Sustained release of corticosterone in rats affects reactivity, but does not affect habituation to immobilization and acoustic stimuli

Depression is often preceded by stressful life events and accompanied with elevated cortisol levels and glucocorticoid resistance. It has been suggested that a major depressive disorder may result from impaired coping with and adaptation to stress. The question is whether or not hypothalamus-pituitary-adrenal (HPA)-axis dysfunction influences the process of adaptation. We examined the effect of a dysregulated HPA-axis on the adaptation to acoustic stimuli in rats with or without preceding restraint stress. HPA-axis function was altered via slow release of corticosterone (CORT, 90 mg) from subcutaneously implanted pellets for 7 or 14 days. The rate of body temperature increases during restraint (10 min) and the response to acoustic stimuli (of 80+120 dB) were used to quantify daily stress reactivity. Rats habituated to either stress regardless of CORT treatment. CORT treatment combined with restraint decreased the initial reactivity and the variability in response, but the rate of habituation was not influenced. These results show that suppressing normal HPA-axis function by chronic exposure to CORT does affect the course of habituation, but not habituation per se. This implies that altered HPA-axis function in depressed patients may not be causally related to stress coping, but instead may influence the course of the disorder.     

Genome-wide microarray expression analysis of CD4+ T Cells from nonobese diabetic congenic mice identifies Cd55 (Daf1) and Acadl as candidate genes for type 1 diabetes

NOD.Idd3/5 congenic mice have insulin-dependent diabetes (Idd) regions on chromosomes 1 (Idd5) and 3 (Idd3) derived from the nondiabetic strains B10 and B6, respectively. NOD.Idd3/5 mice are almost completely protected from type 1 diabetes (T1D) but the genes within Idd3 and Idd5 responsible for the disease-altering phenotype have been only partially characterized. To test the hypothesis that candidate Idd genes can be identified by differential gene expression between activated CD4+ T cells from the diabetes-susceptible NOD strain and the diabetes-resistant NOD.Idd3/5 congenic strain, genome-wide microarray expression analysis was performed using an empirical Bayes method. Remarkably, 16 of the 20 most differentially expressed genes were located in the introgressed regions on chromosomes 1 and 3, validating our initial hypothesis. The two genes with the greatest differential RNA expression on chromosome 1 were those encoding decay-accelerating factor (DAF, also known as CD55) and acyl-coenzyme A dehydrogenase, long chain, which are located in the Idd5.4 and Idd5.3 regions, respectively. Neither gene has been implicated previously in the pathogenesis of T1D. In the case of DAF, differential expression of mRNA was extended to the protein level; NOD CD4+ T cells expressed higher levels of cell surface DAF compared with NOD.Idd3/5 CD4+ T cells following activation with anti-CD3 and -CD28. DAF up-regulation was IL-4 dependent and blocked under Th1 conditions. These results validate the approach of using congenic mice together with genome-wide analysis of tissue-specific gene expression to identify novel candidate genes in T1D.     

Secretory phospholipase A2 increases SR-BI-mediated selective uptake from HDL but not biliary cholesterol secretion

High density lipoprotein cholesterol represents a major source of biliary cholesterol. Secretory phospholipase A2 (sPLA2) is an acute phase enzyme mediating decreased plasma HDL cholesterol levels. Clinical studies reported a link between increased sPLA2 expression and the presence of cholesterol gallstones. The aim of our study was to investigate whether the overexpression of human sPLA2 in transgenic mice affects biliary cholesterol secretion and gallstone formation. Liver weight (P < 0.01) and hepatic cholesterol content (P < 0.01) were significantly increased in sPLA2 transgenic mice compared with controls as a result of increased scavenger receptor class B type I (SR-BI)-mediated hepatic selective uptake of HDL cholesterol (P < 0.01), whereas hepatic SR-BI expression remained unchanged. However, biliary cholesterol secretion as well as fecal neutral sterol and fecal bile salt excretion remained unchanged in sPLA2 transgenic mice. Furthermore, gallstone prevalence in response to a lithogenic diet was identical in both groups. These data demonstrate that i) increased flux of cholesterol from HDL into the liver via SR-BI as a result of phospholipase modification of the HDL particle translates neither into increased biliary and fecal sterol output nor into increased gallstone formation, and ii) increased sPLA2 expression in patients with cholesterol gallstones might be a consequence rather than the underlying cause of the disease.     

Lxralpha deficiency hampers the hepatic adaptive response to fasting in mice

Besides its well established role in control of cellular cholesterol homeostasis, the liver X receptor (LXR) has been implicated in the regulation of hepatic gluconeogenesis. We investigated the role of the major hepatic LXR isoform in hepatic glucose metabolism during the feeding-to-fasting transition in vivo. In addition, we explored hepatic glucose sensing by LXR during carbohydrate refeeding. Lxralpha(-/-) mice and their wild-type littermates were subjected to a fasting-refeeding protocol and hepatic carbohydrate fluxes as well as whole body insulin sensitivity were determined in vivo by stable isotope procedures. Lxralpha(-/-) mice showed an impaired response to fasting in terms of hepatic glycogen depletion and triglyceride accumulation. Hepatic glucose 6-phosphate turnover was reduced in 9-h fasted Lxralpha(-/-) mice as compared with controls. Although hepatic gluconeogenic gene expression was increased in 9-h fasted Lxralpha(-/-) mice compared with wild-type controls, the actual gluconeogenic flux was not affected by Lxralpha deficiency. Hepatic and peripheral insulin sensitivity were similar in Lxralpha(-/-) and wild-type mice. Compared with wild-type controls, the induction of hepatic lipogenic gene expression was blunted in carbohydrate-refed Lxralpha(-/-) mice, which was associated with lower plasma triglyceride concentrations. Yet, expression of "classic" LXR target genes Abca1, Abcg5, and Abcg8 was not affected by Lxralpha deficiency in carbohydrate-refed mice. In summary, these studies identify LXRalpha as a physiologically relevant mediator of the hepatic response to fasting. However, the data do not support a role for LXR in hepatic glucose sensing.     

