Arachidonic acid stimulates glucose uptake in 3T3-L1 adipocytes by increasing GLUT1 and GLUT4 levels at the plasma membrane. Evidence for involvement of lipoxygenase metabolites and peroxisome proliferator-activated receptor gamma

Exposure of insulin-sensitive tissues to free fatty acids can impair glucose disposal through inhibition of carbohydrate oxidation and glucose transport. However, certain fatty acids and their derivatives can also act as endogenous ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor that positively modulates insulin sensitivity. To clarify the effects of externally delivered fatty acids on glucose uptake in an insulin-responsive cell type, we systematically examined the effects of a range of fatty acids on glucose uptake in 3T3-L1 adipocytes. Of the fatty acids examined, arachidonic acid (AA) had the greatest positive effects, significantly increasing basal and insulin-stimulated glucose uptake by 1.8- and 2-fold, respectively, with effects being maximal at 4 h at which time membrane phospholipid content of AA was markedly increased. The effects of AA were sensitive to the inhibition of protein synthesis but were unrelated to changes in membrane fluidity. AA had no effect on total cellular levels of glucose transporters, but significantly increased levels of GLUT1 and GLUT4 at the plasma membrane. While the effects of AA were insensitive to cyclooxygenase inhibition, the lipoxygenase inhibitor, nordihydroguaiaretic acid, substantially blocked the AA effect on basal glucose uptake. Furthermore, adenoviral expression of a dominant-negative PPARgamma mutant attenuated the AA potentiation of basal glucose uptake. Thus, AA potentiates basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes by a cyclooxygenase-independent mechanism that increases the levels of both GLUT1 and GLUT4 at the plasma membrane. These effects are at least partly dependent on de novo protein synthesis, an intact lipoxygenase pathway and the activation of PPARgamma with these pathways having a greater role in the absence than in the presence of insulin.     

Correction of glycaemia and GLUT1 level by mildronate in rat streptozotocin diabetes mellitus model

Anti‐ischaemic drug mildronate suppresses fatty acid metabolism and increases glucose utilization in myocardium. It was proposed that it could produce a favourable effect on metabolic parameters and glucose transport in diabetic animals. Rats with streptozotocin diabetes mellitus were treated with mildronate (100 mg/kg daily, per os, 6 weeks). Therapeutic effect of mildronate was monitored by measuring animal weight, concentrations of blood glucose, insulin, blood triglycerides, free fatty acids, blood ketone bodies and cholesterol, glycated haemoglobin per cent (HbA1c%) and glucose tolerance. GLUT1 mRNA and protein expression in kidneys, heart, liver and muscles were studied by means of real time RT‐PCR and immunohistochemistry correspondingly. In the streptozotocin + mildronate group, mildronate treatment caused a significant decrease in mean blood glucose, cholesterol, free fatty acid and HbA1c concentrations and improved glucose tolerance. Induction of streptozotocin diabetes mellitus provoked increase of both GLUT1 gene and protein expression in kidneys, heart and muscle, mildronate treatment produced normalization of the GLUT1 expression levels. In the liver a similar effect was observed for GLUT1 protein expression, while GLUT1 gene expression was increased by mildronate. Mildronate produces therapeutic effect in streptozotocin diabetes model. Mildronate normalizes the GLUT1 expression up‐regulated by streptozotocin diabetes mellitus in kidneys, heart, muscle and liver. Copyright © 2011 John Wiley & Sons, Ltd.     

Acute activation of glucose uptake by glucose deprivation in L929 fibroblast cells

Glucose is a very important energy source for a wide variety of cells, and the ability of cells to respond to changes in glucose availability or other cell stresses is of critical importance. Many mammalian cells respond to acute stress by increasing the V(max) of transport through GLUT1; the most ubiquitously expressed glucose transporter isoform. This study investigated the acute response of glucose uptake to glucose deprivation in L929 fibroblast cells--a cell line that expresses only the GLUT1 transporter. Results indicated that glucose deprivation of only a minute activated glucose uptake 10-fold and reached a maximum of 20-fold within 10 min. The activation was dose dependent and only partially muted by addition of up to 20mM pyruvate as an alternate energy source. In contrast to the kinetics of acute metabolic stress, glucose deprivation decreased the K(m) of transport, but did not alter the V(max). Maximal activation of glucose transport by glucose deprivation was completely additive to activation of transport by methylene blue--a stimulant that increased the V(max) of transport without a change in the K(m). Glucose-deprived activation of glucose transport was not inhibited by wortmannin or herbimycin A, but was completely inhibited by phenylarsine oxide. Altogether, the data indicate that L929 fibroblast cells respond quickly and robustly to the cell stress of glucose deprivation and methylene blue treatment by two distinct activation pathways.     

