Pharmacological inhibition of TLR4 ameliorates muscle and liver ceramide content after disuse in previously physically active mice.

Toll-like receptor 4 (TLR4) is a proposed mediator of ceramide accumulation, muscle atrophy, and insulin resistance in skeletal muscle. It is currently unknown whether pharmacological inhibition of TLR4, using the TLR4-specific inhibitor TAK-242 during muscle disuse, is able to prevent changes in intracellular ceramide species and consequently preserve muscle size and insulin sensitivity in physically active mice. To address this question, we subjected running wheel-conditioned C57BL/6 male mice (13 wk old; ∼10/group) to 7 days of hindlimb suspension (HS), 7 days of continued wheel running (WR), or daily injections of TAK-242 during HS (HS + TAK242) for 7 days. We measured hindlimb muscle morphology, intramuscular and liver ceramide content, HOMA-IR, mRNA proxies of ceramide turnover and lipid trafficking, and muscle fatty acid and glycerolipid content. As a result, soleus and liver ceramide abundance was greater (P < 0.05) in HS vs. WR but was reduced with TLR4 inhibition (HS + TAK-242 vs. HS). Muscle mass declined (P < 0.01) with HS (vs. WR), but TLR4 inhibition did not prevent this loss (soleus: P = 0.08; HS vs. HS + TAK-242). HOMA-IR was impaired (P < 0.01) in HS versus WR mice, but only fasting blood glucose was reduced with TLR4 inhibition (HS + TAK-242 vs HS, P < 0.05). Robust decreases in muscle Spt2 and Cd36 mRNA and muscle lipidomic trafficking may partially explain reductions in ceramides with TLR4 inhibition. In conclusion, pharmacological TLR4 inhibition in wheel-conditioned mice prevented ceramide accumulation during the early phase of hindlimb suspension (7 days) but had little effect on muscle size and insulin sensitivity.

An accumulation of muscle macrophages is accompanied by altered insulin sensitivity after reduced activity and recovery.

BACKGROUND: Mechanisms underlying physical inactivity-induced insulin resistance are not well understood. In addition to a role in muscle repair, immune cell populations such as macrophages may regulate insulin sensitivity. AIM: The aim of this study was to examine if the dynamic changes in insulin sensitivity during and after recovery from reduced physical activity corresponded to changes in skeletal muscle macrophages. METHODS: In this prospective clinical study, we collected muscle biopsies from healthy older adults (70 ± 2 years, n = 12) before and during a hyperinsulinaemic-euglycaemic clamp and this occurred before (PRE) and after 2-week reduced physical activity (RA), and following 2-week of recovery (REC). Insulin sensitivity (hyperinsulinaemic-euglycaemic clamp), skeletal muscle mRNA expression of inflammatory markers, and immunofluorescent quantification of skeletal muscle macrophages, myofibre-specific satellite cell and capillary content were assessed. RESULTS: Insulin sensitivity was decreased following reduced activity and rebounded following recovery above PRE levels. We observed an increase (P < 0.01) in muscle macrophages (CD68+ CD206+ : 190 [55, 324]; CD11b+ CD206+ : 117 [28, 205]% change from PRE) and CD68 (2.4 [1.4, 3.4]-fold) and CCL2 (1.9 [1.3, 2.5]-fold) mRNA following RA concurrent with increased (P < 0.03) satellite cells (55 [6, 104]%) in slow-twitch myofibres. Moreover, the distance of satellite cells to the nearest capillary was increased 7.7 (1.7, 13.7) µm in fast-twitch myofibres at RA (P = 0.007). Changes in macrophages were positively associated with increased insulin sensitivity following RA (R > 0.57, P < 0.05). CONCLUSION: These findings suggested that a dynamic response of skeletal muscle macrophages following acute changes in physical activity in healthy older adults is related to insulin sensitivity.

Skeletal muscle ceramides and relationship with insulin sensitivity after 2 weeks of simulated sedentary behaviour and recovery in healthy older adults.

KEY POINTS: Insulin sensitivity (as determined by a hyperinsulinaemic-euglyceamic clamp) decreased 15% after reduced activity. Despite not fully returning to baseline physical activity levels, insulin sensitivity unexpectedly, rebounded above that recorded before 2 weeks of reduced physical activity by 14% after the recovery period. Changes in insulin sensitivity in response to reduced activity were primarily driven by men but, not women. There were modest changes in ceramides (nuclear/myofibrillar fraction and serum) following reduced activity and recovery but, in the absence of major changes to body composition (i.e. fat mass), ceramides were not related to changes in inactivity-induced insulin sensitivity in healthy older adults. ABSTRACT: Older adults are at risk of physical inactivity as they encounter debilitating life events. It is not known how insulin sensitivity is affected by modest short-term physical inactivity and recovery in healthy older adults, nor how insulin sensitivity is related to changes in serum and muscle ceramide content. Healthy older adults (aged 64-82 years, five females, seven males) were assessed before (PRE), after 2 weeks of reduced physical activity (RA) and following 2 weeks of recovery (REC). Insulin sensitivity (hyperinsulinaemic-euglyceamic clamp), lean mass, muscle function, skeletal muscle subfraction, fibre-specific, and serum ceramide content and indices of skeletal muscle inflammation were assessed. Insulin sensitivity decreased by 15 ± 6% at RA (driven by men) but rebounded above PRE by 14 ± 5% at REC. Mid-plantar flexor muscle area and leg strength decreased with RA, although only muscle size returned to baseline levels following REC. Body fat did not change and only minimal changes in muscle inflammation were noted across the intervention. Serum and intramuscular ceramides (nuclear/myofibrillar fraction) were modestly increased at RA and REC. However, ceramides were not related to changes in inactivity-induced insulin sensitivity in healthy older adults. Short-term inactivity induced insulin resistance in older adults in the absence of significant changes in body composition (i.e. fat mass) are not related to changes in ceramides.