Patrick Sauvant

CELLULAR RETINOL-BINDING PROTEIN I IS ESSENTIAL FOR VITAMIN A HOMEOSTASIS

The gene encoding cellular retinol (ROL, vitA)‐binding protein type I (CRBPI) has been inactivated. Mutant mice fed a vitA‐enriched diet are healthy and fertile. They do not present any of the congenital abnormalities related to retinoic acid (RA) deficiency, indicating that CRBPI is not indispensable for RA synthesis. However, CRBPI deficiency results in an ∼50% reduction of retinyl ester (RE) accumulation in hepatic stellate cells. This reduction is due to a decreased synthesis and a 6‐fold faster turnover, which are not related to changes in the levels of RE metabolizing enzymes, but probably reflect an impaired delivery of ROL to lecithin:retinol acyltransferase. CRBPI‐null mice fed a vitA‐deficient diet for 5 months fully exhaust their RE stores. Thus, CRBPI is indispensable for efficient RE synthesis and storage, and its absence results in a waste of ROL that is asymptomatic in vitA‐sufficient animals, but leads to a severe syndrome of vitA deficiency in animals fed a vitA‐deficient diet.

ELISA as a new method to measure genistein and daidzein in food and human fluids

Widely distributed in the plant kingdom, phytoestrogens, like soy isoflavones are found in plant protein extracts at varying levels depending on culture conditions and cultivars. Their increasingly used in human, as natural estrogens and their varying levels in raw materials raise the question concerning the measurement of isoflavones both in food or food supplements as well as in human fluid. To validate our new ELISA technique, isoflavones measurements in food-supplements, in soy food and in human fluid from volunteers participating to a kinetic study or a survey, were done. Our method was also compared with other physico-chemical techniques in an inter-laboratory assay (23 laboratories). In conclusion, our new ELISA technique is reliable, sensitive, cheap, rapid and can be used either for a great number of human fluid analysis, for crude matter or food analysis as long as the appropriate extraction technique is performed prior to ELISA procedure. # 2003 Elsevier Ltd. All rights reserved.

PAV-1, a new rat hepatic stellate cell line converts retinol into retinoic acid, a process altered by ethanol

During liver fibrogenesis or long term culture, hepatic stellate cells (HSCs) evolved from quiescent to activated phenotype called myofibroblast-like, a transition prevented by retinoic acid (RA). Little is known about RA generation by HSCs. Our study aimed to check the ability of these cells to produce RA from retinol (Rol) and the alterations of this metabolic step by ethanol. To study this metabolic pathway, primary cultures of HSCs represent the most physiological model but technically suffer several drawbacks. To circumvent these problems, an immortalized rat HSC line (named PAV-1) has been established. We validated PAV-1 cell line as a convenient model to study retinoids metabolism by HSCs. Then, we showed that PAV-1 cells express Rol-binding proteins (RBPs), enzymes and nuclear receptors involved in RA signaling pathway. We also demonstrated in situ generation of functional all-trans-RA (ATRA), using transient transfections with a RA-sensitive reporter gene, in situ modulation of tissue transglutaminase (tTG) activity and HPLC experiments. This production was Rol dose-dependent; 4-methylpyrazole, citral, and ethanol-inhibited which argues in favor of an enzymatic process.In conclusion, we first demonstrate in situ RA generation from Rol in a newly immortalized rat HSC line, named PAV-1. Inhibition of RA production by ethanol in PAV-1 and recent data, suggesting fundamental role of RA to prevent fibrosis development in the liver, allow us to hypothesize that Rol metabolism could be a primary target for ethanol during development of hepatic fibrosis.

Vitamin A and lipid metabolism: relationship between hepatic stellate cells (HSCs) and adipocytes

Vitamin A or retinol plays a major role in the regulation of cellular homeostasis. Retinyl palmitate remains the main chemical form of vitamin A storage and is mainly located in hepatic stellate cells (HSCs) in lipid droplets resembling those found in adipose cells. White adipose tissue (WAT), is essentially involved in the regulation of lipid metabolism, through its role in lipid storage, and might also be considered as a vitamin A storage and metabolism site. WAT contains all the intracellular equipment for vitamin A metabolism and signaling pathways which allows retinol to be metabolized into retinoic acid, known to control genomic expression in WAT. The description of molecular mechanisms involved in the activation of HSCs and the differentiation of preadipocytes reveal similar cellular and molecular mechanisms. Indeed HSCs and adipocytes share a common expression of key transcription factors like PPAR-γ and RXR known to influence perilipin expression, which play fundamental roles in lipid droplet metabolism. Both cells are also sources of important endocrine signaling secretions influencing the expression of these transcription factors. The morphological and functional characteristics of HSCs and adipocytes, including the metabolism of vitamin A and other lipids and their related signaling pathways, are summarized and compared in this review. We highlight the complexity of the interrelationship between lipids and vitamin A metabolism and the role of the complex communication existing between HSCs and WAT in diseases such as non-alcoholic fatty liver disease which is the hepatic manifestation of the metabolic syndrome.

