Plasma Membrane GLUT4 Research Articles

Assay of Insulin‐Stimulated Signaling by Flow Cytometry: Key Points of Regulation

Type 2 Diabetes is an increasingly prevalent disease worldwide that is partially caused by a progressive loss of insulin response in adipose tissue and skeletal muscle, two essential insulin target tissues. Multicolor flow cytometry is a powerful tool that can be used to measure multiple signaling events simultaneously in specific cell types within mixed populations. The objective of this study is to design a sensitive and high‐throughput assay to measure key points of regulation in the insulin signaling pathway for adipocytes and myocytes using flow cytometry. We have developed a multicolor flow cytometry panel to measure the insulin stimulated phosphorylation of Akt (S473) and the transport of the insulin responsive glucose transporter GLUT4 to the plasma membrane. This assay is compatible with cell identity markers, such as CD56, to specifically measure insulin response of muscle cells within mixed populations. C2C12 myoblasts were stained with primary conjugated antibodies for pAkt (S473) and an extracellular region of GLUT4, indicating that GLUT4 is present in the plasma membrane. Both C2C12 myoblasts and non‐human primate primary myoblasts responded to insulin with increased pAkt (S473) and plasma membrane GLUT4 with an EC50 of <10nM, similar to physiological response. Akt phosphorylation and GLUT4 translocation were blocked by the actin depolymerizing agent Cytochalasin D. Future work will expand the panel to measure phosphorylation of insulin receptor substrate (IRS1) and phosphoinosital 3‐kinase (PI3K) activity by determining phosphoinosital (3,4,5) phosphate (PIP3) production. The sensitivity of the assay will be demonstrated with inhibitors to specific signaling events such as inhibition of PI3K by Wortmannin, and inhibition of Akt activation by MK‐2206. We anticipate that this will provide a powerful method to dissect the insulin signaling cascade in mixed populations of cells or to understand the role of specific regulatory elements in difficult to transfect primary cells.Support or Funding InformationSupported by an APS STRIDE Summer Fellowship (SM), the University of Oregon UROP Mini‐Grant (SM), and the National Institute of Diabetes Digestive and Kidney Diseases under Award R01 DK095926 (CEM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carnosol increases skeletal muscle cell glucose uptake via AMPK‐dependent GLUT 4 glucose transporter translocation

Skeletal muscle is highly important in glucose homeostasis since it is quantitatively a major insulin‐target tissue. Insulin action in muscle cells activates the phosphatidylinositol‐3 kinase (PI3K)/Akt signaling pathway causing the translocation of intracellularly stored GLUT4 glucose transporters to the plasma membrane leading to increased glucose uptake. Impaired insulin action in muscle leads to insulin resistance and type 2 diabetes mellitus (T2DM).AMP‐activated kinase (AMPK) is a cellular energy sensor and its activation increases glucose uptake by skeletal muscle cells. Finding AMPK activators is viewed as an effective approach to combat insulin resistance and T2DM. Rosemary extract (RE) has been shown to increase muscle glucose uptake and AMPK activity but the components responsible for these effects have not been identified yet. In the current study, we investigated the effect of carnosol, a polyphenol found in high concentrations in RE. L6 rat muscle cells were used to measure uptake of [3H]‐2‐deoxy‐D‐glucose and the signaling molecules involved were investigated by immunoblotting. Carnosol stimulated glucose uptake in L6 myotubes in a dose‐ and time‐dependent manner. A response comparable to maximum insulin stimulation (196±9.2 % of control) was seen with 50μM of carnosol (2h) (182±7.8 % of control). Carnosol did not affect Akt phosphorylation while it significantly increased AMPK phosphorylation. Furthermore, the increase in glucose uptake in the presence of carnosol was significantly reduced by the AMPK inhibitor compound C (CC) while it was not affected by the PI3K inhibitor wortmannin. Carnosol increased plasma membrane GLUT4 glucose transporter levels in GLUT4myc overexpressing L6 cells and this response was abolished by the AMPK inhibitor CC. Our study is the first to show a significant increase in muscle glucose uptake by carnosol via a mechanism that involves AMPK.Support or Funding InformationSupported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to ET.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Uphill running excessive training increases gastrocnemius glycogen content in C57BL/6 mice.

