Effects of Nutrient Intake Timing on Post-Exercise Glycogen Accumulation and Its Related Signaling Pathways in Mouse Skeletal Muscle

We investigated the effects of nutrient intake timing on glycogen accumulation and its related signals in skeletal muscle after an exercise that did not induce large glycogen depletion. Male ICR mice ran on a treadmill at 25 m/min for 60 min under a fed condition. Mice were orally administered a solution containing 1.2 mg/g carbohydrate and 0.4 mg/g protein or water either immediately (early nutrient, EN) or 180 min (late nutrient, LN) after the exercise. Tissues were harvested at 30 min after the oral administration. No significant difference in blood glucose or plasma insulin concentrations was found between the EN and LN groups. The plantaris muscle glycogen concentration was significantly (p < 0.05) higher in the EN group—but not in the LN group—compared to the respective time-matched control group. Akt Ser473 phosphorylation was significantly higher in the EN group than in the time-matched control group (p < 0.01), while LN had no effect. Positive main effects of time were found for the phosphorylations in Akt substrate of 160 kDa (AS160) Thr642 (p < 0.05), 5′-AMP-activated protein kinase (AMPK) Thr172 (p < 0.01), and acetyl-CoA carboxylase Ser79 (p < 0.01); however, no effect of nutrient intake was found for these. We showed that delayed nutrient intake could not increase muscle glycogen after endurance exercise which did not induce large glycogen depletion. The results also suggest that post-exercise muscle glycogen accumulation after nutrient intake might be partly influenced by Akt activation. Meanwhile, increased AS160 and AMPK activation by post-exercise fasting might not lead to glycogen accumulation.

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title: Postexercise nutrient intake timing in humans is critical to recovery of leg glucose and protein homeostasis.
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title: Glucose transport rate and glycogen synthase activity both limit skeletal muscle glycogen accumulation.
abstract: We varied rates of glucose transport and glycogen synthase I (GS-I) activity (%GS-I) in isolated rat epitrochlearis muscle to examine the role of each process in determining the rate of glycogen accumulation. %GS-I was maintained at or above the fasting basal range during 3 h of incubation with 36 mM glucose and 60 microU/ml insulin. Lithium (2 mM LiCl) added to insulin increased glucose transport rate and muscle glycogen content compared with insulin alone. The glycogen synthase kinase-3beta inhibitor GF-109203 x (GF; 10 microM) maintained %GS-I about twofold higher than insulin with or without lithium but did not increase glycogen accumulation. When %GS-I was lowered below the fasting range by prolonged incubation with 36 mM glucose and 2 mU/ml insulin, raising rates of glucose transport with bpV(phen) or of %GS-I with GF produced additive increases in glycogen concentration. Phosphorylase activity was unaffected by GF or bpV(phen). In muscles of fed animals, %GS-I was approximately 30% lower than in those of fasted rats, and insulin-stimulated glycogen accumulation did not occur unless %GS-I was raised with GF. We conclude that the rate of glucose transport is rate limiting for glycogen accumulation unless %GS-I is below the fasting range, in which case both glucose transport rate and GS activity can limit glycogen accumulation.

title: Determinants of Post-Exercise Glycogen Synthesis During Short-Term Recovery
abstract: AbstractThe pattern of muscle glycogen synthesis following glycogen-depleting exercise occurs in two phases. Initially, there is a period of rapid synthesis of muscle glycogen that does not require the presence of insulin and lasts about 30–60 minutes. This rapid phase of muscle glycogen synthesis is characterised by an exercise-induced translocation of glucose transporter carrier protein-4 to the cell surface, leading to an increased permeability of the muscle membrane to glucose. Following this rapid phase of glycogen synthesis, muscle glycogen synthesis occurs at a much slower rate and this phase can last for several hours. Both muscle contraction and insulin have been shown to increase the activity of glycogen synthase, the rate-limiting enzyme in glycogen synthesis. Furthermore, it has been shown that muscle glycogen concentration is a potent regulator of glycogen synthase. Low muscle glycogen concentrations following exercise are associated with an increased rate of glucose transport and an increased capacity to convert glucose into glycogen.The highest muscle glycogen synthesis rates have been reported when large amounts of carbohydrate (1.0–1.85 g/kg/h) are consumed immediately post-exercise and at 15.60 minute intervals thereafter, for up to 5 hours post-exercise. When carbohydrate ingestion is delayed by several hours, this may lead to ∼50% lower rates of muscle glycogen synthesis. The addition of certain amino acids and/ or proteins to a carbohydrate supplement can increase muscle glycogen synthesis rates, most probably because of an enhanced insulin response. However, when carbohydrate intake is high (≥1.2 g/kg/h) and provided at regular intervals, a further increase in insulin concentrations by additional supplementation of protein and/or amino acids does not further increase the rate of muscle glycogen synthesis. Thus, when carbohydrate intake is insufficient (<1.2 g/kg/h), the addition of certain amino acids and/or proteins may be beneficial for muscle glycogen synthesis. Furthermore, ingestion of insulinotropic protein and/or amino acid mixtures might stimulate post-exercise net muscle protein anabolism. Suggestions have been made that carbohydrate availability is the main limiting factor for glycogen synthesis. A large part of the ingested glucose that enters the bloodstream appears to be extracted by tissues other than the exercise muscle (i.e. liver, other muscle groups or fat tissue) and may therefore limit the amount of glucose available to maximise muscle glycogen synthesis rates. Furthermore, intestinal glucose absorption may also be a rate-limiting factor for muscle glycogen synthesis when large quantities (>1 g/min) of glucose are ingested following exercise.

title: Muscle glucose metabolism following exercise in the rat: increased sensitivity to insulin.
abstract: Muscle glycogen stores are depleted during exercise and are rapidly repleted during the recovery period. To investigate the mechanism for this phenomenon, untrained male rats were run for 45 min on a motor-driven treadmill and the ability of their muscles to utilize glucose was then assessed during perfusion of their isolated hindquarters. Glucose utilization by the hindquarter was the same in exercised and control rats perfused in the absence of added insulin; however, when insulin (30-40,000 muU/ml) was added to the perfusate, glucose utilization was greater after exercise. Prior exercise lowered both, the concentration of insulin that half-maximally stimulated glucose utilization (exercise, 150 muU/ml; control, 480 muU/ml) and modestly increased its maximum effect. The increase in insulin sensitivity persisted for 4 h following exercise, but was not present after 24 h. The rate-limiting step in glucose utilization enhanced by prior exercise appeared to be glucose transport across the cell membrane, as in neither control nor exercised rats did free glucose accumulate in the muscle cell. Following exercise, the ability of insulin to stimulate the release of lactate into the perfusate was unaltered; however its ability to stimulate the incorporation of [(14)C]glucose into glycogen in certain muscles was enhanced. Thus at a concentration of 75 muU/ml insulin stimulated glycogen synthesis eightfold more in the fast-twitch red fibers of the red gastrocnemius than it did in the same muscle of nonexercised rats. In contrast, insulin only minimally increased glycogen synthesis in the fast-twitch white fibers of the gastrocnemius, which were not glycogen-depleted. The uptake of 2-deoxyglucose by these muscles followed a similar pattern suggesting that glucose transport was also differentially enhanced. Prior exercise did not enhance the ability of insulin to convert glycogen synthase from its glucose-6-phosphate-dependent (D) to its glucose-6-phosphate-independent (1) form. On the other hand, following exercise, insulin prevented a marked decrease in muscle glucose-6-phosphate, which could have diminished synthase activity in situ. The possibility that exercise enhanced the ability of insulin to convert glycogen synthase D to an intermediate form of the enzyme, more sensitive to glucose-6-phosphate, remains to be explored. These results suggest that following exercise, glucose transport and glycogen synthesis in skeletal muscle are enhanced due at least in part to an increase in insulin sensitivity. They also suggest that this increase in insulin sensitivity occurs predominantly in muscle fibers that are deglycogenated during exercise.

