Contraction-stimulated Glucose Uptake Research Articles

Time-of-day does not impact skeletal muscle contractile function, fatigability, or contraction-stimulated glucose uptake

Time-of-day impacts different aspects of mammalian physiology, such as the sleep/wake cycle, blood pressure, and body temperature. These time-of-day changes, or “circadian cycles”, are regulated by a central clock in the central suprachiasmatic nucleus of the hypothalamus and peripheral clocks located in other tissues, such as the liver, heart, and skeletal muscle. Interestingly, a growing body of data suggests that exercise performance and capacity also vary by time-of-day, with chronobiological effects on skeletal muscle metabolism and contractile function (i.e., force production) being proposed as important mediators. However, to date, no studies have specifically investigated the effect of time-of-day on skeletal muscle contractile function or glucose metabolism. The objective of this work was to assess ex vivo skeletal muscle contractile function, fatigability, and contraction-stimulated glucose uptake in the extensor digitorum longus (EDL) and soleus from 13-week-old female C57BL/6NJ mice at four different times-of-day (Light cycle time-points: Zeitgeber Time [ZT] ZT1 and ZT7; Dark cycle time-points: ZT13 and ZT19). We used an ex vivo approach as it allowed us to study skeletal muscle in isolation, and thus, independent of other factors that might influence contractile function or glucose metabolism (e.g., nerve function, humoral factors, etc.). We hypothesized that skeletal muscle contractile function, fatigability and contraction-stimulated glucose uptake would not be different by time-of-day. To control for meal-timing, mice were orally gavaged with dextrose (2 g/kg) and then fasted (3 hours) before contractile testing, which consisted of a force-frequency test (1-120 Hz, 0.3 ms pulse, 35 volts; 300 ms and 400 ms train for EDL and soleus, respectively) followed by a 15 minute fatigue test in which the muscles were repeatedly stimulated (EDL: 100 Hz stimulation every 15 seconds; Soleus: 30 Hz stimulation every 2 seconds). Glucose uptake was measured during the fatigue test using the [3H]-2-deoxyglucose (2DOG) uptake approach, with the contralateral muscle being the rested control. Significantly, contractile function, including peak twitch tension and maximal tetanic tension (EDL — ZT1: 281±13; ZT7: 300±21; ZT13: 261±28; ZT19: 280±40 kPa, P = 0.7661, one-way ANOVA; Soleus — ZT1: 117±16; ZT7: 115±18; ZT13: 117±11; ZT19: 111±10 kPa, P = 0.9923, one-way ANOVA) were not different by time-of-day. Similarly, fatigability, as measured as the tension·time integral during the 15 min fatigue test was not different by time-of-day (EDL — ZT1: 640±89; ZT7: 686±92; ZT13: 708±110; ZT19: 683±142 kPa·min, P = 0.9803, one-way ANOVA; Soleus — ZT1: 245±42; ZT7: 261±35; ZT13: 200±41; ZT19: 244±34 kPa·min, P = 0.7019, one-way ANOVA). Finally, while contraction significantly increased 2DOG uptake above basal by ~1.6-fold and ~1.8-fold in the soleus and EDL, respectively, this increase was not different by time-of-day. Taken together, these results demonstrate that time-of-day does not impact skeletal muscle contractile function, fatigability, or contraction-stimulated glucose uptake, regardless of muscle type. By extension, skeletal muscle, per se, is unlikely to be a contributing factor to the documented effects of time-of-day on whole-body exercise performance. This was funded by the UC San Diego Medical Scientist Training Program and NIH R21 AG067495. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle

ObjectiveThe AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. MethodsA recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. ResultsSTO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. ConclusionsWe demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

Conditioning-induced expression of novel glucose transporters in canine skeletal muscle homogenate.

