Contraction-induced Glucose Uptake Research Articles

Exercise ameliorates skeletal muscle insulin resistance by modulating GRK4-mediated D1R expression.

Exercise has been recommended as a nonpharmaceutical therapy to treat insulin resistance (IR). Previous studies showed that dopamine D1-like receptor agonists, such as fenoldopam, could improve peripheral insulin sensitivity, while antipsychotics, which are dopamine receptor antagonists, increased susceptibility to Type 2 diabetes mellitus (T2DM). Meanwhile, exercise has been proved to stimulate dopamine receptors. However, whether the dopamine D1 receptor (D1R) is involved in exercise-mediated amelioration of IR remains unclear. We found that the D1-like receptor antagonist, SCH23390, reduced the effect of exercise on lowering blood glucose and insulin in insulin-resistant mice and inhibited the contraction-induced glucose uptake in C2C12 myotubes. Similarly, the opposite was true for the D1-like receptor agonist, fenoldopam. Furthermore, the expression of D1R was decreased in skeletal muscles from streptozotocin (STZ)- and high-fat intake-induced T2DM mice, accompanied by increased D1R phosphorylation, which was reversed by exercise. A screening study showed that G protein-coupled receptor kinase 4 (GRK4) may be the candidate kinase for the regulation of D1R function, because, in addition to the increased GRK4 expression in skeletal muscles of T2DM mice, GRK4 transgenic T2DM mice exhibited lower insulin sensitivity, accompanied by higher D1R phosphorylation than control mice, whereas the AAV9-shGRK4 mice were much more sensitive to insulin than AAV9-null mice. Mechanistically, the up-regulation of GRK4 expression caused by increased reactive oxygen species (ROS) in IR was ascribed to the enhanced expression of c-Myc, a transcriptional factor of GRK4. Taken together, the present study shows that exercise, via regulation of ROS/c-Myc/GRK4 pathway, ameliorates D1R dysfunction and improves insulin sensitivity.

AMPK activation is sufficient to increase skeletal muscle glucose uptake and glycogen synthesis but is not required for contraction-mediated increases in glucose metabolism

The AMP-activated protein kinase (AMPK) is a cellular sensor of energetics and when activated in skeletal muscle during contraction can impart changes in skeletal muscle metabolism. Therapeutics that selectively activate AMPK have been developed to lower glucose levels through increased glucose disposal rates as an approach to abrogate the hyperglycemic state of diabetes; however, the metabolic fate of glucose following AMPK activation remains unclear. We have used a combination of in vivo evaluation of glucose homeostasis and ex vivo skeletal muscle incubation to systematically evaluate metabolism following pharmacological activation of AMPK with PF-739, comparing this with AMPK activation through sustained intermittent electrical stimulation of contraction. These methods to activate AMPK result in increased glucose uptake but divergent metabolism of glucose: pharmacological activation results in increased glycogen accumulation while contraction-induced glucose uptake results in increased lactate formation and glucose oxidation. These results provide additional evidence to support a role for AMPK in control of skeletal muscle metabolism and additional insight into the potential for AMPK stimulation with small molecule direct activators.

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.

Endurance exercise training-responsive miR-19b-3p improves skeletal muscle glucose metabolism

Skeletal muscle is a highly adaptable tissue and remodels in response to exercise training. Using short RNA sequencing, we determine the miRNA profile of skeletal muscle from healthy male volunteers before and after a 14-day aerobic exercise training regime. Among the exercise training-responsive miRNAs identified, miR-19b-3p was selected for further validation. Overexpression of miR-19b-3p in human skeletal muscle cells increases insulin signaling, glucose uptake, and maximal oxygen consumption, recapitulating the adaptive response to aerobic exercise training. Overexpression of miR-19b-3p in mouse flexor digitorum brevis muscle enhances contraction-induced glucose uptake, indicating that miR-19b-3p exerts control on exercise training-induced adaptations in skeletal muscle. Potential targets of miR-19b-3p that are reduced after aerobic exercise training include KIF13A, MAPK6, RNF11, and VPS37A. Amongst these, RNF11 silencing potentiates glucose uptake in human skeletal muscle cells. Collectively, we identify miR-19b-3p as an aerobic exercise training-induced miRNA that regulates skeletal muscle glucose metabolism.

