Obesogenic diet induces sex-specific alterations of contextual fear memory and associated hippocampal activity in mice.

In addition to metabolic and cardiovascular disorders, obesity is associated with cognitive deficits in humans and animal models. We have previously shown that obesogenic high-fat and sugar diet intake during adolescence (adoHFSD) impairs hippocampus (HPC)-dependent memory in rodents. These results were obtained in males only and it remains to evaluate whether adoHFSD has similar effect in females. Therefore, here, we investigated the effects of adoHFSD consumption on HPC-dependent contextual fear memory and associated brain activation in male and female mice. Exposure to adoHFSD increased fat mass accumulation and glucose levels in both males and females but impaired contextual fear memory only in males. Compared with females, contextual fear conditioning induced higher neuronal activation in the dorsal and ventral HPC (CA1 and CA3 subfields) as well as in the medial prefrontal cortex in males. Also, adoHFSD-fed males showed enhanced c-Fos expression in the dorsal HPC, particularly in the dentate gyrus, and in the basolateral amygdala compared with the other groups. Finally, chemogenetic inactivation of the dorsal HPC rescued adoHFSD-induced memory deficits in males. Our results suggest that males are more vulnerable to the effects of adoHFSD on HPC-dependent aversive memory than females, due to overactivation of the dorsal HPC.

Chemogenetic activation or inhibition of histaminergic neurons bidirectionally modulates recognition memory formation and retrieval in male and female mice.

Several lines of evidence demonstrate that the brain histaminergic system is fundamental for cognitive processes and the expression of memories. Here, we investigated the effect of acute silencing or activation of histaminergic neurons in the hypothalamic tuberomamillary nucleus (TMNHA neurons) in vivo in both sexes in an attempt to provide direct and causal evidence of the necessary role of these neurons in recognition memory formation and retrieval. To this end, we compared the performance of mice in two non-aversive and non-rewarded memory tests, the social and object recognition memory tasks, which are known to recruit different brain circuitries. To directly establish the impact of inactivation or activation of TMNHA neurons, we examined the effect of specific chemogenetic manipulations during the formation (acquisition/consolidation) or retrieval of recognition memories. We consistently found that acute chemogenetic silencing of TMNHA neurons disrupts the formation or retrieval of both social and object recognition memory in males and females. Conversely, acute chemogenetic activation of TMNHA neurons during training or retrieval extended social memory in both sexes and object memory in a sex-specific fashion. These results suggest that the formation or retrieval of recognition memory requires the tonic activity of histaminergic neurons and strengthen the concept that boosting the brain histaminergic system can promote the retrieval of apparently lost memories.

Nucleus accumbens D1- and D2-expressing neurons control the balance between feeding and activity-mediated energy expenditure.

Accumulating evidence points to dysregulations of the Nucleus Accumbens (NAc) in eating disorders (ED), however its precise contribution to ED symptomatic dimensions remains unclear. Using chemogenetic manipulations in male mice, we found that activity of dopamine D1 receptor-expressing neurons of the NAc core subregion facilitated effort for a food reward as well as voluntary exercise, but decreased food intake, while D2-expressing neurons have opposite effects. These effects are congruent with D2-neurons being more active than D1-neurons during feeding while it is the opposite during running. Chronic manipulations of each subpopulations had limited effects on energy balance. However, repeated activation of D1-neurons combined with inhibition of D2-neurons biased behavior toward activity-related energy expenditure, whilst the opposite manipulations favored energy intake. Strikingly, concomitant activation of D1-neurons and inhibition of D2-neurons precipitated weight loss in anorexia models. These results suggest that dysregulations of NAc dopaminoceptive neurons might be at the core of EDs.

Age-dependent effects of estradiol on temporal memory: A role for the type 1 cannabinoid receptor?

