The inflammatory response after an epidermal burn depends on the activities of mouse mast cell proteases 4 and 5

A second-degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to nonmuscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-Kit(W)/Kit(Wv) mice, which lack MCs in a context of other defects due to a mutation of the Kit receptor. To address further an MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury after a second-degree scald burn, whereas mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficient mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first hour postburn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing nonredundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.     

Mast cells that reside at different locations in the jejunum of mice infected with Trichinella spiralis exhibit sequential changes in their granule ultrastructure and chymase phenotype

Whether or not a nontransformed, mature mouse mast cell (MC) or its committed progenitor can change its granule protease phenotype during inflammatory responses, has not been determined. To address this issue, the granule morphology and protease content of the MC in the jejunum of BALB/c mice exposed to Trichinella spiralis were assessed during the course of the infection. Within 1 wk after helminth infection of the mice, increased numbers of MC appeared in the crypts at the base of the villi, and by wk 2 the number of MC throughout the villi increased by approximately 25-fold. Shortly after the peak of the mastocytosis, the intraepithelial population of MC disappeared, followed by a progressive loss of lamina propria MC. The presence of stellate-shaped granules containing crystalline structures in intraepithelial MC at the height of infection and the retention of such granules with fragmented crystals in lamina propria MC during resolution of the mastocytosis suggest that MC migrate during the various phases of the inflammation. As assessed by immunohistochemical analyses of serial sections, predominant chymase phenotypes were observed at the height of the infection in the muscle that expressed mouse MC protease (mMCP) 5 without mMCP-1 or mMCP-2 and in the epithelium that expressed mMCP-1 and mMCP-2 without mMCP-5. Accompanying these two MC populations were transitional forms in the submucosa that expressed mMCP-2 and mMCP-5 without mMCP-1 and in the lamina propria that expressed mMCP-2 alone. These data suggest that jejunal MC sequentially express mMCP-2, cease expressing mMCP-5, and finally express mMCP-1 as the cells progressively appear in the submucosa, lamina propria, and epithelium, respectively. In the recovery phase of the disease, MC sequentially cease expressing mMCP-1, express mMCP-5, and finally cease expressing mMCP-2 as they present at the tips of the villi, the base of the villi, and the submucosa, respectively. That MC can reversibly alter their protease phenotypes suggests that a static nomenclature with fixed functional implications is inadequate to describe MC populations during an inflammatory process within a particular tissue.     

Dual targets for mouse mast cell protease-4 in mediating tissue damage in experimental bullous pemphigoid

Mouse mast cell protease-4 (mMCP-4) has been linked to autoimmune and inflammatory diseases, although the exact mechanisms underlying its role in these pathological conditions remain unclear. Here, we have found that mMCP-4 is critical in a mouse model of the autoimmune skin blistering disease bullous pemphigoid (BP). Mice lacking mMCP-4 were resistant to experimental BP. Complement activation, mast cell (MC) degranulation, and the early phase of neutrophil (PMN) recruitment occurred comparably in mMCP-4(-/-) and WT mice. However, without mMCP-4, activation of matrix metalloproteinase (MMP)-9 was impaired in cultured mMCP-4(-/-) MCs and in the skin of pathogenic IgG-injected mMCP-4(-/-) mice. MMP-9 activation was not fully restored by local reconstitution with WT or mMCP-4(-/-) PMNs. Local reconstitution with mMCP-4(+/+) MCs, but not with mMCP-4(-/-) MCs, restored blistering, MMP-9 activation, and PMN recruitment in mMCP-4(-/-) mice. mMCP-4 also degraded the hemidesmosomal transmembrane protein BP180 both in the skin and in vitro. These results demonstrate that mMCP-4 plays two different roles in the pathogenesis of experimental BP, by both activating MMP-9 and by cleaving BP180, leading to injury of the hemidesmosomes and extracellular matrix of the basement membrane zone.     

