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1.
Eur J Endocrinol ; 153(3): 429-34, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16131606

ABSTRACT

OBJECTIVES: Intrapituitary triiodothyronine (T3) production plays a pivotal role in the control of TSH secretion. Its production is increased in the presence of decreased serum thyroxine (T4) concentrations and the enzyme responsible, deiodinase type 2 (D2), is highest in hypothyroidism. In order to document the role of this enzyme in adult rats we developed an experimental model that inhibited this enzyme using the specific inhibitor, reverse T3 (rT3). METHODS: Hypothyroidism was induced with propylthiouracil (PTU; 0.025 g/l in drinking water) which in addition blocked deiodinase type 1 (D1) activity, responsible for the rapid clearance of rT3 in vivo. During the last 7 days of the experiment, the hypothyroid rats were injected (s.c.) for 4 days with 0.4 or 0.8 nmol T4 per 100 g body weight (bw) per day. For the last 3 days, the same amount of T4 was infused via s.c. minipumps. In additional groups, 25 nmol rT3/100 g bw per day were added to the 3-day infusion of T4. RESULTS: Infusion of 0.4 nmol T4/100 g bw per day did not affect the high serum TSH levels, 0.8 nmol T4/100 g bw per day decreased them to 57% of the hypothyroid values. The infusions of rT3 inhibited D2 activity in all organs where it was measured: the pituitary, brain cortex and brown adipose tissue (BAT). In the pituitary, the activity was 27%, to less than 15% of the activity in hypothyroidism. Despite that, serum TSH levels did not increase, serum T4 concentrations did not change and the changes in serum T3 were minimal. CONCLUSIONS: We conclude that in partly hypothyroid rats, a 3-day inhibition of D2 activity, without concomitant change in serum T4 and minimal changes in serum T3 levels, is not able to upregulate TSH secretion and we postulate that this may be a reflection of absent or only minimal changes in circulating T3 concentrations.


Subject(s)
Enzyme Inhibitors/pharmacology , Hypothyroidism/enzymology , Iodide Peroxidase/antagonists & inhibitors , Thyrotropin/metabolism , Thyroxine/metabolism , Triiodothyronine, Reverse/pharmacology , Animals , Hypothyroidism/blood , Iodide Peroxidase/metabolism , Male , Propylthiouracil , Rats , Rats, Wistar , Thyrotropin/antagonists & inhibitors , Thyrotropin/blood , Thyroxine/blood
2.
Diabetologia ; 48(4): 624-33, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15756538

ABSTRACT

AIMS/HYPOTHESIS: The aims of this work were to determine the effect of hypothyroidism on insulin-stimulated glucose turnover and to unravel the potential mechanisms involved in such an effect. METHODS: Hypothyroidism was induced by administration of propylthiouracil, with partial T4 substitution. Euglycaemic-hyperinsulinaemic clamps, associated with the labelled 2-deoxy-D-glucose technique for measuring tissue-specific glucose utilisation, were used. To assess a possible involvement of leptin in the modulation of glucose metabolism by hypothyroidism, leptin was infused intracerebroventricularly for 6 days. A group of leptin-infused rats was treated with rT3 to determine a potential role of T3 in mediating the leptin effects. RESULTS: Compared with euthyroid rats, hypothyroid animals exhibited decreased overall glucose turnover and decreased glucose utilisation indices in skeletal muscle and adipose tissue. Leptinaemia in hypothyroid rats was lower while resistin mRNA expression in adipose tissue was higher than in euthyroid animals. Intracerebroventricular leptin infusion in hypothyroid rats partially restored overall, muscle and adipose tissue insulin-stimulated glucose utilisation and improved the reduced glycaemic response observed during insulin tolerance tests. The leptin effects were due neither to the observed increase in plasma T3 levels nor to changes in the high adipose tissue resistin expression of hypothyroid rats. The administration of leptin to hypothyroid animals was accompanied by increased expression of muscle and adipose tissue carnitine palmitoyl transferases, decreased plasma NEFA levels and reduced muscle triglyceride content. CONCLUSIONS/INTERPRETATION: Hypothyroidism is characterised by decreased insulin responsiveness, partly mediated by an exaggerated glucose-fatty acid cycle that is partly alleviated by intracerebroventricular leptin administration.


Subject(s)
Energy Metabolism/drug effects , Glucose/metabolism , Hyperthyroidism/metabolism , Leptin/pharmacology , Adipose Tissue/drug effects , Adipose Tissue/metabolism , Animals , Blood Glucose/metabolism , Carnitine O-Palmitoyltransferase/genetics , Fatty Acids, Nonesterified/blood , Gene Expression/genetics , Glucose/pharmacology , Glucose Clamp Technique , Hormones, Ectopic/genetics , Hyperthyroidism/chemically induced , Hyperthyroidism/genetics , Insulin/blood , Insulin/pharmacology , Insulin Resistance/physiology , Iodide Peroxidase/genetics , Iodide Peroxidase/metabolism , Leptin/administration & dosage , Leptin/blood , Male , Muscle, Skeletal/chemistry , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Propylthiouracil , Rats , Rats, Wistar , Resistin , Thyrotropin/blood , Thyroxine/blood , Thyroxine/pharmacology , Triglycerides/analysis , Triglycerides/blood , Triiodothyronine/blood , Triiodothyronine, Reverse/pharmacology , Iodothyronine Deiodinase Type II
3.
Diabetes ; 49(7): 1101-5, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10909965

