Biochemical analysis of female mice urine with reference to endocrine function: a key tool for estrus detection.

Species-specific chemical signals released through urine, sweat, saliva and feces are involved in communication between animals. Urinary biochemical constituents along with pheromones may contribute to variation across reproductive cycles and facilitate to estrus detection. Hence, the present study was designed to analyze such biochemical profiles, such as proteins, carbohydrates, lipids, fatty acids, in response with steroid hormones such as estradiol and progesterone. The experimental groups were normal, prepubertal, ovariectomized, and ovariectomized with estrogentreated female mice. In normal mice, the protein and lipid concentrations in urine were significantly higher in proestrus and estrus phases and the quantity of fatty acids was also comparatively higher in estrus. Furthermore, certain fatty acids, namely tridecanoic, palmitic and oleic acids, were present during proestrus and estrus phases, but were exclusively absent in ovariectomized mice. However, the carbohydrate level was equally maintained throughout the four phases of estrous cycle. For successful communication, higher concentrations of protein and specific fatty acids in estrus are directly involved. The significant increase in estradiol at estrus and progesterone at metestrus seems to be of greater importance in the expression pattern of biochemical constituents and may play a notable role in estrous cycle regulation. Thus, we conclude that the variations observed in the concentration of the biochemical constituents depend on the phase of the reproductive cycle as well as hormonal status of animals. The appearance of protein and specific fatty acids during estrus phase raises the possibility to use these as a urinary indicators for estrus detection.

Decreased expression of peroxisome proliferator-activated receptor alpha and liver fatty acid binding protein after partial hepatectomy of rats and mice.

BACKGROUND AIMS: Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. METHODS: Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. RESULTS: PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. CONCLUSIONS: A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.

Gastric bypass in morbid obese patients is associated with reduction in adipose tissue inflammation via N-oleoylethanolamide (OEA)-mediated pathways.

Paradoxically, morbid obesity was suggested to protect from cardiovascular co-morbidities as compared to overweight/obese patients. We hypothesise that this paradox could be inferred to modulation of the "endocannabinoid" system on systemic and subcutaneous adipose tissue (SAT) inflammation. We designed a translational project including clinical and in vitro studies at Geneva University Hospital. Morbid obese subjects (n=11) were submitted to gastric bypass surgery (GBS) and followed up for one year (post-GBS). Insulin resistance and circulating and SAT levels of endocannabinoids, adipocytokines and CC chemokines were assessed pre- and post-GBS and compared to a control group of normal and overweight subjects (CTL) (n=20). In vitro cultures with 3T3-L1 adipocytes were used to validate findings from clinical results. Morbid obese subjects had baseline lower insulin sensitivity and higher hs-CRP, leptin, CCL5 and anandamide (AEA) levels as compared to CTL. GBS induced a massive weight and fat mass loss, improved insulin sensitivity and lipid profile, decreased C-reactive protein, leptin, and CCL2 levels. In SAT, increased expression of resistin, CCL2, CCL5 and tumour necrosis factor and reduced MGLL were shown in morbid obese patients pre-GBS when compared to CTL. GBS increased all endocannabinoids and reduced adipocytokines and CC chemokines. In morbid obese SAT, inverse correlations independent of body mass index were shown between palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA) levels and inflammatory molecules. In vitro, OEA inhibited CCL2 secretion from adipocytes via ERK1/2 activation. In conclusion, GBS was associated with relevant clinical, metabolic and inflammatory improvements, increasing endocannabinoid levels in SAT. OEA directly reduced CCL2 secretion via ERK1/2 activation in adipocytes.

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice.

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.

Total energy expenditure and patterns of activity in 8-10-year-old obese and nonobese children.

