Receptors In Skeletal Muscle Research Articles

Distinct role of insulin and IGF-I and its receptors in white skeletal muscle during the compensatory growth of gilthead sea bream ( Sparus aurata)

In order to examine compensatory growth (CG) in gilthead sea bream ( Sparus aurata), we used an experimental protocol involving 1-, 3- or 4-week fasting followed by a one- or two-month re-feeding period. After one month of re-feeding, final body weight gain and growth acceleration in groups fasted for 1 or 3 weeks (W1 and W3) were comparable to the control group values (C), indicating that partial CG was achieved. Fasting had a marked effect on depressing circulating glucose plasma levels, which were recovered to C group values after one month re-feeding. Glycogen content in liver and muscle decreased during fasting and was completely recovered after two month re-feeding. FFA plasma levels increased during fasting trial for all the different experimental groups, whereas lipid content in liver and muscle was depressed, indicating that lipid depot utilization contributes to the energy metabolism of gilthead sea bream. After re-feeding for 1 or 2 months, FFA and lipid content in liver and muscle were comparable to C group values. Plasma insulin and IGF-I values decreased during the fasting trial, while normal levels were restored after 1 month of re-feeding IGF-I and insulin binding assays in semi-purifications of soluble proteins from white skeletal muscle showed that IGF-I binding increased with fasting while insulin binding decreased. However, the specific binding for these two peptides was comparable to C group values after re-feeding for 1 or 2 months. In conclusion, after re-feeding insulin and IGF-I recovery was parallel with increases in weight gain and growth acceleration in those groups which showed diminutions in growth during the period of food restriction. Furthermore, the differential regulation of the specific binding for insulin and IGF-I in white skeletal muscle during fasting, indicates that these two receptors play distinct roles in response to the nutritional status of the animal.

Phenotypical spectrum of DOK7 mutations in congenital myasthenic syndromes

Dok ('downstream-of-kinase') family of cytoplasmic proteins play a role in signalling downstream of receptor and non-receptor phosphotyrosine kinases. Recently, a skeletal muscle receptor tyrosine kinase (MuSK)-interacting cytoplasmic protein termed Dok-7 has been identified. Subsequently, we and others identified mutations in DOK7 as a cause of congenital myasthenic syndromes (CMS), providing evidence for a crucial role of Dok-7 in maintaining synaptic structure. Here we present clinical and molecular genetic data of 14 patients from 12 independent kinships with 13 different mutations in the DOK7 gene. The clinical picture of CMS with DOK7 mutations is highly variable. The age of onset may vary between birth and the third decade. However, most of the patients display a characteristic 'limb-girdle' pattern of weakness with a waddling gait and ptosis, but without ophthalmoparesis. Respiratory problems were frequent. Patients did not benefit from long-term therapy with esterase inhibitors; some of the patients even worsened. DOK7 mutations have emerged as one of the major genetic defects in CMS. The clinical picture differs significantly from CMS caused by mutations in other genes, such as the acetylcholine receptor (AChR) subunit genes. None of the patients with DOK7 mutations had tubular aggregates in the muscle biopsy, implying that 'limb-girdle myasthenia (LGM) with tubular aggregates' previously described in literature may be a pathogenic entity distinct from CMS caused by DOK7 mutations.

Essential Role of Insulin and Insulin-Like Growth Factor 1 Receptor Signaling in Cardiac Development and Function

Cardiovascular disease is the leading cause of death in people with type 2 diabetes and is linked to insulin resistance even in the absence of diabetes. Here we show that mice with combined deficiency of the insulin receptor and insulin-like growth factor 1 (IGF-1) receptor in cardiac and skeletal muscle develop early-onset dilated cardiomyopathy and die from heart failure within the first month of life despite having a normal glucose homeostasis. Mice lacking the insulin receptor show impaired cardiac performance at 6 months, and mice lacking the insulin receptor plus one Igf1r allele have slightly increased mortality. By contrast, mice lacking the IGF-1 receptor or the IGF-1 receptor plus one Ir allele appear normal. Morphological characterization and oligonucleotide array analysis of gene expression demonstrate that prior to development of these physiological defects, mice with combined deficiency of both insulin and IGF-1 receptors have a coordinated down-regulation of genes encoding components of the electron transport chain and mitochondrial fatty acid beta-oxidation pathways and altered expression of contractile proteins. Thus, while neither the insulin receptor nor IGF-1 receptor in muscle is critical for glucose homeostasis during the first month of life, signaling from these receptors, particularly the insulin receptor, is required for normal cardiac metabolism and function.

