1/2 (Thr202/Tyr204) and STAT3 (serine [Ser727]) phosphorylations after 30-minute incubation. The addition of palmitate (0.1 mmol/L), associated with 5.6-mmol/L glucose, abolished these latter effects of glucose after 15-minute incubation. Conclusions: Palmitate at physiological concentration associated with 5.6-mmol/L glucose activates IR signaling pathway in pancreatic A cells.

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.

Modulation of rat neutrophil function in vitro by cis- and trans-MUFA

In the present study, the effects of trans-MUFA, elaidic acid (EA; 18 : 1-9t) and vaccenic acid (VA; 18 : 1-11t) on rat neutrophil functions were compared with those of cis-monounsaturated oleic acid (OA) (18 : 1-9c) and saturated stearic acid (SA; 18 : 0) (10-150 mu M). Trans-fatty acids enhanced neutrophil phagocytic capacity, superoxide (O(2)(center dot-)) and hydrogen peroxide production, and candidacidal activity. The same effects were observed for OA. Cells treated with trans-MUFA showed reduced production of NO(center dot), whereas those treated with OA showed an increase in production. Treatment with SA did not provoke significant effect on the parameters investigated. The increase in O(2)(center dot-) production induced by MUFA was not observed when diphenyleneiodonium, an NADPH oxidase inhibitor, was added to the medium. This finding suggests that MUFA stimulate neutrophil NADPH oxidase activity. The addition of 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-inclol-3-yl)-1H-pyrrole-2,5-dione, a protein kinase C (PKC) inhibitor, and wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor, did not affect O(2)(center dot-) production induced by MUFA. Therefore, the mechanisms by which MUFA stimulate NADPH oxidase are not dependent on PKC and do not seem to involve PI3K. Experiments using Zn(2+), an inhibitor of NADPH oxidase H(+) channel, indicated that MUFA activate the NADPH oxidase complex in rat neutrophil due to opening of H(+) channel.

Potential Role of Growth Hormone in Impairment of Insulin Signaling in Skeletal Muscle, Adipose Tissue, and Liver of Rats Chronically Treated with Arginine

We have shown that rats chronically treated with Arginine (Arg), although normoglycemic, exhibit hyperinsulinemia and decreased blood glucose disappearance rate after an insulin challenge. Attempting to investigate the processes underlying these alterations, male Wistar rats were treated with Arg (35 mg/d), in drinking water, for 4 wk. Rats were then acutely stimulated with insulin, and the soleus and extensorum digitalis longus muscles, white adipose tissue (WAT), and liver were excised for total and/or phosphorylated insulin receptor (IR), IR substrate 1/2, Akt, Janus kinase 2, signal transducer and activator of transcription (STAT) 1/3/5, and p85 alpha/55 alpha determination. Muscles and WAT were also used for plasma membrane (PM) and microsome evaluation of glucose transporter (GLUT) 4 content. Pituitary GH mRNA, GH, and liver IGF-I mRNA expression were estimated. It was shown that Arg treatment: 1) did not affect phosphotyrosine-IR, whereas it decreased phosphotyrosine-IR substrate 1/2 and phosphoserine-Akt content in all tissues studied, indicating that insulin signaling is impaired at post-receptor level; 2) decreased PM GLUT4 content in both muscles and WAT; 3) increased the pituitary GH mRNA, GH, and liver IGF-I mRNA expression, the levels of phosphotyrosine-STAT5 in both muscles, phosphotyrosine-Janus kinase 2 in extensorum digitalis longus, phosphotyrosine-STAT3 in liver, and WAT as well as total p85 alpha in soleus, indicating that GH signaling is enhanced in these tissues; and 4) increased p55 alpha total content in muscles, WAT, and liver. The present findings provide the molecular mechanisms by which insulin resistance and, by extension, reduced GLUT4 content in PM of muscles and WAT take place after chronic administration of Arg, and further suggest a putative role for GH in its genesis, considering its diabetogenic effect. (Endocrinology 150: 2080-2086, 2009)

Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure: role of exercise training

