NPY Neuron-Specific Y2 Receptors Regulate Adipose Tissue and Trabecular Bone but Not Cortical Bone Homeostasis in Mice

Background

Y2 receptor signalling is known to be important in neuropeptide Y (NPY)-mediated effects on energy homeostasis and bone physiology. Y2 receptors are located post-synaptically as well as acting as auto receptors on NPY-expressing neurons, and the different roles of these two populations of Y2 receptors in the regulation of energy homeostasis and body composition are unclear.

Methodology/Principal Findings

We thus generated two conditional knockout mouse models, Y2^lox/lox and NPYCre/+;Y2^lox/lox, in which Y2 receptors can be selectively ablated either in the hypothalamus or specifically in hypothalamic NPY-producing neurons of adult mice. Specific deletion of hypothalamic Y2 receptors increases food intake and body weight compared to controls. Importantly, specific ablation of hypothalamic Y2 receptors on NPY-containing neurons results in a significantly greater adiposity in female but not male mice, accompanied by increased hepatic triglyceride levels, decreased expression of liver cartinine palmitoyltransferase (CPT1) and increased expression of muscle phosphorylated acetyl-CoA carboxylase (ACC). While food intake, body weight, femur length, bone mineral content, density and cortical bone volume and thickness are not significantly altered, trabecular bone volume and number were significantly increased by hypothalamic Y2 deletion on NPY-expressing neurons. Interestingly, in situ hybridisation reveals increased NPY and decreased proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus of mice with hypothalamus-specific deletion of Y2 receptors in NPY neurons, consistent with a negative feedback mechanism between NPY expression and Y2 receptors on NPY-ergic neurons.

Conclusions/Significance

Taken together these data demonstrate the anti-obesogenic role of Y2 receptors in the brain, notably on NPY-ergic neurons, possibly via inhibition of NPY neurons and concomitant stimulation of POMC-expressing neurons in the arcuate nucleus of the hypothalamus, reducing lipogenic pathways in liver and/or skeletal muscle in females. These data also reveal as an anti-osteogenic effect of Y2 receptors on hypothalamic NPY-expressing neurons on trabecular but not on cortical bone.     

Protein adsorption and transport in cation exchangers with a rigid backbone matrix with and without polymeric surface extenders

We compare the properties and protein adsorption characteristics of two polymeric cation exchangers: UNOsphere S, which has an open macroporous architecture, and Nuvia S, which is based on a very similar backbone matrix but contains sulfonated polymeric surface extenders. A monoclonal IgG and lysozyme were used as model adsorbates. The characteristic pore sizes, determined by inverse size exclusion chromatography, were about 140 nm for UNOsphere S, and only about 10 nm for Nuvia S, indicating that the polymeric extenders occupy a substantial portion of the base matrix pores. Greater exclusion limits were found for Nuvia S in 1 M NaCl and for a similar matrix containing uncharged surface extenders, suggesting that the polymeric extenders collapse partially at high ionic strength or when they are uncharged. Large equilibrium binding capacities were obtained for Nuvia S, approaching 320 ± 10 mg/mL of particle volume for both proteins in comparison with the UNOsphere S values of 170 ± 10 and 120 ± 10 mg/mL for lysozyme and IgG, respectively. Much higher adsorption rates were also found for Nuvia S, and the rate was nearly independent of protein concentration in solution. Confocal laser scanning microscopy showed very sharp intraparticle protein concentration profiles for UNOsphere S, consistent with a pore diffusion mechanism but diffuse concentration profiles for Nuvia S, consistent with a solid diffusion mechanism. The improved capacity and transport afforded by the polymeric extenders provide substantial potential benefits for bioprocess applications without sacrificing the desirable flow properties of the backbone matrix.     

