Rôle de la ghréline dans la régulation du coactivateur transcriptionnel PGC-1alpha

L’adaptation de l’organisme à son environnement est essentielle à sa survie. L’homéostasie énergétique permet l’équilibre entre les apports, les dépenses et le stockage d’énergie. Un surplus calorique important dérègle ce processus et mène au développement du syndrome métabolique caractérisé, entre autres, par une obésité, un diabète de type II, des maladies cardiovasculaires et des dyslipidémies. La ghréline participe au maintien de l’équilibre énergétique durant le jeûne en stimulant la production de glucose par le foie et le stockage lipidique dans le tissu adipeux. Le coactivateur transcriptionnel PGC-1alpha, surexprimé en situation de jeûne, est impliqué dans l’induction de la production de glucose par le foie et l’oxydation des acides gras. Notre hypothèse est que ces deux acteurs clés du métabolisme énergétique constituent un axe de régulation commun. Dans cette étude, nous montrons que la ghréline participe à la régulation de PGC-1alpha. Son récepteur GHS-R1a, possédant une forte activité constitutive, est également impliqué de façon indépendante au ligand. GHS-R1a réduit l’activité transcriptionnelle de PGC-1alpha tandis que l’ajout du ligand inverse modérément cette action. L’effet de GHS-R1a corrèle avec l’acétylation de PGC-1alpha qui est fortement augmentée de façon dose-dépendante. La stabilité de PGC-1alpha est également augmentée par le GHS-R1a indépendamment de l’ubiquitine. La ghréline diminue la capacité de PGC-1alpha à lier PPARbeta, un récepteur nucléaire partenaire de PGC-1alpha. De plus, la ghréline réduit, de façon ligand-dépendante, la capacité de coactivation de PGC-1alpha sur PPARbeta dans les hépatocytes. L’ensemble de ces résultats identifie PGC-1alpha comme cible du signal de la ghréline et suggère un axe de régulation ghréline/PGC-1alpha/PPARbeta.Une meilleure compréhension de cet axe de régulation va permettre la mise en évidence de nouvelles cibles thérapeutiques pour faire face aux pathologies associées au syndrome métabolique.

Peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha) gene polymorphisms and their relationship to Type 2 diabetes in Asian Indians

AIMS: The objective of the present investigation was to examine the relationship of three polymorphisms, Thr394Thr, Gly482Ser and +A2962G, of the peroxisome proliferator activated receptor-gamma co-activator-1 alpha (PGC-1alpha) gene with Type 2 diabetes in Asian Indians. METHODS: The study group comprised 515 Type 2 diabetic and 882 normal glucose tolerant subjects chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The three polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Haplotype frequencies were estimated using an expectation-maximization (EM) algorithm. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. RESULTS: The three polymorphisms studied were not in linkage disequilibrium. With respect to the Thr394Thr polymorphism, 20% of the Type 2 diabetic patients (103/515) had the GA genotype compared with 12% of the normal glucose tolerance (NGT) subjects (108/882) (P = 0.0004). The frequency of the A allele was also higher in Type 2 diabetic subjects (0.11) compared with NGT subjects (0.07) (P = 0.002). Regression analysis revealed the odds ratio for Type 2 diabetes for the susceptible genotype (XA) to be 1.683 (95% confidence intervals: 1.264-2.241, P = 0.0004). Age adjusted glycated haemoglobin (P = 0.003), serum cholesterol (P = 0.001) and low-density lipoprotein (LDL) cholesterol (P = 0.001) levels and systolic blood pressure (P = 0.001) were higher in the NGT subjects with the XA genotype compared with GG genotype. There were no differences in genotype or allelic distribution between the Type 2 diabetic and NGT subjects with respect to the Gly482Ser and +A2962G polymorphisms. CONCLUSIONS: The A allele of Thr394Thr (G --> A) polymorphism of the PGC-1 gene is associated with Type 2 diabetes in Asian Indian subjects and the XA genotype confers 1.6 times higher risk for Type 2 diabetes compared with the GG genotype in this population.

Prolactin receptor signaling is essential for perinatal brown adipocyte function: a role for insulin-like growth factor-2.

BACKGROUND: The lactogenic hormones prolactin (PRL) and placental lactogens (PL) play central roles in reproduction and mammary development. Their actions are mediated via binding to PRL receptor (PRLR), highly expressed in brown adipose tissue (BAT), yet their impact on adipocyte function and metabolism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: PRLR knockout (KO) newborn mice were phenotypically characterized in terms of thermoregulation and their BAT differentiation assayed for gene expression studies. Derived brown preadipocyte cell lines were established to evaluate the molecular mechanisms involved in PRL signaling on BAT function. Here, we report that newborn mice lacking PRLR have hypotrophic BAT depots that express low levels of adipocyte nuclear receptor PPARgamma2, its coactivator PGC-1alpha, uncoupling protein 1 (UCP1) and the beta3 adrenoceptor, reducing mouse viability during cold challenge. Immortalized PRLR KO preadipocytes fail to undergo differentiation into mature adipocytes, a defect reversed by reintroduction of PRLR. That the effects of the lactogens in BAT are at least partly mediated by Insulin-like Growth Factor-2 (IGF-2) is supported by: i) a striking reduction in BAT IGF-2 expression in PRLR KO mice and in PRLR-deficient preadipocytes; ii) induction of cellular IGF-2 expression by PRL through JAK2/STAT5 pathway activation; and iii) reversal of defective differentiation in PRLR KO cells by exogenous IGF-2. CONCLUSIONS: Our findings demonstrate that the lactogens act in concert with IGF-2 to control brown adipocyte differentiation and growth. Given the prominent role of brown adipose tissue during the perinatal period, our results identified prolactin receptor signaling as a major player and a potential therapeutic target in protecting newborn mammals against hypothermia.

Regulation of lipid metabolism in adipocytes and hepatocytes by hexarelin through scavenger receptor CD36

Les sécrétines de l’hormone de croissance (GHRPs) sont de petits peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance à partir de l’hypophyse via leur liaison au récepteur de la ghréline GHS-R1a. Le GHRP hexaréline a été utilisé afin d’étudier la distribution tissulaire de GHS-R1a et son effet GH-indépendant. Ainsi, par cette approche, il a été déterminé que l’hexaréline était capable de se lier à un deuxième récepteur identifié comme étant le récepteur scavenger CD36. Ce récepteur possède une multitude de ligands dont les particules oxLDL et les acides gras à longue chaîne. CD36 est généralement reconnu pour son rôle dans l’athérogénèse et sa contribution à la formation de cellules spumeuses suite à l’internalisation des oxLDL dans les macrophages/monocytes. Auparavant, nous avions démontré que le traitement des macrophages avec l’hexaréline menait à l’activation de PPARƔ via sa liaison à GHS-R1a, mais aussi à CD36. De plus, une cascade d’activation impliquant LXRα et les transporteurs ABC provoquait également une augmentation de l’efflux du cholestérol. Une stimulation de la voie du transport inverse du cholestérol vers les particules HDL entraînait donc une diminution de l’engorgement des macrophages de lipides et la formation de cellules spumeuses. Puisque CD36 est exprimé dans de multiples tissus et qu’il est également responsable du captage des acides gras à longue chaîne, nous avons voulu étudier l’impact de l’hexaréline uniquement à travers sa liaison à CD36. Dans le but d’approfondir nos connaissances sur la régulation du métabolisme des lipides par CD36, nous avons choisi des types cellulaires jouant un rôle important dans l’homéostasie lipidique n’exprimant pas GHS-R1a, soient les adipocytes et les hépatocytes. L’ensemble de mes travaux démontre qu’en réponse à son interaction avec l’hexaréline, CD36 a le potentiel de réduire le contenu lipidique des adipocytes et des hépatocytes. Dans les cellules adipeuses, l'hexaréline augmente l’expression de plusieurs gènes impliqués dans la mobilisation et l’oxydation des acides gras, et induit également l’expression des marqueurs thermogéniques PGC-1α et UCP-1. De même, hexaréline augmente l’expression des gènes impliqués dans la biogenèse mitochondriale, un effet accompagné de changements morphologiques des mitochondries; des caractéristiques observées dans les types cellulaires ayant une grande capacité oxydative. Ces résultats démontrent que les adipocytes blancs traités avec hexaréline ont la capacité de se transformer en un phénotype similaire aux adipocytes bruns ayant l’habileté de brûler les acides gras plutôt que de les emmagasiner. Cet effet est également observé dans les tissus adipeux de souris et est dépendant de la présence de CD36. Dans les hépatocytes, nous avons démontré le potentiel de CD36 à moduler le métabolisme du cholestérol. En réponse au traitement des cellules avec hexaréline, une phosphorylation rapide de LKB1 et de l’AMPK est suivie d’une phosphorylation inhibitrice de l’HMG-CoA réductase (HMGR), l’enzyme clé dans la synthèse du cholestérol. De plus, la liaison d'hexaréline à CD36 provoque le recrutement d’insig-2 à HMGR, l’étape d’engagement dans sa dégradation. La dégradation de HMGR par hexaréline semble être dépendante de l’activité de PPARƔ et de l’AMPK. Dans le but d’élucider le mécanisme d’activation par hexaréline, nous avons démontré d’une part que sa liaison à CD36 provoque une déphosphorylation de Erk soulevant ainsi l’inhibition que celui-ci exerce sur PPARƔ et d’autre part, un recrutement de l’AMPK à PGC-1α expliquant ainsi une partie du mécanisme d’activation de PPARƔ par hexaréline. Les résultats générés dans cette thèse ont permis d’élucider de nouveaux mécanismes d’action de CD36 et d'approfondir nos connaissances de son influence dans la régulation du métabolisme des lipides.

