Intermittent sucrose solution intake and its schedule of access modulate energy intake and weight gain in response to chronic variable stress in mice.

There is a strong relationship between stress and the intake of calorically-dense palatable food. Additionally, intake of sodas is an important contributory factor to obesity, and is often associated with palatable food consumption. We studied the effects of 2-h intermittent access to sucrose-sweetened water (SSW, 12.3%, soda-like) and its schedule of administration on the response to chronic variable stress in mice fed a high-fat, high-sugar diet. C57BL/6 mice (n = 64) had access to water or to both water and 2-h SSW during 5 weeks, in addition to their diet. After the first two weeks, half of the animals from each group were stressed daily using a chronic variable stress (CVS) paradigm, while the other half were kept undisturbed. During the CVS exposure period, 2-h SSW access was either scheduled randomly, right before the stressors or right after the stressors. The effects of SSW and its schedule of administration on dietary intake, stress hormones and adiposity were analyzed. Results showed a larger consumption of SSW and higher bodyweight gain in mice receiving SSW after the stressor. In addition, SSW consumption was shown to affect appetite regulation by reducing CCK sensitivity. The present study suggests that SSW leads to overconsumption and weight gain only if provided after exposure to stress. These findings may implicate a relation between exposure to stress, binge-drinking behaviors of sugar sweetened beverages that ensues, and weight gain in humans consuming a western diet.

Environmental enrichment and cafeteria diet attenuate the response to chronic variable stress in rats.

Exposure to an enriched environment (EE) or the intake of a highly palatable diet may reduce the response to chronic stress in rodents. To further explore the relationships between EE, dietary intake and stress, male Sprague-Dawley rats were fed one of two diets for 5 weeks: high carbohydrate (HC) or "cafeteria" (CAF) (Standard HC plus a choice of highly palatable cafeteria foods: chocolate, biscuits, and peanut butter). In addition, they were either housed in empty cages or cages with EE. After the first two weeks, half of the animals from each group were stressed daily using a chronic variable stress (CVS) paradigm, while the other half were kept undisturbed. Rats were sacrificed at the end of the 5-week period. The effects of stress, enrichment and dietary intake on animal adiposity, serum lipids, and stress hormones were analyzed. Results showed an increase in intra-abdominal fat associated with the CAF diet and an increase in body weight gain associated with both the CAF diet and EE. Furthermore, the increase in ACTH associated with CVS was attenuated in the presence of EE and the CAF diet independently while the stress-induced increase in corticosterone was reduced by the combination of EE and CAF feeding. The present study provides evidence that the availability of a positive environment combined to a highly palatable diet increases resilience to the effects of CVS in rats. These results highlight the important place of palatable food and supportive environments in reducing central stress responses.

A cafeteria diet modifies the response to chronic variable stress in rats.

Stress is known to lead to metabolic and behavioral changes. To study the possible relationships between stress and dietary intake, male Sprague-Dawley rats were fed one of three diets for 6 weeks: high carbohydrate (HC), high fat (HF), or "Cafeteria" (CAF) (Standard HC plus a choice of highly palatable cafeteria foods: chocolate, biscuits, and peanut butter). After the first 3 weeks, half of the animals from each group (experimental groups) were stressed daily using a chronic variable stress (CVS) paradigm, while the other half of the animals (control groups) were kept undisturbed. Rats were sacrificed at the end of the 6-week period. The effects of stress and dietary intake on animal adiposity, serum lipids, and corticosterone were analyzed. Results showed that both chronic stress and CAF diet resulted in elevated total cholesterol, increased low-density lipoprotein (LDL), and lower high-density lipoprotein (HDL). In addition, increases in body weight, food intake, and intra-abdominal fat were observed in the CAF group compared with the other dietary groups. In addition, there was a significant interaction between stress and diet on serum corticosterone levels, which manifest as an increase in corticosterone levels in stressed rats relative to non-stressed controls in the HC and HF groups but not in the CAF group. These results show that a highly palatable diet, offering a choice of food items, is associated with a reduction in the response to CVS and could validate a stressor-induced preference for comfort food that in turn could increase body weight.

Inhibition of food intake induced by acute stress in rats is due to satiation effects.

Acute mild stress induces an inhibition of food intake in rats. In most studies, the cumulative daily food intake is measured but this only provides a quantitative assessment of ingestive behavior. The present study was designed to analyze the reduction in food intake induced by acute stress and to understand which behavioral and central mechanisms are responsible for it. Two different stressors, restraint stress (RS) and forced swimming stress (FSS), were applied acutely to male Wistar rats. We first measured corticosterone and ACTH in plasma samples collected immediately after acute RS and FSS in order to validate our stress models. We measured food intake after RS and FSS and determined meal patterns and behavioral satiety sequences. The expressions of CRF, NPY and POMC in the hypothalamus were also determined immediately after acute RS and FSS. The rise in corticosterone and ACTH levels after both acute RS and FSS validated our models. Furthermore, we showed that acute stress induced a reduction in cumulative food intake which lasted the whole day for RS but only for the first hour after FSS. For both stressors, this stress-induced food intake inhibition was explained by a decrease in meal size and duration, but there was no difference in ingestion speed. The behavioral satiety sequence was preserved after RS and FSS but grooming was markedly increased, which thus competed with, and could reduce, other behaviors, including eating. Lastly, we showed that RS induced an increase in hypothalamic POMC expression. These results suggest that acute stress may affect ingestive behavior by increasing satiation and to some extent by enhancing grooming, and this may be due to stimulation of the hypothalamic POMC neurons.

Effects of four inorganic lead compounds on the proliferation and junctional coupling of cultured REL liver cells.

BACKGROUND: Chronic, low-level exposure to inorganic lead (Pb) has been involved in a number of human diseases, including tumors. In this study, the effect of four different inorganic Pb compounds (acetate, chloride, monoxide, and sulfate) was evaluated, in vitro, on liver-derived REL cells, known to be very sensitive to tumor promoters. METHODS: Cytotoxicity and effects on intercellular communication (GJIC) were evaluated, respectively, by cell- density/proliferation and dye-transfer assays. Pb concentration in the media solutions used for each treatment was quantified by atomic absorption spectroscopy-electrothermal atomization. RESULTS: Each of the Pb compounds we tested showed a typical dose- and time-related effect on REL cell proliferation, this effect not being related to the free metal concentration. Contrary to classical tumor promoters, none of the compounds significantly affected REL GJIC (1-hour treatment). CONCLUSIONS: Our results are indicative of specificity in the effects of the different Pb compounds. The mechanism(s) of their action need further investigations.

The polarized hepatic human/rat hybrid WIF 12-1 and WIF-B cells communicate efficiently in vitro via connexin 32-constituted gap junctions.

Gap junction intercellular communication (GJIC) plays an essential role in the control of growth, differentiation, and functions of different tissues. The expression of connexins (Cxs), the structural proteins of gap junctions, is developmentally regulated and tissue-specific. In vivo hepatocytes express Cx32 and Cx26. Most currently available in vitro hepatic cell systems express Cx43 instead of the expected Cxs. This work analyzes the GJIC competence and Cx expression of the highly differentiated and polarized hepatoma-derived hybrid cell lines, WIF 12-1 and WIF-B. It shows (using two dye transfer assays) that both lines communicate efficiently and that the acquisition of GJIC competence precedes the formation of bile canaliculi. Interestingly, these cells communicate via Cx32 expression, whereas Cx26 and Cx43 are not expressed, as demonstrated by Western and Northern blotting, immunocytochemistry, and confocal microscopy. The human fibroblast W138 parent communicates via Cx43, whereas the rat hepatoma parent Fao and the subclone WIF 12-1 TGdelta, that has lost the human X chromosome, do not communicate, the expression of Cx32 being restricted to the mRNA in these two lines. The GJIC competence of WIF cells could thus result from the activation of the human X chromosome-linked Cx32 gene.

Flavonoids (apigenin, tangeretin) counteract tumor promoter-induced inhibition of intercellular communication of rat liver epithelial cells.

We have shown previously that two flavonoids, apigenin and tangeretin, enhance gap junctional intercellular communication (GJIC) in rat liver epithelial cells, named REL cells. Here, we show that these two flavones also antagonize the inhibition of GJIC induced by tumor promoters like 12-O-tetradecanoyl-phorbol-acetate (TPA) and 3,5,di-tertio-butyl-4-hydroxytoluene (BHT). Their preventive effect is rapid. It does not seem to involve any change of the amount of the connexin expressed in REL cells, connexin 43 (Cx 43), and in its phosphorylation state. Other flavonoids tested including naringenin, myricetin, catechin and chrysin did not enhance GJIC nor counteract TPA-induced inhibition of GJIC.

Altered function, localization and phosphorylation of gap junctions in rat liver epithelial, IAR 20, cells after treatment with PCBs or TCDD.