This study investigated in male mice how age modulates the effects of acute 17ß-estradiol (E2) on dorsal CA1 (dCA1)-dependent retention of temporal associations, which are critical for declarative memory. E2 was systemically injected to young (3-4 months old) and aged (22-24 months old) adult mice either (i) 1 h before the acquisition of an auditory trace fear conditioning (TFC) procedure allowing the assessment of temporal memory retention 24 h later or (ii) during in vivo electrophysiological recordings of CA3 to dCA1 synaptic efficacy under anesthesia. In young mice, E2 induced parallel dose-dependent reductions in memory and synaptic efficacy, i.e. an impairment in TFC retention and a long-term (NMDA receptor-dependent) depression of dCA1 synaptic efficacy as assessed by field excitatory postsynaptic potentials. In contrast, E2 tended to improved TFC retention whilst failing to change synaptic efficacy in aged mice. Age-dependent effects of E2 treatment were confirmed by immunohistochemical analyses of TFC acquisition-elicited dCA1 Fos activation. Thus, such an activation was respectively reduced and enhanced in young and aged E2-treated mice, compared to vehicle treatments. Hippocampal mRNA expression of estrogen receptors by RT-PCR analyses revealed an age-related increase in each receptor mRNA expression. In keeping with the key role of the endocannabinoid system in memory processes and CA3 to dCA1 synaptic plasticity, we next examined the role of cannabinoid type 1 receptors (CB1-R) in the aforementioned age-dependent effects of E2. Having confirmed that mRNA expression of CB1-R diminishes with age, we then observed that the deleterious effects of E2 on both memory and synaptic efficacy were both prevented by the CB1-R antagonist Rimonabant whilst being absent in CB1-R knock out mice. This study (i) reveals age-dependent effects of acute E2 on temporal memory and CA3 to dCA1 synaptic efficacy and (ii) suggests a key role of CB1-R in mediating E2 deleterious effects in young adulthood. Aging-related reductions in CB1-R might thus underlie E2 paradoxical effects across age.

Age-related impairment of declarative memory: linking memorization of temporal associations to GluN2B redistribution in dorsal CA1.

GluN2B subunits of NMDA receptors have been proposed as a target for treating age-related memory decline. They are indeed considered as crucial for hippocampal synaptic plasticity and hippocampus-dependent memory formation, which are both altered in aging. Because a synaptic enrichment in GluN2B is associated with hippocampal LTP in vitro, a similar mechanism is expected to occur during memory formation. We show instead that a reduction of GluN2B synaptic localization induced by a single-session learning in dorsal CA1 apical dendrites is predictive of efficient memorization of a temporal association. Furthermore, synaptic accumulation of GluN2B, rather than insufficient synaptic localization of these subunits, is causally involved in the age-related impairment of memory. These challenging data identify extra-synaptic redistribution of GluN2B-containing NMDAR induced by learning as a molecular signature of memory formation and indicate that modulating GluN2B synaptic localization might represent a useful therapeutic strategy in cognitive aging.

Protocols to Study Declarative Memory Formation in Mice and Humans:Optogenetics and Translational Behavioral Approaches.

Declarative memory formation depends on the hippocampus and declines in aging. Two functions of the hippocampus, temporal binding and relational organization (Rawlins and Tsaltas, 1983; Eichenbaum et al., 1992 ; Cohen et al., 1997 ), are known to decline in aging (Leal and Yassa, 2015). However, in the literature distinct procedures have been used to study these two functions. Here, we describe the experimental procedures used to investigate how these two processes are related in the formation of declarative memory and how they are compromised in aging ( Sellami et al., 2017 ). First, we studied temporal binding using a one-trial learning procedure: trace fear conditioning. It is classical Pavlovian conditioning requiring temporal binding since a brief temporal gap separates the conditioned stimulus (CS) and unconditioned stimulus (US) presentations. We combined the trace fear condition procedure with an optogenetic approach, and we showed that the temporal binding relies on dorsal (d)CA1 activity over temporal gaps. Then, we studied the interaction between temporal binding and relational organization in declarative memory formation using a two-phase radial-maze task in mice and its virtual analog in humans. The behavioral procedure comprises an initial learning phase where subjects learned the constant rewarding /no rewarding valence of each arm, followed by a test phase where the reward contingencies among the arms remained unchanged but where the arms were recombined to assess flexibility, a cardinal property of declarative memory. We demonstrated that dCA1-dependent temporal binding is necessary for the development of a relational organization of memories that allows flexible declarative memory expression. Furthermore, in aging, the degradation of declarative memory is due to a reduction of temporal binding capacity that prevents relation organization.

