Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.

Reduced Insulin Clearance And Lower Insulin-degrading Enzyme Expression In The Liver Might Contribute To The Thrifty Phenotype Of Protein-restricted Mice.

Nutrient restriction during the early stages of life usually leads to alterations in glucose homeostasis, mainly insulin secretion and sensitivity, increasing the risk of metabolic disorders in adulthood. Despite growing evidence regarding the importance of insulin clearance during glucose homeostasis in health and disease, no information exists about this process in malnourished animals. Thus, in the present study, we aimed to determine the effect of a nutrient-restricted diet on insulin clearance using a model in which 30-d-old C57BL/6 mice were exposed to a protein-restricted diet for 14 weeks. After this period, we evaluated many metabolic variables and extracted pancreatic islet, liver, gastrocnemius muscle (GCK) and white adipose tissue samples from the control (normal-protein diet) and restricted (low-protein diet, LP) mice. Insulin concentrations were determined using RIA and protein expression and phosphorylation by Western blot analysis. The LP mice exhibited lower body weight, glycaemia, and insulinaemia, increased glucose tolerance and altered insulin dynamics after the glucose challenge. The improved glucose tolerance could partially be explained by an increase in insulin sensitivity through the phosphorylation of the insulin receptor/protein kinase B and AMP-activated protein kinase/acetyl-CoA carboxylase in the liver, whereas the changes in insulin dynamics could be attributed to reduced insulin secretion coupled with reduced insulin clearance and lower insulin-degrading enzyme (IDE) expression in the liver and GCK. In summary, protein-restricted mice not only produce and secrete less insulin, but also remove and degrade less insulin. This phenomenon has the double benefit of sparing insulin while prolonging and potentiating its effects, probably due to the lower expression of IDE in the liver, possibly with long-term consequences.

Reduced Plasma Angiotensin Ii Levels Are Reversed By Hydroxyurea Treatment In Mice With Sickle Cell Disease.

Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin-angiotensin system (RAS) may be altered in an animal model of SCD. Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 (AT1R and AT2R) and the angiotensin-converting enzymes (ACE1 and ACE2) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50-75mg/kg/day, 4weeks) treatment on these parameters were also determined. Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart. Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.

Antioxidant And Anti-diabetic Potential Of Passiflora Alata Curtis Aqueous Leaves Extract In Type 1 Diabetes Mellitus (nod-mice).

Leaves of Passiflora alata Curtis were characterized for their antioxidant capacity. Antioxidant analyses of DPPH, FRAP, ABTS, ORAC and phenolic compounds were made in three different extracts: aqueous, methanol/acetone and ethanol. Aqueous extract was found to be the best solvent for recovery of phenolic compounds and antioxidant activity, when compared with methanol/acetone and ethanol. To study the anti-inflammatory properties of this extract in experimental type 1 diabetes, NOD mice were divided into two groups: the P. alata group, treated with aqueous extract of P. alata Curtis, and a non-treated control group, followed by diabetes expression analysis. The consumption of aqueous extract and water ad libitum lasted 28 weeks. The treated-group presented a decrease in diabetes incidence, a low quantity of infiltrative cells in pancreatic islets and increased glutathione in the kidney and liver (p<0.05), when compared with the diabetic and non-diabetic control-groups. In conclusion, our results suggest that the consumption of aqueous extract of P. alata may be considered a good source of natural antioxidants and compounds found in its composition can act as anti-inflammatory agents, helping in the control of diabetes.

Cardioprotective Mechanism Of S-nitroso-n-acetylcysteine Via S-nitrosated Betadrenoceptor-2 In The Ldlr-/- Mice.