Regulation of direct transintestinal cholesterol excretion in mice

Biliary secretion is generally considered to be an obligate step in the pathway of excess cholesterol excretion from the body. We have recently shown that an alternative route exists. Direct transintestinal cholesterol efflux (TICE) contributes significantly to cholesterol removal in mice. Our aim was to investigate whether the activity of this novel pathway can be influenced by dietary factors. In addition, we studied the role of cholesterol acceptors at the luminal side of the enterocyte. Mice were fed a Western-type diet (0.25% wt/wt cholesterol; 16% wt/wt fat), a high-fat diet (no cholesterol; 24% wt/wt fat), or high-cholesterol diet (2% wt/wt), and TICE was measured by isolated intestinal perfusion. Bile salt-phospholipid mixtures served as cholesterol acceptor. Western-type and high-fat diet increased TICE by 50 and 100%, respectively. In contrast, the high-cholesterol diet did not influence TICE. Intestinal scavenger receptor class B type 1 (Sr-B1) mRNA and protein levels correlated with the rate of TICE. Unexpectedly, although confirming a role for Sr-B1, TICE was significantly increased in Sr-B1-deficient mice. Apart from the long-term effect of diets on TICE, acute effects by luminal cholesterol acceptors were also investigated. The phospholipid content of perfusate was the most important regulator of TICE; bile salt concentration or hydrophobicity of bile salts had little effect. In conclusion, TICE can be manipulated by dietary intervention. Specific dietary modifications might provide means to stimulate TICE and, thereby, to enhance total cholesterol turnover.     

Age-related changes in the biomechanics of left ventricular twist measured by speckle tracking echocardiography

The increasing number and proportion of aged individuals in the population warrants knowledge of normal physiological changes of left ventricular (LV) biomechanics with advancing age. LV twist describes the instantaneous circumferential motion of the apex with respect to the base of the heart and has an important role in LV ejection and filling. This study sought to investigate the biomechanics behind age-related changes in LV twist by determining a broad spectrum of LV rotation parameters in different age groups, using speckle tracking echocardiography (STE). The final study population consisted of 61 healthy volunteers (16-35 yr, n=25; 36-55 yr, n=23; 56-75 yr, n=13; 31 men). LV peak systolic rotation during the isovolumic contraction phase (Rot(early)), LV peak systolic rotation during ejection (Rot(max)), instantaneous LV peak systolic twist (Twist(max)), the time to Rot(early), Rot(max), and Twist(max), and rotational deformation delay (defined as the difference of time to basal Rot(max) and apical Rot(max)) were determined by STE using QLAB Advanced Quantification Software (version 6.0; Philips, Best, The Netherlands). With increasing age, apical Rot(max) (P<0.05), time to apical Rot(max) (P<0.01), and Twist(max) (P<0.01) increased, whereas basal Rot(early) (P<0.001), time to basal Rot(early) (P<0.01), and rotational deformation delay (P<0.05) decreased. Rotational deformation delay was significantly correlated to Twist(max) (R(2)=0.20, P<0.05). In conclusion, Twist(max) increased with aging, resulting from both increased apical Rot(max) and decreased rotational deformation delay between the apex and the base of the LV. This may explain the preservation of LV ejection fraction in the elderly.     

Calcium signaling induces a partial EMT

Epithelial plasticity, or epithelial‐to‐mesenchymal transition (EMT), is a well‐recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial–mesenchymal (E‐M) states and that cells exhibiting such partial EMT (P‐EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P‐EMT program operating in vivo by which carcinoma cells lose their epithelial state through post‐translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P‐EMT characterized by the internalization of membrane‐associated E‐cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq‐associated G‐protein‐coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin‐Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial–mesenchymal states in carcinoma cells.     

The neurokinin A receptor activates calcium and cAMP responses through distinct conformational states.

G protein-coupled receptors are thought to mediate agonist-evoked signal transduction by interconverting between discrete conformational states endowed with different pharmacological and functional properties. In order to address the question of multiple receptor states, we monitored rapid kinetics of fluorescent neurokinin A (NKA) binding to tachykinin NK2 receptors, in parallel with intracellular calcium, using rapid mixing equipment connected to real time fluorescence detection. Cyclic AMP accumulation responses were also monitored. The naturally truncated version of neurokinin A (NKA-(4-10)) binds to the receptor with a single rapid phase and evokes only calcium responses. In contrast, full-length NKA binding exhibits both a rapid phase that correlates with calcium responses and a slow phase that correlates with cAMP accumulation. Furthermore, activators (phorbol esters and forskolin) and inhibitors (Ro 31-8220 and H89) of protein kinase C or A, respectively, exhibit differential effects on NKA binding and associated responses; activated protein kinase C facilitates a switch between calcium and cAMP responses, whereas activation of protein kinase A diminishes cAMP responses. NK2 receptors thus adopt multiple activatable, active, and desensitized conformations with low, intermediate, or high affinities and with distinct signaling specificities.