Three different PCB-congeners 3,4,5,3',4'-pentachlorobiphenyl (IUPAC no. 126), 2,4,5,2',4',5'-hexachlorobiphenyl (IUPAC no. 153) and 2,4,5,3',4'-pentachlorobiphenyl (IUPAC no. 118) were investigated for possible structure-activity relationships in altering gap junction intercellular proteins. All tested PCB-congeners and TCDD decreased the gap junctional intercellular communication in IAR 20 cells, but at different treatment periods, suggesting different modes of action. The presence of the Cx43-P(2) band, a phosphorylated isoform of Cx43, was associated with a functional communication. A reduced Cx43 mRNA level was noted after 48 h of exposure with PCB 126, PCB 118 and TCDD. In summary, the non dioxin-like PCB 153 can decrease gap junctional intercellular communication rapidly by reducing the phosphorylated isoform of Cx43, whereas the dioxin-like PCB 126 and TCDD reduce the communication slowly by decreasing the mRNA level of Cx43, resulting in a reduced Cx43 protein level (which includes the P(2)-band). The mixed inducing PCB-congener, PCB 118, can act both as the dioxin-like and the non dioxin-like PCBs in gap junction regulation.

Calcitriol and lexicalcitol (KH1060) inhibit the growth of human breast adenocarcinoma cells by enhancing transforming growth factor-beta production.

The mechanisms involved in the antiproliferative action of calcitriol (1 alpha, 25(OH)2D3) were investigated using human breast carcinoma epithelial cells (the MCF-7 cell line). Calcitriol and KH1060, a synthetic analog, inhibited cell growth in a time-and dose-dependent way. The substances similarly stimulated total TGF-beta secretion after 24 hours, and Northern blot analyses showed that mRNA levels for TGF-beta 1 were increased, as well. When MCF-7 cells were co-incubated with calcitriol and a neutralizing anti TGF-beta 1, beta 2, beta 3 antibody, growth inhibition was completely abrogated. With KH1060, the antibody could only partly block growth inhibition. This study shows that TGF-beta is involved in the growth response to calcitriol and KH1060 in MCF-7 cells.

Lack of tumor-promoting effects of flavonoids: studies on rat liver preneoplastic foci and on in vivo and in vitro gap junctional intercellular communication.

Possible tumor-promoting activity of four flavonoids, quercetin (QC), tangeretin (TG), flavone (FO), and flavanone (FN), was examined in a rat liver short-term carcinogenesis assay as well as with in vivo and in vitro assays of inhibition of gap junctional intercellular communication (GJIC). Rat hepatocarcinogenesis was induced by aflatoxin B1 treatment followed by a selection phase (2-acetylaminofluorene treatment and partial hepatectomy), then treatment with or without test chemicals (in vivo studies of antipromotion were not performed). Using glutathione S-transferase placental form (GST-P)-positive foci, we compared the effects of flavonoids (at 1,000 ppm in the diet) with the effects of phenobarbital (PB) on the occurrence of liver preneoplastic lesions. In addition, we studied the effects of flavonoids on GJIC in the livers derived from these experiments and in two types of cultured cells. No significant difference in the number and area of GST-P-positive foci was found after one or three months of treatment between any flavonoid group and control group. In the positive control group, PB markedly increased the numbers and areas of preneoplastic lesions at three months. Whereas PB also decreased by 60% the average size of lucifer yellow dye spread in slices of liver parenchyma free of preneoplastic lesions among the different flavonoids, only TG decreased the dye transfer in vivo: by 30% at one month and 50% at three months. With the dye transfer assay applied to a rat liver epithelial cell line (REL) and the Chinese hamster V79 metabolic cooperation assay, none of the tested flavonoids (< or = 25 microM) inhibited GJIC. Conversely, protective properties were seen for some of the compounds in antipromotion in vitro studies, because TG and FN enhanced the dye transfer in REL cells and FO, TG, and QC partly prevented the inhibition of metabolic cooperation by 12-O-tetradecanoylphorbol-13-acetate. Thus, taken together, our results suggest that QC, FO, and FN do not show tumor-promoting activity. Concerning TG, some discrepancies in the in vivo data are observed. Some of them (GJIC inhibition in liver slices) are probably more relevant to promotion of hepatocarcinogenesis.

Retinoic acid enhances connexin43 expression at the post-transcriptional level in rat liver epithelial cells.

The mechanism by which all-trans retinoic acid (RA) stimulates gap junctional intercellular communication (GJIC) in the rat liver epithelial cell line. IAR203, was investigated. When RA, at 0.1 microM for 24-48 h, enhanced the dye transfer in IAR203 cells (x 1.4), it increased the amount of connexin43 (Cx43) in the cell-cell contact regions of the plasma membrane, as evidenced by analysis by Western blot and by immunofluorescence. It had no effect on the level of Cx43 mRNA. Freeze-fracture analysis of the size of gap junctions revealed an increase of the proportion of small gap junctions in RA-treated cells. We conclude that, in IAR203 cells, RA stimulates GJIC by acting at the post-transcriptional level of Cx43 regulation. The possibility that RA acts indirectly on the regulation of Cx43 expression, and increases the half-life of Cx43 by inducing adhesion molecules is discussed.