The effect of BPA exposure on insulin resistance and type 2 diabetes – The impact of muscle contraction

Type 2 diabetes (T2D) is considered one of the leading causes of death worldwide. In addition to physical inactivity and obesity, established risk factors for T2D, chemical contaminants consumed in industrialized food such as BPA might also be a contributor to the development of T2D. Epidemiological studies have shown that BPA concentrations are higher in human specimens of T2D when compared to healthy subjects, while experimental studies suggested that bisphenol A (BPA) impairs the pathway by which insulin stimulates glucose uptake. In skeletal muscle and adipocytes, insulin resistance is developed by the impairment of the insulin pathway to stimulate the translocation of glucose transporter, GLUT4, to the cell membrane. Recent results demonstrated that BPA impairs several components of insulin-induced glucose uptake pathway and affect the expression of GLUT4. Regular physical exercise delays or inhibits the development of T2D due to the physiologic processes taking place during muscle contraction, and the fact that skeletal muscle is the site for almost 80% of the glucose transported under insulin stimulation. In fact, the mechanism by which contraction induces glucose uptake in skeletal muscle is partially independent of the insulin pathway, therefore, the effect of BPA on this mechanism is unknown. We hypothesize that during the development of insulin resistance, BPA contributes to the impairment of the molecular pathway by which insulin induces glucose uptake while contraction-induced glucose uptake is not impaired. At the late stages of T2D, BPA may affect GLUT4 expression that will decrease the ability of muscle contraction to induce glucose uptake.

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

None for Perspective.

Cardiovascular and Muscular Consequences of Work-Matched Interval-Type of Concentric and Eccentric Pedaling Exercise on a Soft Robot.

Eccentric types of endurance exercise are an acknowledged alternative to conventional concentric types of exercise rehabilitation for the cardiac patient, because they reduce cardiorespiratory strain due to a lower metabolic cost of producing an equivalent mechanical output. The former contention has not been tested in a power- and work-matched situation of interval-type exercise under identical conditions because concentric and eccentric types of exercise pose specific demands on the exercise machinery, which are not fulfilled in current practice. Here we tested cardiovascular and muscular consequences of work-matched interval-type of leg exercise (target workload of 15 sets of 1-min bipedal cycles of knee extension and flexion at 30 rpm with 17% of maximal concentric power) on a soft robotic device in healthy subjects by concomitantly monitoring respiration, blood glucose and lactate, and power during exercise and recovery. We hypothesized that interval-type of eccentric exercise lowers strain on glucose-related aerobic metabolism compared to work-matched concentric exercise, and reduces cardiorespiratory strain to levels being acceptable for the cardiac patient. Eight physically active male subjects (24.0 years, 74.7 kg, 3.4 L O2 min−1), which power and endurance performance was extensively characterized, completed the study, finalizing 12 sets on average. Average performance was similar during concentric and eccentric exercise (p = 0.75) but lower than during constant load endurance exercise on a cycle ergometer at 75% of peak aerobic power output (126 vs. 188 Watt) that is recommended for improving endurance capacity. Peak oxygen uptake (−17%), peak ventilation (−23%), peak cardiac output (−16%), and blood lactate (−37%) during soft robotic exercise were lower during eccentric than concentric exercise. Glucose was 8% increased after eccentric exercise when peak RER was 12% lower than during concentric exercise. Muscle power and RFD were similarly reduced after eccentric and concentric exercise. The results highlight that the deployed interval-type of eccentric leg exercise reduces metabolic strain of the cardiovasculature and muscle compared to concentric exercise, to recommended levels for cardio-rehabilitation (i.e., 50–70% of peak heart rate). Increases in blood glucose concentration indicate that resistance to contraction-induced glucose uptake after the deployed eccentric protocol is unrelated to muscle fatigue.

The role of TBC1D1 and TBC1D4 in contraction-induced glucose uptake in mouse skeletal muscle

The role of TBC1D1 and TBC1D4 in contraction-induced glucose uptake in mouse skeletal muscle

Exercise ameliorates insulin resistance via Ca2+ signals distinct from those of insulin for GLUT4 translocation in skeletal muscles.