Temporal binding function of dorsal CA1 is critical for declarative memory formation.

Temporal binding, the process that enables association between discontiguous stimuli in memory, and relational organization, a process that enables the flexibility of declarative memories, are both hippocampus-dependent and decline in aging. However, how these two processes are related in supporting declarative memory formation and how they are compromised in age-related memory loss remain hypothetical. We here identify a causal link between these two features of declarative memory: Temporal binding is a necessary condition for the relational organization of discontiguous events. We demonstrate that the formation of a relational memory is limited by the capability of temporal binding, which depends on dorsal (d)CA1 activity over time intervals and diminishes in aging. Conversely, relational representation is successful even in aged individuals when the demand on temporal binding is minimized, showing that relational/declarative memory per se is not impaired in aging. Thus, bridging temporal intervals by dCA1 activity is a critical foundation of relational representation, and a deterioration of this mechanism is responsible for the age-associated memory impairment.

Derivatization-free LC-MS/MS method for estrogen quantification in mouse brain highlights a local metabolic regulation after oral versus subcutaneous administration.

17ß-Estradiol (17ß-E2) is a steroid with pleiotropic actions. In addition to being a sexual hormone, it is also produced in the brain where it modulates the reproductive axis. It has been shown that 17ß-E2 also acts on synaptic plasticity and plays a role in neurological pathways and in neurodegenerative diseases. Assaying this steroid in the brain is thus interesting to improve our knowledge of 17ß-E2 effects in the brain. However, 17ß-E2 concentration in the central nervous system has been reported to be of a few nanograms per gram wet weight (nanomolar range concentration); therefore, its quantification requires both an efficient extraction process and a sensitive detection method. Herein is presented a derivatization-free procedure based on solid-phase extraction followed by LC-MS/MS analysis, targeted on 17ß-E2, its isomer17α-E2, and its metabolites estrone (E1) and estriol (E3). This extraction process allowed reaching 96% 17ß-E2 recovery from the mouse brain. Limit of detection (LOD) and limit of quantification (LOQ) values of 0.5 and 2.5 pmol mL-1, respectively, were reached for both 17α-E2 and 17ß-E2. LOD values for E1 and E3 were 0.01 and 0.025 pmol mL-1, respectively. The variation coefficients for intra- and inter-assays were 6 and 14%, respectively, for both estradiol forms. The method was applied to assess estrogen levels in the mouse brain and hippocampus after 17ß-E2 acute (subcutaneous injection) and chronic (drinking water) physiological administration. Total estrogen levels were determined after enzymatic deconjugation and compared to free estrogen levels. While 17α-E2 was not detected in biological samples, 17ß-E2 and metabolite measurements highlight a local biotransformation of estrogens after physiological administration via drinking water. Graphical abstract Method workflow: After oral or subcutaneous Estradiol administration, mouse brain or hippocampus was removed. Samples were homogenized and prepared according to a liquid-liquid extraction, followed by a solid-phase extraction. Then, LC-MS/MS was optimized to quantify 17ß-E2, its isomer17α-E2, its metabolites estrone (E1) and estriol (E3) and their conjugates.

Estradiol enhances retention but not organization of hippocampus-dependent memory in intact male mice.