title: Enhanced muscle glucose metabolism after exercise in the rat: the two phases.
abstract: Thirty minutes after a treadmill run, glucose utilization and glycogen synthesis in perfused rat skeletal muscle are enhanced due to an increase in insulin sensitivity (Richter et al.,  1982). The exercise used in these studies was of moderate intensity, and muscle glycogen was substantially repleted at the time (30 min postexercise) that glucose metabolism was examined. When rats were run at twice the previous rate (36 m/min), muscle glycogen was still substantially diminished 30 min after the run. At this time the previously noted increase in insulin sensitivity was still observed in perfused muscle; however, glucose utilization was also increased in the absence of added insulin (1.5 vs. 4.2 mumol X g-1 X h-1). In contrast 2.5 h after the run, muscle glycogen had returned to near preexercise values, and only the insulin-induced increase in glucose utilization was evident. The data suggest that the restoration of muscle glycogen after exercise occurs in two phases. In phase I, muscle glycogen is depleted and insulin-stimulated glucose utilization and glucose utilization in the absence of added insulin may both be enhanced. In phase II glycogen levels have returned to near base-line values and only the increase in insulin sensitivity persists. It is proposed that phase I corresponds to the period of rapid glycogen repletion that immediately follows exercise and phase II to the period of supercompensation.

title: Enhanced muscle glucose metabolism after exercise: modulation by local factors.
abstract: Studies in the rat suggest that after voluntary exercise there are two phases of glycogen repletion in skeletal muscle (preceding study). In phase I glucose utilization and glycogen synthesis are enhanced both in the presence and absence of insulin, whereas in phase II only the increase in the presence of insulin is found. To determine whether these alterations and in particular those mediated by insulin are due to local or systemic factors, one hindlimb of an anesthetized rat was electrically stimulated, and both hindlimbs were perfused immediately thereafter. Glucose and glycogen metabolism in the stimulated leg closely mimicked that observed previously after voluntary exercise on a treadmill. With no insulin added to the perfusate, glucose incorporation into glycogen was markedly enhanced in muscles that were glycogen depleted as were the uptake of 2-deoxyglucose and 3-O-methylglucose. Likewise, the stimulation of these processes by insulin was enhanced and continued to be so 2 h later when the muscles of the stimulated leg had substantially repleted their glycogen stores. The results suggest that the increases in insulin-mediated glucose utilization and glycogen synthesis in muscle after exercise are modulated by local contraction-induced factors.

title: Skeletal muscle plasma membrane glucose transport and glucose transporters after exercise.
abstract: Recent reports have shown that immediately after an acute bout of exercise the glucose transport system of rat skeletal muscle plasma membranes is characterized by an increase in both glucose transporter number and intrinsic activity. To determine the duration of the exercise response we examined the time course of these changes after completion of a single bout of exercise. Male rats were exercised on a treadmill for 1 h (20 m/min, 10% grade) or allowed to remain sedentary. Rats were killed either immediately or 0.5 or 2 h after exercise, and red gastrocnemius muscle was used for the preparation of plasma membranes. Plasma membrane glucose transporter number was elevated 1.8- and 1.6-fold immediately and 30 min after exercise, although facilitated D-glucose transport in plasma membrane vesicles was elevated 4- and 1.8-fold immediately and 30 min after exercise, respectively. By 2 h after exercise both glucose transporter number and transport activity had returned to nonexercised control values. Additional experiments measuring glucose uptake in perfused hindquarter muscle produced similar results. We conclude that the reversal of the increase in glucose uptake by hindquarter skeletal muscle after exercise is correlated with a reversal of the increase in the glucose transporter number and activity in the plasma membrane. The time course of the transport-to-transporter ratio suggests that the intrinsic activity response reverses more rapidly than that involving transporter number.

title: Increased GLUT-4 translocation mediates enhanced insulin sensitivity of muscle glucose transport after exercise.
abstract: The purpose of this study was to determine whether the increase in insulin sensitivity of skeletal muscle glucose transport induced by a single bout of exercise is mediated by enhanced translocation of the GLUT-4 glucose transporter to the cell surface. The rate of 3-O-[3H]methyl-D-glucose transport stimulated by a submaximally effective concentration of insulin (30 microU/ml) was approximately twofold greater in the muscles studied 3.5 h after exercise than in those of the sedentary controls (0.89 +/- 0.10 vs. 0.43 +/- 0.05 micromol . ml-1 . 10 min-1; means +/- SE for n = 6/group). GLUT-4 translocation was assessed by using the ATB-[2-3H]BMPA exofacial photolabeling technique. Prior exercise resulted in greater cell surface GLUT-4 labeling in response to submaximal insulin treatment (5.36 +/- 0.45 dpm x 10(3)/g in exercised vs. 3.00 +/- 0.38 dpm x 10(3)/g in sedentary group; n = 10/group) that closely mirrored the increase in glucose transport activity. The signal generated by the insulin receptor, as reflected in the extent of insulin receptor substrate-1 tyrosine phosphorylation, was unchanged after the exercise. We conclude that the increase in muscle insulin sensitivity of glucose transport after exercise is due to translocation of more GLUT-4 to the cell surface and that this effect is not due to potentiation of insulin-stimulated tyrosine phosphorylation.

title: Regulation of muscle glycogen synthase phosphorylation and kinetic properties by insulin, exercise, adrenaline and role in insulin resistance
abstract: In mammals, excess carbohydrate is stored as glycogen and glycogen synthase is the enzyme that incorporates glucose units into the glycogen particle. Glycogen synthase activity is regulated by phosphorylation and allosterically activated by glucose 6-phosphate. Phosphorylation of nine serines by different kinases regulates glycogen synthase affinity for glucose 6-phosphate and its substrate UDP-glucose. Glucose 6-phosphate increases both enzyme activity and substrate affinity. Insulin and exercise increase glycogen synthase affinity for glucose 6-phosphate and activity whereas high glycogen content and adrenaline decrease affinity for glucose 6-phosphate and activity. However, insulin, exercise and adrenaline also regulate intracellular concentration of glucose 6-phosphate which will influence in vivo glycogen synthase activity. Importantly, type 2 diabetes is associated with reduced insulin-stimulated glycogen synthase activation. The nine phosphorylation sites theoretically allow 512 combinations of phosphorylation configurations of glycogen synthase with different kinetic properties. However, due to hierarchal phosphorylation, the number of configurations in vivo is most likely much lower. Unfortunately, many studies only report data on glycogen synthase activity measured with high concentration of UDP-glucose which holds back information about changes in substrate affinity. In this paper we discuss the physiological regulation of glycogen synthase phosphorylation and how the phosphorylation pattern regulates glycogen synthase kinetic properties.

title: Allosteric regulation of glycogen synthase controls glycogen synthesis in muscle.
abstract: Glycogen synthase (GS), a key enzyme in glycogen synthesis, is activated by the allosteric stimulator glucose-6-phosphate (G6P) and by dephosphorylation through inactivation of GS kinase-3 with insulin. The relative importance of these two regulatory mechanisms in controlling GS is not established, mainly due to the complex interplay between multiple phosphorylation sites and allosteric effectors. Here we identify a residue that plays an important role in the allosteric activation of GS by G6P. We generated knockin mice in which wild-type muscle GS was replaced by a mutant that could not be activated by G6P but could still be activated normally by dephosphorylation. We demonstrate that knockin mice expressing the G6P-insensitive mutant display an ∼80% reduced muscle glycogen synthesis by insulin and markedly reduced glycogen levels. Our study provides genetic evidence that allosteric activation of GS is the primary mechanism by which insulin promotes muscle glycogen accumulation in vivo.

title: Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle.
abstract: The main purpose of this study was to determine whether the increased glucose transport (GT) found immediately postexercise (IPEX) or 4 h postexercise (4hPEX) is accompanied by increased phosphorylation of Akt substrate of 160 kDa (AS160, a protein regulator of GLUT4 translocation). Paired epitrochlearis muscles were dissected from rats (sedentary or IPEX, 2-h swim) and used to measure protein phosphorylation and insulin-independent GT. IPEX values exceeded sedentary values for GT and phosphorylations of AS160, AMP-activated protein kinase (pAMPK) and acetyl-CoA carboxylase (pACC) but not for AS160 abundance or phosphorylation of Akt serine (pSerAkt), Akt threonine (pThrAkt), or glycogen synthase kinase-3 (pGSK3). AS160 phosphorylation was significantly correlated with GT (R=0.801, P<0.01) and pAMPK (R=0.655, P<0.05). Muscles from other rats were studied 4hPEX along with sedentary controls. One muscle per rat was incubated without insulin, and the contralateral muscle was incubated with insulin. 4hPEX values exceeded sedentary values for insulin-stimulated GT. The elevated pAMPK and pACC found IPEX had reversed by 4hPEX. Insulin caused a significant increase in pSerAkt,  phosphorylation with or without exercise. Exercise significantly increased AS160 phosphorylation, regardless of insulin, with unchanged AS160 abundance. Among the signaling proteins studied, insulin-stimulated GT was significantly correlated only with insulin-stimulated pThrAkt (R=0.720, P<0.0005). The results are consistent with a role for increased AS160 phosphorylation in the increased insulin-independent GT IPEX, and the exercise effects on AS160 phosphorylation and/or pThrAkt at 4hPEX are potentially relevant to the increased insulin-stimulated glucose transport at this time.

