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J. Biol. Chem., Vol. 279, Issue 19, 19832-19838, May 7, 2004
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**
From the
High Throughput Biology, Bioscience Support, ¶Genetics Research, and ||High Throughput Chemistry, GlaxoSmithKline, Research Triangle Park, North Carolina 27709
Received for publication, December 11, 2003 , and in revised form, February 27, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A significant and discouraging factor for obese individuals trying to lose weight is that homeostatic resistance mechanisms operate to resist weight loss (2, 3). A major homeostatic barrier is decreased basal metabolic rate during periods of reduced caloric intake. Thyroid hormones (THs)1 (thyroxine (T4) and triiodothyronine (T3)) are the predominant regulators of basal metabolic rate. Serum levels of TH show a direct correlation with energy expenditure and caloric loss (4), decreasing significantly during fasting. The most potent thyroid hormone, T3, has been observed to drop by as much as 33% in subjects in weight loss programs after 10 weeks (5). The mechanisms that regulate thyroid hormone during weight loss are not well understood. Understanding the molecular basis of this homeostatic resistance pathway could create pharmacological opportunities for the treatment of obesity.
TH exerts its effects in virtually every tissue, and its action is mediated by binding and activation of the nuclear receptors TR
(NR1A1) and TR
(NR1A2) (6). T3 is derived by enzymecatalyzed deiodination of the prohormone thyroxine (T4) (7) (Fig. 1). Hepatic and extrahepatic metabolism of T4 regulates the level of T3 in the systemic circulation and other tissues. Type I deiodinase, the major deiodinase in liver and kidney, catalyzes outer ring deiodination to produce T3 as well as inner ring deiodination to produce the inactive metabolite reverse-T3. Remarkably, outer ring deiodination becomes undetectable when T4 is sulfated, whereas the rate of inner ring deiodinase activity increases over 130-fold (8). Thus, the hepatic phase II sulfotransferases are of particular significance in TH metabolism because they control the preferred site of deiodination of T4, effectively catalyzing a reaction that ultimately leads to a rapid and irreversible inactivation of TH (Fig. 1).
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The nuclear receptor CAR (NR1I2) is expressed mainly in liver and has been considered a xenobiotic receptor like its closest relative, the pregnane X receptor (PXR, NR1I2) (12–14). Global gene expression analysis has shown that CAR regulates phase I and II pathways of oxidative metabolism, conjugation, and transport (15). Recent studies utilizing phenobarbital have also demonstrated a CAR-dependent repression of genes involved in aspects of energy metabolism, including glucose synthesis and fatty acid oxidation (16). To understand the action of CAR on genes involved in energy homeostasis, we sought to identify a physiological function beyond its role as a xenosensor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Differential Gene Expression Analysis—Male 10–12-week-old Car–/– and wild-type mice (4–5 mice/group) were utilized for gene expression analysis. For the 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) studies, wild-type or CAR-null mice were treated with the CAR-selective ligand TCPOBOP i.p. at 0.3 mg/kg once daily for 7 days, at which time blood and livers were harvested. For the fasting studies, food was removed from the mice (from both wild-type and Car–/– groups) at midnight on day 1, and the mice were fasted for 24 h. Livers and blood were harvested at 24 h. Total RNA from liver samples was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Northern blot analysis was performed exactly as described previously (12). Blots were hybridized to 32P-labeled cDNAs corresponding to CYP2B10 (bases 7–1527) of the published cDNA (Gen-BankTM accession number NM_009998
[GenBank]
), mSULT1A1 (bases 561–1300) of the published cDNA (GenBankTM accession number NM_001055
[GenBank]
), mSULT-N (bases 98–985) of the published cDNA (GenBankTM accession number AF026073
[GenBank]
), and mUGT1a1 (bases 51–606) of the published cDNA (GenBankTM accession number NM_145079
[GenBank]
). The blots were subsequently probed with radiolabeled r18S (bases 293–970) of the published cDNA (GenBankTM accession number X01117
[GenBank]
) or -actin (Clontech). Signal intensity was quantitated using IMAGEQUANT software (Amersham Biosciences). Values were normalized for expression of either -actin (TCPOBOP study) or r18S RNA (fasting study). Real-time quantitative PCR (RT-QPCR) analysis was performed using an ABI PRISM 7700 Sequence Detection System instrument and software (PE Applied Biosystems, Inc., Foster City, CA) exactly as described previously (15). Primers and probes were designed using Primer Express Version 2.0.0 (Applied Biosystems) and synthesized by Keystone Laboratories (Camarillo, CA). All primers and probes were entered into the NCBI Blast program to ensure specificity. Fold induction values were calculated by subtracting the mean threshold cycle number (Ct) for each treatment group from the mean Ct for the vehicle group and raising 2 to the power of this difference. For animal studies, the average of each treatment group (4–5 animals/group) was used.