ABSTRACT

We have shown previously that continuous (6 days) intracerebroventricular (ICV) leptin infusion in normal rats resulted in decreases in food intake and body weight. A reduction of food intake imposed on control rats (pair-feeding), aimed at mimicking leptin-induced hyperphagia, produced a marked decrease in the expression of muscle uncoupling protein-3 (UCP-3), whereas ICV infusion of leptin prevented such a decrease in UCP-3. To investigate an involvement of thyroid hormones in this effect of leptin, plasma levels of these hormones were determined in ICV leptin-infused, ICV vehicle-infused ad libitum fed or pair-fed controls. ICV leptin infusion and pair-feeding resulted in decreased plasma thyroid-stimulating hormone (TSH) and T4 levels relative to ad libitum fed controls. ICV leptin infusion maintained plasma levels of T3, but the levels were decreased by pair-feeding. The activity of the enzyme (hepatic 5'-monodeiodinase) responsible for T4/T3 conversion was measured. In the leptin-infused group, the activity of 5'-monodeiodinase was maintained at the values measured in ad libitum fed rats; in pair-fed rats, activity was reduced. Thus, conversion of T4 to T3 is decreased by pair-feeding, whereas such is not the case during leptin infusion. To further substantiate an involvement of thyroid hormones in the effect of leptin on muscle UCP-3 expression, hypothyroid rats were ICV infused with leptin or vehicle. It was observed that in hypothyroid rats, ICV leptin was unable to maintain muscle UCP-3 expression at values measured in ad libitum fed controls. These results suggest that central leptin stimulates T3 production via an activation of T4 to T3 conversion, and that this stimulation could be responsible for the effect of leptin on muscle UCP-3 expression. Thyroid hormones could thus be important mediators of the effect of leptin on energy expenditure.


Subject(s)
Carrier Proteins/genetics , Cerebral Ventricles/physiology , Leptin/pharmacology , Muscle, Skeletal/metabolism , Thyrotropin/blood , Thyroxine/blood , Triiodothyronine/blood , Animals , Cerebral Ventricles/drug effects , Energy Intake , Gene Expression Regulation/drug effects , Hypothyroidism/metabolism , Infusions, Parenteral , Iodide Peroxidase/metabolism , Ion Channels , Leptin/administration & dosage , Liver/enzymology , Male , Mitochondrial Proteins , Rats , Rats, Sprague-Dawley , Thyroidectomy , Uncoupling Protein 3
4.
Eur J Endocrinol ; 142(1): 71-8, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10633225

ABSTRACT

OBJECTIVE: Examination of the pattern of expression of peroxisome proliferator-activated receptor (PPAR) isoforms alpha and gamma in a model of obesity. DESIGN: Examination of adipose tissue and primary adipocyte cultures from lean and obese Zucker rats at different ages (28 days and 12 weeks). METHODS: mRNA levels were measured by RNase protection assay. RESULTS: The highest levels of PPARalpha and gamma mRNA were present in brown adipose tissue (BAT), followed by liver and white adipose tissue (WAT) for the alpha and gamma subtypes, respectively, at both ages examined. PPARalpha was expressed 100-fold higher in BAT compared with WAT, and PPARgamma mRNA levels were 2-fold higher in the WAT of obese compared with lean rats. PPARalpha and gamma expression was minimal in m. soleus, although higher levels of PPARgamma were found in the diaphragm. In marked contrast to the findings in vivo, virtually no PPARalpha mRNA could be detected in BAT cultures differentiated in vitro. CONCLUSION: PPARalpha and gamma are most highly expressed in BAT in vivo. However, PPARalpha is undetectable in brown adipose cells in vitro, suggesting that the expression of this receptor is induced by some external stimuli. In addition, the expression of PPARgamma was increased in WAT from young obese animals, compatible with an early adaptive phenomenon. Finally, the presence of PPARgamma mRNA is detectable only in particular muscles, such as the diaphragm, suggesting the possibility of an influence of fiber type on its expression, although exercise did not influence the expression of PPARgamma in other skeletal muscles.


