The measurement of GLUT4 translocation in 3T3-L1 adipocytes

Type 2 diabetes (T2D) is one of the fastest growing threats to human health in westernised and developing countries and is associated with central obesity, atherosclerosis, dyslipidaemia, hyperinsulinaemia and  hypertension. Insulin resistance, defined as a diminished response to ordinary levels of circulating insulin in one or more peripheral tissues, is an integral feature of T2D pathophysiology. This includes an impairment of insulin to inhibit hepatic glucose output and to stimulate glucose disposal into muscle and fat. While insulin is responsible for a number of specific biological responses, stimulation of glucose transport is critical for the maintenance of glucose homeostasis. The primary mechanism for insulin stimulation of glucose uptake into muscle and fat is the translocation of glucose transporter 4 (GLUT4) to the cell surface from intracellular storage vesicles within the cell. A major advantage in focussing on insulin regulation of glucose transport is that this represents the endpoint of multiple upstream signalling pathways. This chapter describes the measurement of GLUT4 translocation in cultured cells and its potential application for both  mechanistic and therapeutic studies.

Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.

The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

Visualization and quantitation of GLUT4 translocation in human skeletal muscle following glucose ingestion and exercise

Insulin- and contraction-stimulated increases in glucose uptake into skeletal
muscle occur in part as a result of the translocation of glucose transporter 4
(GLUT4) from intracellular stores to the plasma membrane (PM). This study
aimed to use immunofluorescence microscopy in human skeletal muscle to
quantify GLUT4 redistribution from intracellular stores to the PM in response
to glucose feeding and exercise. Percutaneous muscle biopsy samples were
taken from the m. vastus lateralis of ten insulin-sensitive men in the basal
state and following 30 min of cycling exercise (65% VO2 max). Muscle biopsy
samples were also taken from a second cohort of ten age-, BMI- and VO2 maxmatched insulin-sensitive men in the basal state and 30 and 60 min following
glucose feeding (75 g glucose). GLUT4 and dystrophin colocalization, measured
using the Pearson’s correlation coefficient, was increased following
30 min of cycling exercise (baseline r = 0.47 0.01; post exercise
r = 0.58 0.02; P < 0.001) and 30 min after glucose ingestion (baseline
r = 0.42 0.02; 30 min r = 0.46 0.02; P < 0.05). Large and small GLUT4
clusters were partially depleted following 30 min cycling exercise, but not
30 min after glucose feeding. This study has, for the first time, used immunofluorescence microscopy in human skeletal muscle to quantify increases in
GLUT4 and dystrophin colocalization and depletion of GLUT4 from large
and smaller clusters as evidence of net GLUT4 translocation to the PM.

Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins

A major physiological role of insulin is the regulation of glucose uptake into skeletal and cardiac muscle and adipose tissue, mediated by an insulin-stimulated translocation of GLUT4 glucose transporters from an intracellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells, a process that involves SNARE-complex proteins. Recently, several SNARE proteins were found in adipocytes: vesicle-associated membrane protein (VAMP-2), its related homologue cellubrevin, and syntaxin-4. In this report we show that treatment of permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which selectively cleaves VAMP-2 and cellubrevin, inhibited the ability of insulin to stimulate translocation of GLUT4 vesicles to the plasma membrane. Furthermore, treatment of the permeabilized adipocytes with glutathione S-transferase fusion proteins encoding soluble forms of VAMP-2 or syntaxin-4 also effectively blocked insulin-regulated GLUT4 translocation. These results provide evidence of a functional role for SNARE-complex proteins in insulin-stimulated glucose uptake and suggest that adipocytes utilize a mechanism of regulating vesicle docking and fusion analogous to that found in neuroendocrine tissues.

Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

The acute effects of contraction and insulin on the glucose transport and GLUT4 glucose transporter translocation were investigated in rat soleus muscles by using a 3-O-methylglucose transport assay and the sensitive exofacial labeling technique with the impermeant photoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-y loxy)-2- propylamine (ATB-BMPA), respectively. Addition of wortmannin, which inhibits phosphatidylinositol 3-kinase, reduced insulin-stimulated glucose transport (8.8 +/- 0.5 mumol per ml per h vs. 1.4 +/- 0.1 mumol per ml per h) and GLUT4 translocation [2.79 +/- 0.20 pmol/g (wet muscle weight) vs. 0.49 +/- 0.05 pmol/g (wet muscle weight)]. In contrast, even at a high concentration (1 microM), wortmannin had no effect on contraction-mediated glucose uptake (4.4 +/- 0.1 mumol per ml per h vs. 4.1 +/- 0.2 mumol per ml per h) and GLUT4 cell surface content [1.75 +/- 0.16 pmol/g (wet muscle weight) vs. 1.52 +/- 0.16 pmol/g (wet muscle weight)]. Contraction-mediated translocation of the GLUT4 transporters to the cell surface was closely correlated with the glucose transport activity and could account fully for the increment in glucose uptake after contraction. The combined effects of contraction and maximal insulin stimulation were greater than either stimulation alone on glucose transport activity (11.5 +/- 0.4 mumol per ml per h vs. 5.6 +/- 0.2 mumol per ml per h and 9.0 +/- 0.2 mumol per ml per h) and on GLUT4 translocation [4.10 +/- 0.20 pmol/g (wet muscle weight) vs. 1.75 +/- 0.25 pmol/g (wet muscle weight) and 3.15 +/- 0.18 pmol/g (wet muscle weight)]. The results provide evidence that contraction stimulates translocation of GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

Cigarette smoke exposure severely reduces peripheral insulin sensitivity without changing GLUT4 expression in oxidative muscle of Wistar rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

T-3 Rapidly Increases SLC2A4 Gene Expression and GLUT4 Trafficking to the Plasma Membrane in Skeletal Muscle of Rat and Improves Glucose Homeostasis

Background: Glucose transporter 4 (GLUT4) is highly expressed in muscle and fat tissue, where triiodothyronine (T-3) induces solute carrier family 2 facilitated glucose transporter member 4 (SLC2A4) gene transcription. T-3 was also shown to rapidly increase glucose uptake in myocytes exposed to cycloheximide, indicating that it might act nongenomically to regulate GLUT4 availability. We tested this hypothesis by evaluating, in thyroidectomized rats (Tx rats), the acute and/or chronic T-3 effects on GLUT4 mRNA expression and polyadenylation, protein content, and trafficking to the plasma membrane (PM) in skeletal muscle, as well as on blood glucose disappearance rate (kITT) after insulin administration. Methods: Rats were surgically thyroidectomized and treated with T-3 (0.3 to 100 mu g/100 g body weight) from 10 minutes to 5 days, and killed thereafter. Sham-operated (SO) rats were used as controls. Total RNA was extracted from the skeletal muscles (soleus [SOL] and extensorum digitalis longus [EDL]) and subjected to Northern blotting analysis using rat GLUT4 cDNA probe. Total protein was extracted and subjected to specific centrifugations for subcellular fractionation, and PM as well as microsomal (M) fractions were subjected to Western blotting analysis, using anti-GLUT4 antiserum as a probe. GLUT4 mRNA polyadenylation was examined by a rapid amplification of cDNA ends-poly(A) test (RACE-PAT). Results: Thyroidectomy reduced skeletal muscle GLUT4 mRNA, mRNA poly(A) tail length, protein content, and trafficking to the PM, as well as the kITT. The acute T-3 treatment rapidly (30 minutes) increased all these parameters compared with Tx rats. The 5-day T-3 treatment increased GLUT4 mRNA and protein expression, and restored GLUT4 trafficking to the PM and kITT to SO values. Conclusions: The results presented here show for the first time that, in parallel to its transcriptional action on the SLC2A4 gene, T-3 exerts a rapid post-transcriptional effect on GLUT4 mRNA polyadenylation, which might increase transcript stability and translation efficiency, leading to the increased GLUT4 content and availability to skeletal muscle, as well as on GLUT4 translocation to the PM, improving the insulin sensitivity, as shown by the kITT.

Development of an in vitro reconstitution assay for glucose transporter 4 translocation

In an attempt to define the mechanism of insulin-regulated glucose transporter 4 (Glut4) translocation, we have developed an in vitro reconstitution assay. Donor membranes from 3T3-L1 adipocytes transfected with mycGlut4 were incubated with plasma membrane (PM) from nontransfected 3T3-L1 cells, and the association was assessed by using two types of centrifugation assays. Association of mycGlut4 vesicles derived from donor membranes with the PM was concentration-, temperature-, time-, and Ca2+-dependent but ATP-independent. Addition of a syntaxin 4 fusion protein produced a biphasic response, increasing association at low concentration and inhibiting association at higher concentrations. PM from insulin-stimulated cells showed an enhanced association as compared with those from untreated cells. Use of donor membranes from insulin-stimulated cells further enhanced the association and also enhanced association to the PM from isolated rat adipocytes. Addition of cytosol, GTP, or guanosine 5′-[γ-thio]triphosphate decreased the association. In summary, insulin-induced Glut4 translocation can be reconstituted in vitro to a limited extent by using isolated membranes. This association appears to involve protein–protein interactions among the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. Finally, the ability of insulin to enhance association depends on insulin-induced changes in the PM and, to a lesser extent, in the donor membranes.

