Association study in eating disorders: TPH2 associates with anorexia nervosa and self‐induced vomiting

Twin studies suggest that genetic factors play a substantial role in anorexia nervosa (AN) and self‐induced vomiting (SV), a key symptom that is shared among different types of eating disorders (EDs). We investigated the association of 25 single nucleotide polymorphisms (SNPs), capturing 71–91% of the common variance in candidate genes, stathmin (STMN1), serotonin receptor 1D (HTR1D), tryptophan hydroxylase 2 (TPH2) and brain‐derived neurotrophic factor (BDNF), with AN and EDs characterized by regular SV. The first allele frequencies of all the SNPs were compared between a Dutch case group (182 AN, 149 EDs characterized by SV) and 607 controls. Associations rendering P‐values < 0.05 from this initial study were then tested for replication in a meta‐analysis with two additional independent ED case–control samples, together providing 887 AN cases, 306 cases with an ED characterized by SV and 1914 controls. A significant effect for the minor C‐allele of tryptophan hydroxylase 2 rs1473473 was observed for both AN [odds ratio (OR) = 1.30, 95% CI 1.08–1.57, P < 0.003] and EDs characterized by SV (OR = 1.52, 95% CI 1.28–2.04, P < 0.006). In the combined case group, a dominant effect was observed for rs1473473 (OR = 1.38, 95% CI 1.16–1.64, P < 0.0003). The meta‐analysis revealed that the tryptophan hydroxylase 2 polymorphism rs1473473 was associated with a higher risk for AN, EDs characterized by SV and for the combined group.     

Melanocortin-4 receptor gene mutations in a Dutch cohort of obese children

The most common monogenic form of obesity is caused by mutations in the gene encoding the melanocortin-4 receptor (MC4R). We have screened the MC4R coding sequence in 291 patients of a Dutch outpatient pediatric obesity clinic. We analyzed the minimal promoter region of the gene in a random subgroup of 217 children. Our aims were (i) to determine the frequency of MC4R mutations in a cohort of Dutch clinically obese children and (ii) to search for mutations in the promoter of the gene. Eleven MC4R coding variants were detected. Five children had mutations that have been shown to affect receptor function by other research groups (p.Y35X, p.I251fs, p.G231S). These children did not have earlier onset of obesity or higher BMI-SDS than the remainder of the cohort. One child had a novel nonsynonymous coding mutation (p.L304F). This variant showed a markedly decreased cell surface expression in in vitro experiments and is thus expected to be pathogenic. We detected 12 variants in the MC4R flanking regions. Five of these were not previously described (c.-1101C>T, c.-705A>T, c.-461A>G, c.-312T>C, c.-213A>G). We investigated these mutations by family studies and a bioinformatic approach. We conclude that rare heterozygous mutations in the coding sequence of MC4R account for some severe obesity cases in the Dutch population. These patients are difficult to recognize in a clinical setting. We generated a list of all MC4R variants that were described in the literature so far, which can aid the interpretation of mutations found in a diagnostic setting.     

Adverse effects of members of the Enterobacteriaceae family on boar sperm quality

Semen samples collected in 2012 from 1785 boars belonging to five different breeds were recruited from the quality control laboratory of Magapor SL, Spain. These samples came from 43 boar studs and resulted from diluting the ejaculates in commercial semen extenders. Evaluation of the semen sample characteristics (color, smell, pH, osmolality, concentration, motility of sperm cells, agglutination, acrosome integrity, short hypoosmotic swelling test, and abnormal forms) revealed that they met the international standards. The samples were also tested for the presence of aerobic bacterial contamination. In the present study, 14.73% (n = 263) of the semen samples were contaminated above 3 × 10² colony-forming units/mL with at least one type of bacteria. The Enterobacteriaceae family was by far the major contaminant, being present in 40.68% of the contaminated samples (n = 107). Bacterial strains of the Enterobacteriaceae family isolated from boar semen samples were in order of incidence (percentage of the contaminated samples): Serratia marcescens (12.55%), Klebsiella oxytoca (11.79%), Providencia stuartii (9.12%), Morganella morganii (3.80%), Proteus mirabilis (1.90%), and Escherichia coli (1.52%). We have seen that the presence in semen samples of S. marcescens, K. oxytoca, M. morganii, or P. mirabilis, but not P. stuartii or E. coli, was negatively associated with sperm motility (P < 0.05). The mean sperm concentration (P < 0.05), the mean percentage of spermatozoa with curled tails after the short hypoosmotic swelling test (P < 0.01), and the incidence of morphologically normal acrosomes (P < 0.05) were also lower in semen samples infected with M. morganii compared with uninfected ones. Moreover, P. mirabilis was negatively associated with the presence of abnormal forms. Thus, on the basis of the pathological effects that some of these strains may have on boar sperm quality, bacterial contamination should always be examined in semen samples prepared for artificial insemination.     