Fire Season Effects on Flowering Characteristics and Germination of Longleaf Pine (Pinus palustris) Savanna Grasses

Pinus palustris (longleaf pine) savannas depend on contiguous grass cover to facilitate frequent surface fires. Enhanced flowering, seed production, and germination may be linked to season of fires. We assessed the effect of month of prescribed fire (February, April, May, and July) on percentage of plants with flowering culms (FCs%), FC/area, FC/plant, seed production, and germination for five warm seasons, fall‐flowering grasses. Multivariate analysis indicated the response of flowering and fruiting to burn month varied among the grass species. The dominant species, Sporobolus junceus and Schizachyrium scoparium var. stoloniferum, had greater numbers of most flowering characteristics when burnt during April, May, and July. Aristida purpurascens had increased FC/plant after May and July burns. In contrast, Ar. mohrii had the fewest FC/plant and seeds/FC when burnt in July. Germination was greatest (26–60%) for Ar. purpurascens. Seeds collected following July burns for Ar. purpurascens and Ar. ternarius and after May burns for S. junceus were within the highest germination values recorded. Germination of Sc. scoparium var. stoloniferum was very low after February and July burns (≤5%). With Ar. mohrii, only seed collected following February (2%) and April burns (3%) germinated. April, May, and July fires increased seed production of dominant matrix grasses, thus facilitating the potential for recruitment of these species and facilitating seed collection from potential donor sites for ground‐layer restoration projects. Varying prescribed fire burn month captured variation in flowering characteristics among these grasses.     

Survival and Growth of Planted Uniola paniculata and Dune Building Using Surrogate Wrack on Perdido Key Florida,U.S.A.

The increasing number of stresses on coastal dune ecosystems requires the use of more effective restoration strategies to enhance dune‐building and increase vegetation reestablishment. In this study, the use of a wheat straw as a surrogate wrack was an effective method to improve growth of spring planted Uniola paniculata (sea oats). Approximately 1,000 U. paniculata plugs were planted within 21 × 4 m plots at six replicate sites. Two weeks later, plantings were divided into 11 × 4 m subplots with half of the subplots receiving five bales of wheat straw and the remaining subplots receiving no wheat straw. This surrogate wrack layer measured approximately 10 cm in depth. Mean aboveground biomass of U. paniculata 6 months after planting with surrogate wrack was 9.25 ± 1.00 g compared with 2.18 ± 0.24 g without surrogate wrack. Number of tillers, tiller height, and basal width were also greater at the end of the first growing season for plants treated with surrogate wrack (p < 0.05). Two years after planting, significantly more inflorescences occurred and aboveground biomass (g/m²) was greater with than without surrogate wrack. Sand accumulation was notably greater with surrogate wrack (11.16 cm) than without wrack (7.78 cm) 8 months after planting (p = 0.1093). However, relative sand accumulation was significantly greater with than without surrogate wrack 2 years after planting. Increased sand accumulation suggests surrogate wrack either directly or indirectly traps more sand by creating an additional obstacle or promoting the growth of dune grasses.     

Prognostic factors of improvement in health-related quality of life in atomoxetine-treated children and adolescents with attention-deficit/hyperactivity disorder,based on a pooled analysis

The objective of this study is to identify prognostic factors of treatment response to atomoxetine in improvement of health-related quality of life (HR-QoL), measured by the Child Health and Illness Profile-Child Edition Parent Report Form (CHIP-CE PRF) Achievement and Risk Avoidance domains, in children and adolescents with attention-deficit/hyperactivity disorder (ADHD). Pooled data from 3 placebo-controlled trials and separate data from 3 open-label trials of atomoxetine in children and adolescents with ADHD were analyzed using logistic regression methods. Based on baseline impairment in the Achievement and/or Risk Avoidance domains (CHIP-CE PRF < 40 points), 2 subsamples of subjects were included. Treatment outcome was categorized as <5 points or ≥5 points increase in the CHIP-CE PRF Achievement and Risk Avoidance domains. Data of 190 and 183 subjects from the pooled sample, and 422 and 355 subjects from the open-label trials were included in the analysis of Achievement and Risk Avoidance domains. Baseline CHIP-CE subdomain scores proved to be the most robust prognostic factors for treatment outcome in both domains, based on data from the pooled sample of double-blind studies and from the individual open-label studies (odds ratios [OR] 0.74–1.56, p < 0.05; OR < 1, indicating a worse baseline score associated with worse odds of responding). Initial treatment response (≥25 % reduction in ADHD Rating Scale scores in the first 4–6 weeks) was another robust prognostic factor, based on data from the open-label studies (OR 2.99–6.19, p < 0.05). Baseline impairment in HR-QoL and initial treatment response can be early prognostic factors of atomoxetine treatment outcome in HR-QoL in children and adolescents with ADHD.     

