Liver glycogen metabolism during short-term insulin-induced hypoglycemia in fed rats

The activities of glycogen phosphorylase and synthase during infusions of glucagon, isoproterenol, or cyanide in isolated liver of fed rats submitted to short-term insulin-induced hypoglycemia (IIH) was investigated. A condition of hyperinsulinemia/hypoglycemia was obtained with an intraperitoneal injection of regular insulin (1.0 U kg(-1)). The control group received ip saline. The experiments were carried out 60 min after insulin (IIH group) or saline (COG group) injection. The rats were anesthetized and after laparotomy, blood was collected from the vena cava for glucose and insulin measurements. The liver was their infused with glucagon (1 nM), isoproterenot (2 mu M), or cyanide (0.5 mM) during 20 min and a sample of the organ was collected for determination of the activities of glycogen phosphorylase and synthase 5 min after starting and 10 min after stopping the infusions. The infusions of cyanide, glucagons, and isoproterenol did not change the activities of glycogen synthase and glycogen phosphorylase. However, glycogen catabolism was decreased during the infusions of glucagon and isoproterenol in IIH rats, being more intense with isoproterenol (p < 0.05), than glucagon. It was concluded that short-term IIH promoted changes in the liver responsiveness of glycogen degradation induced by glucagon and isoproterenol without a change in the activities of glycogen phosphorylase and synthase. Copyright (c) 2008 John Wiley & Sons, Ltd.

Reduced cytokine production by glycogen-elicited peritoneal cells from diabetic rats

IL-1 beta, TNF-alpha, cytokine-induced neutrophil chemoattractant-2 alpha/beta, and IL-10 measurements were performed in elicited peritoneal cells from control, diabetic, and insulin-treated diabetic rats. Production/liberation of these cytokines was decreased in elicited peritoneal cells from diabetic rats. These changes were abolished by insulin treatment of diabetic rats. The alterations observed might be involved in the impaired inflammatory response and high occurrence of apoptosis observed in neutrophils under diabetic states.

Changes of glycogen content in liver, skeletal muscle, and heart from fasted rats

Glycogen content of white and red skeletal muscles, cardiac muscle, and liver was investigated in conditions where changes in plasma levels of non-esterified fatty acids (NEFA) occur. The experiments were performed in fed and 12 and 48 h-fasted rats. The animals were also submitted to swimming for 10 and 30 min. Glycogen content was also investigated in both pharmacologically induced low plasma NEFA levels fasted rats and pharmacologically induced high plasma NEFA levels fed rats. The participation of Akt and glycogen synthase kinase-3 (GSK-3) in the changes observed was investigated. Plasma levels of NEFA, glucose, and insulin were determined in all conditions. Fasting increased plasma NEFA levels and reduced glycogen content in the liver and skeletal muscles. However, an increase of glycogen content was observed in the heart under this condition. Akt and GSK-3 phosphorylation was reduced during fasting in the liver and skeletal muscles but it remained unchanged in the heart. Our results suggest that in conditions of increased plasma NEFA levels, changes in insulin-stimulated phosphorylation of Akt and GSK-3 and glycogen content vary differently in liver, skeletal muscles, and heart. Akt and GSK-3 phosphorylation and glycogen content are decreased in liver and skeletal Muscles, but in the heart it remain unchanged (Akt and GSK-3 phosphorylation) or increased (glycogen content) due to consistent increase of plasma NEFA levels. Copyright (C) 2009 John Wiley & Sons, Ltd.

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.

Exogenous insulin stimulates glycogen accumulation in Rhipicephalus (Boophilus) microplus embryo cell line BME26 via PI3K/AKT pathway

Ticks are obligatory blood-feeding arthropods and important vectors of both human and animal disease agents. Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis, development and cell survival. In such cascade, Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). To study the insulin-triggered pathway and its possible roles during embryogenesis we used a culture of embryonic Rhipicephalus microplus cells (BME26). Exogenous insulin elevated cell glycogen content in the absence of fetal calf serum (FCS) when compared to cells without treatment. Moreover, in the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were blocked. We observed an increase in the relative expression level of PI3K`s regulatory subunit (p85), as determined by qRT-PCR. In the presence of PI3K inhibitors these effects on transcription were also reversed. Additionally, treatment with Wortmannin increased the expression level of the insulin-regulated downstream target glycogen synthase kinase 3 beta (GSK3 beta). The p85 subunit showed elevated transcription levels in ovaries from fully engorged females, but was differentially expressed during tick embryogenesis. These results strongly suggest the presence of an insulin responsive machinery in BME26 cells, and its correlation with carbohydrate/glycogen metabolism also during embryogenesis. (C) 2009 Published by Elsevier Inc.

