The effects of a soluble activin type IIB receptor on obesity and insulin sensitivity.

BACKGROUND: Myostatin, also known as Growth and Differentiation Factor 8, is a secreted protein that inhibits muscle growth. Disruption of myostatin signaling increases muscle mass and decreases glucose, but it is unclear whether these changes are related. We treated mice on chow and high-fat diets with a soluble activin receptor type IIB (ActRIIB, RAP-031), which is a putative endogenous signaling receptor for myostatin and other ligands of the TGF-beta superfamily. RESULTS: After 4 weeks, RAP-031 increased lean and muscle mass, grip strength and contractile force. RAP-031 enhanced the ability of insulin to suppress glucose production under clamp conditions in high-fat fed mice, but did not significantly change insulin-mediated glucose disposal. The hepatic insulin-sensitizing effect of RAP-031 treatment was associated with increased adiponectin levels. RAP-031 treatment for 10 weeks further increased muscle mass and drastically reduced fat content in mice on either chow or high-fat diet. RAP-031 suppressed hepatic glucose production and increased peripheral glucose uptake in chow-fed mice. In contrast, RAP-031 suppressed glucose production with no apparent change in glucose disposal in high-fat-diet mice. CONCLUSION: Our findings show that disruption of ActRIIB signaling is a viable pharmacological approach for treating obesity and diabetes.

Harnessing the potential of dystrophin-related proteins for ameliorating Duchenne's muscular dystrophy.

Duchenne's muscular dystrophy (DMD) is a fatal disease caused by mutations in the DMD gene that lead to quantitative and qualitative disturbances in dystrophin expression. Dystrophin is a member of the spectrin superfamily of proteins. Dystrophin itself is closely related to three proteins that constitute a family of dystrophin-related proteins (DRPs): the chromosome 6-encoded DRP or utrophin, the chromosome-X encoded, DRP2 and the chromosome-18 encoded, dystrobrevin. These proteins share sequence similarity and functional motifs with dystrophin. Current attempts at somatic gene therapy of DMD face numerous technical problems. An alternative strategy for DMD therapy, that circumvents many of these problems, has arisen from the demonstration that the DRP utrophin can functionally substitute for the missing dystrophin and its overexpression can rescue dystrophin-deficient muscle. Currently, a promising avenue of research consists of identifying molecules that would increase the expression of utrophin and the delivery of these molecules to dystrophin-deficient tissues as a means of DMD therapy. In this review, we will focus on DRPs from the perspective of strategies and issues related to upregulating utrophin expression for DMD therapy. Additionally, we will address the techniques used for anatomical, biochemical and physiological evaluation of the potential benefits of this and other forms of DMD therapy in dystrophin-deficient animal models.

The role of cGMP hydrolysing phosphodiesterases 1 and 5 in cerebral artery dilatation.

The aim was to investigate the presence and activity of cGMP hydrolysing phosphodiesterases in guinea pig basilar arteries and the effect of selective and non-selective phosphodiesterase inhibitors on cerebral artery dilatation involving the nitric oxide (NO)-guanosine cyclic 3'5-monophosphate (cGMP) pathway. Immunoreactivity to phosphodiesterases 1A, 1B and 5, but not phosphodiesterase 1C was found in fractions of homogenised cerebral arteries eluted by high-pressure liquid chromatography (HPLC). Both the phosphodiesterase 1 inhibitor 8-methoxymethyl-1-methyl-3-(2methylpropyl)-xanthine (8-MM-IBMX) and the phosphodiesterase 5 inhibitors zaprinast and dipyridamole induced dilatation of cerebral arteries. The dilatory response to 8-MM-IBMX was reduced by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 microM) and endothelial removal and restored by sodium nitroprusside (0.1 microM) pretreatment, indicating a close relation to the nitric oxide-cGMP pathway. The responses to zaprinast and dipyridamole, however, were not only moderately affected, but also restored by sodium nitroprusside (0.1 microM) pretreatment. At high concentrations, the dilatory effects of zaprinast and dipyridamole were partly caused by cGMP-independent mechanisms. Targeting the phosphodiesterases present in cerebral arteries, with selective inhibitors or activators of phosphodiesterase, may be a possible new way of treating cerebrovascular disease.

Identification of genes that are differentially expressed in extraocular and limb muscle.

