Jean Chambaz

A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction

The adipocyte-specific hormone leptin, the product of the obese (ob) gene,regulates adipose-tissue mass through hypothalamic effects on satiety and energy expenditure. Leptin acts through the leptin receptor, a single-transmembrane-domain receptor of the cytokine-receptor family. In rodents, homozygous mutations ingenes encoding leptin or the leptin receptor cause early-onsetmorbid obesity, hyperphagia and reduced energy expenditure. These rodents also show hypercortisolaemia, alterations in glucose homeostasis, dyslipidaemia, and infertility due to hypogonadotropic hypogonadism. In humans, leptin deficiency due to a mutation in the leptin gene is associated with early-onset obesity. Here we describe a homozygous mutation in the human leptin receptor gene that results in a truncated leptin receptor lacking both the transmembrane and the intracellular domains. In addition to their early-onset morbid obesity, patients homozygous for this mutation have no pubertal development and their secretion of growth hormone and thyrotropin is reduced. These results indicate that leptin is an important physiological regulator of several endocrine functions in humans.

Resistance of colon cancer cells to long-term 5-fluorouracil exposure is correlated to the relative level of Bcl-2 and Bcl-XL in addition to Bax and p53 status

Defects in apoptosis have been implicated in chemoresistance of colon cancer cells. We report here the ability to resist to 5‐fluorouracil‐induced apoptosis of 8 colon cancer cell lines differing in p53 and bax status: p53−/0bax+/+ for TC7, SW480, HT‐29; p53+/+bax−/− for LS174T, LoVo; p53+/+ bax+/− for HCT116; p53+/+ or p53+/0bax+/+ for LS513 or HCT‐EB, respectively. To approximate to the in vivo therapy, the cell lines were exposed to a long‐term treatment of 5‐FU. The analysis of proteins implicated in the apoptotic pathway has shown that the independent analysis of p53 or bax status was not sufficient to predict the extent of drug‐resistance of all cell lines. In p53+/+ cell lines but not in p53−/0 cell lines, a low level of the pro‐apoptotic Bax protein was correlated with a greater resistance of cells to 5‐FU. In addition, we found that high levels of anti‐apoptotic Bcl‐2 and Bcl‐xL proteins combined with a low level of Bax were correlated to high 5‐FU resistance of wild‐type p53 cell lines. The same correlation was obtained for 2 out of 3 mutated p53 cell lines. In conclusion, the relative levels of Bcl‐2, Bcl‐xL and Bax may altogether contribute to determine the resistance of a majority of colon tumor cells to long‐term 5‐FU treatment, whatever their p53 status.

Essential Fatty Acids Interconversion in the Human Fetal Liver

In order to study the role played by the fetoplacental unit in providing the human fetus with arachidonic acid, delta 5- and delta 6-desaturase activities were studied in microsomes from human fetal liver and placenta after 18 and 22 weeks of gestation. We evidenced for the first time delta 5- and delta 6-desaturase activities in fetal liver microsomes. As in adult liver, delta 6-desaturation is the rate-limiting step of arachidonic acid synthesis. No activity was found in the placenta. Arachidonic acid concentrations were higher in fetal serum than in maternal serum while the opposite was observed for linoleic acid. The fetal liver microsomal content in arachidonic acid was low. Taken together the data suggest that arachidonic acid is supplied to the fetus through a preferential transfer across the placenta.

The nonfibrillar amyloid beta-peptide induces apoptotic neuronal cell death: Involvement of its C-terminal fusogenic domain.

Abstract : The toxicity of the nonaggregated amyloid β‐peptide (1‐40) [Aβ(1‐40)] on the viability of rat cortical neurons in primary culture was investigated. We demonstrated that low concentrations of Aβ peptide, in a nonfibrillar form, induced a time‐ and dose‐dependent apoptotic cell death, including DNA condensation and fragmentation. We compared the neurotoxicity of the Aβ(1‐40) peptide with those of several Aβ‐peptide domains, comprising the membrane‐destabilizing C‐terminal domain of Aβ peptide (e.g., amino acids 29‐40 and 29‐42). These peptides reporduced the effects of the (1‐40) peptide, whereas mutant nonfusogenic Aβ peptides and the central region of the Aβ peptide (e.g., amino acids 13‐28) had no effect on cell viability. We further demonstrated that the neurotoxicity of the nonaggregated Aβ peptide paralleled a rapid and stable interaction between the Aβ peptide and the plasma membrane of neurons, preceding apoptosis and DNa fragmentation. By contrast, the peptide in a fibrillar form induced a rapid and dramatic neuronal death mainly through a necrotic pathway, under our conditions. Taken together, our results suggest that Aβ induces neuronal cell death by either apoptosis and necrosis and that an interaction between the nonfibrillar C‐terminal domain of the Aβ peptide and the plasma membrane of cortical neurons might represent an early event in a cascade leading to neurodegeneration.

