Introduction
Increased circulation of fatty acids (FAs) and accumulation of lipids in muscle contributes to glucose intolerance [
1,
2]. Several FA-induced mechanisms of this metabolic defect have been described [
3]. Although it is possible that one of these dominates, a consensus is that these mechanisms are interdependent [
3]. Similar to the negative consequences of FAs, hyperinsulinaemia causes metabolic derangement [
4,
5], possibly because of impaired insulin-responsive GLUT4 regulation. In fact, hyperinsulinaemia-induced GLUT4 dysregulation has been documented in 3T3-L1 adipocytes and L6 myotubes [
6,
7]. Although the details remain to be defined, data from these cells suggest cortical filamentous actin (F-actin) loss, not defects in insulin signalling, as a basis of hyperinsulinaemia-induced GLUT4 dysregulation [
6,
7]. A comparable loss of F-actin is also observed in skeletal muscle obtained from insulin-resistant obese Zucker rats [
7]. These findings complement an emerging appreciation in the field that, while defects in proximal insulin signalling may occur in various insulin-resistant models, distal GLUT4 defects may represent another critical node of impaired glucose metabolism [
8‐
10].
It is well documented that insulin elicits a rapid dynamic remodelling of actin filaments into a cortical mesh, and that this mesh is necessary for GLUT4 translocation [
9]. Also relevant are data that place GLUT4 storage vesicles (GSVs) in the F-actin meshwork, suggesting that this cytoskeletal structure tethers GSVs in the region beneath the plasma membrane (PM), where the final steps of GSV/PM docking and fusion are critically regulated [
9]. Interestingly, cholesterol-enriched regions of the PM have been implicated in regulating F-actin structure [
11‐
13]. While underexplored, cholesterol regulation at the PM is likely to play a very relevant role in GLUT4 regulation. For example, extraction of small (≤30%) amounts of PM cholesterol enhances insulin-stimulated GLUT4 translocation [
14,
15]. The cholesterol-dependent gain in PM GLUT4 resulting from a small decrease in PM cholesterol is not associated with an inhibition of endocytosis [
14,
15], but this does occur with extraction of larger amounts [
14‐
17].
These findings prompted this study to examine membrane/cytoskeletal features under in vivo and in vitro conditions of FA-induced insulin resistance. Herein, data suggest that excess FAs induce the accrual of skeletal muscle membrane cholesterol and that this provokes a loss in F-actin that plays a critical role in GLUT4 translocation. Data further indicate that the accumulation of membrane cholesterol results from increased hexosamine biosynthesis pathway (HBP) activity engaging a cholesterologenic transcriptional response. Adding to the notion that defects other than those in proximal insulin signalling contribute to insulin resistance, it was found that cholesterol lowering rescues F-actin and GLUT4 responsiveness, but not defects in insulin signalling. Together, these data highlight the hitherto unappreciated importance of membrane/cytoskeletal derangement in GLUT4 dysfunction in insulin resistance.
Discussion
While the derangements that contribute to glucose intolerance are certainly numerous and complex, work throughout the field has highlighted the detrimental effects of saturated FAs, and particularly palmitate, on glucose metabolism in skeletal muscle. Data presented here suggest that membrane cholesterol accrual may play a contributing role in obesity-associated glucose intolerance. Mechanistically, the cholesterol-laden membrane compromises F-actin structure, documented by several laboratories to be an essential feature of insulin and GLUT4 action [
6,
7,
37‐
39]. Data also suggest increased HBP activity as a molecular basis for the membrane cholesterol accrual via engagement of a cholesterolgenic programme.
Interestingly, the palmitate-induced decrease in insulin signalling to Akt2 and AS160 was not as damaging as would be predicted or not yet advanced to a level to compromise GLUT4 regulation. Surprisingly, correction of the membrane/cytoskeletal defect completely restored the palmitate-induced defect in GLUT4/glucose transport regulation, but not the Akt2/AS160 impairment. This view is supported by recent analyses showing that the maximal effect of insulin on GLUT4 translocation in L6 myotubes occurs at insulin concentrations where only 5% of the total Akt pool is phosphorylated [
8]. Moreover, treatments with lower palmitate concentrations (<300 μmol/l) were found to still impair insulin-regulated GLUT4 translocation, yet insulin signalling remained intact. Therefore, these data support a membrane/cytoskeletal defect beyond proximal insulin signalling as a major GLUT4/glucose transport system deregulator.
With regard to the cholesterolgenic model of insulin resistance suggested herein, while we found a significant elevation in the expression of
Hmgcr with palmitate, the expression of other SREBP-1-regulated genes (e.g.
Acaca,
Fasn and
Hmgcs1) did not consistently follow the same pattern of expression (ESM Fig.
3). For example, whereas the transcript level of
Acaca was increased by palmitate, levels of
Fasn and
Hmgcs1 were not. These variations may result from the differing contributions of the two splice variants of SREBP-1 (1a and 1c) in the control of lipogenic transcription, as it has been documented that SREBP-1a and SREBP-1c have differing levels of control over these genes [
40,
41]. These experiments also found that DON was ineffective in preventing the palmitate-induced increase in
Acaca. Unfortunately, we did not measure diacylglycerol or triacylglycerol levels. Nevertheless, DON treatment prevented the palmitate-induced cholesterolgenic response, lending support to a membrane cholesterol-induced, rather than a lipid-induced, state of insulin resistance. Moreover, removal of the excess membrane cholesterol with βCD prevented the membrane/cytoskeletal defect and GLUT4/glucose transport dysregulation. Interestingly, this tactic did not restore insulin signalling, perhaps suggesting that an increase in intramyocellular lipids is occurring and is the basis for the palmitate-induced impairment in insulin signalling. These observations suggest high-fat-feeding-induced cholesterol, but not diacylglycerol/triacylglycerol, accrual compromises membrane/cytoskeletal mechanisms important for glucose transport.
