Inhibition of myostatin prevents microgravity-induced loss of skeletal muscle mass and strength.

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.

The metabolic effects of GDF15 are mediated by the orphan receptor GFRAL.

Growth/differentiation factor 15 (GDF15), also known as MIC-1, is a distant member of the transforming growth factor-ß (TGF-ß) superfamily and has been implicated in various biological functions, including cancer cachexia, renal and heart failure, atherosclerosis and metabolism. A connection between GDF15 and body-weight regulation was initially suggested on the basis of an observation that increasing GDF15 levels in serum correlated with weight loss in individuals with advanced prostate cancer. In animal models, overexpression of GDF15 leads to a lean phenotype, hypophagia and other improvements in metabolic parameters, suggesting that recombinant GDF15 protein could potentially be used in the treatment of obesity and type 2 diabetes. However, the signaling and mechanism of action of GDF15 are poorly understood owing to the absence of a clearly identified cognate receptor. Here we report that GDNF-family receptor α-like (GFRAL), an orphan member of the GFR-α family, is a high-affinity receptor for GDF15. GFRAL binds to GDF15 in vitro and is required for the metabolic actions of GDF15 with respect to body weight and food intake in vivo in mice. Gfral-/- mice were refractory to the effects of recombinant human GDF15 on body-weight, food-intake and glucose parameters. Blocking the interaction between GDF15 and GFRAL with a monoclonal antibody prevented the metabolic effects of GDF15 in rats. Gfral mRNA is highly expressed in the area postrema of mouse, rat and monkey, in accordance with previous reports implicating this region of the brain in the metabolic actions of GDF15 (refs. 4,5,6). Together, our data demonstrate that GFRAL is a receptor for GDF15 that mediates the metabolic effects of GDF15.

Myostatin Neutralization Results in Preservation of Muscle Mass and Strength in Preclinical Models of Tumor-Induced Muscle Wasting.

Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non-tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass- and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non-tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting.

Increased brain bio-distribution and chemical stability and decreased immunogenicity of an engineered variant of GDNF.

Several lines of evidence indicate that Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for dopaminergic neurons. Direct parenchymal administration of GDNF is robustly neuroprotective and neurorestorative in multiple neurotoxin-based animal models (rat and non-human primate (NHP)) of Parkinson's Disease (PD), suggesting its potential as a therapeutic agent. Although small, open-label clinical trials of intra-putamenal administration of bacteria-derived, full length, wild type GDNF (GDNFwt) were efficacious in improving standardized behavioral scores, a double-blinded, randomized controlled trial failed to do so. We hypothesize that the lack of clinical efficacy of GDNFwt in the larger randomized trial was due to poor bio-distribution in the putamen and/or poor chemical stability while in the delivery device for prolonged time periods at 37°C. The development of neutralizing antibodies in some patients may also have been a contributing factor. GDNFv is an engineered form of GDNFwt, expressed and purified from mammalian cells, designed to overcome these limitations, including removal of the N-terminal heparin-binding domain to improve its diffusivity in brain parenchyma by reducing its binding to extracellular matrix (ECM), and key amino acid substitutions to improve chemical stability. Intra-striatal administration of a single injection of GDNFv in the rat produced significantly greater brain distribution than GDNFwt, consistent with reduced binding to ECM. Using liquid chromatography/mass spectrometry (LS/MS) methods GDNFv was shown to have improved chemical stability compared to GDNFwt when stored at 37°C for 4weeks. In addition, GDNFv resulted in lower predicted clinical immunogenicity compared to GDNFwt, as demonstrated by reduced CD4+ T cell proliferation and reduced IL-2-induced secretion in peripheral blood mononucleated cells collected from volunteers representing the world's major histocompatibility complex (MHC) haplotypes. GDNFv was demonstrated to be pharmacologically equivalent to GDNFwt in the key parameters in vitro of GFRα1 receptor binding, c-Ret phosphorylation, neurite outgrowth, and in vivo in its ability to increase dopamine turnover (DA). GDNFv protected dopamine nerve terminals and neurons in a 6-hydroxy-dopamine (6-OHDA) rat model. In summary, we empirically demonstrate the superior properties of GDNFv compared to GDNFwt through enhanced bio-distribution and chemical stability concurrently with decreased predicted clinical immunogenicity while maintaining pharmacological and neurotrophic activity. These data indicate that GDNFv is an improved version of GDNF suitable for clinical assessment as a targeted regenerative therapy for PD.

Segment-specific regulation of the Drosophila AP-2 gene during leg and antennal development.

