The glucose-lowering agent sodium tungstate increases the levels and translocation of GLUT4 in L6 myotubes through a mechanism associated with ERK1/2 and MEF2D.

AIMS/HYPOTHESIS: The aim of this study was to investigate the action of the glucose-lowering compound sodium tungstate on glucose transport in muscle myotubes and to unravel the molecular events underlying the effects observed. METHODS: We studied the effects of tungstate on 2-deoxy-D: -glucose uptake, levels and translocation of the glucose transporters GLUT4 and GLUT1, and Glut4 (also known as Slc2a4) promoter activity. We also measured the modifications of individual components of the signalling pathways involved in the effects observed. RESULTS: Tungstate increased 2-deoxy-D: -glucose uptake in differentiated L6 myotubes through an increase in the total amount and translocation of GLUT4 transporter. The effects on glucose uptake were additive to those of insulin. Tungstate activated transcription of the Glut4 promoter, as shown by an increase in Glut4 mRNA, and by a promoter reporter assay. The assay of deletions of the Glut4 promoter indicated that the effect of tungstate is mediated by the myocyte enhancer factor 2 (MEF2)-binding domain. Accordingly, MEF2 levels and DNA binding activities were increased in response to the treatment. Tungstate-induced glucose uptake and GLUT4 transcriptional activation were dependent on the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), while no changes were observed in the phosphorylation state of the beta subunit of the insulin receptor, in the phosphatidylinositol 3-kinase pathway or in the activation of 5'AMP-activated protein kinase. CONCLUSIONS/INTERPRETATION: Tungstate activates glucose uptake in myotubes through a novel ERK1/2-dependent mechanism. This effect is exerted by an increase in the content and translocation of the GLUT4 transporter. This is the first report of a glucose-lowering compound activating Glut4 transcription through an ERK1/2-dependent increase in MEF2 levels.

Modulation of glucose transporters in rat diaphragm by sodium tungstate.

Oral administration of sodium tungstate is an effective treatment for diabetes in animal models. We examined the effects of 6 weeks of oral administration of tungstate on glucose transporters (GLUT) in streptozotocin-induced diabetic rat diaphragm. Diabetes decreased GLUT4 expression while tungstate treatment normalized not only GLUT4 protein but also GLUT4 mRNA in the diabetic rats. Furthermore, treatment increased GLUT4 protein in plasma and internal membranes, suggesting a stimulation of its translocation to the plasma membrane. Tungstate had no effect on healthy animals. There were no differences in the total amount of GLUT1 transporter in any group. We conclude that the normoglycemic effect of tungstate may be partly due to a normalization of the levels and subcellular localization of GLUT4, which should result in an increase in muscle glucose uptake.

Experimental ulcerative colitis impairs antioxidant defense system in rat intestine.

Increasing attention has been given recently to the role of free radicals in the pathogenesis of ulcerative colitis, since the inflamed intestine is exposed to oxidative stress generated by infiltrating macrophages and neutrophils within the lamina propia. The overall goal of this study was to evaluate whether experimental ulcerative colitis induces significant changes in the antioxidant defense system in an experimental model induced by the intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid. Twenty rats were treated with 80 mg/kg body weight of trinitrobenzenesulfonic acid and 20 with the same volume of 0.9% NaCl. Rats were killed at one and two weeks after treatment to evaluate colon damage by light and electron transmission microscopy. The degree of tissue injury and inflammation was determined by measuring alkaline phosphatase, gamma-glutamyltranspeptidase, and myeloperoxidase activities and prostaglandin E2 and leukotriene B4. Glutathione levels and the activity of the enzymes of the antioxidant defense system were determined. Enzymatic markers of colon injury showed higher activities in rats with ulcerative colitis. Concentrations of prostaglandin E2 and leukotriene B4 were higher in the groups treated for one week with trinitrobenzenesulfonic acid and markers decreased after two weeks of treatment. All antioxidant enzyme activities were higher at one and two weeks after treatment; however, a significant decrease in total glutathione content was also observed. In conclusion, ulcerative colitis induced by trinitrobenzenesulfonic acid damages the intestinal mucosa and is accompanied by a shift in the antioxidant enzyme activities, and low levels of glutathione. This deficiency in glutathione could be a target for new therapies to treat ulcerative colitis.