Athletic conditioning can increase the capacity for insulin-stimulated skeletal muscle glucose uptake through increased sarcolemmal expression of GLUT4 and potentially additional novel glucose transporters. We used a canine model that has previously demonstrated conditioning-induced increases in basal, insulin- and contraction-stimulated glucose uptake to identify whether expression of glucose transporters other than GLUT4 was upregulated by athletic conditioning. Skeletal muscle biopsies were obtained from 12 adult Alaskan Husky racing sled dogs before and after a full season of conditioning and racing, and homogenates from those biopsies were assayed for expression of GLUT1, GLUT3, GLUT4, GLUT6, GLUT8, and GLUT12 using western blots. Athletic conditioning resulted in a 1.31 ± 0.70 fold increase in GLUT1 (p <0.0001), 1.80 ± 1.99 fold increase in GLUT4 (p = 0.005), and 2.46 ± 2.39 fold increase in GLUT12 (p = 0.002). The increased expression of GLUT1 helps explain the previous findings of conditioning-induced increases in basal glucose clearance in this model, and the increase in GLUT12 provides an alternative mechanism for insulin- and contraction-mediated glucose uptake and likely contributes to the substantial conditioning-induced increases in insulin sensitivity in highly trained athletic dogs. Furthermore, these results suggest that athletic dogs can serve as a valuable resource for the study of alternative glucose transport mechanisms in higher mammals.

Kalirin mediates Rac1 activation downstream of calcium/calmodulin-dependent protein kinase II to stimulate glucose uptake during muscle contraction.

In this study, we investigated the role of calcium/calmodulin-dependent protein kinase II (CaMKII) in contraction-stimulated glucose uptake in skeletal muscle. C2C12 myotubes were contracted by electrical pulse stimulation (EPS), and treadmill running was used to exercise mice. The activities of CaMKII, the small G protein Rac1, and the Rac1 effector kinase PAK1 were elevated in muscle by running exercise or EPS, while they were lowered by the CaMKII inhibitor KN-93 and/or small interfering RNA (siRNA)-mediated knockdown. EPS induced the mRNA and protein expression of the Rac1-GEF Kalirin in a CaMKII-dependent manner. EPS-induced Rac1 activation was lowered by the Kalirin inhibitor ITX3 or siRNA-mediated Kalirin knockdown. KN-93, ITX3, and siRNA-mediated Kalirin knockdown reduced EPS-induced glucose uptake. These findings define a CaMKII-Kalirin-Rac1 signaling pathway that contributes to contraction-stimulated glucose uptake in skeletal muscle myotubes and tissue.

CaMKII and Kalirin, a Rac1-GEF, regulate Akt phosphorylation involved in contraction-induced glucose uptake in skeletal muscle cells

Rac1 plays an important role in contraction-stimulated muscle glucose uptake, but the mechanism is not fully elucidated. We previously identified Rac1-dependent activation of Akt played a partial role in contraction-stimulated GLUT4 translocation to the cell surface of C2C12 myotubes. Recognizing that contraction activates CaMKII in muscle and CaMKII is known to regulate Rac1 activity in other systems, here we investigated the relationship between CaMKII, Akt and contraction-stimulated glucose uptake. Expression of a constitutively-active mutant of CaMKIIδ stimulated Akt phosphorylation that was inhibited by Rac1 inhibitor II. C2C12 myotubes were contracted by electrical pulse stimulation (EPS). We observed the CaMKII inhibitor, KN-93 and CaMKIIδ siRNA-mediated knockdown, reduced EPS-induced Akt phosphorylation in C2C12 myotubes. ITX3, an inhibitor of the Rac-GTPase Kalirin and Kalirin siRNA-mediated knockdown reduced EPS-stimulated Akt phosphorylation in myotubes. In addition, the Akt inhibitor MK2206 partly reduced EPS-stimulated glucose uptake without simultaneously affecting CaMKII phosphorylation and Kalirin protein abundance. Our findings demonstrate EPS leads to Akt activation through a CaMKII-Kalirin-Rac1 signaling pathway and partly regulates contraction-stimulated glucose uptake in muscle cells.

