Syntaxin 4 Transgenic Mice Exhibit Enhanced Insulin-Mediated Glucose Uptake in Skeletal Muscle

  1. Beth A. Spurlin1,
  2. So-Young Park2,
  3. Angela K. Nevins1,
  4. Jason K. Kim2 and
  5. Debbie C. Thurmond1
  1. 1Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana
  2. 2Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut
  1. Address correspondence and reprint requests to Debbie C. Thurmond, PhD, Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, IN 46202. E-mail: dthurmon{at}iupui.edu

Abstract

Insulin-stimulated translocation of GLUT4 vesicles from an intracellular compartment to the plasma membrane in 3T3L1 adipocytes is mediated through a syntaxin 4 (Syn4)- and Munc18c-dependent mechanism. To investigate the impact of increasing Syn4 protein abundance on glucose homeostasis in vivo, we engineered tetracycline-repressible transgenic mice to overexpress Syn4 by fivefold in skeletal muscle and pancreas and threefold in adipose tissue. Increases in Syn4 caused increases in Munc18c protein, indicating that Syn4 regulates Munc18c expression in vivo. An important finding was that female Syn4 transgenic mice exhibited an increased rate of glucose clearance during glucose tolerance tests that was repressible by the administration of tetracycline. Insulin-stimulated glucose uptake in skeletal muscle was increased by twofold in Syn4 transgenic mice compared with wild-type mice as assessed by hyperinsulinemic-euglycemic clamp analysis, consistent with a twofold increase in insulin-stimulated GLUT4 translocation in skeletal muscle. Hepatic insulin action was unaffected. Moreover, insulin content and glucose-stimulated insulin secretion by islets isolated from Syn4 transgenic mice did not differ from that of wild-type mice. In sum, these data suggest that increasing the number of Syn4-Munc18c “fusion sites” at the plasma membrane of skeletal muscle increases the amount of GLUT4 available to increase the overall rate of insulin-mediated glucose uptake in vivo.

Insulin resistance is a major factor in the pathogenesis of type 2 diabetes, although the mechanism by which this occurs remains unclear. Insulin resistance affects multiple insulin-sensitive tissue types (skeletal muscle, adipose, and liver); however, it remains uncertain as to whether a defect in one or multiple tissues is required to lead to the progression to diabetes. The majority of insulin-stimulated glucose uptake in skeletal muscle and adipose tissue is attributed to the insulin-responsive glucose transporter GLUT4 (1). In the basal non–insulin-stimulated state, GLUT4 localizes to tubulovesicular elements and small intracellular vesicles throughout the cell cytoplasm (2,3). Upon stimulation with insulin, these GLUT4-containing compartments undergo a series of regulated steps, leading to their eventual fusion with the plasma membrane (49). This ultimately results in a large increase in the number of functional glucose transporters on the cell surface (a process termed “translocation”), which accounts for nearly all of the insulin-stimulated increase in glucose uptake.

Insulin-stimulated translocation of GLUT4 vesicles is mediated by the binding of plasma membrane soluble N-ethylmaleimide−sensitive factor attachment protein (SNAP) receptor (SNARE) proteins syntaxin 4 (Syn4) and SNAP-23 with the GLUT4 vesicle v-SNARE protein vesicle-associated membrane protein 2 (VAMP2). Studies that substantiate this show that GLUT4 vesicles copurify with the VAMP2 v-SNARE, and that specific proteolytic cleavage of VAMP2 and expression of a dominant-interfering VAMP2 mutant or inhibitory peptides all impair insulin-stimulated GLUT4 translocation (1015). Using similar techniques, Syn4 and SNAP-23 have been identified as the target membrane t-SNARE proteins required for insulin-stimulated GLUT4 translocation (1619). Thus, these data provide compelling evidence that VAMP2 functions by directing the association of the GLUT4-containing vesicles with Syn4 and SNAP-23.