Insulin Action on GLUT4 Traffic Visualized in Single 3T3-L1 Adipocytes by Using Ultra-fast MicroscopyV⃞

A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20–30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5–7 min at 37°C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.

Biological effects of resveratrol on skeletal muscle cells

Resveratrol, a polyphenol found in red wine, has been reported to have antithrombotic, antiatherogenic, and anticancer properties both in vitro and III VIVO. However, possible antidiabetic properties of resveratrol have not been examined. The objective of this study was to investigate the direct effects of resveratrol on basal and insulin-stimulated glucose uptake and to elucidate its mechanism of action in skeletal muscle cells. In addition, the effects of resveratrol on basal and insulin- stimulated amino acid transport and mitogenesis were also examined. Fully differentiated L6 rat skeletal muscle cells were incubated with resveratrol concentrations ranging from 1 to 250 IlM for 15 to 120 min. Maximum stimulation, 201 ± 8.90% of untreated control, (p<0.001), of2eH] deoxy- D- glucose (2DG) uptake was seen with 100 IlM resveratrol after 120 min. Acute, 30 min, exposure of the cells to 100 nM insulin stimulated 2DG uptake to 226 ± 12.52% of untreated control (p<0.001). This appears to be a specific property of resveratrol that is not shared by structurally similar antioxidants such as quercetin and rutin, both of which did not have any stimulatory effect. Resveratrol increased the response of the cells to submaximal insulin concentrations but did not alter the maximum insulin response. Resveratrol action did not require insulin and was not blocked by the protein synthesis inhibitor cycloheximide. L Y294002 and wortmannin, inhibitors of PI3K, abolished both insulin and resveratrolstimulated glucose uptake while phosphorylation of AktlPKB, ERK1I2, JNK1I2, and p38 MAPK were not increased by resveratrol. Resveratrol did not stimulate GLUT4 transporter translocation in GLUT4cmyc overexpressing cells, in contrast to the significant translocation observed with insulin. Furthermore, resveratrol- stimulated glucose transport was not blocked by the presence of the protein kinase C (PKC) inhibitors BIMI and G06983. Despite that, resveratrol- induced glucose transport required an intact actin network, similar to insulin. In contrast to the stimulatory effect seen with resveratrol for glucose transport, e4C]methylaminoisobutyric acid (MeAIB) transport was inhibited. Significant reduction of MeAIB uptake was seen only with 100uM resveratrol (74.2 ± 6.55% of untreated control, p<0.05), which appeared to be maximum. In parallel experiments, insulin (100 nM, 30 min) increased MeAIB transport by 147 ± 5.77% (p<0.00l) compared to untreated control. In addition, resveratrol (100 JlM, 120 min) completely abolished insulin- stimulated amino acid transport (103 ± 7.35% of untreated control,p>0.05). Resveratrol also inhibited cell proliferation in L6 myoblasts with maximal inhibition of eH]thymidine incorporation observed with resveratrol at 50 J.LM after 24 hours (8 ± 1.59% of untreated control, pGLUT4 translocation. However, unlike glucose transport, resveratrol inhibits both basal and insulin- stimulated amino acid transport and mitogenesis.

The effect of insulin and exercise on c-Cbl protein abundance and phosphorylation in insulin-resistant skeletal muscle in lean and obese Zucker rats.