Dietary trans fatty acids affect docosahexaenoic acid concentrations in plasma and liver but not brain of pregnant and fetal rats.

The aim of the present study was to investigate the maternal-fetal transport, incorporation, and effects on liver delta-6 fatty-acid desaturase activity of dietary trans fatty acids in pregnant rats. Three groups of six rats each were fed three experimental diets containing approximately 0%, 15%, and 30% of trans fatty acids but containing the same proportion of linoleic (18:2 n-6) and a-linolenic (18:3 n-3) acids for 10 wk. On d 20 of pregnancy, the animals from each group were killed. We determined the fatty acid profiles in plasma, brain, and liver microsomes of pregnant rats, as well as in placenta and fetal liver and brain. No changes were found in the number of fetuses of the pregnant rats. Trans fatty acids were incorporated in high concentrations in placenta and in maternal and fetal tissues, except brain, strongly elevating the linoleic acid proportion and lowering that of docosahexaenoic acid. The delta-6 fatty-acid desaturase activity in the liver microsomes of the pregnant rats was inhibited by trans isomers. In conclusion, high intakes of trans fatty acids partially inhibit liver delta-6 fatty-acid desaturase in pregnant rats, which may explain, in part, the low concentrations of docosahexaenoic acid in pregnant and fetal tissues. However, the fatty acid composition of both fetal and pregnant rat brain remains mostly unaffected regardless of the dietary trans fatty acid content.

Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney.

The evolution of pyruvate carboxylase has been studied in rat liver and kidney during perinatal development. The pyruvate carboxylase activity, amount of enzyme and mRNA levels have been assayed from 2 days before delivery to weaning. In liver, there is a peak of activity and amount of enzyme 24 h before delivery and 2 peaks, at 12 h and 6 days, after parturition. The transcription of the enzyme gene followed a similar pattern, with mRNA peaks preceding those of activity and amount of enzyme. However, in kidney, pyruvate carboxylase activity, amount and mRNA remain low until weaning. These results confirm the limited role of renal gluconeogenesis during the perinatal development. Since all carboxylases contain biotin as prosthetic group, the biotinylation of pyruvate carboxylase during the perinatal period was investigated by western-blot using streptavidin-biotin peroxidase. In the mitochondrial samples from liver and kidney, all the pyruvate carboxylase detected was fully biotinylated, indicating an early development of the holocarboxylase synthetase activity in the perinatal period. This Western-blot technique also allowed us the detection of other biotin-enzymes based on their molecular weight. In liver, during the perinatal development propionyl-coA and 3-methyl-crotonyl-coA carboxylases followed a pattern of induction similar to pyruvate carboxylase. In kidney, the expression of mitochondrial carboxylases was lower compared to liver and propionyl-coA carboxylase was not detected during the studied period.

Increased diaphragm expression of GLUT4 in control and streptozotocin-diabetic rats by fish oil-supplemented diets.

Dietary fat intake influences plasma glucose concentration through modifying glucose uptake and utilization by adipose and skeletal muscle tissues. In this paper, we studied the effects of a low-fat diet on diaphragm GLUT4 expression and fatty acid composition in control and streptozotocin-induced diabetic rats. Control as well as diabetic rats were divided into three different dietary groups each. Either 5% olive oil, 5% sunflower oil, or 5% fish oil was the only fat supplied by the diet. Feeding these low-fat diets for 5 wk induced major changes in fatty acid composition, both in control and in diabetic rats. Arachidonic acid was higher in diabetic olive and sunflower oil-fed rats with respect to fish oil-fed, opposite to docosahexaenoic acid which was higher in diabetic fish oil-fed rats with respect to the other two groups. Animals receiving a fish oil diet had the lowest plasma glucose concentration. GLUT4 expression in diaphragm, as indicated by GLUT4 protein and mRNA, is modulated both by diabetes and by diet fatty acid composition. Diabetes induced a decrease in expression in all dietary groups. Plasma glucose levels correlated well with the increased amount of GLUT4 protein and mRNA found in fish oil-fed groups. Results are discussed in terms of the influence that arachidonic and n-3 polyunsaturated fatty acids may exert on the transcriptional and translational control of the GLUT4 gene.

Modulation of hepatic and intestinal glutathione S-transferases and other antioxidant enzymes by dietary lipids in streptozotocin diabetic rats.

Antioxidant enzymes in liver and small intestine were investigated using control and streptozotocin diabetic rats fed diets with 5% olive, sunflower or fish oil for five weeks. In liver, Glutathione Peroxidase and Superoxide Dismutase decreased and in intestine Glutathione-S-transferase (GST) increased by diabetes. In isolated jejunum and ileum, this increase in GST activity was due to an increase in GST-alpha and -mu isoenzymes in jejunum and GST-alpha, mu and -pi in ileum. Since GST plays an important role in protecting tissues from oxidative damage, our results highlight the role of the intestine against free radicals in physiological or pathological situations.

Effects of dietary fatty acids on lipid metabolism in streptozotocin-induced diabetic rats.

We measured the activity of liver delta9- and delta6-desaturases and examined plasma and liver microsome phospholipid fatty acid composition in control and diabetic rats fed a basal diet supplemented with 5% (by weight) olive oil (OO), sunflower oil (SO), or fish oil (FO), respectively. Plasma glucose, cholesterol, triacylglyceride, and phospholipid levels were also measured. An increase in plasma and liver microsome oleic acid and a decrease in arachidonic acid were found in diabetes. In the liver, docosahexaenoic acid levels were higher in diabetic versus control rats. Diabetes increased liver delta9-desaturase in OO-fed rats and did not modify delta6-desaturase activity in OO- or SO-fed rats. Both enzymatic activities were decreased in diabetic rats fed the FO diet. As a main conclusion, it appears that diet-induced alterations in membrane composition provide a mechanism for improving the diabetic condition in animals and overcoming the effect of insulin deficiency on desaturase activities. Plasma cholesterol was not modified either by diabetes or by diet. In diabetes, OO-fed rats showed the lowest levels of triglycerides. Plasma phospholipids were significantly higher in OO-fed versus FO-fed rats. These findings suggest that OO contributes to a better control of the hypertriglyceridemia accompanying diabetes as compared with the other two diets in this rat model.

Circulating CLA+ lymphocytes from children with atopic dermatitis contain an increased percentage of cells bearing staphylococcal-related T-cell receptor variable segments.

BACKGROUND: Atopic dermatitis is an allergic T-cell mediated skin inflammation. Staphylococcus aureus colonization is very common in cutaneous atopic dermatitis lesions. The cutaneous lymphocyte-associated antigen (CLA) is a T cell skin homing receptor that defines T lymphocytes associated with the cutaneous immune response. OBJECTIVE: To study whether CLA+ T cells from atopic dermatitis children present a selective expression for Staphylococcus aureus-related TCR Vbeta segments. METHODS: Peripheral blood T cells were stained with HECA-452 (anti-CLA) and a panel of TCR Vbeta specific monoclonal antibodies and analysed by flow cytometry. RESULTS: Atopic dermatitis patients have a higher percentage of circulating CLA+ CD3+ lymphocytes compared with healthy controls. Patients with active atopic dermatitis during the study expressed a higher percentage of cells positive for the TCR Vbeta2 and Vbeta5.1 segments in the CLA+ but not in the CLA- subset. These TCR Vbetas are recognized by staphylococcal superantigens. Moreover, there was an increased percentage of HLA-DR+ expression by CLA+ Vbeta5.1+ T cells in patients with active atopic dermatitis, but those patients whose eczema was inactive had very similar values to healthy controls regarding TCR Vbeta and HLA-DR phenotype in circulating CLA+ T lymphocytes. CONCLUSION: Our data indicate that circulating skin-homing T cells of patients with active atopic dermatitis contain an increased percentage of cells bearing TCR Vbeta segments related with Staphylococcus aureus. Staphylococcus superantigens may therefore trigger expansion or at least circulation of appropriate CLA+ T cells.

Sequencing of two alternatively spliced mRNAs corresponding to the extracellular domain of the rat receptor for advanced glycosylation end products (RAGE).

The receptor for advanced glycosylation end products (RAGE) is an integral membrane protein responsible for the recognition and internalization of those extensively modified proteins. The receptor has an extracellular domain that binds to the advanced glycosylation end products. By reverse-transcription and polymerase chain reaction amplification, we have identified in rat liver and kidney two amplified products that correspond to cDNA coding for a part of the extracellular domain of the receptor. Sequencing of these products showed that these amplified molecules were similar except for a 27-bp fragment that was absent in the smaller product. This spliced region is located close to the transmembrane region of the receptor. We have confirmed the possibility of the alternative splicing in the generation of these mRNA isoforms by cloning a fragment of the rat gene for RAGE. This fragment has a distribution of introns and exons fully compatible with the proposed alternative splicing.