Membrane lipid profile alterations are associated with the metabolic adaptation of the Caco-2 cells to aglycemic nutritional condition

Cancer cells can adapt their metabolic activity under nutritional hostile conditions in order to ensure both bioenergetics and biosynthetic requirements to survive. In this study, the effect of glucose deprivation on Caco-2 cells bioenergetics activity and putative relationship with membrane lipid changes were investigated. Glucose deprivation induces a metabolic remodeling characterized at mitochondrial level by an increase of oxygen consumption, arising from an improvement of complex II and complex IV activities and an inhibition of complex I activity. This effect is accompanied by changes in cellular membrane phospholipid profile. Caco-2 cells grown under glucose deprivation show higher phosphatidylethanolamine content and decreased phosphatidic acid content. Considering fatty acid profile of all cell phospholipids, glucose deprivation induces a decrease of monounsaturated fatty acid (MUFA) and n-3 polyunsaturated fatty acids (PUFA) simultaneously with an increase of n-6 PUFA, with consequent drop of n-3/n-6 ratio. Additionally, glucose deprivation affects significantly the fatty acid profile of all individual phospholipid classes, reflected by an increase of peroxidability index in zwitterionic phospholipids and a decrease in all anionic phospholipids, including mitochondrial cardiolipin. These data indicate that Caco-2 cells metabolic remodeling induced by glucose deprivation actively involves membrane lipid changes associated with a specific bioenergetics profile which ensure cell survival.     

Distinct regulation of glucose transport and GLUT1/GLUT3 transporters by glucose deprivation and IGF-I in chromaffin cells

Effects of prolonged metabolic (glucose deprivation) and hormonal [insulin-like growth factor I (IGF-I)] challenge on regulation of glucose transporter (GLUT) expression, glucose transport rate and possible signaling pathways involved were studied in the neuroendocrine chromaffin cell. The results show that bovine chromaffin cells express both GLUT1 and GLUT3. Glucose deprivation and IGF-I activation led to an elevation of GLUT1 and GLUT3 mRNA, the strongest effect being that of IGF-I on GLUT3 mRNA. Both types of stimulus increased the GLUT1 protein content in a cycloheximide (CHX)-sensitive manner, and the glucose transport rate was elevated by 3- to 4-fold after 48 h under both experimental conditions. IGF-I-induced glucose uptake was totally suppressed by CHX. In contrast, only approximately 50% of transport activation in glucose-deprived cells was sensitive to the protein synthesis inhibitor. Specific inhibitors of mTOR/FRAP and p38 MAPK each partially blocked IGF-I-stimulated glucose transport, but had no effect on transport rate in glucose-deprived cells. The results are consistent with IGF-I-activated transport being completely dependent on new GLUT protein synthesis while the enhanced transport in glucose-deprived cells was partially achieved independent of new synthesis of proteins, suggesting a mechanism relying on preexisting transporters.     

SCD1 activity in muscle increases triglyceride PUFA content,exercise capacity,and PPARδ expression in mice

