Losartan Affects Glomerular AKT and mTOR Phosphorylation in an Experimental Model of Type 1 Diabetic Nephropathy

The AKT-mTOR pathway is activated in diabetic nephropathy. Renin-angiotensin system modulators exert beneficial effects on the diabetic kidney. We explored the action of losartan on AKT-mTOR phosphorylation in glomeruli and podocytes. Diabetes mellitus was induced to Sprague-Dawley rats by streptozotocin. Five months later, the rats were commenced on losartan and euthanized 2 months later. Kidneys were processed for immunofluorescence studies. Glomeruli were isolated for Western blot analysis. Diabetes increased activated forms of AKT and mTOR both in glomeruli and podocytes. In diabetic rats, losartan decreased phosphorylated/activated forms of AKT (Thr308) and mTOR (Ser2448) in glomeruli but decreased only activated mTOR in podocytes. However, in both glomeruli and podocytes of healthy animals, an inverse pattern was evident. In conclusion, a new body of evidence indicates the differential activation of AKT-mTOR in glomeruli and podocytes of healthy and diabetic animals in response to losartan.     

A guide to analysis of mouse energy metabolism

We present a consolidated view of the complexity and challenges of designing studies for measurement of energy metabolism in mouse models, including a practical guide to the assessment of energy expenditure, energy intake and body composition and statistical analysis thereof. We hope this guide will facilitate comparisons across studies and minimize spurious interpretations of data. We recommend that division of energy expenditure data by either body weight or lean body weight and that presentation of group effects as histograms should be replaced by plotting individual data and analyzing both group and body-composition effects using analysis of covariance (ANCOVA).     

A proposed mechanism for the inhibitory effect of the anticancer agent titanocene dichloride on tumour gelatinases and other proteolytic enzymes

Titanocene dichloride, the most studied metallocene, exhibits antiproliferative activity in a wide spectrum of murine and human tumours. In this article it is demonstrated that titanocene dichloride inhibits tumour gelatinases in a dose-dependent manner. Substrate saturation experiments and the fact that the IC₅₀ values were increased in correlation with collagen substrate concentrations indicate that the titanocene dichloride induced inhibition is of a competitive type. Titanocene dichloride also specifically inhibits clostridium collagenase and trypsin, particularly when collagens are used as substrates. Binding experiments demonstrate that cyclopentadiene–Ti(IV) moieties, resulting from titanocene dichloride at physiological pH, are bound mainly to different types of collagens and to a lesser extent to casein or bovine serum albumin, forming soluble and stable adducts. These results indicate that titanocene dichloride behaves as a competitive inhibitor against various proteolytic enzymes by binding to the substrate rather than to the enzyme active site. This property may be responsible for the antiangiogenic effect of titanocene dichloride and additionally contributes to its anticancer action.     

Inside the brain of a neuron

For many decades, neurons were considered to be the elementary computational units of the brain and were assumed to summate incoming signals and elicit action potentials only in response to suprathreshold stimuli. Although modelling studies predicted that single neurons constitute a much more powerful computational entity, able to perform an array of nonlinear calculations, this possibility was not explored experimentally until the discovery of active mechanisms in the dendrites of most neuron types. Here, we review several modelling studies that have addressed information processing in single neurons, starting with those characterizing the arithmetic of different dendritic components, to those tackling neuronal integration at the cell body and, finally, those analysing the computational abilities of the axon. We present modelling predictions along with supporting experimental data in an effort to highlight the significant contribution of modelling work to enhancing our understanding of single-neuron arithmetic.     

CCRaVAT and QuTie - enabling analysis of rare variants in large-scale case control and quantitative trait association studies

Background  

Genome-wide association studies have been successful in finding common variants influencing common traits. However, these associations only account for a fraction of trait heritability. There has been a shift in the field towards studying low frequency and rare variants, which are now widely recognised as putative complex trait determinants. Despite this increasing focus on examining the role of low frequency and rare variants in complex disease susceptibility, there is a lack of user-friendly analytical packages implementing powerful association tests for the analysis of rare variants.     

A HIGH‐DENSITY SCAN OF THE Z CHROMOSOME IN FICEDULA FLYCATCHERS REVEALS CANDIDATE LOCI FOR DIVERSIFYING SELECTION

Theoretical and empirical data suggest that genes located on sex chromosomes may play an important role both for sexually selected traits and for traits involved in the build‐up of hybrid incompatibilities. We investigated patterns of genetic variation in 73 genes located on the Z chromosomes of two species of the flycatcher genus Ficedula, the pied flycatcher and the collared flycatcher. Sequence data were evaluated for signs of selection potentially related to genomic differentiation in these young sister species, which hybridize despite reduced fitness of hybrids. Seven loci were significantly more divergent between the two species than expected under neutrality and they also displayed reduced nucleotide diversity, consistent with having been influenced by directional selection. Two of the detected candidate regions contain genes that are associated with plumage coloration in birds. Plumage characteristics play an important role in species recognition in these flycatchers suggesting that the detected genes may have been involved in the evolution of sexual isolation between the species.     