Contraction-Mediated Glucose Transport in Skeletal Muscle Is Regulated by a Framework of AMPK, TBC1D1/4, and Rac1.

The two closely related RabGTPase-activating proteins (RabGAPs) TBC1D1 and TBC1D4, both substrates for AMPK, play important roles in exercise metabolism and contraction-dependent translocation of GLUT4 in skeletal muscle. However, the specific contribution of each RabGAP in contraction signaling is mostly unknown. In this study, we investigated the cooperative AMPK-RabGAP signaling axis in the metabolic response to exercise/contraction using a novel mouse model deficient in active skeletal muscle AMPK combined with knockout of either Tbc1d1, Tbc1d4, or both RabGAPs. AMPK deficiency in muscle reduced treadmill exercise performance. Additional deletion of Tbc1d1 but not Tbc1d4 resulted in a further decrease in exercise capacity. In oxidative soleus muscle, AMPK deficiency reduced contraction-mediated glucose uptake, and deletion of each or both RabGAPs had no further effect. In contrast, in glycolytic extensor digitorum longus muscle, AMPK deficiency reduced contraction-stimulated glucose uptake, and deletion of Tbc1d1, but not Tbc1d4, led to a further decrease. Importantly, skeletal muscle deficient in AMPK and both RabGAPs still exhibited residual contraction-mediated glucose uptake, which was completely abolished by inhibition of the GTPase Rac1. Our results demonstrate a novel mechanistic link between glucose transport and the GTPase signaling framework in skeletal muscle in response to contraction.

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice.

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake. Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the methodapplied possesses some caveats under certain conditions when studying muscle energy metabolism.

Sex‐based differences in contraction‐stimulated glucose uptake by mouse skeletal muscle

Contraction potently stimulates glucose uptake by skeletal muscle. While insulin-stimulated glucose uptake by skeletal muscle is well known to be higher is females compared with males, whether this sex difference exists with contraction is unknown. The objective of this study was to address this gap in knowledge, and based on findings related to insulin, we hypothesized that contraction-stimulated glucose uptake would be higher in female versus male mouse skeletal muscle. To address this hypothesis, in this study we used a radioactive 2-deoxyglucose uptake (2DGU) approach to measure ex vivo glucose uptake in unstimulated (NoSTIM; i.e. rested) and electrically-stimulated (STIM; 100 Hz, 2s train, 0.2ms pulse, 35V every 15 sec for 10 min) soleus and extensor digitorum longus (EDL) from ~15 week old male and female mice. Force production was measured throughout the protocol and 2DGU was measured in the NoSTIM and STIM muscles immediately after completing the stimulation protocol. As expected, force output decreased over the 10 min stimulation protocol, although there were no sex differences in maximal force or the rate of fatigue. Related to 2DGU, within each sex, 2DGU in the EDL and soleus was ~2.5-fold higher in STIM vs. NoSTIM. Interestingly, contraction-stimulated 2DGU (i.e. STIM 2DGU minus NoSTIM 2DGU) was ~1.3 fold higher in female vs. male, and this occurred in both soleus and EDL; this difference was due to greater 2DGU in STIM muscles, as 2DGU in NoSTIM muscles was not different between sexes. This difference in contraction-stimulated 2DGU was not related total muscle stress during stimulation protocol, or the degree of activation/phosphorylation of the contraction-activated kinases, AMP-activated protein kinase and p38 mitogen-activated protein kinase. Taken together, in line with previous work focused on insulin-stimulated glucose uptake, this study demonstrates that contraction-stimulated uptake by mouse skeletal muscle is greater in females compared to males, regardless of muscle type. This suggests that female muscle has an inherently greater capacity for contraction-stimulated glucose uptake and future work will investigate the potential mechanisms underlying this sex-based difference.

Sirtuin 1 is not required for contraction-stimulated glucose uptake in mouse skeletal muscle.

