Dietary weight loss in insulin-resistant non-obese humans: Metabolic benefits and relationship to adipose cell size.

BACKGROUND AND AIMS: Overweight and obesity increase risk for diabetes and cardiovascular disease, largely through development of insulin resistance. Benefits of dietary weight loss are documented for obese individuals with insulin resistance. Similar benefits have not been shown in overweight individuals. We sought to quantify whether dietary weight loss improves metabolic risk profile in overweight insulin-resistant individuals, and evaluated potential mediators between weight loss and metabolic response. METHODS AND RESULTS: Healthy volunteers with BMI 25-29.9 kg/m2 underwent detailed metabolic phenotyping including insulin-mediated-glucose disposal, fasting/daylong glucose, insulin, triglycerides, FFA, and cholesterol. Subcutaneous fat biopsies were performed for measurement of adipose cell size. After 14 weeks of hypocaloric diet and 2 weeks of weight maintenance, cardiometabolic measures and biopsies were repeated. Changes in weight, % body fat, waist circumference, adipose cell size and FFA were evaluated as predictors of change in insulin resistance. Weight loss (4.3 kg) yielded significant improvements in insulin resistance and all cardiovascular risk markers except glucose, HDL-C, and LDL-C. Improvement in insulin sensitivity was greater among those with <2 vs >2 cardiovascular risk factors at baseline. Decrease in adipose cell size and waist circumference, but not weight or body fat, independently predicted improvement in insulin resistance. CONCLUSIONS: Weight loss yields metabolic health benefits in insulin-resistant overweight adults, even in the absence of classic cardiovascular risk factors. Weight loss-related improvement in insulin sensitivity may be mediated through changes in adipose cell size and/or central distribution of body fat. The insulin-resistant subgroup of overweight individuals should be identified and targeted for dietary weight loss. CLINICAL TRIALS IDENTIFIER: NCT00186459.

Subcutaneous adipose cell size and distribution: relationship to insulin resistance and body fat.

OBJECTIVE: Metabolic heterogeneity among obese individuals may be attributable to differences in adipose cell size. We sought to clarify this by quantifying adipose cell size distribution, body fat, and insulin-mediated glucose uptake in overweight to moderately-obese individuals. METHODS: A total of 148 healthy nondiabetic subjects with BMI 25-38 kg/m2 underwent subcutaneous adipose tissue biopsies and quantification of insulin-mediated glucose uptake with steady-state plasma glucose (SSPG) concentrations during the modified insulin suppression test. Cell size distributions were obtained with Beckman Coulter Multisizer. Primary endpoints included % small adipose cells and diameter of large adipose cells. Cell-size and metabolic parameters were compared by regression for the whole group, according to insulin-resistant (IR) and insulin-sensitive (IS) subgroups, and by body fat quintile. RESULTS: Both large and small adipose cells were present in nearly equal proportions. Percent small cells was associated with SSPG (r = 0.26, P = 0.003). Compared to BMI-matched IS individuals, IR counterparts demonstrated fewer, but larger large adipose cells, and a greater proportion of small-to-large adipose cells. Diameter of the large adipose cells was associated with % body fat (r = 0.26, P = 0.014), female sex (r = 0.21, P = 0.036), and SSPG (r = 0.20, P = 0.012). In the highest versus lowest % body fat quintile, adipose cell size increased by only 7%, whereas adipose cell number increased by 74%. CONCLUSIONS: Recruitment of adipose cells is required for expansion of body fat mass beyond BMI of 25 kg/m2 . Insulin resistance is associated with accumulation of small adipose cells and enlargement of large adipose cells. These data support the notion that impaired adipogenesis may underlie insulin resistance.

Pioglitazone increases the proportion of small cells in human abdominal subcutaneous adipose tissue.