The main aim of the present investigation was to verify the effects of three overtraining (OT) protocols performed in downhill (OTR/down), uphill (OTR/up) and without inclination (OTR) on the protein levels of Akt (Ser473), AMPKalpha (Thr172), PGC-1alpha, plasma membrane GLUT-1 and GLUT-4 as well as on the glycogen contents in mice gastrocnemius. A trained (TR) protocol was used as positive control. Rodents were divided into naive (N, sedentary mice), control (CT, sedentary mice submitted to the performance evaluations), TR, OTR/down, OTR/up and OTR groups. At the end of the experimental protocols, gastrocnemius samples were removed and used for immunoblotting analysis as well as for glycogen measurements. There was no significant difference between the experimental groups for the protein levels of pAkt (Ser473), pAMPKalpha (Thr172), PGC-1alpha, plasma membrane GLUT-1 and GLUT-4. However, the OTR/up protocol exhibited higher contents of glycogen compared to the CT and TR groups. In summary, the OTR/up group increased the gastrocnemius glycogen content without significant changes of pAkt (Ser473), pAMPKalpha (Thr172), PGC-1alpha, plasma membrane GLUT-1 and GLUT-4.

β-arrestin-2 is involved in irisin induced glucose metabolism in type 2 diabetes via p38 MAPK signaling

Type 2 diabetes mellitus (T2DM) is a common metabolic disease worldwide. It has been reported that irisin play regulatory role in glucose metabolism in T2DM. However, the underlying mechanism involved in that is not completely known. Herein, we determined the novel role of β-arrestin-2 in irisin-induced glucose utilization in diabetes. Effects of irisin and β-arrestin-2 on glucose utilization were investigated in a rat model of diabetes and in diabetic C2C12 cells in vitro. Results showed that irisin had positive role in glucose metabolism via regulating glucose tolerance as well as uptake in cardiac and skeletal muscle tissues, as evidenced by IPGTT, 2-deoxyglucose uptake and plasma membrane GLUT-4 assay. β-arrestin-2 also improved glucose utilization in diabetes by increasing the glucose uptake and insulin sensitivity, as shown in mice overexpressing β-arrestin-2. In diabetic C2C12 myocytes, irisin-induced GLUT4 and glucose uptake were restrained by β-arrestin-2 inhibition, but was enhanced by β-arrestin-2 overexpression. Additionally, irisin and β-arrestin-2 increased the activation of p38 MAPK in diabetic C2C12 cells, and the repression of p38 MAPK activation decreased the glucose uptake and plasma membrane GLUT-4 was enhanced by irisin and β-arrestin-2 overexpression in diabetic C2C12 cells. In conclusion, we demonstrated that β-arrestin-2 has a crucial role in irisin induced glucose metabolism in T2DM by regulating the p38 MAPK signaling. This might present a novel therapeutic target of treatment for human diabetes.

Estradiol-induced regulation of GLUT4 in 3T3-L1 cells: involvement of ESR1 and AKT activation.

