Skeletal Muscle Reprogramming by Activation of Calcineurin Improves Insulin Action on Metabolic Pathways*

The protein phosphatase calcineurin is a signaling intermediate that induces the transformation of fast-twitch skeletal muscle fibers to a slow-twitch phenotype. This reprogramming of the skeletal muscle gene expression profile may have therapeutic applications for metabolic disease. Insulin-stimulated glucose uptake in skeletal muscle is both impaired in individuals with type II diabetes mellitus and positively correlated with the percentage of slow-versus fast-twitch muscle fibers. Using transgenic mice expressing activated calcineurin in skeletal muscle, we report that skeletal muscle reprogramming by calcineurin activation leads to improved insulin-stimulated 2-deoxyglucose uptake in extensor digitorum longus (EDL) muscles compared with wild-type mice, concomitant with increased protein expression of the insulin receptor, Akt, glucose transporter 4, and peroxisome proliferator-activated receptor-γ co-activator 1. Transgenic mice exhibited elevated glycogen deposition, enhanced amino acid uptake, and increased fatty acid oxidation in EDL muscle. When fed a high-fat diet, transgenic mice maintained superior rates of insulin-stimulated glucose uptake in EDL muscle and were protected against diet-induced glucose intolerance. These results validate calcineurin as a target for enhancing insulin action in skeletal muscle.

Skeletal muscle fibers can be separated into two major classifications: type I slow-twitch and type II fast-twitch fibers (1). The knowledge that type I and type II muscle fibers exhibit distinctively different contractile and metabolic properties has been accepted for decades (1,2). However, the intracellular signaling mechanisms controlling skeletal muscle fiber type have not been clearly elucidated. The serine/threonine protein phosphatase calcineurin has been recognized as a major determinate of skeletal muscle fiber type. Early evidence for the involvement of calcineurin in skeletal muscle fiber type selection comes from studies implementing the use of the immunosuppressant drug cyclosporin A, a potent inhibitor of calcineurin activity. Cyclosporin A blockade of calcineurin activity promotes the expression of fast-twitch fibers in rat soleus mus-cle (3). Conversely, expression of a constitutively active form of calcineurin in cultured muscle cells induces the expression of reporter genes linked to promoters of slow-twitch muscle-specific genes such as myoglobin and slow troponin I (3). Based on these observations, activation of calcineurin was proposed to be involved in the induction of slow muscle genes and the regulation of skeletal muscle fiber type. Consistent with this hypothesis, transgenic expression of a constitutively active form of calcineurin in skeletal muscle increases the percentage of slowtwitch muscle fibers and promotes the expression of myoglobin and slow troponin I (4).
The identification of calcineurin as a regulator of skeletal muscle fiber type may have clinical implications for improving insulin action in individuals with type II diabetes mellitus. A major contributing factor to the progressive development of type II diabetes is reduced insulin-stimulated whole-body glucose disposal, with the greatest defects attributed to skeletal muscle (5). Skeletal muscle insulin resistance in individuals with type II diabetes is associated with impaired insulin signal transduction (6,7) and defects in glucose transporter 4 (GLUT4) 1 trafficking (8). Therefore, strategies to improve insulin signal transduction or enhance insulin-stimulated mobilization of GLUT4 vesicles to the cell surface are likely to correct glucose transport defects and lead to improved glucose homeostasis in diabetic individuals. One means of achieving improved glucose transport in skeletal muscle may be through activation of the calcineurin signaling pathway. Activation of calcineurin may promote metabolic adaptations within muscle cells, resulting in improved insulin-stimulated glucose transport. This hypothesis is supported in part by the observation that insulin action on glucose transport is greater in rat skeletal muscles that are highly enriched in slow-versus fasttwitch muscle fibers (9). Fiber type specificity of insulin-stimulated glucose transport appears to be due to increased expression and/or function of proteins involved in the insulinsignaling cascade (10), as well as a greater abundance of GLUT4 (9,11). Furthermore, insulin-stimulated glucose transport is positively correlated with the percentage of slow-twitch muscle fibers in human skeletal muscle (12). Thus, activation of calcineurin in skeletal muscle represents a novel strategy to improve insulin-stimulated glucose transport and may counterbalance insulin resistance in type II diabetes. We utilized transgenic mice expressing an activated form of calcineurin in skeletal muscle (MCK-CnA* mice) to determine whether skel-etal muscle reprogramming via activation of calcineurin improves insulin-stimulated glucose transport and prevents the development of diet-induced insulin resistance.