Gene expression in skeletal muscle of coronary artery disease patients after concentric and eccentric endurance training

Low-intensity concentric (CET) and eccentric (EET) endurance-type training induce specific structural adaptations in skeletal muscle. We evaluated to which extent steady-state adaptations in transcript levels are involved in the compensatory alterations of muscle mitochondria and myofibrils with CET versus EET at a matched metabolic exercise intensity of medicated, stable coronary patients (CAD). Biopsies were obtained from vastus lateralis muscle before and after 8 weeks of CET (n=6) or EET (n=6). Transcript levels for factors involved in mitochondrial biogenesis (PGC-1alpha, Tfam), mitochondrial function (COX-1, COX-4), control of contractile phenotype (MyHC I, IIa, IIx) as well as mechanical stress marker (IGF-I) were quantified using an reverse-transcriptase polymerase chain reaction approach. After 8 weeks of EET, a reduction of the COX-4 mRNA level by 41% and a tendency for a drop in Tfam transcript concentration (-33%, P=0.06) was noted. This down-regulation corresponded to a drop in total mitochondrial volume density. MyHC-IIa transcript levels were specifically decreased after EET, and MyHC-I mRNA showed a trend towards a reduction (P=0.08). Total fiber cross-sectional area was not altered. After CET and EET, the IGF-I mRNA level was significantly increased. The PGC-1alpha significantly correlated with Tfam, and both PGC-1alpha and Tfam significantly correlated with COX-1 and COX-4 mRNAs. Post-hoc analysis identified significant interactions between the concurrent medication and muscular transcript levels as well as fiber size. Our findings support the concept that specific transcriptional adaptations mediate the divergent mitochondrial response of muscle cells to endurance training under different load condition and indicate a mismatch of processes related to muscle hypertrophy in medicated CAD patients.

Acetyl-L-carnitine feeding to unloaded rats triggers in soleus muscle the coordinated expression of genes involved in mitochondrial biogenesis

The expressional profile of mitochondrial transcripts and of genes involved in the mitochondrial biogenesis pathway induced by ALCAR daily supplementation in soleus muscle of control and unloaded 3-month-old rats has been analyzed. It has been found that ALCAR treatment is able to upregulate the expression level of mitochondrial transcripts (COX I, ATP6, ND6, 16 S rRNA) in both control and unloaded animals. Interestingly, ALCAR feeding to unloaded rats resulted in the increase of transcript level for master factors involved in mitochondrial biogenesis (PGC-1alpha, NRF-1, TFAM). It also prevented the unloading-induced downregulation of mRNA levels for kinases able to transduce metabolic (AMPK) and neuronal stimuli (CaMKIIbeta) into mitochondrial biogenesis. No significant effect on the expressional level of such genes was found in control ALCAR-treated rats. In addition, ALCAR feeding was able to prevent the loss of mitochondrial protein content due to unloading condition. Correlation analysis revealed a strong coordination in the expression of genes involved in mitochondrial biogenesis only in ALCAR-treated suspended animals, supporting a differentiated effect of ALCAR treatment in relation to the loading state of the soleus muscle. In conclusions, we demonstrated the ability of ALCAR supplementation to promote only in soleus muscle of hindlimb suspended rats an orchestrated expression of genes involved in mitochondrial biogenesis, which might counteract the unloading-induced metabolic changes, preventing the loss of mitochondrial proteins.