Changes in plasma and colonic mucosa fatty acid profiles in rats with ulcerative colitis induced by trinitrobenzene sulfonic acid.

Polyunsaturated fatty acids have a key role in the pathogenesis of inflammatory bowel disease since some of the arachidonic acid-derived eicosanoids have been found to be increased in inflamed intestinal mucosa in the acute phase of human disease. The aim of this study was to prospectively assess plasma and colon mucosa fatty acid patterns in rats with experimental ulcerative colitis. Twenty rats were treated with trinitrobenzene sulfonic acid and 20 with NaCl; two groups were killed after one week and two after two weeks to evaluate colon damage. Plasma was obtained by aortic puncture and colonic mucosa was scraped off and the fatty acid pattern was determined by gas-liquid chromatography. Total, saturated, and monounsaturated plasma fatty acids were significantly higher in both periods of ulcerative colitis as compared to controls. Plasma n-6 fatty acids were increased after treatment, but no significant changes were observed concerning to n-3 fatty acids. With regard to colon mucosa, saturated and monounsaturated fatty acids did not change because of the disease; however, n-6 fatty acids decreased in the first week and increased in the second week and n-3 fatty acids were increased. Changes on the fatty acid distribution in plasma did not parallel to those of colonic mucosa except for 22:6(n-3). We have also found that experimental ulcerative colitis induced by trinitrobenzene sulfonic acid reproduces many of the features related to changes in plasma and colon mucosa fatty acids observed in the human disease.

Short-term effects of dietary fats on the lipid composition and desaturase activities of rat liver microsomes.

Diets supplemented with 10% coconut, olive or sunflower oil were given to rats at weaning. After two, four and six days, the lipid composition and desaturase activities of liver microsomes were measured. The percentage of oleic acid and delta 9-desaturase activity were increased in animals fed an olive oil diet while animals given sunflower oil showed the highest content of linoleic and polyunsaturated n-6 fatty acids. On day 6, olive oil-fed rats had the highest levels of 22:6 n-3 in the phosphatidylethanolamine fraction. Saturated fatty acids were similar among dietary groups, despite the marked differences in the saturated fatty acid content of the three oils. Polyunsaturated n-6 and n-3 fatty acids decreased during the six days of feeding with coconut oil. Our results show that liver microsome membranes respond to different dietary fatty acids sources by changes in enzyme activities and relative content of some fatty acids after a period of dietary manipulation as brief as 6 days.

Changes in the fatty acid pattern of plasma fractions of rats fed coconut, olive or sunflower oil.

The fatty acid composition of plasma phospholipids, triacylglycerols and cholesteryl esters from rats fed diets supplemented (10% w/v) with coconut, olive or sunflower oil during six days has been studied. Rats fed the olive oil diet showed an increased amount of oleic acid whereas the animals fed the sunflower oil diet showed a higher content of linoleic acid than that of the other two groups. n-6 polyunsaturated fatty acids were mainly carried in phospholipid and cholesteryl-ester fractions. There were no differences in the amount of n-6 and n-3 polyunsaturated fatty acids. The effect of diet supplementation was shown after only six days of treatment and the results were similar to those reported in longer treatments.

The short-term effect of dietary fats on the brain fatty acid composition in rats.

The effects of dietary fats on brain fatty acid composition were studied in weanling rats. Three groups of rats were fed for six days a basal diet supplemented with a 10% (w/w) of fat as coconut, olive or sunflower oil. There were no differences in the saturated and monounsaturated fatty acid content among the different groups. The more abundant polyunsaturated fatty acids were arachidonic (20:4 n-6) and docosahexaenoic (22:6 n-3) acids. No effect of the diet on their amount in brain membranes has been found. We can conclude that the brain does not modify its fatty acid composition after a short-time administration of these lipids.

Mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase function in alpha-toxin-perforated cells.