EXPERIMENTAL PROCEDURES
Transgenic Mice-A colony of transgenic mice expressing a constitutively active form of calcineurin (13) under control of the muscle creatine kinase promoter/enhancer was established at the Department of Physiology and Pharmacology, Karolinska Institutet, using MCK-CnA* mice that were originally developed at the Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas (4). Mice were bred on a C57/BL6JBom background. Wild-type littermates served as controls in all experiments. Mice were maintained on a 12-h light-dark cycle and allowed free access to food and water. Food was removed from the cages of 12-14-week-old female mice 4 h prior to study unless indicated otherwise. When examining effects of a high-fat diet, female mice were placed on either a standard chow or a high-fat diet (14) from 4 to 20 weeks of age. The Ethics Committee on Animal Research in Northern Stockholm approved all experimental protocols.
Muscle Incubations-Incubation media were prepared from pregassed (95% O 2 , 5% CO 2 ) stocks of Krebs-Henseleit buffer (KHB) supplemented with 5 mM HEPES and 0.1% bovine serum albumin (radioimmunoassay grade). Mice were anesthetized via an intraperitoneal injection of 2.5% avertin (0.02 ml/g body weight). Extensor digitorum longus (EDL) and soleus muscles were excised and incubated in 1 ml of KHB supplemented with 2 mM pyruvate and 18 mM mannitol. Incubations were performed at 30°C in a shaking water bath under a constant gas phase (95% O 2 , 5% CO 2 ) unless stated otherwise. For 2-deoxyglucose uptake and ␣-aminomethylisobutyrate (MeAIB) uptake experiments, muscles were preincubated for 40 min in the absence or presence of 12 nM insulin. For insulin signaling studies, muscles were incubated in KHB containing 2 mM pyruvate for a total of 60 min. Insulin (120 nM) was added for the final 5 min (for insulin receptor tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity) or 40 min (for Akt or glycogen synthase kinase 3 (GSK3) phosphorylation).
Glucose Transport Activity-Following preincubation, muscles were transferred to vials containing 1 mM 2-deoxy-D-[1,2-3 H]glucose (2.5 Ci/ml) and 19 mM [U-14 C]mannitol (0.7 Ci/ml). Insulin was added at concentrations identical to preincubation conditions. 2-Deoxyglucose uptake was assessed for 20 min. After incubation, muscles were digested in 0.5 M NaOH. Sample aliquots were used for protein determination using a commercially available kit (Coomassie Plus, Pierce). Extracellular space and intracellular 2-deoxyglucose concentrations were determined by liquid scintillation counting. Glucose transport activity was expressed as nmol of 2-deoxyglucose/mg of protein/20 min.
Amino Acid Transport Activity-Following preincubation, muscles were transferred to vials containing 0.1 mM [ 14 C]MeAIB (0.4 Ci/ml) and 19.9 mM D-[2-3 H]mannitol (2 Ci/ml) without or with insulin at concentrations identical to preincubation conditions. MeAIB uptake was assessed for 20 min. Muscles were processed as described above for 2-deoxyglucose uptake. Amino acid transport activity was expressed as pmol of MeAIB/mg of protein/20 min.
Oleate Oxidation-Muscles were trimmed of excess tendon and weighed. Muscles were preincubated for 40 min in KHB containing 5 mM HEPES, 3.5% fatty acid-free bovine serum albumin, and 5 mM glucose. Thereafter, muscles were transferred to vials containing 1 ml of identical media with the addition of 0.3 mM [1-14 C]oleate (0.2 Ci/ml) and incubated for 60 min. Vials were sealed with a rubber stopper, which was fitted with a center well. Muscles were oxygenated for the first 15 min of the incubation period via a needle inserted through the stopper, after which time the oxygen needle was removed to close the system. After 60 min, 0.2 ml of Protosol (PerkinElmer Life Sciences) was injected through the rubber stopper into the center well, and the medium was acidified by injection of 0.5 ml of 15% perchloric acid. Liberated CO 2 was collected for 60 min, and center wells were transferred to vials for liquid scintillation counting. Results were expressed as pmol of oxidized oleate/mg of wet weight/h.