Bacurau AV, Jardim MA, Ferreira JC, Bechara LR, Bueno CR Jr, Alba-Loureiro TC, Negrao CE, Casarini DE, Curi R, Ramires PR, Moriscot AS, Brum PC. Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure: role of exercise training. J Appl Physiol 106: 1631-1640, 2009. First published January 29, 2009; doi:10.1152/japplphysiol.91067.2008.-Sympathetic hyperactivity (SH) is a hallmark of heart failure (HF), and several lines of evidence suggest that SH contributes to HF-induced skeletal myopathy. However, little is known about the influence of SH on skeletal muscle morphology and metabolism in a setting of developing HF, taking into consideration muscles with different fiber compositions. The contribution of SH on exercise tolerance and skeletal muscle morphology and biochemistry was investigated in 3- and 7-mo-old mice lacking both alpha(2A)- and alpha(2C)-adrenergic receptor subtypes (alpha(2A)/alpha(2C)ARKO mice) that present SH with evidence of HF by 7 mo. To verify whether exercise training (ET) would prevent skeletal muscle myopathy in advanced-stage HF, alpha(2A)/alpha(2C)ARKO mice were exercised from 5 to 7 mo of age. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF and preserved exercise tolerance and muscular norepinephrine with no changes in soleus morphology. In contrast, plantaris muscle of alpha(2A)/alpha(2C)ARKO mice displayed hypertrophy and fiber type shift (IIA -> IIX) paralleled by capillary rarefaction, increased hexokinase activity, and oxidative stress. At 7 mo, alpha(2A)/alpha(2C)ARKO mice displayed exercise intolerance and increased muscular norepinephrine, muscular atrophy, capillary rarefaction, and increased oxidative stress. ET reestablished alpha(2A)/alpha(2C)ARKO mouse exercise tolerance to 7-mo-old wild-type levels and prevented muscular atrophy and capillary rarefaction associated with reduced oxidative stress. Collectively, these data provide direct evidence that SH is a major factor contributing to skeletal muscle morphological changes in a setting of developing HF. ET prevented skeletal muscle myopathy in alpha(2A)/alpha(2C)ARKO mice, which highlights its importance as a therapeutic tool for HF.

Microcystins -LA, -YR, and -LR action on neutrophil migration

Microcystins (MCs) produced by some freshwater cyanobacterial species possess potent liver toxicity as evidenced by acute neutrophil infiltration. Here, we investigate the ability of three structurally distinct toxins (MC-LA, MC-LR, and MC-YR) to evoke neutrophil recruitment per se and their effects on migration pathways. Intravital Microscopic Studies showed that topical application of only MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression Of L-selectin and beta(2)-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to NO, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. In conclusion, our results show that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation. (C) 2009 Elsevier Inc. All rights reserved.

Proton flux induced by free fatty acids across phospholipid bilayers: New evidences based on short-circuit measurements in planar lipid membranes

Free fatty acids (FFA) are important mediators of proton transport across membranes. However, information concerning the influence of the Structural features of both FFA and the membrane environment on the proton translocation mechanisms across phospholipid membranes is relatively scant. The effects of FFA chain length, unsaturation and membrane composition on proton transport have been addressed in this study by means of electrical measurements in planar lipid bilayers. Proton conductance (G(H)(+)) was calculated from open-circuit voltage and short-circuit current density measurements. We found that cis-unsaturated FFA caused a more pronounced effect on proton transport as compared to Saturated and trans-unsaturated FFA. Cholesterol and cardiolipin decreased membrane leak conductance. Cardiolipin also decreased proton conductance. These effects indicate a dual modulation of protein-independent proton transport by FFA: through a flip-flop mechanism and by modifying a proton diffusional pathway. Moreover the membrane phospholipid composition was shown to importantly affect both processes. (C) 2009 Elsevier Inc. All rights reserved.