Subject(s)
Obesity/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , Adipocytes/metabolism , Adipose Tissue/metabolism , Adipose Tissue, Brown/metabolism , Aging/metabolism , Animals , Cells, Cultured , Male , Physical Conditioning, Animal/physiology , Physical Endurance/physiology , RNA, Messenger/metabolism , Rats , Rats, Zucker , Receptors, Cytoplasmic and Nuclear/genetics , Reference Values , Transcription Factors/genetics
5.
Toxicol Sci ; 51(1): 36-43, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10496675

ABSTRACT

To evaluate the toxicological profile of the phenolic antioxidant ethylene-bis-(oxyethylene)-bis-(3-tert-butyl-4-hydroxy-5-methyl- hydrocinnamate) (EOC) in a non-human primate, male cynomolgus monkeys (Macaca fascicularis) were treated for 4 weeks by oral administration of 0, 200, or 1000 mg/kg body weight/day. Special attention was directed to parameters of the pituitary-thyroid-liver axis. Moderately increased liver weights and minimal to moderate hepatocellular hypertrophy were observed in treated animals. Otherwise, no treatment-related changes were detected in hematological, clinical chemistry, or urinalysis parameters or upon histopathological examination. Except for a slight induction of microsomal testosterone 16beta-hydroxylation, liver xenobiotic-metabolising enzyme activities and peroxisomal fatty acid beta-oxidation remained unchanged. Likewise, serum levels of thyroid stimulating hormone, thyroxine, 3,3',5-triiodothyronine and 3,3',5'-triiodothyronine as well as 5'-monodeiodinase type 1 mRNA levels in the liver, heart, cerebral cortex, and thyroid were found unchanged. The results demonstrate that, in the Cynomolgus monkey, EOC is only a very weak inducer of liver xenobiotic-metabolizing enzymes and has no effect on thyroid function. In contrast, upon feeding rats at dose levels up to 1000 ppm (equivalent to between 50 and 100 mg/kg body weight/day), EOC has been identified as a strong phenobarbital- and peroxisome proliferator-type inducer of hepatic xenobiotic-metabolizing enzymes, interfering with thyroid hormone homeostasis, causing thyroid follicular hypertrophy, and, upon chronic treatment, inducing thyroid gland follicular cell tumors (Thomas et al., 1995. In Toxicology of Industrial Compounds, pp. 319-339. Taylor and Francis). Thus, the results of this study with EOC in the cynomolgus monkey show that effects of xenobiotics on the pituitary-thyroid-liver axis as frequently observed in rodents can not necessarily be extrapolated to primates including man.


Subject(s)
Antioxidants/toxicity , Aryl Hydrocarbon Hydroxylases , Liver/drug effects , Pituitary Gland/drug effects , Polyethylene Glycols/toxicity , Thyroid Gland/drug effects , Animals , Body Weight/drug effects , Cytochrome P-450 Enzyme System/biosynthesis , Iodide Peroxidase/genetics , Iodide Peroxidase/metabolism , Liver/enzymology , Liver/pathology , Macaca fascicularis , Male , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Organ Size/drug effects , Pituitary Gland/metabolism , Pituitary Gland/pathology , RNA, Messenger/metabolism , Rats , Species Specificity , Steroid Hydroxylases/biosynthesis , Thyroid Gland/metabolism , Thyroid Gland/pathology , Thyroid Hormones/blood , Thyrotropin/blood , Toxicity Tests
6.
Mol Cell Endocrinol ; 147(1-2): 37-47, 1999 Jan 25.
Article in English | MEDLINE | ID: mdl-10195690

ABSTRACT

The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. These ligand-activated transcription factors are implicated in the regulation of lipid metabolism and adipocyte differentiation and in the regulation of anti-inflammatory processes. In order to bind to DNA and activate transcription PPAR requires the formation of heterodimers with the retinoid X receptor (RXR). We have previously reported that replacement of a single leucine by an arginine at position 433 of hPPAR alpha (L433R), located in a highly conserved region of the ninth heptad repeat of a leucine-zipper-like motif in the ligand binding domain, abolished heterodimerization of PPAR with RXR and hence its trans-activating capacity. The aim of our present work was to investigate if other conserved amino acids of the ligand binding domain are important for heterodimerization of PPAR with RXR. We found that conserved leucines, L370 and L391, in a leucine-zipper-like motif of hPPAR alpha, as well as a highly conserved aspartic acid (D304) in the tau(i) domain are necessary for heterodimerization with RXR. In contrast, mutations of non-conserved amino acids within the leucine-zipper-like motif do not affect PPAR:RXR heterodimerization. Surprisingly, we found that some mutants deficient in heterodimerization with RXR (hPPAR alpha-L370R and -L391R) were still functional on specific peroxisome proliferator-activator response elements (PPREs). Both mutants could trans-activate on a PPRE from the P450 cytochrome promoter CYP4A1, whereas only the hPPAR alpha-L391R mutant could trans-activate from the acyl-CoA oxidase PPRE (ACOA) and, when stimulated with the peroxisome proliferator Wy14643, also from the bifunctional enzyme PPRE. We therefore hypothesize either that: (i) these mutants might be able to heterodimerize with a protein other than RXR and the affinity for this novel partner may depend on the nature of the PPRE and to some degree on the choice of the activator, or alternatively; (ii) that additional nuclear proteins might compensate in vivo for the decreased binding of RXR to these mutant PPARs observed in vitro.


Subject(s)
Conserved Sequence/physiology , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/metabolism , Transcription Factors/metabolism , Acyl-CoA Oxidase , Amino Acid Sequence , Aspartic Acid/genetics , Aspartic Acid/metabolism , Conserved Sequence/genetics , Cytochrome P-450 CYP4A , Cytochrome P-450 Enzyme System/genetics , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimerization , Leucine/genetics , Leucine/metabolism , Leucine Zippers/genetics , Ligands , Mixed Function Oxygenases/genetics , Molecular Sequence Data , Mutation , Oxidoreductases/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Pyrimidines/pharmacology , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/genetics , Response Elements/genetics , Retinoid X Receptors , Trans-Activators/chemistry , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics
7.
J Biol Chem ; 274(15): 10505-10, 1999 Apr 09.
Article in English | MEDLINE | ID: mdl-10187842

ABSTRACT

The peroxisome proliferator-activated receptors (PPARs) are a subgroup of nuclear receptors activated by fatty acids and eicosanoids. In addition, they are subject to phosphorylation by insulin, resulting in the activation of PPARalpha, while inhibiting PPARgamma under certain conditions. However, it was hitherto unclear whether the stimulatory effect of insulin on PPARalpha was direct and by which mechanism it occurs. We now demonstrate that amino acids 1-92 of hPPARalpha contain an activation function (AF)-1-like domain, which is further activated by insulin through a pathway involving the mitogen-activated protein kinases p42 and p44. Further analysis of the amino-terminal region of PPARalpha revealed that the insulin-induced trans-activation occurs through the phosphorylation of two mitogen-activated protein kinase sites at positions 12 and 21, both of which are conserved across evolution. The characterization of a strong AF-1 region in PPARalpha, stimulating transcription one-fourth as strongly as the viral protein VP16, is compatible with the marked basal transcriptional activity of this isoform in transfection experiments. However, it is intriguing that the activity of this AF-1 region is modulated by the phosphorylation of two serine residues, both of which must be phosphorylated in order to activate transcription. This is in contrast to PPARgamma2, which was previously shown to be phosphorylated at a single site in a motif that is not homologous to the sites now described in PPARalpha. Although the molecular details involved in the phosphorylation-dependent enhancement of the transcriptional activity of PPARalpha remain to be elucidated, we demonstrate that the effect of insulin on the AF-1 region of PPARalpha can be mimicked by the addition of triiodothyronine receptor beta1, a strong binder of corepressor proteins. In addition, a triiodothyronine receptor beta1 mutant deficient in interacting with corepressors is unable to activate PPARalpha. These observations suggest that the AF-1 region of PPARalpha is partially silenced by corepressor proteins, which might interact in a phosphorylation-dependent manner.


Subject(s)
Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Consensus Sequence , Humans , Insulin/metabolism , Ligands , Mice , Molecular Sequence Data , Nuclear Proteins/metabolism , Nuclear Receptor Co-Repressor 1 , Phosphorylation , Rats , Receptors, Cytoplasmic and Nuclear/genetics , Repressor Proteins/metabolism , Sequence Alignment , Transcription Factors/genetics
8.
Eur J Endocrinol ; 137(5): 537-44, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9405035

ABSTRACT

In order to compare the effect of 3,5,3'-triiodothyroacetic acid (TRIAC) with those of triiodothyronine (T3) and thyroxine (T4), severely hypothyroid rats (n=56) were infused over 13 days with 1, 2 or 4 nmol/100 g body weight (BW) per day of T3 or 2, 4 or 8 nmol/100 g BW per day of T4 or TRIAC. The 8 nmol/100 g BW per day of T4 or TRIAC induced the same increase in resting metabolic rate, yet 4 nmol/100 g BW per day of T3 was more potent (P < 0.05). For inhibiting serum TSH levels, 2 nmol/100 g BW per day of TRIAC were significantly less active than 2 nmol/100 g BW per day of T4 or 1 nmol/100 g BW per day of T3 (TRIAC, serum TSH 35.5 +/- 5.7; T3 2.58 +/- 0.91; T4 2.12 +/- 0.59 ng/ml). At higher doses serum TSH and beta-TSH mRNA were unmeasurable. Using serum T3 levels as covariate, the action of T3 and T4 was identical on cardiac monodeiodinase type 1 (5'D1) activity and hepatic malic enzyme (Me) mRNA levels and similar for hepatic 5'D1 activity. The effect of TRIAC was compared with T3 by using increasing doses of 1, 2 and 4 nmol/100 g BW per day of T3 and 2, 4 and 8 nmol/100 g BW per day of TRIAC. ANOVA indicated that there was no major difference between the effects of the hormones since with increasing doses the response of hepatic 5'D1 mRNA levels and enzyme activity and Me mRNA remained parallel. However, when studying the effect on cardiac 5'D1 activity there was not only a difference for type of treatment (T3 > TRIAC) but this difference became greater with each increment in dose. Interestingly there was also only a small effect of TRIAC on increase in heart weight compared with T3 and T4. Brain cortex monodeiodinase type 2 (5'D2) was mainly inhibited by T4 infusions. Monodeiodinase type 3 (5'D3) was stimulated by T4, less so by TRIAC and least by T3, expressing probably the local T3 and TRIAC concentrations. In conclusion, despite apparently similar effects of TRIAC and T3 and T4 on hepatic parameters of thyroid hormone action, TRIAC differs considerably in terms of its effects on cardiac 5'D1 activity and possibly on other fundamental effects of thyroid hormones on the heart since heart weight increased significantly less with TRIAC than with T3 or T4.


