07-1404 Sigma-AldrichAnti-GLUT-4 Antibody, C-terminus

We explored whether altered expression of factors tuning mitochondrial metabolism contributes to muscular adaptations with endurance training in the condition of lowered ambient oxygen concentration (hypoxia) and whether these adaptations relate to oxygen transfer as reflected by subsarcolemmal mitochondria and oxygen metabolism in muscle.Male volunteers completed 30 bicycle exercise sessions in normoxia or normobaric hypoxia (4,000 m above sea level) at 65% of the respective peak aerobic power output. Myoglobin content, basal oxygen consumption, and re-oxygenation rates upon reperfusion after 8 min of arterial occlusion were measured in vastus muscles by magnetic resonance spectroscopy. Biopsies from vastus lateralis muscle, collected pre and post a single exercise bout, and training, were assessed for levels of transcripts and proteins being associated with mitochondrial metabolism.Hypoxia specifically lowered the training-induced expression of markers of respiratory complex II and IV (i.e. SDHA and isoform 1 of COX-4; COX4I1) and preserved fibre cross-sectional area. Concomitantly, trends (p less than 0.10) were found for a hypoxia-specific reduction in the basal oxygen consumption rate, and improvements in oxygen repletion, and aerobic performance in hypoxia. Repeated exercise in hypoxia promoted the biogenesis of subsarcolemmal mitochondria and this was co-related to expression of isoform 2 of COX-4 with higher oxygen affinity after single exercise, de-oxygenation time and myoglobin content (r ≥ 0.75). Conversely, expression in COX4I1 with training correlated negatively with changes of subsarcolemmal mitochondria (r less than -0.82).Hypoxia-modulated adjustments of aerobic performance with repeated muscle work are reflected by expressional adaptations within the respiratory chain and modified muscle oxygen metabolism.

Carbohydrate-response element binding protein (ChREBP) plays a key role in regulating glucose metabolism and de novo lipogenesis in metabolic tissues and cancer cells. Here we report that ChREBP is also a critical regulator of the metabolic alterations induced during human cytomegalovirus (HCMV) infection. The expression of both ChREBP-α and ChREBP-β is robustly induced in HCMV-infected human fibroblasts; this induction is required for efficient HCMV infection. Depletion of ChREBP in HCMV-infected cells results in reduction of HCMV-induced glucose transporter 4 and glucose transporter 2 expression, leading to inhibition of glucose uptake, lactate production, nucleotide biosynthesis, and NADPH generation. We previously reported that HCMV infection induces lipogenesis through the activation of sterol regulatory element binding protein 1, which is mediated by the induction of PKR-like endoplasmic reticulum kinase. Data from the present study show that HCMV-induced lipogenesis is also controlled by the induction of ChREBP, in a second mechanism involved in the regulation of HCMV-induced de novo lipogenesis. These results suggest that ChREBP plays a key role in reprogramming glucose and lipid metabolism in HCMV infection.

The Zucker fa/fa rat has been widely used as an animal model to study obesity, since it recapitulates most of its behavioral and metabolic dysfunctions, such as hyperphagia, hyperglycemia and insulin resistance. Although it is well established that olfaction is under nutritional and hormonal influences, little is known about the impact of metabolic dysfunctions on olfactory performances and glucose-sensing in the olfactory system of the obese Zucker rat. In the present study, using a behavioral paradigm based on a conditioned olfactory aversion, we have shown that both obese and lean Zucker rats have a better olfactory sensitivity when they are fasted than when they are satiated. Interestingly, the obese Zucker rats displayed a higher olfactory sensitivity than their lean controls. By investigating the molecular mechanisms involved in glucose-sensing in the olfactory system, we demonstrated that sodium-coupled glucose transporters 1 (SGLT1) and insulin dependent glucose transporters 4 (GLUT4) are both expressed in the olfactory bulb (OB). By comparing the expression of GLUT4 and SGLT1 in OB of obese and lean Zucker rats, we found that only SGLT1 is regulated in genotype-dependent manner. Next, we used glucose oxidase biosensors to simultaneously measure in vivo the extracellular fluid glucose concentrations ([Gluc]ECF) in the OB and the cortex. Under metabolic steady state, we have determined that the OB contained twice the amount of glucose found in the cortex. In both regions, the [Gluc]ECF was 2 fold higher in obese rats compared to their lean controls. Under induced dynamic glycemia conditions, insulin injection produced a greater decrease of [Gluc]ECF in the OB than in the cortex. Glucose injection did not affect OB [Gluc]ECF in Zucker fa/fa rats. In conclusion, these results emphasize the importance of glucose for the OB network function and provide strong arguments towards establishing the OB glucose-sensing as a key factor for sensory olfactory processing.

