Down-regulation of the ubiquitin�Cproteasome proteolysis system by amino acids and insulin involves the adenosine monophosphate-activated protein kinase and mammalian target of rapamycin pathways in rat hepatocytes

The purpose of this work was to examine whether changes in dietary protein levels could elicit differential responses of tissue proteolysis and the pathway involved in this response. In rats fed with a high protein diet (55%) for 14?days, the liver was the main organ where adaptations occurred, characterized by an increased protein pool and a strong, meal-induced inhibition of the protein breakdown rate when compared to the normal protein diet (14%). This was associated with a decrease in the key-proteins involved in expression of the ubiquitin-proteasome and autophagy pathway gene and a reduction in the level of hepatic ubiquitinated protein. In hepatocytes, we demonstrated that the increase in amino acid (AA) levels was sufficient to down-regulate the ubiquitin proteasome pathway, but this inhibition was more potent in the presence of insulin. Interestingly, AICAR, an adenosine monophosphate-activated protein kinase (AMPK) activator, reversed the inhibition of protein ubiquination induced by insulin at high AA concentrations. Rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, reversed the inhibition of protein ubiquination induced by a rise in insulin levels with both high and low AA concentrations. Moreover, in both low and high AA concentrations in the presence of insulin, AICAR decreased the mTOR phosphorylation, and in the presence of both AICAR and rapamycin, AICAR reversed the effects of rapamycin. These results demonstrate that the inhibition of AMPK and the activation of mTOR transduction pathways, are required for the down-regulation of protein ubiquitination in response to high amino acid and insulin concentrations.     

Colonic luminal ammonia and portal blood l-glutamine and l-arginine concentrations: a possible link between colon mucosa and liver ureagenesis

The highest ammonia concentration in the body is found in the colon lumen and although there is evidence that this metabolite can be absorbed through the colonic epithelium, there is little information on the capacity of the colonic mucosa to transfer and metabolize this compound. In the present study, we used a model of conscious pig with a canula implanted into the proximal colon to inject endoluminally increasing amounts of ammonium chloride and to measure during 5 h the kinetics of ammonia and amino acid concentration changes in the portal and arterial blood. By injecting as a single dose from 1 to 5 g ammonia into the colonic lumen, a dose-related increase in ammonia concentration in the portal blood was recorded. Ammonia concentration remained unchanged in the arterial blood except for the highest dose tested, i.e. 5 g which thus apparently exceeds the hepatic ureagenesis capacity. By calculating the apparent net ammonia absorption, it was determined that the pig colonic epithelium has the capacity to absorb 4 g ammonia. Ammonia absorption through the colonic epithelium was concomitant with increase of l-glutamine and l-arginine concentrations in the portal blood. This coincided with the expression of both glutamate dehydrogenase and glutamine synthetase in isolated colonic epithelial cells. Since l-glutamine and l-arginine are known to represent activators for liver ureagenesis, we propose that increased portal concentrations of these amino acids following increased ammonia colonic luminal concentration represent a metabolic link between colon mucosa and liver urea biosynthesis.     

In vivo treatment by diallyl disulfide increases histone acetylation in rat colonocytes

Diallyl disulfide (DADS) is an organosulfur compound from garlic which exhibits various anticarcinogenic properties including inhibition of tumor cell proliferation. DADS antiproliferative effects were previously associated with an increase in histone acetylation in two human tumor colon cell lines, suggesting that DADS-induced histone hyperacetylation could be one of the mechanisms involved in its protective properties on colon carcinogenesis. The effects of DADS on histone H4 and H3 acetylation levels were investigated in vivo in colonocytes isolated from non-tumoral rat. Administrated by intracaecal perfusion or gavage, DADS increases histone H4 and H3 acetylation in colonocytes. Moreover, data generated using cDNA expression arrays suggest that DADS could modulate the expression of a subset of genes. These results suggest the involvement of histone acetylation in modulation of gene expression by DADS in normal rat colonocytes, which might play a role in its biological effects as well as in its anticarcinogenic properties in vivo.     

Scratch-wounding renders cultivated cells less permissive to prion infection

Using permissive cell lines of epithelial or neuroglial origin, we found that scratch-wounding a small proportion of the recipient cells prior to prion exposure strongly reduced the cell culture's susceptibility to infection. We provide evidence suggesting that wound-triggered inhibition of prion infection was mediated by the release of nucleotides in the extracellular medium of injured cultures. While cell wounding or ATP treatment of unwounded target cells inhibited de novo infection, we found that they had no effect on steady-state infected cultures, indicating that these treatments affect the early stages of infection. These findings support the view that cells have the capacity to modulate their permissiveness to prion infection in response to external stimuli, such as a signalling molecule.     

The Carbohydrate Sensitive Rat as a Model of Obesity

Background

Sensitivity to obesity is highly variable in humans, and rats fed a high fat diet (HFD) are used as a model of this inhomogeneity. Energy expenditure components (basal metabolism, thermic effect of feeding, activity) and variations in substrate partitioning are possible factors underlying the variability. Unfortunately, in rats as in humans, results have often been inconclusive and measurements usually made after obesity onset, obscuring if metabolism was a cause or consequence. Additionally, the role of high carbohydrate diet (HCD) has seldom been studied.