title: Increased AS160 phosphorylation, but not TBC1D1 phosphorylation, with increased postexercise insulin sensitivity in rat skeletal muscle.
abstract: A single exercise bout can increase insulin-independent glucose transport immediately postexercise and insulin-dependent glucose transport (GT) for several hours postexercise. Akt substrate of 160 kDa (AS160) and TBC1D1 are paralog Rab GTPase-activating proteins that have been proposed to contribute to these exercise effects. Previous research demonstrated greater AS160 and Akt threonine phosphorylation in rat skeletal muscle at 3-4 h postexercise concomitant with enhanced insulin-stimulated GT. To further probe whether these signaling events or TBC1D1 phosphorylation were important for the enhanced postexercise insulin-stimulated GT, male Wistar rats were studied using four experimental protocols (2-h swim exercise, differing with regard to timing of muscle sampling and whether food was provided postexercise) that were known to vary in their influence of insulin-independent and insulin-dependent GT postexercise. The results indicated that, in isolated rat epitrochlearis muscle, 1) elevated phosphorylation of AS160 (measured using anti-phospho-Akt substrate, PAS-AS160, and phosphospecific anti-Thr(642)-AS160, pThr(642)-AS160) consistently tracked with elevated insulin-stimulated GT; 2) PAS-TBC1D1 was not different from sedentary values at 3 or 27 h postexercise, when insulin sensitivity was increased; 3) insulin-stimulated Akt activity was not increased postexercise in muscles with increased insulin sensitivity; 4) PAS-TBC1D1 was increased immediately postexercise, when insulin-independent GT was elevated, and reversed at 3 and 27 h postexercise, when insulin-independent GT was also reversed; and 5) there was no significant effect of exercise or insulin on total abundance of AS160, TBC1D1, Akt, or GLUT4 protein with any of the protocols. The results are consistent with increased AS160 phosphorylation (PAS-AS160 or pThr(642)-AS160) but not increased PAS-TBC1D1 or Akt activity, which is important for increased postexercise insulin-stimulated GT in rat skeletal muscle. They also support the idea that increased TBC1D1 phosphorylation may play a role in the insulin-independent increase in GT postexercise.

title: Sustained postexercise increases in AS160 Thr642 and Ser588 phosphorylation in skeletal muscle without sustained increases in kinase phosphorylation.
abstract: Prior exercise by rats can induce a sustained increase in muscle Akt substrate of 160 kDa (AS160) phosphorylation on Thr(642) (pAS160(Thr642)). Because phosphorylation of AS160 on both AS160(Thr642) and AS160(Ser588) is important for insulin-stimulated glucose transport (GT), we determined if exercise would also induce a sustained increase in pAS160(Ser588) concomitant with persistently elevated pAS160(Thr642) and GT. Given that the mechanisms for sustained postexercise (PEX) effects on pAS160 were uncertain, we also studied the four kinases known to phosphorylate AS160 (Akt, AMPK, RSK, and SGK1). In addition, because the serine/threonine phosphatase(s) that dephosphorylate muscle AS160 were previously unidentified, we assessed the ability of four serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) to dephosphorylate AS160. We also evaluated exercise effects on posttranslational modifications (Tyr(307) and Leu(309)) that regulate PP2A. In isolated epitrochlearis muscles from rats, GT at 3hPEX with insulin significantly (P < 0.05) exceeded SED controls. Muscles from 0hPEX vs. 0hSED and 3hPEX vs. 3hSED rats had greater pAS160(Thr642) and pAS160(Ser588). AMPK was the only kinase with greater phosphorylation at 0hPEX vs. 0hSED, and none had greater phosphorylation at 3hPEX vs. 3hSED. Each phosphatase was able to dephosphorylate pAS160(Thr642) and pAS160(Ser588) in cell-free assays. Exercise did not alter posttranslational modifications of PP2A. Our results revealed: 1) pAMPK as a potential trigger for increased pAS160(Thr642) and pAS160(Ser588) at 0hPEX; 2) PP1, PP2A, PP2B, and PP2C were each able to dephosphorylate AS160; and 3) sustained PEX-induced elevations of pAS160(Thr642) and pAS160(Ser588) were attributable to mechanisms other than persistent phosphorylation of known AS160 kinases or altered posttranslational modifications of PP2A.

title: Distinct Signals Regulate AS160 Phosphorylation in Response to Insulin, AICAR, and Contraction in Mouse Skeletal Muscle
abstract: Insulin and contraction increase GLUT4 translocation in skeletal muscle via distinct signaling mechanisms. Akt substrate of 160 kDa (AS160) mediates insulin-stimulated GLUT4 translocation in L6 myotubes, presumably through activation of Akt. Using in vivo, in vitro, and in situ methods, insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR all increased AS160 phosphorylation in mouse skeletal muscle. Insulin-stimulated AS160 phosphorylation was fully blunted by wortmannin in vitro and in Akt2 knockout (KO) mice in vivo. In contrast, contraction-stimulated AS160 phosphorylation was only partially decreased by wortmannin and unaffected in Akt2 KO mice, suggesting additional regulatory mechanisms. To determine if AMPK mediates AS160 signaling, we used AMPK α2-inactive (α2i) transgenic mice. AICAR-stimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the AMPK α2i transgenic mice. Combined AMPK α2 and Akt inhibition by wortmannin treatment of AMPK α2 transgenic mice did not fully ablate contraction-stimulated AS160 phosphorylation. Maximal insulin, together with either AICAR or contraction, increased AS160 phosphorylation in an additive manner. In conclusion, AS160 may be a point of convergence linking insulin, contraction, and AICAR signaling. While Akt and AMPK α2 activities are essential for AS160 phosphorylation by insulin and AICAR, respectively, neither kinase is indispensable for the entire effects of contraction on AS160 phosphorylation.

title: In vivo exercise followed by in vitro contraction additively elevates subsequent insulin-stimulated glucose transport by rat skeletal muscle.
abstract: The cellular mechanisms whereby prior exercise enhances insulin-stimulated glucose transport (GT) are not well understood. Previous studies suggested that a prolonged increase in phosphorylation of Akt substrate of 160 kDa (AS160) may be important for the postexercise increase in insulin sensitivity. In the current study, the effects of in vivo exercise and in vitro contraction on subsequent insulin-stimulated GT were studied separately and together. Consistent with results from previous studies, prior exercise resulted in an increase in AS160 (642)Thr phosphorylation immediately after exercise in rat epitrochlearis muscles, and this increase remained 3 h postexercise concomitant with enhanced insulin-stimulated GT. For experiments with in vitro contraction, isolated rat epitrochlearis muscles were electrically stimulated to contract in the presence or absence of rat serum. As expected, insulin-stimulated GT measured 3 h after electrical stimulation in serum, but not after electrical stimulation without serum, exceeded resting controls. Immediately after electrical stimulation with or without serum, phosphorylation of both AS160 (detected by phospho-Akt substrate, PAS, antibody, or phospho-(642)Thr antibody) and its paralog TBC1D1 (detected by phospho-(237)Ser antibody) was increased. However, both AS160 and TBC1D1 phosphorylation had reversed to resting values at 3 h poststimulation with or without serum. Increasing the amount of exercise (from 1 to 2 h) or electrical stimulation (from 5 to 10 tetani) did not further elevate insulin-stimulated GT. In contrast, the combination of prior exercise and electrical stimulation had an additive effect on the subsequent increase in insulin-stimulated GT, suggesting that these exercise and electrical stimulation protocols may amplify insulin-stimulated GT through distinct mechanisms, with a persistent increase in AS160 phosphorylation potentially important for increased insulin sensitivity after exercise, but not after in vitro contraction.

title: Muscle Glycogen Remodeling and Glycogen Phosphate Metabolism following Exhaustive Exercise of Wild Type and Laforin Knockout Mice*
abstract: Glycogen contains a low level of covalent phosphate that is significantly increased in Lafora disease.  Exercise removes phosphate from muscle glycogen, but not in a Lafora disease mouse model. Restoration of glycogen level and structure is much faster than phosphate accumulation.  Absence of laforin affects glycogen remodeling.  Glycogen phosphorylation is slow, consistent with the teenage onset of Lafora disease. Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease.

