Accumulation of autophagosomes because of impaired autophagy during valosin-containing protein (VCP)–linked dementia is explained by the absence or reduced activity of VCP.

To determine whether uncoupling respiration from oxidative phosphorylation in skeletal muscle is a suitable treatment for obesity and type 2 diabetes, we generated transgenic mice expressing the mitochondrial uncoupling protein (Ucp) in skeletal muscle. Skeletal muscle oxygen consumption was 98% higher in Ucp-L mice (with low expression) and 246% higher in Ucp-H mice (with high expression) than in wild-type mice. Ucp mice fed a chow diet had the same food intake as wild-type mice, but weighed less and had lower levels of glucose and triglycerides and better glucose tolerance than did control mice. Ucp-L mice were resistant to obesity induced by two different high-fat diets. Ucp-L mice fed a high-fat diet had less adiposity, lower levels of glucose, insulin and cholesterol, and an increased metabolic rate at rest and with exercise. They were also more responsive to insulin, and had enhanced glucose transport in skeletal muscle in the setting of increased muscle triglyceride content. These data suggest that manipulating respiratory uncoupling in muscle is a viable treatment for obesity and its metabolic sequelae.

R eliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure "autophagic flux" in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein 1 light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3-II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3-II protein may render possible misinterpretations since LC3-II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3-II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3-II, a technique aptly named the "autophagometer." In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3-II protein levels in mouse skeletal muscle by >100%. The addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3-II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a "colchicine block." Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an "in vivo autophagometer" study using colchicine in skeletal muscle.

Mutations in p97/VCP cause the multisystem disease inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD). p97/VCP is a member of the AAA؉ (ATPase associated with a variety of activities) protein family and has been implicated in multiple cellular processes. One pathologic feature in IBMPFD is ubiquitinated inclusions, suggesting that mutations in p97/VCP may affect protein degradation. The present study shows that IBMPFD mutant expression increases ubiquitinated proteins and susceptibility to proteasome inhibition. Co-expression of an aggregate prone protein such as expanded polyglutamine in IBMPFD mutant cells results in an increase in aggregated protein that localizes to small inclusions instead of a single perinuclear aggresome. These small inclusions fail to co-localize with autophagic machinery. IBMPFD mutants avidly bind to these small inclusions and may not allow them to traffic to an aggresome. This is rescued by HDAC6, a p97/VCP-binding protein that facilitates the autophagic degradation of protein aggregates. Expression of HDAC6 improves aggresome formation and protects IBMPFD mutant cells from polyglutamine-induced cell death. Our study emphasizes the importance of protein aggregate trafficking to inclusion bodies in degenerative diseases and the therapeutic benefit of inclusion body formation.Ubiquitinated inclusions (UBIs) 2 are a common pathology in many degenerative disorders associated with brain and muscle (1). In many cases the molecular constituents of the UBIs are known and are a result of the misfolding of an aggregate prone or mutant protein such as ␣-synuclein in autosomal dominant Parkinson disease or expanded polyglutamine containing proteins in Huntington disease. UBIs in other degenerative disorders may be due to a global impairment in protein degradation. For example, after a misfolded substrate overwhelms the ubiquitin proteasome system (UPS) or as a result of mutations in proteins involved in specific protein degradation pathways. One example is the autosomal dominantly inherited disorder inclusion body myopathy, Paget disease of the bone and frontotemporal dementia (IBMPFD) associated with mutations in the UPS chaperone p97/VCP (2). Affected tissue in IBMPFD contains prominent cytoplasmic and intranuclear UBIs (3-5). In some cases these UBIs contain tubulofilamentous inclusions and insoluble protein aggregates (4). p97/VCP belongs the AAAϩ (ATPases associated with a variety of activities) protein family and participates in the degradation of proteins via the UPS (6). p97/VCP has a clear role in endoplasmic reticulumassociated degradation (ERAD) of misfolded proteins (6). In association with co-factors derlin-1, Ufd1, and Npl4, p97/VCP participates in the retrotranslocation of misfolded endoplasmic reticulum lumen and transmembrane proteins facilitating their delivery to the UPS. We have previously shown that IBMPFD mutant p97/VCP fails to degrade the misfolded ERAD substrate ⌬F508-CFTR as efficiently as wild type p97/VCP in cultured myoblasts (7). ...

Distal arthrogryposis type I (DA1) is a disorder characterized by congenital contractures of the hands and feet for which few genes have been identified. Here we describe a five-generation family with DA1 segregating as an autosomal dominant disorder with complete penetrance. Genome-wide linkage analysis using Affymetrix GeneChip Mapping 10K data from 12 affected members of this family revealed a multipoint LOD(max) of 3.27 on chromosome 12q. Sequencing of the slow-twitch skeletal muscle myosin binding protein C1 (MYBPC1), located within the linkage interval, revealed a missense mutation (c.706T>C) that segregated with disease in this family and causes a W236R amino acid substitution. A second MYBPC1 missense mutation was identified (c.2566T>C)(Y856H) in another family with DA1, accounting for an MYBPC1 mutation frequency of 13% (two of 15). Skeletal muscle biopsies from affected patients showed type I (slow-twitch) fibers were smaller than type II fibers. Expression of a green fluorescent protein (GFP)-tagged MYBPC1 construct containing WT and DA1 mutations in mouse skeletal muscle revealed robust sarcomeric localization. In contrast, a more diffuse localization was seen when non-fused GFP and MYBPC1 proteins containing corresponding MYBPC3 amino acid substitutions (R326Q, E334K) that cause hypertrophic cardiomyopathy were expressed. These findings reveal that the MYBPC1 is a novel gene responsible for DA1, though the mechanism of disease may differ from how some cardiac MYBPC3 mutations cause hypertrophic cardiomyopathy.

