Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1

Bile acids play essential roles in the absorption of dietary lipids and in the regulation of bile acid biosynthesis. Recently, a G protein-coupled receptor, TGR5, was identified as a cell-surface bile acid receptor. In this study, we show that bile acids promote glucagon-like peptide-1 (GLP-1) secretion through TGR5 in a murine enteroendocrine cell line STC-1. In STC-1 cells, bile acids promoted GLP-1 secretion in a dose-dependent manner. As STC-1 cells express TGR5 mRNA, we examined whether bile acids induce GLP-1 secretion through TGR5. RNA interference experiments showed that reduced expression of TGR5 resulted in reduced secretion of GLP-1. Furthermore, transient transfection of STC-1 cells with an expression plasmid containing TGR5 significantly enhanced GLP-1 secretion, indicating that bile acids promote GLP-1 secretion through TGR5 in STC-1 cells. Bile acids induced rapid and dose-dependent elevation of intracellular cAMP levels in STC-1 cells. An adenylate cyclase inhibitor, MDL12330A, significantly suppressed bile acid-promoted GLP-1 secretion, suggesting that bile acids induce GLP-1 secretion via intracellular cAMP production in STC-1 cells.     

Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120

Diabetes, a disease in which the body does not produce or use insulin properly, is a serious global health problem. Gut polypeptides secreted in response to food intake, such as glucagon-like peptide-1 (GLP-1), are potent incretin hormones that enhance the glucose-dependent secretion of insulin from pancreatic beta cells. Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules in various cellular processes, including the secretion of gut incretin peptides. Here we show that a G-protein-coupled receptor, GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain FFAs. Furthermore, we show that the stimulation of GPR120 by FFAs promotes the secretion of GLP-1 in vitro and in vivo, and increases circulating insulin. Because GLP-1 is the most potent insulinotropic incretin, our results indicate that GPR120-mediated GLP-1 secretion induced by dietary FFAs is important in the treatment of diabetes.     

Green tea polyphenol epigallocatechin gallate activates TRPA1 in an intestinal enteroendocrine cell line, STC-1

A characteristic astringent taste is elicited by polyphenols. Among the polyphenols, catechins and their polymers are the most abundant polyphenols in wine and tea. A typical green tea polyphenol is epigallocatechin gallate (EGCG). Currently, the mechanism underlying the sensation of astringent taste is not well understood. We observed by calcium imaging that the mouse intestinal endocrine cell line STC-1 responds to the astringent compound, EGCG. Among major catechins of green tea, EGCG was most effective at eliciting a response in this cell line. This cellular response was not observed in HEK293T or 3T3 cells. Further analyses demonstrated that the 67-kDa laminin receptor, a known EGCG receptor, is not directly involved. The Ca(2+) response to EGCG in STC-1 cells was decreased by inhibitors of the transient receptor potential A1 (TRPA1) channel. HEK293T cells transfected with the mouse TRPA1 (mTRPA1) cDNA showed a Ca(2+) response upon application of EGCG, and their response properties were similar to those observed in STC-1 cells. These results indicate that an astringent compound, EGCG, activates the mTRPA1 in intestinal STC-1 cells. TRPA1 might play an important role in the astringency taste on the tongue.     

TRPM5 is critical for linoleic acid-induced CCK secretion from the enteroendocrine cell line, STC-1

Fatty acid-induced stimulation of enteroendocrine cells leads to release of the hormones such as cholecystokinin (CCK) that contribute to satiety. Recently, the fatty acid activated G protein-coupled receptor GPR120 has been shown to mediate long-chain unsaturated free fatty acid-induced CCK release from the enteroendocrine cell line, STC-1, yet the downstream signaling pathway remains unclear. Here we show that linoleic acid (LA) elicits membrane depolarization and an intracellular calcium rise in STC-1 cells and that these responses are significantly reduced when activity of G proteins or phospholipase C is blocked. LA leads to activation of monovalent cation-specific transient receptor potential channel type M5 (TRPM5) in STC-1 cells. LA-induced TRPM5 currents are significantly reduced when expression of TRPM5 or GPR120 is reduced using RNA interference. Furthermore, the LA-induced rise in intracellular calcium and CCK secretion is greatly diminished when expression of TRPM5 channels is reduced using RNA interference, consistent with a role of TRPM5 in LA-induced CCK secretion in STC-1 cells.     

