Mécanismes d'induction de l'apoptose par le choc thermique et effet protecteur de la thermotolérance induite à 40°C

Il est généralement admis que l'expertise scientifique et technique des laboratoires de recherche ainsi que l'expertise médicale des services cliniques contribuent toutes à mieux connaître et à améliorer la santé de l'homme en favorisant les interfaces entre recherche fondamentale et recherche médicale cognitive et clinique. Ce présent projet vient soutenir cette contribution en soutenant l'intégration de l'hyperthermie comme possibilité thérapeutique dans la lutte contre le cancer au sein des établissements de santé canadiens. Ce travail de recherche comprend deux volets. En premier lieu, nous nous sommes intéressés à la toxicité du choc thermique et à sa capacité d'induire différentes voies de signalisation de l'apoptose. En second lieu, nous avons évalué l'effet protecteur de la thermotolérance induite à des températures douces contre la toxicité d'un choc thermique subséquent létal. Plus précisément, nous avons étudié les mécanismes moléculaires d'induction de la voie des récepteurs de mort Fas par le choc thermique (1), ainsi que la tendance du choc thermique à activer les voies de signalisation cellulaire impliquant les protéines MAP kinases et la protéine p53(2) et à générer un stress du réticulum endoplasmique (3). Dans les trois chapitres de l'étude, nous avons également investigué le rôle de la thermotolérance et des protéines de choc thermique dans la protection des cellules contre l'induction de l'apoptose par l'hyperthermie létale. L'exposition des cellules à des températures élevées létales (42-45°C) a conduit les cellules vers une mort majoritairement apoptotique. Les trois voies de l'apoptose (voie mitochondriale, voie du récepteur de mort Fas et voie du RE) ont été induites. Cependant, l'inhibition spécifique de la voie du récepteur de mort Fas nous a permis de conclure que cette dernière était responsable de l'induction de la voie intrinsèque de l'apoptose médiée par la mitochondrie. Pour sa part, la voie du réticulum endoplasmique (RE) est induite suite à un stress prolongé du RE. Il s'est avéré aussi que l'induction de cette voie n'est pas indispensable pour l'induction de l'apoptose par choc thermique létal. Durant ces cascades signalétiques, les MAP kinases, en particulier les protéines JNK et p38, jouent un rôle crucial dans la phosphorylation et l'activation de divers facteurs pro-apoptotiques tels que Bax, Puma, Noxa et p53. Ceci a conduit à l'arrêt du cycle cellulaire en phase G0/S, et à la translocation de Bax à la mitochondrie et la dégradation de Bcl-2. D'autre part, suite à l'induction de la thermotolérance à la température de la fièvre (40°), nous étions capables de protéger les cellules contre la cytotoxicité d'un choc thermique létal subséquent. Malgré qu'aucunes évidences n'aient été établi, cet effet protecteur de la thermotolérance semble être en parfaite corrélation avec l'augmentation du niveau d'expression des HSPs. L'utilisation conjointe de l'hyperthermie létale et non létale (la thermotolérance) pourrait constituer une possibilité à envisager lors du traitement du cancer par les méthodes classiques. La pertinence et l'originalité de ce projet réside dans le fait que cette alternative est avant tout naturelle et biologique, peu coûteuse, et exemptée de la plupart des effets secondaires observés lors de l'utilisation des méthodes classiques de traitement, qui sont généralement toxiques et couteuses. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Hyperthermie, HSPs, Thermotolérance, Apoptose, Nécrose, Caspases, Calcium, ROS, Bcl-2, Fas, Mitochondrie, Réticulum Endoplasmique, p53, MAP kinases, Calpaïne