Subject(s)
Triiodothyronine/analogs & derivatives , Animals , Body Weight/drug effects , Growth Hormone/genetics , Hypothyroidism/metabolism , Hypothyroidism/pathology , Hypothyroidism/physiopathology , Iodide Peroxidase/metabolism , Myocardium/enzymology , Myocardium/pathology , Organ Size/drug effects , RNA, Messenger/metabolism , Rats , Thyroid Hormones/blood , Thyrotropin/blood , Thyrotropin/genetics , Thyroxine/pharmacology , Triiodothyronine/pharmacology
9.
J Biol Chem ; 272(40): 25252-9, 1997 Oct 03.
Article in English | MEDLINE | ID: mdl-9312141

ABSTRACT

The three subtypes of the peroxisome proliferator-activated receptors (PPARalpha, beta/delta, and gamma) form heterodimers with the 9-cis-retinoic acid receptor (RXR) and bind to a common consensus response element, which consists of a direct repeat of two hexanucleotides spaced by one nucleotide (DR1). As a first step toward understanding the molecular mechanisms determining PPAR subtype specificity, we evaluated by electrophoretic mobility shift assays the binding properties of the three PPAR subtypes, in association with either RXRalpha or RXRgamma, on 16 natural PPAR response elements (PPREs). The main results are as follows. (i) PPARgamma in combination with either RXRalpha or RXRgamma binds more strongly than PPARalpha or PPARbeta to all natural PPREs tested. (ii) The binding of PPAR to strong elements is reinforced if the heterodimerization partner is RXRgamma. In contrast, weak elements favor RXRalpha as heterodimerization partner. (iii) The ordering of the 16 natural PPREs from strong to weak elements does not depend on the core DR1 sequence, which has a relatively uniform degree of conservation, but correlates with the number of identities of the 5'-flanking nucleotides with respect to a consensus element. This 5'-flanking sequence is essential for PPARalpha binding and thus contributes to subtype specificity. As a demonstration of this, the PPARgamma-specific element ARE6 PPRE is able to bind PPARalpha only if its 5'-flanking region is exchanged with that of the more promiscuous HMG PPRE.


Subject(s)
DNA-Binding Proteins/metabolism , Receptors, Pituitary Hormone/metabolism , 3T3 Cells , Animals , Base Sequence , Binding Sites , Chloramphenicol O-Acetyltransferase/biosynthesis , Consensus Sequence , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/chemistry , Dimerization , Genes, Reporter , Humans , Mice , Protein Biosynthesis , Rabbits , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide , Receptors, Pituitary Hormone/biosynthesis , Receptors, Pituitary Hormone/chemistry , Receptors, Retinoic Acid/biosynthesis , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Retinoid X Receptors , Sequence Alignment , Substrate Specificity , Transcription Factors/biosynthesis , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, Genetic , Transfection , Tumor Cells, Cultured
10.
Oncology ; 54(3): 214-9, 1997.
Article in English | MEDLINE | ID: mdl-9143402

ABSTRACT

The cellular homologues of the viral oncogene c-erbA encode for the nuclear thyroid hormone receptor c-erbA (TR). The gene for the human TR beta subtype is located on chromosome 3p24, and a loss of heterozygosity around this region has been reported in breast and small cell lung cancers, suggesting that TR beta might act as a tumor suppressor gene. In the present study, we used PCR-based restriction fragment length polymorphisms to examine the 3p24 region of 19 patients with gastrointestinal tumors for loss of heterozygosity (LOH). Interestingly, only 1 of the patients had an LOH at this locus, while 4 patients had a microdeletion of both alleles in the 3p24 region. These results suggest that, in contrast to previous reports on lung and breast cancers, a loss of heterozygosity of the TR locus at 3p24 is a rare event. A critical review of the literature, however, suggests, that some of the earlier studies have used markers whose location is only imprecisely mapped and may hence point to a tumor suppressor gene candidate other than TR beta. However, a selective microdeletion of both alleles was detected in the tumors of 4 of the 19 patients, indicating that this region on chromosome 3p may be genetically unstable in gastrointestinal tumors.


Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 3 , Esophageal Neoplasms/genetics , Gastrointestinal Neoplasms/genetics , Genes, Tumor Suppressor/genetics , Genes, Viral/genetics , Receptors, Thyroid Hormone/genetics , Aged , Aged, 80 and over , DNA Probes , Female , Heterozygote , Humans , Male , Middle Aged , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length
12.
J Clin Invest ; 100(11): 2858-64, 1997 Dec 01.
Article in English | MEDLINE | ID: mdl-9389752