Insulin Receptor Tyrosine Phosphorylation-Muscles were homogenized as described above for Western blot analysis. Muscle homogenate (500 g) was immunoprecipitated overnight at 4°C using a polyclonal antiphosphotyrosine antibody (BD Transduction Laboratories, Lexington, KY) coupled to protein A-Sepharose. Immunoprecipitates were washed (16), suspended in Laemmli sample buffer, and heated at 95°C for 5 min. Proteins were separated by SDS-PAGE, and Western blot analysis was performed using a monoclonal antiphosphotyrosine antibody (BD Transduction Laboratories).
PI 3-Kinase Activity-Muscles were homogenized as described for Western blot analysis. Muscle homogenate (500 g) was immunoprecipitated overnight at 4°C using a polyclonal IRS1 antibody coupled to protein A-Sepharose. Washing of immunoprecipitates and kinase reactions were performed as described (16). Results were quantified using a PhosphorImager.
Glycogen Analysis-Muscles were weighed, and glycogen content was determined fluorometrically on HCl extracts as described previously (17).
Glucose Tolerance and Blood Chemistry-Glucose (2 g/kg body weight) was administered by intraperitoneal injection. Blood samples were obtained via the tail vein prior to and 15, 30, 60, 90, and 120 min following glucose injection. Blood glucose levels were measured using a One Touch Profile glucose meter (Lifescan, Milpitas, CA). Lactate was determined using an Accusport lactate analyzer (Roche Diagnostics, Basel, Switzerland). Free fatty acid measurements were determined spectophotometrically using a commercially available kit (Wako Chemicals GmbH, Neuss, Germany). Plasma insulin was determined by enzyme-linked immunosorbent assay (Crystal Chem Inc., Downers Grove, IL).
Statistical Analysis-Data are reported as means Ϯ S.E. Differences were determined by Student's t test. Significance was accepted at p Ͻ 0.05.

RESULTS
Calcineurin Protein Expression-Protein expression of endogenous calcineurin and the truncated constitutively active transgene product was assessed in EDL and soleus muscles of wild-type and MCK-CnA* mice. Endogenous levels of calcineurin protein were reduced in both muscle types in transgenic mice (Fig. 1, Table I); levels were greater in EDL compared with soleus muscle in both wild-type and MCK-CnA* mice. Constitutively active calcineurin was 4.5-fold greater in EDL compared with soleus muscle from MCK-CnA* mice. Constitutively active calcineurin accounted for ϳ34 and 21% of the total calcineurin pool (endogenous plus transgenic) in MCK-CnA* EDL and soleus muscle, respectively.
Alterations in Metabolism of MCK-CnA* Mice-In wild-type mice, insulin increased 2-deoxyglucose uptake 2.9-fold in EDL muscle and 4.2-fold in soleus muscle ( Fig. 2A). In EDL muscle from MCK-CnA* mice, basal and insulin-stimulated glucose uptake was increased 36 and 60%, respectively, compared with wild-type EDL muscle ( Fig. 2A). Glucose transport rates in soleus muscle were not significantly different between MCK-CnA* and wild-type mice.
System A amino acid uptake was determined using the nonmetabolizable amino acid analog MeAIB. Insulin increased amino acid uptake 2-fold in EDL muscle from wild-type mice. In wild-type mice, basal and insulin-stimulated MeAIB uptake was 2-and 1.8-fold greater, respectively, in soleus muscle com-pared with EDL muscle (Fig. 2B). As observed for 2-deoxyglucose uptake, amino acid uptake was enhanced in EDL muscle from MCK-CnA* compared with wild-type mice. Basal and insulin-stimulated MeAIB uptake was 2-and 1.8-fold greater under basal and insulin-stimulated conditions, respectively, in EDL muscle from MCK-CnA* compared with wild-type mice (Fig. 2B).
Oleate oxidation was determined in skeletal muscle from wild-type and MCK-CnA* mice. In wild-type mice, oleate oxi-dation was elevated 69% in soleus compared with EDL muscle (Fig. 2C). In MCK-CnA* mice, activation of calcineurin in EDL muscle resulted in a 63% increase in fatty oxidation compared with wild-type mice (Fig. 2C). No further increase was observed in soleus mice from MCK-CnA* mice.
Protein Expression of Molecules Involved in the Regulation of Insulin Action-Protein expression of insulin receptor, IRS1, Akt, GLUT4, and PGC-1 was determined in EDL and soleus muscles from wild-type and MCK-CnA* mice. The abundance of each of these proteins in wild-type mice was 2-5-fold greater in soleus than in EDL muscle (Fig. 3, Table I). In EDL muscle from MCK-CnA* mice, expression of insulin receptor, Akt, GLUT4, and PGC-1 was up-regulated 2-3-fold compared with wild-type mice (Fig. 3, Table I). Protein expression of these molecules in soleus muscle was not different between wild-type and MCK-CnA* mice. IRS1 protein expression was not significantly altered by transgenic activation of calcineurin.