Mast cell mediation of muscle and pulmonary injury following hindlimb ischemia-reperfusion.

We have observed extensive mast cell degranulation in the reperfused hindlimb muscle of the mouse, accompanied by pathological changes within the muscle. As quantitated by the tissue:blood (125)I permeability ratio, both the hindlimbs and lungs exhibited a significant increment in permeability during hindlimb reperfusion. In lungs of the same mice, mast cell-derived chymase mMCP-1 coats alveolar macrophages, an event noted by us in acid-induced direct lung injury. Mast cells in the lung contain mMCP-1, whereas those in the muscle do not. Neither extensive muscle injury nor an increased pulmonary permeability index occurs in the mast cell-deficient W/W(v) mice. We conclude that the mast cell is a key mediator in both local ischemia-reperfusion injury (I-R) of muscle and consequent remote lung injury.     

Mast cell restricted mouse and human tryptase·heparin complexes hinder thrombin-induced coagulation of plasma and the generation of fibrin by proteolytically destroying fibrinogen

The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-β. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-β· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.     

Mechanisms mediating insulin resistance in transgenic mice overexpressing mouse apolipoprotein A-II

We previously demonstrated that transgenic mice overexpressing mouse apolipoprotein A-II (apoA-II) exhibit several traits associated with the insulin resistance (IR) syndrome, including increased atherosclerosis, hypertriglyceridemia, obesity, and IR. The skeletal muscle appeared to be the insulin-resistant tissue in the apoA-II transgenic mice. We now demonstrate a decrease in FA oxidation in skeletal muscle of apoA-II transgenic mice, consistent with reports that decreased skeletal muscle FA oxidation is associated with increased skeletal muscle triglyceride accumulation, skeletal muscle IR, and obesity. The decrease in FA oxidation is not due to decreased carnitine palmitoyltransferase 1 activity, because oxidation of palmitate and octanoate were similarly decreased. Quantitative RT-PCR analysis of gene expression demonstrated that the decrease in FA oxidation may be explained by a decrease in medium chain acyl-CoA dehydrogenase. We previously demonstrated that HDLs from apoA-II transgenic mice exhibit reduced binding to CD36, a scavenger receptor involved in FA metabolism. However, studies of combined apoA-II transgenic and CD36 knockout mice suggest that the major effects of apoA-II are independent of CD36. Rosiglitazone treatment significantly ameliorated IR in the apoA-II transgenic mice, suggesting that the underlying mechanisms of IR in this animal model may share common features with certain types of human IR.     

The C5a receptor on mast cells is critical for the autoimmune skin-blistering disease bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune skin-blistering disease characterized by the presence of autoantibodies against the hemidesmosomal proteins BP230 and BP180. In the IgG passive transfer mouse model of BP, subepidermal blistering is triggered by anti-BP180 antibodies and depends on the complement system, mast cell (MC) degranulation, and neutrophil infiltration. In this study, we have identified the signaling events that connect the activation of the complement system and MC degranulation. We found that mice deficient in MCs or the C5a receptor (C5aR) injected with pathogenic anti-BP180 IgG failed to develop subepidermal blisters and exhibited a drastic reduction in p38 MAPK phosphorylation compared with WT mice. Local reconstitution with MCs from WT but not C5aR-deficient mice restored high levels of p38 MAPK phosphorylation and subepidermal blistering in MC-deficient mice. Local injection of recombinant C5a induced phosphorylation of p38 MAPK in WT but not MC-deficient mice. Cultured mouse MCs treated with recombinant C5a exhibited a significant increase in p38 MAPK phosphorylation and MC degranulation. Taken together, these data demonstrate that C5a interacts with C5aR on MCs and that this C5a-C5aR interaction triggers activation of the p38 MAPK pathway, subsequent MC degranulation, and ultimately BP blistering.     