Oxidative Phenotype Protects Myofibers from Pathological Insults Induced by Chronic Heart Failure in Mice

The fiber specificity of skeletal muscle abnormalities in chronic heart failure (CHF) has not been defined. We show here that transgenic mice (8 weeks old) with cardiac-specific overexpression of calsequestrin developed CHF (50.9% decrease in fractional shortening and 56.4% increase in lung weight, P<0.001), cachexia (37.8% decrease in body weight, P<0.001), and exercise intolerance (69.3% decrease in running distance to exhaustion, P<0.001) without a significant change in muscle fiber-type composition. Slow oxidative soleus muscle maintained muscle mass, whereas fast glycolytic tibialis anterior and plantaris muscles underwent atrophy (11.6 and 13.3%, respectively; P<0.05). In plantaris muscle, glycolytic type IId/x and IIb, but not oxidative type I and IIa, fibers displayed significant decreases in cross-sectional area (20.3%, P<0.05). Fast glycolytic white vastus lateralis muscle showed sarcomere degeneration and decreased cytochrome c oxidase IV (39.5%, P<0.01) and peroxisome proliferator-activated receptor gamma co-activator 1alpha protein expression (30.3%, P<0.01) along with a dramatic induction of the MAFbx/Atrogin-1 mRNA. These findings suggest that exercise intolerance can occur in CHF without fiber type switching in skeletal muscle and that oxidative phenotype renders myofibers resistant to pathological insults induced by CHF.

Histamine levels during postexercise hypotension assessed by skeletal muscle microdialysis in inactive limbs

The activation of histamine receptors in skeletal muscle has been implicated as a major mediator of postexercise hypotension. The purpose of this study was to examine the role that histamine plays in postexercise hypotension in humans. Histamine detection by microdialysis was tested by infusing compound 48–80 (a mast cell degranulator) through microdialysis probes inserted into the biceps muscle of resting humans. Interstitial histamine levels increased (P<0.05) from 2.46±.96 pre-infusion to 6.81±0.74 ng/ml post-infusion, confirming our ability to measure changes in endogenous histamine concentrations. Subsequently, six subjects had microdialysis probes inserted into their biceps muscle (inactive) 75 min prior to lower limb cycling exercise at 60% Vo2 peak for 60 min. Dialysate histamine concentrations were 2.22±0.21 before, 1.71±0.19 during, and 1.09±0.18 ng/ml after exercise and were not significantly different across time. These data indicate that muscle interstitial histamine levels do not increase during or following exercise in inactive limb muscles. Therefore, the next step will be to examine whether histamine levels are altered in the active limb muscles during the postexercise period by directly measuring histamine concentrations via microdialysis in the exercised muscle. Supported by AHA 0555623Z.

A Novel Skeletal Muscle Cytoprotective Action of Adenosine A3 Receptors

Adenosine (Ado) can protect skeletal muscle from ischemic injury, in addition to its cardio- and vasculo-protective effect. However, identity of the Ado receptor and the mechanism mediating skeletal muscle protection is unknown. Using a quantitative mouse hindlimb ischemia-reperfusion injury model, the objective here was to assess the protective role of Ado receptor subtypes. Ado A3 receptor-selective agonist Cl-IBMECA reduced skeletal muscle injury, an effect that was blocked by the A3 receptor antagonist MRS1191 (% injury, vehicle: 28±5.9% vs. Cl-IBMECA: 5.4±3.8%, Cl-IBMECA & MRS1191: 21.5±3.5%, mean±SE, 7–22 mice, ANOVA & comparisons, Cl-IBMECA vs. all others, P<0.05). The A1 receptor agonist CCPA was also protective and and was selectively antagonized by the A1 receptor blocker DPCPX (CCPA: 10.8±1.8%, DPCPX &CCPA: 22±3.3%, 7–12 mice, P<0.05). The protection induced by Cl-IBMECA and another A3 receptor agonist MRS3558 was completely abrogated in phospholipase C β2/3-null mice while that caused by CCPA remained unaffected in these animals (8–10 mice). The study demonstrated a novel potent protective effect of A3 receptors in skeletal muscle and implicates phospholipase C β in mediating this protection. The data add to the cyto-protective actions of adenosine and suggest that the A3 receptor is a novel therapeutic target in ameliorating skeletal muscle injury.