Syntaxin proteins are regulated by interaction with Sec1/Munc18 proteins (SM proteins). SM proteins act as critical modulators of SNARE interactions in S. cerevisiae (n-Sec1/rbSec1), C. elegans (unc18), and D. melanogaster (Rop) (2024) through specific and high-affinity binding to their cognate syntaxins (21,2527). Null mutations in the genes for the SM proteins cause dramatic reductions in vesicle exocytosis, suggesting that these proteins are essential for normal SNARE function (26,28). In all, three Sec1 homologs have been identified in mammalian plasma membranes: Munc18a, -18b, and -18c. Munc18a is predominantly expressed in neurons, where it inhibits the association of VAMP2 and SNAP-25 with syntaxin 1 (23,29). Munc18b and -18c are expressed ubiquitously, and only Munc18c binds Syn4 with high affinity (30,31). Munc18c binds to Syn4 in a manner mutually exclusive of either Syn4 binding proteins (SNAP-23 and VAMP2) in adipocytes and competes for Syn4 when overexpressed (32). Functionally, Munc18c overexpression inhibits GLUT4 vesicle translocation in vitro and in vivo (3336). However, we have shown that the Munc18c-Syn4 complex is specifically required for insulin-stimulated GLUT4 vesicle fusion and is not necessary for proximal trafficking steps (37). These results indicate that the balance of Munc18c and Syn4 proteins directly affects whole-body glucose homeostasis through alterations in insulin action more so than absolute abundance of either protein alone.

Thus, although it is clear that Syn4 is involved in insulin action, it is unclear as to whether increasing its abundance would have a positive or negative effect on glucose homeostasis. Therefore, to investigate the physiological relevance of Syn4 protein overabundance with respect to insulin-stimulated GLUT4 translocation and glucose homeostasis in vivo, we generated transgenic mice that overexpress Syn4 protein under the control of a tetracycline-repressible promoter in peripheral insulin-sensitive tissues (skeletal muscle and fat) and the pancreas. We demonstrated that Syn4 protein overexpression results in increased Munc18c protein abundance, and that the increased abundance of Syn4 and Munc18c together results in enhanced insulin sensitivity. Hyperinsulinemic-euglycemic clamp analyses showed an increased rate of peripheral glucose disposal, consistent with an increase in skeletal muscle insulin-stimulated GLUT4 translocation. Oral administration of tetracycline to the Syn4 transgenic mice restored glucose tolerance back to normal, in coordination with the downregulation of Syn4 transgene expression and Munc18c expression. Taken together, these data suggest that increasing the abundance of Syn4-Munc18c complexes in skeletal muscle leads to increased insulin sensitivity in vivo.

RESEARCH DESIGN AND METHODS

The GLUT4, Syn4, VAMP2, and Munc18c antibodies were obtained as previously described (17,33). The SNAP-23 antibody was purchased from Affinity Bioreagents (Golden, CO). Goat anti-rabbit horseradish peroxidase secondary antibody was purchased from Bio-Rad (Hercules, CA). The sensitive and standard rat insulin radioimmunoassay kits were acquired from Linco Research (St. Charles, MO).

Generation of pCOMBICMV-Syn4 transgenic mice.

All studies involving mice followed the Guidelines for the Use and Care of Laboratory Animals. The pUC-COMBICMV plasmid for generation of tetracycline-repressible transgenic mice was a gift from Dr. Ulli Certa (Hoffman la Roche, Basel, Switzerland) (38). Full-length rat Syn4 cDNA (33) was inserted into the pCombi-CMV vector, and founder mice were generated on a C57BL/6J background strain by the University of Iowa Transgenic Animal Facility (Iowa City, IA), as previously described (36), and the positive animals were bred with C57BL/6J stock.

Tissue homogenization and immunoblotting.

Tissues were homogenized in a 1% Igepal detergent buffer (25 mmol/l Tris [pH 7.4], 1% Igepal, 10% glycerol, 50 mmol/l sodium fluoride, 10 mmol/l sodium pyrophosphate, 137 mmol/l sodium chloride, 1 mmol/l sodium vanadate, 1 mmol/l phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml aprotinin, 1 μg/ml pepstatin, and 5 μg/ml leupeptin) for 15 s and centrifuged at 2,000g for 5 min. Subsequent supernatants were microcentrifuged at 13,500g for 20 min at 4°C. Proteins were separated on 10 or 12% SDS-PAGE, then transferred to polyvinylidine fluoride or nitrocellulose membrane for immunoblotting.

Intraperitoneal glucose tolerance test.

Female Syn4 transgenic and wild-type mice (age 4–6 months) were fasted overnight for 18 h. Blood was collected from the tail vein, and blood glucose was monitored (Hemocue). After samples of fasted blood were collected, mice were administered glucose (2 g/kg body wt) by intraperitoneal injection. Subsequent blood glucose readings were taken at 30-min intervals over 120 min.

Hyperinsulinemic-euglycemic clamp.

