RESUMEN
BACKGROUND: The plasma concentration of 5-hydroxytryptamine (5-HT) in diabetic patients is higher than that in normal subjects. Since recent reports have demonstrated the presence of 5-HT2A receptor in glomerular mesangial cells, it is possible that 5-HT may be involved in the development of diabetic nephropathy through the 5-HT2A receptor in mesangial cells. Because expansion of the glomerular mesangial lesion is a characteristic feature of diabetic nephropathy, we examined the effect of 5-HT on the production of type IV collagen by human mesangial cells. METHODS: Human mesangial cells were incubated with 5-HT with or without 5-HT receptor antagonists, protein kinase C (PKC) inhibitor or transforming growth factor-beta (TGF-beta) antibody. Type IV collagen mRNA and protein concentration in medium were measured by Northern blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. TGF-beta mRNA and bioactivity in the medium were measured by Northern blot analysis and bioassay using mink lung epithelial cells, respectively. RESULTS: 5-HT stimulated the production of type IV collagen by human mesangial cells, which was inhibited by ketanserin and sarpogrelate hydrochloride, 5-HT2A receptor antagonists, but not by ondansetron, a 5-HT3 receptor antagonist. 5-HT increased the bioactivities of both active and total TGF-beta. However, the 5-HT-enhanced production of type IV collagen was completely inhibited by an anti-TGF-beta antibody. Furthermore, a PKC inhibitor, calphostin C, inhibited the 5-HT-induced increase in type IV collagen secretion, and the activity of membrane PKC was increased by 5-HT. Phorbol ester activated type IV collagen production as well as active and total TGF-beta. Calphostin C completely inhibited the 5-HT-enhanced activity of active TGF-beta, but did not inhibit exogenous TGF-beta-induced increase in type IV collagen secretion. CONCLUSIONS: Our results suggest that 5-HT-enhanced production of type IV collagen by human mesangial cells is mediated by activation of PKC and subsequent increase in active TGF-beta activity.
Asunto(s)
Colágeno/biosíntesis , Mesangio Glomerular/efectos de los fármacos , Mesangio Glomerular/metabolismo , Serotonina/farmacología , Células Cultivadas , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/metabolismo , Humanos , Ketanserina/farmacología , Cinética , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Serotonina 5-HT2A , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Succinatos/farmacología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
To elucidate the mechanism of triglyceride (TG) accumulation in adipocytes induced by TG-rich lipoproteins, we examined the effect of beta-very low density lipoprotein (beta-VLDL) on TG accumulation in 3T3-L1 adipocytes. Beta-VLDL did not induce TG accumulation in 3T3-L1 preadipocytes but in 3T3-L1 adipocytes. TG accumulation was significantly inhibited by cytochalasin B, an inhibitor of receptor mediated endocytosis. In contrast, cytochalasin B did not inhibit free fatty acid induced TG accumulation in adipocytes. The binding of [125I]beta-VLDL to preadipocytes was inhibited completely by both beta-VLDL and LDL. In sharp contrast, the binding of [125I]beta-VLDL to adipocytes was inhibited completely by beta-VLDL, but partially by LDL. The VLDL receptor mRNA was only expressed in adipocytes. These results suggest that beta-VLDL induced TG accumulation in adipocytes may be mediated through the VLDL receptor pathway.
Asunto(s)
Adipocitos/metabolismo , Adipocitos/fisiología , Endocitosis , Lipoproteínas VLDL/fisiología , Receptores de LDL/fisiología , Transducción de Señal , Triglicéridos/metabolismo , Células 3T3 , Adipocitos/citología , Animales , Northern Blotting , Diferenciación Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Ratones , Unión Proteica/efectos de los fármacos , Conejos , Transducción de Señal/efectos de los fármacos , Células Madre/citologíaRESUMEN
To elucidate the mechanism of foam cell formation in the mesangial region of a kidney observed in a familial type III hyperlipoproteinemic patient presenting with diabetes mellitus and nephrotic syndrome, we have examined, in the present study, the effect of human beta-VLDL (apo E2/E2) on foam cell formation in human mesangial cells, since an increase in beta-VLDL is a characteristic feature of this patient. Human beta-VLDL (apo E2/E2) induced foam cell formation in human mesangial cells. The binding of [125I]LDL to human mesangial cells was inhibited completely by both LDL and beta-VLDL. On the other hand, the binding of [125I]beta-VLDL was completely inhibited by beta-VLDL, but partially by LDL. The LDL receptor, but not the VLDL receptor was down-regulated by accumulation of cholesteryl esters. These results suggest that human beta-VLDL (apo E2/E2)-induced foam cell formation in mesangial cells is mediated through both the LDL receptor pathway and the beta-VLDL specific pathway, in which the VLDL receptor is one of the candidates.
