J.L. Sadoul

The inflammatory C-reactive protein is increased in both liver and adipose tissue in severely obese patients independently from metabolic syndrome, Type 2 diabetes, and NASH.

OBJECTIVE:C-Reactive Protein (CRP), a nonspecific marker of inflammation that is moderately elevated in obesity, metabolic syndrome (MS), and type 2 diabetes, has been proposed as a surrogate marker of nonalcoholic steatohepatitis (NASH). Its clinical usefulness in the diagnosis of NASH was evaluated in severely obese patients without or with MS, diabetes, and NASH and the potential roles of the liver and of the adipose tissue in CRP production were characterized.METHODS:Severely obese patients without NASH (without MS [N = 13], with MS [N = 11], or with MS and diabetes [N = 7]) and with NASH (without [N = 8] or with [N = 7] MS) were studied. For each patient, liver and adipose tissue biopsies were collected during a bariatric surgery and were used to determine the CRP gene expression by real-time PCR. The role of interleukin-6 (IL6) and lipopolysaccharide in CRP expression was also evaluated in subcutaneous adipose tissue obtained during cosmetic abdominoplasty.RESULTS:Plasma CRP levels were elevated in severely obese patients independently from the presence or absence of MS, diabetes, or NASH. CRP gene expression was not only increased in livers but also in adipose tissues of obese patients compared with controls subjects. In human adipose tissue, CRP mRNA levels were positively correlated with those of IL-6 and the CRP expression was enhanced in vitro by IL-6 and lipopolysaccharide.CONCLUSION:Plasma CRP levels are not predictive of the diagnosis of NASH in severely obese patients. The liver but also the adipose tissue can produce CRP, a process which could be dependent on IL6. Therefore, both tissues might contribute to the elevated plasma CRP levels found in obesity. In addition, the large amount of body fat may well produce an important part of the circulating CRP, further limiting its clinical usefulness in the evaluation of NASH in severely obese patients.

Loss of high affinity neurotensin receptors in substantia nigra from parkinsonian subjects

Monoiodo [125I-Tyr3]-Neurotensin binding was studied in post mortem substantia nigra from 17 control and 15 parkinsonian subjects. Binding to individual homogenates was decreased by 58%, 49% and 26% at 0.36, 1.4, 5.5 M(-9) concentration of ligand, respectively. Saturation analysis using pooled substantia nigra demonstrated an almost complete loss of the high affinity component of the neurotensin receptor complex, yielding a 24% loss of the total binding capacity, with no alteration of the low affinity component. Similarly an important loss of binding was observed in monoiodo[125I-Tyr3]-Neurotensin autoradiograms of two substantia nigra from parkinsonian subjects. These results support the hypothesis of neurotensin receptors occurring on dopamine cell bodies and/or dendrites in human substantia nigra. Role of neurotensin may be of importance in the regulation of dopamine pathway involved in parkinsonism.

Characterization and visualization of neurotensin binding to receptor sites in human brain

The binding of monoiodo [125I-Tyr3]-neurotensin to human brain was characterized and visualized using radioreceptorassay and autoradiographic techniques. Specific binding to homogenates of human substantia nigra at 25 degrees C was maximal at 20 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two populations of binding sites with Kd values of 0.26 nM and 4.3 nM. Corresponding binding capacities were 26 and 89 fmol/mg of protein. Neurotensin analogs inhibited the binding of iodinated neurotensin with relative potencies that demonstrated the crucial role of the C-terminal hexapeptide portion of neurotensin for binding to its receptors. Autoradiography of human substantia nigra sections incubated with iodinated neurotensin revealed high levels of specific binding in the nucleus paranigralis and substantia nigra, pars compacta, and low levels in the substantia nigra, pars reticulata.

Fasting, postprandial, and post-methionine-load homocysteinaemia and methylenetetrahydrofolate reductase polymorphism in vascular disease.

Hyperhomocysteinaemia is an independent risk factor for cardiovascular disease. The C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) is a common genetic cause of increased homocysteine (HCY) levels. Post-methionine-load HCY concentrations allow identification of certain cases of hyperhomocysteinaemia not demonstrated by fasting levels. This study investigated the relationship between MTHFR polymorphism and (1) fasting HCY levels (77 patients); (2) post-methionine HCY levels (54 patients); and (3) postprandial HCY concentrations (36 patients) in cardiovascular disease. As expected, mean fasting HCY value was higher in the +/+ patients. Moreover, patients who were homozygous for the mutation exhibited significantly increased mean post-methionine-load HCY; in contrast, literature results are conflicting. Mean postprandial HCY, which is not known to be increased in controls, was also increased in the (+/+) patients, although the difference did not reach statistical significance, probably owing to the small size of the sample. MTFHR polymorphism is known to be aggravated by a drop in circulating folate. Additional risk factors may be more prevalent in patients with cardiovascular disease.

