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1.
PLoS One ; 3(4): e2063, 2008 Apr 30.
Article in English | MEDLINE | ID: mdl-18446241

ABSTRACT

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.


Subject(s)
Recovery of Function , Salivary Glands/cytology , Salivary Glands/radiation effects , Stem Cell Transplantation , Animals , Cell Differentiation/radiation effects , Cell Separation , Cells, Cultured , Female , Humans , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-kit/metabolism , Rats , Recovery of Function/radiation effects , Salivary Ducts/cytology , Salivary Ducts/radiation effects , Spheroids, Cellular/cytology , Spheroids, Cellular/radiation effects , Stem Cells/cytology , Stem Cells/radiation effects , X-Rays
2.
Clin Cancer Res ; 12(6): 1804-12, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16551865

ABSTRACT

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.


Subject(s)
Bone Marrow Cells/drug effects , Bone Marrow Transplantation , Granulocyte Colony-Stimulating Factor/pharmacology , Salivary Glands/drug effects , Animals , Bone Marrow Cells/chemistry , Bone Marrow Cells/metabolism , Female , Gene Expression/drug effects , Granulocyte Colony-Stimulating Factor/administration & dosage , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hematopoietic Stem Cell Mobilization , Immunohistochemistry , In Situ Hybridization , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Granulocyte Colony-Stimulating Factor/analysis , Receptors, Granulocyte Colony-Stimulating Factor/genetics , Reverse Transcriptase Polymerase Chain Reaction , Salivary Glands/pathology , Salivary Glands/radiation effects , Time Factors
3.
J Biol Chem ; 279(10): 8930-7, 2004 Mar 05.
Article in English | MEDLINE | ID: mdl-14688286

ABSTRACT

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.


Subject(s)
Glucose-6-Phosphate/biosynthesis , Glucose/metabolism , Glycogen/metabolism , Hypoglycemia/metabolism , Liver/metabolism , Receptors, Cytoplasmic and Nuclear/deficiency , Transcription Factors/deficiency , Animals , Down-Regulation , Fasting , Gene Expression Regulation , Mice , Oxidation-Reduction , RNA, Messenger/analysis , Radioisotopes , Receptors, Cytoplasmic and Nuclear/genetics , Time Factors , Transcription Factors/genetics
4.
J Biol Chem ; 278(43): 41930-7, 2003 Oct 24.
Article in English | MEDLINE | ID: mdl-12917447

ABSTRACT

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.


Subject(s)
Bile Acids and Salts/metabolism , DNA-Binding Proteins/physiology , Enterohepatic Circulation/physiology , Hydroxysteroid Dehydrogenases , Membrane Glycoproteins , Receptors, Cytoplasmic and Nuclear/physiology , Transcription Factors/physiology , Animals , Carrier Proteins/analysis , Cholesterol 7-alpha-Hydroxylase/biosynthesis , Cholesterol 7-alpha-Hydroxylase/genetics , Cholic Acid/biosynthesis , Cholic Acid/metabolism , DNA-Binding Proteins/genetics , Feedback, Physiological , Homeostasis , Intestinal Absorption , Mice , Mice, Knockout , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics
5.
Gastroenterology ; 124(1): 160-71, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12512040

ABSTRACT

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.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Fasting/physiology , Liver/metabolism , Receptors, Cytoplasmic and Nuclear/physiology , Transcription Factors/physiology , ATP Binding Cassette Transporter, Subfamily B/deficiency , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/physiology , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/physiology , Animal Feed , Animals , Bile/physiology , Lipid Metabolism , Male , Mice , Mice, Knockout/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Reference Values
6.
Biochem J ; 369(Pt 3): 539-47, 2003 Feb 01.
Article in English | MEDLINE | ID: mdl-12381268

ABSTRACT

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.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Clofibric Acid/analogs & derivatives , Hepatocytes/physiology , Membrane Transport Proteins , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , ATP Binding Cassette Transporter 1 , ATP Binding Cassette Transporter, Subfamily B/drug effects , ATP Binding Cassette Transporter, Subfamily B, Member 11 , ATP Binding Cassette Transporter, Subfamily G, Member 5 , ATP Binding Cassette Transporter, Subfamily G, Member 8 , ATP-Binding Cassette Transporters/drug effects , Animals , Bile/chemistry , Bile/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cells, Cultured , Clofibric Acid/pharmacology , Fibric Acids , Lipid Metabolism , Lipoproteins/genetics , Lipoproteins/metabolism , Liver/drug effects , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Mutant Strains , Organ Size/drug effects , Organic Anion Transporters, Sodium-Dependent , Organic Anion Transporters, Sodium-Independent/genetics , Organic Anion Transporters, Sodium-Independent/metabolism , Peroxisome Proliferators/pharmacology , Pyrimidines/pharmacology , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Cytoplasmic and Nuclear/genetics , Symporters , Taurochenodeoxycholic Acid/administration & dosage , Taurochenodeoxycholic Acid/pharmacology , Transcription Factors/drug effects , Transcription Factors/genetics , ATP-Binding Cassette Sub-Family B Member 4
7.
J Biol Chem ; 277(37): 34182-90, 2002 Sep 13.
Article in English | MEDLINE | ID: mdl-12097330

ABSTRACT

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.


Subject(s)
Lipids/biosynthesis , Lipoproteins, VLDL/biosynthesis , Phospholipid Transfer Proteins , Receptors, Cytoplasmic and Nuclear , Receptors, Retinoic Acid/physiology , Receptors, Thyroid Hormone/physiology , Triglycerides/biosynthesis , Animals , Anticholesteremic Agents/pharmacology , Apolipoproteins E/physiology , Carrier Proteins/physiology , Cell Line , DNA-Binding Proteins , Hydrocarbons, Fluorinated , Hypertriglyceridemia/etiology , Liver/metabolism , Liver X Receptors , Male , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Orphan Nuclear Receptors , Rats , Receptors, LDL/physiology , Sulfonamides
8.
J Biol Chem ; 277(37): 33870-7, 2002 Sep 13.
Article in English | MEDLINE | ID: mdl-12105210

ABSTRACT

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.


Subject(s)
Bile/metabolism , Cholesterol/metabolism , Feces/chemistry , Liver/metabolism , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Retinoic Acid/physiology , Receptors, Thyroid Hormone/physiology , Animals , Anticholesteremic Agents/pharmacology , Biological Transport , DNA-Binding Proteins , Fatty Liver/etiology , Hydrocarbons, Fluorinated , Lipoproteins, LDL/blood , Lipoproteins, VLDL/metabolism , Liver X Receptors , Male , Mice , Mice, Inbred C57BL , Orphan Nuclear Receptors , Sulfonamides
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