Fat-induced membrane cholesterol accrual provokes cortical filamentous actin destabilisation and glucose transport dysfunction in skeletal muscle

Abstract

Aims/hypothesis: Diminished cortical filamentous actin (F-actin) has been implicated in skeletal muscle insulin resistance, yet the mechanism(s) is unknown. Here we tested the hypothesis that changes in membrane cholesterol could be a causative factor, as organised F-actin structure emanates from cholesterol-enriched raft microdomains at the plasma membrane.

Methods: Skeletal muscle samples from high-fat-fed animals and insulin-sensitive and insulin-resistant human participants were evaluated. The study also used L6 myotubes to directly determine the impact of fatty acids (FAs) on membrane/cytoskeletal variables and insulin action.

Results: High-fat-fed insulin-resistant animals displayed elevated levels of membrane cholesterol and reduced F-actin structure compared with normal chow-fed animals. Moreover, human muscle biopsies revealed an inverse correlation between membrane cholesterol and whole-body glucose disposal. Palmitate-induced insulin-resistant myotubes displayed membrane cholesterol accrual and F-actin loss. Cholesterol lowering protected against the palmitate-induced defects, whereas characteristically measured defects in insulin signalling were not corrected. Conversely, cholesterol loading of L6 myotube membranes provoked a palmitate-like cytoskeletal/GLUT4 derangement. Mechanistically, we observed a palmitate-induced increase in O-linked glycosylation, an end-product of the hexosamine biosynthesis pathway (HBP). Consistent with HBP activity affecting the transcription of various genes, we observed an increase in Hmgcr, a gene that encodes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. In line with increased HBP activity transcriptionally provoking a membrane cholesterol-based insulin-resistant state, HBP inhibition attenuated Hmgcr expression and prevented membrane cholesterol accrual, F-actin loss and GLUT4/glucose transport dysfunction.

Conclusions/interpretation: Our results suggest a novel cholesterolgenic-based mechanism of FA-induced membrane/cytoskeletal disorder and insulin resistance.

Fig. 1

Glucose-intolerant humans and animal models display skeletal muscle membrane cholesterol accrual. Skeletal muscle membrane cholesterol from control and high-fat-fed C57/B6 mice (a) and Ossabaw swine (b). HOMA values in the Ossabaw swine (c). Plot of glucose disposal rate and skeletal membrane cholesterol from humans with a range of insulin sensitivities assessed by hyperinsulinaemic–euglycaemic clamp (d); r = −0.7857; p = 0.048. Cortical F-actin from skeletal muscle from control and high-fat-fed C57/B6 mice (e). Values are means ± SE from three mice and nine swine per experimental group, and three to five images of F-actin/area from three to five separate experiments. *p < 0.05 vs control group. C, control; HF, high-fat; IF, immunofluorescence. Scale bar, 10 μm

Fig. 2

Palmitate induces insulin resistance in L6 myotubes. Incubation of L6 myotubes in the presence of palmitate (C16:0) for 16 h impairs insulin-stimulated GLUT4 translocation (a, b) and 2-deoxyglucose uptake (c). White bars, basal; black bars, insulin-stimulated for 20 min. Values are means ± SE from 3–12 separate experiments. *p < 0.05 vs control group; ^† p < 0.05 vs control insulin group. 2-DG, 2-deoxyglucose; IF, immunofluorescence

Fig. 3

Cholesterol lowering improves cortical F-actin and insulin responsiveness impaired by palmitate. Membrane cholesterol (a), cortical F-actin (b), L6-GLUT4myc (c) and 2-deoxyglucose uptake (d) from control and palmitate-treated L6 myotubes co-treated without or with βCD. White bars, basal; black bars, insulin-stimulated for 20 min. Values are means ± SE from 3–28 separate experiments. *p < 0.05 vs control basal group; ^† p < 0.05 vs control insulin group. 2-DG, 2-deoxyglucose; IF, immunofluorescence

Fig. 4

Exogenous cholesterol membrane loading suppresses insulin-stimulated PM GLUT4 and glucose transport. Membrane cholesterol (a), F-actin (b), L6-GLUT4myc (c) and 2-deoxylglucose uptake (d, e) in L6 myotubes treated with or without 5 mmol/l βCD:cholesterol. White bars, basal; black bars, insulin-stimulated for 20 min. Values are means ± SE from 3–14 separate experiments. *p < 0.05 vs control basal; ^† p < 0.05 vs. control insulin group; ^‡ p = 0.1338. The significance of difference between fold insulin-stimulated 2-deoxyglucose transport values was evaluated by a paired one-tailed t test. 2-DG, 2-deoxyglucose; Chol, cholesterol

Fig. 5

Impaired insulin signalling is not rescued by cholesterol lowering. Basal and insulin-stimulated Akt2 phosphorylation in control and palmitate-treated L6 myotubes co-treated without or with βCD. White bars, basal; black bars, insulin-stimulated for 5 min. Values are means ± SE from five to seven separate experiments. *p < 0.05 vs control basal; ^† p < 0.05 vs control insulin group

Fig. 6

Palmitate induces HBP flux and Hmgr transcription in L6 myotubes. a, b Anti-RL2-detected O-linked glycosylation immunofluorescence in: control myotubes, palmitate-treated myotubes and palmitate and DON-treated myotubes. Hmgr transcription (c), membrane cholesterol (d), cortical F-actin (e) and insulin-stimulated 2-deoxyglucose transport (f) from control and palmitate-treated L6 myotubes in the absence or presence of DON. Images were taken at ×60 magnification. Values are means ± SE from 3–13 separate experiments. *p < 0.05 vs non-treated control; ^† p < 0.05 vs non-treated palmitate group