Muscle contraction and insulin induce glucose uptake in skeletal muscle through GLUT4 membrane translocation. Beneficial effects of exercise on glucose homeostasis in insulin-resistant individuals are known to be due to their distinct mechanism between contraction and insulin action on glucose uptake in skeletal muscle. However, the underlying mechanisms are not clear. Here we show that in skeletal muscle, distinct Ca(2+) second messengers regulate GLUT4 translocation by contraction and insulin treatment; d-myo-inositol 1,4,5-trisphosphate/nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose/NAADP are main players for insulin- and contraction-induced glucose uptake, respectively. Different patterns of phosphorylation of AMPK and Ca(2+)/calmodulin-dependent protein kinase II were shown in electrical stimuli (ES)- and insulin-induced glucose uptake pathways. ES-induced Ca(2+) signals and glucose uptake are dependent on glycolysis, which influences formation of NAD(P)-derived signaling messengers, whereas insulin-induced signals are not. High-fat diet (HFD) induced a defect in only insulin-mediated, but not ES-mediated, Ca(2+) signaling for glucose uptake, which is related to a specifically lower NAADP formation. Exercise decreases blood glucose levels in HFD-induced insulin resistance mice via NAADP formation. Thus we conclude that different usage of Ca(2+) signaling in contraction/insulin-stimulated glucose uptake in skeletal muscle may account for the mechanism by which exercise ameliorates glucose homeostasis in individuals with type 2 diabetes.

PS10 - 3. Contraction-induced glucose uptake requires dual signaling input: Consequence for muscular glucose utilization in diabetes

In the insulin resistant/diabetic skeletal and cardiac muscle glucose uptake is greatly impaired, and cell surface localization of the glucose transporter GLUT4 is diminished. Diabetic muscular dysfunction and cardiomyopathy may be averted by strategies that increase glucose uptake.

AMPK regulates contraction‐induced glucose uptake in situ but not ex vivo

The use of transgenic models studying the role of 5′AMP‐activated protein kinase (AMPK) in contraction‐induced glucose uptake (CTGU) has led to inconclusive results. Drawbacks of using less appropriate models displaying incomplete abrogation of AMPKα catalytic activity may explain these differences. The purpose of this study was to determine whether a total lack of both AMPK α1 and α2 catalytic subunits abolishes CTGU in muscle under ex vivo and in situ conditions.We generated a skeletal muscle specific AMPK‐α1 and‐α2 double knock‐out (dKO) mouse model. Electrical stimulation was applied directly to the muscle (ex vivo, 10 min (either 1 or 2 second trains/15 seconds)) or to the sciatic nerve (in situ, 15 min (1 train/second) +30 min recovery) to stimulate contraction. Muscle glucose uptake was measured using radioactively labeled isotopes. Ex vivo CTGU in AMPKα WT and dKO soleus and extensor digitorum longus (EDL) muscles was similar between genotypes. Equal force production was registered under all of the above conditions. Contrary to the ex vivo data, the CTGU in situ was impaired by ~50% in the tibialis anterior, soleus and EDL muscles of AMPKα dKO mice compared to WT mice (p<0.05). Basal non stimulated glucose uptake was similar between genotypes, both ex vivo and in situ.The results suggest that AMPK's role in CTGU may be modulated by a factor(s) related to muscle perfusion and/or stimulation methodology.

Rac1 Is a Novel Regulator of Contraction-Stimulated Glucose Uptake in Skeletal Muscle

In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (∼60–100%) and humans (∼40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20–58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.

ContRac1ion-Mediated Glucose Uptake: A Central Role for Rac1

Skeletal muscle glucose uptake can be regulated in response to different stimuli, with insulin and contraction being central. While the processes underlying insulin-induced glucose uptake have been extensively characterized, the processes underlying contraction-induced glucose uptake remain elusive. In this issue of Diabetes , Sylow et al. (1) provide another piece to this puzzle. They report that Rac1 is essential for contraction-induced GLUT4 translocation. Contraction induces glucose uptake in skeletal muscle both independently (2) and synergistically (3) with insulin. Several different mechanisms have been proposed, which appear to be activated in parallel following muscle contraction (Fig. 1). Key pathways include activation of the AMP-sensitive protein kinase (AMPK) through changes in the AMP:ATP ratio in response to increasing energy demands (4). Increased Ca2+/calmodulin-dependent protein kinase I (CaMKI) influx during muscle depolarization results in activation of calcium signaling cascades via CaMKI (5) and protein kinase C (PKC) (6). Downstream candidates include the Rab-GTPase-activating protein TBC1D1 (7), and evidence supports a role of increased free radical content in contraction-mediated glucose uptake …

Protein Kinase D1 Is Essential for Contraction-induced Glucose Uptake but Is Not Involved in Fatty Acid Uptake into Cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.