Insulin Signal Transduction-Insulin markedly increased insulin receptor ␤-subunit tyrosine autophosphorylation to an equal extent in EDL and soleus muscle from wild-type mice (Fig. 4A, Table I). Insulin receptor phosphorylation was not  Table I.  2. Muscle metabolism is altered in MCK-CnA* mice. A, EDL or soleus muscles from wild-type or MCK-CnA* mice were incubated in the absence (open bars) or presence (closed bars) of insulin, and 2-deoxyglucose uptake was assessed. Data are means Ϯ S.E. for n ϭ 11-12 muscles. *, p Ͻ 0.05, **, p Ͻ 0.0001 compared with wild-type. B, EDL or soleus muscles from wild-type or MCK-CnA* mice were incubated in the absence (open bars) or presence (closed bars) of insulin, and MeAIB uptake was assessed. Data are means Ϯ S.E. for n ϭ 3-4 muscles. **, p Ͻ 0.0005 compared with wild-type. C, oleate oxidation in EDL and soleus muscle from wild-type or MCK-CnA* mice. Data are means Ϯ S.E. for n ϭ 5-6 muscles. **, p Ͻ 0.0001 compared with wild type. different in skeletal muscle from MCK-CnA* compared with wild-type mice. Similar to the pattern observed for insulin receptor phosphorylation, insulin led to a robust stimulation of IRS1-associated PI 3-kinase activity, with comparable effects observed in both muscle types of each genotype (Fig. 4B). Insulin-stimulated Akt phosphorylation in skeletal muscle of wild-type mice was not different between EDL and soleus muscles (Fig. 4C). However, insulin-stimulated Akt phosphorylation in EDL muscle from MCK-CnA* mice was increased 2-fold compared with wild-type mice (Fig. 4C).
Blood Chemistry-Blood glucose, blood lactate, non-esterified free fatty acid levels, and plasma insulin were determined in wild-type and MCK-CnA* mice (Table II). No significant differences were found in any of these parameters.
Elevated Skeletal Muscle Glycogen Content in MCK-CnA* Mice-Muscle glycogen content was increased 125 and 64% in EDL and soleus muscle from MCK-CnA* mice, respectively, compared with wild-type mice (Fig. 5A). Activation of the calcineurin transgene induced a 60% increase in glycogen synthase protein expression in EDL muscle from MCK-CnA* compared with wild-type mice (Fig. 5B, Table I). Expression of glycogen synthase was not increased in soleus muscle from MCK-CnA* mice. Insulin-stimulated phosphorylation of GSK3 ␣/␤ was markedly increased in skeletal muscle from wild-type and MCK-CnA* mice, with similar responses observed between genotypes (Fig. 5C, Table I).
MCK-CnA* Mice Are Protected against Deleterious Effects of a High-fat Diet-2-Deoxyglucose uptake was assessed in skeletal muscle from wild-type and MCK-CnA* mice maintained on either a chow or high-fat diet from 4 to 20 weeks of age. Insulin-stimulated glucose uptake was 72% greater in EDL muscle from chow-fed MCK-CnA* compared with wild-type mice (Fig. 6A). High-fat feeding for 16 weeks resulted in a 21% reduction in insulin-stimulated glucose uptake in EDL muscle from both wild-type and MCK-CnA* mice (Fig. 6A). However, the rate of glucose uptake in high-fat-fed MCK-CnA* mice was 70% higher than that observed in high-fat-fed wild-type mice. Fat feeding did not alter the expression of endogenous or transgenic calcineurin in quadriceps muscle (data not shown).