Serglycin proteoglycan is required for secretory granule integrity in mucosal mast cells

SG (serglycin) PGs (proteoglycans) are strongly implicated in the assembly of MC (mast cell) granules. However, this notion has mainly been on the basis of studies of MCs of the connective tissue subtype, whereas the role of SG PG in mucosal MCs has not been explored. In the present study, we have addressed the latter issue by using mice with an inactivated SG gene. Bone marrow cells were differentiated in vitro into the mucosal MC phenotype, expressing the markers mMCP (mouse MC protease) -1 and -2. Biosynthetic labelling experiments performed on these cells revealed an approximately 80% reduction of 35SO4(2-) incorporation into PGs recovered from SG-/- cells as compared with SG+/+ counterparts, indicating that SG is the dominating cell-associated PG of mucosal MCs. Moreover, the absence of SG led to defective metachromatic staining of mucosal MCs, both in vivo and in the in vitro-derived mucosal MCs. Ultrastructural analysis showed that granules were present in similar numbers in SG+/+ and SG-/- cells, but that their morphology was markedly affected by the absence of SG, e.g. with electron-dense core formation only seen in SG+/+ granules. Analysis of the MC-specific proteases showed that mMCP-1 and mMCP-7 were completely independent of SG for storage, whereas mMCP-2 showed a partial dependence. In contrast, mMCP-4 and -6, and carboxypeptidase A were strongly dependent on SG for storage. Together, our data indicate that SG PG is of crucial importance for assembly of mature mucosal MC granules, but that the specific dependence on SG for storage varies between individual granule constituents.     

Senescent jejunal mast cells and eosinophils in the mouse preferentially translocate to the spleen and draining lymph node, respectively, during the recovery phase of helminth infection

Because mice infected with Trichinella spiralis experience a pronounced, but transient, mastocytosis and eosinophilia in their intestine, this disease model was used to follow the fate of senescent T cell-dependent mast cells (MCs) and eosinophils. Very few MCs or eosinophils undergoing apoptosis were found in the jejunum during the resolution phase of the infection, even though apoptotic MCs were common in the large intestine. Although the mesenteric draining lymph nodes contained large numbers of apoptotic eosinophils, MCs were rarely found at this location. During the recovery phase, large numbers of MCs were present in the spleen, and many of these cells possessed segmented nuclei. These splenic MCs were not proliferating. Although MCs from the jejunum and spleen of noninfected mice failed to express mouse MC protease (mMCP) 9, essentially all of the MCs in the jejunal submucosa and spleen of T. spiralis-infected mice expressed this serine protease during the recovery phase. The MCs in the jejunum expressed mMCP-9 before any mMCP-9-containing cells could be detected in the spleen. The fact that mMCP-9-containing MCs were detected in splenic blood vessels as these cells began to disappear from the jejunum supports the view that many jejunal MCs translocate to the spleen during the recovery phase of the infection. During this translocation process, some senescent jejunal MCs undergo nuclear segmentation. These studies reveal for the first time different exit and disposal pathways for T cell-dependent eosinophils and MCs after their expansion in the jejunum during a helminth infection.     

Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.     

In vivo requirement of the alpha-syntrophin PDZ domain for the sarcolemmal localization of nNOS and aquaporin-4

alpha-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of alpha-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of alpha-syntrophin in vivo by generating transgenic mouse lines expressing full-length alpha-syntrophin or a mutated alpha-syntrophin lacking the PDZ domain (Delta PDZ). The Delta PDZ alpha-syntrophin displaced endogenous alpha- and beta 1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the alpha-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an alpha-syntrophin-null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length alpha-syntrophin, not the Delta PDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound alpha-syntrophin PDZ domain.     