MARK4 is a Novel CaMKKα Substrate and Contraction‐Regulated Kinase in Mouse Skeletal Muscle

Ca2+/calmodulin‐dependent protein kinase kinase α (CaMKKα) regulates contraction‐induced glucose uptake in skeletal muscle, but the signaling pathway underlying this effect is not known. Microtubule affinity regulated kinases 1–4 (MARK1‐4) are homologous to known CaMKKα substrates, yet to date no studies have assessed MARKs as CaMKKα substrates. Our goal was to determine if MARKs are CaMKKα substrates in vitro and in skeletal muscle. Recombinant CaMKKα phosphorylated recombinant MARK4, but not MARK1, 2 or 3 in vitro. Expression of active CaMKKα in mouse tibialis anterior muscle increased MARK4 activity ~2‐fold, but did not affect MARK1 or MARK2/3 activities. In female, ICR mice (a standard outbred strain), 5 and 10 min of in situ contraction increased MARK4 activity 2‐ and 3‐fold, respectively, but did not affect MARK1 or MARK2/3 activities. In female, CaMKKα knockout mice, in situ contraction did not increase MARK4 activity. These results demonstrate that: 1] CaMKKα phosphorylates MARK4, but not MARK1, 2 or 3 in vitro; 2] Expression of active CaMKKα in muscle only stimulates MARK4 activity in skeletal muscle; 3] Contractile activity only increases MARK4 activity in skeletal muscle; and 4] CaMKKα is the main MARK4 kinase during muscle contraction. In conclusion, MARK4 is a newly identified CaMKKα substrate and contraction‐regulated kinase in mouse skeletal muscle.

CaMKII regulates contraction- but not insulin-induced glucose uptake in mouse skeletal muscle

Studies using chemical inhibitors have suggested that the Ca(2+)-sensitive serine/threonine kinase Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a key regulator of both insulin- and contraction-stimulated glucose uptake in skeletal muscle. However, due to nonspecificity of these inhibitors, the specific role that CaMKII may play in the regulation of glucose uptake is not known. We sought to determine whether specific inhibition of CaMKII impairs insulin- and/or contraction-induced glucose uptake in mouse skeletal muscle. Expression vectors containing green fluorescent protein conjugated to a CaMKII inhibitory (KKALHRQEAVDCL) or control (KKALHAQERVDCL) peptide were transfected into tibialis anterior muscles by in vivo electroporation. After 1 wk, muscles were assessed for peptide expression, CaMK activity, insulin- and contraction-induced 2-[(3)H]deoxyglucose uptake, glycogen concentrations, and changes in intracellular signaling proteins. Expression of the CaMKII inhibitory peptide decreased muscle CaMK activity approximately 35% compared with control peptide. Insulin-induced glucose uptake was not changed in muscles expressing the inhibitory peptide. In contrast, expression of the inhibitory peptide significantly decreased contraction-induced muscle glucose uptake (approximately 30%). Contraction-induced decreases in muscle glycogen were not altered by the inhibitory peptide. The CaMKII inhibitory peptide did not alter expression of the glucose transporter GLUT4 and did not impair contraction-induced increases in the phosphorylation of AMP-activated protein kinase (Thr(172)) or TBC1D1/TBC1D4 on phospho-Akt substrate sites. These results demonstrate that CaMKII does not regulate insulin-stimulated glucose uptake in skeletal muscle. However, CaMKII plays a critical role in the regulation of contraction-induced glucose uptake in mouse skeletal muscle.