Erythropoietin and its receptor in human skeletal muscle

Erythropoietin receptor (EPOR) expression in adult skeletal muscle has not been reported to date. Our aims were to investigate if human skeletal muscle expresses EPOR and whether there is an exercise-induced uptake of erythropoietin (EPO) over the exercising leg. Ten healthy male subjects performed 65 min of cycle exercise. Vastus lateralis muscle biopsies were obtained and arterio-venous concentration differences were measured over the exercising leg. EPOR protein was expressed in the skeletal muscle in areas corresponding to the sarcolemmal region and EPOR mRNA expression was increased by exercise. There was a small uptake of EPO at rest. In contrast, during exercise there was a small release of EPO from the exercising leg. In conclusion, these findings indicate that skeletal muscle should be integrated into the wider context of the biological role of EPO. In a clinical context, activation of EPOR in skeletal muscle could be an additive mechanism, complementary to increased amount of erythrocytes, behind the enhanced exercise capacity seen in patients treated with recombinant EPO.

EXCITATION–CONTRACTION COUPLING FROM THE 1950s INTO THE NEW MILLENNIUM

1. Excitation-contraction coupling is broadly defined as the process linking the action potential to contraction in striated muscle or, more narrowly, as the process coupling surface membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum. 2. We now know that excitation-contraction coupling depends on a macromolecular protein complex or 'calcium release unit'. The complex extends the extracellular space within the transverse tubule invaginations of the surface membrane, across the transverse tubule membrane into the cytoplasm and then across the sarcoplasmic reticulum membrane and into the lumen of the sarcoplasmic reticulum. 3. The central element of the macromolecular complex is the ryanodine receptor calcium release channel in the sarcoplasmic reticulum membrane. The ryanodine receptor has recruited a surface membrane L-type calcium channel as a 'voltage sensor' to detect the action potential and the calcium-binding protein calsequestrin to detect in the environment within the sarcoplasmic reticulum. Consequently, the calcium release channel is able to respond to surface depolarization in a manner that depends on the Ca(2+) load within the calcium store. 4. The molecular components of the 'calcium release unit' are the same in skeletal and cardiac muscle. However, the mechanism of excitation-contraction coupling is different. The signal from the voltage sensor to ryanodine receptor is chemical in the heart, depending on an influx of external Ca(2+) through the surface calcium channel. In contrast, conformational coupling links the voltage sensor and the ryanodine receptor in skeletal muscle. 5. Our current understanding of this amazingly efficient molecular signal transduction machine has evolved over the past 50 years. None of the proteins had been identified in the 1950s; indeed, there was debate about whether the molecules involved were, in fact, protein. Nevertheless, a multitude of questions about the molecular interactions and structures of the proteins and their interaction sites remain to be answered and provide a challenge for the next 50 years.