Autoantibodies to the insulin receptor are infrequent findings in Type 1 (insulin-dependent) diabetes mellitus of recent onset

SummaryTo determine whether autoantibodies to the insulin receptor may represent markers of Type 1 (insulin-dependent) diabetes, the prevalence of such antibodies was investigated in sera of 60 newly diagnosed untreated Type 1 diabetic patients. A sensitive assay, based on enzyme linked immunosorbent assay has been set up which detects antibodies to the insulin receptor irrespective of their potentially inhibiting effect on insulin binding. Moreover, this method allows easy determination of the immunoglobulin class involved in the anti-receptor activity. Among the 60 sera examined, only one was found to contain anti-insulin receptor autoantibodies (IgG class). In view of our data, we conclude that autoantibodies to the insulin receptor are infrequent findings in Type 1 diabetes of recent onset.

O21 Expression différentielle de gènes de l’inflammation et de L’immunité spécifiquement associés à l’obésité et à la NASH dans le foie des patients obèses morbides

Introduction Les modifications d’expression hepatique des genes de l’inflammation et de l’immunite au cours de la progression du foie normal vers la steatose et ensuite vers la NASH sont mal caracterisees. Notre but a ete d’identifier des genes de l’inflammation et de l’immunite dont l’expression est specifiquement alteree par l’obesite et la NASH dans le foie de patients obeses morbides. Patients et Methodes L’expression de 222 genes de l’inflammation et de l’immunite a ete evaluee par PCR quantitative en temps reel dans le foie de 3 groupes de patients obeses morbides : (1) patients sans atteintes hepatiques (NAS = 0, n = 6), (2) patients presentant une steatose severe (NAS = 3, n = 6), (3) patients presentant une NASH associee avec une steatose severe (NAS≥5, n = 6) et dans le foie de sujets controles (n = 5). Resultats Parmi les 222 genes de l’inflammation et de l’immunite etudies, l’expression hepatique de 58 genes etait augmentee chez les patients NASH. Les principaux changements ont lieu sur les genes codant pour a) les chimiokines et leurs recepteurs impliques dans le recrutement des leucocytes (CXCL 1, 3, 8–11 ; CXCR 1–2, CCL 2–5 ; CCR5), b) les CD et cytokines impliques dans l’activation des lymphocytes vers un phenotype Th1 (IFNγ, TNFα, IL1β, IL6, CD 18, 28, 54, 80, 86) mais aussi c) les semaphorines immunes (SEMA 4A, 7A, 4D). 47 autres genes de l’inflammation et de l’immunite sont deja augmentes chez les patients obeses independamment des complications hepatiques (e.g. CRP ; TLR2, 4, 6 ; CD14). Conclusion Nous avons identifie des genes de l’inflammation et de l’immunite dont l’expression est deja modifiee dans le foie des patients obeses morbides ne presentant aucun signe histologique d’inflammation, suggerant que des facteurs extra hepatiques (adipokines, FFA, LPS) puissent modifier les fonctions hepatiques. Par ailleurs, la NASH est caracterisee par une signature genique specifique independante de l’obesite et de la steatose. Ces resultats pourraient aider a mieux comprendre la pathogenese des steatopathies metaboliques et permettre d’identifier des cibles therapeutiques pour la prise en charge des complications hepatiques associees a l’obesite.

P220 Étude de l’autophagie dans la mort cellulaire hépatocytaire in vitro et in vivo

Introduction L’obesite s’accompagne de nombreuses complications dont les complications hepatiques comprenant la steatose, l’inflammation et la mort hepatocytaire. Les mecanismes moleculaires responsables de cette apoptose hepatocytaire sont encore mal definis. Materiels et methodes La mort cellulaire a ete etudiee par cytometrie en flux. L’expression des genes a ete mesuree par PCR quantitative en temps reel. Les modeles d’etude sont des lignees hepatocytaires, des cultures primaires d’hepatocytes humains et des biopsies hepatiques de souris et de patients obeses morbides. Resultats Dans cette etude, nous avons pu mettre en evidence que le traitement de lignees hepatocytaires ou de cultures primaires d’hepatocytes humains par un acide gras sature (le palmitate) conduisait a une activation de l’autophagie, evaluee par une accumulation de la proteine LC3-II. Ce mecanisme n’est pas induit lors d’un traitement par un acide gras insature (l’oleate). L’activation de l’autophagie est directement associee a l’induction de l’apoptose hepatocytaire (plus de 50 % de cellules mortes a 16 heures). Cette activation de l’autophagie et de l’apoptose est totalement prevenue par le co-traitement de ces hepatocytes avec un melange palmitate/oleate (1/1). Par contre, un traitement par la bafilomycine, un inhibiteur specifique de l’autophagie, augmente de 30 % la mort cellulaire induite par le palmitate. Enfin, nous avons pu mettre en evidence une augmentation de l’expression genique des ATG dans le foie de souris ou patients avec une steatose hepatique severe par rapport aux souris controles et patients obeses sans atteintes hepatiques. Conclusion Ces resultats indiquent que l’activation de l’autophagie a un role protecteur sur l’apoptose hepatocytaire induit par les acides gras satures. Ce mecanisme de protection est present dans les foies steatosiques mais son niveau d’activation pourrait etre insuffisant pour prevenir l’apoptose hepatocytaire. Une activation de l’autophagie pourrait constituer une nouvelle cible therapeutique des complications hepatiques de l’obesite.