Higher bioavailability of isoflavones after a single ingestion of a soya-based supplement than a soya-based food in young healthy males

Soya isoflavones, genistein and daidzein, are the focus of numerous studies investigating their potential effects on health and results remain controversial. Bioavailability is clearly a crucial factor influencing their bioefficacy and could explain these discrepancies. This study aimed at assessing: (1) the isoflavone content of sixty-nine European soya-derivative products sold on the French market; (2) the bioavailability of isoflavones comparing supplement with food. Twelve healthy volunteers were recruited in a randomized two-way crossover trial and received 35 mg isoflavones equivalent aglycone either through supplements or through cheese, both containing different patterns of isoflavone conjugates and different daidzein:genistein ratios. A specific ELISA method was used to assess the plasma and urinary concentrations of isoflavones and thus the pharmacokinetic parameters, which were then normalized to mg of each isoflavone ingested. Results showed that the normalized Cmax of daidzein (P = 0.002) and similarly the normalized AUC0 --> infinity and Cmax of genistein (P = 0.002) from soya-based capsules were higher than that from soya-based cheese. In conclusion, this work completes studies on isoflavone bioavailability and presents new data regarding isoflavone concentrations in soya-derivative products. Assuming that isoflavone conjugation patterns do not influence isoflavone bioavailability, this study shows that isoflavones contained in capsules are more bioavailable than those contained in soya-based cheese. Although the supplement is more bioavailable, the relative importance of this is difficult to interpret as there is little evidence that supplements are biologically active in human subjects to date and further studies will be necessary for this specific supplement to prove its efficacy.

Growth Arrest and Decrease of α-SMA and Type I Collagen Expression by Palmitic Acid in the Rat Hepatic Stellate Cell Line PAV-1

Liver fibrosis is characterized by an activation of hepatic stellate cells (HSC). During primary culture HSC evolve from a quiescent into an activated phenotype which is characterized by α-smooth muscle actin (α-SMA) up-regulation, increase in cell growth, and extracellular matrix secretion. HSC culture with trans-resveratrol can lead to deactivation of myofibroblast-like HSC. We used an HSC line, PAV-1, to check the role of retinol and palmitic acid in the deactivation process of HSC. Using mass and metabolic-based methods, Western blot and immunocytochemistry assays, we demonstrated that treatment with palmitic acid (75xa0μM) alone or in combination with retinol (2xa0μM) significantly decreased cell proliferation and α-SMA expression. We also established that the association of both compounds strongly decreased collagen type I expression. Our results suggest the potential use of palmitic acid alone or in combination with retinol to induce HSC deactivation.

Retinol mobilization from cultured rat hepatic stellate cells does not require retinol binding protein synthesis and secretion

Retinol mobilization from retinyl esters stores of hepatic stellate cells (HSCs) is a key step in the regulation of mammalian retinol homeostasis, but the precise mechanisms of such a mobilization are still poorly understood. Using primary cultures of HSCs, we first demonstrated that HSCs expressed immunoreactivity against retinol-binding-protein (RBP) when cultured in a medium containing RBP but were unable to synthesize RBP transcripts and proteins. Using pulse and chase-type experiments, we demonstrated that radioactive retinol was released in culture medium without binding proteins. Inhibition of protein secretion by brefeldin A did not modify quantitatively retinol release. This data ruled out, for the first time, the direct involvement of RBP in retinol mobilization from HSCs. Moreover, HSCs co-cultured with primary isolated hepatocytes displayed an increase of retinol transfer from HSCs to hepatocytes when they established direct physical contacts, as compared with co-cultures without contact. Based on this latter data, a mechanism of retinol mobilization from HSCs via the hepatocytes using retinol transfer through cellular membranes is proposed.