Early Loss of E-cadherin from Cell-Cell Contacts Is Involved in the Onset of Anoikis in Enterocytes

Anoikis, i.e. apoptosis induced by detachment from the extracellular matrix, is thought to be involved in the shedding of enterocytes at the tip of intestinal villi. Mechanisms controlling enterocyte survival are poorly understood. We investigated the role of E-cadherin, a key protein of cell-cell adhesion, in the control of anoikis of normal intestinal epithelial cells, by detaching murine villus epithelial cells from the underlying basement membrane while preserving cell-cell interactions. We show that upon the loss of anchorage, normal enterocytes execute a program of apoptosis within minutes, via a Bcl-2-regulated and caspase-9-dependent pathway. E-cadherin is lost early from cell-cell contacts. This process precedes the execution phase of detachment-induced apoptosis as it is only weakly modulated by Bcl-2 overexpression or caspase inhibition. E-cadherin loss, however, is efficiently prevented by lysosome and proteasome inhibitors. We also found that a blocking anti-E-cadherin antibody increases the rate of anoikis, whereas the activation of E-cadherin using E-cadherin-Fc chimera proteins reduces anoikis. In conclusion, our results stress the striking sensitivity of normal enterocytes to the loss of anchorage and the contribution of E-cadherin to the control of their survival/apoptosis balance. They open new perspectives on the key role of this protein, which is dysregulated in the intestinal epithelium in both inflammatory bowel disease and cancer.

Bovine Prion Is Endocytosed by Human Enterocytes via the 37 kDa/67 kDa Laminin Receptor

Some forms of transmissible spongiform encephalopathies result from oral infection. We have thus analyzed the early mechanisms that could account for an uptake of infectious prion particles by enterocytes, the major cell population of the intestinal epithelium. Human Caco-2/TC7 enterocytes cultured on microporous filters were incubated with different prion strains and contaminated brain homogenates in the apical compartment. Internalization of infectious particles was analyzed by Western blotting and immunofluorescence. We observed internalization by enterocytes of prion particles from bovine spongiform encephalopathy brain homogenates but not from mouse-adapted scrapie-strain brain homogenates or purified bovine spongiform encephalopathy scrapie-associated fibrils. Bovine prion particles were internalized via endocytosis within minutes of infection and were associated with subapical vesicular structures related to early endosomes. The endocytosis of the infectious bovine PrP(Sc) was reduced by preincubating the cells with an anti-LRP/LR blocking antibody, identifying the 37 kDa/67 kDa laminin receptor (LRP/LR), which is apically expressed in Caco-2/TC7 cells, as the receptor for the infectious prion protein. Altogether, our results underscore a potential role of enterocytes in the absorption of bovine prions during oral infection through specific LRP/LR-dependent endocytosis.

Apoptotic Neuronal Cell Death Induced by the Non-fibrillar Amyloid-β Peptide Proceeds through an Early Reactive Oxygen Species-dependent Cytoskeleton Perturbation

In the present study, we have determined the nature and the kinetics of the cellular events triggered by the exposure of cells to non-fibrillar amyloid-β peptide (Aβ). When cortical neurons were treated with low concentrations of soluble Aβ (1–40), an early reactive oxygen species (ROS)-dependent cytoskeleton disruption precedes caspase activation. Indeed, caspase activation and neuronal cell death were prevented by the microtubule-stabilizing drug taxol. A perturbation of the microtubule network was noticeable after being exposed to Aβ for 1 h, as revealed by electron microscopy and immunocytochemistry. Microtubule disruption and neuronal cell death induced by Aβ were inhibited in the presence of antioxidant molecules, such as probucol. These data highlight the critical role of ROS production in Aβ-mediated cytoskeleton disruption and neuronal cell death. Finally, using FRAP (fluorescence recoveryafter photo bleaching) analysis, we observed a time-dependent biphasic modification of plasma membrane fluidity, as early as microtubule disorganization. Interestingly, molecules that inhibited neurotubule perturbation and cell death did not affect the membrane destabilizing properties of Aβ, suggesting that the lipid phase of the plasma membrane might represent the earliest target for Aβ. Altogether our results convey the idea that upon interaction with the plasma membrane, the non-fibrillar Aβ induces a rapid ROS-dependent disorganization of the cytoskeleton, which results in apoptosis.