(-)-Epicatechin improves insulin sensitivity in high fat diet-fed mice

Obesity constitutes a major public health concern, being frequently associated with type 2 diabetes (T2D). Evidence from studies in humans and experimental animals suggest that consumption of the flavan-3-ol (-)-epicatechin (EC) and of EC-rich foods may improve insulin sensitivity. To further understand the potential benefits of dietary EC consumption on insulin resistance, this study investigated the capacity of EC supplementation to prevent high fat diet (HFD)-induced insulin resistance in mice. To assess the underlying mechanisms, the effects of HFD and EC consumption on the activation of the insulin cascade and of its negative modulators were evaluated. HFD consumption for 15 w caused obesity and insulin resistance in C57BL/6J mice as evidenced by high fasted and fed plasma glucose and insulin levels, and impaired ITT and GTT tests. This was associated with alterations in the activation of components of the insulin-triggered signaling cascade (insulin receptor, IRS1, ERK1/2, Akt) in adipose and liver tissues. EC supplementation prevented/ameliorated all these parameters. EC acted improving insulin sensitivity in the HFD-fed mice in part through a downregulation of the inhibitory molecules JNK, IKK, PKC and protein tyrosine phosphatase 1B (PTP1B). Thus, the above results suggest that consumption of EC-rich foods could constitute a dietary strategy to mitigate obesity-associated insulin resistance.Fil: Cremonini, Eleonora. University of California at Davis; Estados UnidosFil: Bettaieb, Ahmed. University of California at Davis; Estados Unidos. University of Tennessee; Estados UnidosFil: Haj, Fawaz G.. University of California at Davis; Estados UnidosFil: Fraga, César Guillermo. University of California at Davis; Estados Unidos. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analitica y Fisicoquímica. Cátedra de Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Oteiza, Patricia Isabel. University of California at Davis; Estados Unido

Shc depletion stimulates brown fat activity in vivo and in vitro.

Adipose tissue is an important metabolic organ that integrates a wide array of homeostatic processes and is crucial for whole-body insulin sensitivity and energy metabolism. Brown adipose tissue (BAT) is a key thermogenic tissue with a well-established role in energy expenditure. BAT dissipates energy and protects against both hypothermia and obesity. Thus, BAT stimulation therapy is a rational strategy for the looming pandemic of obesity, whose consequences and comorbidities have a huge impact on the aged. Shc-deficient mice (ShcKO) were previously shown to be lean, insulin sensitive, and resistant to high-fat diet and obesity. We investigated the contribution of BAT to this phenotype. Insulin-dependent BAT glucose uptake was higher in ShcKO mice. Primary ShcKO BAT cells exhibited increased mitochondrial respiration; increased expression of several mitochondrial and lipid-oxidative enzymes was observed in ShcKO BAT. Levels of brown fat-specific markers of differentiation, UCP1, PRDM16, ELOVL3, and Cox8b, were higher in ShcKO BAT. In vitro, Shc knockdown in BAT cell line increased insulin sensitivity and metabolic activity. In vivo, pharmacological stimulation of ShcKO BAT resulted in higher energy expenditure. Conversely, pharmacological inhibition of BAT abolished the improved metabolic parameters, that is the increased insulin sensitivity and glucose tolerance of ShcKO mice. Similarly, in vitro Shc knockdown in BAT cell lines increased their expression of UCP1 and metabolic activity. These data suggest increased BAT activity significantly contributes to the improved metabolic phenotype of ShcKO mice

Concurrent regulation of LKB1 and CaMKK2 in the activation of AMPK in castrate-resistant prostate cancer by a well-defined polyherbal mixture with anticancer properties

Abstract Background Zyflamend, a blend of herbal extracts, effectively inhibits tumor growth using preclinical models of castrate-resistant prostate cancer mediated in part by 5′-adenosine monophosphate-activated protein kinase (AMPK), a master energy sensor of the cell. Clinically, treatment with Zyflamend and/or metformin (activators of AMPK) had benefits in castrate-resistant prostate cancer patients who no longer responded to treatment. Two predominant upstream kinases are known to activate AMPK: liver kinase B1 (LKB1), a tumor suppressor, and calcium-calmodulin kinase kinase-2 (CaMKK2), a tumor promotor over-expressed in many cancers. The objective was to interrogate how Zyflamend activates AMPK by determining the roles of LKB1 and CaMKK2. Methods AMPK activation was determined in CWR22Rv1 cells treated with a variety of inhibitors of LKB1 and CaMKK2 in the presence and absence of Zyflamend, and in LKB1-null HeLa cells that constitutively express CaMKK2, following transfection with wild type LKB1 or catalytically-dead mutants. Upstream regulation by Zyflamend of LKB1 and CaMKK2 was investigated targeting protein kinase C-zeta (PKCζ) and death-associated protein kinase (DAPK), respectively. Results Zyflamend’s activation of AMPK appears to be LKB1 dependent, while simultaneously inhibiting CaMKK2 activity. Zyflamend failed to rescue the activation of AMPK in the presence of pharmacological and molecular inhibitors of LKB1, an effect not observed in the presence of inhibitors of CaMKK2. Using LKB1-null and catalytically-dead LKB1-transfected HeLa cells that constitutively express CaMKK2, ionomycin (activator of CaMKK2) increased phosphorylation of AMPK, but Zyflamend only had an effect in cells transfected with wild type LKB1. Zyflamend appears to inhibit CaMKK2 by DAPK-mediated phosphorylation of CaMKK2 at Ser511, an effect prevented by a DAPK inhibitor. Alternatively, Zyflamend mediates LKB1 activation via increased phosphorylation of PKCζ, where it induced translocation of PKCζ and LKB1 to their respective active compartments in HeLa cells following treatment. Altering the catalytic activity of LKB1 did not alter this translocation. Discussion Zyflamend’s activation of AMPK is mediated by LKB1, possibly via PKCζ, but independent of CaMKK2 by a mechanism that appears to involve DAPK. Conclusions Therefore, this is the first evidence that natural products simultaneously and antithetically regulate upstream kinases, known to be involved in cancer, via the activation of AMPK.https://deepblue.lib.umich.edu/bitstream/2027.42/144515/1/12906_2018_Article_2255.pd