title: Effects of Glucose with Casein Peptide Supplementation on Post-Exercise Muscle Glycogen Resynthesis in C57BL/6J Mice
abstract: Numerous studies have reported that post-exercise ingestion of carbohydrates with protein supplementation can enhance glycogen recovery. However, few reports have focused on the degrees of degradation of the ingested proteins due to post-exercise glycogen resynthesis. Accordingly, the aim of this study was to clarify the effects of differences in protein degradation on muscle glycogen recovery. Male seven-week-old C57BL/6J mice performed a single bout of 60-min treadmill running exercise and were then orally administered glucose (Glu; 1.5 mg/g body weight (BW)), glucose with casein peptide (Glu + Pep; 1.5 + 0.5 mg/g BW) or its constituent amino acid mixture (Glu + AA; 1.5 + 0.5 mg/g BW). At 120 min after supplementation, the soleus muscle glycogen content in the Glu and Glu + AA groups was significantly higher than that immediately after exercise; however, no such difference was observed in the Glu + Pep group. Blood substrate concentration and insulin signaling did not differ among the three groups. Furthermore, energy expenditure during the recovery period in the Glu + Pep group was significantly higher than that in the Glu and Glu + AA groups. These findings suggest that post-exercise co-ingestion of glucose and casein peptide might delay glycogen resynthesis, at least in part through increased energy expenditure caused by casein peptide ingestion.

title: Effects of β-hydroxybutyrate treatment on glycogen repletion and its related signaling cascades in epitrochlearis muscle during 120 min of post-exercise recovery.
abstract: We investigated the effects of β-hydroxybutyrate (β-HB), the most abundant type of ketone body in mammals, on post-exercise glycogen recovery in skeletal muscle by using an in vitro experimental model. Male ICR mice swam for 60 min, and then their epitrochlearis muscles were removed and incubated with either physiological levels of glucose (8 mM) and insulin (60 μU/mL), or glucose and insulin plus 1, 2 or 4 mM of sodium β-HB. Four mM β-HB had a significant positive effect on glycogen repletion in epitrochlearis muscle at 120 min after exercise (p<0.01), while 2 mM of β-HB showed a tendency to increase the glycogen level (p<0.09) and 1 mM of β-HB had no significant effect. We further investigated the effect of 4 mM β-HB treatment on the signaling cascade related to glycogen repletion in the epitrochlearis muscles throughout a 120 min recovery period. After incubating the muscles in 4 mM of β-HB for 15 min post-exercise, the Akt substrate of 160 kDa Thr642 (p<0.05) and Akt Thr308 (p<0.05) phosphorylations were significantly increased compared to the control treatment. At the same time point, 5'-AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylations were significantly lower (p<0.05) in the epitrochlearis muscle incubated with 4 mM of β-HB than in the control muscle. Our results demonstrate that post-exercise 4 mM β-HB administration enhanced glycogen repletion in epitrochlearis muscle. Four mM β-HB treatment was associated with alternation of the phosphorylated status of several proteins involved in glucose uptake and metabolic/energy homeostasis at the early stage of post-exercise.

title: Effects of Royal Jelly Administration on Endurance Training-Induced Mitochondrial Adaptations in Skeletal Muscle
abstract: We investigated the effect of royal jelly (RJ), a natural secretion from worker bees, on the endurance training-induced mitochondrial adaptations in skeletal muscles of ICR mice. Mice received either RJ (1.0 mg/g body weight) or distilled water for three weeks. The mice in the training group were subjected to endurance training (20 m/min; 60 min; 5 times/week). There was a main effect of endurance training on the maximal activities of the mitochondrial enzymes, citrate synthase (CS), and β-hydroxyacyl coenzyme Adehydrogenase (β-HAD), in the plantaris and tibialis anterior (TA) muscles, while no effect of RJ treatment was observed. In the soleus muscle, CS and β-HAD maximal activities were significantly increased by endurance training in the RJ-treated group, while there was no effect of training in the control group. Furthermore, we investigated the effects of acute RJ treatment on the signaling cascade involved in mitochondrial biogenesis. In the soleus, phosphorylation of 5′-AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were additively increased by a single RJ treatment and endurance exercise, while only an exercise effect was found in the plantaris and TA muscles. These results indicate that the RJ treatment induced mitochondrial adaptation with endurance training by AMPK activation in the soleus muscles of ICR mice.

title: Interaction between glucose and free fatty acid metabolism in human skeletal muscle.
abstract: The mechanism by which FFA metabolism inhibits intracellular insulin-mediated muscle glucose metabolism in normal humans is unknown. We used the leg balance technique with muscle biopsies to determine how experimental maintenance of FFA during hyperinsulinemia alters muscle glucose uptake, oxidation, glycolysis, storage, pyruvate dehydrogenase (PDH), or glycogen synthase (GS). 10 healthy volunteers had two euglycemic insulin clamp experiments. On one occasion, FFA were maintained by lipid emulsion infusion; on the other, FFA were allowed to fall. Leg FFA uptake was monitored with [9,10-3H]-palmitate. Maintenance of FFA during hyperinsulinemia decreased muscle glucose uptake (1.57 +/- 0.31 vs 2.44 +/- 0.39 mumol/min per 100 ml tissue, P < 0.01), leg respiratory quotient (0.86 +/- 0.02 vs 0.93 +/- 0.02, P < 0.05), contribution of glucose to leg oxygen consumption (53 +/- 6 vs 76 +/- 8%, P < 0.05), and PDH activity (0.328 +/- 0.053 vs 0.662 +/- 0.176 nmol/min per mg, P < 0.05). Leg lactate balance was increased. The greatest effect of FFA replacement was reduced muscle glucose storage (0.36 +/- 0.20 vs 1.24 +/- 0.25 mumol/min per 100 ml, P < 0.01), accompanied by decreased GS fractional velocity (0.129 +/- 0.26 vs 0.169 +/- 0.033, P < 0.01). These results confirm in human skeletal muscle the existence of competition between glucose and FFA as oxidative fuels, mediated by suppression of PDH. Maintenance of FFA levels during hyperinsulinemia most strikingly inhibited leg muscle glucose storage, accompanied by decreased GS activity.

title: Mechanisms of fatty acid-induced inhibition of glucose uptake.
abstract: Increased plasma FFA reduce insulin-stimulated glucose uptake. The mechanisms responsible for this inhibition, however, remain uncertain. It was the aim of this study to determine whether the FFA effect was dose dependent and to investigate its mechanism. We have examined in healthy volunteers (13 male/1 female) the effects of three steady state plasma FFA levels (approximately 50, approximately 550, approximately 750 microM) on rates of glucose uptake, glycolysis (both with 3-3H-glucose), glycogen synthesis (determined with two independent methods), carbohydrate (CHO) oxidation (by indirect calorimetry), hepatic glucose output, and nonoxidative glycolysis (glycolysis minus CHO oxidation) during euglycemic-hyperinsulinemic clamping. Increasing FFA concentration (from approximately 50 to approximately 750 microM) decreased glucose uptake in a dose-dependent fashion (from approximately 9 to approximately 4 mg/kg per min). The decrease was caused mainly (approximately 2/3) by a reduction in glycogen synthesis and to a lesser extent (approximately 1/3) by a reduction in CHO oxidation. We have identified two independent defects in glycogen synthesis. The first consisted of an impairment of muscle glycogen synthase activity. It required high FFA concentration (approximately 750 microM), was associated with an increase in glucose-6-phosphate, and developed after 4-6 h of fat infusion. The second defect, which preceded the glycogen synthase defect, was seen at medium (approximately 550 microM) FFA concentration, was associated with a decrease in muscle glucose-6-phosphate concentration, and was probably due to a reduction in glucose transport/phosphorylation. In addition, FFA and/or glycerol increased insulin-suppressed hepatic glucose output by approximately 50%. We concluded that fatty acids caused a dose-dependent inhibition of insulin-stimulated glucose uptake (by decreasing glycogen synthesis and CHO oxidation) and that FFA and/or glycerol increased insulin-suppressed hepatic glucose output and thus caused insulin resistance at the peripheral and the hepatic level.