Effects of four-week high-fructose diet on gene expression in skeletal muscle of healthy men

AIMS: A high-fructose diet (HFrD) may play a role in the obesity and metabolic disorders epidemic. In rodents, HFrD leads to insulin resistance and ectopic lipid deposition. In healthy humans, a four-week HFrD alters lipid homoeostasis, but does not affect insulin sensitivity or intramyocellular lipids (IMCL). The aim of this study was to investigate whether fructose may induce early molecular changes in skeletal muscle prior to the development of whole-body insulin resistance. METHODS: Muscle biopsies were taken from five healthy men who had participated in a previous four-week HFrD study, during which insulin sensitivity (hyperinsulinaemic euglycaemic clamp), and intrahepatocellular lipids and IMCL were assessed before and after HFrD. The mRNA concentrations of 16 genes involved in lipid and carbohydrate metabolism were quantified before and after HFrD by real-time quantitative PCR. RESULTS: HFrD significantly (P<0.05) increased stearoyl-CoA desaturase-1 (SCD-1) (+50%). Glucose transporter-4 (GLUT-4) decreased by 27% and acetyl-CoA carboxylase-2 decreased by 48%. A trend toward decreased peroxisomal proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was observed (-26%, P=0.06). All other genes showed no significant changes. CONCLUSION: HFrD led to alterations of SCD-1, GLUT-4 and PGC-1alpha, which may be early markers of insulin resistance.

The impact of antioxidant supplements and endurance exercise on genes of the carbohydrate and lipid metabolism in skeletal muscle of mice

To ascertain whether reactive oxygen species (ROS) contribute to training-induced adaptation of skeletal muscle, we administered ROS-scavenging antioxidants (AOX; 140 mg/l of ascorbic acid, 12 mg/l of coenzyme Q10 and 1% N-acetyl-cysteine) via drinking water to 16 C57BL/6 mice. Sixteen other mice received unadulterated tap water (CON). One cohort of both groups (CON(EXE) and AOX(EXE) ) was subjected to treadmill exercise for 4 weeks (16-26 m/min, incline of 5°-10°). The other two cohorts (CON(SED) and AOX(SED) ) remained sedentary. In skeletal muscles of the AOX(EXE) mice, GSSG and the expression levels of SOD-1 and PRDX-6 were significantly lower than those in the CON(EXE) mice after training, suggesting disturbance of ROS levels. The peak power related to the body weight and citrate synthase activity was not significantly influenced in mice receiving AOX. Supplementation with AOX significantly altered the mRNA levels of the exercise-sensitive genes HK-II, GLUT-4 and SREBF-1c and the regulator gene PGC-1alpha but not G6PDH, glycogenin, FABP-3, MCAD and CD36 in skeletal muscle. Although the administration of AOX during endurance exercise alters the expression of particular genes of the ROS metabolism, it does not influence peak power or generally shift the metabolism, but it modulates the expression of specific genes of the carbohydrate and lipid metabolism and PGC-1alpha within murine skeletal muscle.

Altering the redox state of skeletal muscle by glutathione depletion increases the exercise-activation of PGC-1α

We investigated the relationship between mitochondrial biogenesis, cell signalling and antioxidant enzymes by depleting skeletal muscle glutathione with diethyl maleate (DEM) which resulted in a demonstrable increase in oxidative stress during exercise. Animals were divided into six groups: (1) sedentary control rats; (2) sedentary rats treated with DEM; (3) exercise control rats euthanized immediately after exercise; (4) exercise rats + DEM; (5) exercise control rats euthanized 4 h after exercise, and; (6) exercise rats + DEM euthanized 4 h after exercise. Exercising animals ran on the treadmill at a 10% gradient at 20 m/min for the first 30 min. The speed was then increased every 10 min by 1.6 m/min until exhaustion. There was a reduction in total glutathione in the skeletal muscle of DEM treated animals compared to the control animals (P<0.05). Within the control group, total glutathione was higher in the sedentary group compared to after exercise (P<0.05). DEM treatment also significantly increased oxidative stress, as measured by increased plasma F2-isoprostanes (P<0.05). Exercising animals given DEM showed a significantly greater increase in peroxisome proliferator activated receptor γ coactivator-1α(PGC-1α) mRNA compared to the control animals that were exercised (P<0.05). This study provides novel evidence that by reducing the endogenous antioxidant glutathione in skeletal muscle and inducing oxidative stress through exercise, PGC-1α gene expression was augmented. These findings further highlight the important role of exercise induced oxidative stress in the regulation of mitochondrial biogenesis.

