Heparanase affects food intake and regulates energy balance in mice

Mutation of the melanocortin-receptor 4 (MC4R) is the most frequent cause of severe obesity in humans. Binding of agouti-related peptide (AgRP) to MC4R involves the co-receptor syndecan-3, a heparan sulfate proteoglycan. The proteoglycan can be structurally modified by the enzyme heparanase. Here we tested the hypothesis that heparanase plays a role in food intake behaviour and energy balance regulation by analysing body weight, body composition and food intake in genetically modified mice that either lack or overexpress heparanase. We also assessed food intake and body weight following acute central intracerebroventricular administration of heparanase; such treatment reduced food intake in wildtype mice, an effect that was abolished in mice lacking MC4R. By contrast, heparanase knockout mice on a high-fat diet showed increased food intake and maturity-onset obesity, with up to a 40% increase in body fat. Mice overexpressing heparanase displayed essentially the opposite phenotypes, with a reduced fat mass. These results implicate heparanase in energy balance control via the central melanocortin system. Our data indicate that heparanase acts as a negative modulator of AgRP signaling at MC4R, through cleavage of heparan sulfate chains presumably linked to syndecan-3.     

Ethnic Differences in Disability Prevalence and Their Determinants Studied over a 20-Year Period: A Cohort Study

Background

To compare disability prevalence rates in the major ethnic groups in the UK and understand the risk factors contributing to differences identified. It was hypothesised that Indian Asian and African Caribbean people would experience higher rates of disability compared with Europeans.

Methods

Data was collected from 888 European, 636 Indian Asian and 265 African Caribbean men and women, aged 58–88 years at 20-year follow-up of community-based cohort study, based in West London. Disability was measured using a performance-based locomotor function test and self-reported questionnaires on functional limitation, and instrumental (IADL) and basic activities of daily living (ADL).

Results

The mean (SD) age of participants at follow-up was 69.6 (6.2) years. Compared with Europeans, Indian Asian people were significantly more likely to experience all of the disability outcomes than Europeans; this persisted after adjustment for socioeconomic, behavioural, adiposity and chronic disease risk factors measured at baseline (locomotor dysfunction: adjusted odds ratio (OR) 2.20, 95% CI 1.56–3.11; functional limitation: OR 2.77, 2.01–3.81; IADL impairment: OR 3.12, 2.20–4.41; ADL impairment: OR 1.58, 1.11–2.24). In contrast, a modest excess risk of disability was observed in African Caribbeans, which was abolished after adjustment (e.g. locomotor dysfunction: OR 1.37, 0.90–1.91); indeed a reduced risk of ADL impairment appeared after multivariable adjustment (OR from 0.99, 0.68–1.45 to 0.59, 0.38–0.93), compared with Europeans.

Conclusions

Substantially elevated risk of disability was observed among Indian Asian participants, unexplained by known factors. A greater understanding of determinants of disability and normative functional beliefs of healthy aging is required in this population to inform intervention efforts to prevent disability.     

The wild side of life: Drosophila reproduction in nature

Drosophila species vary in the rates at which females remate and the number of sperm they receive in the laboratory. In species such as D. melanogaster and D. pseudoobscura, in which females receive thousands of sperm and remate infrequently compared with species such as D. hydei and D. nigrospiracula, where females receive only a few hundred sperm and remate many times in a day, wild caught females should produce far more progeny. We tested this prediction by collecting, directly from nature, females of six species whose remating rates and number of sperm received vary from high to low and assessing the proportion of females with sperm and the number of progeny females produce. Over 95% of D. pseudoobscura and D. melanogaster females were inseminated while far fewer of the other species contained any sperm. In addition, D, pseudoobscura females produced progeny for over two weeks, D. melanogaster for over a week, while D. hydei and D. nigrospiracula females ran out of sperm after 1-2 d. These observations suggest extreme sperm limitation in these latter species.     

Glutathione serves an extracellular defence function to decrease arsenite accumulation and toxicity in yeast

Arsenic is an environmental toxin and a worldwide health hazard. Since this metalloid is ubiquitous in nature, virtually all living organisms require systems for detoxification and tolerance acquisition. Here, we show that during chronic exposure to arsenite [As(III)], Saccharomyces cerevisiae (budding yeast) exports and accumulates the low‐molecular‐weight thiol molecule glutathione (GSH) outside of cells. Extracellular accumulation of the arsenite triglutathione complex As(GS)₃ was also detected and direct transport assays demonstrate that As(GS)₃ does not readily enter cells. Yeast cells with increased extracellular GSH levels accumulate less arsenic and display improved growth when challenged with As(III). Conversely, cells defective in export and extracellular accumulation of GSH are As(III) sensitive. Taken together, our data are consistent with a novel detoxification mechanism in which GSH is exported to protect yeast cells from arsenite toxicity by preventing its uptake.     