Stearoyl-CoA desaturase (SCD)1 converts saturated fatty acids into monounsaturated fatty acids. Using muscle overexpression, we sought to determine the role of SCD1 expression in glucose and lipid metabolism and its effects on exercise capacity in mice. Wild-type C57Bl/6 (WT) and SCD1 muscle transgenic (SCD1-Tg) mice were generated, and expression of the SCD1 transgene was restricted to skeletal muscle. SCD1 overexpression was associated with increased triglyceride (TG) content. The fatty acid composition of the muscle revealed a significant increase in polyunsaturated fatty acid (PUFA) content of TG, including linoleate (18:2n6). Untrained SCD1-Tg mice also displayed significantly increased treadmill exercise capacity (WT = 6.6 ± 3 min, Tg = 71.9 ± 9.5 min; P = 0.0009). SCD1-Tg mice had decreased fasting plasma glucose, glucose transporter (GLUT)1 mRNA, fatty acid oxidation, mitochondrial content, and increased peroxisome proliferator-activated receptor (PPAR)δ and Pgc-1 protein expression in skeletal muscle. In vitro studies in C2C12 myocytes revealed that linoleate (18:2n6) and not oleate (18:1n9) caused a 3-fold increase in PPARδ and a 9-fold increase in CPT-1b with a subsequent increase in fat oxidation. The present model suggests that increasing delta-9 desaturase activity of muscle increases metabolic function, exercise capacity, and lipid oxidation likely through increased PUFA content, which increases PPARδ expression and activity. However, the mechanism of action that results in increased PUFA content of SCD1-Tg mice remains to be elucidated.     

Ensemble Modeling of Hepatic Fatty Acid Metabolism with a Synthetic Glyoxylate Shunt

The liver plays a central role in maintaining whole body metabolic and energy homeostasis by consuming and producing glucose and fatty acids. Glucose and fatty acids compete for hepatic substrate oxidation with regulation ensuring glucose is oxidized preferentially. Increasing fatty acid oxidation is expected to decrease lipid storage in the liver and avoid lipid-induced insulin-resistance. To increase hepatic lipid oxidation in the presence of glucose, we previously engineered a synthetic glyoxylate shunt into human hepatocyte cultures and a mouse model and showed that this synthetic pathway increases free fatty acid β-oxidation and confers resistance to diet-induced obesity in the mouse model. Here we used ensemble modeling to decipher the effects of perturbations to the hepatic metabolic network on fatty acid oxidation and glucose uptake. Despite sampling of kinetic parameters using the most fundamental elementary reaction models, the models based on current metabolic regulation did not readily describe the phenotype generated by glyoxylate shunt expression. Although not conclusive, this initial negative result prompted us to probe unknown regulations, and malate was identified as inhibitor of hexokinase 2 expression either through direct or indirect actions. This regulation allows the explanation of observed phenotypes (increased fatty acid degradation and decreased glucose consumption). Moreover, the result is a function of pyruvate-carboxylase, mitochondrial pyruvate transporter, citrate transporter protein, and citrate synthase activities. Some subsets of these flux ratios predict increases in fatty acid and decreases in glucose uptake after glyoxylate expression, whereas others predict no change. Altogether, this work defines the possible biochemical space where the synthetic shunt will produce the desired phenotype and demonstrates the efficacy of ensemble modeling for synthetic pathway design.     

Methamphetamine inhibits the glucose uptake by human neurons and astrocytes: stabilization by acetyl-L-carnitine

Methamphetamine (METH), an addictive psycho-stimulant drug exerts euphoric effects on users and abusers. It is also known to cause cognitive impairment and neurotoxicity. Here, we hypothesized that METH exposure impairs the glucose uptake and metabolism in human neurons and astrocytes. Deprivation of glucose is expected to cause neurotoxicity and neuronal degeneration due to depletion of energy. We found that METH exposure inhibited the glucose uptake by neurons and astrocytes, in which neurons were more sensitive to METH than astrocytes in primary culture. Adaptability of these cells to fatty acid oxidation as an alternative source of energy during glucose limitation appeared to regulate this differential sensitivity. Decrease in neuronal glucose uptake by METH was associated with reduction of glucose transporter protein-3 (GLUT3). Surprisingly, METH exposure showed biphasic effects on astrocytic glucose uptake, in which 20 μM increased the uptake while 200 μM inhibited glucose uptake. Dual effects of METH on glucose uptake were paralleled to changes in the expression of astrocytic glucose transporter protein-1 (GLUT1). The adaptive nature of astrocyte to mitochondrial β-oxidation of fatty acid appeared to contribute the survival of astrocytes during METH-induced glucose deprivation. This differential adaptive nature of neurons and astrocytes also governed the differential sensitivity to the toxicity of METH in these brain cells. The effect of acetyl-L-carnitine for enhanced production of ATP from fatty oxidation in glucose-free culture condition validated the adaptive nature of neurons and astrocytes. These findings suggest that deprivation of glucose-derived energy may contribute to neurotoxicity of METH abusers.     