(-)-Epicatechin mitigates high-fructose-associated insulin resistance by modulating redox signaling and endoplasmic reticulum stress

We investigated the capacity of dietary (-)-epicatechin (EC) to mitigate insulin resistance through the modulation of redox-regulated mechanisms in a rat model of metabolic syndrome (MetS). Adolescent rats were fed a regular chow diet without or with high fructose (HFr) (10% (w/v)) in drinking water for 8 weeks, and a group of HFr-fed rats was supplemented with EC in the diet. HFr-fed rats developed insulin resistance which was mitigated by EC supplementation. Accordingly, the activation of components of the insulin signaling cascade (insulin receptor (IR), IRS-1, Akt and ERK1/2) was impaired, while negative regulators (PKC, IKK, JNK and PTP1B) were upregulated in the liver and adipose tissue of HFr rats. These alterations were partially or totally prevented by EC supplementation. In addition, EC inhibited events which contribute to insulin resistance: HFr-associated increased expression and activity of NADPH oxidase , activation of redox-sensitive signals , expression of NF-kB-regulated pro-inflammatory cytokines and chemokines, and some sub-arms of endoplasmic reticulum stress signaling. Collectively, these findings indicate that EC supplementation can mitigate HFr-induced insulin resistance and are relevant to define interventions that can prevent/mitigate MetS-associated insulin resistance.Fil: Bettaieb, Ahmed. University of California at Davis; Estados UnidosFil: Vazquez, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Rodriguez Lanzi, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Miatello, Roberto Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Haj, Fawaz G.. University of California at Davis; Estados UnidosFil: Fraga, César Guillermo. University of California at Davis; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Oteiza, Patricia Isabel. University of California at Davis; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Role for zinc transporter gene SLC39A12 in the nervous system and beyond

The SLC39A12 gene encodes the zinc transporter protein ZIP12, which is expressed across many tissues and is highly abundant in the vertebrate nervous system. As a zinc transporter, ZIP12 functions to transport zinc across cellular membranes, including cellular zinc influx across the plasma membrane. Genome-wide association and exome sequencing studies have shown that brain susceptibility-weighted magnetic resonance imaging (MRI) intensity is associated with ZIP12 polymorphisms and rare mutations. ZIP12 is required for neural tube closure and embryonic development in Xenopus tropicalis. Frog embryos depleted of ZIP12 by antisense morpholinos develop an anterior neural tube defect and lack viability. ZIP12 is also necessary for neurite outgrowth and mitochondrial function in mouse neural cells. ZIP12 mRNA is increased in brain regions of schizophrenic patients. Outside of the nervous system, hypoxia induces ZIP12 expression in multiple mammalian species, including humans, which leads to endothelial and smooth muscle thickening in the lung and contributes towards pulmonary hypertension. Other studies have associated ZIP12 with other diseases such as cancer. Given that ZIP12 is highly expressed in the brain and that susceptibility-weighted MRI is associated with brain metal content, ZIP12 may affect neurological diseases and psychiatric illnesses such as Parkinson's disease, Alzheimer's disease, and schizophrenia. Furthermore, the induction of ZIP12 and resultant zinc uptake under pathophysiological conditions may be a critical component of disease pathology, such as in pulmonary hypertension. Drug compounds that bind metals like zinc may be able to treat diseases associated with impaired zinc homeostasis and altered ZIP12 function.Human Sciences Research and Graduate StudiesGraduate CollegeNutritional Science