Because estrogens have mostly been studied in gonadectomized females, effects of chronic exposure to environmental estrogens in the general population are underestimated. Estrogens can enhance hippocampus-dependent memory through the modulation of information storage. However, declarative memory, the hippocampus-dependent memory of facts and events, demands more than abilities to retain information. Specifically, memory of repetitive events of everyday life such as "where I parked" requires abilities to organize/update memories to prevent proactive interference from similar memories of previous "parking events". Whether such organizational processes are estrogen-sensitive is unknown. We here studied, in intact young and aged adult mice, drinking-water (1µM) estradiol effects on both retention and organizational components of hippocampus-dependent memory, using a radial-maze task of everyday-like memory. Demand on retention vs organization was manipulated by varying the time-interval separating repetitions of similar events. Estradiol increased performance in young and aged mice under minimized organizational demand, but failed to improve the age-associated memory impairment and diminished performance in young mice under high organizational demand. In fact, estradiol prolonged mnemonic retention of successive events without improving organization abilities, hence resulted in more proactive interference from irrelevant memories. c-Fos imaging of testing-induced brain activations showed that the deterioration of young memory was associated with dentate gyrus dysconnectivity, reminiscent of that seen in aged mice. Our findings support the view that estradiol is promnesic but also reveal that such property can paradoxically impair memory. These findings have important outcomes regarding health issues relative to the impact of environmental estrogens in the general population.

Temporal Memory and Its Enhancement by Estradiol Requires Surface Dynamics of Hippocampal CA1 N-Methyl-D-Aspartate Receptors.

BACKGROUND: Identifying the underlying cellular mechanisms of episodic memory is an important challenge, since this memory, based on temporal and contextual associations among events, undergoes preferential degradation in aging and various neuropsychiatric disorders. Memory storage of temporal and contextual associations is known to rely on hippocampal N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity, which depends ex vivo on dynamic organization of surface NMDARs. Whether NMDAR surface trafficking sustains the formation of associative memory, however, remains unknown. METHODS: We tested this hypothesis, using single nanoparticle imaging, electrophysiology, and behavioral approaches, in hippocampal networks challenged with a potent modulator of NMDAR-dependent synaptic plasticity and memory, 17ß-estradiol (E2). RESULTS: We demonstrate that E2 modulates NMDAR surface trafficking, a necessary condition for E2-induced potentiation at hippocampal cornu ammonis 1 synapses. Strikingly, cornu ammonis 1 NMDAR surface trafficking controls basal and E2-enhanced mnemonic retention of temporal, but not contextual, associations. CONCLUSIONS: NMDAR surface trafficking and its modulation by the sex hormone E2 is a cellular mechanism critical for a major component of episodic memory, opening a new and noncanonical research avenue in the physiopathology of cognition.

Naringin, the major grapefruit flavonoid, specifically affects atherosclerosis development in diet-induced hypercholesterolemia in mice.

Naringin (NAR) from grapefruit has exhibited potential protective effects against atherosclerosis development. However, specific mechanisms responsible for such effects are poorly understood. Thus, we aimed to investigate the antiatherogenic effects of NAR in different mouse models of hypercholesterolemia and decipher its molecular targets in the aorta using transcriptomic approach. Two mouse models of hypercholesterolemia, wild-type mice fed a high-fat/high-cholesterol diet and apolipoprotein E-deficient mice fed a semisynthetic diet, were studied. Mice were fed a respective control diets supplemented or not for 18 weeks with 0.02% of NAR, that is, nutritional supplementation. NAR supplementation reduced plaque progression only in wild-type mice fed the high-fat/high-cholesterol diet (-41%). Consistent with this protective effect, NAR reduced plasma non-high-density lipoprotein cholesterol concentrations as well as biomarkers of endothelial dysfunction. Microarray studies performed on aortas demonstrated differentially expressed genes encoding proteins involved in cell adhesion, actin cytoskeleton organization and cell division. Thus, the changes in gene expression induced by NAR could suggest a limited atherosclerosis progression by preventing immune cell adhesion and infiltration in the intima of vascular wall, as well as smooth muscle cell proliferation. Furthermore, this hypothesis was strengthened by in vitro experiments, which showed the ability of naringenin to reduce monocyte adhesion to endothelial cells and smooth muscle cell proliferation. In conclusion, this study revealed the antiatherogenic effect of NAR supplemented at a nutritionally achievable dose, specifically toward diet-induced atherosclerosis, and depicted its multitarget mode of action at the vascular level.