Energy substrate utilization during nightly vocal activity in three species of Scinax (Anura/Hylidae)

Anuran amphibians exhibit different patterns of energy substrate utilization that correlate with the intensity of vocal and locomotor activities. Given the remarkable differences among species in breeding and feeding strategies, and the different ways energy is used in the whole animal, the suggested correlations between calling and locomotor behavior and the level of energy substrates in the muscles responsible for such activities are more complex than previously reported. We explored the relationships between calling and locomotor behavior and energy supply to trunk and hindlimb muscles, respectively, within the ecologically diverse tree-frog genus Scinax. Specifically, we measured the relative amount of carbohydrates and lipids in these two groups of muscles, and in the liver of three species of Scinax that differ in vocal and locomotor performance, and compared our results with those of two other species for which comparable data are available. We also compared the contents of lipids and carbohydrates of conspecific males collected at the beginning and after 4 h of calling activity. The stomach content to potential feeding opportunities across species was also assessed in both groups of males. Scinax hiemalis and S. rizibilis exhibit comparatively low and episodic calling during long periods of activity whereas S. crospedospilus calls at higher rates over shorter periods. Male S. hiemalis had highest levels of trunk muscle glycogen followed by those of S. rizilbilis and S. crospedospilus, respectively. There was no correlation between total lipid content in trunk muscle and calling rate among different species, suggesting that other metabolic aspects may be responsible for the energetic support for vocal activity. The levels of lipids and carbohydrates in trunk and hindlimb muscles and liver of males collected at the beginning and 4 h into the calling period were similar across species, so the extent of energetic reserves does not appear to constrain vocal or locomotor activity. Finally, we found exceptionally high levels of carbohydrates and lipids in the liver of S. rizibilis, a trait perhaps related to a long and demanding breeding period.

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.

Prolonged exposure to palmitate impairs fatty acid oxidation despite activation of AMP-activated protein kinase in skeletal muscle cells

The aim of this study was to investigate the chronic effects of palmitate on fatty acid (FA) oxidation, AMPK/ACC phosphorylation/activation, intracellular lipid accumulation, and the molecular Mechanisms involved in these processes in skeletal muscle cells. Exposure of L6 myotubes for 8 h to 200, 400, 600, and 800 mu M of palmitate did rot affect cel viability but significantly reduced FA oxidation by similar to 26.5%, similar to 43.5%, similar to 50%, and similar to 47%, respectively. Interestingly, this occurred despite significant increases in AMPK (similar to 2.5-fold) and ACC (similar to 3-fold) phosphorylation and in malonyl-CoA decarboxylase activity (similar to 38-60%). Low concentrations of palmitate (50-100 mu M) caused an increase (similar to 30%) in CPT-I activity. However, as the concentration of palmitate increased, CPT-I activity decreased by similar to 32% after exposure for 8 h to 800 mu M of palmitate. Although FA uptake was reduced (similar to 35%) in cells exposed to increasing, palmitate concentrations, intracellular lipid accumulation increased in a dose-dependent manner, reaching values similar to 2.3-, similar to 3-, and 4-fold higher than control in muscle cells exposed to 400, 600, and 800 mu M palmitate, respectively. Interestingly, myotubes exposed to 400 mu M of palmitate for 1h increased basal glucose uptake and glycogen synthesis by similar to 40%. However, as time of incubation in the presence of palmitate progressed from 1 to 8h, these increases were abolished and a time-dependent inhibition of insulin-stimulated glucose uptake (similar to 65%) and glycogen synthesis (30%) was observed in myotubes. These findings may help explain the dysfunctional adaptations that occur in glucose and FA Metabolism in skeletal muscle under conditions of chronically elevated circulating levels of non-esterified FAs. Such as in obesity and Type 2 Diabetes.

