The SNARE Protein SNAP23 and the SNARE-Interacting Protein Munc18c in Human Skeletal Muscle Are Implicated in Insulin Resistance/Type 2 Diabetes

Details of reagents, Western blot and RT-PCR analyses, quantification of lipid droplets, human skeletal myotubes, time-lapse studies, the skeletal muscle cell line L6 G4m, and coprecipitation studies are available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1503/DC1.

The main study population consisted of eight healthy, lean subjects and eight obese, nondiabetic subjects carefully matched to eight obese patients with type 2 diabetes. These subjects were recruited by the Diabetes Research Centre at Odense University Hospital, Denmark. In addition, six monozygotic twin pairs discordant for type 2 diabetes were recruited by the Diabetes Research Centre at Odense University Hospital, Denmark, and five monozygotic twin pairs discordant for type 2 diabetes were recruited from the Swedish twin register. See online appendix for medication details and eligibility criteria.

The subjects underwent a 4-h euglycemic hyperinsulinemic clamp and routine analysis, and muscle biopsies were taken from the subjects before and after the clamp as described (11). See online appendix for further details.

Muscle biopsy sample (50 mg) was homogenized in 300 μl of 10 mmol/l NaHCO₃, pH 7.5, with 5 mmol/l NaN₃, 100 μmol/l phenylmethylsulfonylfluoride, and the Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics AB) using the Polytron 1300D (Kinematica AG) homogenizer at speed 20 for 30 s. Homogenates were then centrifuged for 1 min at 500g and 4°C, and the supernatants were transferred to new tubes and recentrifuged for 10 min at 20,000g and 4°C. The pellet and the supernatant were recovered and dissolved in SDS-gel electrophoresis sample buffer.

To assess the recovery of cytosol, microsomes, and plasma membrane during the procedure, we followed the recovery of marker enzymes present in the homogenate of the skeletal muscle biopsies. The recovery of these marker enzymes in the supernatant after the 500g centrifugation was as follows: GAPDH, 60%; α-tubulin, 63%; GRP78, 53%; Golgi protein 58k, 66%; and Na/K ATPase, 58%; the recovery of SNAP23 was 75% (all results are the mean of two experiments). The recovery of the marker enzymes present in the 500g supernatant after the subsequent 20,000g centrifugation was as follows: GAPDH, 97%; α-tubulin, 100%; GRP78, 68%; Golgi protein 58k, 100%; and Na/K ATPase, 54%; the recovery of SNAP23 was 59% (all results are the mean of two experiments).

These results indicate a good recovery of the cytosol and microsomes present in the supernatant of the skeletal muscle homogenate obtained after the 20,000g centrifugation. Thus, we conclude that the cytosol and microsomes recovered in this subcellular fraction are representative of the cytosol and microsomes present in the skeletal muscle biopsy sample.

The distribution of marker proteins between the supernatant (microsomes/cytosol) and the pellet (containing the markers for plasma membrane and t-tubules) obtained after the 20,000g centrifugation is shown in supplementary Fig. 1 (available in an online appendix). The supernatant was highly enriched in markers for microsomes and cytosol, while only traces of plasma membrane and t-tubules were present in this fraction. We therefore conclude that we isolated a microsomal/cytosolic fraction that was not contaminated to any significant degree with plasma membranes or t-tubules. The pellet was highly enriched in markers for plasma membranes and t-tubules and was only marginally contaminated by markers for cytosol and microsomes. However, this fraction also contained most of the cellular organelles, and as indicated above, the recovery of the plasma membrane markers was lower than the recovery of the microsomal/cytosolic markers. These circumstances should be taken into account when evaluating results about SNAP23 in the plasma membrane.

Comparisons of mean values from multiple groups were made using one-way ANOVA and Tukey post hoc testing. ANOVA and Dunnett post hoc testing was used to compare different groups with a control group. Comparison between two groups was carried out by t test. Correlation tests were performed using Pearson two-tailed correlation test. A P value of <0.05 was considered significant. Presented P values are noncorrected.