Serum Analysis—Total serum T3 and T4 analysis was performed by Anilytics, Inc. (Gaithersburg, MD). Total serum T4 was measured by enzyme-linked immunosorbent assay (Alpha Diagnostic International, San Antonio, TX).
Quantitative Nuclear Magnetic Resonance—Quantitative nuclear magnetic resonance was performed on individual, unanesthetized animals using an EchoMRI Whole Body Composition Analyzer (EchoMedical Systems, Houston, TX).
Caloric Restriction Studies—Car–/– mice and wild-type siblings (8 animals/group) were fed regular chow ad libitum for 3 months. Mice were then single-caged and fed an AIN-93 M diet (Bio-Serv) in 1-g pellets using a pellet feeding tube. Semipurified diet (17) was used rather than nonpurified (i.e. lab chow) because the latter is comprised of less refined foodstuffs than that found in a semipurified diet and more prone to being contaminated by nonnutritive substances. The animals ad libitum regular chow average consumption (total kcal) was calculated to be 13.3 kcal/day, and this was used as the measured normal diet control value in the caloric restriction studies. The animals were then divided into two groups, with half being fed a 40% reduced calorie diet of 8 kcal/day (16 x 1-g pellets/week). The number of calories we calculated for a 40% reduced calorie diet is in good agreement with averages from other studies (18). The animals were weighed weekly for 3 months.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice were treated with TCPOBOP (i.p., 0.3 mg/kg) for 7 days, and total serum T3 and T4 levels were measured by enzyme-linked immunosorbent assay. After a 7-day TCPOBOP treatment, decreases in the average concentrations of serum T3 and T4 levels were seen in the wild-type mice (Fig. 2A), but only the decrease in T4 levels was statistically significant. Average T4 levels decreased 27% (p < 0.005 in the T4 samples) in these animals. In contrast, no statistically significant changes in serum T3 or T4 were observed in TCPOBOP-treated Car–/– mice (Fig. 2A). The serum levels of both T3 and T4 in untreated wild-type mice are consistent with previously published values (21, 22). Serum levels of TSH were also measured after this study and shown to increase by 33% (p = 0.03) in wild-type mice, whereas no significant increases were seen in Car–/– mice (data not shown). These results are consistent with the fact that T4 (a key regulator of the negative feedback loop controlling thyroid hormone homeostasis) is decreased only in wild-type mice. These results are also consistent with the hypothesis that TCPOBOP is effecting decreases in T4 by causing increased metabolism, rather than decreasing its synthesis.
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20-fold) after the 7-day TCPOBOP treatment in wild-type mice, but not in Car–/– mice (Fig. 2C). TH Levels during Fasting Are Dysregulated in Car–/– Mice—To identify a physiological role for CAR in TH metabolism, the effect of food deprivation was studied. Serum T3 and T4 levels in Car–/– mice and sibling wild-type mice were compared under standard feeding and 24-h fasted conditions. After a 24-h fast, a significant difference in serum T4 and T3 levels was observed in wild-type versus Car–/– mice (Fig. 3A). T3 and T4 levels decreased by 39% (p < 0.005) and 36% (p < 0.05), respectively, after a 24-h fast in wild-type mice. The magnitude of these changes is consistent with previously published mouse studies (23). In contrast, the fasted Car–/– mice did not show statistically significant changes in either T3 or T4. The Car–/– mice had an average T3 relative decrease of only 24% and an average T4 decrease of 12%, but p values for both were >0.05 (Fig. 3A). These experiments suggested that CAR activity is required for the full response in TH metabolism during fasting.
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7-fold over basal levels after 24 h of fasting (Fig. 3B). No increase in Cyp2b10 was observed in Car–/– mice. These results are consistent with previous studies that showed the CYP2B gene is induced upon fasting in rats (26).