Previous studies from our group have demonstrated the protective effect of S-nitroso-N-acetylcysteine (SNAC) on the cardiovascular system in dyslipidemic LDLr-/- mice that develop atheroma and left ventricular hypertrophy after 15 days on a high fat diet. We have shown that SNAC treatment attenuates plaque development via the suppression of vascular oxidative stress and protects the heart from structural and functional myocardial alterations, such as heart arrhythmia, by reducing cardiomyocyte sensitivity to catecholamines. Here we investigate the ability of SNAC to modulate oxidative stress and cell survival in cardiomyocytes during remodeling and correlation with β₂-AR signaling in mediating this protection. Ventricular superoxide (O₂⁻) and hydrogen peroxide (H₂O₂) generation was measured by HPLC methods to allow quantification of dihydroethidium (DHE) products. Ventricular histological sections were stained using terminal dUTP nick-end labeling (TUNEL) to identify nuclei with DNA degradation (apoptosis) and this was confirmed by Western blot for cleaved caspase-3 and caspase-7 protein expression. The findings show that O₂⁻ and H₂O₂ production and also cell apoptosis were increased during left ventricular hypertrophy (LVH). SNAC treatment reduced oxidative stress during on cardiac remodeling, measured by decreased H₂O₂ and O₂⁻ production (65% and 52%, respectively), and a decrease in the ratio of p-Ser1177 eNOS/total eNOS. Left ventricle (LV) from SNAC-treated mice revealed a 4-fold increase in β₂-AR expression associated with coupling change to Gi; β₂-ARs-S-nitrosation (β₂-AR-SNO) increased 61%, while apoptosis decreased by 70%. These results suggest that the cardio-protective effect of SNAC treatment is primarily through its anti-oxidant role and is associated with β₂-ARs overexpression and β₂-AR-SNO via an anti-apoptotic pathway.

NEURAL CORRELATES OF SCENT MARKING BEHAVIOR IN C57BL/6J MICE: DETECTION AND RECOGNITION OF A SOCIAL STIMULUS

Mice show urinary scent marking behavior as a form of social communication. Marking to a conspecific stimulus mouse or odor varies with stimulus familiarity, indicating discrimination of novel and familiar animals. This study investigated Fos immunoreactivity in inbred C57BL/6J (C57) males following scent marking behavior in response to detection of a social stimulus, or discrimination between a familiar and an unfamiliar conspecific. In Experiment 1 C57 mice were exposed for four daily trials to an empty chamber; on a test day they were exposed to the same chamber or to a male CD-1 mouse in that chamber. Increased scent marking to the CD-1 mouse was associated with increased Fos-immunoreactive cells in the basolateral amygdala, medial amygdala, and dorsal and ventral premammillary nuclei. In Experiment 2 C57 mice were habituated to a CD-1 male for 4 consecutive days and, on the 5th day, exposed to the same CD-1 male, or to a novel CD-1 male. Mice exposed to a novel CD-1 displayed a significant increase in scent marking compared to their last exposure to the familiar stimulus, indicating discrimination of the novelty of this social stimulus. Marking to the novel stimulus was associated with enhanced activation of several telencephalic, as well as hypothalamic and midbrain, structures in which activation had not been seen in the detection paradigm (Experiment 1). These included medial prefrontal and piriform cortices, and lateral septum; the paraventricular nuclei, ventromedial nuclei, and lateral area of the hypothalamus, and the ventrolateral column of the periaqueductal gray. These data suggest that a circumscribed group of structures largely concerned with olfaction is involved in detection of a conspecific olfactory stimulus, whereas discrimination of a novel vs. a familiar conspecific stimulus engages a wider range of forebrain structures encompassing higher-order processes and potentially providing an interface between cognitions and emotions. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Different strains of mice present distinct lung tissue mechanics and extracellular matrix composition in a model of chronic allergic asthma

The impact of genetic factors on asthma is well recognized but poorly understood. We tested the hypothesis that different mouse strains present different lung tissue strip mechanics in a model of chronic allergic asthma and that these mechanical differences may be potentially related to changes of extracellular matrix composition and/or contractile elements in lung parenchyma. Oscillatory mechanics were analysed before and after acetylcholine (ACh) in C57BL/10, BALB/c, and A/J mice, subjected or not to ovalbumin sensitization and challenge. In controls, tissue elastance (E) and resistance (R), collagen and elastic fibres` content, and alpha-actin were higher in A/J compared to BALB/c mice, which, in turn, were more elevated than in C57BL/10. A similar response pattern was observed in ovalbumin-challenged animals irrespective of mouse strain. E and R augmented more in ovalbumin-challenged A/J [E: 22%, R: 18%] than C57BL/10 mice [E: 9.4%, R: 11 %] after ACh In conclusion, lung parenchyma remodelled differently yielding distinct in vitro mechanics according to mouse strain. (C) 2008 Elsevier B.V. All rights reserved.