Omega-3 fatty acid deficiency increases stearoyl-CoA desaturase expression and activity indices in rat liver: positive association with non-fasting plasma triglyceride levels

Although omega-3 (n-3) fatty acids negatively regulate triglyceride biosynthesis, the mechanisms mediating this effect are poorly understood, and emerging evidence suggests that stearoyl-CoA desaturase (Scd1) is required for de novo triglyceride biosynthesis. To investigate this mechanism, we determined the effects of perinatal n-3 deficiency and postnatal repletion on rat liver Scd1 mRNA expression and activity indices (liver 16:1/16:0 and 18:1/18:0 ratios), and determined relationships with postprandial (non-fasting) plasma triglyceride levels. Rats were fed conventional diets with or without the n-3 fatty acid precursor α-linolenic acid (ALA, 18:3n-3) during perinatal development (E0-P100), and a subset of rats fed the ALA- diet were switched to the ALA+ diet post-weaning (P21-P100, repletion). Compared with controls, rats fed the ALA- diet exhibited significantly lower liver long-chain n-3 fatty acid compositions and elevations in monounsaturated fatty acid composition, both of which were normalized in repleted rats. Liver Scd1 mRNA expression and activity indices (16:1/16:0 and 18:1/18:0 ratios) were significantly greater in n-3 deficient rats compared with controls and repleted rats. Among all rats, liver Scd1 mRNA expression was positively correlated with liver 18:1/18:0 and 16:1/16:0 ratios. Plasma triglyceride levels, but not glucose or insulin levels, were significantly greater in n-3 deficient rats compared with controls and repleted rats. Liver Scd1 mRNA expression and activity indices were positively correlated with plasma triglyceride levels. These preclinical findings demonstrate that n-3 fatty acid status is an important determinant of liver Scd1 mRNA expression and activity, and suggest that down-regulation of Scd1 is a mechanism by which n-3 fatty acids repress constitutive triglyceride biosynthesis.     

Standardized LC-MS/MS based steroid hormone profile-analysis

In order to overcome many limitations of immunoassays, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has the potential to find its place in the clinical laboratory medicine for quantification of steroid hormones. A prerequisite for the application of a new analytical procedure in clinical diagnostics is standardization to minimize analytical intra- and interlaboratory variability and inaccuracy. We evaluate a newly standardized HPLC-MS/MS assay in kit-format, developed for routine determination of 16 steroid hormones in human serum samples. Fifteen metabolites can be measured quantitatively, which include aldosterone, androstenedione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17β-estradiol (E2), estrone (E1), etiocholanolone, 17α-hydroxyprogesterone (17OHP), progesterone, and testosterone. 11-Deoxycorticosterone is the only compound rated as semi-quantitative in this kit. The sample preparation is performed by solid phase extraction (SPE) on a 96-well plate. The standardized assay has been validated for human serum in terms of lower and upper limit of quantification (LLOQ 0.01-32 ng/mL, ULOQ 5-8000 ng/mL), linear correlation coefficient of calibration (R(2)>0.9966), intra- and inter-day precision (intra-day 1.1-8.8%, inter-day 5.2-14.8% and 8.2-18.6% for 11-deoxycorticosterone), accuracy (intra-day 88.3-115.5% and 109.3-128.2% for 11-deoxycorticosterone, inter-day 91.4-117.2% and 102.3-137.1% for 11-deoxycorticosterone), analytical total error (3.6-17.8%), proficiency test accuracy (85.4-113.4%), recovery (68-99%), and metabolite stability (freeze/thaw stability 95.5-108.1%, short term stability 86.9-107.2%). Inter-assay comparison with a routine reference HPLC-MS/MS assay and seven immunoassays demonstrates the outstanding high performance of this HPLC-MS/MS based kit by improvements in accuracy for progesterone, androstenedione, and 17OHP. Finally, results of two metyrapone tests demonstrate the potential of the standardized HPLC-MS/MS assay for the analysis of a comprehensive steroid hormone profile in clinical diagnostics.     

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady-state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox systems have distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H₂O₂ bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol-rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.     

In situ characterization of antibody grafting on porous monolithic supports

The efficient immobilization of antibodies on monolithic support is one of the most critical steps when preparing immunoaffinity supports. In this work, the ADECA (amino density estimation by colorimetric assay) method was adapted to tridimensional supports (in a dynamic mode) and proved to be efficient to characterize the antibodies grafting efficiency on 15.3±0.9mg porous glycidyl methacrylate (GMA)-co-ethylene dimethacrylate (EDMA) monolithic columns. The amount of grafted antibodies measured in situ on the monolith by ADECA (8.2±0.2μg of antibodies per milligram of monolith) was consistent with values obtained by bicinchoninic acid assay (BCA) after crushing the monolith. ADECA was shown to be less time-consuming and more versatile than BCA. The ADECA method was further implemented to thoroughly study and optimize the antibody grafting conditions (influence of pH and kinetics of the grafting step) on GMA-based monoliths and to check the covalent nature of the antibody/surface linking and its stability. Using the total amount of grafted antibodies and the amount of recognized antigen, we found that 65±6% of antibodies were able to capture their antigen. Finally, the grafting of Fab and F(ab')(2) fragments demonstrated that no significant improvement of the global binding capacity of the monolith was obtained.     

Moose (Alces alces) calf survival rates in the presence of wolves (Canis lupus) in southeast Norway

Wolves (Canis lupus) are recolonizing Scandinavia and becoming a new limiting factor that should be taken into account in the management of moose (Alces alces). However, there is a lack of empirical estimates of moose survival after wolf recolonization. We investigated the effects of wolf abundance, moose litter size (single/twin calves), and climatic factors on annual and seasonal calf survival rates in a moose population in southeast Norway. We used data that were obtained over 7?years by radio-tracking and regular visual observations of 68 moose cows to determine the presence or absence of calves at heel. Annual and winter calf survival rates were 20–40 percentage points lower in the wolf territory compared with previous estimates of moose calf survival in similar areas that lacked wolves. Cause-specific studies of mortality would further enhance our ability to determine the relative role of various limiting factors. Our study suggests that moose managers should regulate quotas to buffer the lower survival rates after wolf recolonization.