Bernard Bouscarel

Bile acids and signal transduction: role in glucose homeostasis.

Bile acids are mainly recognized for their role in dietary lipid absorption and cholesterol homeostasis. However, recent progress in bile acid research suggests that bile acids are important signaling molecules that play a role in glucose homeostasis. Among the various supporting evidence, several reports have demonstrated an improvement of the glycemic index of type 2 diabetic patients treated with diverse bile acid binding resins. Herein, we review the diverse interactions of bile acids with various signaling/response pathways, including calcium mobilization and protein kinase activation, membrane receptor-mediated responses, and nuclear receptor responses. Some of the effects of the bile acids are direct through the activation of specific receptors, i.e., TGR5, CAR, VDR, and FXR, while others imply modulation of the hormonal, growth factor and/or neuromediator responses, i.e., glucagon, EGF, and acetylcholine. We also discuss recent evidence implicating the interaction of bile acids with glucose homeostasis mechanisms, with the integration of our understanding of how the signaling mechanisms modulated by bile acid could regulate glucose metabolism.

pH-dependent uptake of irinotecan and its active metabolite, SN-38, by intestinal cells.

Irinotecan (CPT‐11) and its active metabolite, 7‐ethyl‐10‐hydroxycamptothecin (SN‐38), are believed to be reabsorbed by intestinal cells and to enter the entero‐hepatic circulation, but there is little information to date. Our objective was to investigate the intestinal transport of CPT‐11 and SN‐38 in correlation with their associated cytotoxicity. Using either isolated hamster intestinal epithelial cells or/and human colon carcinoma HT29 cells, the uptake rates of [14C]CPT‐11 and [14C]SN‐38, both as respective non‐ionic lactone form at acidic pH and anionic carboxylate form at basic pH, were investigated by the rapid vacuum filtration technique. The effect of physiologic intestinal luminal pH (6.2–8.0) on the uptake rate and cytotoxicity of SN‐38 were estimated by the above method and the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay, respectively. The lactone forms of CPT‐11 and SN‐38 were transported passively, while the respective carboxylate form was absorbed actively. Uptake rates of both lactones were significantly higher than those of their carboxylates. Under physiologic pH, the respective uptake rates of CPT‐11 and SN‐38 were pH sensitive and decreased significantly by around 65%, at pH greater than 6.8. Furthermore, with decreasing pH, a higher uptake rate of SN‐38 into HT29 cells correlates with a greater cytotoxic effect (r = 0.987). CPT‐11 and SN‐38 have absorption characteristics of weakly basic drugs such as short‐chain fatty acids, suggesting that alkalization of the intestinal lumen may be critical to reduce their reabsorption and associated side effects. Int. J. Cancer 83:491–496, 1999.

Optimal and effective oral dose of taurine to prolong exercise performance in rat

Summary.The aim of this study was to determine the effective and optimum dose of taurine for exercise performance and to maintain tissue taurine concentration. Rats received a respective daily dose of 0, 20, 100, and 500 mg/kg body weight of taurine (EC and ET-1, -2, -3 groups, respectively) for two weeks, and then, were subjected to treadmill until exhaustion. The running time to exhaustion was significantly prolonged by 25% and 50% in the ET-2 and -3 groups, respectively, compared to that in the EC group accompanied with maintenance of taurine tissue concentrations. Furthermore, the oxidative glutathione per total glutathione ratio in tissues was inhibited in the ET-2 and -3 groups whereas it was higher in the EC group than in both the no exercise and taurine-administered groups. Therefore the effective and optimal doses of oral taurine administration for two weeks on a transient exercise performance were between 100 and 500 mg/kg/day.