Rodent and in vitro studies suggest that thiazolidinediones promote adipogenesis but there are few studies in humans to corroborate these findings. The purpose of this study was to determine whether pioglitazone stimulates adipogenesis in vivo and whether this process relates to improved insulin sensitivity. To test this hypothesis, 12 overweight/obese nondiabetic, insulin-resistant individuals underwent biopsy of abdominal subcutaneous adipose tissue at baseline and after 12 weeks of pioglitazone treatment. Cell size distribution was determined via the Multisizer technique. Insulin sensitivity was quantified at baseline and postpioglitazone by the modified insulin suppression test. Regional fat depots were quantified by computed tomography (CT). Insulin resistance (steady-state plasma insulin and glucose (SSPG)) decreased following pioglitazone (P < 0.001). There was an increase in the ratio of small-to-large cells (1.16 +/- 0.44 vs. 1.52 +/- 0.66, P = 0.03), as well as a 25% increase in the absolute number of small cells (P = 0.03). The distribution of large cell diameters widened (P = 0.009), but diameter did not increase in the case of small cells. The increase in proportion of small cells was associated with the degree to which insulin resistance improved (r = -0.72, P = 0.012). Visceral abdominal fat decreased (P = 0.04), and subcutaneous abdominal (P = 0.03) and femoral fat (P = 0.004) increased significantly. Changes in fat volume were not associated with SSPG change. These findings demonstrate a clear effect of pioglitazone on human subcutaneous adipose cells, suggestive of adipogenesis in abdominal subcutaneous adipose tissue, as well as redistribution of fat from visceral to subcutaneous depots, highlighting a potential mechanism of action for thiazolidinediones. These findings support the hypothesis that defects in subcutaneous fat storage may underlie obesity-associated insulin resistance.

Inflammation in subcutaneous adipose tissue: relationship to adipose cell size.

AIMS/HYPOTHESIS: Inflammation is associated with increased body mass and purportedly with increased size of adipose cells. We sought to determine whether increased size of adipose cells is associated with localised inflammation in weight-stable, moderately obese humans. METHODS: We recruited 49 healthy, moderately obese individuals for quantification of insulin resistance (modified insulin suppression test) and subcutaneous abdominal adipose tissue biopsy. Cell size distribution was analysed with a multisizer device and inflammatory gene expression with real-time PCR. Correlations between inflammatory gene expression and cell size variables, with adjustment for sex and insulin resistance, were calculated. RESULTS: Adipose cells were bimodally distributed, with 47% in a 'large' cell population and the remainder in a 'small' cell population. The median diameter of the large adipose cells was not associated with expression of inflammatory genes. Rather, the fraction of small adipose cells was consistently associated with inflammatory gene expression, independently of sex, insulin resistance and BMI. This association was more pronounced in insulin-resistant than insulin-sensitive individuals. Insulin resistance also independently predicted expression of inflammatory genes. CONCLUSIONS/INTERPRETATION: This study demonstrates that among moderately obese, weight-stable individuals an increased proportion of small adipose cells is associated with inflammation in subcutaneous adipose tissue, whereas size of mature adipose cells is not. The observed association between small adipose cells and inflammation may reflect impaired adipogenesis and/or terminal differentiation. However, it is unclear whether this is a cause or consequence of inflammation. This question and whether small vs large adipose cells contribute differently to inflammation in adipose tissue are topics for future research. TRIAL REGISTRATION: ClinicalTrials.gov NCT00285844.

Insulin resistance is associated with a modest increase in inflammation in subcutaneous adipose tissue of moderately obese women.