Impaired insulin-stimulated glucose uptake involves reduced expression of the GLUT4 (solute carrier family 2 facilitated glucose transporter member 4, SLC2A4 gene). 17β-estradiol (E2) modulates SLC2A4/GLUT4 expression, but the involved mechanisms are unclear. Although E2 exerts biological effects by binding to estrogen receptors 1/2 (ESR1/2), which are nuclear transcriptional factors; extranuclear effects have also been proposed. We hypothesize that E2 regulates GLUT4 through an extranuclear ESR1 mechanism. Thus, we investigated the effects of E2 upon (1) subcellular distribution of ESRs and the proto-oncogene tyrosine-protein kinases (SRC) involvement; (2) serine/threonine-protein kinase (AKT) activation; (3) Slc2a4/GLUT4 expression and (4) GLUT4 subcellular distribution and glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were cultivated or not with E2 for 24 h, and additionally treated or not with ESR1-selective agonist (PPT), ESR1-selective antagonist (MPP) or selective SRC inhibitor (PP2). Subcellular distribution of ESR1, ESR2 and GLUT4 was analyzed by immunocytochemistry; Slc2a4 mRNA and GLUT4 were quantified by qPCR and Western blotting, respectively; plasma membrane GLUT4 translocation and glucose uptake were analyzed under insulin stimulus for 20 min or not. E2 induced (1) translocation of ESR1, but not of ESR2, from nucleus to plasma membrane and AKT phosphorylation, effects mimicked by PPT and blocked by MPP and PP2; (2) increased Slc2a4/GLUT4 expression and (3) increased insulin-stimulated GLUT4 translocation and glucose uptake. In conclusion, E2 treatment promoted a SRC-mediated nucleus-plasma membrane shuttle of ESR1, and increased AKT phosphorylation, Slc2a4/GLUT4 expression and plasma membrane GLUT4 translocation; consequently, improving insulin-stimulated glucose uptake. These results unravel mechanisms through which estrogen improves insulin sensitivity.

Carnosic acid as a component of rosemary extract stimulates skeletal muscle cell glucose uptake via AMPK activation.

Compounds that increase the activity of the energy sensor AMP-activated kinase (AMPK) have the potential to regulate blood glucose levels. Although rosemary extract (RE) has been reported to activate AMPK and reduce blood glucose levels in vivo, the chemical components responsible for these effects are not known. In the present study, we measured the levels of the polyphenol carnosic acid (CA) in RE and examined the effects and the mechanism of action of CA on glucose transport system in muscle cells. High performance liquid chromatography (HPLC) was used to measure the levels of CA in RE. Parental and GLUT4myc or GLUT1myc overexpressing L6 rat myotubes were used. Glucose uptake was assessed using [3 H]-2-deoxy-d-glucose. Total and phosphorylated levels of Akt and AMPK were measured by immunoblotting. Plasma membrane GLUT4myc and GLUT1myc levels were examined using a GLUT translocation assay. Statistics included analysis of variance (ANOVA) followed by Tukey's post-hoc test. At concentrations found in rosemary extract, CA stimulated glucose uptake in L6 myotubes. At 2.0μmol/L CA a response (226±9.62% of control, P=.001), similar to maximum insulin (201±7.86% of control, P=.001) and metformin (213±10.74% of control, P=.001) was seen. Akt phosphorylation was not affected by CA while AMPK and ACC phosphorylation was increased and the CA-stimulated glucose uptake was significantly reduced by the AMPK inhibitor compound C. Plasma membrane GLUT4 or GLUT1 glucose transporter levels were not affected by CA. Our study shows increased muscle cell glucose uptake and AMPK activation by low CA concentrations, found in rosemary extract, indicating that CA may be responsible for the antihyperglycemic properties of rosemary extract seen in vivo.

Treatment to streptozotocin hyperglycemic rats with Ibervillea sonorae root extract increases GLUT4 plasma membrane translocation in thigh muscle tissue

Treatment to streptozotocin hyperglycemic rats with Ibervillea sonorae root extract increases GLUT4 plasma membrane translocation in thigh muscle tissue

Short-Term Detraining does not Change Insulin Sensitivity and RBP4 in Rodents Previously Submitted to Aerobic Exercise.

Elevated serum retinol binding protein 4 (RBP4) levels were previously described in insulin-resistance states. Exercise training can improve insulin sensitivity and RBP4, but the time-response effect of exercise detraining on RBP4 has not been studied. Thus, we examined the effects of exercise training and short-term detraining on insulin resistance, serum RBP4 levels, and GLUT4 expression in spontaneously hypertensive rats (SHR). Thirty-two male SHR, 4 months old, were submitted to 10-week treadmill training, 5 times/week or kept sedentary, followed by a 2- and 4-day detraining period. Body weight, insulin tolerance test, maximum speed in a maximal exercise test, serum RBP4 (ELISA), and epididymal fat GLUT4 expression (Western blot) were measured. Although all rats gained weight (43%, p=0.004) only the trained group showed a reduction (p<0.001) of epididymal fat weight. Detraining did not change these parameters. Exercise training increased insulin sensitivity (26%, p=0.001) and maximum exercise capacity (80%, p<0.001), benefits not lost after detraining. RBP4 levels were reduced in response to exercise training (45%, p<0.001); detraining did not change these benefits. Trained rats had increased GLUT4 expression (microsomal, ~226%; p<0.001 and plasma membrane, ~55%; p=0.011). A slight reduction in GLUT4 expression in the plasma membrane (~28%, p=0.041), but not in the microsomal fraction, was observed after 4 days of detraining. Exercise training is associated with reduced RBP4 levels, increased insulin sensitivity, and epididymal fat GLUT4 expression. Even short periods of detraining (4 days) were shown to be associated with reversal of higher plasma membrane GLUT4.