Methodology/Findings

Rats (n=24) were fed for 3 weeks on HCD and then 3 weeks on HFD. Body composition was tracked by MRI and compared to energy expenditure components measured prior to obesity. Results: 1) under HFD, as expected, by adiposity rats were variable enough to be separable into relatively fat resistant (FR) and sensitive (FS) groups, 2) under HCD, and again by adiposity, rats were also variable enough to be separable into carbohydrate resistant (CR) and sensitive (CS) groups, the normal body weight of CS rats hiding viscerally-biased fat accumulation, 3) HCD adiposity sensitivity was not related to that under HFD, and both HCD and HFD adiposity sensitivities were not related to energy expenditure components (BMR, TEF, activity cost), and 4) only carbohydrate to fat partitioning in response to an HCD test meal was related to HCD-induced adiposity.

Conclusions/Significance

The rat model of human obesity is based on substantial variance in adiposity gains under HFD (FR/FS model). Here, since we also found this phenomenon under HCD, where it was also linked to an identifiable metabolic difference, we should consider the existence of another model: the carbohydrate resistant (CR) or sensitive (CS) rat. This new model is potentially complementary to the FR/FS model due to relatively greater visceral fat accumulation on a low fat high carbohydrate diet.     

The vitamin E analog tocopherol succinate strongly inhibits gap junctional intercellular communication in rat liver epithelial cells (IAR203)

Vitamin E is a scavenger molecule trapping free radicals in biological membranes. However, it has also been shown to elicit the formation of reactive oxygen species and apoptosis in cancer cells. In this study, we tested the ability of alpha-tocopherol, tocopherol acetate, tocopherol phosphate and tocopherol succinate (TS) to modulate gap junctional intercellular communication in the rat liver epithelial cell line IAR203, as measured by the transfer of Lucifer yellow. While alpha-tocopherol, tocopherol acetate and tocopherol phosphate moderately reduced the dye transfer, TS at 10 and 25 microM strongly inhibited it, probably via the induction of the hypophosphorylation of connexin 43. Our results show that, besides their interesting antioxidant properties, vitamin E analogs, especially TS, can exert adverse effects on gap junctional intercellular communication, which could explain their controversial effects in carcinogenesis.     

Butyrate metabolism in human colon carcinoma cells: implications concerning its growth-inhibitory effect

Butyrate and acetate are bacterial metabolites present in the large intestine lumen. Although butyrate is well known to inhibit the in vitro proliferation of human colon carcinoma cells in a process involving the hyperacetylation of specific nuclear histones, little is known about the possible link between butyrate metabolism and its growth-inhibitory effect. In a previous study (Leschelle et al., 2000, Eur J Biochem 267: 6435-6442), we showed that butyrate accumulates and is metabolized in HT-29 Glc(-/+) cells without increasing oxygen consumption. In the present study, using the same cell line incubated with (14)C-labeled butyrate, we determined that a minor part of (14)C from butyrate was recovered in nuclear histones. Unlike butyrate, acetate exerted no effect on cell growth but was a precursor for overall net histone acetylation. Although butyrate was able to increase the cellular AMP/ADP ratio, it did not affect the ATP cell content or the adenylate charge or the oxidation of endogenous L-glutamine. Butyrate oxidation was found to be markedly sensitive to the presence of other substrates with D-glucose decreasing this oxidation and L-malate stimulating it. Furthermore, in the presence of L-malate, the growth-inhibitory effect of butyrate was significantly weaker than in its absence. From these data, we conclude that the metabolism of butyrate downstream acetyl-CoA synthesis is not involved in the butyrate antiproliferative effect. The suggestion that butyrate metabolism in mitochondria is not used in these cells as a fuel but acts as a regulator of butyrate free concentrations (thus limiting its action upon cellular targets), is discussed.     

Expression of mitochondrial HMGCoA synthase and glutaminase in the colonic mucosa is modulated by bacterial species.

The expression of the colonic mitochondrial 3-hydroxy 3-methyl glutaryl CoA (mHMGCoA) synthase, a key control site of ketogenesis from butyrate, is lower in germ-free (GF) than in conventional (CV) rats. In contrast, the activity of glutaminase is higher. The objective of this study was to investigate whether the intestinal flora can affect gene expression through short chain fatty acid (SCFA) and butyrate production. GF rats were inoculated with a conventional flora (Ino-CV) or with a bacterial strain producing butyrate (Clostridium paraputrificum, Ino-Cp) or not (Bifidobacterium breve, Ino-Bb). In the Ino-CV rats, mHMGCoA synthase expression was restored to the CV values 2 days after the inoculation, i.e. concomitantly with SCFA production. In the Ino-Cp group, but not in the Ino-Bb group, mHMGCoA synthase and glutaminase were expressed at the level observed in the CV rats. These data suggest that the intestinal flora, through butyrate production, could control the expression of colonic mHMGCoA synthase and glutaminase. These modifications in gene expression by butyrate in vivo seem unrelated to a modification of histone acetylation.