The satiety effect of disguised liquid preloads administered acutely and differing only in their nutrient content tended to be weaker for lipids but did not differ between proteins and carbohydrates in human subjects.

Whether protein is the macronutrient with the strongest satiety effect remains a matter of debate because of the diversity of study designs employed. The aim of the present study was to compare the effect of different liquid preloads made up of proteins, fats or carbohydrates only, under stringently controlled conditions, on satiety. Fifty-six subjects participated in the present study which consisted of four randomised test days, i.e. 1 d per macronutrient and one control day. During each test day, the subjects were required to consume the preload in full, and then their subsequent food intake was measured. The volunteers were divided into two groups: the first (T0) group, which consumed the preload immediately before lunch, and the second (T1) group, which consumed it 1 h beforehand. The main results showed that the participants consumed significantly less at lunch following the consumption of all three preloads than on the no-preload day, and consumed less after the consumption of the carbohydrate preload than after the consumption of the lipid preload. When energy from the preload was included, overall energy intake was significantly greater in all the three preload conditions than in the situation involving no preload, with only partial compensation for preload energy in all conditions. Total daily energy intake was highest after the lipid preload ingestion, but this could be a chance finding since it was not significantly higher than that observed after protein or carbohydrate preload ingestion. No significant effects of the interval between the preload and test meal ingestion were found. These results do not confirm the greater satiety effect of proteins than of carbohydrates, but partially confirm the weaker effect of fats.

Comparative effects of R- and S-equol and implication of transactivation functions (AF-1 and AF-2) in estrogen receptor-induced transcriptional activity.

Equol, one of the main metabolites of daidzein, is a chiral compound with pleiotropic effects on cellular signaling. This property may induce activation/inhibition of the estrogen receptors (ER) a or b, and therefore, explain the beneficial/deleterious effects of equol on estrogen-dependent diseases. With its asymmetric centre at position C-3, equol can exist in two enantiomeric forms (R- and S-equol). To elucidate the yet unclear mechanisms of ER activation/inhibition by equol, we performed a comprehensive analysis of ERa and ERb transactivation by racemic equol, as well as by enantiomerically pure forms. Racemic equol was prepared by catalytic hydrogenation from daidzein and separated into enantiomers by chiral HPLC. The configuration assignment was performed by optical rotatory power measurements. The ER-induced transactivation by R- and S-equol (0.1-10 µM) and 17b-estradiol (E2, 10 nM) was studied using transient transfections of ERα and ERß in CHO, HepG2 and HeLa cell lines. R- and S-equol induce ER transactivation in an opposite fashion according to the cellular context. R-equol and S-equol are more potent in inducing ERα in an AF-2 and AF-1 permissive cell line, respectively. Involvement of ERα transactivation functions (AF-1 and AF-2) in these effects has been examined. Both AF-1 and AF-2 are involved in racemic equol, R-equol and S-equol induced ERα transcriptional activity. These results could be of interest to find a specific ligand modulating ER transactivation and could contribute to explaining the diversity of equol actions in vivo.

A high-protein, moderate-energy, regular cheesy snack is energetically compensated in human subjects.

Snacking is often regarded as a cause of overweight. However, the main issue is to determine whether the consumption of snacks leads to an increase in energy intake or whether a compensation phenomenon exists and maintains daily energy intake at a constant level. The objective of the present study was to determine whether the repeated consumption of a high-protein, moderate-energy, cheesy snack given as a preload 1 h before a meal altered energy intake at the next meal and then throughout the day, and if this kind of snack was energetically compensated. Normal-weight women (n 27) were recruited for the study. All subjects were healthy non-smokers, aged 18-60 years. The snacks consisted of portions of cheese containing 22 g protein, with an energy value of 836 kJ. Two types of snack were compared, differing in terms of the type of milk proteins they contained: the first contained casein only (CAS), while the second contained a mixture of casein and whey proteins (WHEY+CAS; 2:1). The principal finding of the present study was that the ingestion of the two snacks 1 h before lunch led to energy compensation of 83.1 (SEM 9.4) and 67.0 (SEM 16.4) % for WHEY+CAS and CAS respectively, at lunch, and 121.6 (SEM 36.5) and 142.1 (SEM 29.7) % for WHEY+CAS and CAS respectively, regarding the whole-day energy intake. In conclusion, the repeated consumption of a high-protein, moderate-energy, regular cheesy snack should not promote overweight because energy intake appears to be regulated during subsequent meals on the same day.