Rab GAPs AS160 and Tbc1d1 play nonredundant roles in the regulation of glucose and energy homeostasis in mice.

The related Rab GTPase-activating proteins (Rab GAPs) AS160 and Tbc1d1 regulate the trafficking of the glucose transporter GLUT4 that controls glucose uptake in muscle and fat cells and glucose homeostasis. AS160- and Tbc1d1-deficient mice exhibit different adipocyte- and skeletal muscle-specific defects in glucose uptake, GLUT4 expression and trafficking, and glucose homeostasis. A recent study analyzed male mice with simultaneous deletion of AS160 and Tbc1d1 (AS160(-/-)/Tbc1d1(-/-) mice). Herein, we describe abnormalities in male and female AS160(-/-)/Tbc1d1(-/-) mice on another strain background. We confirm the earlier observation that GLUT4 expression and glucose uptake defects of single-knockout mice join in AS160(-/-)/Tbc1d1(-/-) mice to affect all skeletal muscle and adipose tissues. In large mixed fiber-type skeletal muscles, changes in relative basal GLUT4 plasma membrane association in AS160(-/-) and Tbc1d1(-/-) mice also combine in AS160(-/-)/Tbc1d1(-/-) mice. However, we found different glucose uptake abnormalities in isolated skeletal muscles and adipocytes than reported previously, resulting in different interpretations of how AS160 and Tbc1d1 regulate GLUT4 translocation to the cell surface. In support of a larger role for AS160 in glucose homeostasis, in contrast with the previous study, we find similarly impaired glucose and insulin tolerance in AS160(-/-)/Tbc1d1(-/-) and AS160(-/-) mice. However, in vivo glucose uptake abnormalities in AS160(-/-)/Tbc1d1(-/-) skeletal muscles differ from those observed previously in AS160(-/-) mice, indicating additional defects due to Tbc1d1 deletion. Similar to AS160- and Tbc1d1-deficient mice, AS160(-/-)/Tbc1d1(-/-) mice show sex-specific abnormalities in glucose and energy homeostasis. In conclusion, our study supports nonredundant functions for AS160 and Tbc1d1.

Periapical lesions decrease Akt serine phosphorylation and plasma membrane GLUT4 content in rat skeletal muscle.

Periapical lesion (PL) promotes insulin resistance; however, the mechanisms underlying this alteration are not fully understood. Therefore, in this study, we aimed to evaluate the Akt serine phosphorylation status and GLUT4 expression levels in the gastrocnemius muscle (GM) of rats with PL. Male Wistar rats (n = 42) were distributed equally into control (CN) and PL groups. The pulpal tissue of the PL group rats was exposed to the oral environment for 30days. Thereafter, glucose and insulin levels were assessed, followed by homeostasis model assessment of insulin resistance (HOMA-IR). The Akt serine phosphorylation and GLUT4 levels of microsomal (M) and plasma membrane (PM) fractions were evaluated by western blotting and analyzed statistically. Compared to CN group rats, PL group rats had lower insulin sensitivity (as observed by HOMA-IR), lower Akt serine phosphorylation status after insulin stimulus, and lower GLUT4 levels in the PM fraction. However, the M fraction in the PL group did not differ significantly from that of the CN group. PL decreases insulin sensitivity, Akt phosphorylation, and PM GLUT4 content. The present study indicates that preventing endodontic disease can thwart insulin resistance.