Changes in cardiac substrate transporters and metabolic proteins mirror the metabolic shift in patients with aortic stenosis

In the hypertrophied human heart, fatty acid metabolism is decreased and glucose utilisation is increased. We hypothesized that the sarcolemmal and mitochondrial proteins involved in these key metabolic pathways would mirror these changes, providing a mechanism to account for the modified metabolic flux measured in the human heart. Echocardiography was performed to assess in vivo hypertrophy and aortic valve impairment in patients with aortic stenosis (n = 18). Cardiac biopsies were obtained during valve replacement surgery, and used for western blotting to measure metabolic protein levels. Protein levels of the predominant fatty acid transporter, fatty acid translocase (FAT/CD36) correlated negatively with levels of the glucose transporters, GLUT1 and GLUT4. The decrease in FAT/CD36 was accompanied by decreases in the fatty acid binding proteins, FABPpm and H-FABP, the β-oxidation protein medium chain acyl-coenzyme A dehydrogenase, the Krebs cycle protein α-ketoglutarate dehydrogenase and the oxidative phosphorylation protein ATP synthase. FAT/CD36 and complex I of the electron transport chain were downregulated, whereas the glucose transporter GLUT4 was upregulated with increasing left ventricular mass index, a measure of cardiac hypertrophy. In conclusion, coordinated downregulation of sequential steps involved in fatty acid and oxidative metabolism occur in the human heart, accompanied by upregulation of the glucose transporters. The profile of the substrate transporters and metabolic proteins mirror the metabolic shift from fatty acid to glucose utilisation that occurs in vivo in the human heart.     

Changes in gene expression involved in energy utilization during chicken follicle development

Ovarian follicle development in egg-laying species is characterized by rapid growth in 7 days prior to ovulation when DNA and protein synthesis is markedly increased in the granulosa and theca cells. However, energy and substrate sources to facilitate the extensive DNA and protein synthesis necessary for folliculogenesis have not been identified in avian species. The current study was undertaken to investigate the expression profiles of regulatory genes involved in glucose transport, glycolysis and fatty acid oxidation in the follicle membranes from the small white follicle (SWF) to follicle 1 (F1) stages of follicle development. In our analysis of glucose transporter (GLUT) isoform expression, the level of GLUT1 mRNA increased with follicle development while GLUT2, GLUT3 and GLUT8 mRNA levels were unaffected by follicle development. In contrast, the expression patterns of proteins involved in metabolism down-stream of glucose transport, including hexokinase (HK), pyruvate dehydrogenase E1alpha (PDH E1alpha) and citrate synthase (CS), did not vary with the developmental stage of the follicle, even during rapid follicle growth. Expression of genes related to beta-oxidation of fatty acids (carnitine palmityl CoA transferase I and II, l-3-hydroxyacyl CoA dehydrogenase and long-chain acyl-CoA dehydrogenase), for which expression in the ovarian follicles of mammalian species has not previously been studied, was not changed consistently with the follicle development. These results suggest that both glucose and fatty acids might work as energy sources to ensure rapid follicle development in the chicken ovary, even though glycolysis and beta-oxidation are not modulated by follicle development.     

Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody

An increase in circulating levels of specific NEFAs (non-esterified fatty acids) has been implicated in the pathogenesis of insulin resistance and impaired glucose disposal in skeletal muscle. In particular, elevation of SFAs (saturated fatty acids), such as palmitate, has been correlated with reduced insulin sensitivity, whereas an increase in certain MUFAs and PUFAs (mono- and poly-unsaturated fatty acids respectively) has been suggested to improve glycaemic control, although the underlying mechanisms remain unclear. In the present study, we compare the effects of palmitoleate (a MUFA) and palmitate (a SFA) on insulin action and glucose utilization in rat L6 skeletal muscle cells. Basal glucose uptake was enhanced approx. 2-fold following treatment of cells with palmitoleate. The MUFA-induced increase in glucose transport led to an associated rise in glucose oxidation and glycogen synthesis, which could not be attributed to activation of signalling proteins normally modulated by stimuli such as insulin, nutrients or cell stress. Moreover, although the MUFA-induced increase in glucose uptake was slow in onset, it was not dependent upon protein synthesis, but did, nevertheless, involve an increase in the plasma membrane abundance of GLUT1 and GLUT4. In contrast, palmitate caused a substantial reduction in insulin signalling and insulin-stimulated glucose transport, but was unable to antagonize the increase in transport elicited by palmitoleate. Our findings indicate that SFAs and MUFAs exert distinct effects upon insulin signalling and glucose uptake in L6 muscle cells and suggest that a diet enriched with MUFAs may facilitate uptake and utilization of glucose in normal and insulin-resistant skeletal muscle.     

Rapid Inhibition of Pyruvate Dehydrogenase: An Initiating Event in High Dietary Fat-Induced Loss of Metabolic Flexibility in the Heart

Cardiac function depends on the ability to switch between fatty acid and glucose oxidation for energy production in response to changes in substrate availability and energetic stress. In obese and diabetic individuals, increased reliance on fatty acids and reduced metabolic flexibility are thought to contribute to the development of cardiovascular disease. Mechanisms by which cardiac mitochondria contribute to diet-induced metabolic inflexibility were investigated. Mice were fed a high fat or low fat diet for 1 d, 1 wk, and 20 wk. Cardiac mitochondria isolated from mice fed a high fat diet displayed a diminished ability to utilize the glycolytically derived substrate pyruvate. This response was rapid, occurring within the first day on the diet, and persisted for up to 20 wk. A selective increase in the expression of pyruvate dehydrogenase kinase 4 and inhibition of pyruvate dehydrogenase are responsible for the rapid suppression of pyruvate utilization. An important consequence is that pyruvate dehydrogenase is sensitized to inhibition when mitochondria respire in the presence of fatty acids. Additionally, increased expression of pyruvate dehydrogenase kinase 4 preceded any observed diet-induced reductions in the levels of glucose transporter type 4 and glycolytic enzymes and, as judged by Akt phosphorylation, insulin signaling. Importantly, diminished insulin signaling evident at 1 wk on the high fat diet did not occur in pyruvate dehydrogenase kinase 4 knockout mice. Dietary intervention leads to a rapid decline in pyruvate dehydrogenase kinase 4 levels and recovery of pyruvate dehydrogenase activity indicating an additional form of regulation. Finally, an overnight fast elicits a metabolic response similar to that induced by high dietary fat obscuring diet-induced metabolic changes. Thus, our data indicate that diet-induced inhibition of pyruvate dehydrogenase may be an initiating event in decreased oxidation of glucose and increased reliance of the heart on fatty acids for energy production.     

Glucose transport and utilization are altered in the brain of rats deficient in n-3 polyunsaturated fatty acids

Long-chain polyunsaturated (n-3) fatty acids have been reported to influence the efficiency of membrane receptors, transporters and enzymes. Because the brain is particularly rich in docosahexaenoic acid (DHA, 22:6 n-3), the present study addresses the question of whether the 22:6 n-3 fatty acid deficiency induces disorder in regulation of energy metabolism in the CNS. Three brain regions that share a high rate of energy metabolism were studied: fronto-parietal cortex, hippocampus and suprachiasmatic nucleus. The effect of the diet deficient in n-3 fatty acids resulted in a 30-50% decrease in DHA in membrane phospholipids. Moreover, a 30% decrease in glucose uptake and a 20-40% decrease in cytochrome oxidase activity were observed in the three brain regions. The n-3 deficient diet also altered the immunoreactivity of glucose transporters, namely GLUT1 in endothelial cells and GLUT3 in neurones. In n-3 fatty acid deficient rats, GLUT1-immunoreactivity readily detectable in microvessels became sparse, whereas the number of GLUT3 immunoreactive neurones was increased. However, western blot analysis showed no significant difference in GLUT1 and GLUT3 protein levels between rats deficient in n-3 fatty acids and control rats. The present results suggest that changes in energy metabolism induced by n-3 deficiency could result from functional alteration in glucose transporters.