AIMS/HYPOTHESIS: We have previously described differences in adipose cell size distribution and expression of genes related to adipocyte differentiation in subcutaneous abdominal fat obtained from insulin-sensitive (IS) and -resistant (IR) persons, matched for degree of moderate obesity. To determine whether other biological properties also differ between IR and IS obese individuals, we quantified markers of inflammatory activity in adipose tissue from overweight IR and IS individuals. METHODS: Subcutaneous abdominal tissue was obtained from moderately obese women, divided into IR (n = 14) and IS (n = 19) subgroups by determining their steady-state plasma glucose (SSPG) concentrations during the insulin suppression test. Inflammatory activity was assessed by comparing expression of nine relevant genes and by immunohistochemical quantification of CD45- and CD68-containing cells. RESULTS: SSPG concentrations were approximately threefold higher in IR than in IS individuals. Expression levels of CD68, EMR1, IL8, IL6 and MCP/CCL2 mRNAs were modestly but significantly increased (p < 0.05) in IR compared with IS participants. Results of immunohistochemical staining were consistent with gene expression data, demonstrating modest differences between IR and IS individuals. Crown-like structures, in which macrophages surround single adipocytes, were rarely seen in tissue from either subgroup. CONCLUSIONS/INTERPRETATION: A modest increase in inflammatory activity was seen in subcutaneous adipose tissue from IR compared with equally obese IS individuals. Together with previous evidence of impaired adipose cell differentiation in IR vs equally obese individuals, it appears that at least two biological processes in subcutaneous adipose tissue characterize the insulin-resistant state independent of obesity per se.

Enhanced proportion of small adipose cells in insulin-resistant vs insulin-sensitive obese individuals implicates impaired adipogenesis.

AIMS/HYPOTHESIS: The biological mechanism by which obesity predisposes to insulin resistance is unclear. One hypothesis is that larger adipose cells disturb metabolism via increased lipolysis. While studies have demonstrated that cell size increases in proportion to BMI, it has not been clearly shown that adipose cell size, independent of BMI, is associated with insulin resistance. The aim of this study was to test this widely held assumption by comparing adipose cell size distribution in 28 equally obese, otherwise healthy individuals who represented extreme ends of the spectrum of insulin sensitivity, as defined by the modified insulin suppression test. SUBJECTS AND METHODS: Subcutaneous periumbilical adipose tissue biopsy samples were fixed in osmium tetroxide and passed through the Beckman Coulter Multisizer to obtain cell size distributions. Insulin sensitivity was quantified by the modified insulin suppression test. Quantitative real-time PCR for adipose cell differentiation genes was performed for 11 subjects. RESULTS: All individuals exhibited a bimodal cell size distribution. Contrary to expectations, the mean diameter of the larger cells was not significantly different between the insulin-sensitive and insulin-resistant individuals. Moreover, insulin resistance was associated with a higher ratio of small to large cells (1.66 +/- 1.03 vs 0.94 +/- 0.50, p = 0.01). Similar cell size distributions were observed for isolated adipose cells. The real-time PCR results showed two- to threefold lower expression of genes encoding markers of adipose cell differentiation (peroxisome proliferator-activated receptor gamma1 [PPARgamma1], PPARgamma2, GLUT4, adiponectin, sterol receptor element binding protein 1c) in insulin-resistant compared with insulin-sensitive individuals. CONCLUSIONS/INTERPRETATION: These results suggest that after controlling for obesity, insulin resistance is associated with an expanded population of small adipose cells and decreased expression of differentiation markers, suggesting that impairment in adipose cell differentiation may contribute to obesity-associated insulin resistance.

Characterisation of insulin-resistant phenotype of cultured rat primary adipose cells.