α1A-Adrenergic receptor prevents cardiac ischemic damage through PKCδ/GLUT1/4-mediated glucose uptake

While α1-adrenergic receptors (ARs) have been previously shown to limit ischemic cardiac damage, the mechanisms remain unclear. Most previous studies utilized low oxygen conditions in addition to ischemic buffers with glucose deficiencies, but we discovered profound differences if the two conditions are separated. We assessed both mouse neonatal and adult myocytes and HL-1 cells in a series of assays assessing ischemic damage under hypoxic or low glucose conditions. We found that α1-AR stimulation protected against increased lactate dehydrogenase release or Annexin V+ apoptosis under conditions that were due to low glucose concentration not to hypoxia. The α1-AR antagonist prazosin or nonselective protein kinase C (PKC) inhibitors blocked the protective effect. α1-AR stimulation increased 3H-deoxyglucose uptake that was blocked with either an inhibitor to glucose transporter 1 or 4 (GLUT1 or GLUT4) or small interfering RNA (siRNA) against PKCδ. GLUT1/4 inhibition also blocked α1-AR-mediated protection from apoptosis. The PKC inhibitor rottlerin or siRNA against PKCδ blocked α1-AR stimulated GLUT1 or GLUT4 plasma membrane translocation. α1-AR stimulation increased plasma membrane concentration of either GLUT1 or GLUT4 in a time-dependent fashion. Transgenic mice overexpressing the α1A-AR but not α1B-AR mice displayed increased glucose uptake and increased GLUT1 and GLUT4 plasma membrane translocation in the adult heart while α1A-AR but not α1B-AR knockout mice displayed lowered glucose uptake and GLUT translocation. Our results suggest that α1-AR activation is anti-apoptotic and protective during cardiac ischemia due to glucose deprivation and not hypoxia by enhancing glucose uptake into the heart via PKCδ-mediated GLUT translocation that may be specific to the α1A-AR subtype.

Tropomodulin3 is a novel Akt2 effector regulating insulin-stimulated GLUT4 exocytosis through cortical actin remodeling.

Akt2 and its downstream effectors mediate insulin-stimulated GLUT4-storage vesicle (GSV) translocation and fusion with the plasma membrane (PM). Using mass spectrometry, we identify actin-capping protein Tropomodulin 3 (Tmod3) as an Akt2-interacting partner in 3T3-L1 adipocytes. We demonstrate that Tmod3 is phosphorylated at Ser71 on insulin-stimulated Akt2 activation, and Ser71 phosphorylation is required for insulin-stimulated GLUT4 PM insertion and glucose uptake. Phosphorylated Tmod3 regulates insulin-induced actin remodelling, an essential step for GSV fusion with the PM. Furthermore, the interaction of Tmod3 with its cognate tropomyosin partner, Tm5NM1 is necessary for GSV exocytosis and glucose uptake. Together these results establish Tmod3 as a novel Akt2 effector that mediates insulin-induced cortical actin remodelling and subsequent GLUT4 membrane insertion. Our findings suggest that defects in cytoskeletal remodelling may contribute to impaired GLUT4 exocytosis and glucose uptake.

Chromium chloride increases insulin-stimulated glucose uptake in the perfused rat hindlimb.

To determine the effect of chromium chloride (CrCl3 ) on healthy skeletal muscle glucose uptake in the absence and presence of submaximal insulin using the rat hindlimb perfusion technique. Sprague-Dawley rats were randomly assigned to an experimental group: basal (Bas), chromium chloride (Cr), submaximal insulin (sIns) or chromium chloride plus submaximal insulin (Cr-sIns). Insulin significantly increased [H(3)]-2 deoxyglucose (2-DG) uptake in the gastrocnemius muscles. Additionally, Cr-sIns displayed greater rates of 2-DG uptake than sIns (Cr-sIns 6.86 ± 0.74 μmol g h(-1) vs. sIns 4.83 ± 0.42 μmol g h(-1)). There was no difference between Cr and Bas treatment groups. It has been speculated that chromium works to increase glucose uptake by increasing insulin signalling. We found that Akt and AS160 phosphorylation was increased in the sINS treatment group, while chromium treatment had no additional effect on Akt or AS160 phosphorylation in the absence or presence of insulin. Cr-sIns significantly increased plasma membrane GLUT4 concentration above that of sIns (Cr-sIns 72.22 ± 12.7%, sIns 53.4 ± 6.1%), but in the absence of insulin, chromium had no effect. Exposure of healthy skeletal muscle to chromium may increase skeletal muscle insulin-stimulated GLUT4 translocation and glucose uptake. However, these effects do not appear to result from enhanced insulin signalling proximal to AS160.