The regulation of mevalonic acid synthesis requires both nonsterol isopentenoid and sterol regulatory signal molecules. A primary target of this multivalent control process is the enzyme which catalyzes mevalonate synthesis: 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34). In this report Staphylococcus aureus alpha-toxin perforated Chinese hamster ovary cells were used to facilitate the identification of isopentenoidogenic reactions and metabolites required for mevalonate-mediated loss of HMG-CoA reductase activity. alpha-Toxin-perforated cells retained the capacity to decrease, upon demand, HMG-CoA reductase activity and protein in response to mevalonate or isopentenoid pyrophosphate esters. Also, it was deduced with highly specific metabolic inhibitors, that conversion of farnesyl 1-diphosphate to squalene was required for mevalonate-mediated suppression of reductase activity. Since squalene (2 microM) did not downregulate reductase activity, pre-squalene pyrophosphate or a derivative, or polyprenyl-1-pyrophosphate-generated inorganic pyrophosphate, or a combination of these metabolites are proposed as candidate regulatory nonsterol isopentenoid signal molecules.

Isopentenoid synthesis in eukaryotic cells. An initiating role for post-translational control of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Using insect (Drosophila) and rodent mammalian cell cultures we demonstrate that acute mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A was initiated in the absence of protein synthesis. In addition, insect and mammalian cells depleted (1 h) of putative regulatory post-isopentenyl-1-pyrophosphate metabolites by incubation with an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A synthase (L-659,699) accumulated 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme units. This accumulation resulted primarily from a decrease in the loss of 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme units rather than from an increase in enzyme synthesis. These observations suggest that the initial phase of mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was governed primarily by post-translational processes. An unknown rapidly turning over post-isopentenyl-1-pyrophosphate metabolite(s) is proposed as the agonist for these post-translational processes.

Long-term effects of dietary monounsaturated and polyunsaturated fatty acids on plasma lipids in dogs.

To compare the effect of monounsaturated vs. polyunsaturated fatty acids on plasma lipids, three months old dogs were studied for a period of six months. The dogs were fed a basal diet supplemented with either 14% olive oil or sunflower oil. Blood samples were taken fortnightly during this period. We have found changes in the various unsaturated fatty acids in response to the diet. Oleic and 20:3 n-9 acids were higher in the olive oil group while linoleic acid was increased in the dogs fed the sunflower oil diet. Arachidonic acid and PUFA n-3 > 18C index were nearly similar with both diets. The cholesterol levels were similar to those found in adult humans and no significant differences were brought about diets at any time. Thus a diet rich in monounsaturated fatty acids is as efficient as a polyunsaturated rich in relation to total cholesterol levels but more beneficial because of the antiatherogenic effect of HDL-cholesterol which is increased with this type of dietary fat.

Long-term effects of dietary monounsaturated and polyunsaturated fatty acids on the lipid composition of erythrocyte membranes in dogs.

1. Lipid analyses were conducted on erythrocyte membranes from dogs fed for 6 months with a normal defatted diet supplemented with either olive or sunflower oil. 2. Levels of palmitic, stearic and arachidonic acids were only slightly affected by dietary fat. 3. The unsaturated fatty acids of n-9 series were elevated in all the phospholipid fractions analysed for olive oil-fed dogs while the n-6 fatty acids, with the exception of arachidonic acid, were elevated in sunflower oil-fed dogs. 4. In the olive oil group the 20:5 (n-3) acid was higher than in the sunflower oil group. 5. The unsaturation index and the cholesterol/phospholipid ratio increased along the time course in the sunflower oil group. Both increases are complementary in order to maintain the constant fluidity of membranes.

Changes in lipid composition and desaturase activities of duodenal mucosa induced by dietary fat.

In the present work we have studied the effects of feeding either olive or sunflower oil on lipid composition and desaturase activities of duodenal mucosa microsomes. Duodenal microsomes prepared from dogs fed the sunflower oil diet showed higher percentages of saturated, of linoleic and of n - 3 polyunsaturated fatty acids as well as lower levels of oleic, dihomo-gamma-linolenic and arachidonic acids in phosphatidylcholine and phosphatidylethanolamine than those prepared from animals fed the olive oil diet. In sphingomyelin, the dietary supplementation did not produce significant differences between the two groups. The cholesterol/phospholipid molar ratio was higher in the sunflower oil group than in the olive oil group. The in vitro delta 9-desaturase activity was higher in microsomes from the olive oil dogs. The delta 6-desaturase activity was similar in microsomes from the two groups and lower than that found for delta 9-desaturase activity. Desaturase activities were higher in duodenal microsomes than those previously found for liver microsomes.