While it has long been known that contraction robustly stimulates skeletal muscle glucose uptake, the molecular steps regulating this increase remain incompletely defined. The mammalian ortholog of Sir2, sirtuin 1 (SIRT1), is an NAD+-dependent protein deacetylase that is thought to link perturbations in energy flux associated with exercise to subsequent cellular adaptations. Nevertheless, its role in contraction-stimulated glucose uptake has not been described. The objective of this study was to determine the importance of SIRT1 to contraction-stimulated glucose uptake in mouse skeletal muscle. Using a radioactive 2-deoxyglucose uptake (2DOGU) approach, we measured ex vivo glucose uptake in unstimulated (rested) and electrically stimulated (100 Hz contraction every 15 s for 10 min; contracted) extensor digitorum longus (EDL) and soleus from ∼15-wk-old male and female mice with muscle-specific knockout of SIRT1 deacetylase activity and their wild-type littermates. Skeletal muscle force decreased over the contraction protocol, although there were no differences in the rate of fatigue between genotypes. In EDL and soleus, loss of SIRT1 deacetylase activity did not affect contraction-induced increase in glucose uptake in either sex. Interestingly, the absolute rate of contraction-stimulated 2DOGU was ∼1.4-fold higher in female compared with male mice, regardless of muscle type. Taken together, our findings demonstrate that SIRT1 is not required for contraction-stimulated glucose uptake in mouse skeletal muscle. Moreover, to our knowledge, this is the first demonstration of sex-based differences in contraction-stimulated glucose uptake in mouse skeletal muscle.NEW & NOTEWORTHY Here, we demonstrate that glucose uptake in response to ex vivo contractions is not affected by the loss of sirtuin 1 (SIRT1) deacetylase function in muscle, regardless of sex or muscle type. Interestingly, however, similar to studies on insulin-stimulated glucose uptake, we demonstrate that contraction-stimulated glucose uptake is robustly higher in female compared with the male skeletal muscle. To our knowledge, this is the first demonstration of sex-based differences in contraction-stimulated glucose uptake in skeletal muscle.

Tiam1 mediates Rac1 activation and contraction‐induced glucose uptake in skeletal muscle cells

Contraction-stimulated glucose uptake in skeletal muscle requires Rac1, but the molecular mechanism of its activation is not fully understood. Treadmill running was applied to induce C57BL/6 mouse hind limb skeletal muscle contraction in vivo and electrical pulse stimulation contracted C2C12 myotube cultures in vitro. The protein levels or activities of AMPK or the Rac1-specific GEF, Tiam1, were manipulated by activators, inhibitors, siRNA-mediated knockdown, and adenovirus-mediated expression. Activated Rac1 was detected by a pull-down assay and immunoblotting. Glucose uptake was measured using the 2-NBD-glucose fluorescent analog. Electrical pulse stimulated contraction or treadmill exercise upregulated the expression of Tiam1 in skeletal muscle in an AMPK-dependent manner. Axin1 siRNA-mediated knockdown diminished AMPK activation and upregulation of Tiam1 protein expression by contraction. Tiam1 siRNA-mediated knockdown diminished contraction-induced Rac1 activation, GLUT4 translocation, and glucose uptake. Contraction increased Tiam1 gene expression and serine phosphorylation of Tiam1 protein via AMPK. These findings suggest Tiam1 is part of an AMPK-Tiam1-Rac1 signaling pathway that mediates contraction-stimulated glucose uptake in skeletal muscle cells and tissue.

The RabGAPs TBC1D1 and TBC1D4 Control Uptake of Long-Chain Fatty Acids Into Skeletal Muscle via Fatty Acid Transporter SLC27A4/FATP4.