After an overnight fast, 2-h hyperinsulinemic-euglycemic clamp experiments were performed using a primed continuous infusion of insulin (15 pmol · kg −1 · min−1; Humulin; Eli Lilly, Indianapolis, IN) and [3-3H]glucose (0.1 mCi/min; PerkinElmer, Boston, MA) in awake transgenic and wild-type mice, as previously described (36,39). To assess insulin-stimulated glucose uptake in individual tissues, 2-deoxy-d-[1-14C]glucose (PerkinElmer) was administered as a bolus (10 mCi) during clamps, and tissues were taken at the end of clamps for biochemical assays, as previously described (36,39). Whole-body fat and lean mass were measured in awake mice using 1H-MRS (Bruker Mini-Spec).

Skeletal muscle subcellular fractionation.

Hindlimb skeletal muscle was subfractionated into plasma membrane and intracellular membrane components, as previously described (35,40). Syn4 transgenic and wild-type mice (age 4–6 months) were fasted overnight for 16 h, injected intraperitoneally with 21 units/kg body wt Humulin or vehicle, and killed after 40 min for removal of the soleus and hindquarter muscles into homogenization buffer (20 mmol/l HEPES [pH 7.4], 250 mmol/l sucrose, 1 mmol/l EDTA, 5 mmol/l benzamidine, 10 μg/ml aprotinin, 5 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mmol/l PMSF) for Polytron homogenization. Homogenates were centrifuged at 2,000g for 5 min at 4°C. The supernatant was then centrifuged at 9,000g for 20 min at 4°C. The resulting supernatant was centrifuged at 180,000g for 90 min. Pellets were resuspended and combined with sample buffer for separation by 10% SDS-PAGE and subsequent immunoblotting for GLUT4.

Isolation, culture, and stimulation of insulin secretion of mouse islets.

Pancreatic mouse islets were isolated using a modification of the previously described method (41). Briefly, pancreases from 8- to 12-week-old female or male mice were digested with collagenase and purified using a Ficoll density gradient. After being isolated, islets were cultured overnight in CMRL-1066 medium. Islets were hand picked into groups of 10, preincubated in Krebs-Ringer bicarbonate buffer (10 mmol/l HEPES [pH 7.4], 134 mmol/l NaCl, 5 mmol/l NaHCO3, 4.8 mmol/l KCl, 1 mmol/l CaCl2, 1.2 mmol/l MgSO4, and 1.2 mmol/l KH2PO4) containing 2.8 mmol/l glucose and 0.1% BSA for 2 h, then stimulated with 20 mmol/l glucose for 2 h. The medium was collected to measure insulin secretion, and islets were harvested in NP-40 lysis buffer to determine cellular insulin content by radioimmunoassay.

Quantitation of SNARE proteins in tissue homogenates.

Skeletal muscle homogenate proteins were subjected to electrophoresis alongside known quantities of glutathione S-transferase (GST) fusion proteins GST-Munc18c, GST-Syn4, or GST-SNAP-23 recombinant proteins on 10% SDS-PAGE, then immunoblotted for Munc18c, Syn4, or SNAP-23, as previously described (36). Proteins were detected using enhanced chemiluminescence using exposures well within the linear range of the film and quantitated using the BioRad Quantity One software package.

RESULTS

Syn4 and Munc18c levels are increased in Syn4 transgenic mice.

To determine the importance of Syn4 protein on insulin-stimulated GLUT4 translocation in vivo, we sought to generate transgenic mice that overexpressed Syn4 in skeletal muscle. The initial use of this particular vector described differential levels of transgenic protein expression among various tissues, with skeletal muscle exhibiting the largest increase in expression and no changes in brain tissue (38). Of the six founders, five lines transmitted the transgene and one line showed overexpression of the Syn4 protein in skeletal muscle (by approximately fivefold). The overexpression of Syn4 protein in the transgenic mice was compared with the endogenous levels in a wild-type littermate expressed in heart, liver, pancreas, skeletal muscle, and epididymal fat (determined by immunoblot analysis). Of these tissues, only pancreas (Fig. 1A, lanes 5 and 6), skeletal muscle (Fig. 1A, lanes 7 and 8), and fat (Fig. 1A, lanes 9 and 10) showed a repeated significant increase in Syn4 protein (five-, five-, and threefold, respectively), without altering SNAP-23 or VAMP2 abundance. Syn4 abundance was unchanged in lung, brain, kidney, and spleen (data not shown).