Influence of Genetic Background on Fluoride Metabolism in Mice

A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis, due to their genetic backgrounds. This study tested whether these differences are due to variations in water intake and/or F metabolism. A/J (susceptible to dental fluorosis) and 129P3/J mice (resistant) received drinking water containing 0, 10, or 50 ppm F. Weekly F intake, excretion and retention, and terminal plasma and femur F levels were determined. Dental fluorosis was evaluated clinically and by quantitative fluorescence (QF). Data were tested by two-way ANOVA. Although F intakes by the strains were similar, excretion by A/J mice was significantly higher due to greater urinary F excretion, which resulted in lower plasma and femur F levels. Compared with 129P3/J mice given 50 ppm F, significantly higher QF scores were recorded for A/J mice. In conclusion, these strains differ with respect to several features of F metabolism, and amelogenesis in the 129P3/J strain seems to be unaffected by high F exposure.

Subcutaneous immunization against Leishmania major - infection in mice: efficacy of formalin-killed promastigotes combined with adjuvants

Formalin-killed promastigotes (FKP) of Leishmania major, in combination with Montanide ISA 720 (MISA), BCG or alum were used in vaccination of an inbred murine model against cutaneous leishmaniasis (CL). Significant and specific increases in anti-FKP IgG responses were detected for both alum-FKP and BCG-FKP compared to MISA-FKP (p < 0.001). Significant increases in splenic lymphocyte recall proliferation was obtained in the MISA-FKP vaccinated mice compared to alum-FKP or BCG-FKP vaccinated groups (p < 0.01). The highest interferon-γ responses were observed in the BCG-FKP group followed by the MISA-FKP while the alum-FKP gave the least responses. Significantly reduced lesion sizes were obtained in the MISA-FKP group compared to the BCG/alum adjuvants-FKP vaccinated groups. Although the BCG-FKP group showed the highest IFN-γ responses, it failed to control cutaneous lesions. Significant reductions in parasite numbers were observed in the MISA-FKP and BCG-FKP vaccinated groups (p < 0.001). There was a good correlation between parasite burden and IFN-γ level indicating IFN-γ response as a sensitive parameter of the immune status. In conclusion, MISA-FKP is the most efficacious vaccine formulation against murine cutaneous leishmaniasis.

Host factors influencing outcome of Leishmania mexicana infection in mice

Studies were undertaken to determine the influence of several host-related parameters on the course of Leishmania mexicana mexicana infection in inbred C57B1/10 (C57) and outbred albino (OA) mice. An important influence of the following variables was demonstrated: Host strain: lesions in C57s were significantly less variable in size and outcome than those of OAs under the conditions studied and even when persistent developed at a slower rate. Host age: Subcutanous injection of 2 x 10 [raised to the power of 4] to 2 x 10 [raised to the power of 6] amastigotes into the dorsum of the rear paw produced significantly larger lesions which healed more slowly in 2 mo. old C57s than in 4 mo. old mice. Reduced healing ability was observed in older (8 mo. old) female C57s, and low mortality occurred after 15 months of age in infected mice of both sexes. Lesion site: Following amastigote infection, lesions in paws of most C57s regress within 15 - 25 wks. In contrast, perinasal legions produced with the same number of parasites tend to persist for the life of the animal as slowly spreading irregular nodules. In animals infected in both locations, each lesion site behaves similarly to that in singly infected animals of the same age, i.e. regression in the two sites is independent. Our results indicate that while host strain may strongly influence infection outcoem, such variables as lesion site and host age play important roles and may explain, in part, reported inter- and intraexperimental variability in responses of murine hosts to a given leishmanial parasite.