Involvement of integrin-linked kinase in carbon tetrachloride-induced hepatic fibrosis in rats

Integrin‐linked kinase (ILK) is a multidomain focal adhesion protein implicated in signal transduction between integrins and growth factor receptors. Although its expression is upregulated in pulmonary and renal fibrosis, its role in the development of hepatic fibrosis remains to be determined. Therefore, we considered it important to investigate whether ILK is involved in activation of hepatic stellate cells and thus plays a role in the development of hepatic fibrosis. Immunohistochemical analysis of liver sections obtained from rats with CCl4‐induced cirrhosis revealed increased expression and colocalization of ILK and alpha‐smooth muscle actin in hepatic stellate cells in perisinusoidal areas. In addition, hepatic stellate cells isolated from fibrotic livers expressed high levels of ILK and alpha‐smooth muscle actin, and their expression was sustained in culture. In contrast, hepatic stellate cells (HSCs) isolated from normal rat liver did not express ILK, but its expression was increased when the cells were activated in culture. Our studies also showed that ILK is involved in the phosphorylation of ERK 1/2, p38 MAPK, JNK, and PKB and that selective inhibition of ILK expression by siRNA results in a significant decrease in their phosphorylation. These changes were accompanied by significant inhibition of cell spreading and migration without affecting cell proliferation. In conclusion, ILK plays a key role in HSC activation and could be a possible target for antifibrogenic therapy. (HEPATOLOGY 2006;44:612–622.)

Decreased taurine concentration in skeletal muscles after exercise for various durations.

PURPOSE To examine the changes of taurine concentrations in blood and skeletal muscles after transient exercise. METHODS Rats were placed on a treadmill set at 25 m.min-1. The animals were divided into four groups: control (no exercise) and exercise groups 1, 2, and 3. The exercise duration for groups 1, 2, and 3 was 30, 60, and 100 +/- 12.5 min (to exhaustion: mean +/- SD), respectively. We examined the plasma concentrations of taurine and lactate, the serum concentrations of sodium and chloride ions, as well as the skeletal muscle taurine content in the soleus (slow-twitch fiber dominant type), gastrocnemius (slow- and fast-twitch fiber mix type), and plantaris and extensor digitorum longus (fast-twitch fiber dominant type) muscles. RESULTS Although the plasma taurine concentration was not affected by the increased exercise duration, that in skeletal muscles was significantly decreased. The gastrocnemius and plantaris muscles from the exercise group 3 had a significantly lower concentration of taurine than those of the control group. The extensor digitorum longus taurine concentration from the different exercise groups was significantly decreased compared with that from the control group. However, there was no significant difference among the exercise groups. CONCLUSION Taurine concentration was decreased in all skeletal muscles after exercise, regardless of the duration. Moreover, this decrease was specific to fast-twitch dominant fibers. However, under these conditions, the plasma taurine concentration remained unchanged.

Suppressive effect of ursodeoxycholic acid on type IIA phospholipase A2 expression in HepG2 cells

Phospholipase A2 IIA (PLA2IIA), which plays a crucial role in arachidonic acid metabolism and in inflammation, is upregulated under various pathological conditions, including in the gallbladder and gallbladder bile from patients with multiple cholesterol gallstones, in the liver and kidney of rats with cirrhosis, as well as in the colonic tissue of animals treated with a chemical carcinogen. The administration of ursodeoxycholic acid (UDCA) partially attenuated the PLA2IIA expression level in these different models. The aim of this study was to investigate the modulatory effect of UDCA on the PLA2IIA expression level at the cellular level. The HepG2 cells were selected to investigate the direct inhibitory effect of UDCA on PLA2IIA expression level. The proinflammatory cytokines (interleukin‐6 and tumor necrosis factor α) ‐induced PLA2IIA expression in HepG2 cells was partially inhibited by the presence of UDCA in a dose‐dependent fashion. The effect of UDCA on proinflammatory cytokines‐induced PLA2IIA expression occurred at the transcriptional level. In addition, among the bile acids tested, this inhibitory effect was UDCA‐specific. In conclusion, this study supports the possible alteration of arachidonic acid metabolism and PLA2IIA expression level, in particular, as the protective action of UDCA in patients with chronic liver disease. (HEPATOLOGY 2005;41:896–905.)