Free fatty acids in the serum of patients with alcoholism

Blood free fatty acids and their composition were investigated in 25 alcohol abusers hospitalized for detoxication. Blood samples were collected at the admission to the hospital and following detoxication therapy. Free fatty acids were assayed with Dole's technique whereas their composition with gas chromatography. Liver functioning was evaluated with the aid of the following tests: AspAT, AlAT, AP, bilirubin concentration, and thymol test. An increase in free fatty acids concentration was seen in 12 out of 25 patients prior to detoxication. Blood serum free fatty acids were within normal limits in all except one patient after detoxication. A decrease in linoleic acid levels was seen in both total fatty acids and free fatty acids before detoxication. It is more marked in total fatty acids than in free fatty acids and persists after detoxication. Linoleic acid content in free fatty acids returns to the normal values following detoxication. An increase in oleic acid level accompanying a decrease in linoleic acid concentration is probably secondary to the stimulation with hydrogen excess formed during alcohol fermentation.     

Roles of GPR41 and GPR43 in leptin secretory responses of murine adipocytes to short chain fatty acids

GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Gα_i. Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Gα_i signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Gα_i signalling mediated by GPR43 in SCFA-stimulated leptin secretion.     

Role of GPR40 in fatty acid action on the beta cell line INS-1E

GPR40 is a G protein-coupled receptor expressed preferentially in beta cells, that has been implicated in mediating free fatty acid-stimulated insulin release. GPR40 RNAi impaired the ability of palmitic acid (PA) to increase both insulin secretion and intracellular calcium ([Ca2+]i). The PA-dependent [Ca2+]i increase was attenuated by inhibitors of Galphaq, PLC, and SERCA. Thus GPR40 activates the Galphaq pathway, leading to release of Ca2+ from the ER. Yet the GPR40-dependent [Ca2+]i rise was dependent on extracellular Ca2+ and elevated glucose, and was blocked by inhibition of L-type calcium channels (LTCC) or opening of the K(ATP) channel; this suggests that GPR40 promotes Ca2+ influx through up-regulation of LTCC pre-activated by glucose and membrane depolarization. Taken together, the data indicate that GPR40 mediates the increase in [Ca2+]i and insulin secretion through the Galphaq-PLC pathway, resulting in release of Ca2+ from the ER and leading to up-regulation of Ca2+ influx via LTCC.     

Tissue-type plasminogen activator rescues neurones from serum deprivation-induced apoptosis through a mechanism independent of its proteolytic activity

Although the mechanism of action of tissue-type plasminogen activator (tPA) in excitotoxic necrosis is well documented, whether this serine protease can influence the apoptotic cascade remains a subject of debate. Here, we report that tPA protects cultured cortical neurones against apoptotic cell death induced by serum deprivation, an effect associated with a reduction of caspase-3 activation. Interestingly, blocking tPA proteolytic activity by either tPA stop or neuroserpin did not prevent this neuroprotection. Similarly, prevention of the interaction between tPA and its receptor low-density lipoprotein receptor-related protein (LRP) could not alter tPA anti-apoptotic activity. Interestingly, the survival-promoting effect of tPA was blocked by the phosphatidylinositol-3 (PI-3) kinase inhibitor, LY294002, but not by the mitogen-activated protein (MAP) kinase inhibitor, U0126. In conclusion, the present demonstration of an anti-apoptotic effect of tPA, independent of its enzymatic activity, reveals an additional level of complexity in our understanding of this critical mediator of brain physiology and pathology.     

Daxx enhances Fas-mediated apoptosis in a murine pro-B cell line,BAF3

Daxx has been shown to play an essential in type I interferon (IFN-/β)-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and nuclear translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the mechanism of Daxx-mediated apoptosis signaling in B lymphocyte progenitors, here we introduced an efficient suicide switch in a murine pro-B cell line, BAF3, by expressing FK506-binding protein-fused Fas intracellular domain (FKBP-Fas) and Daxx. It allows us to monitor Fas/Daxx-mediated signal by induction of Fas dimerization with the dimerizer drug AP20187. AP20187-mediated Fas dimerization induced not only apoptosis but also Jun N-terminal kinase (JNK) activation. However, AP20187 had no effect on cells expressing either Fas or Daxx only. Furthermore, expression of a JNK inhibitor, the JNK-binding domain of JIP-1, resulted in resistance to AP20187-mediated apoptosis in cells expressing FKBP-Fas and Daxx. These results imply that our novel suicide switch system may provide a powerful tool to delineate or identify the signaling molecules for Daxx-mediated apoptotic machinery in B lymphocyte progenitors through JNK activation.     