Grb10 Deletion Enhances Muscle Cell Proliferation, Differentiation and GLUT4 Plasma Membrane Translocation

Grb10 is an intracellular adaptor protein which binds directly to several growth factor receptors, including those for insulin and insulin-like growth factor receptor-1 (IGF-1), and negatively regulates their actions. Grb10-ablated (Grb10(-/-) ) mice exhibit improved whole body glucose homeostasis and an increase in muscle mass associated specifically with an increase in myofiber number. This suggests that Grb10 may act as a negative regulator of myogenesis. In this study, we investigated in vitro, the molecular mechanisms underlying the increase in muscle mass and the improved glucose metabolism. Primary muscle cells isolated from Grb10(-/-) mice exhibited increased rates of proliferation and differentiation compared to primary cells isolated from wild-type mice. The improved proliferation capacity was associated with an enhanced phosphorylation of Akt and ERK in the basal state and changes in the expression of key cell cycle progression markers involved in regulating transition of cells from the G1 to S phase (e.g., retinoblastoma (Rb) and p21). The absence of Grb10 also promoted a faster transition to a myogenin positive, differentiated state. Glucose uptake was higher in Grb10(-/-) primary myotubes in the basal state and was associated with enhanced insulin signaling and an increase in GLUT4 translocation to the plasma membrane. These data demonstrate an important role for Grb10 as a link between muscle growth and metabolism with therapeutic implications for diseases, such as muscle wasting and type 2 diabetes.

Strain‐Dependent Differences for Suppression of Insulin‐Stimulated Glucose Uptake in Skeletal and Cardiac Muscle by Ethanol

Chronic ethanol (EtOH) consumption impairs the ability of insulin to suppress hepatic glucose production in a strain-dependent manner, with hepatic insulin resistance being greater in Long-Evans (LE) than Sprague-Dawley (SD) rats. We assessed whether strain differences exist for whole-body and tissue glucose uptake under basal and insulin-stimulated conditions and whether they were associated with coordinate strain-dependent elevations in muscle cytokines. Male rats (160 g) were provided the Lieber-DeCarli EtOH-containing (36% total energy) diet or pair-fed a control diet for 8 weeks. Rats were studied in the basal state or during a euglycemic hyperinsulinemic clamp, and whole-body glucose flux assessed using (3) H-glucose and in vivo tissue glucose uptake by (14) C-2-deoxyglucose. EtOH impaired whole-body insulin-mediated glucose uptake (IMGU) more in SD than LE rats. This difference was due to impaired IMGU by gastrocnemius and heart in EtOH-fed SD versus LE rats. However, decreased IMGU in adipose tissue (epididymal and perirenal) produced by EtOH was comparable between strains. EtOH-induced insulin resistance in muscle from SD rats was associated with reduced AKT and AS160 phosphorylation and plasma membrane-localized GLUT4 protein as well as enhanced phosphorylation of c-Jun N-terminal kinase (JNK) and IRS-1 (S307), changes which were absent in muscle from LE rats. EtOH increased tumor necrosis factor alpha (TNFα) mRNA in gastrocnemius and fat under basal conditions in both SD and LE rats; however, hyperinsulinemia decreased TNFα in skeletal muscle from LE, but not SD rats. Interleukin (IL)-6 mRNA in gastrocnemius was increased under basal conditions and increased further in response to insulin in SD rats, but no EtOH- or insulin-induced change was detected in muscle IL-6 of LE rats. These data indicate strain-dependent differences in EtOH-induced IMGU in skeletal and cardiac muscle, but not fat, associated with sustained increases in TNFα and IL-6 mRNA and JNK activation and decreased plasma membrane GLUT4 in response to insulin.