AIMS/HYPOTHESIS: We characterised insulin resistance, metabolic defects and endocrine dysfunction in cultured adipose cells and examined the autocrine or paracrine roles of cytokines/adipokines in the progression of insulin resistance. MATERIALS AND METHODS: Rat primary adipose cells were prepared and cultured for 24 and 48 h. Insulin resistance and gene expression were examined by glucose uptake assay, cDNA microarray and real-time RT-PCR. RESULTS: After 24 h in culture, the fold increase of insulin-stimulated glucose uptake in adipose cells was markedly reduced; after 48 h the response of the cells to insulin decreased. cDNA microarray analysis showed that the expression of 514 genes was altered in adipose cells after 24 h in culture. The dysregulated genes included those involved in the citric acid cycle and in fatty acid and pyruvate metabolism. Specifically, the following genes were all downregulated: genes encoding lipolytic and lipogenic enzymes; uncoupling protein 1 and 2 genes; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha gene. This indicates that lipolytic and lipogenic activity, as well as mitochondria capacity decline in adipose cells cultured for 24 h. The mRNAs encoding 40 adipokines were also dysregulated in cultured cells. Strikingly, the dysregulated adipokines in cultured cells and in freshly isolated adipose cells from insulin-resistant Zucker fa/fa rats displayed a similar pattern with regard to protein functions. Also striking was the fact that progression of insulin resistance was promoted by the adipokines secreted from insulin-resistant adipose tissue or cells. CONCLUSIONS/INTERPRETATION: Our data demonstrate that the impairment of metabolism and endocrine dysfunction in cultured adipose cells mimics the insulin resistance occurring in vivo. Cytokines and adipokines appear to play a critical role in the progression of insulin resistance in adipose cells.

Insulin-regulated trafficking of dual-labeled glucose transporter 4 in primary rat adipose cells.

In isolated rat adipose cells, physiologically relevant insulin target cells, glucose transporter 4 (GLUT4) subcellular trafficking can be assessed by transfection of exofacially HA-tagged GLUT4. To simultaneously visualize the transfected GLUT4, we fused GFP with HA-GLUT4. With the resulting chimeras, GFP-HA-GLUT4 and HA-GLUT4-GFP, we were able to visualize for the first time the cell-surface localization, total expression, and intracellular distribution of GLUT4 in a single cell. Confocal microscopy reveals that the intracellular proportions of both GFP-HA-GLUT4 and HA-GLUT4-GFP are properly targeted to the insulin-responsive aminopeptidase-positive vesicles. Dynamic studies demonstrate close similarities in the trafficking kinetics between the two constructs and with native GLUT4. However, while the basal subcellular distribution of HA-GLUT4-GFP and the response to insulin are indistinguishable from those of HA-GLUT4 and endogenous GLUT4, most of the GFP-HA-GLUT4 is targeted to the plasma membrane with little further insulin response. Thus, HA-GLUT4-GFP will be useful to study GLUT4 trafficking in vivo while GFP on the N-terminus interferes with intracellular retention.

Targeting of GLUT6 (formerly GLUT9) and GLUT8 in rat adipose cells.

The subcellular targeting of the two recently cloned novel mammalian glucose transporters, GLUT6 [previously referred to as GLUT9 [Doege, Bocianski, Joost and Schürmann (2000) Biochem. J. 350, 771-776] and GLUT8, was analysed by expression of haemagglutinin (HA)-epitope-tagged GLUTs in transiently transfected primary rat adipose cells. Similar to HA-GLUT4, both transporters, HA-GLUT6 and HA-GLUT8, were retained in intracellular compartments in non-stimulated cells. In contrast, mutation of the N-terminal dileucine motifs in both constructs led to constitutive expression of the proteins on the plasma membrane. Likewise, when endocytosis was blocked by co-expression of a dominant-negative mutant of the dynamin GTPase, wild-type HA-GLUT6 and HA-GLUT8 accumulated on the cell surface. However, in contrast with HA-GLUT4, no translocation of HA-GLUT6 and HA-GLUT8 to the plasma membrane was observed when the cells were stimulated with insulin, phorbol ester or hyperosmolarity. Thus GLUT6 and GLUT8 appear to recycle in a dynamin-dependent manner between internal membranes and the plasma membrane in rat adipose cells, but are unresponsive to stimuli that induce translocation of GLUT4.

GLUT4 overexpression in db/db mice dose-dependently ameliorates diabetes but is not a lifelong cure.

We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-lepr(db/+) (db/+) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(D-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10-12 weeks), both lower (two- to threefold) and higher (four- to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/+ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo.