title: Five-hour fatty acid elevation increases muscle lipids and impairs glycogen synthesis in the rat.
abstract: Insulin-mediated muscle glycogen synthesis is impaired after several weeks of high-fat feeding in rats, but not by short-term (2-hour) nonesterified fatty acids (NEFA) elevation induced by intravenous triglyceride/heparin infusion (TG/H). We examined whether a longer TG/H infusion induces defective glycogen synthesis. Five-hour hyperinsulinemic (700 pmol/L) euglycemic clamps with either TG/H or saline infusion were performed. TG/H-infused rats developed insulin resistance, but only after 2 to 3 hours. Red gastrocnemius glycogen synthesis rate decreased by 50% (P < .01 v saline) associated with decreased glycogen synthase activity (GSa; assessed at several glucose-6-phosphate [G-6-P] levels; two-way ANOVA, P=.02) and increased muscle TG and total long-chain acyl coenzyme A (LCAC) content (twofold; P < .05 v saline). Thus a 3- to 5-hour NEFA elevation in the rat produced significant impairment of insulin-stimulated muscle glycogen synthesis, associated with muscle lipid accumulation. These effects were similar to those observed after several weeks of fat feeding. The 5-hour TG/H-infused rat is a useful model for studying lipid-induced muscle insulin resistance.

title: Postexercise whole body heat stress additively enhances endurance training-induced mitochondrial adaptations in mouse skeletal muscle.
abstract: A recent study demonstrated that heat stress induces mitochondrial biogenesis in C2C12 myotubes, thereby implying that heat stress may be an effective treatment to enhance endurance training-induced mitochondrial adaptations in skeletal muscle. However, whether heat stress actually induces mitochondrial adaptations in skeletal muscle in vivo is unclear. In the present study, we report the novel findings that 1) whole body heat stress produced by exposure of ICR mice to a hot environment (40°C, 30 min/day, 5 days/wk, 3 wk) induced mitochondrial adaptations such as increased mitochondrial enzyme activity (citrate synthase and 3-hydroxyacyl CoA dehydrogenase) and respiratory chain protein content (complexes I-V) in skeletal muscle in vivo and 2) postexercise whole body heat stress additively enhanced endurance training-induced mitochondrial adaptations (treadmill running, 25 m/min, 30 min/day, 5 days/wk, 3 wk). Moreover, to determine the candidate mechanisms underlying mitochondrial adaptations, we investigated the acute effects of postexercise whole body heat stress on the phosphorylation status of cellular signaling cascades that subsequently induce mitochondrial gene transcription. We found that whole body heat stress boosted the endurance exercise-induced phosphorylation of p38 MAPK, increased the phosphorylation status of p70S6K, a biomarker of mammalian target of rapamycin complex 1 activity, and unexpectedly dephosphorylated AMP-activated protein kinase and its downstream target acetyl-CoA carboxylase in skeletal muscle. Our present observations suggest that heat stress can act as an effective postexercise treatment. Heat stress treatment appeared to be clinically beneficial for people who have difficulty participating in sufficient exercise training, such as the elderly, injured athletes, and patients.

title: Exercise Capacity of Mice Genetically Lacking Muscle Glycogen Synthase
abstract: The glucose storage polymer glycogen is generally considered to be an important source of energy for skeletal muscle contraction and a factor in exercise endurance. A genetically modified mouse model lacking muscle glycogen was used to examine whether the absence of the polysaccharide affects the ability of mice to run on a treadmill. The MGSKO mouse has the GYS1 gene, encoding the muscle isoform of glycogen synthase, disrupted so that skeletal muscle totally lacks glycogen. The morphology of the soleus and quadriceps muscles from MGSKO mice appeared normal. MGSKO-null mice, along with wild type littermates, were exercised to exhaustion. There were no significant differences in the work performed by MGSKO mice as compared with their wild type littermates. The amount of liver glycogen consumed during exercise was similar for MGSKO and wild type animals. Fasting reduced exercise endurance, and after overnight fasting, there was a trend to reduced exercise endurance for the MGSKO mice. These studies provide genetic evidence that in mice muscle glycogen is not essential for strenuous exercise and has relatively little effect on endurance.

title: Impaired glucose metabolism and exercise capacity with muscle-specific glycogen synthase 1 (gys1) deletion in adult mice
abstract: Muscle glucose storage and muscle glycogen synthase (gys1) defects have been associated with insulin resistance. As there are multiple mechanisms for insulin resistance, the specific role of glucose storage defects is not clear. The aim of this study was to examine the effects of muscle-specific gys1 deletion on glucose metabolism and exercise capacity.  Tamoxifen inducible and muscle specific gys-1 KO mice were generated using the Cre/loxP system. Mice were subjected to glucose tolerance tests, euglycemic/hyperinsulinemic clamps and exercise tests. Results gys1-KO mice showed ≥85% reduction in muscle gys1 mRNA and protein concentrations, 70% reduction in muscle glycogen levels, postprandial hyperglycaemia and hyperinsulinaemia and impaired glucose tolerance. Under insulin-stimulated conditions, gys1-KO mice displayed reduced glucose turnover and muscle glucose uptake, indicative of peripheral insulin resistance, as well as increased plasma and muscle lactate levels and reductions in muscle hexokinase II levels. gys1-KO mice also exhibited markedly reduced exercise and endurance capacity.  Thus, muscle-specific gys1 deletion in adult mice results in glucose intolerance due to insulin resistance and reduced muscle glucose uptake as well as impaired exercise and endurance capacity. In brief This study demonstrates why the body prioritises muscle glycogen storage over liver glycogen storage despite the critical role of the liver in supplying glucose to the brain in the fasting state and shows that glycogen deficiency results in impaired glucose metabolism and reduced exercise capacity.

title: Astaxanthin improves muscle lipid metabolism in exercise via inhibitory effect of oxidative CPT I modification.
abstract: Intracellular redox balance may affect nutrient metabolism in skeletal muscle. Astaxanthin, a carotenoid contained in various natural foods, exerts high antioxidative capacity in the skeletal muscles. The present study investigated the effect of astaxanthin on muscle lipid metabolism in exercise. ICR mice (8 weeks old) were divided into four different groups: sedentary, sedentary treated with astaxanthin, running exercise, and exercise treated with astaxanthin. After 4 weeks of treatment, exercise groups performed treadmill running. Astaxanthin increased fat utilization during exercise compared with mice on a normal diet with prolongation of the running time to exhaustion. Colocalization of fatty acid translocase with carnitine palmitoyltransferase I (CPT I) in skeletal muscle was increased by astaxanthin. We also found that hexanoyl-lysine modification of CPT I was increased by exercise, while astaxanthin prevented this increase. In additional experiment, we found that astaxanthin treatment accelerated the decrease of body fat accumulation with exercise training. Our results suggested that astaxanthin promoted lipid metabolism rather than glucose utilization during exercise via CPT I activation, which led to improvement of endurance and efficient reduction of adipose tissue with training.

title: Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism
abstract: Nrf2 (NF-E2-related factor 2) contributes to the maintenance of glucose homeostasis in vivo. Nrf2 suppresses blood glucose levels by protecting pancreatic β cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1 knockout (Keap1MuKO) mice that express abundant Nrf2 in their SkM and then examined Nrf2 target gene expression in that tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated and the levels of the glycogen branching enzyme (Gbe1) and muscle-type PhKα subunit (Phka1) mRNAs, along with those of the glycogen branching enzyme (GBE) and the phosphorylase b kinase α subunit (PhKα) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2 inducers promoted Gbe1 and Phka1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 and Phka1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2 induction in SkM increases GBE and PhKα expression and reduces muscle glycogen content, resulting in improved glucose tolerance. Our results also indicate that Nrf2 differentially regulates glycogen metabolism in SkM and the liver.