Defective elongation of fatty acids in a recessive 25-hydroxycholesterol-resistant mutant cell line

The Chinese hamster ovary recessive mutant, crB, has been selected for its resistance to the cytotoxic effects of 25-hydroxycholesterol in sterol-free media (Sinensky, M., Logel, J., and Torget, R. (1982) J. Cell. Physiol. 113, 314-319). Growth of crB in a chemically defined lipid-poor medium is very slow and is enhanced by a mixture of saturated and unsaturated fatty acids. Incorporation of [3H]acetate into total fatty acids is 4-fold lower in crB compared to that in parental Chinese hamster ovary K1 and in contrast to the wild-type cells, crB cells are unable to synthesize either stearate or oleate. In addition, crB cells can not elongate exogenous palmitate, while they are capable of desaturating exogenous stearate. The mutant cells are also pleiotropically defective in the regulation of mRNA levels for the enzymes of cholesterol biosynthesis. 25-Hydroxycholesterol is a poor regulator of the synthesis and degradation of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase in crB in comparison to the wild-type Chinese hamster ovary K1 cells. The defect in the elongation of fatty acids is reversed in revertants of crB selected for their ability to grow in lipid-poor medium. Such revertants exhibit normal regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity by 25-hydroxycholesterol. Regulation of reductase activity in crB cells can also be restored by supplementing the culture medium with a mixture of fatty acids that restores normal growth rate. The defective regulation of reductase in crB does not appear to be due to nonspecific adverse effects of fatty acid starvation nor is it due to any gross change in the fatty acid composition of cellular phospholipids. These results strongly suggest a direct relationship between the fatty acid auxotrophy of crB and defective regulation of the enzymes of cholesterol biosynthesis.     

Retinoic acids inhibit activation-induced apoptosis in T cell hybridomas and thymocytes.

Apoptosis is induced in immature thymocytes and T cell hybridomas upon stimulation via the TCR/CD3 complex. This phenomenon appears to be related to negative selection of T cell clones in the thymus. In T cell hybridomas, it has been shown that glucocorticoids inhibit TCR/CD3-mediated apoptosis, whereas glucocorticoids alone induce apoptosis. All-trans-retinoic acid (RA) at 0.1 to 10 microM also inhibited TCR/CD3-mediated apoptosis assessed by DNA fragmentation and cytolysis, but RA alone hardly induced apoptosis. RA enhanced the effects of glucocorticoids to induce apoptosis and to inhibit TCR/CD3-mediated apoptosis. TCR/CD3-mediated stimulation can be mimicked by the combination of ionomycin, a calcium ionophore, and PMA, an activator of protein kinase C, and the combination-induced DNA fragmentation was also inhibited by RA. RA, however, failed to inhibit the combination-induced increase in intracellular Ca2+ concentration or the combination-induced translocation of protein kinase C from the cytosolic fraction to the particulate fraction. Time course studies of RA addition into the culture indicated that a 3- to 6-h delay in the addition of RA did not reduce its inhibitory effect on anti-CD3-induced DNA fragmentation. These results suggest that RA interferes with the apoptotic process at some point after its initiation stage. It has been suggested that negative selection involves not only TCR/CD3-mediated signals but also LFA-1-mediated signals. RA at 0.01 to 1 microM significantly inhibited the induction of thymocyte apoptosis by co-immobilized mAb to CD3 and LFA-1 molecules. RA by itself hardly induced apoptosis, but enhanced glucocorticoid-induced apoptosis. The results suggest that thymic selection might be influenced by RA at near-physiologic concentrations. The receptors of glucocorticoids and RA belong to the erbA oncogene-related steroid hormone receptor superfamily. Thyroid hormones and 1 alpha,25-dihydroxy vitamin D3, whose receptors also belong to the superfamily, failed to modulate apoptosis in both T cell hybridomas and thymocytes.     