The effect of exercise training on adiponectin receptor expression in KKAy obese/diabetic mice

Adiponectin is an adipocyte-derived factor that plays a pivotal role in lipid and glucose metabolism. Recently, two types of adiponectin receptors (AdipoR1 and AdipoR2) were identified. We investigated whether exercise training (ET) or dietary restriction (DR) affects the expression of adiponectin receptors in skeletal muscle and liver, thereby improving glucose and lipid metabolism in KKAy mice. KKAy mice were subjected to 8 weeks of exercise training or food restriction. Following the experimental protocol, an intravenous glucose tolerance test and an intraperitoneal insulin tolerance test were performed in addition to the measurement of blood lipid and adiponectin concentrations. The mRNA levels of adiponectin, adiponectin receptors and genes that are putatively regulated by the adiponectin receptors were also analyzed. Both the 8-week exercise training and food restriction protocol improved insulin resistance in KKAy mice but did not alter plasma adiponectin concentration nor its mRNA expression. In comparison with C57BL/6 mice, AdipoR1 expression level was significantly decreased in skeletal muscle and AdipoR2 expression level was significantly increased in the liver in KKAy mice. After the 8-week experimental protocol, the expression level of AdipoR1 mRNA was approximately 1.8-fold greater in the skeletal muscle and 1.3-fold greater in the liver, and the level of AdipoR2 mRNA was 30% less in the liver of the ET group as compared with the control group. Additionally, in the ET group, mRNA expression of acyl coenzyme A-oxidase and carnitine palmitoyl transferase 1 (CPT1) was greater in the liver but not in skeletal muscle. In contrast, no significant changes were observed in the expression of genes encoding the adiponectin receptors in addition to other genes except for CPT1 in the DR group. These findings suggest that chronic exercise training affects the expression level of adiponectin receptors thereby improving insulin resistance in KKAy mice.

Kinetic evidence that desensitized nAChR may promote transitions of active nAChR to desensitized states during sustained exposure to agonists in skeletal muscle

During prolonged exposure of postjunctional nicotinic acetylcholine receptors (nAChR) of skeletal muscle to acetylcholine (ACh), agonist-activated nAChR (nAChRa) gradually fall into a refractory "desensitized" state (nAChRd), which no longer supports the high-conductance channel openings characteristic of the initially active nAChRa. In the present study, the possibility was examined that nAChRd, rather than simply constituting a passive "trap" for nAChRa, may actively promote further conversions of nAChRa to nAChRd in a formally autocatalytic manner. Single-ion whole-cell voltage-clamp currents (Na+ and Li+ in separate trials) were measured using two KCl-filled capillary electrodes (5-10 MOmega) implanted at the postjunctional locus of single frog skeletal muscle fibers (Rana pipiens) equilibrated in 30 mM K+ bath media to eliminate mechanical responses. Various nAChR agonists (carbamylcholine, acetylcholine, suberyldicholine) at different concentrations were delivered focally by positive pressure microjet. It was found that the decline of postmaximal agonist-induced currents under these different conditions (driven by the growth of the subpool of nAChRd) consistently followed an autocatalytic logistic rule modified for population growth of fixed units in a planar array: [Formula: see text] (where y represents the remaining agonist-induced current at time t, A=initial maximum current, and n is a constant). Some further experimental features that might result from a self-promoting growth of nAChRd were also tested, namely, (1) the effect of increased nAChRa and (2) the effect of increased nAChRd. Increase in agonist concentration of the superfusate, by increasing the planar density of active nAChRa at the outset, should enhance the probability of autocatalytic interactions with emerging nAChRd, hence, the rate of decline of agonist-induced current, and this was a consistent finding under all conditions tested. Raising the initial level of desensitized nAChRd by pretreatment of fibers with very low concentrations of agonist would be another way to increase autocatalytic interactions with active nAChRa, and this was also found to produce increased rates of decline of agonist-induced currents when tested in additional trials. It is concluded that several kinetic features of nAChR desensitization in skeletal muscle are consistent with an action of nAChRd to promote further transitions of nAChRa to desensitized forms. This could occur by a direct effect of nAChRd on contiguous nAChRa or perhaps through some intermediary membrane component or local intracellular pathway.