DHHC17 Palmitoylates ClipR-59 and Modulates ClipR-59 Association with the Plasma Membrane

ClipR-59 interacts with Akt and regulates Akt compartmentalization and Glut4 membrane trafficking in a plasma membrane association-dependent manner. The association of ClipR-59 with plasma membrane is mediated by ClipR-59 palmitoylation at Cys534 and Cys535. To understand the regulation of ClipR-59 palmitoylation, we have examined all known mammalian DHHC palmitoyltransferases with respect to their ability to promote ClipR-59 palmitoylation. We found that, among 23 mammalian DHHC palmitoyltransferases, DHHC17 is the major ClipR-59 palmitoyltransferase, as evidenced by the fact that DHHC17 interacted with ClipR-59 and palmitoylated ClipR-59 at Cys534 and Cys535. By palmitoylating ClipR-59, DHHC17 directly regulates ClipR-59 plasma membrane association, as ectopic expression of DHHC17 increased whereas silencing of DHHC17 reduced the levels of ClipR-59 associated with plasma membrane. We have also examined the role of DHHC17 in Akt signaling and found that silencing of DHHC17 in 3T3-L1 adipocytes decreased the levels of Akt as well as ClipR-59 on the plasma membrane and impaired insulin-dependent Glut4 membrane translocation. We suggest that DHHC17 is a ClipR-59 palmitoyltransferase that modulates ClipR-59 plasma membrane binding, thereby regulating Akt signaling and Glut4 membrane translocation in adipocytes.

Heart energy metabolism impairment in Western-diet induced obese mice

Nutritional transition has contributed to growing obesity, mainly by changing eating habits of the population. The mechanisms by which diet-induced obesity leads to cardiac injury are not completely understood, but it is known that obesity is associated to impaired cardiac function and energy metabolism, increasing morbidity and mortality. Therefore, our study aimed to investigate the mechanisms underlying cardiac metabolism impairment related to Western diet-induced obesity. After weaning, male Swiss mice were fed a Western diet for 16 weeks in order to induce obesity. After this period, the content of proteins involved in heart energy metabolism GLUT1, cytosolic lysate and plasma membrane GLUT4, AMPK, pAMPK, IRβ, IRS-1, PGC-1α, CPT1 and UCP2 was evaluated. Also, the oxidative phosphorylation of myocardial fibers was measured by high-resolution respirometry. Mice in the Western diet group (WG) presented altered biometric parameters compared to those in control group, including higher body weight, increased myocardial lipid deposition and glucose intolerance, which demonstrate the obesogenic role of Western diet. WG presented increased CPT1 and UCP2 contents and decreased IRS-1, plasma membrane GLUT4 and PGC-1α contents. In addition, WG presented cardiac mitochondrial dysfunction and reduced biogenesis, demonstrating a lower capacity of carbohydrates and fatty acid oxidation and also decreased coupling between oxidative phosphorylation and adenosine triphosphate synthesis. Cardiac metabolism impairment related to Western diet-induced obesity is probably due to damaged myocardial oxidative capacity, reduced mitochondrial biogenesis and mitochondria uncoupling, which compromise the bioenergetic metabolism of heart.

Impaired Tethering and Fusion of GLUT4 Vesicles in Insulin-Resistant Human Adipose Cells