We determined the effect of a high-fat diet on glucose toler-ance in wild-type and MCK-CnA* mice. Wild-type mice developed glucose intolerance when maintained on a high-fat diet (Fig. 6B). Blood glucose levels in high-fat-fed wild-type mice were significantly elevated compared with chow-fed wild-type mice at all measured time points between 30 and 120 min. In contrast, MCK-CnA* mice were protected against the development of diet-induced glucose intolerance. DISCUSSION We hypothesized that activation of calcineurin in transgenic mice could alter the gene expression program of fast-twitch skeletal muscle, thereby resulting in a slow-twitch muscle phenotype with enhanced insulin-stimulated glucose transport. We studied EDL and soleus muscles because they exhibit distinctively different contractile and metabolic properties. EDL muscles from wild-type mice are predominantly composed of fast-twitch fibers and contain as little as 2% type I (slowtwitch) fibers, whereas soleus muscles contain ϳ50% type I fibers, with the balance accounted for by type IIa/b (fast-twitch) muscle fibers in each case (18). MCK-CnA* mice have been  Table I. B, IRS1-associated PI 3-kinase activity in wild-type or MCK-CnA* EDL or soleus muscles incubated in the absence (open bars) or presence (closed bars) of insulin. Data are means Ϯ S.E. for n ϭ 4 muscles. No significant differences were observed. C, EDL or soleus muscles from wild-type or MCK-CnA* mice were incubated in the absence (open bars) or presence (closed bars) of insulin, and Akt phosphorylation was determined by Western blot analysis. Values are means Ϯ S.E. for n ϭ 6 muscles. A representative image is shown (inset). *, p Ͻ 0.005 compared with wild type. utilized to demonstrate that activation of calcineurin promotes fast-to-slow-twitch muscle fiber type transformation (4). Here we provide direct evidence that muscle fiber type transformation is associated with a shift in the metabolic parameters to a more insulin-responsive phenotype. Insulin-stimulated glucose uptake in EDL muscle from MCK-CnA* mice was increased 60% compared with wild-type mice, concomitant with increased expression of proteins involved in the regulation of glucose transport. In addition, amino acid transport and fatty acid oxidation were increased in fast-twitch muscle from MCK-CnA* mice to levels characteristic of slow-twitch soleus muscle. Thus, multiple fiber type-specific physiological processes are under the control of calcineurin. Importantly, activated calcineurin protected against the development of diet-induced glucose intolerance. Our results provide evidence to suggest that calcineurin is a plausible target for improving insulin action in skeletal muscle.
We examined whether changes in insulin signal transduction contribute to the increased insulin responsiveness to glucose transport in EDL muscle from MCK-CnA* mice. Although insulin receptor protein expression was increased in EDL muscle from MCK-CnA* mice, insulin-stimulated receptor autophosphorylation was unchanged. Furthermore, insulin signal transduction at the level of PI 3-kinase activity, a key component of the insulin-signaling cascade (19), was not altered in skeletal muscle from MCK-CnA* mice. Although components of the signaling cascade between PI 3-kinase and GLUT4 translocation have not been fully elucidated, the serine/threonine A, intramuscular glycogen levels in EDL or soleus muscle from wild-type or MCK-CnA* mice. Data are means Ϯ S.E. for n ϭ 6 -7 mice. *, p Ͻ 0.05, **, p Ͻ 0.0001 compared with wild-type mice. B, glycogen synthase (GS) protein expression in wild-type and MCK-CnA* EDL and soleus muscle as determined by Western blot analysis. Quantification of results is presented in Table I. C, GSK3␣/␤ phosphorylation. EDL or soleus muscles from wild-type or MCK-CnA* mice were incubated in the absence or presence of insulin, and GSK3␣/␤ phosphorylation was determined by Western blot analysis. No significant differences were observed. Quantification of results is presented in Table I. protein kinase Akt is a common step in several PI 3-kinase-dependent signaling pathways that regulate metabolism and cell growth. Nevertheless, the direct role of Akt in the regulation of glucose transport is a matter of debate (20 -22). Overexpression of activated calcineurin was associated with a 2-fold increase in Akt protein expression and insulin-stimulated phosphorylation in EDL muscle. This finding is consistent with studies in cardiomyocytes, whereby calcineurin signaling has been proposed to activate the hypertrophic program, which may then indirectly lead to Akt activation (23). Although the functional significance of the elevation in Akt protein expression and insulinstimulated phosphorylation in EDL muscle from MCK-CnA* mice as related to glucose transport is not clear, collectively our data provide evidence that adaptive mechanisms independent of changes in early components of the insulin signal transduction pathway are involved in improving glucose transport in response to calcineurin activation.