Enhancement of DNA topoisomerase I inhibitor–induced apoptosis by ursodeoxycholic acid

Certain hydrophobic bile acids, including deoxycholic acid and chenodeoxycholic acid, exert toxic effects not only in the liver but also in the intestine. Moreover, ursodeoxycholic acid (UDCA), which has protective actions against apoptosis in the liver, may have both protective and toxic effects in the intestine. The goal of the present study was to clarify the mechanisms responsible for the toxic effect of UDCA in intestinal HT-29 cells. Here, we show that UDCA potentiated both phosphatidylserine externalization and internucleosomal DNA fragmentation induced by SN-38, the most potent metabolite of the DNA topoisomerase I inhibitor, CPT-11. Furthermore, the loss of mitochondrial membrane potential as well as mitochondrial membrane permeability transition induced by SN-38 was enhanced in the presence of UDCA, resulting in an increased lethality determined by colony-forming assay. This UDCA-induced increased apoptosis was not due to alteration of either intracellular accumulation of SN-38 or cell cycle arrest by SN-38. The increased apoptosis was best observed when UDCA was present after SN-38 stimulation and was independent of caspase-8 but dependent on caspase-9 and caspase-3 activation. Furthermore, UDCA enhanced SN-38-induced c-Jun NH2-terminal kinase activation. In conclusion, UDCA increases the apoptotic effects while decreasing the necrotic effects of SN-38 when added after the topoisomerase I inhibitor, showing potential clinical relevance as far as targeted cell death and improved wound healing are concerned. However, the use of this bile acid as an enhancer in antitumor chemotherapy should be further evaluated clinically. [Mol Cancer Ther 2006;5(1):68–79]

Selective inhibition of CYP27A1 and of chenodeoxycholic acid synthesis in cholestatic hamster liver

The aim of this study was to explore the regulation of serum cholic acid (CA)/chenodeoxycholic acid (CDCA) ratio in cholestatic hamster induced by ligation of the common bile duct for 48 h. The serum concentration of total bile acids and CA/CDCA ratio were significantly elevated, and the serum proportion of unconjugated bile acids to total bile acids was reduced in the cholestatic hamster similar to that in patients with obstructive jaundice. The hepatic CA/CDCA ratio increased from 3.6 to 11.0 (P<0.05) along with a 2.9-fold elevation in CA concentration (P<0.05) while the CDCA level remained unchanged. The hepatic mRNA and protein level as well as microsomal activity of the cholesterol 7alpha-hydroxylase, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase and 5beta-cholestane-3alpha,7alpha,12alpha-triol 25-hydroxylase were not significantly affected in cholestatic hamsters. In contrast, the mitochondrial activity and enzyme mass of the sterol 27-hydroxylase were significantly reduced, while its mRNA levels remained normal in bile duct-ligated hamster. In conclusion, bile acid biosynthetic pathway via mitochondrial sterol 27-hydroxylase was preferentially inhibited in bile duct-ligated hamsters. The suppression of CYP27A1 is, at least in part, responsible for the relative decreased production of CDCA and increased CA/CDCA ratio in the liver, bile and serum of cholestatic hamsters.

Changes in G protein expression account for impaired modulation of hepatic cAMP formation after BDL