Systemic glucose homeostasis is profoundly influenced by adipose cell function. Here we investigated GLUT4 dynamics in living adipose cells from human subjects with varying BMI and insulin sensitivity index (Si) values. Cells were transfected with hemagglutinin (HA)-GLUT4-green fluorescent protein (GFP)/mCherry (red fluorescence), and were imaged live using total internal reflection fluorescence and confocal microscopy. HA-GLUT4-GFP redistribution to the plasma membrane (PM) was quantified by surface-exposed HA epitope. In the basal state, GLUT4 storage vesicle (GSV) trafficking to and fusion with the PM were invariant with donor subject Si, as was total cell-surface GLUT4. In cells from insulin-sensitive subjects, insulin augmented GSV tethering and fusion approximately threefold, resulting in a corresponding increase in total PM GLUT4. However, with decreasing Si, these effects diminished progressively. All insulin-induced effects on GLUT4 redistribution and trafficking correlated strongly with Si and only weakly with BMI. Thus, while basal GLUT4 dynamics and total cell-surface GLUT4 are intact in human adipose cells, independent of donor Si, cells from insulin-resistant donors show markedly impaired GSV tethering and fusion responses to insulin, even after overnight culture. This altered insulin responsiveness is consistent with the hypothesis that adipose cellular dysfunction is a primary contributor to systemic metabolic dysfunction.

SLC2A4 Gene: A Promising Target for Pharmacogenomics of Insulin Resistance

The SLC2A4 gene The GLUT4 protein, encoded by the solute carrier SLC2A4 gene in humans and Slc2a4 in mice and rats, is preferentially expressed in differentiated myotubes and adipocytes. Described in the early 1990s, GLUT4 is considered the insulin-sensitive glucose transporter based on its particular characteristic of being inserted in membranes of intracellular vesicles, which under insulin stimulus translocate to the plasma membrane, increasing the glucose uptake by these cells; the basis of postprandial glycemic control. Insulin-induced GLUT4 translocation is described as a much more robust phenomenon than it really is in vivo. In vitro translocation compares maximal insulin effect with the socalled basal condition in which insulin is absent. Whenever one tries to compare maximal insulin effect with basal physiological insulin concentrations, GLUT4 translocation is very low [1]. Besides, in skeletal muscle, GLUT4 translocation is also stimulated by muscle contraction, and contractile tonus is enough to induce great levels of translocation. However, a small relative translocation of GLUT4 (e.g., small percentage related to the total content) is able to significantly increase glucose uptake, avoiding impaired postprandial hyperglycemia. Unbelievably, several studies concerning GLUT4 translocation simply analyze the absolute amount of GLUT4 in plasma membrane. We point out that insulin induces vesicle translocation; and reduction in SLC2A4 expression (decreasing GLUT4 density in the vesicles) can explain decreased insulin-stimulated plasma membrane GLUT4 content, despite a preserved translocation system [2]. Finally, although some studies have proposed that molecular changes in the GLUT4 protein might alter its kinetics of transport, no consistent data has confirmed this hypothesis [3,4].

Expression and phosphorylation of the AS160_v2 splice variant supports GLUT4 activation and the Warburg effect in multiple myeloma

BackgroundMultiple myeloma (MM) is a fatal plasma cell malignancy exhibiting enhanced glucose consumption associated with an aerobic glycolytic phenotype (i.e., the Warburg effect). We have previously demonstrated that myeloma cells exhibit constitutive plasma membrane (PM) localization of GLUT4, consistent with the dependence of MM cells on this transporter for maintenance of glucose consumption rates, proliferative capacity, and viability. The purpose of this study was to investigate the molecular basis of constitutive GLUT4 plasma membrane localization in MM cells.FindingsWe have elucidated a novel mechanism through which myeloma cells achieve constitutive GLUT4 activation involving elevated expression of the Rab-GTPase activating protein AS160_v2 splice variant to promote the Warburg effect. AS160_v2-positive MM cell lines display constitutive Thr642 phosphorylation, known to be required for inactivation of AS160 Rab-GAP activity. Importantly, we show that enforced expression of AS160_v2 is required for GLUT4 PM translocation and activation in these select MM lines. Furthermore, we demonstrate that ectopic expression of a full-length, phospho-deficient AS160 mutant is sufficient to impair constitutive GLUT4 cell surface residence, which is characteristic of MM cells.ConclusionsThis is the first study to tie AS160 de-regulation to increased glucose consumption rates and the Warburg effect in cancer. Future studies investigating connections between the insulin/IGF-1/AS160_v2/GLUT4 axis and FDG-PET positivity in myeloma patients are warranted and could provide rationale for therapeutically targeting this pathway in MM patients with advanced disease.