The endogenous caspase-8 inhibitor c-FLIPL regulates ER morphology and crosstalk with mitochondria

Components of the death receptors-mediated pathways like caspase-8 have been identified in complexes at intracellular membranes to spatially restrict the processing of local targets. In this study, we report that the long isoform of the cellular FLICE-inhibitory protein (c-FLIPL), a well- known inhibitor of the extrinsic cell death initiator caspase-8, localizes at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). ER morphology was disrupted and ER Ca2+-release as well as ER-mitochondria tethering were decreased in c-FLIP-/- mouse embryonic fibroblasts (MEFs). Mechanistically, c-FLIP ablation resulted in enhanced basal caspase-8 activation and in caspase-mediated processing of the ER-shaping protein reticulon-4 (RTN4) that was corrected by re-introduction of c-FLIPL and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIPL emerges as a component of the MAMs signaling platforms, where caspases appear to regulate ER morphology and ER-mitochondria crosstalk by impinging on ER-shaping proteins like the RTN4

Effects of preoperative feeding with a whey protein plus carbohydrate drink on the acute phase response and insulin resistance. A randomized trial

<p>Abstract</p> <p>Background</p> <p>Prolonged preoperative fasting increases insulin resistance and current evidence recommends carbohydrate (CHO) drinks 2 hours before surgery. Our hypothesis is that the addition of whey protein to a CHO-based drink not only reduces the inflammatory response but also diminish insulin resistance.</p> <p>Methods</p> <p>Seventeen patients scheduled to cholecystectomy or inguinal herniorraphy were randomized and given 474 ml and 237 ml of water (CO group) or a drink containing CHO and milk whey protein (CHO-P group) respectively, 6 and 3 hours before operation. Blood samples were collected before surgery and 24 hours afterwards for biochemical assays. The endpoints of the study were the insulin resistance (IR), the prognostic inflammatory and nutritional index (PINI) and the C-reactive protein (CRP)/albumin ratio. A 5% level for significance was established.</p> <p>Results</p> <p>There were no anesthetic or postoperative complications. The post-operative IR was lower in the CHO-P group when compared with the CO group (2.75 ± 0.72 vs 5.74 ± 1.16; p = 0.03). There was no difference between the two groups in relation to the PINI. The CHO-P group showed a decrease in the both CRP elevation and CRP/albumin ratio (p < 0.05). The proportion of patients who showed CRP/albumin ratio considered normal was significantly greater (p < 0.05) in the CHO-P group (87.5%) than in the CO group (33.3%).</p> <p>Conclusions</p> <p>Shortening the pre-operative fasting using CHO and whey protein is safe and reduces insulin resistance and postoperative acute phase response in elective moderate operations.</p> <p>Trial registration</p> <p>ClinicalTrail.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT01354249">NCT01354249</a></p

MCT Expression and Lactate Influx/Efflux in Tanycytes Involved in Glia-Neuron Metabolic Interaction

Metabolic interaction via lactate between glial cells and neurons has been proposed as one of the mechanisms involved in hypothalamic glucosensing. We have postulated that hypothalamic glial cells, also known as tanycytes, produce lactate by glycolytic metabolism of glucose. Transfer of lactate to neighboring neurons stimulates ATP synthesis and thus contributes to their activation. Because destruction of third ventricle (III-V) tanycytes is sufficient to alter blood glucose levels and food intake in rats, it is hypothesized that tanycytes are involved in the hypothalamic glucose sensing mechanism. Here, we demonstrate the presence and function of monocarboxylate transporters (MCTs) in tanycytes. Specifically, MCT1 and MCT4 expression as well as their distribution were analyzed in Sprague Dawley rat brain, and we demonstrate that both transporters are expressed in tanycytes. Using primary tanycyte cultures, kinetic analyses and sensitivity to inhibitors were undertaken to confirm that MCT1 and MCT4 were functional for lactate influx. Additionally, physiological concentrations of glucose induced lactate efflux in cultured tanycytes, which was inhibited by classical MCT inhibitors. Because the expression of both MCT1 and MCT4 has been linked to lactate efflux, we propose that tanycytes participate in glucose sensing based on a metabolic interaction with neurons of the arcuate nucleus, which are stimulated by lactate released from MCT1 and MCT4-expressing tanycytes

Early maternal deprivation affects dentate gyrus structure and emotional learning in adult female rats

Rationale: Stress elicits functional and structural changes in the hippocampus. Early life stress is one of the major risk factors for stress-related pathologies like depression. Patients suffering from depression show a reduced hippocampal volume, and in women, this occurs more often when depression is preceded by childhood trauma. However, the underlying mechanisms that account for a reduced hippocampal volume are unknown. Objective: We examined the effects of maternal absence on structure and function of the hippocampus in female offspring. Methods: We studied whether 24 h of maternal deprivation (MD) on postnatal day 3 altered adult neurogenesis, individual neuronal morphology and dentate gyrus (DG) structure in young adult female rats. In addition, functional alterations were addressed by studying synaptic plasticity in vitro, and spatial as well as emotional learning was tested. Results: Adult females that were subjected to MD revealed significant reductions in DG granule cell number and density. In addition, DG neurons were altered in their dendritic arrangement. No effects on the rate of adult neurogenesis were found. Furthermore, MD did not alter synaptic plasticity in vitro, neither under normal nor high-stress conditions. In addition, spatial learning and contextual fear conditioning were comparable between control and MD animals. However, MD animals showed an improved amygdala-dependent fear memory. Conclusion: Although early life stress exposure did not impair hippocampus-dependent functioning in female offspring, it irreversibly affected DG structure by reducing cell numbers. This may be relevant for the reduced hippocampal volume observed in depression and the increased vulnerability of women to develop depression

Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27×10−9 M versus 2.69×10−7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol

Exercise therapy in Type 2 diabetes

Structured exercise is considered an important cornerstone to achieve good glycemic control and improve cardiovascular risk profile in Type 2 diabetes. Current clinical guidelines acknowledge the therapeutic strength of exercise intervention. This paper reviews the wide pathophysiological problems associated with Type 2 diabetes and discusses the benefits of exercise therapy on phenotype characteristics, glycemic control and cardiovascular risk profile in Type 2 diabetes patients. Based on the currently available literature, it is concluded that Type 2 diabetes patients should be stimulated to participate in specifically designed exercise intervention programs. More attention should be paid to cardiovascular and musculoskeletal deconditioning as well as motivational factors to improve long-term treatment adherence and clinical efficacy. More clinical research is warranted to establish the efficacy of exercise intervention in a more differentiated approach for Type 2 diabetes subpopulations within different stages of the disease and various levels of co-morbidity

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment