Geoffrey D. Holman

Emerging role for AS160/TBC1D4 and TBC1D1 in the regulation of GLUT4 traffic

Vesicular traffic of the glucose transporter GLUT4 occurs in response to insulin, muscle contraction, and metabolic stimuli that lead to changes in the energy status of the cell. These stimuli are associated with linked kinase cascades that lead to changes in glucose uptake that meet the energy challenges imposed on the highly regulated cell types in insulin-responsive tissues. The need to mechanistically link these kinase-associated stimuli to identifiable intermediates in vesicular traffic has long been known but has been difficult to fulfill. The Rab-GTPase-activating proteins AS160 and TBC1D1 have now emerged as strong candidates to fill this void. Here we review the initial discovery of these proteins as phosphorylated substrates for Akt and the more recent emerging data that indicate that these proteins are substrates for additional kinases that are downstream of contraction and energy status signaling. The mechanism of coupling these phosphorylated proteins to vesicle traffic appears to be dependent on linking to small GTPase of the Rab family. We examine the current state of a hypothesis that suggests that phosphorylation of the Rab-GTPase-activating proteins leads to increased GTP loading of Rab proteins on GLUT4 vesicles and subsequently to increased interaction with Rab effectors that control GLUT4 vesicle translocation.

Blood-brain barrier glucose transporter: effects of hypo- and hyperglycemia revisited.

Abstract : The transport of glucose across the blood‐brain barrier (BBB) is mediated by the high molecular mass (55‐kDa) isoform of the GLUT1 glucose transporter protein. In this study we have utilized the tritiated, impermeant photolabel 2‐N‐[4‐(1‐azi‐2,2,2‐trifluoroethyl)[2‐3H]propyl]‐1,3‐bis(d‐mannose‐4‐yloxy)‐2‐propylamine to develop a technique to specifically measure the concentration of GLUT1 glucose transporters on the luminal surface of the endothelial cells of the BBB. We have combined this methodology with measurements of BBB glucose transport and immunoblot analysis of isolated brain microvessels for labeled luminal GLUT1 and total GLUT1 to reevaluate the effects of chronic hypoglycemia and diabetic hyperglycemia on transendothelial glucose transport in the rat. Hypoglycemia was induced with continuous‐release insulin pellets (6 U/day) for a 12‐ to 14‐day duration ; diabetes was induced by streptozotocin (65 mg/kg i.p.) for a 14‐ to 21‐day duration. Hypoglycemia resulted in 25‐45% increases in regional BBB permeability‐surface area (PA) values for d‐[14C]glucose uptake, when measured at identical glucose concentration using the in situ brain perfusion technique. Similarily, there was a 23 ± 4% increase in total GLUT1/mg of microvessel protein and a 52 ± 13% increase in luminal GLUT1 in hypoglycemic animals, suggesting that both increased GLUT1 synthesis and a redistribution to favor luminal transporters account for the enhanced uptake. A corresponding (twofold) increase in cortical GLUT1 mRNA was observed by in situ hybridization. In contrast, no significant changes were observed in regional brain glucose uptake PA, total microvessel 55‐kDa GLUT1, or luminal GLUT1 concentrations in hyperglycemic rats. There was, however, a 30‐40% increase in total cortical GLUT1 mRNA expression, with a 96% increase in the microvessels. Neither condition altered the levels of GLUT3 mRNA or protein expression. These results show that hypoglycemia, but not hyperglycemia, alters glucose transport activity at the BBB and that these changes in transport activity result from both an overall increase in total BBB GLUT1 and an increased transporter concentration at the luminal surface.

Identification of Mammalian Vps24p as an Effector of Phosphatidylinositol 3,5-Bisphosphate-dependent Endosome Compartmentalization

Phosphatidylinositol 3,5-bisphosphate is a membrane lipid found in all eukaryotes so far studied but downstream effector proteins of this lipid have yet to be identified. Here we report the use of cDNA phage libraries in conjunction with synthetic biotinylated derivatives of phosphatidylinositol 3,5-bisphosphate in the identification of a mammalian phosphatidylinositol 3,5-bisphosphate-binding protein, mVps24p. This protein is orthologous to the Saccharomyces cerevisiae protein, Vps24p, a class-E vacuolar protein-sorting protein. Using in vitro liposome binding and competition assays, we demonstrate that mVps24p selectively binds to phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate in preference to other phosphoinositides tested. When expressed in cultured mammalian cells, full-length mVps24p is cytosolic. However, when cells expressing the full-length mVps24p are co-transfected with a mutated form of mVps4p (which is defective in ATP hydrolysis), or when a N-terminal construct of mVps24p is expressed, the class-E cellular phenotype with swollen vacuoles is induced and mVps24p is membrane-associated. Furthermore, the accumulation of the N-terminal mVps24p construct on the swollen endosomal membranes is abrogated when phosphatidylinositol 3,5-bisphosphate synthesis is blocked with wortmannin. These data provide the first direct link between phosphatidylinositol 3,5-bisphosphate and the protein machinery involved in the production of the class-E cellular phenotype. We hypothesize that accumulation of Vps24 on membranes occurs when membrane association (dependent on interaction of phosphatidylinositol 3,5-bisphosphate with the N-terminal domain of the protein) is uncoupled from membrane disassociation (driven by Vps4p).

Repeat Treatment of Obese Mice With BRL 49653, a New Potent Insulin Sensitizer, Enhances Insulin Action in White Adipocytes: Association With Increased Insulin Binding and Cell-Surface GLUT4 as Measured by Photoaffinity Labeling

(±)-5-([4-[2-Methyl-2(pyridylamino)ethoxy]phenyl]methyl) 2,4-thiazolidinedione (BRL 49653) is a new potent antidiabetic agent that improves insulin sensitivity in animal models of NIDDM. In C57BL/6 obese (ob/ob) mice, BRL 49653, included in the diet for 8 days, improved glucose tolerance. The half-maximal effective dose was 3 μmol/kg diet, which is equivalent to ∼0.1 mg/kg body wt. Improvements in glucose tolerance were accompanied by significant reductions in circulating triacylglycerol, nonesterified fatty acids, and insulin. The insulin receptor number of epididymal white adipocytes prepared from obese mice treated with BRL 49653 (30 μmol/kg diet) for 14 days was increasedtwofold. The affinity of the receptor for insulin was unchanged. In the absence of added insulin, the rates of glucose transport in adipocytes from untreated and BRL 49653-treated obese mice were similar. Insulin (73 nmol/l) produced only a 1.5-fold increase in glucose transport in adipocytes from control obese mice, whereas after BRL 49653 treatment, insulin stimulated glucose transport 2.8-fold. BRL 49653 did not alter the sensitivity of glucose transport to insulin. The increase in insulin responsiveness was accompanied by a 2.5-fold increase in the total tissue content of the glucose transporter GLUT4. Glucose transport in adipocytes from lean littermates was not altered by BRL 49653. To establish the contribution of changes in glucose transporter trafficking to the BRL 49653-mediated increase in insulin action, the cell-impermeant bis-mannose photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-yloxy)-2-[2-3H]-propylamine was used to measure adipocyte cell-surface–associated glucose transporters. In these experiments, the increase in maximal insulin-stimulated glucose transport (4.2-fold) produced after BRL 49653 treatment was correlated with a 2.6-fold increase in cell-surface–associated GLUT4. Photolabeled cell-sur-face GLUT1 was not detectable in any adipocyte preparation. These results suggest that the improvement in glycemic control produced by repeated administration of BRL 49653 to obese mice is mediated by increased insulin responsiveness of target tissues. BRL 49653 potentiates insulin-stimulated glucose transport in adipocytes from insulin-resistant obese mice, both by increasing insulin receptor number and by facilitating translocation of GLUT4, from an expanded intracellular pool, to the cell surface. In addition, the increased intrinsic activity of cell-surface glucose transporters may also contribute to an increased insulin responsiveness of adipose tissue.