Asunto(s)
Células Espumosas/patología , Mesangio Glomerular/patología , Lipoproteínas VLDL/fisiología , Northern Blotting , Células Cultivadas , Ésteres del Colesterol/metabolismo , Células Espumosas/efectos de los fármacos , Células Espumosas/metabolismo , Mesangio Glomerular/metabolismo , Humanos , Ligandos , Lipoproteínas VLDL/metabolismo , Unión Proteica/efectos de los fármacosRESUMEN
We investigated whether low density lipoprotein (LDL) under oxidative stress might induce the release of fructose, glucose-6-phosphate and fructose-6-phosphate from perivascular cells, and also whether these substances might accelerate the formation of advanced glycation end products (AGE) from proteins in vitro. When vascular smooth muscle cells were incubated with LDL in Ham's F10 at 37 degrees C for 48 h. release of all these substances was increased dose-dependently by oxidized LDL. Fructose release was increased in a dose-dependent manner by glucose. Indomethacin (20 microM) significantly (P < 0.01) suppressed the release of fructose (25.4 +/- 15.7% of control) and hexose phosphates (29.4 +/- 4.0) with the inhibition of release of lactate dehydrogenase (35.5 +/- 4.9) as well as probucol, whereas an aldose reductase inhibitor, epalrestat, significantly (P < 0.001) inhibited only the fructose release (0.9 +/- 0.8). Release of fructose and hexose phosphates from vascular endothelial cells was also induced by oxidized LDL. AGE immunoreactivities and AGE-related fluorescence formed from proteins and glucose were significantly increased (P < 0.001) in the presence of small amounts of the cellular glucose metabolites (6.6%) with glucose (93.4%). These data suggest that release of potent AGE initiators, fructose and hexose phosphates, from perivascular cells induced by oxidized LDL may be an important phenomenon for vascular complications.
Asunto(s)
Endotelio Vascular/metabolismo , Fructosa/metabolismo , Glicoproteínas/metabolismo , Hexosafosfatos/metabolismo , Lipoproteínas LDL/farmacología , Músculo Liso Vascular/metabolismo , Factor de Activación Plaquetaria/análisis , Aldehído Reductasa/antagonistas & inhibidores , Animales , Aorta Torácica , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glicosilación , Humanos , Cinética , L-Lactato Deshidrogenasa , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo , Probucol/farmacología , Conejos , Rodanina/análogos & derivados , Rodanina/farmacología , Tiazolidinas , Sustancias Reactivas al Ácido Tiobarbitúrico , Venas UmbilicalesRESUMEN
To elucidate whether beta-migrating very low density lipoproteins (beta-VLDL) induce foam cell formation in mesangial cells or not, surface binding and foam cell formation with beta-VLDL were studied in mouse mesangial cells. Specific binding kinetics for beta-VLDL and low density lipoproteins (LDL) on the mesangial cells were observed with Kd = 3.8 and 13.7 micrograms/ml, and Bmax = 65.9 and 71.9 ng/ml cell protein at 4 degrees C, respectively. The binding of beta-VLDL was inhibited by excess amounts of LDL or beta-VLDL, but not by acetyl-low density lipoproteins. Ligand blotting using beta-VLDL or LDL and immunoblotting using anti-human LDL receptor monoclonal antibody detected the same apparent single protein (approx. 130 kDa). Incorporation of [14C]oleate into cholesteryl ester in mouse mesangial cells was enhanced by beta-VLDL to 3-fold higher than that by LDL, and it was inhibited by chloroquine or anti-human LDL receptor monoclonal antibody. The light microscopic findings also demonstrated that cholesteryl ester deposition increased in these cells incubated with beta-VLDL, but not with LDL. In conclusion, beta-VLDL was specifically taken up by receptor-mediated endocytosis in mouse mesangial cells through LDL receptors, resulting in foam cell formation.
Asunto(s)
Células Espumosas/citología , Mesangio Glomerular/efectos de los fármacos , Lipoproteínas VLDL/farmacología , Receptores de LDL/metabolismo , Animales , Unión Competitiva , Células Cultivadas , Ésteres del Colesterol/metabolismo , LDL-Colesterol/metabolismo , LDL-Colesterol/farmacología , Femenino , Células Espumosas/metabolismo , Mesangio Glomerular/citología , Immunoblotting , Lipoproteínas VLDL/metabolismo , Ratones , Ratones Endogámicos , Ensayo de Unión RadioliganteRESUMEN
In a chase study using double-radiolabeled apolipoprotein (apo) A-I-containing lipoproteins (14C-labeled cholesteryl ester and 125I-labeled apolipoprotein) with or without apo A-II (Lp A-I/A-II particle and Lp A-I particle), these lipoproteins internalized into HepG2 cells were demonstrated to be time-dependently released into the medium as trichloroacetic acid (TCA)-precipitable fraction. The molar ratio of 14C/125I-radioactivity of TCA-precipitable fraction in the medium was time-dependently decreased. In Sephacryl S-300 HR chromatography of both circulating mature and resecreted apo A-I-containing lipoproteins in the medium after the chase period, a single major protein peak corresponding to that of high density lipoproteins was detected by absorbance at 280 nm. The 14C-radioactivity in apo A-I-containing lipoproteins resecreted from HepG2 cells after 3-h chase was approximately one-fourth of that in circulating mature apo A-I-containing lipoproteins. Cholesterol mass in resecreted apo A-I-containing lipoproteins was three-tenths of that in circulating mature apo A-I-containing lipoproteins. In a cholesterol efflux experiment using macrophage foam cells labeled with [3H]cholesterol, apo A-I-containing lipoproteins resecreted significantly decreased cholesteryl ester radioactivity in macrophage foam cells, as compared with circulating mature apo A-I-containing lipoproteins. There were no remarkable differences in the metabolic fates and cholesterol efflux from macrophage foam cells between Lp A-I and Lp A-I/A-II particles. These results suggest that a part of apo A-I-containing lipoproteins internalized into HepG2 cells may be resecreted in the form of intact lipoproteins with lower cholesterol content, and apo A-I-containing lipoproteins resecreted may be a potent inducer for cholesterol efflux through the processes of reverse cholesterol transport.