RESUMEN
PURPOSE: One of the major reasons for failure of radiotherapeutic cancer treatment is the limitation in dose that can be applied to the tumor because of coirradiation of the normal healthy tissue. Late radiation-induced damage reduces the quality of life of the patient and may even be life threatening. Replacement of the radiation-sterilized stem cells with unirradiated autologous stem cells may restore the tissue function. Here, we assessed the potential of granulocyte colony-stimulating factor (G-CSF)-mobilized bone marrow-derived cells (BMC) to regenerate and functionally restore irradiated salivary glands used as a model for normal tissue damage. EXPERIMENTAL DESIGN: Male-eGFP+ bone marrow chimeric female C57BL/6 mice were treated with G-CSF, 10 to 60 days after local salivary gland irradiation. Four months after irradiation, salivary gland morphology and flow rate were assessed. RESULTS: G-CSF treatment induced homing of large number of labeled BMCs to the submandibular glands after irradiation. These animals showed significant increased gland weight, number of acinar cells, and salivary flow rates. Donor cells expressed surface markers specific for hematopoietic or endothelial/mesenchymal cells. However, salivary gland acinar cells neither express the G-CSF receptor nor contained the GFP/Y chromosome donor cell label. CONCLUSIONS: The results show that BMCs home to damaged salivary glands after mobilization and induce repair processes, which improve function and morphology. This process does not involve transdifferentiation of BMCs to salivary gland cells. Mobilization of BMCs could become a promising modality to ameliorate radiation-induced complications after radiotherapy.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Trasplante de Médula Ósea , Factor Estimulante de Colonias de Granulocitos/farmacología , Glándulas Salivales/efectos de los fármacos , Animales , Células de la Médula Ósea/química , Células de la Médula Ósea/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Movilización de Célula Madre Hematopoyética , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/patología , Glándulas Salivales/efectos de la radiación , Factores de TiempoRESUMEN
Head and neck cancer is the fifth most common malignancy and accounts for 3% of all new cancer cases each year. Despite relatively high survival rates, the quality of life of these patients is severely compromised because of radiation-induced impairment of salivary gland function and consequential xerostomia (dry mouth syndrome). In this study, a clinically applicable method for the restoration of radiation-impaired salivary gland function using salivary gland stem cell transplantation was developed. Salivary gland cells were isolated from murine submandibular glands and cultured in vitro as salispheres, which contained cells expressing the stem cell markers Sca-1, c-Kit and Musashi-1. In vitro, the cells differentiated into salivary gland duct cells and mucin and amylase producing acinar cells. Stem cell enrichment was performed by flow cytrometric selection using c-Kit as a marker. In vitro, the cells differentiated into amylase producing acinar cells. In vivo, intra-glandular transplantation of a small number of c-Kit(+) cells resulted in long-term restoration of salivary gland morphology and function. Moreover, donor-derived stem cells could be isolated from primary recipients, cultured as secondary spheres and after re-transplantation ameliorate radiation damage. Our approach is the first proof for the potential use of stem cell transplantation to functionally rescue salivary gland deficiency.