The two closely related RabGTPase-activating proteins (RabGAPs) TBC1D1 and TBC1D4 play a crucial role in the regulation of GLUT4 translocation in response to insulin and contraction in skeletal muscle. In mice, deficiency in one or both RabGAPs leads to reduced insulin- and contraction-stimulated glucose uptake and to elevated fatty acid (FA) uptake and oxidation in both glycolytic and oxidative muscle fibers without altering mitochondrial copy number and the abundance of proteins for oxidative phosphorylation. Here we present evidence for a novel mechanism of skeletal muscle lipid utilization involving the two RabGAPs and the FA transporter SLC27A4/FATP4. Both RabGAPs control the uptake of saturated and unsaturated long-chain FAs (LCFAs) into skeletal muscle and knockdown (Kd) of a subset of RabGAP substrates, Rab8, Rab10, or Rab14, decreased LCFA uptake into these cells. In skeletal muscle from Tbc1d1 and Tbc1d4 knockout animals, SLC27A4/FATP4 abundance was increased and depletion of SLC27A4/FATP4 but not FAT/CD36 completely abrogated the enhanced FA oxidation in RabGAP-deficient skeletal muscle and cultivated C2C12 myotubes. Collectively, our data demonstrate that RabGAP-mediated control of skeletal muscle lipid metabolism converges with glucose metabolism at the level of downstream RabGTPases and involves regulated transport of LCFAs via SLC27A4/FATP4.

It's well and truly time to stop stating that AMPK regulates glucose uptake and fat oxidation during exercise.

None for Perspective.

An AMPK/Axin1-Rac1 signaling pathway mediates contraction-regulated glucose uptake in skeletal muscle cells.

Contraction stimulates skeletal muscle glucose uptake predominantly through activation of AMP-activated protein kinase (AMPK) and Rac1. However, the molecular details of how contraction activates these signaling proteins are not clear. Recently, Axin1 has been shown to form a complex with AMPK and liver kinase B1 during glucose starvation-dependent activation of AMPK. Here, we demonstrate that electrical pulse-stimulated (EPS) contraction of C2C12 myotubes or treadmill exercise of C57BL/6 mice enhanced reciprocal coimmunoprecipitation of Axin1 and AMPK from myotube lysates or gastrocnemius muscle tissue. Interestingly, EPS or exercise upregulated total cellular Axin1 levels in an AMPK-dependent manner in C2C12 myotubes and gastrocnemius mouse muscle, respectively. Also, direct activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide treatment of C2C12 myotubes or gastrocnemius muscle elevated Axin1 protein levels. On the other hand, siRNA-mediated Axin1 knockdown lessened activation of AMPK in contracted myotubes. Further, AMPK inhibition with compound C or siRNA-mediated knockdown of AMPK or Axin1 blocked contraction-induced GTP loading of Rac1, p21-activated kinase phosphorylation, and contraction-stimulated glucose uptake. In summary, our results suggest that an AMPK/Axin1-Rac1 signaling pathway mediates contraction-stimulated skeletal muscle glucose uptake.

Dexamethasone rapidly inhibits glucose uptake via non-genomic mechanisms in contracting myotubes

Glucocorticoids (GCs) are a class of steroid hormones that regulate multiple aspects of glucose homeostasis. In skeletal muscle, it is well established that prolonged GC excess inhibits glucose uptake and utilization through glucocorticoid receptor (GR)-mediated transcriptional changes. However, it remains obscure that whether the rapid non-genomic effects of GC on glucose uptake are involved in acute exercise stress. Therefore, we used electric pulse stimulation (EPS)-evoked contracting myotubes to determine whether the non-genomic actions of GC were involved and its underlying mechanism(s). Pretreatment with dexamethasone (Dex, 10 μM) significantly prevented contraction-stimulated glucose uptake and glucose transporter 4 (Glut4) translocation within 20 min in C2C12 myotubes. Neither GC nuclear receptor antagonist (RU486) nor protein synthesis inhibitor (cycloheximide, Chx) affected the rapid inhibition effects of Dex. AMPK and CaMKII-dependent signaling pathways were associated with the non-genomic effects of Dex. These results provide evidence that GC rapidly suppresses glucose uptake in contracting myotubes via GR-independent non-genomic mechanisms. AMPK and CaMKII-mediated Glut4 translocation may play a critical role in GC-induced rapid inhibition of glucose uptake.