The extraocular muscles (EOM) are anatomically and physiologically distinct from other striated muscles in mammals. Among other differences, they can be driven to generate individual twitch contractions at an extremely high frequency and are resistant to [Ca(2+)]-induced myonecrosis. While EOM are preferentially targeted in some neuromuscular diseases such as myasthenia gravis and congenital fibrosis of the extraocular muscles, they are enigmatically spared in Duchenne's muscular dystrophy, despite the widespread damage seen in all other skeletal muscle groups during the course of this disease. To address the molecular mechanisms that specify the EOM-phenotype, we characterized the transcriptional profile of genes expressed in rat EOM versus limb muscle using a differential display strategy. Ninety-five putative differentially expressed cDNA tags were cloned, from which fourteen were confirmed as being differentially expressed by RNA slot blot and Northern blot analysis. Ten of these cDNAs were homologous to known human or murine genes and ESTs, while four genes that were upregulated in EOM were novel, and have been named expressed in ocular muscle (eom) 1-4. The identification of these differentially expressed genes may provide mechanistic clues toward understanding the unique patho-physiological phenotype of EOM.

Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS).

The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).

Activation of utrophin promoter by heregulin via the ets-related transcription factor complex GA-binding protein alpha/beta.

Utrophin/dystrophin-related protein is the autosomal homologue of the chromosome X-encoded dystrophin protein. In adult skeletal muscle, utrophin is highly enriched at the neuromuscular junction. However, the molecular mechanisms underlying regulation of utrophin gene expression are yet to be defined. Here we demonstrate that the growth factor heregulin increases de novo utrophin transcription in muscle cell cultures. Using mutant reporter constructs of the utrophin promoter, we define the N-box region of the promoter as critical for heregulin-mediated activation. Using this region of the utrophin promoter for DNA affinity purification, immunoblots, in vitro kinase assays, electrophoretic mobility shift assays, and in vitro expression in cultured muscle cells, we demonstrate that ets-related GA-binding protein alpha/beta transcription factors are activators of the utrophin promoter. Taken together, these results suggest that the GA-binding protein alpha/beta complex of transcription factors binds and activates the utrophin promoter in response to heregulin-activated extracellular signal-regulated kinase in muscle cell cultures. These findings suggest methods for achieving utrophin up-regulation in Duchenne's muscular dystrophy as well as mechanisms by which neurite-derived growth factors such as heregulin may influence the regulation of utrophin gene expression and subsequent enrichment at the neuromuscular junction of skeletal muscle.

Expression patterns of transmembrane and released forms of neuregulin during spinal cord and neuromuscular synapse development.

We mapped the distribution of neuregulin and its transmembrane precursor in developing, embryonic chick and mouse spinal cord. Neuregulin mRNA and protein were expressed in motor and sensory neurons shortly after their birth and levels steadily increased during development. Expression of the neuregulin precursor was highest in motor and sensory neuron cell bodies and axons, while soluble, released neuregulin accumulated along early motor and sensory axons, radial glia, spinal axonal tracts and neuroepithelial cells through associations with heparan sulfate proteoglycans. Neuregulin accumulation in the synaptic basal lamina of neuromuscular junctions occurred significantly later, coincident with a reorganization of muscle extracellular matrix resulting in a relative concentration of heparan sulfate proteoglycans at endplates. These results demonstrate an early axonal presence of neuregulin and its transmembrane precursor at developing synapses and a role for heparan sulfate proteoglycans in regulating the temporal and spatial sites of soluble neuregulin accumulation during development.

Neuregulin and erbB receptors play a critical role in neuronal migration.

The migration of neuronal precursors along radial glial fibers is a critical step in the formation of the nervous system. In this report, we show that neuregulin-erbB receptor signaling plays a crucial role in the migration of cerebellar granule cells along radial glial fibers. Granule cells express neuregulin (NRG), and radial glia cells express erbB4 in the developing cerebellum and in vitro. When the glial erbB receptors are blocked, neurons fail to induce radial glia formation, and their migration along radial glial fibers is impaired. Moreover, soluble NRG is as effective as neuron-glia contact in the induction of radial glia formation. These results suggest that the activation of glial erbB4 by NRG is an early critical step in the neuronal migration program.

The concomitant use of dystrophin and utrophin/dystrophin related protein antibodies to reduce misdiagnosis of Duchenne/Becker muscular dystrophy.

Antibodies to dystrophin have increased accuracy in the diagnosis of Duchenne/Becker muscular dystrophy (D/BMD). Both typical and 'atypical' presentations of this disease can be confirmed by demonstrating qualitative and quantitative defects in the expression of dystrophin protein. However, owing to the propensity for dystrophin degradation in vitro, caution needs to be applied while performing and interpreting antibody-based dystrophin analysis. Here we identify two cases where in vitro protein degradation caused diagnostic confusion. We demonstrate the use of utrophin/dystrophin related protein (DRP) as sensitive control for sample degradation, since it is more labile than dystrophin. We suggest that the concomitant or sequential usage of antibodies specific for dystrophin along with utrophin/DRP can help reduce the misdiagnosis of D/BMD.