The endogenous abundance of Munc18c protein was also elevated in parallel with Syn4 levels in skeletal muscle, adipose, and pancreatic tissues in the Syn4 transgenic mice (Fig. 1A, lanes 510). No changes in GLUT4 protein or the GLUT4 vesicle cargo protein insulin-responsive aminopeptidase (IRAP) were detected in heart or skeletal muscle from Syn4 transgenic mice compared with that from wild-type mice (Fig. 1B). Thus, the transgenic mouse lines overexpressed the transgenic Syn4 protein, with parallel endogenous overexpression of Munc18c in insulin-secreting and insulin-responsive tissues, but without altering GLUT4 protein levels.

Increased expression of Syn4 and Munc18c enhances glucose tolerance.

To determine the effects of Syn4 overexpression in vivo, Syn4 transgenic and wild-type littermate mice were fasted overnight and subjected to an intraperitoneal injection of glucose (2 g/kg); glucose disposal was monitored over a 2-h period (Fig. 2). Peak blood glucose was reached 30 min after the injection in wild-type mice and was cleared over the remaining 90 min. Although the blood glucose levels of female Syn4 transgenic mice also peaked by 30 min, this glucose level was dramatically reduced compared with that of wild-type mice, and remained significantly lower after 90 min of the test. Quantitation of the area under the curve for the initial 60 min of the glucose tolerance test confirmed that this observed difference was significant (P < 0.05). These data clearly demonstrated that the female Syn4 transgenic mice had an enhanced rate of glucose clearance from the circulation compared with wild-type mice when assessed by intraperitoneal glucose tolerance test.

To investigate the mechanism of the enhanced rate of glucose clearance in the male and female Syn4 transgenic mice, we used the more sensitive and accurate hyperinsulinemic-euglycemic clamp method. The plasma glucose concentrations were maintained at 7 mmol/l, and plasma insulin concentrations were raised from 246 ± 30 pmol/l for wild-type and 241 ± 35 pmol/l for transgenic mice during the clamps. The glucose infusion rate necessary to maintain euglycemia under conditions of constant infusion of insulin (2.5 mU · kg−1 · min−1) in Syn4 transgenic mice was increased to ∼115% of that of wild-type mice. Female Syn4 transgenic mice exhibited an even larger 27% increase in insulin-stimulated whole-body glucose uptake, as compared with the female wild-type mice (Fig. 3A). In addition, male Syn4 transgenic mice had a 21% increase in whole-body glucose uptake as compared with male wild-type mice (Fig. 3B). This finding was initially surprising, as we were unable to detect enhanced glucose uptake in the male Syn4 transgenic mice by glucose tolerance testing (36); it also supported the argument for using the hyperinsulinemic-euglycemic clamp analysis. In addition, neither basal hepatic glucose production nor insulin’s ability to suppress hepatic glucose production was significantly affected in the Syn4 transgenic mice compared with wild-type mice (Fig. 3C and D). These data indicated that the insulin sensitivity in the Syn4 transgenic mice was due to enhanced peripheral glucose disposal in response to insulin and not significant alterations in hepatic insulin action.

Enhanced glucose uptake in skeletal muscle of Syn4 transgenic mice.

Because glucose uptake into skeletal muscle and adipose tissue accounts for the majority of peripheral glucose disposal, we examined tissue-specific glucose uptake in vivo during hyperinsulinemic-euglycemic clamps. Insulin-stimulated glucose uptake and glycolysis in skeletal muscle (gastrocnemius) was increased by ∼45% in the female Syn4 transgenic mice as compared with in the female wild-type littermate mice (Fig. 4A). Furthermore, male Syn4 transgenic mice showed an ∼35% increase in skeletal muscle glucose uptake and glycolysis (Fig. 4B). By contrast, there were no significant increases in glucose uptake in white adipose tissue in the Syn4 transgenic mice compared with the wild-type littermates of female or male mice (Fig. 4C and D). Similarly, glucose uptake in brown adipose tissue of male Syn4 transgenic mice did not significantly differ from that of wild-type mice (Fig. 4E). Overall, the enhanced whole-body glucose tolerance in the Syn4 transgenic mice was accounted for primarily by the increases in skeletal muscle glucose uptake in these mice.