Asunto(s)
Recuperación de la Función , Glándulas Salivales/citología , Glándulas Salivales/efectos de la radiación , Trasplante de Células Madre , Animales , Diferenciación Celular/efectos de la radiación , Separación Celular , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-kit/metabolismo , Ratas , Recuperación de la Función/efectos de la radiación , Conductos Salivales/citología , Conductos Salivales/efectos de la radiación , Esferoides Celulares/citología , Esferoides Celulares/efectos de la radiación , Células Madre/citología , Células Madre/efectos de la radiación , Rayos XRESUMEN
Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that controls expression of genes involved in lipid metabolism and is activated by fatty acids and hypolipidaemic fibrates. Fibrates induce the hepatic expression of murine multidrug resistance 2 ( Mdr2 ), encoding the canalicular phospholipid translocator. The physiological role of PPARalpha in regulation of Mdr2 and other genes involved in bile formation is unknown. We found no differences in hepatic expression of the ATP binding cassette transporter genes Mdr2, Bsep (bile salt export pump), Mdr1a / 1b, Abca1 and Abcg5 / Abcg8 (implicated in cholesterol transport), the bile salt-uptake systems Ntcp (Na(+)-taurocholate co-transporting polypeptide gene) and Oatp1 (organic anion-transporting polypeptide 1 gene) or in bile formation between wild-type and Ppar alpha((-/-)) mice. Upon treatment of wild-type mice with ciprofibrate (0.05%, w/w, in diet for 2 weeks), the expression of Mdr2 (+3-fold), Mdr1a (+6-fold) and Mdr1b (+11-fold) mRNAs was clearly induced, while that of Oatp1 (-5-fold) was reduced. Mdr2 protein levels were increased, whereas Bsep, Ntcp and Oatp1 were drastically decreased. Exposure of cultured wild-type mouse hepatocytes to PPARalpha agonists specifically induced Mdr2 mRNA levels and did not affect expression of Mdr1a / 1b. Altered transporter expression in fibrate-treated wild-type mice was associated with a approximately 400% increase in bile flow: secretion of phospholipids and cholesterol was increased only during high-bile-salt infusions. No fibrate effects were observed in Ppar alpha((-/-)) mice. In conclusion, our results show that basal bile formation is not affected by PPARalpha deficiency in mice. The induction of Mdr2 mRNA and Mdr2 protein levels by fibrates is mediated by PPARalpha, while the induction of Mdr1a / 1b in vivo probably reflects a secondary phenomenon related to chronic PPARalpha activation.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Ácido Clofíbrico/análogos & derivados , Hepatocitos/fisiología , Proteínas de Transporte de Membrana , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transportador 1 de Casete de Unión a ATP , Subfamilia B de Transportador de Casetes de Unión a ATP/efectos de los fármacos , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5 , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Animales , Bilis/química , Bilis/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Ácido Clofíbrico/farmacología , Ácidos Fíbricos , Metabolismo de los Lípidos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Mutantes , Tamaño de los Órganos/efectos de los fármacos , Transportadores de Anión Orgánico Sodio-Dependiente , Transportadores de Anión Orgánico Sodio-Independiente/genética , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Proliferadores de Peroxisomas/farmacología , Pirimidinas/farmacología , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/genética , Simportadores , Ácido Tauroquenodesoxicólico/administración & dosificación , Ácido Tauroquenodesoxicólico/farmacología , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUND & AIMS: Fatty acids are natural ligands of the peroxisome proliferator-activated receptor alpha (PPARalpha). Synthetic ligands of this nuclear receptor, i.e., fibrates, induce the hepatic expression of the multidrug resistance 2 gene (Mdr2), encoding the canalicular phospholipid translocator, and affect hepatobiliary lipid transport. We tested whether fasting-associated fatty acid release from adipose tissues alters hepatic transporter expression and bile formation in a PPARalpha-dependent manner. METHODS: A 24-hour fasting/48-hour refeeding schedule was used in wild-type and Pparalpha((-/-)) mice. Expression of genes involved in the control of bile formation was determined and related to secretion rates of biliary components. RESULTS: Expression of Pparalpha, farnesoid X receptor, and liver X receptor alpha genes encoding nuclear receptors that control hepatic bile salt and sterol metabolism was induced on fasting in