Insulin action in cultured human skeletal muscle cells during differentiation: assessment of cell surface GLUT4 and GLUT1 content

Abstract: In mature human skeletal muscle, insulin-stimulated glucose transport is mediated primarily via the GLUT4 glucose transporter. However, in contrast to mature skeletal muscle, cultured muscle expresses significant levels of the GLUT1 glucose transporter. To assess the relative contribution of these two glucose transporters, we used a novel photolabelling techniques to assess the cell surface abundance of GLUT1 and GLUT4 specifically in primary cultures of human skeletal muscle. We demonstrate that insulin-stimulated glucose transport in cultured human skeletal muscle is mediated by GLUT4, as no effect on GLUT1 appearance at the plasma membrane was noted. Furthermore, GLUT4 mRNA and protein increased twofold (p < 0.05), after differentiation, whereas GLUT1 mRNA and protein decreased 55% (p < 0.005). Incubation of differentiated human skeletal muscle cells with a non-peptide insulin mimetic significantly (p < 0.05) increased glucose uptake and glycogen synthesis. Thus, cultured myotubes are a useful tool to facilitate biological and molecular validation of novel pharmacological agents aimed to improve glucose metabolism in skeletal muscle.

Moving the insulin-regulated glucose transporter GLUT4 into and out of storage

The glucose transporter isoform GLUT4 is unique among the glucose transporter family of proteins in that, in resting cells, it is sequestered very efficiently in a storage compartment. In insulin-sensitive cells, such as fat and muscle, insulin stimulation leads to release of GLUT4 from this reservoir and its translocation to the plasma membrane. This process is crucial for the control of blood and tissue glucose levels. Investigations of the composition and structure of the GLUT4 storage compartment, together with the targeting motifs that direct GLUT4 to this compartment, have been extensive but have been controversial. Recent findings have now provided a clearer consensus of opinion on the mechanisms involved in the formation of this storage compartment. However, another controversy has now emerged, which is unresolved. This concerns the issue of whether the insulin-regulated step occurs at the level of release of GLUT4 from the storage compartment or at the level at which released vesicles fuse with the plasma membrane.

The causal role of breakfast in energy balance and health: a randomized controlled trial in obese adults

Background: The causal nature of associations between breakfast and health remain unclear in obese individuals. Objective: We sought to conduct a randomized controlled trial to examine causal links between breakfast habits and components of energy balance in free-living obese humans. Design: The Bath Breakfast Project is a randomized controlled trial with repeated measures at baseline and follow-up among a cohort in South West England aged 21–60 y with dual-energy X-ray absorptiometry–derived fat mass indexes of ≥13 kg/m2 for women (n = 15) and ≥9 kg/m2 for men (n = 8). Components of energy balance (resting metabolic rate, physical activity thermogenesis, diet-induced thermogenesis, and energy intake) were measured under free-living conditions with random allocation to daily breakfast (≥700 kcal before 1100) or extended fasting (0 kcal until 1200) for 6 wk, with baseline and follow-up measures of health markers (e.g., hematology/adipose biopsies). Results: Breakfast resulted in greater physical activity thermogenesis during the morning than when fasting during that period (difference: 188 kcal/d; 95% CI: 40, 335) but without any consistent effect on 24-h physical activity thermogenesis (difference: 272 kcal/d; 95% CI: −254, 798). Energy intake was not significantly greater with breakfast than fasting (difference: 338 kcal/d; 95% CI: −313, 988). Body mass increased across both groups over time but with no treatment effects on body composition or any change in resting metabolic rate (stable within 8 kcal/d). Metabolic/cardiovascular health also did not respond to treatments, except for a reduced insulinemic response to an oral-glucose-tolerance test over time with daily breakfast relative to an increase with daily fasting (P = 0.05). Conclusions: In obese adults, daily breakfast leads to greater physical activity during the morning, whereas morning fasting results in partial dietary compensation (i.e., greater energy intake) later in the day. There were no differences between groups in weight change and most health outcomes, but insulin sensitivity increased with breakfast relative to fasting. This trial was registered at www.isrctn.org as ISRCTN31521726.