The mechanism for glucose uptake into skeletal muscle involves the fusion of GLUT4-containing vesicles via Syn4. To investigate the subcellular distribution of GLUT4 in skeletal muscle from wild-type or Syn4 transgenic mice, we used skeletal muscle homogenization coupled with sucrose velocity sedimentation to separate sarcolemma/transverse tubule–enriched fractions (P1 and P2) from the fractions enriched for intracellular membranes (35,40). As seen in Fig. 5A, insulin stimulation resulted in increased levels of GLUT4 in the P1 and P2 surface membrane fractions and decreased levels of GLUT4 in the intracellular membrane sucrose fractions. The distribution of marker proteins GLUT1 in P1 and P2 compartments and transferrin receptor (P1, P2, and early sucrose fractions) showed the expected localization patterns, as previously described (40). Remarkably, the amount of GLUT4 in the P2 fraction from Syn4 transgenic muscle was consistently greater than that that in wild-type muscle (Fig. 5B). Optical density quantitation of GLUT4 immunoblots confirmed that the Syn4 transgenic mice translocated twofold more GLUT4 to the P2 fraction of skeletal muscle in response to insulin compared with wild-type mice, whereas all mice showed similar quantities of GLUT4 protein in the P2 fraction under basal conditions (Fig. 5C and D). Syn4 protein was localized with the P1 and P2 fractions, with little-to-none detected in the sucrose fractions (as has been previously described) (35), and was increased in P1 and P2 fractions from Syn4 transgenic mice (Fig. 5E). Proximal insulin signaling was unaffected, however, as both female and male Syn4 transgenic and wild-type mice injected with insulin showed equivalently elevated levels of serine phosphorylation of protein kinase B/Akt in skeletal muscle tissue homogenates (Fig. 5F). Taken together, these data indicated that Syn4 overexpression enhanced insulin-stimulated GLUT4 translocation and was independent of effects on proximal insulin-signaling events.

Tetracycline-repression of Syn4 transgene expression normalizes glucose tolerance.

The Syn4 transgene is under the regulation of the tetracycline operator, so that oral administration of tetracycline causes downregulation of the transgene (38). To evaluate the effectiveness of the tetracycline-repressible system, 4- to 6-month-old female Syn4 transgenic and wild-type littermate mice were pair-fed tetracycline in drinking water (1 mg/ml) for 7 days. Their tissues were subsequently homogenized and immunoblotted for protein expression. Tetracycline administration downregulated Syn4 protein overexpression to levels observed in wild-type mice in pancreas, skeletal muscle, and fat (Fig. 6A). Moreover, tetracycline feeding also reduced expression of Munc18c protein back to levels present in wild-type mice and had no effect on expression of SNAP-23 or VAMP2 (data not shown). Thus, the tetracycline exerted its repressive effect specifically on the tetracycline-sensitive Syn4 transgene, which in turn altered expression of Munc18c.

To determine whether the enhanced glucose tolerance in the Syn4 transgenic mice was directly caused by the increased Syn4 and/or Munc18c protein, we administered tetracycline to the Syn4 transgenic and wild-type mice used in the data set for Fig. 2 for 1 week and repeated the intraperitoneal glucose tolerance test. Consistent with the immunoblotting results of Fig. 6A, normalization of Syn4 and Munc18c protein content in the transgenic mice directly correlated with normalization of glucose tolerance to levels indistinguishable from those of wild-type mice fed tetracycline (Fig. 6B). Moreover, tetracycline treatment did not induce any changes in metabolic components of the serum such as glucose, insulin, cholesterol, triglyceride, or nonesterified fatty acid content in wild-type or Syn4 transgenic mice (Table 1). These data confirmed that alterations in glucose tolerance directly correlated with increases in Syn4 and/or Munc18c protein in skeletal muscle and adipose tissue.

Syn4 overexpression does not alter glucose-stimulated insulin secretion in isolated islets.

The general physical and metabolic characteristics of Syn4 transgenic and wild-type mice were examined. Female Syn4 transgenic mice showed no significant differences in overall body weight (20 ± 1 g), lean body mass (16 ± 0.1 g), or fat body mass (2.0 ± 0.1 [transgenic] vs. 1.7 ± 0.1g [wild-type]) compared with female wild-type mice. Similarly, no differences in body weight (26 ± 1 g), lean body mass (22 ± 0.5 g), or fat body mass (2.1 ± 0.3 [transgenic] vs. 1.9 ± 0.2 g [wild-type]) were observed for male Syn4 transgenic or wild-type mice. Furthermore, there were no differences in weight of heart, lung, liver, spleen, kidney, or hindlimb skeletal muscle between Syn4 transgenic and wild-type mice (data not shown). However, there was a significant decrease in adipose mass in ovarian depots from the female Syn4 transgenic mice compared with wild-type mice (0.24 ± 0.01 [wild-type] vs. 0.15 ± 0.03 g [transgenic]; P < 0.02). The decreased weight of the fat pad of the female Syn4 transgenic mice correlated with a slight reduction in serum nonesterified fatty acid content under control conditions (Table 1). In addition, Syn4 transgenic mice showed no significant alterations in fasting blood glucose compared with wild-type mice, and no statistically significant differences were observed for serum triglycerides or cholesterol (Table 1). Despite the overexpression of Syn4 and Munc18c in transgenic islets (data not shown), no significant changes were observed in serum insulin levels.