Total energy expenditure (TEE) and patterns of activity were measured by means of a heart rate (HR)-monitoring method in a group of 8-10-year-old children including 13 obese children (weight, 46 +/- 10 kg; fat mass: 32 +/- 9%) and 16 nonobese children (weight, 31 +/- 5 kg; fat mass, 18 +/- 5%). Time for sleeping was not statistically different in the two groups of children (596 +/- 33 vs. 582 +/- 43 min; p = NS). Obese children spent more time doing sedentary activities (400 +/- 129 vs. 295 +/- 127 min; p < 0.05) and less time in nonsedentary activities (449 +/- 126 vs. 563 +/- 135 min; p < 0.05) than nonobese children. Time spent in moderate or vigorous activity-i.e., time spent at a HR between 50% of the maximal O2 uptake (peak VO2) and 70% peak VO2 (moderate) and at a HR > or = 70% peak VO2 (vigorous)-was not statistically different in obese and nonobese children (88 +/- 69 vs. 52 +/- 35 min and 20 +/- 21 vs. 16 +/- 13 min, respectively; p = NS). TEE was significantly higher in the obese group than in the nonobese group (9.46 +/- 1.40 vs. 7.51 +/- 1.67 MJ/day; p < 0.01). The energy expenditure for physical activity (plus thermogenesis) was significantly higher in the obese children (3.98 +/- 1.30 vs. 2.94 +/- 1.39 MJ/day; p < 0.05). The proportion of TEE daily devoted to physical activity (plus thermogenesis) was not significantly different in the two groups, as shown by the ratio between TEE and the postabsorptive metabolic rate (PMR): 1.72 +/- 0.25 obese vs 1.61 +/- 0.28 non-obese. In conclusion, in free-living conditions obese children have a higher TEE than do nonobese children, despite the greater time devoted to sedentary activities. The higher energy cost to perform weight-bearing activities as well as the higher absolute PMR of obese children help explain this apparent paradox.

Unstable shoes increase energy expenditure of obese patients.

BACKGROUND: Ergonomic unstable shoes, which are widely available to the general population, could increase daily non-exercise activity thermogenesis as the result of increased muscular involvement. We compared the energy expenditure of obese patients during standing and walking with conventional flat-bottomed shoes versus unstable shoes.¦METHODS: Twenty-nine obese patients were asked to stand quietly and to walk at their preferred walking speed while wearing unstable or conventional shoes. The main outcome measures were metabolic rate of standing and gross and net energy cost of walking, as assessed with indirect calorimetry.¦RESULTS: Metabolic rate of standing was higher while wearing unstable shoes compared with conventional shoes (1.11±0.20 W/kg(-1)vs 1.06±0.23 W/kg(-1), P=.0098). Gross and net energy cost of walking were higher while wearing unstable shoes compared with conventional shoes (gross: 4.20±0.42 J/kg(-1)/m(-1)vs 4.01±0.39 J/kg(-1)/m(-1), P=.0035; net: 3.37±0.41 J/kg(-1)/m(-1)vs 3.21±0.37 J/kg(-1)/m(-1); P=.032).¦CONCLUSION: In obese patients, it is possible to increase energy expenditure of standing and walking by means of ergonomic unstable footwear. Long-term use of unstable shoes may eventually prevent a positive energy balance.

Regulatory T cell defects are associated with autoimmunity in Wiskott-Aldrich Syndrome protein deficient mice

Abstract : The Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive human primary immunodeficiency. It is caused by mutations in the gene encoding the hermatopoietic specific regulator of the actin cytoskeleton Wiskott-Aldrich Syndrome Protein (WASP). Importantly, a majority of affected patients develop autoimmunity including an inflammatory bowel disease (IBD)-like disease. WASP deficient mice share many similarities with the human WAS. One of these similarities is the spontaneous development of colitis. I have focused my dissertation studies on the pathogenesis of colitis in WASP deficient mice. Prior work from our laboratory had shown that lymphocytes were required and that CD4+ T cells sufficient for colitis development. This colitis was associated with a predominant Th2-cytokine skewing. I have contributed in exploring whether the Th2 cytokine IL-4 plays a role in disease maintenance. Using two approaches to neutralize IL-4, we found that this cytokine plays a role in disease maintenance. Natural CD4*CD25*Foxp3* regulatory T cells (nTreg cells) have been implicated in the pathogenesis of several autoimmune disorders. We found that WASP deficient mice have reduced nTreg cell numbers in peripheral lymphoid organs. This was associated with functional defects in suppressing T cell proliferation and preventing colitis induced by transfer of naïve T cells into SCID recipient, which lack lymphocytes. WASP deficiency affected homing of nTreg cells to lymphoid compartments, IL-2-mediated activation and secretion of the immunomodulatory cytokine IL-10. Finally, we could prevent colitis onset via adoptive transfer of WT nTreg cells prior to colitis development. This suggests that nTreg cells dysfunction is one of the mechanisms underlying colitis development in WASP deficient mice. Future directions will aim at deciphering the role of other immune cell types, the bacterial flora, and various cytokines in colitis development in this murine model of colitis. In addition, we believe that colitis in WASP deficient mice could serve as a useful tool to evaluate nTreg cells manipulation as novel therapeutic approach for IBD.