Influence of ethnic origin (Asian v . Caucasian) and background diet on the bioavailability of dietary isoflavones

Soya isoflavones: genistein and daidzein are increasingly consumed in Western countries. Their beneficial effects are discussed considering nutrition and health in Asia. The present study aimed to check whether chronic ingestions, ethnic origin and dietary context can influence soya phyto-oestrogen bioavailability. Two prospective trials were carried out to blindly assess the pharmacokinetics after acute and chronic intake of soya-based cheese (45.97 (sd1.57) mg isoflavones) taken once a day for 10 d. Twelve healthy young Asians immersed for 2 months in France were randomised in a cross-over design to compare the influence of a Western v. Asian dietary context. The second trial partly nested in the first one, compared Asians under the Western diet to twelve healthy young male Caucasians under the same diet. All volunteers were non-equol producers. After an acute intake of soya in Western diet, Asians exhibited higher maximum concentration measured in plasma (Cmax) and area under the plasma concentration-time curve (AUC) for genistein and daidzein than Caucasians (P = 0.005, 0.006, 0.032 and 0.008, respectively). In Caucasians under Western diet, AUC and Cmax values significantly increased after chronic intake. This was not the case for daidzein in Asians whatever the dietary context. For the first time, it is evidenced that on acute intake of soya cheese, Asians absorb soya phyto-oestrogens better than Caucasians, regardless of whether the background diet is Western or Asian. On chronic ingestions, AUC and Cmax values were increased for daidzein and genistein in Caucasians but not in Asians. There are ethnic differences in isoflavone pharmacokinetic and bioavailability. This may influence health outcomes.

Possible mechanisms of weight loss of Siberian hamsters (Phodopus sungorus sungorus) exposed to short photoperiod

Several weeks of short day photoperiod (SD) exposure promote a dramatic decrease of white adipose tissue (WAT) mass in Siberian hamsters(Phodopus sungorus sungorus). This slimming effect is accompanied by changes in the adipocyte responsiveness to adrenergic stimulation that are still under debate. We investigated whether possible changes in the antilipolytic responses, and/or lipogenic activities could be involved in such lipid deposition/mobilisation imbalance. Male Siberian hamsters were exposed for 11 weeks to SD or long day photoperiod and basal or stimulated lipolytic and lipogenic activities were measured on white adipocytes. As expected, the body mass of SD-animals was decreased. Besides a slight reduction in the basal lipolysis and in the maximal response to dibutyryl-cAMP, the responses to adrenergic and non-adrenergic lipolytic agents (forskolin, adenosine deaminase) were similar in both groups. Fat mass loss was likely not resulting from changes in the lipolytic responses of adipocytes to biogenic amines (e.g. octopamine), which were unaltered, or to a direct lipolytic stimulation by melatonin or histamine, which were inactive. Antilipolytic responses to insulin or tyramine were slightly decreased in SD-adipocytes. Basal or insulin-stimulated lipid accumulation in WAT, measured by glucose incorporation into lipids, did not change after SD-exposure. However, a significant decrease in the lipoprotein lipase activity was observed in the WAT of SDanimals. Despite the observed changes, the weight loss of SD-exposed Siberian hamsters was likely not resulting only from impaired antilipolytic orde novo lipogenic activities in white adipocytes, but either from other dramatic changes occurring during seasonal photoperiod-sensitive body weight regulation.

Synthesis of haptens and conjugates for ELISA of glycitein: development and validation of an immunological test.

Two carboxylic acid haptens of glycitein were synthesized, with a spacer arm at the C2 position. They differed in the length of the spacer arm, with the length of the spacer arms being three or four carbon atoms, and were named Delta3-glycitein and Delta4-glycitein haptens, respectively. The different haptens were coupled to bovine serum albumin (BSA), and the coupling efficiency was assessed by MALDI mass spectrometry. Polyclonal antibodies were generated against the BSA conjugates. An additional conjugate of Delta4-glycitein hapten was generated with swine thyroglobulin (Thyr). Enzyme-linked immunosorbent assays (ELISAs) based on the competition between free glycitein and Delta4-glycitein-Thyr conjugates for specific antibodies were developed. The IC50 of the standard curves was 15.6 ng mL(-1) with anti-Delta3-glycitein and 62.5 ng mL(-1) with anti-Delta4-glycitein, that is, 10.9 and 44 pmol/well, respectively. With the Delta3-glycitein antibody, interassay and intra-assay variations were 12.2 and 11.5%, respectively. Specificity tests did not show any significant cross-reaction with any other soy isoflavone. This specificity is not influenced by the length of the spacer arm. The assay was validated by measurements performed on plasma samples as well as on soy-based foodstuffs and on soy-based food supplements.