The export of major histocompatibility complex class I molecules from the endoplasmic reticulum of rat brown adipose cells is acutely stimulated by insulin.

Major histocompatibility complex class I (MHC-I) molecules have been implicated in several nonimmunological functions including the regulation and intracellular trafficking of the insulin-responsive glucose transporter GLUT4. We have used confocal microscopy to compare the effects of insulin on the intracellular trafficking of MHC-I and GLUT4 in freshly isolated rat brown adipose cells. We also used a recombinant vaccinia virus (rVV) to express influenza virus hemagglutinin (HA) as a generic integral membrane glycoprotein to distinguish global versus specific enhancement of protein export from the endoplasmic reticulum (ER) in response to insulin. In the absence of insulin, MHC-I molecules largely colocalize with the ER-resident protein calnexin and remain distinct from intracellular pools of GLUT4. Surprisingly, insulin induces the rapid export of MHC-I molecules from the ER with a concomitant approximately three-fold increase in their level on the cell surface. This ER export is blocked by brefeldin A and wortmannin but is unaffected by cytochalasin D, indicating that insulin stimulates the rapid transport of MHC-I molecules from the ER to the plasma membrane via the Golgi complex in a phosphatidyl-inositol 3-kinase-dependent and actin-independent manner. We further show that the effect of insulin on MHC-I molecules is selective, because insulin does not affect the intracellular distribution or cell-surface localization of rVV-expressed HA. These results demonstrate that in rat brown adipose cells MHC-I molecule export from the ER is stimulated by insulin and provide the first evidence that the trafficking of MHC-I molecules is acutely regulated by a hormone.

Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin.

Although uncoating of clathrin-coated vesicles is a key event in clathrin-mediated endocytosis it is unclear what prevents uncoating of clathrin-coated pits before they pinch off to become clathrin-coated vesicles. We have shown that the J-domain proteins auxilin and GAK are required for uncoating by Hsc70 in vitro. In the present study, we expressed auxilin in cultured cells to determine if this would block endocytosis by causing premature uncoating of clathrin-coated pits. We found that expression of auxilin indeed inhibited endocytosis. However, expression of auxilin with its J-domain mutated so that it no longer interacted with Hsc70 also inhibited endocytosis as did expression of the clathrin-assembly protein, AP180, or its clathrin-binding domain. Accompanying this inhibition, we observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which provided us with an opportunity to determine whether the absence of clathrin from clathrin-coated pits affected the distribution of the clathrin assembly proteins AP1 and AP2. Surprisingly we found almost no change in the association of AP2 and AP1 with the plasma membrane and the trans-Golgi network, respectively. This was particularly obvious when auxilin or GAK was expressed with functional J-domains since, in these cases, almost all of the clathrin was sequestered in granules that also contained Hsc70 and auxilin or GAK. We conclude that expression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it is no longer available to bind to nascent pits but that assembly proteins bind to these pits independently of clathrin.

Immunoelectron microscopic evidence that GLUT4 translocation explains the stimulation of glucose transport in isolated rat white adipose cells.

We used an improved cryosectioning technique in combination with quantitative immunoelectron microscopy to study GLUT4 compartments in isolated rat white adipose cells. We provide clear evidence that in unstimulated cells most of the GLUT4 localizes intracellularly to tubulovesicular structures clustered near small stacks of Golgi and endosomes, or scattered throughout the cytoplasm. This localization is entirely consistent with that originally described in brown adipose tissue, strongly suggesting that the GLUT4 compartments in white and brown adipose cells are morphologically similar. Furthermore, insulin induces parallel increases (with similar magnitudes) in glucose transport activity, approximately 16-fold, and cell-surface GLUT4, approximately 12-fold. Concomitantly, insulin decreases GLUT4 equally from all intracellular locations, in agreement with the concept that the entire cellular GLUT4 pool contributes to insulin-stimulated exocytosis. In the insulin-stimulated state, GLUT4 molecules are not randomly distributed on the plasma membrane, but neither are they enriched in caveolae. Importantly, the total number of GLUT4 C-terminal epitopes detected by the immuno-gold method is not significantly different between basal and insulin-stimulated cells, thus arguing directly against a reported insulin-induced unmasking effect. These results provide strong morphological evidence (1) that GLUT4 compartments are similar in all insulin-sensitive cells and (2) for the concept that GLUT4 translocation almost fully accounts for the increase in glucose transport in response to insulin.