Longitudinal Changes in the Physical Activity of Adolescents with Anorexia Nervosa and Their Influence on Body Composition and Leptin Serum Levels after Recovery

Objective

Patients with anorexia nervosa (AN) are often observed to have high levels of physical activity, which do not necessarily diminish after a successful therapy. Previous studies have shown that body fat tissue recovery in these patients is associated with a disproportional restoration of the adipocyte hormone, leptin. Therefore, we wondered whether the individual variation in physical activity in AN patients prior to treatment may be related to body fat percentage and plasma leptin level outcome.

Method

Body fat percentage, leptin serum, and physical activity levels (accelerometer) were measured in adolescents with an (n=37, age 13 to 17.5 years) at initial assessment, at the end of study participation (median 12 months), and at one-year follow-up.

Results

Accelerometer data were used to split the patients in two groups: those with low (n=26) and those with high levels of physical activity (HLPA, n=11). These groups did not differ in terms of age, IQ, presence of menses, BMI and season of admission. The HLPA group was characterized by a longer total duration of illness. Physical activity levels during therapy decreased for the group with initially HLPA and increased for the group with low levels of physical activity (to comparable levels). Physical activity remained stable after one year. The increase in body fat percentage and leptin levels were dependent on the recovery status; however, recovered patients with initially HLPA had significantly higher fat mass during the follow-up.

Discussion

HLPA, an important modulator of AN progression in adolescents, can be successfully diminished by therapeutic intervention. Among recovered patients, those with initially HLPA had higher fat mass levels than those with low levels of physical activity. This finding suggests that HLPA are an important modulator of the body composition recovery mechanism.     

Differential changes of neuroactive amino acids in samples obtained from discrete rat brain regions after systemic administration of saxitoxin

Aspartic acid, glutamic acid, γ-amino-n-butyric acid (GABA) and 2-aminoethanesulfonic acid are neuroactive amino acids. They are found in the central rat nervous system. Here, we have studied if a relationship exists between the presence of saxitoxin (STX) a paralytic poisoning shellfish (PSP) and the neuroactive amino acids. Samples of striatum (S), hypothalamus (H), mid brain (MB), frontal cortex (FC), brain stem (BS), right hemisphere (RH) and left hemisphere (LH) of rat brain were collected and analyzed for neuroactive amino acids (AAn_t) by Aswad method (1984). Experiments, consisting of intraperitoneal injection of SXT (5 and 10 μg kg^?1 body weight) to young male rats, evoked significant changes in AAn_t above basal values. Aspartic and glutamic acid significantly increased for RH and LH (after 30 min the increased was 116% and 210%, P ≤ 0.001 over basal values, respectively). On the other hand, aspartic, glutamic, taurine and GABA significantly decreased for S (after 30 min the decreased was 77.4%; 84%; 93.8% and 95.3%, P ≤ 0.001 over basal values, respectively). These results suggest that STX alters AAn_t. It is produced at least in part, because STX blocks voltage-gated sodium channels and this blockade could decrease AAn_t release by exocytotic dependent mechanism of depolarization.     

Ligands for FKBP12 Increase Ca2+ Influx and Protein Synthesis to Improve Skeletal Muscle Function

Rapamycin at high doses (2–10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 μg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca²⁺ influx to enhance refilling of sarcoplasmic reticulum Ca²⁺ stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function.     