Protein, amino acids and the control of food intake.

PURPOSE OF REVIEW: The present review presents recent findings on peripheral and central pathways involved in protein and amino acid-induced satiety. RECENT FINDINGS: A high-protein load leads to a higher decrease of energy intake at the next meal than carbohydrate and fat. A protein-enriched diet induces satiety, improves body composition and results in weight loss. At the peripheral level, proteins seem to induce the release of anorexigenic gut hormones cholecystokinin, glucagon-like peptide-1 and peptide YY, whereas the involvement of ghrelin remains uncertain. Energy expenditure and glucose are probably involved as metabolic signals in protein-induced satiety. Moreover, there is some evidence that the circulating level of leucine could impact food intake. Leucine has been shown to modulate the activity of the energy and nutrient sensor pathways controlled by AMPK and mTOR in the hypothalamus. Moreover, high-protein diets lead to activation of the noradrenergic/adrenergic neuronal pathway in the nucleus of the solitary tract and in melanocortin neurons in the arcuate nucleus. SUMMARY: Complex and redundant pathways are involved in protein and amino acid-induced satiety. Significant advances have recently allowed a better understanding of the involved cellular and molecular mechanisms. The involvement of some specific area of the brain including the hypothalamus and the nucleus of the solitary tract has to be further analyzed.

Respective contribution exerted by AF-1 and AF-2 transactivation functions in estrogen receptor alpha induced transcriptional activity by isoflavones and equol: consequence on breast cancer cell proliferation.

Estrogens used in hormone replacement therapy regimens may increase the risk of developing breast cancer. Paradoxically, high consumption of plant-derived phytoestrogens, particularly soybean isoflavones, is associated with a low incidence of breast cancer. To explore the molecular basis for these potentially different experimental/clinical outcomes, we investigated whether soybean isoflavones elicit distinct transcriptional actions from estrogens by performing transient transfections in different cell lines. Our results demonstrate that 17beta estradiol (E2), isoflavones, and equol (EQ) effectively trigger the transcriptional activation with both estrogen receptors (ER), ER alpha and ER beta. ER alpha transcriptional activity is mediated through two transactivation domains AF-1 and AF-2, whose activity is tightly regulated in a cell-type and promoter-specific manner. Isoflavones, genistein, and daidzein (DAI), and EQ, the main estrogenic metabolite of DAI, are ER alpha agonists for transcriptional activation. The molecular mechanisms for ER alpha-induced transcriptional activity by isoflavones and EQ involve their capacity to act mainly through AF-1 regardless of the cell type. Therefore, our data indicate that estrogenic ligands, such as isoflavones and EQ, exert their effects on ER alpha transactivation similarly to the endogenous ligand E2, and suggest that the risk of estrogen-related diseases might not be reduced by soy-rich food and/or isoflavone- or EQ-based supplements.

Comparison of digestibility and quality of intact proteins with their respective hydrolysates.

Quality of proteins depends on their composition in essential amino acids and on the availability of amino acids. Great interest has been shown in the role played by hydrolysates of proteins in clinical diets for pathologies with reduced absorptive capacity and food allergies caused by intact protein epitopes. Milk proteins are the most important protein source used in the development of protein hydrolysates designed for nutritional support of patients. Several studies have shown that casein and whey hydrolysates have a composition in amino acids equivalent to that in native milk proteins and that digestibility is similar or better. Among plant proteins, soy is the major source of hydrolysates. Soy hydrolysates are also used in infant formulas. Plant hydrolysates have good functional properties and a nutritional quality similar to that of starting material. Some technical improvements in production of hydrolysates, particularly for plants, are nevertheless necessary to improve product palatability.