To more thoroughly investigate the possibility that Syn4 transgenic islets had altered capacity to secreted insulin in response to glucose under static incubation conditions, islets were isolated from Syn4 transgenic or wild-type mice (Table 2). Levels of insulin secreted under basal conditions (2.8 mmol/l glucose) were similar between the female Syn4 transgenic and wild-type islets, and glucose stimulation (20 mmol/l) resulted in a similar 10- to 14-fold increase in insulin release from islets of wild-type and Syn4 transgenic mice. Moreover, islets isolated from male Syn4 transgenic mice showed nearly identical levels of insulin secretion under basal and glucose-stimulated conditions. No significant alterations of total insulin content of wild-type or Syn4 transgenic islets incubated with or without glucose were detected. These data demonstrated that increased expression of Syn4 in pancreatic islet cells did not alter the overall responsiveness to glucose under static conditions and had no effect on insulin content.

DISCUSSION

We have documented the generation and characterization of a transgenic mouse model in which the level of Syn4 protein can be modulated by tetracycline. In addition, our data have shown that alteration of Syn4 expression induces parallel changes in Munc18c protein levels, and that Syn4 overexpression in skeletal muscle, adipose tissue, and pancreas leads to increased glucose tolerance, which could be normalized to wild-type levels after tetracycline administration. Enhanced glucose tolerance resulted from significant increases in whole-body glucose uptake and similar increases in skeletal muscle glucose uptake and glycolysis. An important finding was that increased glucose tolerance in the Syn4 transgenic mice was independent of any significant effect on hepatic insulin action, indicating that the observed increase in whole-body glucose uptake resulted from enhancements in peripheral glucose uptake. The increased glucose clearance of the Syn4 transgenic mice was fully reversed after 1 week of oral tetracycline administration to ablate transgene expression, indicating that the accelerated rate of glucose uptake by the transgenic skeletal muscle was indeed the result of Syn4 transgene expression.

Our study has demonstrated that increasing the amount of Syn4 in skeletal muscle cells leads to an accelerated rate of glucose uptake in vivo, consistent with the proposed role of Syn4 as a positive regulator of insulin-stimulated GLUT4 translocation. These data are complemented by data from recent studies citing the downregulation of Syn4 gene expression in STZ-induced diabetic mice in correlation with a decreased insulin response (42). Moreover, our finding that increased Syn4 leads to enhanced glucose uptake specifically in skeletal muscle is consistent with the skeletal muscle insulin-resistant phenotype of the gene-targeted Syn4 heterozygous (+/−) knockout mice (43). However, these data contradict those from a study (44) that has suggested that a 2.2-fold increase in Syn4 abundance correlates with insulin resistance in the ZDF rat. Although Maier et al. (44) suggested that this may be an adaptive response to chronic hyperinsulinemia and/or hyperglycemia, another possible explanation for this discrepancy is an unbalanced abundance of Munc18c. This hypothesis is supported by the increased expression of endogenous Munc18c in the Syn4 transgenic mice and also by the decreased expression of endogenous Munc18c in the Syn4 (−/+) mice. Thus, further studies of the expression patterns of SNARE proteins of the ZDF rat will need to include evaluation of Munc18c abundance.

Although increased expression of Syn4 and Munc18c resulted in altered insulin action, Syn4 transgenic mice exhibited no significant alterations in serum insulin levels or insulin secretion from islets isolated therefrom. These results are consistent with Syn4 overexpression in β-cell lines (45), indicating that Syn4 overexpression is not deleterious to insulin granule exocytosis. These findings contrast with the results of a recent study that showed a significant reduction in insulin secretion in βHC9 cells electroporated with the soluble form of Syn4 (46). This difference is likely due to the inability of the soluble Syn4 to localize to the plasma membrane, which thus effectively sequesters the exocytotic machinery otherwise needed at the plasma membrane for secretory function.

