Y2 and Y4 receptor signaling synergistically act on energy expenditure and physical activity

Neuropeptide Y receptors are critical regulators of energy homeostasis and are well known for their powerful influence on feeding, but their roles in other important aspects of energy homeostasis, such as energy expenditure and their functional interactions in these processes, are largely unknown. Here we show that mice lacking both Y2 and Y4 receptors exhibited a reduction in adiposity, more prominent in intra-abdominal vs. subcutaneous fat, and an increase in lean mass as determined by dual-energy X-ray absorptiometry. These changes were more pronounced than those seen in mice with Y2 or Y4 receptor single deletion, demonstrating the important roles and synergy of Y2 and Y4 signaling in the regulation of body composition. These changes in body composition occurred without significant changes in food intake, but energy expenditure and physical activity were significantly increased in Y4(-/-) and particularly in Y2(-/-)Y4(-/-) but not in Y2(-/-) mice, suggesting a critical role of Y4 signaling and synergistic interactions with Y2 signaling in the regulation of energy expenditure and physical activity. Y2(-/-) and Y4(-/-) mice also exhibited a decrease in respiratory exchange ratio with no further synergistic decrease in Y2(-/-)Y4(-/-) mice, suggesting that Y2 and Y4 signaling each play important and independent roles in the regulation of substrate utilization. The synergy between Y2 and Y4 signaling in regulating fat mass may be related to differences in mitochondrial oxidative capacity, since Y2(-/-)Y4(-/-) but not Y2(-/-) or Y4(-/-) mice showed significant increases in muscle protein levels of peroxisome proliferator-activated receptor (PPAR)γ coactivator (PGC)-1α, and mitochondrial respiratory chain complexes I and III. Taken together, this work demonstrates the critical roles of Y2 and Y4 receptors in the regulation of body composition and energy metabolism, highlighting dual antagonism of Y2 and Y4 receptors as a potentially effective anti-obesity treatment.     

The Angiogenic Inhibitor Long Pentraxin PTX3 Forms an Asymmetric Octamer with Two Binding Sites for FGF2

The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility, and vascular biology (e.g. it inhibits FGF2 (fibroblast growth factor 2)-mediated angiogenesis). PTX3 is composed of multiple protomers, each composed of distinct N- and C-terminal domains; however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer), giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer, explaining both its quaternary organization and how this is required for its antiangiogenic function.     

Heterogeneity of Collagen VI Microfibrils: STRUCTURAL ANALYSIS OF NON-COLLAGENOUS REGIONS*

Collagen VI, a collagen with uncharacteristically large N- and C-terminal non-collagenous regions, forms a distinct microfibrillar network in most connective tissues. It was long considered to consist of three genetically distinct α chains (α1, α2, and α3). Intracellularly, heterotrimeric molecules associate to form dimers and tetramers, which are then secreted and assembled to microfibrils. The identification of three novel long collagen VI α chains, α4, α5, and α6, led to the question if and how these may substitute for the long α3 chain in collagen VI assembly. Here, we studied structural features of the novel long chains and analyzed the assembly of these into tetramers and microfibrils. N- and C-terminal globular regions of collagen VI were recombinantly expressed and studied by small angle x-ray scattering (SAXS). Ab initio models of the N-terminal globular regions of the α4, α5, and α6 chains showed a C-shaped structure similar to that found for the α3 chain. Single particle EM nanostructure of the N-terminal globular region of the α4 chain confirmed the C-shaped structure revealed by SAXS. Immuno-EM of collagen VI extracted from tissue revealed that like the α3 chain the novel long chains assemble to homotetramers that are incorporated into mixed microfibrils. Moreover, SAXS models of the C-terminal globular regions of the α1, α2, α4, and α6 chains were generated. Interestingly, the α1, α2, and α4 C-terminal globular regions dimerize. These self-interactions may play a role in tetramer formation.     