The regulation of cAMP synthesis by hormones and bile acids is altered in isolated hamster hepatocytes 2 days after bile duct ligation (BDL) [Y. Matsuzaki, B. Bouscarel, M. Le, S. Ceryak, T. W. Gettys, J. Shoda, and H. Fromm. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G164-G174, 1997]. Therefore, studies were undertaken to elucidate the mechanism(s) responsible for this impaired modulation of cAMP formation. Hepatocytes were isolated 48 h after either a sham operation or BDL. Both preparations were equally devoid of cholangiocyte contamination. Although the basal cAMP level was not affected after BDL, the ability of glucagon to maximally stimulate cAMP synthesis was decreased by approximately 40%. This decreased glucagon effect after BDL was not due to alteration of the total glucagon receptor expression. However, this effect was associated with a parallel 50% decreased expression of the small stimulatory G protein alpha-subunit (GsalphaS). The expression of either the large subunit (GsalphaL) or the common beta-subunit remained unchanged. The expression of Gialpha2 and Gialpha3 was also decreased by 25 and 46%, respectively, and was associated with the failure of ANG II to inhibit stimulated cAMP formation. Therefore, alterations of the expression of GsalphaS and Galphai are, at least in part, responsible for the attenuated hormonal regulation of cAMP synthesis. Because cAMP has been reported to stimulate both bile acid uptake and secretion, impairment of cAMP synthesis and bile acid uptake may represent an initial hepatocellular defense mechanism during cholestasis.The regulation of cAMP synthesis by hormones and bile acids is altered in isolated hamster hepatocytes 2 days after bile duct ligation (BDL) [Y. Matsuzaki, B. Bouscarel, M. Le, S. Ceryak, T. W. Gettys, J. Shoda, and H. Fromm. Am. J. Physiol. 273 ( Gastrointest. Liver Physiol. 36): G164-G174, 1997]. Therefore, studies were undertaken to elucidate the mechanism(s) responsible for this impaired modulation of cAMP formation. Hepatocytes were isolated 48 h after either a sham operation or BDL. Both preparations were equally devoid of cholangiocyte contamination. Although the basal cAMP level was not affected after BDL, the ability of glucagon to maximally stimulate cAMP synthesis was decreased by ∼40%. This decreased glucagon effect after BDL was not due to alteration of the total glucagon receptor expression. However, this effect was associated with a parallel 50% decreased expression of the small stimulatory G protein α-subunit (GsαS). The expression of either the large subunit (GsαL) or the common β-subunit remained unchanged. The expression of Giα2and Giα3was also decreased by 25 and 46%, respectively, and was associated with the failure of ANG II to inhibit stimulated cAMP formation. Therefore, alterations of the expression of GsαSand Giα are, at least in part, responsible for the attenuated hormonal regulation of cAMP synthesis. Because cAMP has been reported to stimulate both bile acid uptake and secretion, impairment of cAMP synthesis and bile acid uptake may represent an initial hepatocellular defense mechanism during cholestasis.

PKCε-dependent and -independent effects of taurolithocholate on PI3K/PKB pathway and taurocholate uptake in HuH-NTCP cell line

The cholestatic bile acid taurolithocholate (TLC) inhibits biliary secretion of organic anions and hepatic uptake of taurocholate (TC). TLC has been suggested to induce retrieval of Mrp2 from the canalicular membrane via the phosphoinositide-3-kinase (PI3K)/PKB-dependent activation of novel protein kinase Cepsilon (nPKCepsilon) in rat hepatocytes. The aim of the present study was to determine whether TLC-induced inhibition of TC uptake may also involve PI3K-dependent activation of PKCepsilon in HuH7 cells stably transfected with human Na(+)-dependent TC-cotransporting polypeptide (NTCP) (HuH-NTCP cells). To avoid direct competition for uptake, cells were pretreated with TLC, washed, and then incubated with (3)H-TC to determine TC uptake. TLC produced time- and dose-dependent inhibition of TC uptake. TLC inhibited TC uptake competitively without affecting NTCP membrane translocation. A PI3K inhibitor failed to reverse TLC-induced TC uptake inhibition and TLC-inhibited PKB phosphorylation. TLC did activate nPKCepsilon as evidenced by increased membrane translocation and nPKCepsilon-Ser(729) phosphorylation. Overexpression of dominant negative-nPKCepsilon reversed TLC-induced inhibition of PKB phosphorylation but not of TC uptake. Finally, cAMP prevented TLC-induced inhibition of TC uptake via the PI3K pathway, and the prevention is due to the sum of cAMP-induced stimulation and TLC-induced inhibition of TC uptake. Taken together, these results suggest that TLC-induced inhibition of PKB, but not of TC uptake, is mediated via nPKCepsilon. Activation of nPKCepsilon and inhibition of TC uptake by TLC are not mediated via the PI3K/PKB pathway.