Anti-leishmania effector functions of CD4+ Th1 cells and early events instructing Th2 cell development and susceptibility to Leishmania major in BALB/c mice.

Resistance and susceptibility to infection with the intracellular parasite, Leishmania major, are mediated by parasite-specific CD4+ Th1 and Th2 cells, respectively. It is well established that the protective effect of parasite-specific CD4+ Th1 cells is largely dependent upon the IFN-gamma produced. However, recent results indicate that the effect of Th1 cells on resolution of lesions induced by L. major in genetically resistant mice also requires a functional Fas-FasL pathway of cytotoxicity. In contrast to resistant mice, susceptible BALB/c mice develop aberrant Th2 responses following infection with L. major and consequently suffer progressive disease. These outcomes clearly depends upon the production of interleukin 4 (IL-4) early after infection. We have shown that a burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hrs after infection, occurs within CD4+ T cells that express V beta 4-V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and Th1 responses occurred following infection. The LACK antigen of L. major induced comparable IL-4 production in V beta 4-V alpha 8 CD4+ T cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4-V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex parasite. The IL-4 produced rapidly by these CD4+ T cells induces within 48 hours a state of unresponsiveness to IL-12 among parasite-specific CD4+ T cell precursors by downregulating the IL-12 receptor beta 2 chain expression.

Rôle et mécanisme d'action de la metformine et du telmisartan sur la régulation de la prise alimentaire

Dans ce travail de thèse, nous avons étudié les mécanismes d'action de deux médicaments connus pour diminuer la prise alimentaire et pondérale : la metformine et le telmisartan. Nous avons dans un premier temps étudié les effets de la metformine, un antidiabétique oral connu pour avoir des effets anorexigènes. Les mécanismes hypothalamiques potentiellement impliqués dans la modulation de la prise alimentaire par la metformine ont été étudiés dans trois groupes de rats : un groupe de rats obèses (DIO), un groupe de rats résistants à l'obésité (DR) ainsi qu'un groupe contrôle. A la fin de la période de prise pondérale de six mois, les rats DIO avaient des taux d'ARNm de NPY hypothalamique plus élevés que leurs congénères résistants et contrôles. Chez les DIO ainsi que chez les DR un traitement par metformine induit une baisse significative de la prise alimentaire accompagnée par une baisse du poids. Nous avons pu d'autre part constater que la perte de poids obtenue par un traitement de metformine était corrélée aux taux circulants de leptine avant le traitement. Cet effet s'accompagne d'une augmentation de l'expression du récepteur ObRb au niveau hypothalamique. Dans un second temps, nous avons étudié les effets du telmisartan, un inhibiteur du récepteur à l'angiotensine II ayant une activité agoniste partielle PPARγ. L'influence du telmisartan associé à la pioglitazone sur la prise alimentaire et pondérale a été examinée en étudiant leur effet sur les neuropeptides hypothalamiques responsables du contrôle de la prise alimentaire. Quatre groupes de souris soumises à un régime riche en graisse ont été formés : un groupe placebo, un groupe pioglitazone, un groupe telmisartan et un groupe pioglitazone-telmisartan. Le telmisartan a aboli la prise pondérale induite par une diète riche en graisse ou par un traitement de pioglitazone. Cette diminution était corrélée à une baisse de la prise alimentaire et de l'expression hypothalamique d'AgRP. Cette étude confirme donc les effets anorexigènes du telmisartan et démontre pour la première fois le rôle fonctionnel du telmisartan sur l'expression hypothalamique d'AgRP. English Abstract : In this work, we investigated the effect of two drugs known to have interessants effects on food intake and body weight. First we investigated the hypothalamic mechanisms potentially implicated in the modulation of feeding by the glucose-lowering drug metformin in three different groups of animals: diet-induced obese (DIO) and diet-resistant (DR) male rats as well as lean controls (CT). At the end of the high fat diet period, despite higher leptin levels, DIO rats had higher levels of hypothalamic NPY expression than DR or CT, suggesting a central leptin resistance. In DIO but also in DR rats, metformin treatment induced significant reductions of food intake accompanied by decreases in body weight. Interestingly, the weight loss achieved by metformin was correlated with pre-treatment plasma leptin levels. This effect was paralleled by a stimulation of the expression of the leptin receptor gene (ObRb) in the arcuate nucleus. Next we investigated the antihypertensive drug Telmisartan, an angiotensin II receptor blocker with PPARγ agonistic properties. The influence of telmisartan, of pioglitazone and of their association on weight gain and food intake was assessed by studying their effects on neuro-endocrine mediators involved in food intake. Mice were fed a high fat diet, weightmatched and randomized in four treatment groups: vehicle, pioglitazone, telmisartan and pioglitazone-telmisartan. Telmisartan treatment was found to abolish weight and fat gain in either vehicle or pioglitazone treated mice. This effect was accompanied by a decrease in food intake. The hypothalamic expression of the agouti-related protein and plasma leptin levels show also a decrease under metformin treatment. This study confirms the anorexigenic effects of telmisartan in mice fed a high fat diet, and suggests for the first time a functional role of telmisartan on hypothalamic orexigenic agouti-related protein regulation.