The concept that increased HBP activity might provoke insulin resistance via a SREBP response is supported by recent data showing that inhibition of SREBP improves insulin resistance in high-fat-fed mice [
42]. Whether this is a SREBP/HMGR response is not clear, but our work would suggest this to be a possibility. In addition to HBP activity possibly increasing SREBP transcript via O-linked
N-acetylglucosylation of Sp1 [
33,
36], the HBP has also been implicated in increased liver X receptor-dependent activation of the SREBP-1c promoter [
43]. Given the complexity of the transcriptional control of cholesterol synthesis, nuclear analyses dissecting how the HBP provokes membrane cholesterol toxicity are currently under way.
It should be noted that the high-fat diets we employed for the swine and mice contained 2% and 0.2% cholesterol, respectively. This difference in dietary cholesterol amount did not equate to a similar difference in muscle membrane cholesterol accrual. For example, the high-fat-fed swine that consumed more cholesterol displayed a lower increase in muscle membrane cholesterol than the mice fed a high-fat diet containing far less cholesterol. As mammalian cells contain an intricate feedback system that senses the level of membrane cholesterol and modulates the transcription of genes that mediate cholesterol synthesis and uptake [
44], it is likely that circulating cholesterol does not contribute to muscle membrane cholesterol content. In fact, these data seem to collectively place the HBP as a central participant in peripheral cholesterol toxicity.
It is possible that the F-actin changes are localised in cholesterol-enriched caveolae microdomain membrane regions. Intriguingly, imaging analyses from this study support the observed reciprocal changes in membrane cholesterol and F-actin. Notably, F-actin labelling has been documented in electron micrographs to be localised in caveolae regions [
45]. While caveolae have been postulated to contribute to many functions in insulin and GLUT4 action through the years [
9], these findings must be cautiously interpreted. Concerns regarding the study of caveolae are associated with each of the numerous strategic approaches used to study these structures. In spite of these caveats, fluorescence confocal labelling of caveolae and F-actin have revealed actin filaments emanating from caveolae microdomains [
11]. Moreover, quantitative electron microscopy and freeze-fracture analyses have revealed that cytoskeletal components, including actin, are highly enriched in the membrane area underlying the neck part of caveolae [
12]. These findings assign caveolae a critical role in the functionality of F-actin organisation. Given the unequivocal importance of F-actin in insulin-regulated GLUT4 translocation, these findings also emphasise the importance of caveolae in GLUT4 regulation.
Of interest to our understanding of caveolae-associated actin regulation are new electron microscopic data showing high concentrations of phosphatidylinositol 4,5-bisphosphate (PIP
2) at the rim of caveolae [
46]. This localisation of PIP
2 is consistent with its regulation of the cytoskeleton where the availability of this lipid is recognised to modulate membrane/cytoskeleton interaction, the stability of F-actin and the turnover of stress fibres [
47]. Interestingly, reduced PM PIP
2 and F-actin structure are observed in hyperinsulinaemia-induced insulin-resistant 3T3-L1 adipocytes and L6 myotubes. In these cells insulin-stimulated GLUT4 translocation is impaired, but can be corrected with exogenous PIP
2 addition to the PM that mediates a restoration of F-actin structure [
6,
7].
In striking similarity to our myotube findings, an approximate 40% reduction in insulin-stimulated muscle glucose transport has been seen as early as 5 weeks in C57Bl/6J mice fed a high-fat diet [
23] and, at the 4 week interval, muscle insulin resistance in these animals is also suggested by a marked decrease in glucose disposal rate with no change in hepatic glucose production [
24]. Interestingly, insulin-stimulated Akt phosphorylation shows a trend to be decreased by 4 weeks of high-fat feeding, though this effect did not reach statistical significance until after 8 weeks; a similar pattern was observed in liver tissue [
25]. Ongoing studies are now specifically evaluating the temporal sequence of membrane/cytoskeletal and signal transduction derangements in skeletal muscle from high-fat fed animals. A prediction we favour is that membrane/cytoskeletal derangement occurs before signal dysfunction and this early event may contribute to the initial loss of insulin sensitivity.
In summary, these data suggest that a contributing factor in the pathogenesis of glucose intolerance might involve an accrual of skeletal muscle membrane cholesterol and a resultant defect in membrane/cytoskeletal function. Interestingly, this membrane/cytoskeletal defect seems to occur concomitantly with impaired insulin signal propagation in the L6 myotube system. Nevertheless, the later derangement was not limiting, as correction of membrane cholesterol excess mitigated cytoskeletal dysfunction and GLUT4 responsiveness, while signalling remained impaired. Collectively, these findings implicate a reversible abnormality (i.e. elevated cholesterol synthesis/accrual) that may be an early defect that could be targeted to improve glucose disposal.
Acknowledgements
The expertise and assistance of A. Baron (University of California, San Diego, CA, USA) are gratefully acknowledged. This work was supported by NIH grants: AT001846 (J. S. Elmendorf); DK082773 (J. S. Elmendorf); DK082773-S1 (J. S. Elmendorf); HL062552 (M. Sturek); and RR013223 (M. Sturek). In addition, this work was funded by the American Diabetes Association (M. Sturek); the Purdue-Indiana University Comparative Medicine Program (M. Sturek); the Fortune-Fry Ultrasound Research Fund of the Department of Cellular & Integrative Physiology at Indiana University School of Medicine; Indiana University Diabetes and Obesity Research Training Program DeVault Fellowship (K. M. Habegger, E. K. Blue); and Indiana Center for Vascular Biology HL079995 (W. Sealls).