Clinical data for patients with type 2 diabetes and lean and obese control subjects are shown in Table 1. The patients with type 2 diabetes had significantly lower insulin sensitivity compared with the control subjects. This was supported by a lower phosphorylation of AKT (Protein Kinase B) in the skeletal muscle biopsies taken at the end of the euglycemic hyperinsulinemic clamp from patients with type 2 diabetes (Fig. 1,A and B; supplementary Fig. 2A and B [available in an online appendix]). In addition, plasma HDL levels were lower and plasma triglyceride levels were higher in the patients with type 2 diabetes. We also showed that the accumulation of neutral lipids (measured as the total area of Oil Red O-stained lipid droplets) was greater in skeletal muscle biopsy samples from patients with type 2 diabetes compared with both lean and obese control subjects (Fig. 1,C). However, the extent of lipid droplet accumulation was small even in the biopsy samples from patients with type 2 diabetes (Fig. 1,C). There were no differences in any of these variables between the control groups (Table 1; Fig. 1 A and C).

The total SNAP23 mRNA levels were similar in skeletal muscle from lean and obese control subjects and patients with type 2 diabetes (Fig. 2,A). However, the total SNAP23 protein levels were higher in skeletal muscle from patients with type 2 diabetes than from the lean control subjects (Fig. 2,B; supplementary Fig. 2B and C). We observed a significant negative correlation between total SNAP23 protein levels in skeletal muscle and insulin sensitivity (determined as glucose infusion rate after the euglycemic hyperinsulinemic clamp) when the results from the three groups were combined (Fig. 3 C). However, although there were negative correlations between total SNAP23 levels in skeletal muscle and insulin sensitivity in the individual groups (lean control subjects, r = −0.54, P = 0.17; obese control subjects, r = −0.69, P = 0.06; patients with type 2 diabetes, r = −0.33, P = 0.43), none of these individual correlations were statistically significant.

We addressed the role of genetic or environmental influences on SNAP23 expression by investigating skeletal muscle from six pairs of monozygotic Danish twins and five pairs of monozygotic Swedish twins discordant for type 2 diabetes (see Table 1 for clinical characteristics). For the Danish group, SNAP23 mRNA levels in skeletal muscle did not differ between the twins with type 2 diabetes and those without (Table 2). However, for the Swedish group, SNAP23 mRNA levels were significantly lower in skeletal muscle from the twins with type 2 diabetes, and this difference remained when the groups were combined (Table 2). By contrast, we did not observe any differences in SNAP23 protein expression between the twins with type 2 diabetes and those without in either the Danish or Swedish group, or in the combined group (Table 2).

To further address the role of environmental factors, we incubated human myotubes (derived from satellite cells from a metabolically healthy person) with high levels of glucose, fatty acids, or insulin or combinations of these to simulate the conditions present in type 2 diabetes. Fatty acids and insulin combined significantly decreased SNAP23 mRNA levels (supplementary Fig. 3A). None of these treatments affected the SNAP23 protein levels (supplementary Fig. 3B).

We have previously shown that oleic acid induces the redistribution of SNAP23 and promotes insulin resistance in HL1 cardiomyocytes in vitro (8). To determine whether these findings translate to an in vivo situation, we investigated if the localization of SNAP23 in skeletal muscle cells differed between patients with type 2 diabetes (i.e., with insulin resistance and increased levels of neutral lipids in their skeletal muscle) and lean control subjects (i.e., without insulin resistance and with low levels of neutral lipids in the skeletal muscle). The patients and control subjects used in this study were randomly selected from the main study population.

Subcellular fractionation of the skeletal muscle biopsy samples showed that SNAP23 levels were higher in the microsomal/cytosol fraction of skeletal muscle isolated from patients with type 2 diabetes compared with that from lean control subjects (Fig. 3,A and B), and these levels were similar in biopsy samples taken before or after the euglycemic hyperinsulinemic clamp (data not shown). SNAP23 levels were decreased in the plasma membrane/t-tubule–containing fraction of skeletal muscle isolated from patients with type 2 diabetes (Fig. 3 C and D), but it is important to note that this fraction also contains cellular organelles. We also showed a clear separation between the insulin-stimulated glucose infusion rates measured in the patients with type 2 diabetes (range ∼40–220 mg/m²/min; n = 3) and the rates measured in the lean control subjects (range 300–400 mg/m²/min; n = 3), which suggests that SNAP23 distribution is related to insulin sensitivity. Together with our earlier in vitro results (8), these data indicate that the diversion of SNAP23 to the interior of the skeletal muscle cell may be important for the induction of insulin resistance/type 2 diabetes.