Segmentation involves subdivision of a developing body part into multiple repetitive units during embryogenesis. In Drosophila and other insects, embryonic segmentation is regulated by genes expressed in the same domain of every segment. Less is known about the molecular basis for segmentation of individual body parts occurring at later developmental stages. The Drosophila transcription factor AP-2 gene, dAP-2, is required for outgrowth of leg and antennal segments and is expressed in every segment boundary within the larval imaginal discs. To investigate the molecular mechanisms generating the segmentally repetitive pattern of dAP-2 expression, we performed transgenic reporter analyses and isolated multiple cis-regulatory elements that can individually or cooperatively recapitulate endogenous dAP-2 expression in different segments of the appendages. We further analyzed an enhancer specific for the proximal femur region which corresponds to the distal-most expression domain of homothorax (hth) in the leg imaginal discs. Hth is known to be responsible for the nuclear localization and, hence, function of the Hox cofactor, Extradenticle (Exd). We show that both Hth and Exd are required for dAP-2 expression in the femur and that a conserved Exd/Hox binding site is essential for enhancer activity. Our loss- and gain-of-function studies further support direct regulation of dAP-2 by Hox proteins and suggest that Hox proteins function redundantly in dAP-2 regulation. Our study reveals that discrete segment-specific enhancers underlie the seemingly simple repetitive expression of dAP-2 and provides evidence for direct regulation of leg segmentation by regional combinations of the proximodistal patterning genes.

FGF21 attenuates lipolysis in human adipocytes - a possible link to improved insulin sensitivity.

Fibroblast growth factor 21 (FGF21) is active in murine adipocytes and has beneficial metabolic effects in animal models of type 2 diabetes mellitus. We assessed whether FGF21 influences lipolysis in human adipocytes and 3T3-L1 cells. FGF21 had no short-time effect (h) while a 3-day incubation with FGF21 attenuated hormone-stimulated lipolysis. FGF21 did not influence the mRNA expression of genes involved in regulating lipolysis, but significantly reduced the expression of the lipid droplet-associated phosphoprotein perilipin without affecting differentiation. Via reduced release of fatty acids into the circulation, the anti-lipolytic effect could be a mechanism through which FGF21 promotes insulin sensitivity in man.

Expression patterns of the transcription factor AP-2alpha during hair follicle morphogenesis and cycling.

AP-2alpha is a member of a family of transcription factors expressed in cells of the epithelial and neural crest lineage. AP-2alpha plays an essential role in embryonic development and in regulation of epithelial gene transcription. To further characterize the role of AP-2alpha in skin biology, we assessed its expression in the skin of C57BL/6J mice during defined stages of hair follicle morphogenesis and cycling. During early hair follicle morphogenesis, AP-2alpha was upregulated in the epidermal placode, in the basal keratinocytes of the hair follicle bud, and then in the inner root sheath. The follicular papilla cells underwent a brief upregulation of AP-2alpha expression during the initiation of hair shaft formation and active hair follicle downward growth. Completion of hair follicle morphogenesis was associated with a marked reduction of AP-2alpha immunoreactivity in the lower portion of the hair follicle including both epithelial and mesenchymal compartments. In adolescent mouse skin, consistently strong AP-2alpha expression was found in the basal keratinocytes of the epidermis, in the hair follicle infundibulum, and in the sebocytes. In the follicular papilla, AP-2alpha was weakly expressed in telogen, significantly upregulated in early anagen, then gradually declined, and reappeared again in middle catagen. In the inner root sheaths, AP-2alpha expression was detected during early and middle anagen and during middle catagen stages. Prominent AP-2alpha expression was also seen in the zone of club hair formation. Therefore, AP-2alpha upregulation in both epithelial and mesenchymal hair follicle compartments was coordinated with initiation of major remodeling processes. Our findings support the use of the hair follicle as a model to explore the role of AP-2alpha in physiologic remodeling of developing organs and in reciprocal ectodermal-mesenchymal interactions.

Insulin-like growth factor I stimulates myoblast expansion and myofiber development in the limb.

Insulin-like growth factor I (IGF-I) is expressed in the anterior and posterior mesodermal cells of the developing limb. However, a definite role for IGF-I during early limb organogenesis is unknown. To determine the inherent participation of IGF-I during limb organ development, a retroviral delivery system (RCAS) was used to overexpress IGF-I throughout the developing hind limb of stage 24 chicken embryos. The area of the belly of the external gastrocnemius muscle in the IGF-I infected limb was an average of 160, 90, 70, and 80% larger than the contralateral control muscle belly, 4, 5, 6, and 7 days postinjection, respectively (all differences P < 0.01). In comparison to the contralateral control muscles, there were a significantly greater number of muscle fibers in the IGF-I infected muscles (P < 0.05), confirming that the majority of IGF-I-mediated muscle enlargement was due to an increase in total fiber numbers (hyperplasia). Four days postinjection, there was a 32% increase in myoblast to myofiber ratio in the muscle of injected limbs compared with the muscle in the contralateral noninjected control limbs (P < 0.05). This result demonstrates that IGF-I acts to expand the undifferentiated myoblast population, and as a result, more myofibers subsequently develop, and the muscles expressing ectopic IGF-I are enlarged by means of hyperplasia. There was no difference in tibiotarsus and fibula length or diameter between the IGF-I injected and control limb, suggesting that ectopic IGF-I expression within the mesoderm was not a nonspecific growth stimulant of all tissues of the developing limb, but specifically enhanced skeletal muscle development and growth. Ectopic IGF-I expression had no significant effect on myostatin mRNA concentrations. Our results support a model where mesodermally expressed IGF-I acts to regulate the number of primary myofibers, and, therefore, size of skeletal muscles, which form during the initial events of limb myogenesis.