title: Postexercise improvement in glucose uptake occurs concomitant with greater γ3-AMPK activation and AS160 phosphorylation in rat skeletal muscle.
abstract: A single exercise session can increase insulin-stimulated glucose uptake (GU) by skeletal muscle, concomitant with greater Akt substrate of 160 kDa (AS160) phosphorylation on Akt-phosphosites (Thr642 and Ser588) that regulate insulin-stimulated GU. Recent research using mouse skeletal muscle suggested that ex vivo 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or electrically stimulated contractile activity-inducing increased γ3-AMPK activity and AS160 phosphorylation on a consensus AMPK-motif (Ser704) resulted in greater AS160 Thr642 phosphorylation and GU by insulin-stimulated muscle. Our primary goal was to determine whether in vivo exercise that increases insulin-stimulated GU in rat skeletal muscle would also increase γ3-AMPK activity and AS160 site-selective phosphorylation (Ser588, Thr642, and Ser704) immediately postexercise (IPEX) and/or 3 h postexercise (3hPEX). Epitrochlearis muscles isolated from sedentary and exercised (2-h swim exercise; studied IPEX and 3hPEX) rats were incubated with 2-deoxyglucose to determine GU (without insulin at IPEX; without or with insulin at 3hPEX). Muscles were also assessed for γ1-AMPK activity, γ3-AMPK activity, phosphorylated AMPK (pAMPK), and phosphorylated AS160 (pAS160). IPEX versus sedentary had greater γ3-AMPK activity, pAS160 (Ser588, Thr642, Ser704), and GU with unaltered γ1-AMPK activity. 3hPEX versus sedentary had greater γ3-AMPK activity, pAS160 Ser704, and GU with or without insulin; greater pAS160 Thr642 only with insulin; and unaltered γ1-AMPK activity. These results using an in vivo exercise protocol that increased insulin-stimulated GU in rat skeletal muscle are consistent with the hypothesis that in vivo exercise-induced enhancement of γ3-AMPK activation and AS160 Ser704 IPEX and 3hPEX are important for greater pAS160 Thr642 and enhanced insulin-stimulated GU by skeletal muscle.

title: Enhanced Muscle Insulin Sensitivity After Contraction/Exercise Is Mediated by AMPK
abstract: Earlier studies have demonstrated that muscle insulin sensitivity to stimulate glucose uptake is enhanced several hours after an acute bout of exercise. Using AICAR, we recently demonstrated that prior activation of AMPK is sufficient to increase insulin sensitivity in mouse skeletal muscle. Here we aimed to determine whether activation of AMPK is also a prerequisite for the ability of muscle contraction to increase insulin sensitivity. We found that prior in situ contraction of m. extensor digitorum longus (EDL) and treadmill exercise increased muscle and whole-body insulin sensitivity in wild-type (WT) mice, respectively. These effects were not found in AMPKα1α2 muscle-specific knockout mice. Prior in situ contraction did not increase insulin sensitivity in m. soleus from either genotype. Improvement in muscle insulin sensitivity was not associated with enhanced glycogen synthase activity or proximal insulin signaling. However, in WT EDL muscle, prior in situ contraction enhanced insulin-stimulated phosphorylation of TBC1D4 Thr649 and Ser711. Such findings are also evident in prior exercised and insulin-sensitized human skeletal muscle. Collectively, our data suggest that the AMPK-TBC1D4 signaling axis is likely mediating the improved muscle insulin sensitivity after contraction/exercise and illuminates an important and physiologically relevant role of AMPK in skeletal muscle.

title: Exercise-induced molecular mechanisms promoting glycogen supercompensation in human skeletal muscle
abstract: A single bout of exercise followed by intake of carbohydrates leads to glycogen supercompensation in prior exercised muscle. Our objective was to illuminate molecular mechanisms underlying this phenomenon in skeletal muscle of man.  We studied the temporal regulation of glycogen supercompensation in human skeletal muscle during a 5 day recovery period following a single bout of exercise. Nine healthy men depleted (day 1), normalized (day 2) and supercompensated (day 5) muscle glycogen in one leg while the contralateral leg served as a resting control. Euglycemic hyperinsulinemic clamps in combination with leg balance technique allowed for investigating insulin-stimulated leg glucose uptake under these 3 experimental conditions. Cellular signaling in muscle biopsies was investigated by global proteomic analyses and immunoblotting. We strengthened the validity of proposed molecular effectors by follow-up studies in muscle of transgenic mice.  Sustained activation of glycogen synthase (GS) and AMPK in combination with elevated expression of proteins determining glucose uptake capacity were evident in the prior exercised muscle. We hypothesize that these alterations offset the otherwise tight feedback inhibition of glycogen synthesis and glucose uptake by glycogen. In line with key roles of AMPK and GS seen in the human experiments we observed abrogated ability for glycogen supercompensation in muscle with inducible AMPK deletion and in muscle carrying a G6P-insensitive form of GS in muscle.  Our study demonstrates that both AMPK and GS are key regulators of glycogen supercompensation following a single bout of glycogen-depleting exercise in skeletal muscle of both man and mouse.

title: Exercise training reduces the insulin‐sensitizing effect of a single bout of exercise in human skeletal muscle
abstract: A single bout of exercise is capable of increasing insulin sensitivity in human skeletal muscle. Whether this ability is affected by training status is not clear. Studies in mice suggest that the AMPK‐TBC1D4 signalling axis is important for the increased insulin‐stimulated glucose uptake after a single bout of exercise. The present study is the first longitudinal intervention study to show that, although exercise training increases insulin‐stimulated glucose uptake in skeletal muscle at rest, it diminishes the ability of a single bout of exercise to enhance muscle insulin‐stimulated glucose uptake. The present study provides novel data indicating that AMPK in human skeletal muscle is important for the insulin‐sensitizing effect of a single bout of exercise.

title: Regulation of glucose transport by insulin: traffic control of GLUT4
abstract: Despite daily fasting and feeding, plasma glucose levels are normally maintained within a narrow range owing to the hormones insulin and glucagon. Insulin increases glucose uptake into fat and muscle cells through the regulated trafficking of vesicles that contain glucose transporter type 4 (GLUT4). New insights into insulin signalling reveal that phosphorylation events initiated by the insulin receptor regulate key GLUT4 trafficking proteins, including small GTPases, tethering complexes and the vesicle fusion machinery. These proteins, in turn, control GLUT4 movement through the endosomal system, formation and retention of specialized GLUT4 storage vesicles and targeted exocytosis of these vesicles. Understanding these processes may help to explain the development of insulin resistance in type 2 diabetes and provide new potential therapeutic targets.

title: Mechanisms for greater insulin-stimulated glucose uptake in normal and insulin-resistant skeletal muscle after acute exercise.
abstract: Enhanced skeletal muscle and whole body insulin sensitivity can persist for up to 24-48 h after one exercise session. This review focuses on potential mechanisms for greater postexercise and insulin-stimulated glucose uptake (ISGU) by muscle in individuals with normal or reduced insulin sensitivity. A model is proposed for the processes underlying this improvement; i.e., triggers initiate events that activate subsequent memory elements, which store information that is relayed to mediators, which translate memory into action by controlling an end effector that directly executes increased insulin-stimulated glucose transport. Several candidates are potential triggers or memory elements, but none have been conclusively verified. Regarding potential mediators in both normal and insulin-resistant individuals, elevated postexercise ISGU with a physiological insulin dose coincides with greater Akt substrate of 160 kDa (AS160) phosphorylation without improved proximal insulin signaling at steps from insulin receptor binding to Akt activity. Causality remains to be established between greater AS160 phosphorylation and improved ISGU. The end effector for normal individuals is increased GLUT4 translocation, but this remains untested for insulin-resistant individuals postexercise. Following exercise, insulin-resistant individuals can attain ISGU values similar to nonexercising healthy controls, but after a comparable exercise protocol performed by both groups, ISGU for the insulin-resistant group has been consistently reported to be below postexercise values for the healthy group. Further research is required to fully understand the mechanisms underlying the improved postexercise ISGU in individuals with normal or subnormal insulin sensitivity and to explain the disparity between these groups after similar exercise.

title: Unravelling the activation mechanisms of protein kinase B/Akt
abstract: Over the past decade, protein kinase B (PKB, also termed Akt) has emerged as an important signaling mediator between extracellular cues and modulation of gene expression, metabolism, and cell survival. The enzyme is tightly controlled and consequences of its deregulation include loss of growth control and oncogenesis. Recent work has better characterized the mechanism of PKB activation, including upstream regulators and secondary binding partners. This minireview refreshes some old concepts with new twists and highlights current outstanding questions.