To further investigate the localization of SNAP23, we also used immunohistochemistry and confocal microscopy to analyze skeletal muscle from patients with type 2 diabetes and lean and obese control subjects (Fig. 4). SNAP23 was mostly diffusely spread in the cytosol of skeletal muscle cells from both groups, but higher levels of immunoreactive SNAP23 were present in the plasma membrane from the lean control subjects.

To clarify the nature of this cytosolic localization, we microinjected a plasmid coding for SNAP23-CFP into human myoblasts and followed SNAP23-CFP over time using confocal microscopy (Fig. 5 and supplementary Movie 1, available in online appendix 2). For comparison, we also followed the membrane-spanning protein syntaxin-4, which is transferred to the plasma membrane through the secretory pathway (supplementary Fig. 4 and supplementary Movie 2, available in online appendix 3). Expression of SNAP23-CFP was observed 130 min after the injection. It appeared diffusely spread in the major part of the cytosol, compatible with synthesis and accumulation in the cytosol. Some fluorescence was also localized to organelle structures, most likely the Golgi apparatus. The protein had reached the plasma membrane after 130–160 min. These results suggest that SNAP23 is synthesized in the cytosol and sorted from this compartment to the plasma membrane.

We also investigated the expression of proteins known to interact with SNARE proteins or participate in the formation of lipid droplets: SNAPAP, a protein known to interact with SNAP23 (12); Synip (13,14) and Munc18c (15), proteins that interact with the SNARE complex involved in GLUT4 translocation; ADRP, TIP47, and LSDP5, lipid droplet–associated proteins (5); and PLD1 and ERK2, enzymes important for the assembly of lipid droplets (16).

Of all the proteins investigated, the only significant change in mRNA expression was noted for Munc18c (Fig. 6,A). Skeletal muscle from patients with type 2 diabetes had higher levels of Munc18c mRNA compared with biopsy samples from both lean and obese control subjects (Fig. 6,A) and higher levels of Munc18c protein compared with that from lean control subjects (Fig. 6 B; supplementary Fig. 2B and D). The levels of Munc18c protein were slightly lower after the clamp (0.86 ± 0.01 fold of levels before the clamp; n = 4; P = 0.0002). No changes in Munc18c mRNA expression were observed in adipose tissue biopsy samples from patients with type 2 diabetes compared with those from lean and obese control subjects (data not shown).

To determine whether an increased flow of lipids to skeletal muscle could affect Munc18c expression, we incubated human myotubes with oleic acid and observed increases in both mRNA and protein levels of Munc18c (supplementary Fig. 5A and B). These results indicate that Munc18c expression may be promoted by environmental influences that result in increased levels of fatty acids, but they do not rule out the importance of genetic influences.

We addressed the role of genetic influences on Munc18c expression by investigating skeletal muscle from the monozygotic twins discordant for type 2 diabetes described above. For the Danish group, neither the mRNA nor protein levels of Munc18c were different in skeletal muscle from the twins with type 2 diabetes compared with that from the nondiabetic twins (Table 2). For the Swedish group, Munc18c mRNA levels were significantly lower in skeletal muscle from the twins with type 2 diabetes, but there were no differences in the Munc18c protein levels (Table 2). Combining the results from the two groups showed that neither the mRNA nor protein levels of Munc18c were significantly different between the twins with type 2 diabetes and the nondiabetic twins (Table 2), indicating that there may be a genetic background at least for the increased expression of Munc18c protein in skeletal muscle from patients with type 2 diabetes.

Overexpression of Munc18c in L6 G4m cells promoted increased expression of total SNAP23 protein (supplementary Fig. 6A). Although we also observed an increase in the amount of SNAP23 in the microsomal/cytosolic fraction (supplementary Fig. 6B), this increase was not significant when expressed as a percentage of the total SNAP23 pool in the cell (data not shown). Transfection of human myotubes with Munc18c siRNA significantly reduced the Munc18c and SNAP23 protein levels (supplementary Fig. 6C and D) but did not affect the distribution of SNAP23 in the cell (data not shown).

Because these results indicated that Munc18c may promote the expression of SNAP23, we tested the possibility that Munc18c increases SNAP23 expression by forming a complex with SNAP23. We showed that Munc18c coprecipitated with syntaxin-4 in L6 G4m cells and that SNAP23 seemed to be completely excluded from the complex between Munc18c and syntaxin-4 (supplementary Fig. 6E).

