This is the first study to report tissue Munc18c, and Syntaxin 4 protein content in humans. We found no significant differences in the content of either protein in obese compared to lean subjects after an overnight fast (Figures and ). Therefore, these data suggest insulin resistance associated with obesity in humans is not explained by constitutive differences in skeletal muscle Munc18c and Syntaxin 4 content.
Prolonged fasting induced a significant decrease in glucose uptake during the clamp in lean and obese subjects after the 48 compared to the 12 hour fast (Table ). Fasting for 48 hours did not change Syntaxin 4 (Figure ), but significantly decreased Munc18c content in lean and obese subjects (Figure ). These data suggest prolonged fasting decreased basal skeletal muscle Munc18c content regardless of body type, which may be one mechanism by which fasting decreased insulin stimulated glucose uptake.
The optimum amount of skeletal muscle Munc18c to promote GLUT4 vesicle fusion is not known. However, it has been demonstrated that deletion of the Munc18c gene is lethal [19
], and that too little or too much Munc18c can alter insulin sensitivity in animals [7
], and cell culture [6
]. Further, the content of Munc18c relative to Syntaxin 4 appears more important than the absolute content of each on whole body glucose disposal in mice [7
]. This study elaborates on previous data and suggests that Munc18c, and not Syntaxin 4, appears to be the regulated of these two proteins in human skeletal muscle.
We have previously reported increased Munc18c with no change in Syntaxin 4 content in mice over expressing skeletal muscle lipoprotein lipase [20
] which correlated with decreased insulin action following high fat feeding. The current data are different from that found in rodents, as both Munc18c and Syntaxin 4 content were unchanged in obese compared to lean humans after an overnight fast despite significant differences in glucose Rd during the clamp.
Moreover, our data demonstrating decreased skeletal muscle Munc18c content after a 48 hour fast are not what was expected from the animal literature where tissue Munc18c content is negatively related to insulin action [6
]. Therefore, it is possible that decreased Munc18c is a compensatory response to maintain insulin action during a prolonged fast. These discrepancies in data in humans vs. mice suggest a species difference in the regulation of Munc18c.
Basal Syntaxin 4 content was not significantly different between lean and obese subjects after 12 and 48 hours of fasting. In mice, decreased Syntaxin 4 content has been associated with insulin resistance during a clamp [9
] and following streptozotocin-induced diabetes [21
]. Our data differ from animal studies and suggest Syntaxin 4 content may not be involved in short-term regulation of insulin action in humans, and may not be important to obesity-induced insulin resistance.
We found a significant relationship between plasma glucose concentration and Munc18c content (Figure , p = 0.0015) in lean and obese subjects after 12 and 48 hr fasts. Whether glucose concentration influences Munc18c content or vice versa
remains to be determined. However, our data indicate close regulation between these 2 parameters. In the fasting state FFA and beta-hydroxybutyrate concentrations change dramatically, and could reduce by an unknown mechanism the Munc18c content of skeletal muscle. Another possible explanation is that alterations in the expression of muscle Munc18c may promote changes in glucose concentration and skeletal muscle AKT content. Animal data suggest that Munc18c may promote insulin resistance [6
]. The relationship between plasma glucose concentration and skeletal muscle Munc18c in this study is consistent with this idea. If Munc18c influences glucose concentration, what else besides FFA and beta-hydroxybutyrate drives changes in Munc18c content? Previous data from our laboratory indicated skeletal muscle LPL overexpression in mice increased Munc18c and intramuscular triglyceride content, along with a decrease in insulin action [20
]. These animal data suggest increased storage of intramuscular triglyceride in muscle may be related to increased Munc18c content. In hindsight, measurement of intramuscular triglyceride concentration would have been informative in this study to determine if there is a relationship with muscle Munc18c content.
The significant relationships between glucose (Figure ), FFA (Figure ) and beta-hydroxybutyrate concentration (Figure ) suggest there are potential associations between plasma substrates and muscle Munc18c. The significant negative relationship between Munc18c and AKT content in skeletal muscle biopsies (Figure ) could indicate that increased Munc18c expression in skeletal muscle may be related to decreased AKT content, and promote insulin resistance. The relationship between Munc18c and AKT is somewhat puzzling because a decrease in Munc18c with fasting (Figure ) corresponded to decreased insulin action (Table ) as well as an increase in AKT content. However, we did not find a significant change in the phosphorylation of AKT relative to the total content, suggesting that the change in total content was not related to activity. Further, it is possible that both Munc18c and AKT content were altered in response to other signals, and that the change in one does not promote a change in the other. This study was not designed to test relationships between Munc18c and metabolic or insulin signaling variables. However, these relationships are intriguing and suggest that the content of Munc18c changes in parallel with blood metabolites and the insulin signaling cascade.
We are hesitant to discuss non-significant relationships, but given that this is the first report of Munc18c and Syntaxin 4 in humans, we feel obligated to show all data that may move the field forward if further studied. We did not find significant relationships between Munc18c and insulin concentration (p = 0.22), as well as the adipokines adiponectin (p = 0.63), and leptin (p = 0.43) which are thought to modulate insulin action [22
]. We found several interesting, but non-significant negative relationships between Munc18c and glucose disposal during the insulin clamp (p = 0.15), and insulin signaling including insulin receptor phosphotyrosine content (p = 0.085), serine307 phosphorylation of IRS-1/total IRS-1 (p = 0.11), and pan85 content (p = 0.11). Given that there was a significant relationship between Munc18c and AKT content, these data suggest that there may be concurrent regulation of Munc18c and the insulin signaling cascade.