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

Effect of Fish Oil Supplementation for Two Generations on Changes of Lymphocyte Function Induced by Walker 256 Cancer Cachexia in Rats

Fish oil supplementation has been shown to improve the cachectic state of tumor-bearing animals and humans. Our previous study showed that fish oil supplementation (1 g per kg body weight per day) for 2 generations had anticancer and anticachetic effects in Walker 256 tumor-bearing rats as demonstrated by reduced tumor growth and body weight loss and increased food intake and survival. In this study, the effect of fish oil supplementation for 2 generations on membrane integrity, proliferation capacity, and CD4/CD8 ratio of lymphocytes isolated from mesenteric lymph nodes, spleen, and thymus of Walker 256 tumor-bearing animals was investigated. We also determined fish oil effect on plasma concentration and ex vivo production of cytokines [tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6, and IL-10]. Lymphocytes from thymus of tumor-bearing rats presented lower viability, but this change was abolished by fish oil supplementation. Tumor growth increased proliferation of lymphocytes from all lymphoid organs, and fish oil supplementation abolished this effect. Ex vivo production of TNF-alpha and IL-6 was reduced in supplemented animals, but IL-4 and IL-10 secretion was stimulated in both nontumor and tumor-bearing rats. IL-10 and IFN-gamma plasma levels was also decreased in supplemented animals. These results suggest that the anticachetic effects of fish oil supplementation for a long period of time (2 generations) in Walker 256 tumor-bearing rats may be associated to a decrease in lymphocyte function as demonstrated by reduced viability, proliferation capacity, and cytokine production.

Evidence that L-glutamine is better than L-alanine as gluconeogenic substrate in perfused liver of weaned fasted rats submitted to short-term insulin-induced hypoglycaemia

Gluconeogenesis in livers from overnight fasted weaned rats submitted to short-term insulin-induced hypoglycaemia (IIH) was investigated. For this purpose, a condition of hyperinsulinemia/hypoglycaemia was obtained with an intraperitoneal (ip) injection of regular insulin (1.0 U kg(-1)). Control group (COG group) received ip saline. The studies were performed 30 min after insulin (IIH group) or saline (COG group) injection. The livers from IIH and COG rats were perfused with L-alanine (5 mM), L-lactate (2 mM)), L-glutamine (10 mM) or glycerol (2 mM). Hepatic glucose, L-lactate and pyruvate production from L-alanine was not affected by IIH. In agreement with this result, the hepatic ability in producing glucose from L-lactate or glycerol remained unchanged (IIH group vs. COG group). However, livers from IIH rats showed higher glucose production from L-glutamine than livers front COG rats and, in the IIH rats, the production of glucose from L-glutamine was higher than that front L-alanine. The higher glucose production in livers from the IIH group. when compared with the COG group was due to its entrance further on gluconeogenic pathway. Taken together. the results suggest that L-glutamine is better than L-alanine, as gluconeogenic substrate in livers of hypoglyceaemic weaned rats. Copyright (C) 2008 John Wiley & Sons. Ltd.

Effects of short chain fatty acids on effector mechanisms of neutrophils

Short chain fatty acids (SCFAs) are metabolic by products of anerobic bacteria fermentation. These fatty acids, despite being an important fuel for colonocytes, are also modulators of leukocyte function. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate, and butyrate) on function of neutrophils, and the possible mechanisms involved. Neutrophils obtained from rats by intraperitoneal lavage 4 h after injection of oyster glycogen solution (1%) were treated with non toxic concentrations of the fatty acids. After that, the following measurements were performed: phagocytosis and destruction of Candida albicans, production of ROS (O(2)(center dot-), H(2)O(2), and HOCl) and degranulation. Gene expression (p47(phox) and p22(phox)) and protein phosphorylation (p47(phox)) were analyzed by real time reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. Butyrate inhibited phagocytosis and killing of C. albicans. This SCFA also had an inhibitory effect on production of O(2)(center dot-), H(2)O(2), and HOCI by neutrophils stimulated with PMA or fMLP. This effect of butyrate was not caused by modulation of expression of NADPH oxidase subunits (p47(phox) and p22(phox)) but it was in part due to reduced levels of p47(phox) phosphorylation and an increase in the concentration of cyclic AMP. Acetate increased the production of O(2)(center dot-) and H(2)O(2), in the absence of stimuli but had no effect on phagocytosis and killing of C. albicans. Propionate had no effect on the parameters studied. These results suggest that butyrate can modulate neutrophil function, and thus could be important in inflammatory neutrophil-associated diseases. Copyright (C) 2008 John Wiley & Sons, Ltd.