Aims/hypothesis: Recruitment of the protein c-Cbl to the insulin receptor (IR) and its tyrosine phosphorylation via a pathway that is independent from phosphatidylinositol 3prime-kinase is necessary for insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. The activation of this pathway by insulin or exercise has yet to be reported in skeletal muscle. Methods: Lean and obese Zucker rats were randomly assigned to one of three treatment groups: (i) control, (ii) insulin-stimulated or (iii) acute, exhaustive exercise. Hind limb skeletal muscle was removed and the phosphorylation state of IR, Akt and c-Cbl measured.  Results:   Insulin receptor phosphorylation was increased 12-fold after insulin stimulation (p<0.0001) in lean rats and threefold in obese rats. Acute exercise had no effect on IR tyrosine phosphorylation. Similar results were found for serine phosphorylation of Akt. Exercise did not alter c-Cbl tyrosine phosphorylation in skeletal muscle of lean or obese rats. However, in contrast to previous studies in adipocytes, c-Cbl tyrosine phosphorylation was reduced after insulin treatment (p<0.001). Conclusions/interpretation: We also found that c-Cbl associating protein expression is relatively low in skeletal muscle of Zucker rats compared to 3T3-L1 adipocytes and this could account for the reduced c-Cbl tyrosine phosphorylation after insulin treatment. Interestingly, basal levels of c-Cbl tyrosine phosphorylation were higher in skeletal muscle from insulin-resistant Zucker rats (p<0.05), but the physiological relevance is not clear. We conclude that the regulation of c-Cbl phosphorylation in skeletal muscle differs from that previously reported in adipocytes.

Nocodazole inhibits insulin-stimulated glusocs transport in 3T3-L1 adipocytes via a microtubule independant mechanism

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 µM nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 µM nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 µM) significantly inhibited basal and insulin-stimulated glucose uptake in adipocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 µM nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 µM nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 °C. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.

The effect of exercise and insulin on AS160 phosphorylation and 14-3-3 binding capacity in human skeletal muscle

AS160 is an Akt substrate of 160 kDa implicated in the regulation of both insulin- and contraction-mediated GLUT4 translocation and glucose uptake. The effects of aerobic exercise and subsequent insulin stimulation on AS160 phosphorylation and the binding capacity of 14-3-3, a novel protein involved in the dissociation of AS160 from GLUT4 vesicles, in human skeletal muscle are unknown. Hyperinsulinemic-euglycemic clamps were performed on seven men at rest and immediately and 3 h after a single bout of cycling exercise. Skeletal muscle biopsies were taken before and after the clamps. The insulin sensitivity index calculated during the final 30 min of the clamp was 8.0 ± 0.8, 9.1 ± 0.5, and 9.2 ± 0.8 for the rest, postexercise, and 3-h postexercise trials, respectively. AS160 phosphorylation increased immediately after exercise and remained elevated 3 h after exercise. In contrast, the 14-3-3 binding capacity of AS160 and phosphorylation of Akt and AMP-activated protein kinase were only increased immediately after exercise. Insulin increased AS160 phosphorylation and 14-3-3 binding capacity and insulin receptor substrate-1 and Akt phosphorylation, but the response to insulin was not enhanced by prior exercise. In conclusion, the 14-3-3 binding capacity of AS160 is increased immediately after acute exercise in human skeletal muscle, but this is not maintained 3 h after exercise completion despite sustained AS160 phosphorylation. Insulin increases AS160 phosphorylation and 14-3-3 binding capacity, but prior exercise does not appear to enhance the response to insulin.

Resistance exercise and insulin regulate AS160 and interaction with 14-3-3 in human skeletal muscle

A single bout of aerobic exercise can enhance insulin action, but whether a similar effect occurs after resistance exercise is unknown. Hyperinsulinemic-euglycemic clamps were performed on eight male subjects at rest and after a single bout and three repeated bouts of resistance exercise over 7 days. Skeletal muscle biopsies were taken before and after the clamp and immediately after a single exercise bout. Whole-body insulin action measured by glucose infusion rate decreased (P < 0.05) after a single exercise bout, whereas in response to repeated bouts of resistance exercise, the glucose infusion rate was similar to the rest trial. In skeletal muscle, Akt substrate of 160 kDa (AS160) phosphorylation, an Akt substrate implicated in the regulation of GLUT4 translocation, and its interaction with 14-3-3 was decreased (P < 0.05) only after a single exercise bout. Insulin increased (P < 0.05) phosphorylation of AS160 and its interaction with 14-3-3, but the insulin response was not influenced by resistance exercise. Phosphorylation of insulin receptor substrate-1 and Akt were similar to changes in AS160 phosphorylation after exercise and/or insulin. In conclusion, a single bout of resistance exercise impairs whole-body insulin action. Regulation of AS160 and interaction with 14-3-3 in skeletal muscle are influenced by resistance exercise and insulin but do not fully explain the effect of resistance exercise on whole-body insulin action.