ABSTRACT

Leptin is thought to exert its actions on energy homeostasis through the long form of the leptin receptor (OB-Rb), which is present in the hypothalamus and in certain peripheral organs, including adipose tissue. In this study, we examined whether leptin has direct effects on the function of brown and white adipose tissue (BAT and WAT, respectively) at the metabolic and molecular levels. The chronic peripheral intravenous administration of leptin in vivo for 4 d resulted in a 1.6-fold increase in the in vivo glucose utilization index of BAT, whereas no significant change was found after intracerebroventricular administration compared with pair-fed control rats, compatible with a direct effect of leptin on BAT. The effect of leptin on WAT fat pads from lean Zucker Fa/ fa rats was assessed ex vivo, where a 9- and 16-fold increase in the rate of lipolysis was observed after 2 h of exposure to 0.1 and 10 nM leptin, respectively. In contrast, no increase in lipolysis was observed in the fat pads from obese fa/fa rats, which harbor an inactivating mutation in the OB-Rb. At the level of gene expression, leptin treatment for 24 h increased malic enzyme and lipoprotein lipase RNA 1.8+/-0.17 and 1.9+/-0.14-fold, respectively, while aP2 mRNA levels were unaltered in primary cultures of brown adipocytes from lean Fa/fa rats. Importantly, however, no significant effect of leptin was observed on these genes in brown adipocytes from obese fa/fa animals. The presence of OB-Rb receptors in adipose tissue was substantiated by the detection of its transcripts by RT-PCR, and leptin treatment in vivo and in vitro activated the specific STATs implicated in the signaling pathway of the OB-Rb. Taken together, our data strongly suggest that leptin has direct effects on BAT and WAT, resulting in the activation of the Jak/STAT pathway and the increased expression of certain target genes, which may partially account for the observed increase in glucose utilization and lipolysis in leptin-treated adipose tissue.


Subject(s)
Adipose Tissue, Brown/drug effects , Adipose Tissue/drug effects , Neoplasm Proteins , Nerve Tissue Proteins , Proteins/pharmacology , Receptors, Cell Surface , Thiazolidinediones , Adipocytes/metabolism , Adipose Tissue/metabolism , Adipose Tissue, Brown/metabolism , Animals , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Cell Nucleus/metabolism , Cells, Cultured , DNA-Binding Proteins/metabolism , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Gene Expression/drug effects , Glucose/metabolism , Hypoglycemic Agents/pharmacology , Janus Kinase 1 , Leptin , Lipoprotein Lipase/biosynthesis , Lipoprotein Lipase/genetics , Malate Dehydrogenase/biosynthesis , Malate Dehydrogenase/genetics , Male , Myelin P2 Protein/biosynthesis , Myelin P2 Protein/genetics , Protein-Tyrosine Kinases/metabolism , Rats , Rats, Sprague-Dawley , Rats, Zucker , Receptors, Leptin , Rosiglitazone , STAT1 Transcription Factor , Thiazoles/pharmacology , Trans-Activators/metabolism
14.
Endocrinology ; 137(10): 4499-502, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8828512

ABSTRACT

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily implicated in adipocyte differentiation. The observations that PPAR alpha is a regulator of hepatic lipid metabolism and that the insulin-sensitizing thiazolidinediones are ligands for PPAR gamma suggest that cross-talk might exist between insulin signaling and PPAR activity, possibly through insulin-induced PPAR phosphorylation. Immunoprecipitation of endogenous PPAR alpha from primary rat adipocytes prelabeled with [32P]-orthophosphate and pretreated for 2 h with vanadate and okadaic acid demonstrated for the first time that PPAR alpha is a phosphoprotein in vivo. Treatment with insulin induced a time-dependent increase in PPAR phosphorylation showing a 3-fold increase after 30 min. Insulin also increased the phosphorylation of human PPAR alpha expressed in CV-1 cells. These changes in phosphorylation were paralleled by enhanced transcriptional activity of PPAR alpha and gamma. Transfection studies in CV-1 cells and HepG2 cells revealed a nearly 2-fold increase of PPAR activity in the presence of insulin. In contrast, insulin had no effect on the transcriptional activity of transfected thyroid hormone receptor in CV-1 cells, suggesting a PPAR-specific effect. Thus, insulin stimulates PPAR alpha phosphorylation and enhances the transcriptional activity of PPAR, suggesting that the transcriptional activity of this nuclear hormone receptor might be modulated by insulin-mediated phosphorylation.


Subject(s)
Insulin/pharmacology , Phosphoproteins/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Transcription Factors/physiology , Animals , Cell Line , Humans , Phosphorylation/drug effects , Rats , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/drug effects
15.
J Endocrinol ; 151(1): 147-57, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8943779