Estradiol‐Induced Anorexia Is Independent of Leptin and Melanin‐Concentrating Hormone

Objective: Treatment of male rodents with estradiol (E₂) is associated with anorexia and weight loss by poorly understood mechanisms. We examined the role of the orexigenic hypothalamic peptide melanin‐concentrating hormone (MCH) and the appetite‐inhibiting, fat‐derived hormone leptin in mediating E₂‐induced anorexia. Research Methods and Procedures: We studied the effect of E₂ treatment (implantation of either E₂ pellet or matching placebo) in male C57Bl/6J mice, as well as in a lean mouse model (MCH knockout mice) and an obese model (leptin‐deficient ob/ob mice). We also studied the effect of E₂ treatment in the context of high‐fat diet. Results: We confirmed E₂ dose‐dependent anorexia in male wild type mice fed a normal chow diet. E₂ treatment was associated with a significant decrease in body fat, serum leptin levels, and arcuate hypothalamic proopiomelanocortin expression. E₂‐implanted mice also showed increased hypothalamic neuropeptide Y and MCH expression. As MCH has been implicated in E₂‐induced hypophagia, we performed E₂ pellet implantation in MCH knockout mice and observed hypophagia and weight loss, indicating that MCH is not an essential mediator of E₂‐induced anorexia. E₂‐implanted ob/ob mice also had hypophagia and weight loss, indicating that leptin is not essential for E₂‐induced anorexia. High‐fat diet significantly exacerbated the effect of E₂ treatment, leading to a 99.6% decrease in food intake at 48 hours and a 30% loss of body weight within 1 week. Discussion: The anorectic effects of E₂ were independent of MCH and leptin. Our results suggested that E₂ may have effects on nutrient preferences.     

Neuropeptides,Including Neuropeptide Y and Melanocortins,Mediate Lipolysis in Murine Adipocytes

Objective: To determine whether key appetite‐regulating neuropeptides such as melanin‐concentrating hormone (MCH), neuropeptide Y (NPY), and α‐melanocyte—stimulating hormone (α‐MSH), which are known to mediate energy balance through centrally mediated pathways, also have direct acute effects on the lipolytic activity of murine adipocytes. Research Methods and Procedures: Fully differentiated 3T3‐L1 adipocytes serum starved overnight in Dulbecco's modified Eagle medium containing 2% bovine serum albumin or freshly isolated mouse adipocytes were incubated for up to 2 hours in the absence and presence of 100 nM each of NPY, MCH, α‐MSH, the melanocortin receptor agonist MTII, or isoproterenol as a control. Free fatty acids secreted into the incubation medium were measured using a commercially available nonesterified fatty acid C test kit. Results: Treatment of 3T3‐L1 cells with 100 nM NPY decreased basal free fatty acid secretion (basal, 0.006 ± 0.001 vs. NPY, 0.001 ± 0.0003 nM at 90 minutes; p < 0.05), whereas both α‐MSH and MTII stimulated up to a 7‐fold increase in free fatty acid release (MTII, 0.238 ± 0.004 vs. basal, 0.024 ± 0.002 nM at 2 hours; p < 0.05; and α‐MSH, 0.22 ± 0.005 vs. basal, 0.04 ± 0.003 nM at 2 hours; p < 0.05). Treatment with 100 nM MCH had no effect on basal free fatty acid release or on α‐MSH—induced lipolysis during concurrent treatment. Conversely, concurrent treatment with 100 nM NPY dramatically inhibited (by ～90%) α‐MSH—induced lipolysis. Similar treatment of freshly isolated mouse adipocytes showed virtually identical results. Discussion: In addition to their centrally mediated actions, appetite‐regulating neuropeptides modulate adipose tissue mass through direct peripheral effects. Systemic administration of pharmacological agents altering the effects of these neuropeptides may form the basis of future obesity therapies. Thus, some of these agents will likely have direct effects on adipocytes that may serve to alter their therapeutic effectiveness.     

Global co-ordination of protein translocation by the SecA IRA1 switch

SecA, the dimeric ATPase subunit of protein translocase, contains a DEAD helicase catalytic core that binds to a regulatory C-terminal domain. We now demonstrate that IRA1, a conserved helix-loop-helix structure in the C-domain, controls C-domain conformation through direct interdomain contacts. C-domain conformational changes are transmitted to the DEAD motor and alter its conformation. These interactions establish DEAD motor/C-domain conformational cross-talk that requires a functional IRA1. IRA1-controlled binding/release cycles of the C-domain to the DEAD motor couple this cross-talk to protein translocation chemistries, i.e. DEAD motor affinities for ligands (nucleotides, preprotein signal peptides, and SecYEG, the integral membrane component of translocase) and ATP turnover. IRA1-mediated global co-ordination of SecA catalysis is essential for protein translocation.     

NPY ablation in C57BL/6 mice leads to mild obesity and to an impaired refeeding response to fasting

Neuropeptide Y (NPY) is an orexigenic (appetite-stimulating) peptide that plays an important role in regulating energy balance. When administered directly into the central nervous system, animals exhibit an immediate increase in feeding behavior, and repetitive injections or chronic infusions lead to obesity. Surprisingly, initial studies of Npy(-/-) mice on a mixed genetic background did not reveal deficits in energy balance, with the exception of an attenuation in obesity seen in ob/ob mice in which the NPY gene was also deleted. Here, we show that, on a C57BL/6 background, NPY ablation is associated with an increase in body weight and adiposity and a significant defect in refeeding after a fast. This impaired refeeding response in Npy(-/-) mice resulted in a deficit in weight gain in these animals after 24 h of refeeding. These data indicate that genetic background must be taken into account when the biological role of NPY is evaluated. When examined on a C57BL/6 background, NPY is important for the normal refeeding response after starvation, and its absence promotes mild obesity.