Cloning and characterization of the human homologue of a dystrophin related phosphoprotein found at the Torpedo electric organ post-synaptic membrane.

Dystrophin is the protein product which is absent in Duchenne muscular dystrophy (DMD). In mammalian skeletal muscle, dystrophin is found in association with several integral and peripheral membrane proteins, forming a complex known as the dystrophin glycoprotein complex (DGC). In an expressed sequence tag (EST) database search to identify new dystrophin related genes, we isolated EST00891 which showed 57% homology to the cysteine-rich domain of dystrophin and localized to 18q12.1-12.2. This EST is also highly homologous (90%) to the Torpedo californica post-synaptic 87 kDa phosphoprotein. Screening human adult brain and skeletal muscle cDNA libraries with this EST resulted in cloning multiple cDNAs which encode several splice forms all homologous to the C-terminal domain of dystrophin. The largest open reading frame isolated shows 94% homology (86% identity) to the Torpedo 87 kDa protein and 50% homology to the cysteine-rich and carboxy-terminal domains of dystrophin. The other cDNAs isolated encode smaller splice forms of this gene which we have named dystrobrevin. The tissue distribution of dystrobrevin mRNA shows five distinct transcripts which are preferentially expressed between different tissues. In addition, antibodies against either the Torpedo 87 kDa protein or human dystrobrevin demonstrate that at least three of the splice forms are translated as proteins in human brain tissue extracts.

Absence of extraocular muscle pathology in Duchenne's muscular dystrophy: role for calcium homeostasis in extraocular muscle sparing.

Duchenne muscular dystrophy (DMD) is characterized by clinical weakness and progressive necrosis of striated muscle as a consequence of dystrophin deficiency. While all skeletal muscle groups are thought to be affected, enigmatically, the extraocular muscles (EOM) appear clinically unaffected. Here we show that dystrophin deficiency does not result in myonecrosis or pathologically elevated levels of intracellular calcium ([Ca2+]i) in EOM. At variance with a previous report, we find no evidence for dystrophin-related protein/utrophin up-regulation in EOM. In vitro experiments demonstrate that extraocular muscles are inherently more resistant to necrosis caused by pharmacologically elevated [Ca2+]i levels when compared with pectoral musculature. We believe that EOM are spared in DMD because of their intrinsic ability to maintain calcium homeostasis better than other striated muscle groups. Our results indicate that modulating levels of [Ca2+]i in muscle may be of potential therapeutic use in DMD.

Interaction of chromosome-6-encoded dystrophin related protein with the extracellular matrix.

Dystrophin-related protein/utrophin is a large, cytoskeletal protein that shares significant sequence similarity with dystrophin. Dystrophin-related protein is known to be enriched where cell-extracellular matrix contacts are well defined; however, the mechanism of dystrophin-related protein enrichment and its functional role(s) at these sites are yet to be defined. Here, we demonstrate that dystrophin-related protein is concentrated in patches of astrocyte membrane in apposition with the extracellular matrix and that the distribution of dystrophin-related protein is temporally modulated by the extracellular matrix constituent laminin. Furthermore, we demonstrate the existence of a specific biochemical association between dystrophin-related protein and laminin in astrocytes. In these astrocytes, the depletion of dystrophin-related protein by the use of antisense dystrophin-related protein oligonucleotides causes marked reduction in the formation of functional substratum-membrane attachments. Taken together, these data suggest that dystrophin-related protein may function in the generation and maintenance of regional substratum-associated membrane specializations, such as those found at the blood-brain barrier.

The subcellular distribution of chromosome 6-encoded dystrophin-related protein in the brain.

Chromosome 6-encoded dystrophin-related-protein (DRP) shows significant structural similarities to dystrophin at the carboxyl terminus, though the two proteins are encoded on different chromosomes. Both DRP and dystrophin are expressed in muscle and brain and show some similarity in their subcellular localization. For example, in skeletal muscle both are expressed at neuromuscular and myotendinous junctions. However, while dystrophin is absent or severely reduced in Duchenne/Becker muscular dystrophy, DRP continues to be expressed. Within the brain, dystrophin is enriched at the postsynaptic regions of specific subsets of neurons, while the distribution of DRP is yet to be described. In this study we demonstrate a distinct though highly specific pattern of distribution of DRP in the brain. DRP is enriched in the choroid plexus, pia mater, intracerebral vasculature, and ependymal lining. Within the parenchyma proper, DRP is located at the inner plasma face of astrocytic foot processes at the abluminal aspect of the blood-brain barrier. The distribution of DRP is conserved across a large evolutionary distance, from mammals to elasmobranchs, suggesting that DRP may play a role in the maintenance of regional specializations in the brain.