ABSTRACT

Interleukin-1 beta has been implicated as a pathogenic factor in the development of autoimmune thyroiditis. When given for 5 days to normal non-diabetes-prone Wistar Kyoto rats, it decreased plasma concentrations of total tri-iodothyronine and thyroxine and increased plasma TSH. These effects were not prevented by co-injection of nitroarginine methyl ester or aminoguanidine, inhibitors of NO synthases. Exposure to interleukin-1 beta dose-dependently reduced iodine uptake in FRTL-5 cells, but had no effect on thyroglobulin secretion. Nitrite was not detected in the FRTL-5 cell culture media after exposure to interleukin-1 beta. However, reverse transcription PCR analysis of mRNA isolated from interleukin-1 beta-exposed FRTL-5 cells revealed a transitory expression of the inducible NO synthase, which was markedly lower than inducible NO synthase induction in interleukin-1 beta-exposed isolated rat islets of Langerhans. Co-incubation with the NO synthase inhibitor NG-monomethylarginine did not ameliorate the effect of interleukin-1 beta on FRTL-5 cell iodine uptake. Furthermore, we demonstrate that daily injections of interleukin-1 beta for 13 weeks aggravated spontaneous thyroiditis and induced severe hypothyroidism in non-diabetic diabetes-prone BB rats. The data suggest that NO does not mediate interleukin-1 beta-induced inhibition of rat thyroid function in vivo or in vitro in FRTL-5 cells, and the induction of hypothyroidism by interleukin-1 beta in diabetes-prone BB rats is speculated to be due to exacerbation of recruitment and activation of intrathyroidal mononuclear cells.


Subject(s)
Hypothyroidism/chemically induced , Interleukin-1/pharmacology , Nitric Oxide , Thyroid Gland/drug effects , Thyroiditis/chemically induced , Animals , Cell Line , Cells, Cultured , Diabetes Mellitus , Male , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Nitroarginine/pharmacology , RNA, Messenger/analysis , Rats , Rats, Brattleboro , Rats, Inbred WKY , Recombinant Proteins/pharmacology
16.
Obes Res ; 4(3): 213-9, 1996 May.
Article in English | MEDLINE | ID: mdl-8732955

ABSTRACT

The present study was designed to explore the thermogenic effect of thyroid hormone administration and the resulting changes in nitrogen homeostasis. Normal male volunteers (n = 7) received thyroxin during 6 weeks. The first 3-week period served to suppress endogenous thyroid secretion (180 micrograms T4/day). This dose was doubled for the next 3 weeks. Sleeping energy expenditure (respiratory chamber) and BMR (hood) were measured by indirect calorimetry, under standardized conditions. Sleeping heart rate was continuously recorded and urine was collected during this 12-hour period to assess nitrogen excretion. The changes in energy expenditure, heart rate and nitrogen balance were then related to the excess thyroxin administered. After 3 weeks of treatment, serum TSH level fell to 0.15 mU/L, indicating an almost complete inhibition of the pituitary-thyroid axis. During this phase of treatment there was an increase in sleeping EE and sleeping heart rate, which increased further by doubling the T4 dose (delta EE: +8.5 +/- 2.3%, delta heart rate +16.1 +/- 2.2%). The T4 dose, which is currently used as a substitutive dose, lead to a borderline hyperthyroid state, with an increase in EE and heart rate. Exogenous T4 administration provoked a significant increase in urinary nitrogen excretion averaging 40%. It is concluded that T4 provokes an important stimulation of EE, which is mostly mediated by an excess protein oxidation.


Subject(s)
Energy Metabolism/drug effects , Sleep/drug effects , Thyroxine/pharmacology , Adult , Body Composition/physiology , Calorimetry, Indirect , Carbohydrate Metabolism , Dose-Response Relationship, Drug , Energy Metabolism/physiology , Heart Rate/drug effects , Heart Rate/physiology , Homeostasis/drug effects , Homeostasis/physiology , Humans , Hypothalamo-Hypophyseal System/drug effects , Hypothalamo-Hypophyseal System/physiology , Lipid Metabolism , Male , Nitrogen/metabolism , Nitrogen/urine , Oxidation-Reduction , Sleep/physiology , Thyroid Gland/drug effects , Thyroid Gland/physiology , Thyrotropin/blood , Thyroxine/administration & dosage , Thyroxine/blood , Time Factors , Triiodothyronine/blood
17.
Eur J Endocrinol ; 134(1): 6, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8590956
18.
J Endocrinol Invest ; 18(10): 767-73, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8787953

ABSTRACT

Thyroid hormones act by binding to nuclear receptor proteins, the thyroid hormone receptors (TR) alpha and beta. Data from cell culture and animal studies indicate that TR expression may be regulated to modulate target organ responsiveness to thyroid hormone. To investigate whether such adaptive changes in TR expression occur in humans, we determined the mRNA levels of the hTR beta 1 in various thyroid states. Patients with overt hypo- or hyperthyroidism were enrolled in the study. Total RNA was isolated from peripheral blood mononuclear cells and hTR beta 1 mRNA levels determined by quantitative competitive reverse transcription PCR. For comparison, hTR beta 1 mRNA levels were determined in lymphocytes and normal thyroid tissue of euthyroid patients. Human TR beta 1 mRNA levels in lymphocytes were 1.8 +/- 0.4, 1.9 +/- 0.5, 1.1 +/- 0.4 10(-18) mol/microgram RNA in hypo-, eu- and hyperthyroid patients, respectively, corresponding to an estimated 0.5 - 2 molecules per cell. Although the mean hTR beta 1 mRNA levels were 40% lower in hyperthyroid than in euthyroid subjects, this difference did not reach statistical significance. Similar levels of hTR beta 1 mRNA levels were detected in thyroid gland from euthyroid patients. In summary, we developed an assay for the quantitative determination of hTR beta 1 mRNA levels in small human tissue samples, containing as little as 50 ng of total RNA. Absolute hTR beta 1 mRNA levels are very low with an estimated one molecule of mRNA being present in a mononuclear blood cell or thyrocyte. No up-regulation of hTR beta 1 was seen in hypothyroid relative to euthyroid patients. However, there is a non-significant trend towards a down-regulation of hTR beta 1 mRNA levels in hyperthyroid patients.