Suppression of oncogene-induced transformation by quercetin and retinoic acid in rat liver epithelial cells.

AP1 is a heterodimeric complex containing products of the Jun and Fos oncogene families. The c-fos and c-jun protooncogenes act as transcriptional activator for numerous cellular genes, and the overexpression of these genes may cause malignant transformation. In this study, to show evidence of a possible inhibition of AP1 transcriptional activity in molecular mechanisms of foodborne molecules, known to be negative modulators of carcinogenesis, we established two rat liver epithelial (REL) cell lines overexpressing either c-fos (43C line) or c-jun (RELcJ1 line) oncoproteins. Contrary to the 43C line, which was spontaneously transformed, the c-jun-transfected REL cells were only transformed in vitro after 12-O-tetra-decanoylphorbol 13-acetate (TPA) exposure. All trans-retinoic acid (RA) abolished the transformation of the 43C line and TPA-treated RELcJ1 cells, suggesting that RA could decrease AP1 activity in these cells despite c-fos or c-jun overexpression. Furthermore, we show for the first time that a flavonoid, quercetin, which is a natural component of vegetables, inhibited only the transformation of the 43C line. The spontaneous transformation of the c-fos-transfected REL cells was associated with the appearance of c-fos/AP1 complexes binding TRE, suggesting that c-fos/AP1 complexes are involved in the antitransforming mechanism of quercetin.

Apigenin and tangeretin enhance gap junctional intercellular communication in rat liver epithelial cells.

Two flavones, apigenin and tangeretin, were studied for their ability to modulate gap junctional intercellular communication (GJIC) in the rat liver epithelial cell line REL. Their cytotoxicity was first determined by cell density and neutral red uptake assays: neither apigenin nor tangeretin are cytotoxic at 10 and 25 microM, the concentrations used in our experiments. We then studied GJIC using the dye transfer assay and we observed that both apigenin and tangeretin enhance it, the maximum stimulation (x 1.7-1.8) being achieved at 25 microM for 24 h. When the dye transfer was enhanced, the amount of connexin 43 increased, which was demonstrated by Western blot and immunofluorescence analysis. For apigenin only, Northern blot analysis showed an accumulation of connexin 43 mRNA. In addition, the incubation of REL cells with the two compounds, for 1 or 24 h, prevented the inhibition of dye transfer by 12-O-tetradecanoylphorbol-13-acetate (1 or 10 ng/ml). The enhancement of GJIC by apigenin could be one of the major mechanisms responsible for apigenin's anti-tumour promoting action in vivo. As for tangeretin, its capacity to enhance GJIC completes its potential protective properties towards the post-initiation process.

Studies on the modulating effects of retinoic acid and retinol acetate using dye transfer and metabolic cooperation assays.

The effects of retinoic acid and retinol acetate on gap junctional communication were examined in two in vitro tests. Rat liver epithelial cell line IAR 203 was used for dye transfer assays, and hamster lung fibroblast V79 cells were used for metabolic cooperation assays. A reversible dose-dependent inhibition of dye transfer was detected after a 1-hr treatment with retinoic acid or retinol acetate at concentrations ranging from 10 to 50 microM. On the other hand, enhancement of dye transfer was observed after a 24-hr treatment with retinoic acid at 0.1 microM. A dose-dependent inhibition of metabolic cooperation was obtained with retinoic acid at noncytotoxic concentrations ranging from 5 to 50 microM. Retinoids and TPA (1 ng/ml) acted synergistically in their inhibition of cell communication. Thus, the assays appear to be complementary: the dye transfer assay was useful in studying the time course and the reversibility of the inhibition or enhancement of dye transfer, whereas the metabolic cooperation assay was effective in quantifying the inhibitory effect of TPA or retinoids and interactions between them.

[In vitro use of a model for studying the effects of tumor promoters in food]. / Utilisation d'un modèle in vitro pour l'étude des effets de promoteurs de tumeurs de l'alimentation.

Two antioxidants, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), which are widely used as food additives, have been proved in vivo to act as tumor promoters in rodent species. In order to study their mechanisms of action, BHT, BHA and phenobarbital (PB) were tested in vitro on liver epithelial cells isolated from 2-acetylaminofluorene-initiated rats. Interactions of BHT 3 x 10(-6) to 3 x 10(-5) M, BHA 10(-5) to 10(-4) M and of PB 10(-4) to 10(-3) M with cell growth on plastic dishes or in agarose, and with the expression of different proteins (gamma-glutamyltranspeptidase, cytoskeletal proteins, fibronectin) were followed.