PGC-1 has been implicated as a regulator of muscle fiber type in skeletal muscle of transgenic mice (24). PGC-1-mediated regulation of skeletal muscle fiber type has been proposed to occur via a mechanism downstream of calcium-sensitive signaling intermediates such as calcineurin and/or the calcium/ calmodulin-dependent protein kinase (CaMK) (24). We observed a fiber type-specific difference in protein expression of PGC-1. Protein expression of PGC-1 was increased in slowtwitch soleus muscle from wild-type mice and up-regulated in EDL muscle from MCK-CnA* mice. Thus, calcineurin is directly implicated in the muscle fiber type-specific regulation of PGC-1. Our results and those of others (24,25) support the hypothesis that a calcineurin/PGC-1 pathway regulates, at least in part, muscle fiber type-specific control of insulin-stimulated glucose transport. Adenoviral expression of PGC-1 in L6 myotubes is sufficient to induce a marked increase in GLUT4 protein expression (25), thus leading to elevated basal and insulin-stimulated glucose transport. Consistent with this, we show that activation of calcineurin in fast-twitch EDL muscle yields an adaptive increase in GLUT4 expression, which may solely explain improvements in insulin-stimulated glucose transport. However, protein expression levels of GLUT4 in the soleus muscle from wild-type mice were considerably higher than those observed in EDL muscle from MCK-CnA* mice, indicating that additional mechanisms cooperate with calcineurin to regulate fiber type-specific expression of GLUT4. A likely candidate would be one or more of the known CaMK isoforms. Transgenic activation of CaMKIV in skeletal muscle is sufficient to promote muscle fiber type transformation and up-regulation of slow muscle genes (26). Calcineurin and CaMKIV synergistically induce MEF2 (myocyte enhancer factor 2) transcriptional activity in C2C12 muscle cells (27). Interestingly, deletion of the MEF2 binding domain of the GLUT4 promoter abolishes expression of a reporter gene in skeletal muscles of transgenic mice (28). Therefore, increased activation of calcineurin and CaMK may be required to achieve the levels of GLUT4 that are observed in slow-twitch skeletal muscle.
Once transported inside the muscle cell, glucose is primarily stored as glycogen. Both the rate of glucose transport (29) and the level of glycogen synthase expression (30) strongly influence skeletal muscle glycogen storage. Because both glucose transport and glycogen synthase protein expression are increased in EDL muscle from MCK-CnA* mice, the increase in glycogen content is not surprising. Nevertheless, activation of calcineurin in EDL muscle did not lead to a slow-twitch phenotype in terms of muscle glycogen storage because intramuscular glycogen was found to be lower in soleus compared with EDL muscle from wild-type mice. Activation of calcineurin in skeletal muscle leads to parallel adaptations induced by contractile activity (31), presenting the possibility that constitutively active calcineurin recapitulates the consequences of exercise training to enhance glycogen storage in a manner that is not evident even in type I muscle of sedentary animals.
To validate calcineurin as a therapeutic target for the treatment of skeletal muscle insulin resistance, we determined whether calcineurin-induced changes in glucose metabolism could protect against insulin resistance commonly associated with high-fat feeding in rodent models (14,32). High-fat feeding reduced insulin-stimulated glucose uptake in EDL muscle from both wild-type and MCK-CnA* mice. However, glucose uptake in high-fat-fed MCK-CnA* mice was 70% higher than that observed in high-fat-fed wild-type mice and 35% higher than for chow-fed wild-type mice, indicating that activated calcineurin preserves insulin action. Skeletal muscle insulin resistance in the high-fat-fed wild-type mice was associated with impaired glucose tolerance. In contrast, high-fat-fed MCK-CnA* mice were protected against the development of glucose intolerance. These data demonstrate that enhanced insulin-stimulated glucose uptake driven by targeted activation of calcineurin in skeletal muscle can protect against the disturbances in whole-body glucose disposal that are commonly associated with skeletal muscle insulin resistance.
In conclusion, activation of the calcineurin pathway drives adaptive responses to enhance insulin action in skeletal muscle. Activated calcineurin leads to the induction of multiple genes involved in the regulation of glucose transport in skeletal muscle. These changes are associated with increased glucose uptake and glycogen deposition in skeletal muscle. Activation of calcineurin also results in increased amino acid uptake and fatty acid oxidation. These metabolic adaptations are coincident with an up-regulation in protein expression of PGC-1. Importantly, MCK-CnA* mice fed a high-fat diet maintain superior rates of insulin-stimulated glucose uptake in EDL muscle and are protected against the development of dietinduced glucose intolerance. Future strategies aimed at activating the calcineurin pathway in skeletal muscle may prove fruitful in the treatment of skeletal muscle insulin resistance in type II diabetes mellitus.