Glimepiride as insulin sensitizer: increased liver and muscle responses to insulin

Aim: Glimepiride, a low-potency insulin secretagogue, is as efficient on glycaemic control as other sulphonylureas, suggesting an additional insulin-sensitizer role. The aim of the present study was to confirm the insulin-sensitizer role of glimepiride and to show extra-pancreatic effects of the drug. Methods: Three-month-old monosodium glutamate (MSG)-induced obese insulin-resistant rats were treated (OG) or not treated (O) with glimepiride for 4 weeks and compared with age-matched non-obese rats (C). Insulin sensitivity in whole body, glucose transporter 4 (GLUT4) protein content, glucose uptake and glycogen synthesis in oxidative skeletal muscle and phospho-glycogen synthase kinase (p-GSK3) and glycogen content in liver were analysed. Results: Insulin sensitivity, analysed by the insulin tolerance test, was 30% lower in O than in C rats (p < 0.05), and OG rats recovered this parameter (p < 0.05). In oxidative muscle, glimepiride increased the GLUT4 protein content (50%, p < 0.001) and recovered the obesity-induced reduction (similar to 20%) of the in vitro insulin-stimulated glucose uptake and incorporation into glycogen. In liver, glimepiride increased p-GSK3 (p < 0.01) and glycogen (p < 0.05) contents. Conclusion: The increased GLUT4 protein expression and glucose utilization in oxidative muscle and the increased insulin sensitivity and glycogen storage in liver evidence the insulin-sensitizer effect of glimepiride, which must be important to enable the glimepiride drug to promote an efficient glycaemic control.

Intensive insulin treatment induces insulin resistance in diabetic rats by impairing glucose metabolism-related mechanisms in muscle and liver

Insulin replacement is the only effective therapy to manage hyperglycemia in type 1 diabetes mellitus (T1DM). Nevertheless, intensive insulin therapy has inadvertently led to insulin resistance. This study investigates mechanisms involved in the insulin resistance induced by hyperinsulinization. Wistar rats were rendered diabetic by alloxan injection, and 2 weeks later received saline or different doses of neutral protamine Hagedorn insulin (1.5, 3, 6, and 9 U/day) over 7 days. Insulinopenic-untreated rats and 6U- and 9U-treated rats developed insulin resistance, whereas 3U-treated rats revealed the highest grade of insulin sensitivity, but did not achieve good glycemic control as 6U- and 9U-treated rats did. This insulin sensitivity profile was in agreement with glucose transporter 4 expression and translocation in skeletal muscle, and insulin signaling, phosphoenolpyruvate carboxykinase/glucose-6-phosphatase expression and glycogen storage in the liver. Under the expectation that insulin resistance develops in hyperinsulinized diabetic patients, we believe insulin sensitizer approaches should be considered in treating T1DM. Journal of Endocrinology (2011) 211, 55-64

Dietary Free Oleic and Linoleic Acid Enhances Neutrophil Function and Modulates the Inflammatory Response in Rats

The high ingestion of oleic (OLA) and linoleic (LNA) acids by Western populations, the presence of inflammatory diseases in these populations, and the importance of neutrophils in the inflammatory process led us to investigate the effects of oral ingestion of unesterified OLA and LNA on rat neutrophil function. Pure OLA and LNA were administered by gavage over 10 days. The doses used (0.11, 0.22 and 0.44 g/kg of body weight) were based on the Western consumption of OLA and LNA. Neither fatty acid affected food, calorie or water intake. The fatty acids were not toxic to neutrophils as evaluated by cytometry using propidium iodide (membrane integrity and DNA fragmentation). Neutrophil migration in response to intraperitoneal injection of glycogen and in the air pouch assay, was elevated after administration of either OLA or LNA. This effect was associated with enhancement of rolling and increased release of the chemokine CINC-2 alpha beta. Both fatty acids elevated l-selectin expression, whereas no effect on beta(2)-integrin expression was observed, as evaluated by flow cytometry. LNA increased the production of proinflammatory cytokines (IL-1 beta and CINC-2 alpha beta) by neutrophils after 4 h in culture and both fatty acids decreased the release of the same cytokines after 18 h. In conclusion, OLA and LNA modulate several functions of neutrophils and can influence the inflammatory process.

Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils

Short chain fatty acids (SCFAs) are fermentation products of anaerobic bacteria. More than just being an important energy source for intestinal epithelial cells, these compounds are modulators of leukocyte function and potential targets for the development of new drugs. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate and butyrate) on production of nitric oxide (NO) and proinflammatory cytokines [tumor necrosis factor alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant-2 (CINC-2 alpha beta)] by rat neutrophils. The involvement of nuclear factor kappa B (NF-kappa B) and histone deacetylase (HDAC) was examined. The effect of butyrate was also investigated in vivo after oral administration of tributyrin (a pro-drug of butyrate). Propionate and butyrate diminished TNF-alpha, CINC-2 alpha beta and NO production by LPS-stimulated neutrophils. We also observed that these fatty acids inhibit HDAC activity and NF-kappa B activation, which might be involved in the attenuation of the LPS response. Products of cyclooxygenase and 5-lipoxygenase are not involved in the effects of SCFAs as indicated by the results obtained with the inhibitors of these enzymes. The recruitment of neutrophils to the peritonium after intraperitoneal administration of a glycogen solution (1%) and the ex vivo production of cytokines and NO by neutrophils were attenuated in rats that previously received tributyrin. These results argue that this triglyceride may be effective in the treatment of inflammatory conditions. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Saturated Fatty Acid-Induced Insulin Resistance Is Associated With Mitochondrial Dysfunction in Skeletal Muscle Cells

Increased plasma levels of free fatty acids (FFA) occur in states of insulin resistance such as obesity and type 2 diabetes mellitus. These high levels of plasma FFA are proposed to play an important role for the development of insulin resistance but the mechanisms involved are still unclear. This study investigated the effects of saturated and unsaturated FFA on insulin sensitivity in parallel with mitochondrial function. C2C12 myotubes were treated for 24 h with 0.1 mM of saturated (palmitic and stearic) and unsaturated (oleic, linoleic, eicosapentaenoic, and docosahexaenoic) FFA. After this period, basal and insulin-stimulated glucose metabolism and mitochondrial function were evaluated. Saturated palmitic and stearic acids decreased insulin-induced glycogen synthesis, glucose oxidation, and lactate production. Basal glucose oxidation was also reduced. Palmitic and stearic acids impaired mitochondrial function as demonstrated by decrease of both mitochondrial hyperpolarization and ATP generation. These FFA also decreased Akt activation by insulin. As opposed to saturated FFA, unsaturated FFA did not impair glucose metabolism and mitochondrial function. Primary cultures of rat skeletal muscle cells exhibited similar responses to saturated FFA as compared to C2C12 cells. These results show that in muscle cells saturated FFA-induced mitochondrial dysfunction associated with impaired insulin-induced glucose metabolism. J. Cell. Physiol. 222: 187-194, 2010. (C) 2009 Wiley-Liss, Inc.

Hypermethioninemia provokes oxidative damage and histological changes in liver of rats

In the present study we evaluated the effect of chronic methionine administration on oxidative stress and biochemical parameters in liver and serum of rats, respectively. We also performed histological analysis in liver. Results showed that hypermethioninemia increased chemiluminescence, carbonyl content and glutathione peroxidase activity, decreased total antioxidant potential, as well as altered catalase activity. Hypermethioninemia increased synthesis and concentration of glycogen, besides histological studies showed morphological alterations and reduction in the glycogen/glycoprotein content in liver. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose were increased in hypermethioninemic rats. These findings suggest that oxidative damage and histological changes caused by methionine may be related to the hepatic injury observed in hypermethioninemia. (C) 2009 Elsevier Masson SAS. All rights reserved.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.