Selected tetrapeptides lead to a GLP-1 release from the human enteroendocrine cell line NCI-H716

Enteroendocrine cells in the intestine sense the luminal contents and have been shown to respond to not only fatty acids, proteins, and monosaccharides but also artificial sweeteners and bitter compounds. Secretion of hormones such as CCK and GLP-1 from these cells is often associated with a rise in intracellular calcium concentration [Ca2+](i). The human NCI-H716 enteroendocrine cell line has been described as a proper model to study GLP-1 secretion in response to amino acids and protein hydrolysates. Here, we describe that NCI-H716 cells specifically respond to selective tetrapeptides such as tetra-glycine, tetra-alanine and Gly-Trp-Gly-Gly with a dose-dependent [Ca2+](i) response and a GLP-1 secretion, whereas selected free amino acids, dipeptides, tripeptides and pentapeptides failed to elicit such a response. Hormone secretion was not associated with changes in cAMP levels in the cells. The calcium-dependence of hormone secretion appears to involve store-operated calcium channels (SOCCs), since the SOCC blocker 2-APB abolished both the [Ca2+](i) response and GLP-1 release upon tetra-glycine stimulation. The nature of the sensor currently remains elusive, and no obvious common structural pattern in tetrapeptides eliciting GLP-1 secretion was identified. This tetrapeptide sensing in NCI-H716 cells may be underlying the effective stimulation of hormone secretion shown for various protein hydrolysates, and could involve a novel G-protein-coupled receptor (GPCR).     

Cytotoxicity of combined essential fatty acids on a human prostate cancer cell line

In this study the effect of single and concomitantly added n-6 or n-3 polyunsaturated fatty acids (PUFAs) was investigated on human prostate cells. Data obtained from the single fatty acids (FAs) experiments showed that except for oleic acid (OA), arachidonic (AA) and linoleic acid (LA), which had very little (less than 10% cells dead) effect on the cells, an increase in dead cells was observed at physiological concentrations of, eicosapentaenoic acid (EPA), gamma-linolenic acid (GLA) and alpha-linolenic acid (ALA). However, this was not the case when combining these acids at physiological concentrations. A slight increase in cell death was only obtained with three combinations of ALA, namely with AA, OA, or GLA. Other combinations with ALA, such as with LA or EPA, had respectively no effect on cell number or increased the cell number by causing less cells to die. Other PUFAs combinations tested, did not show the three groups mentioned with ALA, but only the last two types, namely, no effect, or a decrease in the amount of cell death. The latter might mean that the FA combination had stimulated the cells, since a decrease in the amount of dead cells was observed. Therefore, it is concluded that the characteristics of combined FAs may differ from single FAs, which may explain some controversies in the literature and in response to treatments.     

HIV-1 gp120-induced tubular epithelial cell apoptosis is mediated through p38-MAPK phosphorylation

BACKGROUND: HIV-associated nephropathy is accompanied by significant tubular alterations in the form of tubular cell proliferation, apoptosis, and microcystic dilatation. In the present study we evaluated the role of CD4 receptors in HIV-1-induced tubular cell injury. METHODS: To confirm the presence of CD4 receptors in tubular cells, immunocytochemical, Western and Northern blot studies were carried out. To determine the downstream effect of CD4 and gp120 interaction, we evaluated the effect of gp120 on tubular cell p38 mitogen-activated protein kinase (MAPK) activity and phosphorylation. To establish causal relationships between gp120, CD4, and p38 MAPK pathways, we studied the effect of anti-CD4 antibody and SB 202190 (an inhibitor of p38 MAPK) on gp120-induced tubular cell apoptosis. RESULTS: Proximal tubular cells in culture as well as in intact tissue showed expression of CD4 (immunocytochemical and Western blot studies). Cultured tubular cells also showed mRNA expression for CD4 (Northern blot studies). Gp120, at concentrations of 10-100 ng/ ml, triggered tubular cell apoptosis; however, this effect of gp120 was inhibited by anti-CD4 antibody. SB 202190 also inhibited gp120-induced tubular cell apoptosis. In addition, gp120 promoted tubular cell p38 MAPK phosphorylation in a time- and dose- dependent manner. CONCLUSION: Gp120 through interaction with CD4 triggers tubular cell apoptosis. This effect of gp120 on tubular cells is mediated through phosphorylation of p38 MAPK.