Exercise Alleviates Lipid-Induced Insulin Resistance in Human Skeletal Muscle–Signaling Interaction at the Level of TBC1 Domain Family Member 4

Excess lipid availability causes insulin resistance. We examined the effect of acute exercise on lipid-induced insulin resistance and TBC1 domain family member 1/4 (TBCD1/4)-related signaling in skeletal muscle. In eight healthy young male subjects, 1 h of one-legged knee-extensor exercise was followed by 7 h of saline or intralipid infusion. During the last 2 h, a hyperinsulinemic-euglycemic clamp was performed. Femoral catheterization and analysis of biopsy specimens enabled measurements of leg substrate balance and muscle signaling. Each subject underwent two experimental trials, differing only by saline or intralipid infusion. Glucose infusion rate and leg glucose uptake was decreased by intralipid. Insulin-stimulated glucose uptake was higher in the prior exercised leg in the saline and the lipid trials. In the lipid trial, prior exercise normalized insulin-stimulated glucose uptake to the level observed in the resting control leg in the saline trial. Insulin increased phosphorylation of TBC1D1/4. Whereas prior exercise enhanced TBC1D4 phosphorylation on all investigated sites compared with the rested leg, intralipid impaired TBC1D4 S341 phosphorylation compared with the control trial. Intralipid enhanced pyruvate dehydrogenase (PDH) phosphorylation and lactate release. Prior exercise led to higher PDH phosphorylation and activation of glycogen synthase compared with resting control. In conclusion, lipid-induced insulin resistance in skeletal muscle was associated with impaired TBC1D4 S341 and elevated PDH phosphorylation. The prophylactic effect of exercise on lipid-induced insulin resistance may involve augmented TBC1D4 signaling and glycogen synthase activation

Metabolic and molecular changes associated with the increased skeletal muscle insulin action 24–48 h after exercise in young and old humans

The molecular and metabolic mechanisms underlying the increase in insulin sensitivity (i.e. increased insulin-stimulated skeletal muscle glucose uptake, phosphorylation and storage as glycogen) observed from 12 to 48 h following a single bout of exercise inhumans remain unresolved. Moreover, whether these mechanisms differ with age is unclear. It is well established that a single bout of exercise increases the translocation of the glucose transporter, GLUT4, to the plasma membrane. Previous research using unilateral limb muscle contraction models in combination with hyperinsulinaemia has demonstrated that the increase in insulin sensitivity and glycogen synthesis 24 h after exercise is also associated with an increase in hexokinase II (HKII) mRNA and protein content, suggesting an increase in the capacity of the muscle to phosphorylate glucose and divert it towards glycogen synthesis. Interestingly, this response is altered in older individuals for up to 48 h post exercise and is associated with molecular changes in skeletal muscle tissue that are indicative of reduced lipid oxidation, increased lipogenesis, increased inflammation and a relative inflexibility of changes in intramyocellular lipid (IMCL) content. Reduced insulin sensitivity (insulin resistance) is generally related to IMCL content, particularly in the subsarcolemmal (SSL) region, and both are associated with increasing age. Recent research has demonstrated that ageing per se appears to cause an exacerbated lipolytic response to exercise that may result in SSL IMCL accumulation. Further research is required to determine if increased IMCL content affects HKII expression in the days after exercise in older individuals, and the effect of this on skeletal muscle insulin action

Exercise-induced TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle

TBC1D1 is a Rab-GTPase activating protein involved in regulation of GLUT4 translocation in skeletal muscle. We here evaluated exercise-induced regulation of TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle. In separate experiments healthy men performed all-out cycle exercise lasting either 30 s, 2 min or 20 min. After all exercise protocols, TBC1D1 Ser237 phosphorylation increased (∼70–230%, P < 0.005), with the greatest response observed after 20 min of cycling. Interestingly, capacity of TBC1D1 to bind 14-3-3 protein showed a similar pattern of regulation, increasing 60–250% (P < 0.001). Furthermore, recombinant 5′AMP-activated protein kinase (AMPK) induced both Ser237 phosphorylation and 14-3-3 binding properties on human TBC1D1 when evaluated in vitro. To further characterize the role of AMPK as an upstream kinase regulating TBC1D1, extensor digitorum longus muscle (EDL) from whole body α1 or α2 AMPK knock-out and wild-type mice were stimulated to contract in vitro. In wild-type and α1 knock-out mice, contractions resulted in a similar ∼100% increase (P < 0.001) in Ser237 phosphorylation. Interestingly, muscle of α2 knock-out mice were characterized by reduced protein content of TBC1D1 (∼50%, P < 0.001) as well as in basal and contraction-stimulated (∼60%, P < 0.001) Ser237 phosphorylation, even after correction for the reduced TBC1D1 protein content. This study shows that TBC1D1 is Ser237 phosphorylated and 14-3-3 protein binding capacity is increased in response to exercise in human skeletal muscle. Furthermore, we show that the catalytic α2 AMPK subunit is the main (but probably not the only) donor of AMPK activity regulating TBC1D1 Ser237 phosphorylation in mouse EDL muscle

Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 in human skeletal muscle

We investigated the phosphorylation signatures of two Rab-GTPase activating proteins TBC1D1 and TBC1D4 in human skeletal muscle in response to physical exercise and physiological insulin levels induced by a carbohydrate rich meal using a paired experimental design. Eight healthy male volunteers exercised in the fasted or fed state and muscle biopsies were taken before and immediately after exercise. We identified TBC1D1/4 phospho-sites that (1) did not respond to exercise or postprandial increase in insulin (TBC1D4: S666), (2) responded to insulin only (TBC1D4: S318), (3) responded to exercise only (TBC1D1: S237, S660, S700; TBC1D4: S588, S751), and (4) responded to both insulin and exercise (TBC1D1: T596; TBC1D4: S341, T642, S704). In the insulin-stimulated leg, Akt phosphorylation of both T308 and S473 correlated significantly with multiple sites on both TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Interestingly, in the exercised leg in the fasted state TBC1D1 phosphorylation (S237, T596) correlated significantly with the activity of the α2/β2/γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) correlated with the activity of the α2/β2/γ1 AMPK trimer. Our data show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and support the idea that Akt and AMPK are upstream kinases. TBC1D1 phosphorylation signatures were comparable between in vitro contracted mouse skeletal muscle and exercised human muscle, and we show that AMPK regulated phosphorylation of these sites in mouse muscle. Contraction and exercise elicited a different phosphorylation pattern of TBC1D4 in mouse compared with human muscle, and although different circumstances in our experimental setup may contribute to this difference, the observation exemplifies that transferring findings between species is problematic. KEY POINTS: Phosphorylation signature patterns on TBC1D1 and TBC1D4 proteins in the insulin–glucose pathway were investigated in human skeletal muscle in response to physiological insulin and exercise. In response to postprandial increase in insulin, Akt phosphorylation of T308 and S473 correlated significantly with sites on TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Exercise induced phosphorylation of TBC1D1 (S237, T596) that correlated significantly with activity of the α2/β2/γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) with exercise correlated with activity of the α2/β2/γ1 AMPK trimer. TBC1D1 phosphorylation signatures with exercise/muscle contraction were comparable between human and mouse skeletal muscle, and AMPK regulated phosphorylation of these sites in mouse muscle, whereas contraction and exercise elicited different TBC1D4 phosphorylation patterns in mouse compared with human muscle. Our results show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and indicate that Akt and AMPK may be upstream kinases

Exercise alleviates lipid-induced insulin resistance in human skeletal muscle-signaling interaction at the level of TBC1 domain family member 4

Excess lipid availability causes insulin resistance. We examined the effect of acute exercise on lipid-induced insulin resistance and TBC1 domain family member 1/4 (TBCD1/4)-related signaling in skeletal muscle. In eight healthy young male subjects, 1 h of one-legged knee-extensor exercise was followed by 7 h of saline or intralipid infusion. During the last 2 h, a hyperinsulinemic-euglycemic clamp was performed. Femoral catheterization and analysis of biopsy specimens enabled measurements of leg substrate balance and muscle signaling. Each subject underwent two experimental trials, differing only by saline or intralipid infusion. Glucose infusion rate and leg glucose uptake was decreased by intralipid. Insulin-stimulated glucose uptake was higher in the prior exercised leg in the saline and the lipid trials. In the lipid trial, prior exercise normalized insulin-stimulated glucose uptake to the level observed in the resting control leg in the saline trial. Insulin increased phosphorylation of TBC1D1/4. Whereas prior exercise enhanced TBC1D4 phosphorylation on all investigated sites compared with the rested leg, intralipid impaired TBC1D4 S341 phosphorylation compared with the control trial. Intralipid enhanced pyruvate dehydrogenase (PDH) phosphorylation and lactate release. Prior exercise led to higher PDH phosphorylation and activation of glycogen synthase compared with resting control. In conclusion, lipid-induced insulin resistance in skeletal muscle was associated with impaired TBC1D4 S341 and elevated PDH phosphorylation. The prophylactic effect of exercise on lipid-induced insulin resistance may involve augmented TBC1D4 signaling and glycogen synthase activation