Less mortality but more relapses in experimental allergic encephalomyelitis in CD8-/- mice.

Mice lacking in CD8 were generated from homologous recombination in embryonal stem cells at the CD8 locus and bred with the experimental allergic encephalomyelitis (EAE)-susceptible PL/JH-2u through four backcross generations to investigate the role of CD8+ T cells in this model of multiple sclerosis. The disease onset and susceptibility were similar to those of wild-type mice. However, the mutant mice had a milder acute EAE, reflected by fewer deaths, but more chronic EAE, reflected by a higher frequency of relapse. This suggests that CD8+ T lymphocytes may participate as both effectors and regulators in this animal model.

In Vivo Systematic Analysis of Candida albicans Zn2-Cys6 Transcription Factors Mutants for Mice Organ Colonization.

The incidence of fungal infections in immuno-compromised patients increased considerably over the last 30 years. New treatments are therefore needed against pathogenic fungi. With Candida albicans as a model, study of host-fungal pathogen interactions might reveal new sources of therapies. Transcription factors (TF) are of interest since they integrate signals from the host environment and participate in an adapted microbial response. TFs of the Zn2-Cys6 class are specific to fungi and are important regulators of fungal metabolism. This work analyzed the importance of the C. albicans Zn2-Cys6 TF for mice kidney colonization. For this purpose, 77 Zn2-Cys6 TF mutants were screened in a systemic mice model of infection by pools of 10 mutants. We developed a simple barcoding strategy to specifically detect each mutant DNA from mice kidney by quantitative PCR. Among the 77 TF mutant strains tested, eight showed a decreased colonization including mutants for orf19.3405, orf19.255, orf19.5133, RGT1, UGA3, orf19.6182, SEF1 and orf19.2646, and four an increased colonization including mutants for orf19.4166, ZFU2, orf19.1685 and UPC2 as compared to the isogenic wild type strain. Our approach was validated by comparable results obtained with the same animal model using a single mutant and the revertant for an ORF (orf19.2646) with still unknown functions. In an attempt to identify putative involvement of such TFs in already known C. albicans virulence mechanisms, we determined their in vitro susceptibility to pH, heat and oxidative stresses, as well as ability to produce hyphae and invade agar. A poor correlation was found between in vitro and in vivo assays, thus suggesting that TFs needed for mice kidney colonization may involve still unknown mechanisms. This large-scale analysis of mice organ colonization by C. albicans can now be extended to other mutant libraries since our in vivo screening strategy can be adapted to any preexisting mutants.