Reg IV, a new member of the regenerating gene family, is overexpressed in colorectal carcinomas

A better understanding of the mechanisms by which colon tumor cells are able to survive exposure to drugs would be valuable for the development of new therapeutic strategies. We used differential display‐PCR to compare gene expression in the drug‐sensitive HT‐29 colon cancer cell line and 3 drug‐resistant subpopulations derived from this parental cell line. One of the genes identified is a new gene, Regenerating IV gene (Reg IV), and was strongly overexpressed in HT‐29 drug‐resistant cells. Other drug‐resistant cell lines expressed Reg IV at a high level, whereas a low expression was noted in sensitive cell lines. Northern blot and real‐time PCR analysis showed that Reg IV is more strongly expressed in 71% of colorectal tumors (in particular in mucinous carcinomas) than in normal colon tissues. The comparison of Reg IV expression with that of other REG genes, Regenerating Iα or (Reg Iα), Regenerating Iβ (Reg Iβ) and Pancreatitis‐associated protein (PAP), highlights its predominant expression in colorectal tumors. Reg IV mRNA‐positive tumor cells display different phenotypes: mucus‐secreting, enterocyte‐like or undifferentiated. Interestingly, whereas Reg IV expression is low in normal colon, its level in normal small intestine is similar to that in some colorectal tumors. In normal tissue, Reg IV mRNA‐positive cells are mostly enteroendocrine cells and goblet cells. Our results point out the potential role of Reg IV in colorectal tumors and its subsequent interest as a pronostic indicator of tumor survival.

Molecular basis of Alzheimer's disease

Abstract. Despite an exponential production of data, Alzheimers disease (AD) remains an enigma. Unresolved questions persist in the face of the heterogeneity of this neuropathology. Recent progress in understanding mechanisms for AD results from the study of amyloid precursor protein (APP) metabolism and the involvement of senile plaque-associated proteins. In addition to the amyloid cascade hypothesis, alternative schemes emerge, in which the amyloid peptide is not the primary effector of the disease. Perturbations of vesicular trafficking, the cytoskeletal network, and membrane cholesterol distribution could be central events. Furthermore, since the physiological role of APP, presenilins, and apolipoprotein E in the central nervous system are not completely understood, their involvement in AD etiology remains speculative. New actors have to be found to try to explain sporadic cases and non-elucidated familial cases.

Overexpression of Human Apolipoprotein A-II in Mice Induces Hypertriglyceridemia Due to Defective Very Low Density Lipoprotein Hydrolysis

Two lines of transgenic mice, hAIItg-δ and hAIItg-λ, expressing human apolipoprotein (apo)A-II at 2 and 4 times the normal concentration, respectively, displayed on standard chow postprandial chylomicronemia, large quantities of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) but greatly reduced high density lipoprotein (HDL). Hypertriglyceridemia may result from increased VLDL production, decreased VLDL catabolism, or both. Post-Triton VLDL production was comparable in transgenic and control mice. Postheparin lipoprotein lipase (LPL) and hepatic lipase activities decreased at most by 30% in transgenic mice, whereas adipose tissue and muscle LPL activities were unaffected, indicating normal LPL synthesis. However, VLDL-triglyceride hydrolysis by exogenous LPL was considerably slower in transgenic compared with control mice, with the apparent V max of the reaction decreasing proportionately to human apoA-II expression. Human apoA-II was present in appreciable amounts in the VLDL of transgenic mice, which also carried apoC-II. The addition of purified apoA-II in postheparin plasma from control mice induced a dose-dependent decrease in LPL and hepatic lipase activities. In conclusion, overexpression of human apoA-II in transgenic mice induced the proatherogenic lipoprotein profile of low plasma HDL and postprandial hypertriglyceridemia because of decreased VLDL catabolism by LPL.