wild-type mice only. The expression of Mdr2 was 5-fold increased in fasted wild-type mice and increased only marginally in Pparalpha((-/-)) mice, and it normalized on refeeding. Mdr2 protein levels and maximal biliary phospholipid secretion rates were clearly increased in fasted wild-type mice. Hepatic expression of the liver X receptor target genes ATP binding cassette transporter a1 (Abca1), Abcg5, and Abcg8, implicated in hepatobiliary cholesterol transport, was induced in fasted wild-type mice only. However, the maximal biliary cholesterol secretion rate was reduced by approximately 50%. CONCLUSIONS: Induction of Mdr2 expression and function is part of the PPARalpha-mediated fasting response in mice. Fasting also induces expression of the putative hepatobiliary cholesterol transport genes Abca1, Abcg5, and Abcg8, but, nonetheless, maximal biliary cholesterol excretion is decreased after fasting.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ayuno/fisiología , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/fisiología , Alimentación Animal , Animales , Bilis/fisiología , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Noqueados/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Valores de ReferenciaRESUMEN
The ATP-binding cassette transporter ABCA1 is essential for high density lipoprotein (HDL) formation and considered rate-controlling for reverse cholesterol transport. Expression of the Abca1 gene is under control of the liver X receptor (LXR). We have evaluated effects of LXR activation by the synthetic agonist T0901317 on hepatic and intestinal cholesterol metabolism in C57BL/6J and DBA/1 wild-type mice and in ABCA1-deficient DBA/1 mice. In wild-type mice, T0901317 increased expression of Abca1 in liver and intestine, which was associated with an approximately 60% rise in HDL. Biliary cholesterol excretion rose 2.7-fold upon treatment, and fecal neutral sterol output was increased by 150-300%. Plasma cholesterol levels also increased in treated Abca1(-/-) mice (+120%), but exclusively in very low density lipoprotein-sized fractions. Despite the absence of HDL, hepatobiliary cholesterol output was stimulated upon LXR activation in Abca1(-/-) mice, leading to a 250% increase in the biliary cholesterol/phospholipid ratio. Most importantly, fecal neutral sterol loss was induced to a similar extent (+300%) by the LXR agonist in DBA/1 wild-type and Abca1(-/-) mice. Expression of Abcg5 and Abcg8, recently implicated in biliary excretion of cholesterol and its intestinal absorption, was induced in T0901317-treated mice. Thus, activation of LXR in mice leads to enhanced hepatobiliary cholesterol secretion and fecal neutral sterol loss independent of (ABCA1-mediated) elevation of HDL and the presence of ABCA1 in liver and intestine.
Asunto(s)
Bilis/metabolismo , Colesterol/metabolismo , Heces/química , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Ácido Retinoico/fisiología , Receptores de Hormona Tiroidea/fisiología , Animales , Anticolesterolemiantes/farmacología , Transporte Biológico , Proteínas de Unión al ADN , Hígado Graso/etiología , Hidrocarburos Fluorados , Lipoproteínas LDL/sangre , Lipoproteínas VLDL/metabolismo , Receptores X del Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Nucleares Huérfanos , SulfonamidasRESUMEN
The bile salt-activated farnesoid X receptor (FXR; NR1H4) controls expression of several genes considered crucial in maintenance of bile salt homeostasis. We evaluated the physiological consequences of FXR deficiency on bile formation and on the kinetics of the enterohepatic circulation of cholate, the major bile salt species in mice. The pool size, fractional turnover rate, synthesis rate, and intestinal absorption of cholate were determined by stable isotope dilution and were related to expression of relevant transporters in the livers and intestines of FXR-deficient (Fxr-/-) mice. Fxr-/- mice showed only mildly elevated plasma bile salt concentrations associated with a 2.4-fold higher biliary bile salt output, whereas hepatic mRNA levels of the bile salt export pump were decreased. Cholate pool size and total bile salt pool size were increased by 67 and 39%, respectively, in Fxr-/- mice compared with wild-type mice. The cholate synthesis rate was increased by 85% in Fxr-/- mice, coinciding with a 2.5-fold increase in cholesterol 7alpha-hydroxylase (Cyp7a1) and unchanged sterol 12alpha-hydroxylase (Cyp8b1) expression in the liver. Despite a complete absence of ileal bile acid-binding protein mRNA and protein, the fractional turnover rate and cycling time of the cholate pool were not affected. The calculated amount of cholate reabsorbed from the intestine per day was approximately 2-fold higher in Fxr-/- mice than in wild-type mice. Thus, the absence of FXR in mice is associated with defective feedback inhibition of hepatic cholate synthesis, which leads to enlargement of the circulating cholate pool with an unaltered fractional turnover rate. The absence of ileal bile acid-binding protein does not negatively interfere with the enterohepatic circulation of cholate in mice.