Roles of Skeletal Muscle and Peroxisome Proliferator-Activated Receptors in the Development and Treatment of Obesity

Metabolic disturbances associated with alterations in lipid metabolism, such as obesity, type 2 diabetes, and syndrome X, are becoming more and more prominent in Western societies. Despite extensive research in such pathologies and their molecular basis, we are still far from completely understanding how these metabolic perturbations are produced and interrelate and, consequently, how to treat them efficiently. The discovery that adipose tissue is, in fact, an endocrine tissue able to secrete active molecules related to lipid homeostasis--the adipokines--has dramatically changed our understanding of the molecular events that take place in such diseases. This knowledge has been further improved by the discovery of peroxisome proliferator-activated receptors and their ligands, at present commonly used for the clinical treatment of lipid disturbances. However, a key point remains to be solved, and that is the role of muscle lipid metabolism, notably because of the main role played by this tissue in the development of such pathologies. In addition, a reciprocal regulation between adipose tissue and skeletal muscle has been proposed. New discoveries on the role of peroxisome proliferator-activated receptor-delta in skeletal muscle functions as well as the secretory capabilities of muscle, now considered as an endocrine tissue, have changed the general point of view on lipid homeostasis, opening new and promising doors for the treatment of lipid disorders.

Circulating Adiponectin and Expression of Adiponectin Receptors in Human Skeletal Muscle: Associations with Metabolic Parameters and Insulin Resistance and Regulation by Physical Training

Adiponectin, an adipocyte-secreted hormone, is associated with insulin resistance and the metabolic syndrome. The physiological regulation of circulating adiponectin levels and mRNA expression of its receptors (AdipoR1 and AdipoR2) in skeletal muscle remains to be fully elucidated. We assessed circulating adiponectin and AdipoR1/R2 mRNA expression in human skeletal muscle in a cross-sectional study of 140 subjects with normal or impaired glucose tolerance or type 2 diabetes. In the context of an interventional study, the same measurements were performed in 60 of these subjects (20/glucose tolerance group) before and after 4 wk of physical training. Finally, we measured these same variables in addition to protein levels of AMP kinase (AMPK), acetyl phosphorylated AMPK, coenzyme A carboxylase, phosphorylated coenzyme A carboxylase, and phosphatidylinositol 3-kinase in muscle before and after 3 h of intensive exercise in a subgroup of five subjects. This study was performed at an academic clinical research center. Circulating adiponectin was negatively associated, whereas AdipoR1/R2 mRNA levels were positively associated with obesity, glucose and lipid levels, and insulin resistance. Physical training for 4 wk resulted in increased circulating adiponectin levels and AdipoR1/R2 mRNA expression in muscle. Exercise for 3 h increased AdipoR1/R2 mRNA expression as well as phosphorylation of AMPK and acetyl coenzyme A carboxylase in muscle, but had no effect on circulating adiponectin. Adiponectin, AdipoR1, and AdipoR2 are all associated with body composition, insulin sensitivity, and metabolic parameters. Physical training increases circulating adiponectin and mRNA expression of its receptors in muscle, which may mediate the improvement of insulin resistance and the metabolic syndrome in response to exercise.

Recombinant Human Insulin-Like Growth Factor-I Treatment Inhibits Gluconeogenesis in a Transgenic Mouse Model of Type 2 Diabetes Mellitus

IGF-I and insulin are structurally related polypeptides that mediate a similar pattern of biological effects via receptors that display considerably homology. Administration of recombinant human IGF-I (rhIGF-I) has been proven to improve glucose control and liver and muscle insulin sensitivity in patients with type 2 diabetes mellitus (DM). The effect of rhIGF-I treatment was evaluated in a mouse model of type 2 DM (MKR mouse), which expresses a dominant-negative form of the human IGF-I receptor under the control of the muscle creatine kinase promoter specifically in skeletal muscle. MKR mice have impaired IGF-I and insulin signaling in skeletal muscle, leading to severe insulin resistance in muscle, liver, and fat, developing type 2 DM at 5 wk of age. Six-week-old MKR mice were treated with either saline or rhIGF-I for 3 wk. Blood glucose levels were decreased in response to rhIGF-I treatment in MKR mice. rhIGF-I treatment also increased body weight in MKR with concomitant changes in body composition such as a decrease in fat mass and an increase in lean body mass. Insulin, fatty acid, and triglyceride levels were not affected by rhIGF-I, nor were insulin or glucose tolerance in MKR mice. Hyperinsulinemic-euglycemic clamp analysis demonstrated no improvement in overall insulin sensitivity. Pyruvate and glutamine tolerance tests proved that there was a decrease in the rate of glucose appearance in MKR mice treated with rhIGF-I, suggesting a reduction in the gluconeogenic capacity of liver, kidney, and small intestine. Taken together these results demonstrate that the improvement of the hyperglycemia was achieved by inhibition of gluconeogenesis rather than an improvement in insulin sensitivity. Also, these results suggest that a functional IGF-I receptor in skeletal muscle is required for IGF-I to improve insulin sensitivity in this mouse model of type 2 DM.