Contraction stimulates muscle glucose uptake independent of atypical PKC.

Exercise increases skeletal muscle glucose uptake, but the underlying mechanisms are only partially understood. The atypical protein kinase C (PKC) isoforms λ and ζ (PKC-λ/ζ) have been shown to be necessary for insulin-, AICAR-, and metformin-stimulated glucose uptake in skeletal muscle, but not for treadmill exercise-stimulated muscle glucose uptake. To investigate if PKC-λ/ζ activity is required for contraction-stimulated muscle glucose uptake, we used mice with tibialis anterior muscle-specific overexpression of an empty vector (WT), wild-type PKC-ζ (PKC-ζ(WT)), or an enzymatically inactive T410A-PKC-ζ mutant (PKC-ζ(T410A)). We also studied skeletal muscle-specific PKC-λ knockout (MλKO) mice. Basal glucose uptake was similar between WT, PKC-ζ(WT), and PKC-ζ(T410A) tibialis anterior muscles. In contrast, insitu contraction-stimulated glucose uptake was increased in PKC-ζ(T410A) tibialis anterior muscles compared to WT or PKC-ζ(WT) tibialis anterior muscles. Furthermore, invitro contraction-stimulated glucose uptake was greater in soleus muscles of MλKO mice than WT controls. Thus, loss of PKC-λ/ζ activity increases contraction-stimulated muscle glucose uptake. These data clearly demonstrate that PKC-λζ activity is not necessary for contraction-stimulated glucose uptake.

Fiber type effects on contraction-stimulated glucose uptake and GLUT4 abundance in single fibers from rat skeletal muscle.

To fully understand skeletal muscle at the cellular level, it is essential to evaluate single muscle fibers. Accordingly, the major goals of this study were to determine if there are fiber type-related differences in single fibers from rat skeletal muscle for: 1) contraction-stimulated glucose uptake and/or 2) the abundance of GLUT4 and other metabolically relevant proteins. Paired epitrochlearis muscles isolated from Wistar rats were either electrically stimulated to contract (E-Stim) or remained resting (No E-Stim). Single fibers isolated from muscles incubated with 2-deoxy-d-[(3)H]glucose (2-DG) were used to determine fiber type [myosin heavy chain (MHC) isoform protein expression], 2-DG uptake, and abundance of metabolically relevant proteins, including the GLUT4 glucose transporter. E-Stim, relative to No E-Stim, fibers had greater (P < 0.05) 2-DG uptake for each of the isolated fiber types (MHC-IIa, MHC-IIax, MHC-IIx, MHC-IIxb, and MHC-IIb). However, 2-DG uptake for E-Stim fibers was not significantly different among these five fiber types. GLUT4, tethering protein containing a UBX domain for GLUT4 (TUG), cytochrome c oxidase IV (COX IV), and filamin C protein levels were significantly greater (P < 0.05) in MHC-IIa vs. MHC-IIx, MHC-IIxb, or MHC-IIb fibers. TUG and COX IV in either MHC-IIax or MHC-IIx fibers exceeded values for MHC-IIxb or MHC-IIb fibers. GLUT4 levels for MHC-IIax fibers exceeded MHC-IIxb fibers. GLUT4, COX IV, filamin C, and TUG abundance in single fibers was significantly (P < 0.05) correlated with each other. Differences in GLUT4 abundance among the fiber types were not accompanied by significant differences in contraction-stimulated glucose uptake.

Rac1--a novel regulator of contraction-stimulated glucose uptake in skeletal muscle.