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4-fold, but no increase was seen in Car–/– mice (Fig. 3B). Sult1a1 expression was also shown to increase during fasting (Fig. 3B). SULT1A1 mRNA levels increased 2-fold during fasting but were not induced in the Car–/– mice. Finally, using RT-QPCR, we found that Sult2a1 was induced 4-fold by fasting in wild-type but not Car–/– animals (Fig. 3C). Our results suggest that induction of phase II sulfotransferase enzymes is one of the primary pathways for CAR-dependent increases in T4 metabolism during fasting. The expression of the major mRNA isoforms expressed from the Ugt1a gene (UGT1A1, UGT1A6, UGT1A7, and UGT1A9) was probed by Northern blot. Both the bilirubin UGT1A1 and the phenol UGT1A9 enzymes have been shown to glucuronidate T4 (28). We found that Ugt1a1 was modestly induced during fasting (1.5-fold increase) and that the induction was CAR-dependent (Fig. 3B). Ugt1a6, Ugt1a7, and Ugt1a9 were not induced by fasting in Car–/– mice, although Ugt1a7 and Ugt1a9 exhibited increased basal expression levels in the knockout mice. We conclude that the induction of phase II glucuronidation of TH by Ugt1a1 upon fasting is a CAR-mediated process that may also contribute to the TH phenotype.
Caloric Restriction Study—Lowering of T3 and T4 levels during caloric restriction opposes weight loss. To assess whether the higher levels of thyroid hormones seen in Car–/– during caloric restriction were physiologically significant, we challenged Car–/– and wild-type littermates with a reduced calorie diet (an approximately 40% decrease in daily calorie intake) for 12 weeks. Statistically significant differences in the weights of the animals were observed at the end of the study (Fig. 4A). The wild-type animals lost an average of 2.8 g of weight, whereas the Car–/– lost an average of 6.9 g (p = 0.044), or >20% of their starting body weights. Thus, by 12 weeks, the Car–/– animals lost 2.5x as much weight as wild-type animals. Analysis of the weights of the animals during the course of the study revealed that the Car–/– mice lost weight for a longer period of time before reaching a plateau in weight. No differences in total serum T3 between wild-type and Car–/– mice were observed at the end of the study. At the end of the study, QNMR analysis was used to quantify lean mass and fat mass/total body weight. Changes in fat mass/total body weight were observed after caloric restriction in both wild-type (39% decrease) and Car–/– animals (46% decrease) (Fig. 4B). Similarly, corresponding changes in the lean mass/total body weight were also observed after caloric restriction in wild-type (32% increase) and Car–/– animals (50% increase) (Fig. 4B). Both of the ratios were consistent with the fact that Car–/– animals lost more weight versus wild-type animals. Importantly, these data indicate that the weight loss observed in the Car–/– animals was normal loss of stored fat from adipose tissue and not lean mass.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There are several potential mechanisms for the nutritional regulation of CAR activity. In mouse primary hepatocytes and in mouse liver in vivo, CAR resides in the cytoplasm and translocates to the nucleus upon activation (37). Induction of CAR nuclear translocation does not require ligand binding, and phenobarbital has been shown to be an indirect activator in vivo through a phosphorylation/dephosphorylation cascade (37). A similar mechanism may be utilized to regulate CAR translocation during fasting upon receipt of a signal from the cell surface. However, at present, the nature of this signal is unknown. Interestingly, the increases in CAR target genes during fasting are much less robust compared with the dramatic changes induced by the potent agonist TCPOBOP. This fact indicates that the mode of activation of CAR during fasting is more likely to be induced translocation of the receptor rather than activation by a hormonal ligand or metabolite. Interestingly, CAR activation may be related to the status of PGC-1, a fasting-induced transcriptional cofactor that plays a critical role in regulating multiple aspects of energy metabolism (38). Shiraki et al. (39) have recently shown that there is a functional coupling of CAR and PGC-1 in cells. We have also shown that PGC-1 can interact specifically with CAR in mammalian two-hybrid assays.2 It is possible that CAR activation during fasting may be functionally related to induction of PGC-1 expression.