Modeling Circadian Rhythms of Glucocorticoid Receptor and Glutamine Synthetase Expression in Rat Skeletal Muscle

The circadian rhythm of endogenous corticosterone (CS) may produce fluctuations of downstream gene expression in normal rats. This study examined changes in glucocorticoid receptor (GR) and glutamine synthetase (GS) expression in rat skeletal muscle in relation to plasma CS over a 24-h period. Fifty-four normal male Wistar rats were sacrificed at 18 time points (n = 3) over 24 h. Plasma CS concentrations and gastrocnemius muscle GR and GS mRNA and GS activity were measured. The circadian rhythm of plasma CS was captured by a two-harmonic function. The expression of GR and GS mRNA and GS activity follow a circadian rhythm in normal rat skeletal muscle. GR mRNA reaches a trough at 4 h after the peak of plasma CS and it fluctuates between 0.55 and 0.9 fmol g tissue(-1). GS mRNA and activity reach peaks at 6 and 12 h after the endogenous CS peak. GS mRNA oscillates between 3 and 6 fmol g tissue(-1), whereas GS activity fluctuates between 17 and 23 micromol min(-1) g protein(-1). Mechanistic receptor/gene-mediated pharmacodynamic models were applied to describe the temporal patterns of GR mRNA, GS mRNA, and GS activity within the circadian cycle. The integrated models were able to capture the circadian expression patterns of plasma CS, and GR and GS in normal rat skeletal muscle showing a dependence of tissue gene expression on plasma CS.

Point: The muscle metaboreflex does restore blood flow to contracting muscles

Imposed reductions in oxygen delivery to active skeletal muscle during dynamic exercise in conscious dogs evoke a powerful pressor response, termed the muscle metaboreflex. During submaximal work rates, the major mechanism used by this reflex is to increase cardiac output (CO), which generates a

The mammalian exercise pressor reflex in health and disease

The exercise pressor reflex (a peripheral neural reflex originating in skeletal muscle) contributes significantly to the regulation of the cardiovascular system during exercise. Exercise-induced signals that comprise the afferent arm of the reflex are generated by activation of mechanically (muscle mechanoreflex) and chemically sensitive (muscle metaboreflex) skeletal muscle receptors. Activation of these receptors and their associated afferent fibres reflexively adjusts sympathetic and parasympathetic nerve activity during exercise. In heart failure, the cardiovascular response to exercise is augmented. Owing to the peripheral skeletal myopathy that develops in heart failure (e.g. muscle atrophy, decreased peripheral blood flow, fibre-type transformation and reduced oxidative capacity), the exercise pressor reflex has been implicated as a possible mechanism by which the cardiovascular response to physical activity is exaggerated in this disease. Accumulating evidence supports this conclusion. This review therefore focuses on the role of the exercise pressor reflex in regulating the cardiovascular system during exercise in both health and disease. Updates on our current understanding of the exercise pressor reflex neural pathway as well as experimental models used to study this reflex are presented. In addition, special emphasis is placed on the changes in exercise pressor reflex activity that develop in heart failure, including the contributions of the muscle mechanoreflex and metaboreflex to this pressor reflex dysfunction.