title: Insulin-modulated Akt subcellular localization determines Akt isoform-specific signaling
abstract: The 3 Akt protein kinase isoforms have critical and distinct functions in the regulation of metabolism, cell growth, and apoptosis, yet the mechanisms by which their signaling specificity is achieved remain largely unclear. Here, we investigated potential mechanisms underlying Akt isoform functional specificity by using Akt2-specific regulation of glucose transport in insulin-stimulated adipocytes as a model system. We found that insulin activates both Akt1 and Akt2 in adipocytes, but differentially regulates the subcellular distribution of these Akt isoforms. The greater accumulation of Akt2 at the plasma membrane (PM) of insulin-stimulated adipocytes correlates with Akt2-specific regulation of the trafficking of the GLUT4 glucose transporter. Consistent with this pattern, Akt constructs that do not accumulate at the PM to the same degree as Akt2 fail to regulate GLUT4 translocation to the PM, whereas enhancement of Akt1 PM association through mutation in Akt1 PH domain is sufficient to overcome Akt-isoform specificity in GLUT4 regulation. Indeed, we found that this distinct insulin-induced PM accumulation of Akt kinases is translated into a differential regulation by the Akt isoforms of AS160, a RabGAP that regulates GLUT4 trafficking. Our data show that Akt2 specifically regulates AS160 phosphorylation and membrane association providing molecular basis for Akt2 specificity in the modulation of GLUT4 trafficking. Together, our findings reveal the stimulus-induced subcellular compartmentalization of Akt kinases as a mechanism contributing to specify Akt isoform functions.

title: 5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle.
abstract: It has previously been reported that exercise causes an increase in glucose uptake in skeletal muscle and also an increase in 5' AMP-activated protein kinase (AMPK) activity. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA-riboside), an analog of adenosine, is taken up into cells and phosphorylated to form AICA-riboside monophosphate (ZMP), which can also activate AMPK. This study was designed to determine whether the increase in glucose uptake observed with AMPK activation by AICA-riboside is due to GLUT4 translocation from an intracellular location to the plasma membranes, similar to that seen in response to contraction. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine erythrocytes, 8 mmol/l glucose, and +/-2 mmol/AICA-riboside or +/-60 nmol/l insulin. Perfusion medium containing AICA-riboside was found to significantly increase AMPK activity, glucose uptake, and GLUT4 translocation in skeletal muscle above basal levels. Insulin-perfused muscles showed significant increases in glucose uptake and GLUT4 translocation, but AMPK activation was not significantly changed from basal levels. These results provide evidence that the increased glucose uptake observed with AMPK activation by AICA-riboside in perfused rat hindlimb muscles is due to an increase in the translocation of GLUT4 to surface membranes.

title: AMPK-Mediated AS160 Phosphorylation in Skeletal Muscle Is Dependent on AMPK Catalytic and Regulatory Subunits
abstract: AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 β-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (α1β1γ1 and α2β2γ1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (α2 AMPK knockout [KO], α2 AMPK kinase dead [KD], and γ3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing α2 and γ3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle.  AS160 phosphorylation was also impaired in α2 AMPK KO and KD but not γ3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.

title: Prior AICAR Stimulation Increases Insulin Sensitivity in Mouse Skeletal Muscle in an AMPK-Dependent Manner
abstract: An acute bout of exercise increases glucose uptake in skeletal muscle by an insulin-independent mechanism. In the period after exercise, insulin sensitivity to increased glucose uptake is enhanced. The molecular mechanisms underpinning this phenomenon are poorly understood but appear to involve an increased cell surface abundance of GLUT4. While increased proximal insulin signaling does not seem to mediate this effect, elevated phosphorylation of TBC1D4, a downstream target of both insulin (Akt) and exercise (AMPK) signaling, appears to play a role. The main purpose of this study was to determine whether AMPK activation increases skeletal muscle insulin sensitivity. We found that prior AICAR stimulation of wild-type mouse muscle increases insulin sensitivity to stimulate glucose uptake. However, this was not observed in mice with reduced or ablated AMPK activity in skeletal muscle. Furthermore, prior AICAR stimulation enhanced insulin-stimulated phosphorylation of TBC1D4 at Thr649 and Ser711 in wild-type muscle only. These phosphorylation events were positively correlated with glucose uptake. Our results provide evidence to support that AMPK activation is sufficient to increase skeletal muscle insulin sensitivity. Moreover, TBC1D4 phosphorylation may facilitate the effect of prior AMPK activation to enhance glucose uptake in response to insulin.

title: Genetic disruption of AMPK signaling abolishes both contraction- and insulin-stimulated TBC1D1 phosphorylation and 14-3-3 binding in mouse skeletal muscle.
abstract: TBC1D1 is a Rab-GTPase-activating protein (GAP) known to be phosphorylated in response to insulin, growth factors, pharmacological agonists that activate 5'-AMP-activated protein kinase (AMPK), and muscle contraction. Silencing TBC1D1 in L6 muscle cells by siRNA increases insulin-stimulated GLUT4 translocation, and overexpression of TBC1D1 in 3T3-L1 adipocytes with low endogenous TBC1D1 expression inhibits insulin-stimulated GLUT4 translocation, suggesting a role of TBC1D1 in regulating GLUT4 translocation. Aiming to unravel the regulation of TBC1D1 during contraction and the potential role of AMPK in intact skeletal muscle, we used EDL muscles from wild-type (WT) and AMPK kinase dead (KD) mice. We explored the site-specific phosphorylation of TBC1D1 Ser(237) and Thr(596) and their relation to 14-3-3 binding, a proposed mechanism for regulation of GAP function of TBC1D1. We show that muscle contraction increases 14-3-3 binding to TBC1D1 as well as phosphorylation of Ser(237) and Thr(596) in an AMPK-dependent manner. AMPK activation by AICAR induced similar Ser(237) and Thr(596) phosphorylation of, and 14-3-3 binding to, TBC1D1 as muscle contraction. Insulin did not increase Ser(237) phosphorylation or 14-3-3 binding to TBC1D1. However, insulin increased Thr(596) phosphorylation, and intriguingly this response was fully abolished in the AMPK KD mice. Thus, TBC1D1 is differentially regulated in response to insulin and contraction. This study provides genetic evidence to support an important role for AMPK in regulating TBC1D1 in response to both of these physiological stimuli.

title: Contraction regulates site-specific phosphorylation of TBC1D1 in skeletal muscle
abstract: TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab-GAP (GTPase-activating protein) that is highly expressed in skeletal muscle, but little is known about TBC1D1 regulation and function. We studied TBC1D1 phosphorylation on three predicted AMPK (AMP-activated protein kinase) phosphorylation sites (Ser231, Ser660 and Ser700) and one predicted Akt phosphorylation site (Thr590) in control mice, AMPKα2 inactive transgenic mice (AMPKα2i TG) and Akt2-knockout mice (Akt2 KO). Muscle contraction significantly increased TBC1D1 phosphorylation on Ser231 and Ser660, tended to increase Ser700 phosphorylation, but had no effect on Thr590. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) also increased phosphorylation on Ser231, Ser660 and Ser700, but not Thr590, whereas insulin only increased Thr590 phosphorylation. Basal and contraction-stimulated TBC1D1 Ser231, Ser660 and Ser700 phosphorylation were greatly reduced in AMPKα2i TG mice, although contraction still elicited a small increase in phosphorylation. Akt2 KO mice had blunted insulin-stimulated TBC1D1 Thr590 phosphorylation. Contraction-stimulated TBC1D1 Ser231 and Ser660 phosphorylation were normal in high-fat-fed mice. Glucose uptake in vivo was significantly decreased in tibialis anterior muscles overexpressing TBC1D1 mutated on four predicted AMPK phosphorylation sites. In conclusion, contraction causes site-specific phosphorylation of TBC1D1 in skeletal muscle, and TBC1D1 phosphorylation on AMPK sites regulates contraction-stimulated glucose uptake. AMPK and Akt regulate TBC1D1 phosphorylation, but there must be additional upstream kinases that mediate TBC1D1 phosphorylation in skeletal muscle.

title: Ablating the protein TBC1D1 impairs contraction-induced sarcolemmal glucose transporter 4 redistribution but not insulin-mediated responses in rats
abstract: TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role of TBC1D1 in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased (p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role for TBC1D1 in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle.