Subject(s)
Hyperthyroidism/metabolism , Hypothyroidism/metabolism , Polymerase Chain Reaction , RNA, Messenger/analysis , Receptors, Thyroid Hormone/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Base Sequence , Binding, Competitive , Female , Humans , Lymphocytes/chemistry , Male , Middle Aged , Molecular Sequence Data , RNA-Directed DNA Polymerase , Thyroid Gland/chemistry
19.
J Biol Chem ; 270(30): 18117-22, 1995 Jul 28.
Article in English | MEDLINE | ID: mdl-7629123

ABSTRACT

The peroxisome proliferator-activated receptors (PPAR) and thyroid hormone receptors (TR) are members of the nuclear receptor superfamily, which regulate lipid metabolism and tissue differentiation. In order to bind to DNA and activate transcription, PPAR requires the formation of heterodimers with the retinoid X receptor (RXR). In addition to activating transcription through its own response elements, PPAR is able to selectively down-regulate the transcriptional activity of TR, but not vitamin D receptor. The molecular basis of this functional interaction has not been fully elucidated. By means of site-directed mutagenesis of hPPAR alpha we mapped its inhibitory action on TR to a leucine zipper-like motif in the ligand binding domain of PPAR, which is highly conserved among all subtypes of this receptor and mediates heterodimerization with RXR. Replacement of a single leucine by arginine at position 433 of hPPAR alpha (L433R) abolished heterodimerization of PPAR with RXR and consequently its trans-activating capacity. However, a similar mutation of a leucine residue to arginine at position 422 showed no alteration of heterodimerization, DNA binding, or transcriptional activation. The dimerization deficient mutant L433R was no longer able to inhibit TR action, demonstrating that the selective inhibitory effect of PPAR results from the competition for RXR as well as possibly for other TR-auxiliary proteins. In contrast, abolition of DNA binding by a mutation in the P-box of PPAR (C122S) did not eliminate the inhibition of TR trans-activation, indicating that competition for DNA binding is not involved. Additionally, no evidence for the formation of PPAR:TR heterodimers was found in co-immunoprecipitation experiments. In summary, we have demonstrated that PPAR selectively inhibits the transcriptional activity of TRs by competition for RXR and possibly non-RXR TR-auxiliary proteins. In contrast, this functional interaction is independent of the formation of PPAR:TR heterodimers or competition for DNA binding.


Subject(s)
Leucine Zippers , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/metabolism , Receptors, Thyroid Hormone/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Base Sequence , Binding, Competitive , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides , Polymers , Retinoid X Receptors , Sequence Homology, Amino Acid , Tumor Cells, Cultured
20.
Eur J Endocrinol ; 132(6): 751-8, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7788017

ABSTRACT

The purpose of this study was to investigate the relation between the serum levels of thyroid hormones and their biological effects. For this purpose, hypothyroid rats were studied after stopping treatment with a long-acting thyroid hormone, thyroxine (T4) and a short-acting one, triiodothyroacetic acid (Triac). Based on preliminary experiments with different doses of T4 and Triac, hypothyroid rats (N = 84) received over 6 days' injections of 10 nmol Triac or 2 nmol T4/100 g body wt per day. Biological effects of Triac and T4 were measured in the pituitary, liver and kidney up to 8 days after stopping treatment. With Triac, serum thyrotropin (TSH) levels were inhibited completely 6 h after injection, yet after 24 h they were 4.9 +/- 1.8 micrograms/l (hypothyroid 14.5 +/- 0.8 micrograms/l). The rapid changes in serum TSH levels were followed by a more gradual increase in serum TSH levels were followed by a more gradual increase in serum TSH, which was similar to that after T4 injection. Even 8 days after Triac treatment, serum TSH levels did not reach the hypothyroid control levels. Changes in beta-TSH mRNA levels also showed a prolonged inhibition after both treatments and a slow return to hypothyroid values, which was not complete 8 days after stopping treatment. A second parameter was hepatic 5'-deiodinase type I (5'D-I). The 6-day treatment with Triac had a markedly stronger effect on 5'D-I enzyme activity and mRNA levels than treatment with T4.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Iodide Peroxidase/metabolism , Thyrotropin/blood , Thyroxine/pharmacology , Triiodothyronine/analogs & derivatives , Animals , Hypothyroidism/drug therapy , Hypothyroidism/genetics , Hypothyroidism/metabolism , Iodide Peroxidase/genetics , Kidney/drug effects , Kidney/metabolism , Liver/drug effects , Liver/metabolism , Male , Organ Specificity , Pituitary Gland/drug effects , Pituitary Gland/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Thyrotropin/genetics , Thyroxine/administration & dosage , Triiodothyronine/administration & dosage , Triiodothyronine/pharmacology
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