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Proteínas de Unión al ADN/fisiología , Circulación Enterohepática/fisiología , Hidroxiesteroide Deshidrogenasas , Glicoproteínas de Membrana , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Proteínas Portadoras/análisis , Colesterol 7-alfa-Hidroxilasa/biosíntesis , Colesterol 7-alfa-Hidroxilasa/genética , Ácido Cólico/biosíntesis , Ácido Cólico/metabolismo , Proteínas de Unión al ADN/genética , Retroalimentación Fisiológica , Homeostasis , Absorción Intestinal , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
Apart from impaired beta-oxidation, Pparalpha-deficient (Pparalpha(-/-)) mice suffer from hypoglycemia during prolonged fasting, suggesting alterations in hepatic glucose metabolism. We compared hepatic glucose metabolism in vivo in wild type (WT) and Pparalpha(-/-) mice after a short term fast, applying novel isotopic methods. After a 9-h fast, mice were infused with [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol for 6 h, and blood and urine was collected in timed intervals. Plasma glucose concentrations remained constant and were not different between the groups. Hepatic glycogen content was 69 +/- 11 and 90 +/- 31 microM/g liver after 15 h of fasting in WT and Pparalpha(-/-) mice, respectively. The gluconeogenic flux toward glucose 6-phosphate was not different between the groups (i.e. 157 +/- 9 and 153 +/- 9 microM/kg/min in WT and Pparalpha(-/-) mice, respectively). The gluconeogenic flux toward plasma glucose, however, was decreased in PPARalpha(-/-) mice (i.e. 142 +/- 9 versus 124 +/- 13 microM/kg/min) (p < 0.05), accounting for the observed decrease (-15%) in hepatic glucose production in Pparalpha(-/-) mice. Expression of the gene encoding glucose-6-phosphate hydrolase (G6ph) was lower in the PPARalpha(-/-) mice compared with WT mice. In conclusion, Pparalpha(-/-) mice were able to maintain a normal total gluconeogenic flux to glucose 6-phosphate during moderate fasting, despite their inability to up-regulate beta-oxidation. However, this gluconeogenic flux was directed more toward glycogen, leading to a decreased hepatic glucose output. This was associated with a down-regulation of the expression of G6ph in PPARalpha-deficient mice.
Asunto(s)
Glucosa-6-Fosfato/biosíntesis , Glucosa/metabolismo , Glucógeno/metabolismo , Hipoglucemia/metabolismo , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/deficiencia , Factores de Transcripción/deficiencia , Animales , Regulación hacia Abajo , Ayuno , Regulación de la Expresión Génica , Ratones , Oxidación-Reducción , ARN Mensajero/análisis , Radioisótopos , Receptores Citoplasmáticos y Nucleares/genética , Factores de Tiempo , Factores de Transcripción/genéticaRESUMEN
The oxysterol-activated liver X receptor (LXR) provides a link between sterol and fatty acid metabolism; activation of LXR induces transcription of lipogenic genes. This study shows that induction of the lipogenic genes Srebp-1c, Fas, and Acc1 upon administration of the synthetic LXR agonist T0901317 to C57BL/6J mice (10 mg/kg/day, 4 days) is associated with massive hepatic steatosis along the entire liver lobule and a 2.5-fold increase in very low density lipoprotein-triglyceride (VLDL-TG) secretion. The increased VLDL-TG secretion was fully accounted for by formation of larger (129 +/- 9 nm versus 94 +/- 12 nm, a 2.5-fold increase of particle volume) TG-rich particles. Stimulation of VLDL-TG secretion did not lead to elevated plasma TG levels in C57BL/6J mice, indicating efficient particle metabolism and clearance. However, T0901317 treatment did lead to severe hypertriglyceridemia in mouse models of defective TG-rich lipoprotein clearance, i.e. APOE*3-Leiden transgenic mice (3.2-fold increase) and apoE-/- LDLr-/- double knockouts (12-fold increase). Incubation of rat hepatoma McA-RH7777 cells with T0901317 also resulted in intracellular TG accumulation and enhanced TG secretion. We conclude that, in addition to raising high density lipoprotein cholesterol concentrations, pharmacological LXR activation in mice leads to development of hepatic steatosis and secretion of atherogenic, large TG-rich VLDL particles.