Knockout of the predominant conventional PKC isoform, PKCα, in mouse skeletal muscle does not affect contraction-stimulated glucose uptake

Conventional (c) protein kinase C (PKC) activity has been shown to increase with skeletal muscle contraction, and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC activity is required for contraction-stimulated glucose uptake in mouse muscles, contraction-stimulated glucose uptake ex vivo was first evaluated in the presence of three commonly used cPKC inhibitors (calphostin C, Gö-6976, and Gö-6983) in incubated mouse soleus and extensor digitorum longus (EDL) muscles. All potently inhibited contraction-stimulated glucose uptake by 50-100%, whereas both Gö compounds, but not calphostin C, inhibited insulin-stimulated glucose uptake modestly. AMP-activated protein kinase (AMPK) and eukaryotic elongation factor 2 phosphorylation was unaffected by the blockers. PKCalpha was estimated to account for approximately 97% of total cPKC protein expression in skeletal muscle. However, in muscles from PKCalpha knockout (KO) mice, neither contraction- nor phorbol ester-stimulated glucose uptake ex vivo differed compared with the wild type. Furthermore, the effects of calphostin C and Gö-6983 on contraction-induced glucose uptake were similar in muscles lacking PKCalpha and in the wild type. It can be concluded that PKCalpha, representing approximately 97% of cPKC in skeletal muscle, is not required for contraction-stimulated glucose uptake. Thus the effect of the PKC blockers on glucose uptake is either nonspecific working on other parts of contraction-induced signaling or the remaining cPKC isoforms are sufficient for stimulating glucose uptake during contractions.

Hunting for the SNARK in metabolic disease

dysregulation of energy balance is a primary constituent in the etiology of obesity and type 2 diabetes mellitus, which is manifested by altered metabolic homeostasis and insulin resistance in a variety of tissues, including brain, liver, and skeletal muscle. The discovery of the AMP-activated

Skeletal Muscle Pathways of Contraction-Enhanced Glucose Uptake

Muscle contraction acutely increases glucose transport in both healthy and type 2 diabetic individuals. Since glucose uptake during muscle contraction has been observed in the absence of insulin, the existence of an insulin-independent pathway has been suggested to explain this phenomenon. However, the exact mechanism behind the translocation of GLUT4 vesicles through the sarcolemma during muscle contraction is still unknown. Some substances, such as AMPK and calcium activated proteins, have been suggested as potential mediators but the exact mechanisms of their involvement remain to be elucidated. A hypothetical convergence point between the insulin cascade and the potential pathways triggered by muscle contraction has been suggested. Therefore, the earliest concept that two different routes exist in skeletal muscle has been progressively modified to the notion that glucose uptake is induced by muscle contraction via components of the insulin pathway. With further consideration, increased glucose uptake and enhanced insulin sensitivity observed during/after exercise might be explained by a metabolic- and calcium-dependent activation of several intermediate molecules of the insulin cascade. This paper aimed to review the literature in order to examine in detail these concepts behind muscle contraction-induced glucose uptake.

Identification of protein kinase D as a novel contraction-activated kinase linked to GLUT4-mediated glucose uptake, independent of AMPK

Contraction-induced glucose uptake is only partly mediated by AMPK activation. We examined whether the diacylglycerol-sensitive protein kinase D (PKD; also known as novel PKC isoform μ) is also involved in the regulation of glucose uptake in the contracting heart. As an experimental model, we used suspensions of cardiac myocytes, which were electrically stimulated to contract or treated with the contraction-mimicking agent oligomycin. Induction of contraction at 4 Hz in cardiac myocytes or treatment with 1 μM oligomycin enhanced (i) autophosphorylation of PKD at Ser916 by 5.1- and 3.8-fold, respectively, (ii) phosphorylation of PKD's downstream target cardiac-troponin-I (cTnI) by 2.9- and 2.1-fold, respectively, and (iii) enzymatic activity of immunoprecipitated PKD towards the substrate peptide syntide-2 each by 1.5-fold. Although AMPK was also activated under these same conditions, in vitro phosphorylation assays and studies with cardiac myocytes from AMPKα2 −/− mice indicated that activation of PKD occurs independent of AMPK activation. CaMKKβ, and the cardiac-specific PKC isoforms α, δ, and ε were excluded as upstream kinases for PKD in contraction signaling because none of these kinases were activated by oligomycin. Stimulation of glucose uptake and induction of GLUT4 translocation in cardiac myocytes by contraction and oligomycin each were sensitive to inhibition by the PKC/PKD inhibitors staurosporin and calphostin-C. Together, these data elude to a role of PKD in contraction-induced GLUT4 translocation. Finally, the combined actions of PKD on cTnI phosphorylation and on GLUT4 translocation would efficiently link accelerated contraction mechanics to increased energy production when the heart is forced to increase its contractile activity.