Our studies have also established that Syn4 regulates the expression of Munc18c protein in vivo. As was first observed in Syn4 heterozygous mice, Munc18c abundance is reduced in parallel with Syn4 (43). Furthermore, using real-time PCR analysis of Syn4 heterozygous knockout mice, we recently determined that this effect is posttranscriptional, as Munc18c mRNA levels were not different in these mice compared with Munc18c mRNA from wild-type mice (data not shown). Similarly, quantitative immunoblotting using total homogenates of skeletal muscle (gastrocnemius) from male wild-type and Syn4 transgenic mice revealed that Syn4 was present at ∼50 and 250 nmol/l, respectively. SNAP-23 was identical in wild-type and transgenic mice at 150 nmol/l, whereas Munc18c was present in wild-type mice at 2 nmol/l, which was elevated to 6 nmol/l in Syn4 transgenic mice. This is in contrast to the quantitative immunoblotting of skeletal muscle of our Munc18c transgenic mice, which had 15 nmol/l Munc18c but only 50 nmol/l Syn4 with SNAP-23 at 150 nmol/l (36). Taken together, these data show that Syn4 abundance dictates Munc18c expression and not vice versa. Numerous studies have documented the inherent instability of the SM proteins in the absence of their cognate syntaxin binding partner in cell culture and in vitro (13,37,47) and support the notion that the structure and function of Munc18c and Syn4 proteins are interdependent.

The current study demonstrated that alterations in Syn4 protein levels in adipose tissue do not result in significant alterations in insulin-stimulated glucose uptake, an observation that supports our previously proposed idea that Syn4-mediated docking and fusion may not be the rate-limiting steps of GLUT4 translocation in adipocytes (32). Although we cannot exclude the possibility that the absence of effect in adipose is due to our using an in vivo method to study insulin action rather than using isolated adipocytes, adipocytes isolated from Syn4 heterozygous knockout mice also failed to show significant deficits in glucose uptake, whereas skeletal muscle glucose uptake was clearly impaired (43). Taken together, these data suggest that the number of fusion sites may be rate limiting in skeletal muscle cells but not adipocytes. In support of this, Khan et al. (35) showed that the abundance of Syn4 in the transverse tubule membrane of the skeletal muscle cells was a limiting factor for GLUT4–enhanced green fluorescent protein (EGFP) translocation, and that the increase of GLUT4-EGFP translocation exclusively in muscle is sufficient to account for enhanced whole-body glucose uptake in vivo. Another possibility is that increased Syn4 in the adipose tissue of the Syn4 transgenic mice may have altered the regulated secretion of an adipokine, which in turn altered insulin sensitivity. In sum, our data support the idea that the addition of Syn4-Munc18c fusion sites leads to an increased rate of GLUT4-mediated glucose uptake into skeletal muscle, and that this is sufficient to enhance whole-body insulin sensitivity in vivo.

FIG. 1.

Transgenic protein expression in pancreas, skeletal muscle, and fat of Syn4 transgenic mice. Heart, liver, pancreas (Panc), gastrocnemius skeletal muscle (Musc), and epididymal/ovarian adipose (Fat) tissues were isolated from Syn4 transgenic (Tg) and wild-type (Wt) littermate mice and homogenized as described in research design and methods. Samples (50 μg protein per lane) were resolved by SDS-PAGE and immunoblotted with antibodies for Syn4, Munc18c, SNAP-23, or VAMP2 (A) or IRAP and GLUT4 (20 μg protein per lane) (B). Immunoblots are representative of at least three mice per group.

FIG. 2.

Syn4 transgenic mice clear glucose more rapidly than wild-type mice. Glucose tolerance testing of Syn4 transgenic (Tg) and wild-type (Wt) mice was performed by intraperitoneal injection of d-glucose (2 g/kg body wt) into 4- to 6-month-old female mice fasted for 18 h. Blood glucose was monitored at 0, 30, 60, 90, and 120 min postinjection as described in research design and methods. Data are means ± SE from 8 Syn4 transgenic and 10 wild-type mice. *P < 0.05 vs. wild-type mice by unpaired Student’s t test.

FIG. 3.

Syn4 transgenic mice exhibit an enhanced rate of whole-body glucose flux as assessed by hyperinsulinemic-euglycemic clamp procedures. Shown are insulin-stimulated glucose uptake in female (A) and male (B) wild-type (Wt) and Syn4 transgenic (Tg) mice and hepatic glucose production in female (C) and male (D) mice. Data are means ± SE from five female wild-type mice (□), eight female Syn4 transgenic mice (▪), and seven pairs each of male transgenic and wild-type mice. *P < 0.05 vs. wild-type mice by unpaired Student’s t test.

FIG. 4.

