One exercise session can increase subsequent insulin‐stimulated glucose uptake (ISGU) by skeletal muscle in both sexes. We recently found that muscle expression and phosphorylation of key sites of Akt substrate of 160 kDa (AS160; also called TBC1D4) are essential for the full‐exercise effect on postexercise‐ISGU (PEX‐ISGU) in male rats. In striking contrast, AS160's role in increased PEX‐ISGU has not been rigorously tested in females. Our rationale was to address this major knowledge gap. Wild‐type (WT) and AS160‐knockout (KO) rats were either sedentary or acutely exercised. Adeno‐associated virus (AAV) vectors were engineered to express either WT‐AS160 or AS160 mutated on key serine and threonine residues (Ser588, Thr642, and Ser704) to alanine to prevent their phosphorylation. AAV vectors were delivered to the muscle of AS160‐KO rats to determine if WT‐AS160 or phosphorylation‐inactivated AS160 would influence PEX‐ISGU. AS160‐KO rats have lower skeletal muscle abundance of the GLUT4 glucose transporter protein. This GLUT4 deficit was rescued using AAV delivery of GLUT4 to determine if eliminating muscle GLUT4 deficiency would normalize PEX‐ISGU. The novel results were as follows: (1) AS160 expression was required for greater PEX‐ISGU; (2) rescuing muscle AS160 expression in AS160‐KO rats restored elevated PEX‐ISGU; (3) AS160's essential role for the postexercise increase in ISGU was not attributable to reduced muscle GLUT4 content; and (4) AS160 phosphorylation on Ser588, Thr642, and Ser704 was not essential for greater PEX‐ISGU. In conclusion, these novel findings revealed that three phosphosites widely proposed to influence PEX‐ISGU are not required for this important outcome in female rats.

The current study evaluated the role of Akt substrate of 160 kDa (AS160) phosphorylation on Ser704 in increased insulin-stimulated glucose uptake by skeletal muscle after exercise. Adeno-associated virus vectors were engineered to express either wild-type-AS160 or AS160 mutated so that it could not be phosphorylated on Ser704 in paired muscles from AS160-knockout rats. The results demonstrated that AS160 phosphorylation on Ser704 was not essential for exercise-induced elevation in insulin-stimulated glucose uptake by rats of either sex.

We assessed the effects of two levels of calorie restriction (CR; eating either 15% or 35% less than ad libitum, AL, food intake for 8 weeks) by 24-month-old female and male rats on glucose uptake (GU) and phosphorylation of key signaling proteins (Akt; AMP-activated protein kinase, AMPK; Akt substrate of 160 kDa, AS160) measured in isolated skeletal muscles that underwent four incubation conditions (without either insulin or AICAR, an AMPK activator; with AICAR alone; with insulin alone; or with insulin and AICAR). Regardless of sex: (1) neither CR group versus the AL group had greater GU by insulin-stimulated muscles; (2) phosphorylation of Akt in insulin-stimulated muscles was increased in 35% CR versus AL rats; (3) prior AICAR treatment of muscle resulted in greater GU by insulin-stimulated muscles, regardless of diet; and (4) AICAR caused elevated phosphorylation of acetyl CoA carboxylase, an indicator of AMPK activation, in all diet groups. There was a sexually dimorphic diet effect on AS160 phosphorylation, with 35% CR exceeding AL for insulin-stimulated muscles in male rats, but not in female rats. Our working hypothesis is that the lack of a CR-effect on GU by insulin-stimulated muscles was related to the extended duration of the ex vivo incubation period (290 min compared to 40–50 min that was previously reported to be effective). The observed efficacy of prior treatment of muscles with AICAR to improve glucose uptake in insulin-stimulated muscles supports the strategy of targeting AMPK with the goal of improving insulin sensitivity in older females and males.

Calorie restriction (CR; calorie intake reduced by ∼20%-40% below ad libitum, AL, intake) potentiates skeletal muscle insulin sensitivity during old age by incompletely understood mechanisms. We aimed to identify CR-induced changes in muscle insulin signaling that may explain this enhanced sensitivity. We examined how CR (65% of AL intake for 8-weeks) alters muscle insulin action and signaling in aged rats (24-month old) of both sexes. We assessed insulin-stimulated glucose uptake (ISGU) in muscle together with deep phosphoproteomic profiling. CR enhanced ISGU in both sexes, with higher ISGU in females regardless of diet. We identified 590 diet-responsive phosphosites, indicating extensive CR-induced remodelling of muscle phosphorylation, particularly within structural and contractile pathways. Strikingly, 70% of these sites were sex-specific. Numerous insulin-responsive sites were identified (193 in females; 107 in males) with 60 overlapping sites. The magnitude of the insulin-effects among all significantly regulated sites correlated between sexes. S1443 phosphorylation on EH domain-binding protein 1-like protein-1 (Ehbp1l1; a potential regulator of Rab proteins that control GLUT4 glucose transporter trafficking) was insulin-responsive in both sexes but only associated to ISGU in females. Personalized phosphoproteomic analysis also identified insulin-responsive sites on Leiomodin-1 (Lmod1) that correlated with ISGU across individuals. Both Lmod1 and Ehbp1l1 have strong genetic association with glycemic traits in humans, reinforcing their translational relevance. This study revealed sex-dependent and sex-independent phosphosignaling mechanisms that associate with muscle insulin responsiveness as well as hundreds of sex-specific, CR-responsive phosphosites. These findings provide a rich resource for future research on CR and insulin sensitivity.

To stimulate glucose uptake in muscle, insulin mobilizes GLUT4 glucose transporters to the cell surface. During fasting, GLUT4 and the transmembrane aminopeptidase IRAP are trapped in intracellular, insulin-responsive vesicles bound by TUG, AS160, and Usp25m proteins. Here we show that Usp25m, a protease, is required for the bulk of insulin-stimulated TUG cleavage and consequent vesicle mobilization and glucose uptake. Efficient TUG cleavage also requires AS160. In mice with diet-induced insulin resistance, Usp25m abundance is reduced, IRAP is mislocalized during fasting, and TUG cleavage is impaired; effects of Usp25m and TUG deletion to alter insulin-stimulated and fasting glucose uptake, respectively, are ablated. We conclude that skeletal muscle insulin resistance results in part from altered membrane trafficking of GLUT4 and IRAP during fasting. This alteration depletes the pool of insulin-responsive vesicles marked by TUG and Usp25m. Mistargeting of GLUT4 and IRAP may contribute to distinct aspects of the metabolic syndrome in humans.