Enterodiol and enterolactone, two major diet-derived polyphenol metabolites have different impact on ERalpha transcriptional activation in human breast cancer cells.

Lignans are plant compounds metabolized in the mammalian gut to produce the estrogenic enterolignans, enterodiol (ED) and enterolactone (EL). Because estrogens have been linked to breast cancer etiology, enterolignans could affect breast cancer risk, but to our knowledge, the mechanisms by which they exert their estrogenic and/or anti-estrogenic effects in humans are still unclear. To better understand how estrogenic compounds from the food, such as the enterolignans, might influence breast cancer progression and their mechanisms to interfere with human estrogen receptor (ER) signalling in hormone-dependant diseases, we examined and compared the ability of ED, EL and 17beta-estradiol (E2) to induce the transactivation of ERalpha and ERbeta, to modulate ERalpha target genes, to exert either growth stimulatory or anti-proliferative effects and finally to modulate MCF-7 cell migration by acting on matrix metalloproteases (MMP)-2 and -9, at concentrations that are achievable through a lignan-rich diet. This study indicates that enterolignans show distinct properties for transactivation of ERalpha and ERbeta. ED, as E2, induces ERalpha transcriptional activation through transactivation functions AF-1 and AF-2, while EL is less efficient in inducing AF-1, acting predominantly through AF-2. Furthermore, ED and EL modulate ERalpha mRNA and protein contents as well as MCF-7 cell proliferation and secreted MMP activities in a different way. Enterolignans are compounds of wide interest nowadays and our results help to unveil their mechanisms of action on ER, emphasizing the fact that the dietary load in lignans could be of importance in the balance between being risk or chemopreventive factors for breast cancer and women's health.

Activation of the estrogen receptor contributes to the progression of pulmonary lymphangioleiomyomatosis via matrix metalloproteinase-induced cell invasiveness.

CONTEXT: The role of estrogens in the pathogenesis of lymphangioleiomyomatosis (LAM), an aggressive and destructive, eventually fatal lung disease of women, is poorly understood. OBJECTIVE: The study was conducted to test the hypothesis that the lung disease in LAM is estrogen mediated and to determine whether estrogens contribute to the invasiveness of LAM. DESIGN: In vitro cell culture of spindle-shaped LAM cells (LAMD-SM) were isolated and propagated from affected lungs. Estrogen receptor (ER)-alpha and ERbeta analyses were conducted by RT-PCR. ERalpha and ERbeta, tissue inhibitor of metalloproteinase-2, and matrix metalloproteinases (MMP)-2 had Western blot analysis for protein assessment. Activity assays were performed for MT1-MMP, MMP-2, and tissue inhibitor of metalloproteinase-2. Assessment of MMP-2 promoter function was done via transfection assays. Cell invasion chambers were used to determine and quantitate cell invasiveness. SETTING: The study was conducted at an academic medical center. PATIENTS: Tissue and cells were obtained from patients as outlined in approved institution review board protocol (97/007). INTERVENTION: LAMD-SM cells were treated with a specific MMP-2 antibody or a nonspecific inhibitor, doxycycline. MAIN OUTCOME MEASURES: Activity of MMP-2 and invasiveness of LAMD-SM cells were measured. RESULTS: LAMD-SM cells express functional ERs (ERalpha and ERbeta), which undergo rapid intracellular turnover in their unbound state. 17beta-estradiol (E(2)) enhances the transcriptional ER activity. E(2)-induced ER activation increases synthesis and activity of MMP-2 through posttranscriptional mechanisms in LAMD-SM. The E(2)/ER-mediated increase of MMP-2 promotes LAMD-SM invasiveness, in assays in vitro, which can be inhibited by specific antibodies against MMP-2 or doxycycline, an inhibitor of MMPs. CONCLUSION: The invasion and destruction of lung parenchyma in LAM is, at least partially, an estrogen-MMP-driven process, which has major implications for therapeutic interventions.

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.