Although our data demonstrate that CAR is required for the full decrease in T3 and T4 levels upon fasting, it is notable that a partial response was still observed in the CAR knockout mice, indicating that other mechanisms contribute to the process. The mechanisms leading to reduced plasma T3 appear to differ between humans and rats. For example, rats display an increased reliance on decreased TSH secretion during caloric restriction to effect decreased production of T3 and T3 (40). Thus, in rodents, TSH may play a role in the observed partial decrease in TH even after short-term caloric restriction. Another potential candidate is the related xenosensor PXR, which has also been shown to regulate Ugt and Sult gene expression (15). However, we only observed small changes during fasting in the expression of Cyp3a11 (data not shown), a target gene primarily regulated by PXR in mice (41). Thus, it is unlikely that PXR is responsible for the partial decrease in T3 levels in the fasted CAR knockout mice.
We observed CAR-dependent increases in Ugt1a1 gene expression during fasting. UGT1A1 has been shown to play a role in TH metabolism (22, 42), and both T4 and T3-glucuronides of TH are excreted in the bile (43). In general, Ugt1a1 was not induced strongly by CAR during fasting. Consistent with this observation, treatment of wild-type mice with the potent CAR agonist TCPOBOP induced hepatic Ugt1a1 expression only 2–3-fold (Fig. 2B) (15). Because we could detect only a relatively small change in a single glucuronidase compared with the larger increases in expression of multiple sulfotransferases, it is likely that the latter provide the major pathway for regulation of TH metabolism during fasting in mice. Because we only assessed a single time point after fasting (24 h), other significant gene expression changes may have occurred that were not captured in our analysis. For example, we only detected a modest (<2-fold) increase in hepatic deiodinase type I after a 24-h fast or after our 7-day TCPOBOP treatment (data not shown), but another group has shown that TCPOBOP induces this gene 4.9-fold after 3 h of treatment (44).
CAR is closely related to the xenobiotic receptor PXR, and it is noteworthy that TH metabolism and xenobiotic metabolism share common features. Multiple phase II enzymes show broad substrate specificity and are able to metabolize a range of xenobiotics and thyroid hormones. Furthermore, the major sites of xenobiotic and TH metabolism include liver, intestine, and brain, the same tissues in which CAR expression is highest. PXR and CAR have been shown to activate largely overlapping sets of genes in comparative global gene expression studies in mice (15). Thus, it is the activation signals, not the target genes, that appear to differentiate the physiological roles of these receptors. In head-to-head comparisons, PXR was shown to be directly activated by a diverse spectrum of xenobiotics, whereas CAR was not (45). Significantly, CAR has also been shown to respond to another metabolic stress, hyperbilirubinemia (46, 47), through an indirect mechanism that does not require ligand binding. The ability of CAR to respond to a wide range of non-ligand-mediated signals may have allowed this receptor to develop a specialized function distinct from a PXR-like progenitor. Hence, whereas the predominant role of PXR is to coordinate a response to xenobiotic stress, the role of CAR may be to respond to metabolic and nutritional stress.
Overall, our studies have shown that CAR is activated during fasting and required for the normal decrease in T3 and T4 levels. Thus, CAR inverse agonists represent potentially useful drugs because their activity may provide a therapeutic approach to uncouple metabolic rate from food intake. The caloric restriction studies using Car–/– animals support the concept that inhibiting CAR activity can have significant effects on weight loss. It is likely that decreases in thyroid hormone are ultimately lowered to equal levels by decreases in TSH, a known response to fasting in rodents (40). Because of species differences in thyroid hormone metabolism, it will also be important to test this hypothesis in a chronic weight loss model in primates or human clinical studies. The only CAR inverse agonist active in a human system that has been reported to date is androstanol, and this compound is not sufficiently orally available or receptor-selective for such studies, emphasizing a need to identify new human CAR inverse agonists.
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FOOTNOTES |
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** To whom correspondence should be addressed: GlaxoSmithKline, 5 Moore Dr., Venture116-1b, Research Triangle Park, NC 27709. Tel.: 919-483-3936; Fax: 919-315-6720; E-mail: John.T.Moore{at}gsk.com.
1 The abbreviations used are: TH, thyroid hormone; T3, triiodothyronine; T4, thyroxine; UGT, UDP-glucuronosyltransferase; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; TSH, thyrotropin; RT-QPCR, real-time quantitative PCR.
2 J. M. Maglich, J. Watson, P. J. McMillen, B. Goodwin, T. M. Willson, and J. T. Moore, unpublished data.
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REFERENCES |
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