Bile-acid-mediated decrease in endoplasmic reticulum stress: a potential contributor to the metabolic benefits of ileal interposition surgery in UCD-T2DM rats

Post-operative increases in circulating bile acids have been suggested to contribute to the metabolic benefits of bariatric surgery; however, their mechanistic contributions remain undefined. We have previously reported that ileal interposition (IT) surgery delays the onset of type 2 diabetes in UCD-T2DM rats and increases circulating bile acids, independently of effects on energy intake or body weight. Therefore, we investigated potential mechanisms by which post-operative increases in circulating bile acids improve glucose homeostasis after IT surgery. IT, sham or no surgery was performed on 2-month-old weight-matched male UCD-T2DM rats. Animals underwent an oral fat tolerance test (OFTT) and serial oral glucose tolerance tests (OGTT). Tissues were collected at 1.5 and 4.5 months after surgery. Cell culture models were used to investigate interactions between bile acids and ER stress. IT-operated animals exhibited marked improvements in glucose and lipid metabolism, with concurrent increases in postprandial glucagon-like peptide-1 (GLP-1) secretion during the OFTT and OGTTs, independently of food intake and body weight. Measurement of circulating bile acid profiles revealed increases in circulating total bile acids in IT-operated animals, with a preferential increase in circulating cholic acid concentrations. Gut microbial populations were assessed as potential contributors to the increases in circulating bile acid concentrations, which revealed proportional increases in Gammaproteobacteria in IT-operated animals. Furthermore, IT surgery decreased all three sub-arms of ER stress signaling in liver, adipose and pancreas tissues. Amelioration of ER stress coincided with improved insulin signaling and preservation of β-cell mass in IT-operated animals. Incubation of hepatocyte, adipocyte and β-cell lines with cholic acid decreased ER stress. These results suggest that postoperative increases in circulating cholic acid concentration contribute to improvements in glucose homeostasis after IT surgery by ameliorating ER stress

Bile-acid-mediated decrease in endoplasmic reticulum stress: a potential contributor to the metabolic benefits of ileal interposition surgery in UCD-T2DM rats

Post-operative increases in circulating bile acids have been suggested to contribute to the metabolic benefits of bariatric surgery; however, their mechanistic contributions remain undefined. We have previously reported that ileal interposition (IT) surgery delays the onset of type 2 diabetes in UCD-T2DM rats and increases circulating bile acids, independently of effects on energy intake or body weight. Therefore, we investigated potential mechanisms by which post-operative increases in circulating bile acids improve glucose homeostasis after IT surgery. IT, sham or no surgery was performed on 2-month-old weight-matched male UCD-T2DM rats. Animals underwent an oral fat tolerance test (OFTT) and serial oral glucose tolerance tests (OGTT). Tissues were collected at 1.5 and 4.5 months after surgery. Cell culture models were used to investigate interactions between bile acids and ER stress. IT-operated animals exhibited marked improvements in glucose and lipid metabolism, with concurrent increases in postprandial glucagon-like peptide-1 (GLP-1) secretion during the OFTT and OGTTs, independently of food intake and body weight. Measurement of circulating bile acid profiles revealed increases in circulating total bile acids in IT-operated animals, with a preferential increase in circulating cholic acid concentrations. Gut microbial populations were assessed as potential contributors to the increases in circulating bile acid concentrations, which revealed proportional increases in Gammaproteobacteria in IT-operated animals. Furthermore, IT surgery decreased all three sub-arms of ER stress signaling in liver, adipose and pancreas tissues. Amelioration of ER stress coincided with improved insulin signaling and preservation of β-cell mass in IT-operated animals. Incubation of hepatocyte, adipocyte and β-cell lines with cholic acid decreased ER stress. These results suggest that postoperative increases in circulating cholic acid concentration contribute to improvements in glucose homeostasis after IT surgery by ameliorating ER stress