AICAR prevents heat-induced sudden death in RyR1 mutant mice independent of AMPK activation

Mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation that is associated with malignant hyperthermia in humans, die when exposed to short periods of temperature elevation (≥37 °C). We show here that treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevents this heat-induced sudden death in this mouse model. The protection by AICAR is independent of AMP-activated protein kinase (AMPK) activation and results from a newly identified action of the compound on mutant Ryr1 to reduce Ca(2+) leak from the sarcoplasmic reticulum to the sarcoplasm. AICAR thus prevents Ca(2+)-dependent increases in the amount of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) that act to further increase resting Ca(2+) concentrations. If unchecked, the temperature-driven increases in resting Ca(2+) concentrations and the amounts of ROS and RNS create an amplifying cycle that ultimately triggers sustained muscle contractions, rhabdomyolysis and death. Although antioxidants are effective in reducing this cycle in vitro, only AICAR prevents heat-induced death in vivo. Our findings suggest that AICAR is probably effective in prophylactic treatment of humans with enhanced susceptibility to exercise- and/or heat-induced sudden death associated with RYR1 mutations.     

NPY receptor subtype specification for behavioral adaptive strategies during limited food access

The neuropeptide Y (NPY) system in the brain regulates a wide variety of behavioral, metabolic and hormonal homeostatic processes required for energy balance control. During times of limited food availability, NPY promotes behavioral hyperactivity necessary to explore and prepare for novel food resources. As NPY can act via 5 different receptor subtypes, we investigated the path through which NPY affects different behavioral components relevant for adaptation to such conditions. We tested NPY Y1 and Y2 receptor knockout mice and their wild-type littermate controls in a daily scheduled limited food access paradigm with unlimited access to running wheel. Here we show that NPY Y1 receptor deficient mice lack the expression of appetitive behavior and that NPY Y2 receptors control the level of hyperactive behavior under these conditions. Thus, receptor specificity determines the differential expression of NPY-mediated behavioral adaptations to overcome a negative energy status.     

Nutritional State Affects the Expression of the Obesity‐Associated Genes Etv5, Faim2, Fto,and Negr1

Obesity is a risk factor for type II diabetes, atherosclerosis, and some forms of cancer. Variation in common measures of obesity (e.g., BMI, waist/hip ratio) is largely explained by heritability. The advent of genome‐wide association studies (GWAS) has made it possible to identify several genetic variants that associate with measures of obesity, but how exactly these genetic variants contribute to overweight has remained largely unresolved. One first hint is given by the fact that many of the associated variants reside in or near genes that act in the central nervous system, which implicates neuronal signaling in the etiology of obesity. Although the brain controls both energy intake and expenditure, it has more capacity to regulate energy intake rather than energy expenditure. In environments where food is abundant, this renders the body prone to weight increases. To gain more insight into the neurobiological mechanisms involved, we set out to investigate the effect of dietary exposure on the expression levels of obesity‐associated genes in the ventro‐medial hypothalamus (VMH)/arcuate nucleus (ARC) and the substantia nigra (SN)/ventral tegmental area (VTA), two brain regions that are implicated in feeding behavior. We show that the expression of Etv5, Faim2, Fto, Negr1 but not Sh2b1 is affected by nutritional state in these two areas, thereby providing insight into the relationship between nutritional state and expression levels of obesity‐associated genes in two brain areas relevant to feeding.     

Comparison of the estrogen responsiveness of the rat and bovine oxytocin gene promoters

DNA sequences in the 5'-flanking region of rat and bovine oxytocin genes were examined for their capacity to confer estrogen responsiveness to their homologous promoters. In contrast to the 5'-flanking region of the rat oxytocin gene, upstream promoter sequences up to 3200 bp of the bovine gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene which were transfected in estrogen receptor expressing MCF-7 cells did not respond to estrogen. Testing 5'-deletion mutants of the rat upstream region linked to the luciferase gene in P19 embryocarcinoma cells co-transfected with an estrogen receptor expression plasmid showed that two regions each associated with approximately 15-fold stimulation of promoter activity were located between nucleotides -172 and -149 and between -148 and +16 in the rat gene. The former region contains the imperfect palindrome GGTGACCTTGACC which differs in one nucleotide from the estrogen response element (ERE) consensus. It is concluded that the corresponding motive CATAACCTTGACC of the bovine gene is not a functional ERE. Thus, the estrogen responsiveness of oxytocin genes is species-dependent.