Syn4 transgenic mice show increases in skeletal muscle glucose uptake and glycolysis in hyperinsulinemic-euglycemic clamp procedures. Shown are insulin-stimulated glucose uptake, glycolysis, and glycogen synthesis in gastrocnemius muscles in female (A) and male (B) wild-type (Wt) and Syn4 transgenic (Tg) mice. Data are means ± SE from four female pairs each of wild-type and Syn4 transgenic mice, four male wild-type mice, and seven male transgenic mice. *P < 0.05 vs. wild-type mice by unpaired Student’s t test. Also shown is glucose uptake into white adipose tissue in four pairs each of female (C) and male (D) wild-type or Syn4 transgenic mice, and glucose uptake into brown adipose tissue of four male wild-type and seven male Syn4 transgenic mice (E). □, wild-type; ▪, transgenic.

FIG. 5.

Insulin-stimulated GLUT4 translocation to the plasma membrane is increased in skeletal muscle of Syn4 transgenic (Tg) mice. A: Mice were fasted for 16 h and left untreated or injected with 21 units/kg body wt of insulin as described in research design and methods. Hindquarter muscles were homogenized and centrifuged to partition muscle into sarcolemma/transverse tubule membrane fractions (P1 and P2) and sucrose velocity sedimentation fractions (nine fractions). Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for GLUT4, GLUT1, or transferrin receptor (TnFR). B: Immunoblot of GLUT4 from fractions of insulin-stimulated wild-type and Syn4 transgenic mice. C and D: GLUT4-specific P2 fractions on immunoblots from five independent experiments were quantitated using optical density scanning, under unstimulated (C) and insulin-stimulated (D) conditions. Basal data are presented as percent of wild-type control. Insulin-stimulated GLUT4 translocation into the plasma membrane fraction was normalized to wild-type = 1. *P < 0.02. E: Immunoblot of Syn4 in P1 and P2 fractions from wild-type and Syn4 transgenic mice. F: Whole hindquarter tissue homogenates were prepared from mice stimulated with or without insulin and proteins were separated (as described in Fig. 1) for analysis of insulin signaling by immunoblotting for activation of Akt-1 (P-Akt). Data are representative of two independent experiments in male and female mice.

FIG. 6.

Downregulation of the Syn4 transgene reduces glucose tolerance to normal levels. A: Syn4 transgenic (Tg) and wild-type (Wt) female mice age 4–6 months were fed tetracycline (1 mg/ml) in drinking water for 7 days. Tissue extracts from pancreas (Panc), skeletal muscle (Musc), and ovarian adipose (Fat) tissue were prepared, and protein (50 μg) was separated by 10% SDS-PAGE gel and immunoblotted as described in Fig. 1. B: Before being killed for tissue analysis, mice were fasted for 18 h and their glucose tolerance tested as described in Fig. 2. Data are means ± SE of eight transgenic and seven wild-type mice and are representative of at least three independent sets of tissues.

TABLE 1

Metabolic characteristics of wild-type and syntaxin 4 transgenic mice

TABLE 2

Glucose-stimulated insulin secretion in islets isolated from wild-type and syntaxin 4 transgenic mice

Acknowledgments

This study was supported by a predoctoral fellowship from the Indiana University Diabetes Graduate Training Program (to B.A.S.); a predoctoral training (T32) fellowship from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-064466-01) (to A.K.N.); and a career development award from the American Diabetes Association (1-03-CD-10), a research grant from the Indiana University School of Medicine Showalter Research Trust Fund, and National Institutes of Health Grant DK-067912 (to D.C.T.). The clamp studies were conducted at the Yale Mouse Metabolic Phenotyping Center and supported by grants from the U.S. Public Health Service (U24 DK-59635; to J.K.K.) and the American Diabetes Association (7-01-JF-05; to J.K.K.).

We are very grateful to Dr. Jeffrey E. Pessin for assistance in generating the founding transgenic mice at the University of Iowa Transgenic Animal Facility and to Dr. John Corbett (St. Louis University, St. Louis, MO) for assistance in isolating islets for immunoblotting. We thank Rhonda M. Thomas for her expert technical assistance and Drs. Ulli Certa, Richard Scheller, and Steve Waters for their gifts of the pCOMBI vector, Syn4 cDNA, and IRAP antibody, respectively. We are indebted to Drs. Robert Considine and Robert Harris for their critical review of this manuscript. The Indiana University School of Medicine Analyte Core Facility was invaluable for its assistance with metabolic measurements of serum samples.

Footnotes

    • Accepted May 26, 2004.
    • Received February 17, 2004.

REFERENCES

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