Our previous in vitro studies in cardiomyocytes demonstrated that SNAP23 could have an important role in the development of insulin resistance (8). Here, we investigated the expression and localization of SNAP23 in vivo in skeletal muscle biopsy samples from patients with type 2 diabetes and control subjects.

We first showed that the patients with type 2 diabetes (and thus insulin resistance) had increased accumulation of lipid droplets in their skeletal muscle, in agreement with earlier results (1,17,18). There was very little lipid accumulation in biopsy samples from both lean healthy and obese nondiabetic control subjects, demonstrating that obese people do not necessarily store fat in their skeletal muscles. Furthermore, we did not observe any major difference in variables linked to insulin resistance between these two control groups, consistent with the known overlap in insulin sensitivity between lean control subjects and obese nondiabetic subjects (19).

We did not observe any difference among SNAP23 mRNA levels in skeletal muscle biopsy samples from patients with type 2 diabetes and the two control groups. This result contrasts with a recent study showing a statistically significant decrease in SNAP23 mRNA levels in skeletal muscle from insulin-resistant women compared with that from insulin-sensitive women (20). However, when we compared monozygotic twins discordant for type 2 diabetes, we found a similar decrease in SNAP23 mRNA levels in the twins with type 2 diabetes compared with the nondiabetic twins in the Swedish cohort but not in the Danish cohort. Thus, there may be population variations in the expression of SNAP23 mRNA.

In contrast to mRNA, SNAP23 protein levels were higher in skeletal muscle from patients with type 2 diabetes compared with lean control subjects and did not differ between the monozygotic twins discordant for type 2 diabetes in either the Swedish cohort or the Danish cohort. This lack of reflection between SNAP23 mRNA and protein expression argues for a posttranscriptional regulation of SNAP23 expression.

The decrease in SNAP23 mRNA levels in Swedish twins with type 2 diabetes (which remained when the two cohorts of twins were combined) suggested that SNAP23 mRNA levels may depend on environmental factors, whereas the lack of difference in SNAP23 protein levels between the twins indicated a genetic influence on protein expression. These findings were supported by our observation of decreased SNAP23 mRNA levels and no effect on protein levels in human myotubes incubated under conditions that simulate the situation in type 2 diabetes.

We have previously demonstrated that oleic acid promotes insulin resistance, increases the storage of triglycerides in lipid droplets, and induces the redistribution of SNAP23 from the plasma membrane to the interior of the cell in cardiomyocytes (8). A major aim of the present study was to determine whether SNAP23 also redistributes in vivo in patients with insulin resistance/type 2 diabetes. By comparing patients with type 2 diabetes (i.e., patients with insulin resistance and increased levels of neutral lipids in their skeletal muscle) with lean control subjects (subjects without insulin resistance and with low levels of neutral lipids in the skeletal muscle), we obtained an in vivo situation that resembled the earlier in vitro experiments (8). It should be noted, however, that we cannot separate insulin resistance from type 2 diabetes in this study as we did not include a control group with insulin resistance but without type 2 diabetes.

In agreement with the fatty acid–induced redistribution of SNAP23 in HL-1 cardiomyocytes (8), subcellular fractionation studies showed that SNAP23 was present at higher levels in the microsomal/cytosolic fraction (i.e., interior of the cell) of skeletal muscle from patients with type 2 diabetes compared with that from lean control subjects. Immunohistochemistry and confocal microscopy confirmed that plasma membrane levels of SNAP23 were lower in skeletal muscle from patients with type 2 diabetes, while the majority of the protein was present in a diffuse intracellular pattern compatible with a cytosolic localization.

The cytosolic localization of SNAP23 was supported by time-lapse studies in human myoblasts derived from satellite cells, which showed that SNAP23 was synthesized in the cytosol and, although it reached the plasma membrane, a substantial amount of the total pool remained in the cytosol. These studies explain the cytosolic appearance of SNAP23 in skeletal muscle from both the patients with type 2 diabetes and the control subjects, and are consistent with SNAP23 lacking signal and membrane-spanning sequences; SNAP23 would thus not be cotranslationally targeted to membranes or integrated into these structures in other ways but would associate with membranes by a process that is highly dependent on covalent acylation (21).