Insulin resistance causes increased beta-cell mass but defective glucose-stimulated insulin secretion in a murine model of type 2 diabetes

Although insulin resistance induces compensatory increases in beta cell mass and function to maintain normoglycaemia, it is not clear whether insulin resistance can precipitate beta cell dysfunction and hyperglycaemia without a pre-existing beta cell susceptibility. We therefore examined the beta cell phenotype in the MKR mouse, a model in which expression of a dominant-negative IGF 1 receptor (IGF1R) in skeletal muscle leads to systemic insulin resistance and diabetes. Circulating glucose, insulin and glucagon concentrations were measured. Insulin sensitivity, glucose tolerance and insulin release in vivo were assessed by i.p. insulin and glucose tolerance tests. Beta cell function was assessed via insulin secretion from isolated islets and the glucose gradient in the perfused pancreas. Beta cell morphology was examined via immunohistochemistry. MKR mice were fed a high-fat diet containing sucrose (HFSD) to test metabolic capacity and beta cell function. Insulin-resistant MKR mice developed hyperglycaemia and a loss of insulin responsiveness in vivo. Basal insulin secretion from the perfused pancreas was elevated, with no response to glucose. Despite the demand on insulin secretion, MKR mice had increased pancreatic insulin content and beta cell mass mediated through hyperplasia and hypertrophy. The HFSD worsened hyperglycaemia in MKR mice but, despite increased food intake in these mice, failed to induce the obesity observed in wild-type mice. Our studies demonstrate that insulin resistance of sufficient severity can impair glucose-stimulated insulin secretion, thereby undermining beta cell compensation and leading to hyperglycaemia. Moreover, because insulin stores were intact, the secretory defects reflect an early stage of beta cell dysfunction.

Short-term Caloric Restriction Does Not Modify theIn VivoInsulin Signaling Pathway Leading to Akt Activation in Skeletal Muscle of Ames Dwarf (Prop1df/Prop1df) Mice

The purpose of this study was to analyze the interaction between caloric restriction (CR) and the dwarf mutation at the level of insulin sensitivity and signal transduction. To this end, we analyzed the in vivo status of the insulin signaling system in skeletal muscle from Ames dwarf (df/df) and normal mice fed ad libitum or subjected to short-term (20-day) CR. We measured insulin-stimulated phosphorylation of the IR and IRS-1, IRS-1-p85 association and Akt activation, and the abundance of the IR, IRS-1, p85, GLUT-4 and IGF-1 receptor in skeletal muscle. In terms of glucose homeostasis, the response to CR was different in both groups of animals. In normal animals, CR induced a significant reduction in both circulating insulin and glucose levels, while CR did not modify these parameters in df/df mice. We did not find any significant alteration in either activation or abundance of signaling molecules analyzed after short-term CR in either normal or Ames dwarf mice. We conclude that the initial adaptation to CR in normal mice is an increase in insulin sensitivity without changes in insulin signal transduction, and that this adaptation is not evidenced in df/df mice, probably since they are already hypersensitive to insulin.

Ryanodine Receptor Type 1 (RyR1) Mutations C4958S and C4961S Reveal Excitation-coupled Calcium Entry (ECCE) Is Independent of Sarcoplasmic Reticulum Store Depletion

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

Greater adenosine A 2A receptor densities in cardiac and skeletal muscle in endurance-trained men: a [ 11C]TMSX PET study

We examined the densities of adenosine A 2A receptors in cardiac and skeletal muscles between untrained and endurance-trained subjects using positron emission tomography (PET) and [7- methyl- 11C]-( E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([ 11C]TMSX), a newly developed radioligand for mapping adenosine A 2A receptors. Five untrained and five endurance-trained subjects participated in this study. The density of adenosine A 2A receptors was evaluated as the distribution volume of [ 11C]TMSX in cardiac and triceps brachii muscles in the resting state using PET. The distribution volume of [ 11C]TMSX in the myocardium was significantly greater than in the triceps brachii muscle in both groups. Further, distribution volumes [ 11C]TMSX in the trained subjects were significantly grater than those in untrained subjects (myocardium, 3.6±0.3 vs. 3.1±0.4 ml g −1; triceps brachii muscle, 1.7±0.3 vs. 1.2±0.2 ml g −1, respectively). These results indicate that the densities of adenosine A 2A receptors in the cardiac and skeletal muscles are greater in the endurance-trained men than in the untrained men.