Concepts in modelling N2O emissions from land use

Modelling nitrous oxide (N₂O) emissions from soil is challenging because multiple biological processes are involved that each respond differently to various environmental and soil factors. Soil water content, organic carbon, temperature and pH are often used in models that predict N₂O emissions, yet for each of these factors there are concepts that are not fully understood. Though a ubiquitous measure of soil water for models, the application of functions based on water filled pore space across soils that vary in bulk density is not ideal. Diffusion of gases and solutes in soil are controlled by the volume fractions of air and water present. Across soils with different bulk densities, both of these terms vary at constant water filled pore space. Soil organic carbon influences N₂O emissions in two ways: as a source of energy for denitrifiers and also by driving biological oxygen demand and the creation of anaerobic zones in the soil. Soil temperature influences N₂O emissions through its effect on the activity of microorganisms and enzymes. A variety of temperature response functions have been proposed. The preferred response function should contain a temperature optimum that can be varied in response to climatic conditions to account for microbial adaptation. Soil pH can have direct and indirect influences on rates and product ratios of nitrification and denitrification. The concepts of pH optima and microbial adaptation need to be considered in modelling. Methodological issues such as microsite versus bulk soil measurements and apportioning N₂O fluxes to the various N transformation processes remain an impediment to characterising the influence of pH and other factors on N₂O emissions. Quantifying the response of N₂O emissions to individual factors using regression analysis requires all other factors to be controlled experimentally. Boundary line analysis provides a way of defining the response to a single input variable where other influencing variables are not controlled. Such analyses can aid in the definition of the shape and magnitude of response functions to be incorporated into process simulation models. Process/mechanistic simulation models offer a greater transferability than empirical models but careful consideration of temporal and spatial scale and the availability of data to run these models is critical in developing model structure.     

Conditional deletion of hypothalamic Y2 receptors reverts gonadectomy-induced bone loss in adult mice

Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment.     

Marfan syndrome-causing mutations in fibrillin-1 result in gross morphological alterations and highlight the structural importance of the second hybrid domain

Mutations in fibrillin-1 result in Marfan syndrome, which affects the cardiovascular, skeletal and ocular systems. The multiorgan involvement and wide spectrum of associated phenotypes highlights the complex pathogenesis underlying Marfan syndrome. To elucidate the genotype to phenotype correlations, we engineered four Marfan syndrome causing mutations into a fibrillin-1 fragment encoded by exons 18-25, a region known to interact with tropoelastin. Biophysical and biochemical approaches, including small angle x-ray scattering, analytical ultracentrifugation, and circular dichroism, were used to study the impact of these mutations upon the structure and function of the protein. Mutations G880S, C862R, and C908R, situated within the second hybrid domain, disrupted the ratio of alpha-helix to beta-sheet leading to a more compact conformation. These data clearly demonstrate the importance of the previously uncharacterized hybrid domain in fibrillin-1 structure. In contrast, mutation K1023N situated within the linker region between the third eight cysteine motif and cbEGF 11 markedly extended the length of the fragment. However, none of the mutations affected tropoelastin binding. The profound effects of all four mutations on fragment conformation suggest that they contribute to the pathogenesis of Marfan syndrome by disrupting protein folding and its assembly into fibrillin-rich microfibrils.     

Assembly of the Respiratory Mucin MUC5B: A NEW MODEL FOR A GEL-FORMING MUCIN*

Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D′D3; NT5B) and subdomains (D1, D1-D2, D2-D′-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.     

Prader-Willi Critical Region,a Non-Translated,Imprinted Central Regulator of Bone Mass: Possible Role in Skeletal Abnormalities in Prader-Willi Syndrome

Prader-Willi Syndrome (PWS), a maternally imprinted disorder and leading cause of obesity, is characterised by insatiable appetite, poor muscle development, cognitive impairment, endocrine disturbance, short stature and osteoporosis. A number of causative loci have been located within the imprinted Prader-Willi Critical Region (PWCR), including a set of small non-translated nucleolar RNA’s (snoRNA). Recently, micro-deletions in humans identified the snoRNA Snord116 as a critical contributor to the development of PWS exhibiting many of the classical symptoms of PWS. Here we show that loss of the PWCR which includes Snord116 in mice leads to a reduced bone mass phenotype, similar to that observed in humans. Consistent with reduced stature in PWS, PWCR KO mice showed delayed skeletal development, with shorter femurs and vertebrae, reduced bone size and mass in both sexes. The reduction in bone mass in PWCR KO mice was associated with deficiencies in cortical bone volume and cortical mineral apposition rate, with no change in cancellous bone. Importantly, while the length difference was corrected in aged mice, consistent with continued growth in rodents, reduced cortical bone formation was still evident, indicating continued osteoblastic suppression by loss of PWCR expression in skeletally mature mice. Interestingly, deletion of this region included deletion of the exclusively brain expressed Snord116 cluster and resulted in an upregulation in expression of both NPY and POMC mRNA in the arcuate nucleus. Importantly, the selective deletion of the PWCR only in NPY expressing neurons replicated the bone phenotype of PWCR KO mice. Taken together, PWCR deletion in mice, and specifically in NPY neurons, recapitulates the short stature and low BMD and aspects of the hormonal imbalance of PWS individuals. Moreover, it demonstrates for the first time, that a region encoding non-translated RNAs, expressed solely within the brain, can regulate bone mass in health and disease.