Reaction to novelty and spatial orientation in ALPHA1B(-/-) KO mice

Knockout mice lacking alphalb noradrenergic receptors were tested in behavioural experiments to test a possible effect of the absence of this receptor in reaction to novelty and spatial orientation. Reaction to novelty was tested in two experiments. In the first one the mice' latency to exit the first part of a two compartment set-up was measured. The knockout mice were faster to emerge then their littermate controls. Then they were tested in an open-field, in which new objects were added at the second trial. In the open-field without objects (first trial), the knockout mice showed a greater locomotor activity (path length). Then the same mice showed enhanced exploration of the newly introduced objects, relative to the control. The spatial orientation experiments were done on a homing board and in the water maze. The homing board did not yield a significant difference between the knock-out and the control mice. Both groups showed impaired results when the proximal (olfactory) and distal (visual) cues were disrupted by the rotation of the table. In the water maze however, the alphalb(-/-) mice were unable to solve the task (acquisition and retention), whereas the control mice showed a good acquisition and retention behaviour. The knockout mice' incapacity to learn to reach the submerged platform was not due to an incapacity to swim, as they were as good as their control littermates to reach the platform when it was visible.

In Vivo Modulation of Mitochondrial Activity Determines HSC Engraftment and Post-Transplant Survival in Mice

Cellular metabolism is emerging as a potential fate determinant in cancer and stem cell biology, constituting a crucial regulator of the hematopoietic stem cell (HSC) pool [1-4]. The extremely low oxygen tension in the HSC microenvironment of the adult bone marrow forces HSCs into a low metabolic profile that is thought to enable their maintenance by protecting them from reactive oxygen species (ROS). Although HSC quiescence has for long been associated with low mitochondrial activity, as testified by the low rhodamine stain that marks primitive HSCs, we hypothesized that mitochondrial activation could be an HSC fate determinant in its own right. We thus set to investigate the implications of pharmacologically modulating mitochondrial activity during bone marrow transplantation, and have found that forcing mitochondrial activation in the post-transplant period dramatically increases survival. Specifically, we examined the mitochondrial content and activation profile of each murine hematopoietic stem and progenitor compartment. Long-term-HSCs (LT-HSC, Lin-cKit+Sca1+ (LKS) CD150+CD34-), short-term-HSCs (ST-HSC, LKS+150+34+), multipotent progenitors (MPPs, LKS+150-) and committed progenitors (PROG, Lin-cKit+Sca1-) display distinct mitochondrial profiles, with both mitochondrial content and activity increasing with differentiation. Indeed, we found that overall function of the hematopoietic progenitor and stem cell compartment can be resolved by mitochondrial activity alone, as illustrated by the fact that low mitochondrial activity LKS cells (TMRM low) can provide efficient long-term engraftment, while high mitochondrial activity LKS cells (TMRM high) cannot engraft in lethally irradiated mice. Moreover, low mitochondrial activity can equally predict efficiency of engraftment within the LT-HSC and ST-HSC compartments, opening the field to a novel method of discriminating a population of transitioning ST-HSCs that retain long-term engraftment capacity. Based on previous experience that a high-fat bone marrow microenvironment depletes short-term hematopoietic progenitors while conserving their long-term counterparts [5], we set to measure HSC mitochondrial activation in high-fat diet fed mice, known to decrease metabolic rate on a per cell basis through excess insulin/IGF-1 production. Congruently, we found lower mitochondrial activation as assessed by flow cytometry and RT-PCR analysis as well as a depletion of the short-term progenitor compartment in high fat versus control chow diet fed mice. We then tested the effects of a mitochondrial activator known to counteract the negative effects of high fat diet. We first analyzed the in vitro effect on HSC cell cycle kinetics, where no significant change in proliferation or division time was found. However, HSCs responded to the mitochondrial activator by increasing asynchrony, a behavior that is thought to directly correlate with asymmetric division [6]. As opposed to high-fat diet fed mice, mice fed with the mitochondrial activator showed an increase in ST-HSCs, while all the other hematopoietic compartments were comparable to mice fed on control diet. Given the dependency on short-term progenitors to rapidly reconstitute hematopoiesis following bone marrow transplantation, we tested the effect of pharmacological mitochondrial activation on the recovery of mice transplanted with a limiting HSC dose. Survival 3 weeks post-transplant was 80% in the treated group compared to 0% in the control group, as predicted by faster recovery of platelet and neutrophil counts. In conclusion, we have found that mitochondrial activation regulates the long-term to short-term HSC transition, unraveling mitochondrial modulation as a valuable drug target for post-transplant therapy. Identification of molecular pathways accountable for the metabolically mediated fate switch is currently ongoing.