Resistance exercise with low glycogen increases p53 phosphorylation and PGC-1α mRNA in skeletal muscle

Purpose We determined the effect of reduced muscle glycogen availability on cellular pathways regulating mitochondrial biogenesis and substrate utilization after a bout of resistance exercise. Methods Eight young, recreationally trained men undertook a glycogen depletion protocol of one-leg cycling to fatigue (LOW), while the contralateral (control) leg rested (CONT). Following an overnight fast, subjects completed 8 sets of 5 unilateral leg press repetitions (REX) at 80 % 1 Repetition Maximum (1RM) on each leg. Subjects consumed 500 mL protein/CHO beverage (20 g whey + 40 g maltodextrin) upon completion of REX and 2 h later. Muscle biopsies were obtained at rest and 1 and 4 h after REX in both legs. Results Resting muscle glycogen was higher in the CONT than LOW leg (~384 ± 114 vs 184 ± 36 mmol kg−1 dry wt; P < 0.05), and 1 h and 4 h post-exercise (P < 0.05). Phosphorylation of p53Ser15 increased 1 h post-exercise in LOW (~115 %, P < 0.05) and was higher than CONT at this time point (~87 %, P < 0.05). p38MAPKThr180/Tyr182 phosphorylation increased 1 h post-exercise in both CONT and LOW (~800–900 %; P < 0.05) but remained above rest at 4 h only in CONT (~585 %, P < 0.05; different between legs P < 0.05). Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) mRNA was elevated 4 h post-exercise in LOW (~200 %, P < 0.05; different between legs P < 0.05). There were no changes in Fibronectin type III domain-containing protein 5 (FNDC5) mRNA for CONT or LOW legs post-exercise. Conclusion Undertaking resistance exercise with low glycogen availability may enhance mitochondrial-related adaptations through p53 and PGC-1α-mediated signalling.

光纤激光传感器PGC解调系统数字化实现的研究

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HIF-1alpha role in oxygen-dependent radio- and chemosensitivity

Poor oxygenation (hypoxia) is a common characteristic of human solid tumours, and is associated with cell survival, metastasis and resistance to radio- and chemotherapies. Hypoxia-induced stabilisation of hypoxia-inducible factor-1α (HIF-1α) leads to changes in expression of various genes associated with growth, vascularisation and metabolism. However whether HIF-1α plays a causal role in promoting hypoxic resistance to antitumour therapies remains unclear. In this study we used pharmacological and genetic methods to investigate the HIF-1α contribution to radio- and chemoresistance in four cancer cell lines derived from cervical, breast, prostate and melanoma human tumours. Under normoxia or hypoxia (<0.2% or 0.5% oxygen) the cells were exposed to either a standard irradiation dose (6.2 Gy) or chemotherapeutic drug (cisplatin), and subsequent cell proliferation (after 7 days) was measured in terms of resazurin reduction. Oxygen-dependent radio- and chemosensitivity was evident in all wild type whereas it was reduced or abolished in HIF-1α (siRNA) knockdown cells. The effects of HIF-1α-modulating drugs (EDHB, CoCl2, deferoxamine to stabilise and R59949 to destabilise it) reflected both HIF-1α-dependent and independent mechanisms. Collectively the data show that HIF-1α played a causal role in our in vitro model of hypoxia-induced radioresistance whereas its contribution to oxygendependent sensitivity to cisplatin was less clear-cut. Although this behavior is likely to be conditioned by further biological and physical factors operating in vivo, it is consistent with the hypothesis that interventions directed at HIF-1α may improve the clinical effectiveness of tumour treatments.

A common polymorphism in the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor-1alpha (HIF-1alpha) does not impair Pro-564 hydroxylation

The hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the alpha subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1alpha in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).