Demonstration of insulin-responsive trafficking of GLUT4 and vpTR in fibroblasts.

Insulin-responsive trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) in adipose and muscle cells is well established. Insulin regulation of GLUT4 trafficking in these cells underlies the role that adipose tissue and muscle play in the maintenance of whole body glucose homeostasis. GLUT4 is expressed in a very limited number of tissues, most highly in adipose and muscle, while IRAP is expressed in many tissues. IRAP's physiological role in any of the tissues in which it is expressed, however, is unknown. The fact that IRAP, which traffics by the same insulin-regulated pathway as GLUT4, is expressed in 'non-insulin responsive' tissues raises the question of whether these other cell types also have a specialized insulin-regulated trafficking pathway. The existence of an insulin-responsive pathway in other cell types would allow regulation of IRAP activity at the plasma membrane as a potentially important physiological function of insulin. To address this question we use reporter molecules for both GLUT4 and IRAP trafficking to measure insulin-stimulated translocation in undifferentiated cells by quantitative fluorescence microscopy. One reporter (vpTR), a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor, has been previously characterized. The other is a GLUT4 construct with an exofacial HA epitope and a C-terminal GFP. By comparing these reporters to the transferrin receptor, a marker for general endocytic trafficking, we demonstrate the existence of a specialized, insulin-regulated trafficking pathway in two undifferentiated cell types, neither of which normally express GLUT4. The magnitude of translocation in these undifferentiated cells (approximately threefold) is similar to that reported for the translocation of GLUT4 in muscle cells. Thus, undifferentiated cells have the necessary retention and translocation machinery for an insulin response that is large enough to be physiologically important.

Transient enhancement of GLUT-4 levels in rat epitrochlearis muscle after exercise training.

The purpose of the present study was to examine the effect of detraining on the glucose transport system after short-term swim training (5 days), long-term swim training (5 wk), and treadmill run training (5 wk). Skeletal muscles were isolated from female Wistar rats at 24 or 48 h posttraining. SST produces a 48% increase in GLUT-4 mRNA, a 30% increase in GLUT-4 protein, and a 60% increase in insulin-stimulated glucose transport activity at 24 h posttraining but not at 48 h posttraining. Similar to SST, long-term swim training produces a 60% increase in GLUT-4 mRNA and a 30% increase in GLUT-4 protein content at 24 h posttraining but not at 48 h posttraining. Finally, treadmill run training produces a transient 35% increase in GLUT-4 protein content that is completely reversed at 48 h after the last bout of exercise. These results demonstrate that the increase in GLUT-4 mRNA and GLUT-4 protein occurs during the first week of exercise training and is rapidly lost after training cessation. We believe that the transient enhancement in GLUT-4 protein after exercise training is due to a short GLUT-4 half-life, a process that is primarily regulated by pretranslational mechanisms.

Overexpression of the glucose transporter GLUT4 in adipose cells interferes with insulin-stimulated translocation.