Together, these results are thus consistent with the hypothesis that SNAP23 redistribution plays an important role in the development of insulin resistance and/or type 2 diabetes (8). Moreover, they suggest that the sorting of SNAP23 from the biosynthesis pool in the cytosol to the plasma membrane may be impaired in skeletal muscle from patients with type 2 diabetes. Thus, an important future task is to elucidate the intracellular processes involved in sorting of SNAP23 between the interior of the cell and the plasma membrane and to clarify how these processes are changed in patients with type 2 diabetes. It is also possible that the increased SNAP23 protein expression observed in skeletal muscle from patients with type 2 diabetes represents an attempt to overcome the insulin resistance. Unfortunately, we could not test this possibility as we did not induce a substantial and stable insulin resistance in skeletal muscle cells by fatty acid treatment (supplementary Fig. 7).

We also investigated the expression of proteins that could potentially interact with SNAP23 in skeletal muscle biopsy samples from patients with type 2 diabetes and the lean and obese control subjects. Of the candidates investigated, only Munc18c expression differed between patients with type 2 diabetes and the control subjects, with increased expression in skeletal muscle from patients with type 2 diabetes. In agreement with an earlier study (22), there were no differences in skeletal muscle Munc18c levels between the lean and obese control subjects.

Munc18c binds to syntaxin-4/SNAP23/VAMP2, the SNARE system of importance for fusion of GLUT4 vesicles with the plasma membrane (15). The precise role of Munc18c is not clear, as in some studies it has been shown to inhibit GLUT4 translocation (23,,,–27) and in others it has been shown to promote GLUT4 translocation (28,–30). However, studies in transgenic mice demonstrated that induction of Munc18c expression promotes insulin resistance (31). Our results in humans support the view that Munc18c plays a role in the development of insulin resistance/type 2 diabetes. We did not observe any increase in the expression of Munc18c in adipose tissue biopsy samples from patients with type 2 diabetes, indicating that the potential role of Munc18c in the development of type 2 diabetes may be confined to skeletal muscle.

We showed that Munc18c expression increased in human myotubes incubated with oleic acid. These data indicate that fatty acids may have a role in the regulation of Munc18c expression, and are consistent with a previous study that showed increased Munc18c protein levels in mice fed a high-fat diet (25). Because the patients with type 2 diabetes had higher levels of plasma triglycerides and skeletal muscle lipid accumulation compared with healthy control subjects, we thus propose that an increased inflow of lipids to the skeletal muscle may partly explain the increased expression of Munc18c in these patients.

These data may suggest that environmental influences may be important in promoting Munc18c expression, but they do not exclude a role for genetic influences that may, for example, determine the extent of inflow of lipids to the skeletal muscle cells. To further address this possibility, we compared the expression of Munc18c in skeletal muscle from the two sets of monozygotic twins discussed above. We did not observe a significant increase in Munc18c expression in the twins with type 2 diabetes compared with the nondiabetic cotwins, which argues for a potential genetic influence on the increased expression of Munc18c in patients with type 2 diabetes.

Overexpression and knockdown experiments indicated that Munc18c may regulate the cellular levels of SNAP23 but not the cellular distribution of SNAP23. Thus, we propose that the increased levels of Munc18c in the skeletal muscle from patients with type 2 diabetes could at least partially explain the increase in total SNAP23 levels but not the redistribution to the microsomal/cytosolic fraction. We also showed that SNAP23 did not form a complex with Munc18c, which excludes a heterodimerization between the two proteins as an explanation for the role of Munc18c on SNAP23 expression.

In summary, the main finding from our present study indicates that SNAP23 is redistributed to the cell interior in skeletal muscle from patients with type 2 diabetes. Together with our previous in vitro experiments, these results indicate that the redistribution of SNAP23 could play an important role in the development of insulin resistance/type 2 diabetes. We also propose that Munc18c, which increased in patients with type 2 diabetes, is a potential regulator of SNAP23 expression but not of its redistribution.

This work was supported by grants from the Swedish Research Council, the Swedish Foundation for Strategic Research, the Swedish Heart and Lung Foundation, the NovoNordic Foundation, the Swedish Diabetes Association, the Söderberg foundation, and the EU projects NACARDIO and LipidomicNet. The group of Swedish twins was recruited from The Swedish Twin Registry, which is supported by grants from the Swedish Department of Higher Education and the Swedish Research Council. No other potential conflicts of interest relevant to this article were reported.

Parts of this study were presented orally by K.H. at the 70th Scientific Sessions of the American Diabetes Association, 25–29 June 2010, Orlando, FL.

We thank Dr. Rosie Perkins for expert editing of the manuscript.