Gregory D. Cartee

A single endurance exercise session can elevate subsequent insulin-stimulated glucose uptake by skeletal muscle. Refeeding a high-carbohydrate diet postexercise accelerates the reversal of exercise-enhanced insulin-stimulated glucose uptake. We hypothesize that increased glucose flux through the hexosamine biosynthetic pathway mediates the reversal of postexercise-enhanced insulin-stimulated glucose uptake observed with high dietary carbohydrate intake.

This study used untargeted metabolomics to investigate how calorie restriction (CR) affects skeletal muscle in older female and male rats. Fischer-344 Brown Norway rats of both sexes (22-23-month-old) were randomly assigned to an ad libitum-fed (AL) group or a CR group, which received 65% of the daily food intake of their sex-matched AL counterparts for eight weeks. Epitrochlearis muscles were collected and analyzed using untargeted metabolomics and immunoblotting. CR altered the abundance of 554 out of the 999 detected muscle metabolites. Additionally, 521 metabolites differed between females and males. CR raised the levels of metabolites related to bile acids, with significantly greater CR-induced elevation of secondary bile acids in females, suggesting possible sex-dependent interactions involving the gut, liver, and muscle. Furthermore, CR increased the levels of five glycolytic metabolites. These changes occurred without increases in the abundance of phosphofructokinase (a rate-limiting glycolytic enzyme) or in key allosteric activators (AMP and ADP). There was no evidence for a CR-induced increase in NAD+/NADH ratio: NAD+ levels decreased and NADH levels increased for CR versus AL rats. Approximately 78% of dipeptides were lower in CR rats compared to AL rats, and 38% of dipeptides were greater for females versus males. Levels for 5-methyl-2’-deoxycytidine (a methylated DNA breakdown product and potential marker for changes in DNA remodeling) were significantly reduced by CR only in females. Together, these findings offer important insights for understanding both the shared and sex-specific mechanisms through which CR influences muscle metabolism, health, and function.

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.

Calorie restriction (CR: ∼20–40% below AL: ad libitum intake) can increase insulin sensitivity in various species. We reported that CR did not induce an increase in insulin-stimulated glucose uptake (ISGU) by skeletal muscle from female Akt substrate of 160 kDa knockout (AS160-KO) rats. It seemed possible that the marked decline in muscle GLUT4 abundance, a characteristic of AS160-KO animals, played a role in our observation. We used an adeno-associated virus (AAV) approach to increase muscle GLUT4 abundance in female AS160-KO rats. We injected an AAV-GLUT4 vector into one epitrochlearis muscle. The contralateral epitrochlearis underwent sham injection and served as the control. Rats were randomly assigned to AL or CR (consuming 65% of AL intake) groups for 8-weeks. ISGU was determined in isolated muscles incubated ± insulin. Akt phosphorylation on regulatory sites (Threonine-308, pAkt^Thr308 and Serine-473, pAkt^Ser473) was determined to evaluate a key insulin signaling protein. The abundance of proteins that regulate ISGU (GLUT4 glucose transporter and hexokinase II) was determined by immunoblotting. We found: (1) AAV-delivery of GLUT4 to muscles successfully increased GLUT4 expression compared to sham-treated contralateral muscles in both diet groups; (2) this elevation in muscle GLUT4 did not result in a CR-induced increase in ISGU; (3) no significant diet-related differences were detected for pAkt^Thr308, pAkt^Ser473 or abundance of GLUT4 and hexokinase II in the muscles with either sham treatment or AAV-GLUT4. These observations revealed that the absence of a CR-induced increase in muscle ISGU of female AS160-KO rats was not attributable to low muscle GLUT4 abundance.

One exercise session can increase subsequent insulin‐stimulated glucose uptake (ISGU) by skeletal muscle. Postexercise refeeding induces reversal of postexercise (PEX)‐enhanced ISGU concomitant with attaining high muscle glycogen in rats. To test the relationship between high glycogen and reversal of PEX‐ISGU, we injected one epitrochlearis muscle from each rat with adeno‐associated virus (AAV) small hairpin RNA (shRNA) that targets glycogen synthase 1 (GS1) and injected contralateral muscles with AAV‐shRNA‐Scrambled (Scr). Muscles from PEX and sedentary rats were collected at 3‐hour PEX (3hPEX) or 6‐hour PEX (6hPEX). Rats were either not refed or refed rat‐chow during the recovery period. Isolated muscles were incubated with [ ³ H]‐3‐ O ‐methylglucose, with or without insulin. The results revealed: (1) GS1 abundance was substantially lower for AAV‐shRNA‐GS1‐treated versus AAV‐shRNA‐Scr‐treated muscles; (2) reduced GS1 abundance in refed‐rats induced much lower glycogen in AAV‐shRNA‐GS1‐treated versus AAV‐shRNA‐Scr‐treated muscles at 3hPEX or 6hPEX; (3) PEX‐ISGU was elevated in not refed‐rats at either 3hPEX or 6hPEX versus sedentary controls, regardless of GS1 abundance; (4) PEX‐ISGU was not reversed by 3 h of refeeding, regardless of GS1 abundance; (5) despite substantially lower glycogen in AAV‐shRNA‐GS1‐treated versus AAV‐shRNA‐Scr‐treated muscles, elevated PEX‐ISGU was eliminated at 6hPEX in both of the paired muscles of refed‐rats; and (6) 3hPEX versus sedentary non‐refed rats had greater AMP‐activated protein kinase‐γ3 activity in both paired muscles, but this exercise effect was eliminated in both paired muscles by 3 h of refeeding. In conclusion, the results provided compelling evidence that the reversal of exercise‐enhanced ISGU by refeeding was not attributable to the accumulation of high muscle glycogen concentration.

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.