Localization of dystrophin and dystrophin-related protein at the electromotor synapse and neuromuscular junction in Torpedo marmorata.

The immunological identification of dystrophin isoforms at the neuromuscular junction and Torpedo marmorata electromotor synapse was attempted using various antibodies. A polyclonal antibody raised against electrophoretically purified dystrophin from T. marmorata electrocyte has been thoroughly investigated. This antibody recognized dystrophin in the electric tissue as well as sarcolemmal and synaptic neuromuscular junction dystrophin in all studies species (T. marmorata, rat, mice and human) at serum dilutions as high as 1:10,000. At variance, no staining of either the sarcolemma or neuromuscular junction was observed in Duchenne muscular dystrophy or mdx mice skeletal muscles. In these muscles, other members of the dystrophin superfamily, in particular the dystrophin-related protein(s) encoded by autosomal genes are present. These data thus demonstrate the specificity of our antibodies for dystrophin. Anti-dystrophin-related protein antibodies [Khurana et al. (1991) Neuromusc. Disorders 1, 185-194] which gave a strong immunostaining of the neuromuscular junction in various species, including T. marmorata, cross-reacted weakly with the postsynaptic membrane of the electrocyte. Taken together, these observations are in favor of the existence of a protein very homologous to dystrophin at the electromotor synapse in T. marmorata, whereas both dystrophin and dystrophin-related protein co-localize at the neuromuscular junction as in all species studied. The electrocyte thus offers the unique opportunity to study the interaction of dystrophin with components of the postsynaptic membrane.

Immunolocalization and developmental expression of dystrophin related protein in skeletal muscle.

Dystrophin Related Protein is the recently identified protein product of a large autosomal transcript, showing significant similarity to dystrophin at the carboxyl terminus. Dystrophin related protein and dystrophin share a similar abundance and molecular weight, however, they differ both in their tissue distribution and expression in Duchenne/Becker muscular dystrophy. Here we define the immunolocalization of dystrophin related protein to neuromuscular and myotendinous junctions, along with peripheral nerves and vasculature of skeletal muscle. Groups of regenerating muscle fibres as well as embryonic and neonatal muscle express far greater amounts of dystrophin related protein compared with adult mdx mice. These findings may explain the paradoxical labelling seen using dystrophin antibodies in Duchenne patients and dystrophin deficient mdx mice. Finally, no abnormalities of dystrophin related protein expression were detected in three patients with Duchenne-like autosomal recessive muscular dystrophy.

Different gene loci for hyperkalemic and hypokalemic periodic paralysis.

The periodic paralyses are dominantly inherited disorders in which patients acutely develop muscle weakness in association with changes in the level of blood potassium. We recently reported genetic linkage of hyperkalemic periodic paralysis (HIKPP) to the gene encoding the adult form of the skeletal muscle sodium channel on the long arm of chromosome 17. In this paper, we exclude genetic linkage between hypokalemic periodic paralysis (HOKPP) and this sodium channel gene, demonstrating that there is non-allelic genetic heterogeneity among different forms of periodic paralysis. Electrophysiological abnormalities in muscle sodium conductance have been reported for both HIKPP and HOKPP as well as other muscle disorders characterized by membrane hyperexcitability or myotonia (myotonia congenita, paramyotonia congenita and the Schwartz-Jampel syndrome). The possibility that there may be a family of human muscle diseases arising from mutations in the sodium channel suggests these disorders may be classified by categories of mutations within this critical voltage-sensitive membrane protein.

Hyperkalemic periodic paralysis and the adult muscle sodium channel alpha-subunit gene.

Hyperkalemic periodic paralysis (HYPP) is an autosomal dominant disorder characterized by episodes of muscle weakness due to depolarization of the muscle cell membrane associated with elevated serum potassium. Electrophysiological studies have implicated the adult muscle sodium channel. Here, portions of the adult muscle sodium channel alpha-subunit gene were cloned and mapped near the human growth hormone locus (GH1) on chromosome 17. In a large pedigree displaying HYPP with myotonia, these two loci showed tight linkage to the genetic defect with no recombinants detected. Thus, it is likely that the sodium channel alpha-subunit gene contains the HYPP mutation.