The conduction system of the heart in mice chronically infected with Trypanosoma cruzi: histopathological lesions and electrocardiographic correlations

Chronic focal and diffuse myiocarditis with interstitial fibrosis developed in Swiss outbred mice and in the inbred AKR and A/J strains of mice which were chronically infected with several Trypanosoma cruzi strains belonging to three biological types (Type I, II and III). High incidence of electrocardiographic changes with predominance of intraventricular conduction disturbances, 1st. and 2nd. degree AV block, arrhythmias, comparable with those found in human Chagas' disease, were also present. Morphological study of the conduction tissue of the heart revealed inflammatory and fibrotic changes. The presence of inflammation in the inter-atrial septum almost always coincided with the inflammatory involvement of the ventricular conduction system. Focal inflammation was associated with vacuolization and focal necrosis of the specific fibers. Most of the lesions were seen affecting the His bundel (76.3% of the cases), the right bundle branch (73.3%), AV node (43.9%) and left bundle branch (37.5%). Correlation between morphological changes in the conduction tissue and electrocardiographic alteration occured in 53.0 to 62.5% of the cases, according to the experimental groups.

Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2.

FGF-2 has been implicated in the cardiac response to hypertrophic stimuli. Angiotensin II (Ang II) contributes to maintain elevated blood pressure in hypertensive individuals and exerts direct trophic effects on cardiac cells. However, the role of FGF-2 in Ang II-induced cardiac hypertrophy has not been established. Therefore, mice deficient in FGF-2 expression were studied using a model of Ang II-dependent hypertension and cardiac hypertrophy. Echocardiographic measurements show the presence of dilated cardiomyopathy in normotensive mice lacking FGF-2. Moreover, hypertensive mice without FGF-2 developed no compensatory cardiac hypertrophy. In wild-type mice, hypertrophy was associated with a stimulation of the c-Jun N-terminal kinase, the extracellular signal regulated kinase, and the p38 kinase pathways. In contrast, mitogen-activated protein kinase (MAPK) activation was markedly attenuated in FGF-2-deficient mice. In vitro, FGF-2 of fibroblast origin was demonstrated to be essential in the paracrine stimulation of MAPK activation in cardiomyocytes. Indeed, fibroblasts lacking FGF-2 expression have a defective capacity for releasing growth factors to induce hypertrophic responses in cardiomyocytes. Therefore, these results identify the cardiac fibroblast population as a primary integrator of hypertrophic stimuli in the heart, and suggest that FGF-2 is a crucial mediator of cardiac hypertrophy via autocrine/paracrine actions on cardiac cells.

D-amphetamine fails to increase extracellular dopamine levels in mice lacking alpha 1b-adrenergic receptors: relationship between functional and nonfunctional dopamine release.

It was found recently that locomotor and rewarding effects of psychostimulants and opiates were dramatically decreased or suppressed in mice lacking alpha1b-adrenergic receptors [alpha1b-adrenergic receptor knock-outs (alpha1bAR-KOs)] (Drouin et al., 2002). Here we show that blunted locomotor responses induced by 3 and 6 mg/kg d-amphetamine in alpha1bAR-KO mice [-84 and -74%, respectively, when compared with wild-type (WT) mice] are correlated with an absence of d-amphetamine-induced increase in extracellular dopamine (DA) levels in the nucleus accumbens of alpha1bAR-KO mice. Moreover, basal extracellular DA levels in the nucleus accumbens are lower in alpha1bAR-KO than in WT littermates (-28%; p < 0.001). In rats however, prazosin, an alpha1-adrenergic antagonist, decreases d-amphetamine-induced locomotor hyperactivity without affecting extracellular DA levels in the nucleus accumbens, a finding related to the presence of an important nonfunctional release of DA (Darracq et al., 1998). We show here that local d-amphetamine releases nonfunctional DA with the same affinity but a more than threefold lower amplitude in C57BL6/J mice than in Sprague Dawley rats. Altogether, this suggests that a trans-synaptic mechanism amplifies functional DA into nonfunctional DA release. Our data confirm the presence of a powerful coupling between noradrenergic and dopaminergic neurons through the stimulation of alpha1b-adrenergic receptors and indicate that nonfunctional DA release is critical in the interpretation of changes in extracellular DA levels. These results suggest that alpha1b-adrenergic receptors may be important therapeutic pharmacological targets not only in addiction but also in psychosis because most neuroleptics possess anti-alpha1-adrenergic properties.

Nasal immunization of mice with human papillomavirus type 16 virus-like particles elicits neutralizing antibodies in mucosal secretions.

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 microgram of VLP given at weekly intervals to anesthetized mice induced high (>10(4)) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-microgram VLP systemic priming followed by two 5-microgram VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.