In adipose cells, insulin induces the translocation of GLUT4 by stimulating their exocytosis from a basal intracellular compartment to the plasma membrane. Increasing overexpression of a hemagglutinin (HA) epitope-tagged GLUT4 in rat adipose cells results in a roughly proportional increase in cell surface HA-GLUT4 levels in the basal state, accompanied by a marked reduction of the fold HA-GLUT4 translocation in response to insulin. Using biochemical methods and cotransfection experiments with differently epitope-tagged GLUT4, we show that overexpression of GLUT4 does not affect the intracellular sequestration of GLUT4 in the absence of insulin, but rather reduces the relative insulin-stimulated GLUT4 translocation to the plasma membrane. In contrast, overexpression of GLUT1 does not interfere with the targeting of GLUT4 and vice versa. These results suggest that the mechanism involved in the intracellular sequestration of GLUT4 has a high capacity whereas the mechanism for GLUT4 translocation is readily saturated by overexpression of GLUT4, implicating an active translocation machinery in the exocytosis of GLUT4.

GLUT4 trafficking in insulin-stimulated rat adipose cells: evidence that heterotrimeric GTP-binding proteins regulate the fusion of docked GLUT4-containing vesicles.

Agents that activate the G-protein G(i) (e.g. adenosine) increase, and agents that activate G(s) [e.g. isoprenaline (isoproterenol)] decrease, steady-state insulin-stimulated glucose transport activity and cell-surface GLUT4 in isolated rat adipose cells without changing plasma membrane GLUT4 content. Here we have further examined the effects of R(s)G(s) and R(i)G(i) ligands (in which R(s) and R(i) are G(s)- and G(i)-coupled receptors respectively) on insulin-stimulated cell-surface GLUT4 and the kinetics of GLUT4 trafficking in these same cells. Rat adipose cells were preincubated for 2 min with or without isoprenaline (200 nM) and adenosine deaminase (1 unit/ml), to stimulate G(s) and decrease the stimulation of G(i) respectively, followed by 0-20 min with insulin (670 nM). Treatment with isoprenaline and adenosine deaminase decreased insulin-stimulated glucose transport activity by 58%. Treatment with isoprenaline and adenosine deaminase also resulted in similar decreases in insulin-stimulated cell-surface GLUT4 as assessed by both bis-mannose photolabelling of the substrate-binding site and biotinylation of the extracellular carbohydrate moiety when evaluated under similar experimental conditions. After stimulation with insulin in the absence of G(s) and the presence of G(i) agents, a distinct sequence of plasma membrane events took place, starting with an increase in immunodetectable GLUT4, then an increase in the accessibility of GLUT4 to bis-mannose photolabel, and finally an increase in glucose transport activity. Pretreatment with isoprenaline and adenosine deaminase before stimulation with insulin did not affect the time course of the increase in immunodetectable GLUT4 in the plasma membrane, but did delay both the increase in accessibility of GLUT4 to photolabel and the increase in glucose transport activity. These results suggest that R(s)G(s) and R(i)G(i) modulate insulin-stimulated glucose transport by influencing the extent to which GLUT4 is associated with occluded vesicles attached to the plasma membrane during exocytosis, perhaps by regulating the fusion process through which the GLUT4 in docked vesicles becomes exposed on the cell surface.

1-[N, O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), an inhibitor of calcium-dependent camodulin protein kinase II, inhibits both insulin- and hypoxia-stimulated glucose transport in skeletal muscle.