The role of p53 in vivo during skeletal muscle post-natal development and regeneration: studies in p53 knockout mice

The tumour suppressor gene p53 is recognised as a central regulator of the cell cycle and apoptosis. Post-natally, p53 mutations are associated with many cancers and mice lacking p53 are prone to spontaneous tumour formation. The present study examines skeletal muscle formation in post-natal mice lacking p53 using two different models of skeletal muscle regeneration. The level of endogenous myogenic cell proliferation in mature skeletal muscle was examined and the time course of muscle regeneration after whole muscle transplantation or crush injury were compared in p53 (-/-) and control C57Bl/6J adult mice, using desmin and proliferating cell nuclear antigen (PCNA) immunohistochemistry and histological analysis. The pattern of inflammation, myoblast proliferation and myotube formation in regenerating p53 (-/-) skeletal muscles appears normal and similar to those in control C57Bl/6J muscle. These data indicate that p53 is not required for the regulation of myoblast proliferation, differentiation and myotube formation in vivo during myogenesis of adult skeletal muscle.     

Genetic disruption of calcineurin improves skeletal muscle pathology and cardiac disease in a mouse model of limb-girdle muscular dystrophy

Calcineurin (Cn) is a Ca(2+)/calmodulin-dependent serine/threonine phosphatase that regulates differentiation-specific gene expression in diverse tissues, including the control of fiber-type switching in skeletal muscle. Recent studies have implicated Cn signaling in diminishing skeletal muscle pathogenesis associated with muscle injury or disease-related muscle degeneration. For example, use of the Cn inhibitor cyclosporine A has been shown to delay muscle regeneration following toxin-induced injury and inhibit regeneration in the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy. In contrast, transgenic expression of an activated mutant of Cn in skeletal muscle was shown to increase utrophin expression and reduce overall disease pathology in mdx mice. Here we examine the effect of altered Cn activation in the context of the delta-sarcoglycan-null (scgd(-/-)) mouse model of limb-girdle muscular dystrophy. In contrast to results discussed in mdx mice, genetic deletion of a loxP-targeted calcineurin B1 (CnB1) gene using a skeletal muscle-specific cre allele in the scgd(-/-) background substantially reduced skeletal muscle degeneration and histopathology compared with the scgd(-/-) genotype alone. A similar regression in scgd-dependent disease manifestation was also observed in calcineurin Abeta (CnAbeta) gene-targeted mice in both skeletal muscle and heart. Conversely, increased Cn expression using a muscle-specific transgene increased cardiac fibrosis, decreased cardiac ventricular shortening, and increased muscle fiber loss in the quadriceps. Our results suggest that inhibition of Cn may benefit select types of muscular dystrophy.     

Effect of glycogen synthase overexpression on insulin-stimulated muscle glucose uptake and storage

Insulin-stimulated muscle glucose uptake is inversely associated with the muscle glycogen concentration. To investigate whether this association is a cause and effect relationship, we compared insulin-stimulated muscle glucose uptake in noncontracted and postcontracted muscle of GSL3-transgenic and wild-type mice. GSL3-transgenic mice overexpress a constitutively active form of glycogen synthase, which results in an abundant storage of muscle glycogen. Muscle contraction was elicited by in situ electrical stimulation of the sciatic nerve. Right gastrocnemii from GSL3-transgenic and wild-type mice were subjected to 30 min of electrical stimulation followed by hindlimb perfusion of both hindlimbs. Thirty minutes of contraction significantly reduced muscle glycogen concentration in wild-type (49%) and transgenic (27%) mice, although transgenic mice retained 168.8 +/- 20.5 micromol/g glycogen compared with 17.7 +/- 2.6 micromol/g glycogen for wild-type mice. Muscle of transgenic and wild-type mice demonstrated similar pre- (3.6 +/- 0.3 and 3.9 +/- 0.6 micromol.g(-1).h(-1) for transgenic and wild-type, respectively) and postcontraction (7.9 +/- 0.4 and 7.0 +/- 0.4 micromol.g(-1).h(-1) for transgenic and wild-type, respectively) insulin-stimulated glucose uptakes. However, the [14C]glucose incorporated into glycogen was greater in noncontracted (151%) and postcontracted (157%) transgenic muscle vs. muscle of corresponding wild-type mice. These results indicate that glycogen synthase activity is not rate limiting for insulin-stimulated glucose uptake in skeletal muscle and that the inverse relationship between muscle glycogen and insulin-stimulated glucose uptake is an association, not a cause and effect relationship.     