Muscle contraction stimulates muscle glucose uptake by facilitating translocation of glucose transporter4 from intracellular locations to the cell surface, which allows for diffusion of glucose into the myofibres. The intracellular mechanisms regulating this process are not well understood. The GTPase Rac1 has, until recently, been investigated only with regard to its involvement in insulin-stimulated glucose uptake. However, we recently found that Rac1 is activated during muscle contraction and exercise in mice and humans. Remarkably, Rac1 seems to be necessary for exercise and contraction-stimulated glucose uptake in skeletal muscle, because muscle-specific Rac1 knockout mice display reduced ex vivo contraction- and in vivo exercise-stimulated glucose uptake. The molecular mechanism by which Rac1 regulates glucose uptake is presently unknown. However, recent studies link Rac1 to the actin cytoskeleton, the small GTPase RalA and/or free radical production, which have previously been shown to be regulators of glucose uptake in muscle. We propose a model in which Rac1 is activated by contraction- and exercise-induced mechanical stress signals and that Rac1 in conjunction with other signalling regulates glucose uptake during muscle contraction and exercise.

Roles of TBC1D1 and TBC1D4 in insulin- and exercise-stimulated glucose transport of skeletal muscle.

This review focuses on two paralogue Rab GTPase activating proteins known as TBC1D1 Tre-2/BUB2/cdc 1 domain family (TBC1D) 1 and TBC1D4 (also called Akt Substrate of 160kDa, AS160) and their roles in controlling skeletal muscle glucose transport in response to the independent and combined effects of insulin and exercise. Convincing evidence implicates Akt2-dependent TBC1D4 phosphorylation on T642 as a key part of the mechanism for insulin-stimulated glucose uptake by skeletal muscle. TBC1D1 phosphorylation on several insulin-responsive sites (including T596, a site corresponding to T642 in TBC1D4) does not appear to be essential for in vivo insulin-stimulated glucose uptake by skeletal muscle. In vivo exercise or ex vivo contraction of muscle result in greater TBC1D1 phosphorylation on S237 that is likely to be secondary to increased AMP-activated protein kinase activity and potentially important for contraction-stimulated glucose uptake. Several studies that evaluated both normal and insulin-resistant skeletal muscle stimulated with a physiological insulin concentration after a single exercise session found that greater post-exercise insulin-stimulated glucose uptake was accompanied by greater TBC1D4 phosphorylation on several sites. In contrast, enhanced post-exercise insulin sensitivity was not accompanied by greater insulin-stimulated TBC1D1 phosphorylation. The mechanism for greater TBC1D4 phosphorylation in insulin-stimulated muscles after acute exercise is uncertain, and a causal link between enhanced TBC1D4 phosphorylation and increased post-exercise insulin sensitivity has yet to be established. In summary, TBC1D1 and TBC1D4 have important, but distinct roles in regulating muscle glucose transport in response to insulin and exercise.

Reduced skeletal muscle AMPK and mitochondrial markers do not promote age-induced insulin resistance.

In both rodents and humans, aging-associated reductions in skeletal muscle AMP-activated protein kinase (AMPK) activity and mitochondrial function have been linked to the development of skeletal muscle insulin resistance. However, whether reductions in skeletal muscle AMPK and mitochondrial capacity actually precipitate the development of aging-induced insulin resistance is not known. Mice lacking both isoforms of the AMPK β-subunit in skeletal muscle (AMPK-MKO) have no detectable AMPK activity and are characterized by large reductions in exercise capacity, mitochondrial content, and contraction-stimulated glucose uptake making them an ideal model to determine whether reductions in AMPK and mitochondrial content promote the development of aging-induced insulin resistance. In the current study we find that a lack of skeletal muscle AMPK results in a life-long reduction in mitochondrial activity but does not affect body mass, body composition, glucose tolerance, or insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp in mice of old age (18 mo). These data demonstrate that reductions in skeletal muscle AMPK and mitochondrial activity do not cause the development of age-induced insulin resistance.