Palmitoleic acid was previously shown to improve glucose homeostasis by reducing hepatic glucose production and by enhancing insulin-stimulated glucose uptake in skeletal muscle. Herein we tested the hypothesis that palmitoleic acid positively modulates glucose uptake and metabolism in adipocytes.For this, both differentiated 3 T3-L1 cells treated with either palmitoleic acid (16:1n7, 200 μM) or palmitic acid (16:0, 200 μM) for 24 h and primary adipocytes from mice treated with 16:1n7 (300 mg/kg/day) or oleic acid (18:1n9, 300 mg/kg/day) by gavage for 10 days were evaluated for glucose uptake, oxidation, conversion to lactate and incorporation into fatty acids and glycerol components of TAG along with the activity and expression of lipogenic enzymes.Treatment of adipocytes with palmitoleic, but not oleic (in vivo) or palmitic (in vitro) acids, increased basal and insulin-stimulated glucose uptake and GLUT4 mRNA levels and protein content. Along with uptake, palmitoleic acid enhanced glucose oxidation (aerobic glycolysis), conversion to lactate (anaerobic glycolysis) and incorporation into glycerol-TAG, but reduced de novo fatty acid synthesis from glucose and acetate and the activity of lipogenic enzymes glucose 6-phosphate dehydrogenase and ATP-citrate lyase. Importantly, palmitoleic acid induction of adipocyte glucose uptake and metabolism were associated with AMPK activation as evidenced by the increased protein content of phospho(p)Thr172AMPKα, but no changes in pSer473Akt and pThr308Akt. Importantly, such increase in GLUT4 content induced by 16:1n7, was prevented by pharmacological inhibition of AMPK with compound C.In conclusion, palmitoleic acid increases glucose uptake and the GLUT4 content in association with AMPK activation.

In the brain, glucose homeostasis of extracellular fluid is crucial to the point that systems specifically dedicated to glucose sensing are found in areas involved in energy regulation and feeding behavior. Olfaction is a major sensory modality regulating food consumption. Nutritional status in turn modulates olfactory detection. Recently it has been proposed that some olfactory bulb (OB) neurons respond to glucose similarly to hypothalamic neurons. However, the precise molecular cues governing glucose sensing in the OB are largely unknown. To decrypt these molecular mechanisms, we first used immunostaining to demonstrate a strong expression of two neuronal markers of glucose-sensitivity, insulin-dependent glucose transporter type 4 (GLUT4), and sodium glucose co-transporter type 1 (SGLT1) in specific OB layers. We showed that expression and mapping of GLUT4 but not SGLT1 were feeding state-dependent. In order to investigate the impact of metabolic status on the delivery of blood-borne glucose to the OB, we measured extracellular fluid glucose concentration using glucose biosensors simultaneously in the OB and cortex of anesthetized rats. We showed that glucose concentration in the OB is higher than in the cortex, that metabolic steady-state glucose concentration is independent of feeding state in the two brain areas, and that acute changes in glycemic conditions affect bulbar glucose concentration alone. These data provide new evidence of a direct relationship between the OB and peripheral metabolism, and emphasize the importance of glucose for the OB network, providing strong arguments toward establishing the OB as a glucose-sensing organ.

We evaluated the effects of aerobic exercise training without dietary changes on cardiovascular and metabolic variables and on the expression of glucose transporter Type 4 in rats with metabolic syndrome.Twenty male spontaneously hypertensive rats received monosodium glutamate during the neonatal period. The animals were allocated to the following groups: MS (sedentary metabolic syndrome), MS-T (trained on a treadmill for 1 hour/day, 5 days/week for 10 weeks), H (sedentary spontaneously hypertensive rats) and H-T (trained spontaneously hypertensive rats). The Lee index, blood pressure (tail-cuff system), insulin sensitivity (insulin tolerance test) and functional capacity were evaluated before and after 10 weeks of training. Glucose transporter Type 4 expression was analyzed using Western blotting. The data were compared using analysis of variance (ANOVA) (pless than 0.05).At baseline, the MS rats exhibited lower insulin sensitivity and increased Lee index compared with the H rats. Training decreased the body weight and Lee index of the MS rats (MS-T vs. MS), but not of the H rats (H-T vs. H). There were no differences in food intake between the groups. At the end of the experiments, the systolic blood pressure was lower in the two trained groups than in their sedentary controls. Whole-body insulin sensitivity increased in the trained groups. Glucose transporter Type 4 content increased in the heart, white adipose tissue and gastrocnemius muscle of the trained groups relative to their respective untrained groups.In conclusion, the present study shows that an isolated aerobic exercise training intervention is an efficient means of improving several components of metabolic syndrome, that is, training reduces obesity and hypertension and increases insulin sensitivity.

The development of adipocytes from their progenitor cells requires the action of growth factors signaling to transcription factors to induce the expression of adipogenic proteins leading to the accumulation of lipid droplets, induction of glucose transport, and secretion of adipokines signaling metabolic events throughout the body. Murine 3T3-L1 pre-adipocytes sequentially express all the proteins necessary to become mature adipocytes throughout an 8-10 day process initiated by a cocktail of hormones. We examined the role of Clk/STY or Clk1, a cdc2-like kinase, in adipogenesis since it is known to be regulated by Akt, a pivotal kinase in development. Inhibition of Clk1 by a specific inhibitor, TG003, blocked alternative splicing of PKCβII and expression of PPARγ1 and PPARγ2. SiRNA depletion of Clk1 resulted in early expression of PKCβII and sustained PKCβI expression. Since Clk1 is a preferred Akt substrate, required for phosphorylation of splicing factors, mutation of Clk1 Akt phosphorylation sites was undertaken. Akt sites on Clk1 are in the serine/arginine-rich domain and not the kinase domain. Mutation of single and multiple sites resulted in dysregulation of PKCβII, PKCβI, and PPARγ1&2 expression. Additionally, adipogenesis was blocked as assessed by Oil Red O staining, adiponectin, and Glut1 and 4 expression. Immunofluorescence microscopy revealed that Clk1 triple mutant cDNA, transfected into pre-adipocytes, resulted in excluding SRp40 (SFSR6) from co-localizing to the nucleus with PFS, a perispeckle specific protein. This study demonstrates the role of Akt and Clk1 kinases in the early differentiation of 3T3-L1 cells to adipocytes.