Changes in Skeletal Muscle and Body Weight on Sleeping Beauty Transposon-Mediated Transgenic Mice Overexpressing Pig mIGF-1

Insulin-like growth factor (IGF-I) is an important growth factor in mammals, but the functions of the local muscle-specific isoform of insulin-like growth factor 1 (mIGF-1) to skeletal muscle development have rarely been reported. To determine the effect of pig mIGF-1 on body development and muscle deposition in vivo and to investigate the molecular mechanisms, the transgenic mouse model was generated which can also provide experimental data for making transgenic pigs with pig endogenous IGF1 gene. We constructed a skeletal muscle-specific expression vector using 5′- and 3′-regulatory regions of porcine skeletal α-actin gene. The expression cassette was flanked with Sleeping Beauty transposon (SB)-inverted terminal repeats. The recombinant vector could strongly drive enhanced green fluorescence protein (EGFP) reporter gene expression specifically in mouse myoblast cells and porcine fetal fibroblast cells, but not in porcine kidney cells. The EGFP level driven by α-actin regulators was significantly stronger than that driven by cytomegalovirus promoters. These results indicated that the cloned α-actin regulators could effectively drive specific expression of foreign genes in myoblasts, and the skeletal muscle-specific expression vector mediated with SB transposon was successfully constructed. To validate the effect of pig mIGF-1 on skeletal muscle growth, transgenic mice were generated by pronuclear microinjection of SB-mediated mIGF-1 skeletal expression vector and SB transposase-expressing plasmid. The transgene-positive rates of founder mice and the next-generation F1 mice were 30% (54/180) and 90.1% (64/71), respectively. The mIGF-1 gene could be expressed in skeletal muscle specifically. The levels of mRNA and protein in transgenic mice were 15 and 3.5 times higher, respectively, than in wild-type mice. The body weights of F1 transgenic mice were significantly heavier than wild-type mice from the age of 8 weeks onwards. The paraffin-embedded sections of gastrocnemius from 16-week-old transgenic male mice showed that the numbers of myofibers per unit were increased in comparison with those in the wild-type mice. mIGF-1 overexpression in mice skeletal muscle may promote myofibers hypertrophy and muscle production, and increased the average body weight of adult mice. Transgenic mice models can be generated by the mediation of SB transposon with high transgene efficiency.     

Overexpression of inducible 70-kDa heat shock protein in mouse attenuates skeletal muscle damage induced by cryolesioning

Heat shock protein expression is elevated upon exposure to a variety of stresses and limits the extent of stress-induced damage. To investigate the putative role of inducible 70-kDa heat shock protein (HSP70) in skeletal muscle damage and regeneration, soleus and tibialis anterior (TA) muscles from HSP70-overexpressing transgenic mice were subjected to cryolesioning and analyzed after 1, 10, and 21 days. Histological analysis showed that the muscles from both HSP70 and wild-type mice treated with radicicol (a HSP inducer) had decreased necrosis after cryolesioning compared with controls. The decrease in muscle fiber cross-sectional area in both soleus and TA muscles in 10 days postlesioning was attenuated in HSP70 mice compared with wild-type mice. Glutathione peroxidase activity was increased 1 day after cryolesioning in both HSP70 and control mice and remained elevated for up to 21 days. Immunodetection of neuronal cell adhesion molecule (a satellite cell marker) and developmental/neonatal MHC were significantly lower in cryolesioned HSP70-overexpressing mice than in cryolesioned controls. These results suggest that HSP70 protects skeletal muscle against injury and radicicol might be useful as a skeletal muscle protective agent. regeneration; radicicol; transgenic mouse; myoprotection     

Exercise capacity of mice genetically lacking muscle glycogen synthase: in mice, muscle glycogen is not essential for exercise

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