title: Molecular Mechanism by Which AMP-Activated Protein Kinase Activation Promotes Glycogen Accumulation in Muscle
abstract: During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxically, chronic activation of AMPK causes an increase in glycogen accumulation in skeletal and cardiac muscles, which in some cases is associated with cardiac dysfunction. The aim of this study was to elucidate the molecular mechanism by which AMPK activation promotes muscle glycogen accumulation.  We recently generated knock-in mice in which wild-type muscle GS was replaced by a mutant (Arg582Ala) that could not be activated by glucose-6-phosphate (G6P), but possessed full catalytic activity and could still be activated normally by dephosphorylation. Muscles from GS knock-in or transgenic mice overexpressing a kinase dead (KD) AMPK were incubated with glucose tracers and the AMPK-activating compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) ex vivo. GS activity and glucose uptake and utilization (glycolysis and glycogen synthesis) were assessed.  Even though AICAR caused a modest inactivation of GS, it stimulated muscle glycogen synthesis that was accompanied by increases in glucose transport and intracellular [G6P]. These effects of AICAR required the catalytic activity of AMPK. Strikingly, AICAR-induced glycogen synthesis was completely abolished in G6P-insensitive GS knock-in mice, although AICAR-stimulated AMPK activation, glucose transport, and total glucose utilization were normal.  We provide genetic evidence that AMPK activation promotes muscle glycogen accumulation by allosteric activation of GS through an increase in glucose uptake and subsequent rise in cellular [G6P].

title: Rabbit skeletal muscle glycogen synthase. I. Relationship between phosphorylation state and kinetic properties.
abstract: Nine samples of purified rabbit skeletal muscle glycogen synthase (UDP-glucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) were obtained with alkali-labile phosphate contents ranging from 0.27 to 3.49 residues per 85,000 molecular weight subunit. The enzyme samples appeared essentially homogeneous when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and had relatively constant specific activity under standard conditions with saturating UDP-glucose and glucose-l-P concentrations (37.1 +/- 1.0 mumol of glucose incorporated/mg/min). When the UDP-glucose concentration was varied, deviations from Michaelis-Menten kinetics were observed for all samples (Hill slopes of 0.79 +/- 0.02), but these deviations were virtually abolished by the presence of 5 mM glucose-6-P. Glucose-6-P decreased the S0.5 (concentration required for half-maximal rate) for UDP-glucose. The plots of activity increase caused by glucose-6-P versus glucose-6-P concentration became progressively more sigmoid in shape with enzyme samples of higher phosphate content. Both the S0.5 for UDP-glucose and the M0.5 for glucose-6-P (concentration for half-maximal activation) were sensitive functions of the alkali-labile phosphate content of glycogen synthase. The M0.5 increased from 3.3 muM at 0.27 phosphate/subunit to 2.7 mM at 3.5 phosphates/subunit, and the S0.5 varied from 0.75 mM (0.27 phosphate/subunit) to at least 61 mM (2.3 phosphates/subunit). Both parameters increased continuously with phosphate content, with the greatest absolute changes occurring at values greater than 2 residues of phosphates/subunit. The effects of both phosphorylation and activation by glucose-6-P appeared to be mediated primarily through alteration of the apparent affinity for UDP-glucose. With the phosphorylating conditions used, the results suggested that phosphate could not be introduced into the glycogen synthase molecule without altering its kinetic properties. Further, either the different phosphorylation sites were not equivalent, or else identical sites interacted in determining the kinetic properties of glycogen synthase.

title: Additive effect of contraction and insulin on glucose uptake and glycogen synthase in muscle with different glycogen contents.
abstract: Insulin and contraction regulate glucose uptake and glycogen synthase (GS) via distinct mechanisms in skeletal muscles, and an additive effect has been reported. Glycogen content is known to influence both contraction- and insulin-stimulated glucose uptake and GS activity. Our study reports that contraction and insulin additively stimulate glucose uptake in rat epitrochlearis muscles with normal (NG) and high (HG) glycogen contents, but the additive effect was only partial. In muscles with low glycogen (LG) content no additive effect was seen, but glucose uptake was higher in LG than in NG and HG during contraction, insulin stimulation, and when the two stimuli were combined. In LG, contraction-stimulated AMP-activated protein kinase (AMPK) activity and insulin-stimulated PKB phosphorylation were higher than in NG and HG, but phosphorylation of Akt substrate of 160 kDa was not elevated correspondingly. GLUT4 content was 50% increased in LG (rats fasted 24 h), which may explain the increased glucose uptake. Contraction and insulin also additively increased GS fractional activity in NG and HG but not in LG. GS fractional activity correlated most strongly with GS Ser641 phosphorylation (R -0.94, P<0.001). GS fractional activity also correlated with GS Ser7,10 phosphorylation, but insulin did not reduce GS Ser7,10 phosphorylation. In conclusion, an additive effect of contraction and insulin on glucose uptake and GS activity occurs in muscles with normal and high glycogen content but not in muscles with low glycogen content. Furthermore, contraction, insulin, and glycogen content all regulate GS Ser641 phosphorylation and GS fractional activity in concert.

title: Effect of prior exercise on the partitioning of an intestinal glucose load between splanchnic bed and skeletal muscle.
abstract: Exercise leads to marked increases in muscle insulin sensitivity and glucose effectiveness. Oral glucose tolerance immediately after exercise is generally not improved. The hypothesis tested by these experiments is that after exercise the increased muscle glucose uptake during an intestinal glucose load is counterbalanced by an increase in the efficiency with which glucose enters the circulation and that this occurs due to an increase in intestinal glucose absorption or decrease in hepatic glucose disposal. For this purpose, sampling (artery and portal, hepatic, and femoral veins) and infusion (vena cava, duodenum) catheters and Doppler flow probes (portal vein, hepatic artery, external iliac artery) were implanted 17 d before study. Overnightfasted dogs were studied after 150 min of moderate treadmill exercise or an equal duration rest period. Glucose ([14C]glucose labeled) was infused in the duodenum at 8 mg/kg x min for 150 min beginning 30 min after exercise or rest periods. Values, depending on the specific variable, are the mean +/- SE for six to eight dogs. Measurements are from the last 60 min of the intraduodenal glucose infusion. In response to intraduodenal glucose, arterial plasma glucose rose more in exercised (103 +/- 4 to 154 +/- 6 mg/dl) compared with rested (104 +/- 2 to 139 +/- 3 mg/dl) dogs. The greater increase in glucose occurred even though net limb glucose uptake was elevated after exercise (35 +/- 5 vs. 20 +/- 2 mg/min) as net splanchnic glucose output (5.1 +/- 0.8 vs. 2.1 +/- 0.6 mg/kg x min) and systemic appearance of intraduodenal glucose (8.1 +/- 0.6 vs. 6.3 +/- 0.7 mg/kg x min) were also increased due to a higher net gut glucose output (6.1 +/- 0.7 vs. 3.6 +/- 0.9 mg/kg x min). Adaptations at the muscle led to increased net glycogen deposition after exercise [1.4 +/- 0.3 vs. 0.5 +/- 0.1 mg/(gram of tissue x 150 min)], while no such increase in glycogen storage was seen in liver [3.9 +/- 1.0 vs. 4.1 +/- 1.1 mg/(gram of tissue x 150 min) in exercised and sedentary animals, respectively]. These experiments show that the increase in the ability of previously working muscle to store glycogen is not solely a result of changes at the muscle itself, but is also a result of changes in the splanchnic bed that increase the efficiency with which oral glucose is made available in the systemic circulation.

title: Exercise and exhaustion effects on glycogen synthesis pathways.
abstract: Female Sprague-Dawley rats were infused with [1-13C]glucose to measure the effect of endurance training and the effect of various metabolic conditions on pathways of hepatic glycogen synthesis. Four metabolic states [sedentary (S), trained (T), sedentary exhausted (SE), and trained exhausted (TE)] were studied. T and TE rats were trained on a motor-driven treadmill (30 m/min, 15% grade, 1.0 h/day, 5 days/wk) for 8-10 wk. After a 24-h fast, SE and TE rats were run to exhaustion (sedentary average = 78 min, trained average = 155 min) at a training pace and immediately infused with labeled glucose for 2 h. S and T rats were infused after a 24-h fast. After infusion, tissues were removed and glycogen was isolated and hydrolyzed to glucose. The glucose was measured for distribution of 13C by using nuclear magnetic resonance. Glycogen was synthesized predominantly by the indirect pathway for all metabolic states, indicating that infused glucose was first metabolized primarily in the peripheral tissue. The direct-pathway utilization was greater in rested S than in rested T animals (30 vs. 14%); however, for exhausted animals, the trained use of the direct pathway was greater (22 vs. 9%). Both TE and rested T animals utilize the indirect pathway a comparable amount. Sedentary animals, on the other hand, dramatically decreased utilization of the direct pathway, with exhaustive exercise changing from 30 to 9%. The results indicate that endurance training modifies glucose utilization during glycogen synthesis after fasting and exhaustive exercise.