Inclusion body myopathy associated with Paget's disease of the bone and fronto-temporal dementia (IBMPFD) is a progressive autosomal dominant disorder caused by mutations in p97/VCP (valosin-containing protein). p97/VCP is a member of the AAA1 (ATPase associated with a variety of activities) protein family and participates in multiple cellular processes. One particularly important role for p97/VCP is facilitating intracellular protein degradation. p97/VCP has traditionally been thought to mediate the ubiquitin-proteasome degradation of proteins; however, recent studies challenge this dogma. p97/VCP clearly participates in the degradation of aggregate-prone proteins, a process principally mediated by autophagy. In addition, IBMPFD mutations in p97/VCP lead to accumulation of autophagic structures in patient and transgenic animal tissue. This is likely due to a defect in p97/VCP-mediated autophagosome maturation. The following review will discuss the evidence for p97/VCP in autophagy and how a disruption in this process contributes to IBMPFD pathogenesis.

AMP activated protein kinase (AMPK) plays a key role in the regulatory network responsible for maintaining systemic energy homeostasis during exercise or nutrient deprivation. To understand the function of the regulatory ␤2 subunit of AMPK in systemic energy metabolism, we characterized ␤2 subunit-deficient mice. Using these mutant mice, we demonstrated that the ␤2 subunit plays an important role in regulating glucose, glycogen, and lipid metabolism during metabolic stress. The ␤2 mutant animals failed to maintain euglycemia and muscle ATP levels during fasting. In addition, ␤2-deficient animals showed classic symptoms of metabolic syndrome, including hyperglycemia, glucose intolerance, and insulin resistance when maintained on a high-fat diet (HFD), and were unable to maintain muscle ATP levels during exercise. Cell surface-associated glucose transporter levels were reduced in skeletal muscle from ␤2 mutant animals on an HFD. In addition, they displayed poor exercise performance and impaired muscle glycogen metabolism. These mutant mice had decreased activation of AMPK and deficits in PGC1␣-mediated transcription in skeletal muscle. Our results highlight specific roles of AMPK complexes containing the ␤2 subunit and suggest the potential utility of AMPK isoform-specific pharmacological modulators for treatment of metabolic, cardiac, and neurological disorders. AMP activated protein kinase (AMPK) plays a crucial role in maintaining systemic energy homeostasis through its coordinated actions on the central nervous system and peripheral tissues (54). Loss of AMPK function causes metabolic abnormalities in mice and defects in development and growth, cell polarity, and structure in plants, Drosophila, and rodents (1, 10, 33). Moreover, AMPK is necessary for caloric restriction-mediated longevity in Caenorhabditis elegans (19). AMPK is an intracellular energy sensor. When intracellular levels of ATP decrease, a corresponding increase in AMP leads to activation of AMPK, a step that is vital for restoring intracellular energy balance via AMPK-dependent inhibition of energy-consuming biosynthetic processes and concomitant activation of pathways that increase ATP production.AMPK is a multisubstrate, heterotrimeric serine/threonine kinase consisting of one ␣, one ␤, and one ␥ subunit. The mammalian genome encodes two ␣, two ␤, and three ␥ subunits. The N terminus of the ␣ subunit contains the catalytic domain as well as a phosphorylation site for upstream kinases that regulate its activity (9). The ␥ subunits are nucleotide binding regulatory subunits that bind AMP. The conserved C terminus of the ␤ subunit interacts with both the ␣ and ␥ subunits and plays an obligatory role in AMPK complex formation (10, 46). In addition, ␤ subunits contain a conserved carbohydrate-binding domain that allows AMPK to function as a glycogen sensor (37,42).While AMPK is present in all tissues, the individual AMPK subunits display considerable variation in tissue-specific expression, subunit association, and subcellular localization. Earlie...

Autophagy is dysfunctional in many degenerative diseases including myopathies. Mutations in valosin-containing protein (VCP) cause inclusion body myopathy (IBM) associated with Paget's disease of the bone, fronto-temporal dementia and amyotrophic lateral sclerosis (IBMPFD/ALS). VCP is necessary for protein degradation via the proteasome and lysosome. IBMPFD/ALS mutations in VCP disrupt autophagosome and endosome maturation resulting in vacuolation, weakness and muscle atrophy. To understand the regulation of autophagy in VCP-IBM muscle, we examined the AKT/FOXO3 and mammalian target of rapamycin (mTOR) pathways. Basal Akt and FOXO3 phosphorylation was normal. In contrast, the phosphorylation of mTOR targets was decreased. Consistent with this, global protein translation was diminished and autophagosome biogenesis was increased in VCP-IBM muscle. Further mTORC1 inhibition with rapamycin hastened weakness, atrophy and vacuolation in VCP-IBM mice. This was accompanied by the accumulation of autophagic substrates such as p62, LC3II and ubiquitinated proteins. The decrease in mTOR signaling was partially rescued by insulin and to a lesser extent by amino acid (AA) stimulation in VCP-IBM muscle. Cells expressing catalytically inactive VCP or treated with a VCP inhibitor also failed to activate mTOR upon nutrient stimulation. Expression of a constitutively active Rheb enhanced mTOR activity and increased the fiber size in VCP-IBM mouse skeletal muscle. These studies suggest that VCP mutations may disrupt mTOR signaling and contribute to IBMPFD/ALS disease pathogenesis. Treatment of some autophagic disorders with mTOR inhibitors such as rapamycin may worsen disease.