Increasing evidence suggests that abnormal brain accumulation of amyloid-β(1-42) (Aβ(1-42)) oligomers plays a causal role in Alzheimer's disease (AD), and in particular may cause the cognitive deficits that are the hallmark of AD. In vitro, Aβ(1-42) oligomers impair insulin signaling and suppress neural functioning. We previously showed that endogenous insulin signaling is an obligatory component of normal hippocampal function, and that disrupting this signaling led to a rapid impairment of spatial working memory, while delivery of exogenous insulin to the hippocampus enhanced both memory and metabolism; diet-induced insulin resistance both impaired spatial memory and prevented insulin from increasing metabolism or cognitive function. Hence, we tested the hypothesis that Aβ(1-42) oligomers could acutely impair hippocampal metabolic and cognitive processes in vivo in the rat. Our findings support this hypothesis: Aβ(1-42) oligomers impaired spontaneous alternation behavior while preventing the task-associated dip in hippocampal ECF glucose observed in control animals. In addition, Aβ(1-42) oligomers decreased plasma membrane translocation of the insulin-sensitive glucose transporter 4 (GluT4), and impaired insulin signaling as measured by phosphorylation of Akt. These data show in vivo that Aβ(1-42) oligomers can rapidly impair hippocampal cognitive and metabolic processes, and provide support for the hypothesis that elevated Aβ(1-42) leads to cognitive impairment via interference with hippocampal insulin signaling.

Salivary glucose is often considered as being from glandular origin. Little information is available, however, on the possible role of glucose transporters in the secretion of the hexose by salivary glands. The major aim of the present study was to investigate the expression and localization of several distinct glucose transporters in acinar cells of rat parotid glands.Quantitative real-time PCR analysis, immunohistochemistry and western blotting techniques were used to assess the presence of SGLT1, GLUT1, GLUT2 and GLUT4 in acinar cells of rat parotid glands.Quantitative real-time PCR documented the expression of SGLT1 and GLUT1 in parotid tissues, with a much lower level of GLUT4 mRNA and no expression of GLUT2 mRNA. Western blot analysis revealed the presence of SGLT1, GLUT1 and GLUT4 proteins, but not GLUT2 proteins in the parotid extract. Immunohistochemistry confirmed these findings. SGLT1 was specifically located at the baso-lateral membrane, co-localizing with Na(+)/K(+) ATPase. GLUT1 was found both at the baso-lateral and apical level. GLUT4 appeared to be also located at the baso-lateral level. However, too little GLUT4 was present to allow co-localization labeling.Based on these findings, a model is proposed for the transport of glucose into the acinar cells and thereafter into the acinar lumen.

Contraction and insulin promote glucose uptake in skeletal muscle through GLUT4 translocation to cell surface membranes. Although the signaling mechanisms leading to GLUT4 translocation have been extensively studied in muscle, the cellular transport machinery is poorly understood. Myo1c is an actin-based motor protein implicated in GLUT4 translocation in adipocytes; however, the expression profile and role of Myo1c in skeletal muscle have not been investigated. Myo1c protein abundance was higher in more oxidative skeletal muscles and heart. Voluntary wheel exercise (4 weeks, 8.2 ± 0.8 km/day), which increased the oxidative profile of the triceps muscle, significantly increased Myo1c protein levels by ∼2-fold versus sedentary controls. In contrast, high fat feeding (9 weeks, 60% fat) significantly reduced Myo1c by 17% in tibialis anterior muscle. To study Myo1c regulation of glucose uptake, we expressed wild-type Myo1c or Myo1c mutated at the ATPase catalytic site (K111A-Myo1c) in mouse tibialis anterior muscles in vivo and assessed glucose uptake in vivo in the basal state, in response to 15 min of in situ contraction, and 15 min following maximal insulin injection (16.6 units/kg of body weight). Expression of wild-type Myo1c or K111A-Myo1c had no effect on basal glucose uptake. However, expression of wild-type Myo1c significantly increased contraction- and insulin-stimulated glucose uptake, whereas expression of K111A-Myo1c decreased both contraction-stimulated and insulin-stimulated glucose uptake. Neither wild-type nor K111A-Myo1c expression altered GLUT4 expression, and neither affected contraction- or insulin-stimulated signaling proteins. Myo1c is a novel mediator of both insulin-stimulated and contraction-stimulated glucose uptake in skeletal muscle.