New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle

Despite the pivotal role of creatine (Cr) and phosphocreatine (PCr) in muscle metabolism, relatively little is known about sarcolemmal creatine transport, creatine transporter (CRT) isoforms, and subcellular localization of the CRT proteins. To be able to quantify creatine transport across the sarcolemma, we have developed a new in vitro assay using rat sarcolemmal giant vesicles. The rat giant sarcolemmal vesicle assay reveals the presence of a specific high-affinity and saturable transport system for Cr in the sarcolemma (Michaelis-Menten constant 52.4 +/- 9.4 microM and maximal velocity value 17.3 +/- 3.1 pmol x min(-1) x mg vesicle protein(-1)), which cotransports Cr into skeletal muscle together with Na(+) and Cl(-) ions. The regulation of Cr transport in giant vesicles by substrates, analogs, and inhibitors, as well as by phorbol 12-myristate 13-acetate and insulin, was studied. Two antibodies raised against COOH- and NH(2)-terminal synthetic peptides of CRT sequences both recognize two major polypeptides on Western blots with apparent molecular masses of 70 and 55 kDa, respectively. The highest CRT expression occurs in heart, brain, and kidney, and although creatine kinase is absent in liver cells, CRT is also found in this tissue. Surprisingly, immunofluorescence staining of cultured adult rat heart cardiomyocytes with specific anti-CRT antibodies, as well as cell fractionation and cell surface biotinylation studies, revealed that only a minor CRT species with an intermediate molecular mass of approximately 58 kDa is present in the sarcolemma, whereas the previously identified major CRT-related protein species of 70 and 55 kDa are specifically located in mitochondria. Our studies indicate that mitochondria may represent a major compartment of CRT localization, thus providing a new aspect to the current debate about the existence and whereabouts of intracellular Cr and PCr compartments that have been inferred from [(14)C]PCr/Cr measurements in vivo as well as from recent in vivo NMR studies.     

De novo mutations of the gene encoding the histone acetyltransferase KAT6B cause Genitopatellar syndrome

Genitopatellar syndrome (GPS) is a rare disorder in which patellar aplasia or hypoplasia is associated with external genital anomalies and severe intellectual disability. Using an exome-sequencing approach, we identified de novo mutations of KAT6B in five individuals with GPS; a single nonsense variant and three frameshift indels, including a 4 bp deletion observed in two cases. All identified mutations are located within the terminal exon of the gene and are predicted to generate a truncated protein product lacking evolutionarily conserved domains. KAT6B encodes a member of the MYST family of histone acetyltranferases. We demonstrate a reduced level of both histone H3 and H4 acetylation in patient-derived cells suggesting that dysregulation of histone acetylation is a direct functional consequence of GPS alleles. These findings define the genetic basis of GPS and illustrate the complex role of the regulation of histone acetylation during development.     

Y1 and Y5 receptors are both required for the regulation of food intake and energy homeostasis in mice

Neuropeptide Y (NPY) acting in the hypothalamus is one of the most powerful orexigenic agents known. Of the five known Y receptors, hypothalamic Y1 and Y5 have been most strongly implicated in mediating hyperphagic effects. However, knockout of individual Y1 or Y5 receptors induces late-onset obesity--and Y5 receptor knockout also induces hyperphagia, possibly due to redundancy in functions of these genes. Here we show that food intake in mice requires the combined actions of both Y1 and Y5 receptors. Germline Y1Y5 ablation in Y1Y5(-/-) mice results in hypophagia, an effect that is at least partially mediated by the hypothalamus, since mice with adult-onset Y1Y5 receptor dual ablation targeted to the paraventricular nucleus (PVN) of the hypothalamus (Y1Y5(Hyp/Hyp)) also exhibit reduced spontaneous or fasting-induced food intake when fed a high fat diet. Interestingly, despite hypophagia, mice with germline or hypothalamus-specific Y1Y5 deficiency exhibited increased body weight and/or increased adiposity, possibly due to compensatory responses to gene deletion, such as the decreased energy expenditure observed in male Y1Y5(-/-) animals relative to wildtype values. While Y1 and Y5 receptors expressed in other hypothalamic areas besides the PVN--such as the dorsomedial nucleus and the ventromedial hypothalamus--cannot be excluded from having a role in the regulation of food intake, these studies demonstrate the pivotal, combined role of both Y1 and Y5 receptors in the mediation of food intake.