Previous studies have indicated a role for calmodulin in hypoxia-and insulin-stimulated glucose transport. However, since calmodulin interacts with multiple protein targets, it is unknown which of these targets is involved in the regulation of glucose transport. In the present study, we have used the calcium-dependent calmodulin protein kinase II (CAMKII) inhibitor 1-[N, O-bis-(5-isoquinolinesulphonyl) -N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) to investigate the possible role of this enzyme in the regulation of glucose transport in isolated rat soleus and epitrochlearis muscles. KN-62 did not affect basal 2-deoxyglucose transport, but it did inhibit both insulin- and hypoxia-stimulated glucose transport activity by 46 and 40% respectively. 1-[N,O-Bis-(1, 5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-04), a structural analogue of KN-62 that does not inhibit CAMKII, had no effect on hypoxia-or insulin-stimulated glucose transport. Accordingly, KN-62 decreased the stimulated cell-surface GLUT4 labelling by a similar extent as the inhibition of glucose transport (insulin, 49% and hypoxia, 54%). Additional experiments showed that KN-62 also inhibited insulin- and hypoxia-stimulated transport by 37 and 40% respectively in isolated rat epitrochlearis (a fast-twitch muscle), indicating that the effect of KN-62 was not limited to the slow-twitch fibres of the soleus. The inhibitory effect of KN-62 on hypoxia-stimulated glucose transport appears to be specific to CAMKII, since KN-62 did not inhibit hypoxia-stimulated 45Ca efflux from muscles pre-loaded with 45Ca, or hypoxia-stimulated glycogen breakdown. Additionally, KN-62 affected neither insulin-stimulated phosphoinositide 3-kinase nor Akt activity, suggesting that the effects of KN-62 are not due to non-specific effects of this inhibitor on these regions of the insulin-signalling cascade. The results of the present study suggest that CAMKII might have a distinct role in insulin- and hypoxia-stimulated glucose transport, possibly in the vesicular trafficking of GLUT4.

SNAP-23 participates in SNARE complex assembly in rat adipose cells.

SNARE proteins are required for vesicle docking and fusion in eukaryotic cells in processes as diverse as homotypic membrane fusion and synaptic vesicle exocytosis [SNARE stands for SNAP receptor, where SNAP is soluble NSF attachment protein]. The SNARE proteins syntaxin 4 and vesicle-associated membrane protein (VAMP) 2/3 also participate in the insulin-stimulated translocation of GLUT4 from intracellular vesicles to the plasma membrane in adipose cells. We now report the molecular cloning and characterization of rat SNAP-23, a ubiquitously expressed homologue of the essential neuronal SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Rat SNAP-23 is 86% and 98% identical respectively to human and mouse SNAP-23. Southern blot analysis reveals that the rat, mouse and human SNAP-23 genes encode species-specific isoforms of the same protein. Co-immunoprecipitation of syntaxin 4 and SNAP-23 shows association of these two proteins in rat adipose cell plasma membranes, and insulin stimulation does not alter the SNAP-23/syntaxin 4 complex. In addition, we demonstrate for the first time the participation of SNAP-23, along with syntaxin 4 and VAMP2/3, in the formation of 20S SNARE complexes prepared using rat adipose cell membranes and recombinant alpha-SNAP and NSF proteins. The stoichiometry of the SNARE complexes formed is essentially identical using membranes from either unstimulated or insulin-stimulated adipose cells. These data demonstrate that rat SNAP-23 associates with syntaxin 4 before insulin stimulation and is present in the SNARE complexes known to mediate the translocation of GLUT4 from intracellular vesicles to the plasma membrane of rat adipose cells.

Short-term exercise enhances insulin-stimulated GLUT-4 translocation and glucose transport in adipose cells.

This investigation examined the effects of short-term exercise training on insulin-stimulated GLUT-4 glucose transporter translocation and glucose transport activity in rat adipose cells. Male Wistar rats were randomly assigned to a sedentary (Sed) or swim training group (Sw, 4 days; final 3 days: 2 x 3 h/day). Adipose cell size decreased significantly but minimally (approximately 20%), whereas total GLUT-4 increased by 30% in Sw vs. Sed rats. Basal 3-O-methyl-D-[14C]glucose transport was reduced by 62%, whereas maximally insulin-stimulated (MIS) glucose transport was increased by 36% in Sw vs. Sed rats. MIS cell surface GLUT-4 photolabeling was 44% higher in the Sw vs. Sed animals, similar to the increases observed in MIS glucose transport activity and total GLUT-4. These results suggest that increases in total GLUT-4 and GLUT-4 translocation to the cell surface contribute to the increase in MIS glucose transport with short-term exercise training. In addition, the results suggest that the exercise training-induced adaptations in glucose transport occur more rapidly than previously thought and with minimal changes in adipose cell size.