Glut 4 content in the plasma membrane of rat skeletal muscle: comparative studies of the subcellular fractionation method and the exofacial photolabelling technique using ATB‐BMPA

Employing subcellular membrane fractionation methods it has been shown that insulin induces a 2‐fold increase in the Glut 4 protein content in the plasma membrane of skeletal muscle from rats. Data based upon this technique are, however, impeded by poor plasma membrane recovery and cross‐contamination with intracellular membrane vesicles. The present study was undertaken to compare the subcellular fractionation technique with the technique using [3H]ATB‐BMPA exofacial photolabelling and immunoprecipitation of Glut 4 on soleus muscles from 3‐week‐old Wistar rats. Maximal insulin stimulation resulted in a 6‐fold increase in 3‐O‐methylglucose uptake, and studies based on the subcellular fractionation method showed a 2‐fold increase in Glut 4 content in the plasma membrane, whereas the exofacial photolabelling demonstrated a 6‐ to 7‐fold rise in cell surface associated Glut 4 protein. Glucose transport activity was positively correlated with cell surface Glut 4 content as estimated by exofacial labelling. In conclusion: (1) the increase in glucose uptake in muscle after insulin exposure is caused by an augmented concentration of Glut 4 protein on the cell surface membrane, (2) at maximal insulin stimulation (20 mU/ml) approximately 40% of the muscle cell content of Glut 4 is at the cell surface, and (3) the exofacial labelling technique is more sensitive than the subcellular fractionation technique in measuring the amount of glucose transporters on muscle cell surface.

Specificity and kinetics of hexose transport in Trypanosoma brucei

Transport of 6-deoxy-D-glucose was studied in Trypanosoma brucei in order to characterise the kinetics of hexose transport in this organism using a nonphosphorylated sugar. Kinetic parameters for efflux and entry, measured using zero-trans and equilibrium exchange protocols, indicate that the transporter is probably kinetically symmetrical. Comparison of the kinetic constants of D-glucose metabolism with those for 6-deoxy-D-glucose transport shows that transport across the plasma membrane is likely to be the rate-limiting step of glucose utilisation. The transport rate is nevertheless very fast and 6-deoxy-D-glucose, at concentrations below Km, enters the cells with a half filling time of less than 2 s at 20 degrees C. Thus the high metabolic capacity of these organisms is matched by a high transport rate. The structural requirements for the trypanosome hexose transporter were explored by measuring inhibition constants (Ki) for a range of D-glucose analogues including fluoro and deoxy sugars as well as epimeric hexoses. The relative affinities shown by these analogues indicated H-bonds from the carrier to the C-3, C-4 and C-5 hydroxyl oxygens and from the C-1 and C-3 hydroxyl hydrogens to the binding site. Hydrophobic interactions are likely at the C-2 and C-6 regions of the glucose molecule. Spatial constraints appear to occur around C-4 indicating that the transport site at this position is not freely open to the external solution as is the case with the mammalian hexose transporter. However, the trypanosome transporter appears to accept D-fructose but the common mammalian (erythrocyte type) hexose transporter does not.

Interleukin-3-mediated cell survival signals include phosphatidylinositol 3-kinase-dependent translocation of the glucose transporter GLUT1 to the cell surface

Maintenance of glucose uptake is a key component in the response of hematopoietic cells to survival factors. To investigate the mechanism of this response we employed the interleukin-3 (IL-3)-dependent murine mast cell line IC2.9. In these cells, hexose uptake decreased markedly upon withdrawal of IL-3, whereas its readdition led to rapid (t½ ∼ 10 min) stimulation of transport, associated with an ∼4-fold increase in Vmax but no change in Km. Immunocytochemistry and photoaffinity labeling revealed that IL-3 caused translocation of intracellular GLUT1 transporters to the cell surface, whereas a second transporter isoform, GLUT3, remained predominantly intracellular. The inhibitory effects of latrunculin B and jasplakinolide, and of nocodazole and colchicine, respectively, revealed a requirement for both the actin and microtubule cytoskeletons in GLUT1 translocation and transport stimulation. Both IL-3 stimulation of transport and GLUT1 translocation were also prevented by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. The time courses for activation of phosphatidylinositol 3-kinase and its downstream target, protein kinase B, by IL-3 were consistent with a role in IL-3-induced transporter translocation and enhanced glucose uptake. We conclude that one component of the survival mechanisms elicited by IL-3 involves the subcellular redistribution of glucose transporters, thus ensuring the supply of a key metabolic substrate.