The Study Of Thyroid-stimulating Hormone In Regulating Hepatic Cholesterol Conversion

Background:As the change of people’s life way, the prevalence of hyperlipidemia increased year by year. It was reported that the prevalence of hypercholesterolemia was 9.0%, the prevalence of borderline hige total cholesterol was 22.5% in Chinese adults aged >20 years. Thyroid dysfunction is often associated with the lipid disorder. For example, serum total cholesterol (TC) level is decreased in hyperthyroidism, or conversely increased in hypothyroidism. The change of thyroid hormone levels has been historically and widely considered to be the mechanism for lipid disorder in thyroid disease. For example, serum TC level is decreased in hyperthyroidism (thyroid hormone increases and TSH level decreases), or conversely increased in hypothyroidism (thyroid hormone decreases and TSH level increases).More and more clinical studies have confirmed that the serum TSH levels are associated with serum cholesterol levels. It is wll known that the liver is the most important organ on cholesterol metabolism. Our previous studies have confirmed that functional TSH receptor was expressed on the surface of hepatocytes, and TSH can elevate liver cholesterol content via hepatocytic TSHR by up-regulating the expression of HMG-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis.The conversion of cholesterol to bile acids (BAs) is the predominant pathway for the elimination of cholesterol and plays a central role in the regulation of whole-body cholesterol homeostasis. The above findings have prompted several questions. For example, it is not known why the TSH-induced excess cholesterol is not converted to BAs to maintain normal cholesterol homeostasis; additionally, whether TSH also affects the conversion of cholesterol for the regulation of increased cholesterol remains unknown. Thus far, no studies have evaluated these issues.There are only very little studies on the relationship between thyroid function and bile acids. PAULETZKI reported that the cholic acid (CA) synthesis rate decreased by 34% (5.8 ± 2.8 vs.7.9 ± 4.2, P< 0.02), and CA/CDCA in bile acid lower 52%(0.57± 0.13 vs.1.19 ± 0.32, P< 0.01) in hyperthyroidism patients compared with the control group. While Kosuge demonstrated that serum total bile acid levels increased in hyperthyroidism patients and decreased in hypothyroidism, although the diference has no stastical significance.and this study found that the component of bile acids also changed in patients with abnormal thyroid function, the ratio of DCA+CA/LCA+CDCA increased in hypothyroidism patients (1.45±0.42 vs 0.95±0.48, P<0.01), and decreased in hyperthyroidism patients (0.50±0.19 vs 0.95±0.48, P<0.05). Cholesterol 7a-hydroxylase (CYP7A1) is the rate-limiting enzyme for cholesterol conversion.Tiangang Li reported that the gallbladder bile acid composition changed from predominantly cholic acid (57%) in wild-type to chenodeoxycholic acid (54%) in Cyp7a1-transgenic mice which suggested that the change of bile acid composition in thyroid disease is contributed to the changed of CYP7A1 activity. Sauter reported that the change of bile acid in hyperthyroidism and hypothyroidism can exclude the effect of thyroid, which suggests that TSH may regulate bile acid metabolism via CYP7A1.In this study, using a series of in vivo and in vitro experiments, we focused on cholesterol 7a-hydroxylase (CYP7A1), the rate-limiting enzyme for cholesterol conversion, and found a novel role of TSH in the inhibition of cholesterol conversion through binding to TSH receptors on the surfaces of hepatocytes.Objective:To demonstrate the effect and mechanism of TSH on CYP7A1, the the rate-limiting enzyme for cholesterol conversion in liver, furtherly help us obtain a better understanding on the physio-pathologic effects of TSH outside the thyroid gland, and a better interpretation of the atherosclerosis and cardiovascular events resulting from hypercholesterolemia through hypothyroidism.Methods:1. To examine the dose and time dependent effect of TSH on CYP7A1, HepG2 cells were treated with bovine TSH (bTSH) with different dose for different time.2. To further investigate the role of TSH in the regulation of cholesterol conversion, we performed the model of surgically thyroidectomized rats, in which the effects of TH were absent because of the removal of the thyroid gland. So, the serum TH level can be easily maintained through supplementation with exogenous TH, particularly when attempting to change the TSH levels in vivo through the administration of exogenous TSH without altering the TH levels. We only chose those rats with a complete Tx as demonstrated by the disappearance of thyroid hormone (undetectable T4) for further experimental manipulations with administration of exogenous T4 and TSH.3. To determine whether the responses of TSH on bile acid synthesis occurred via TSHR in liver, we used the mice that the TSHR gene was knockout. Because of the absence of TSHR gene expression, serum T4 was undetectable in TSHR(-/-) mice. To exclude the effect of thyroid hormones, the mice were placed on a hormone-replacement diet supplemented with 100 ppm desiccated thyroid powder. Wild-type [TSHR(+/+)] C57BL/6 mice were used as a control. Next, we used a lentivirus-based RNAi delivery system to infect cultured hepatocytes with short interfering RNA targeting human TSHR (TSHR-RNAi-lentivirus), and CYP7A1 expression changes after detecting TSH stimulation.4. To determine the mechanism underlying the perturbation of BA homeostasis induced by TSH, we analyzed the expression of a panel of genes involved in the regulation of liver CYP7A1 expression in TSHR(-/-) mice.5. In order to determine the regulation of CYP7A1 expression of TSH stimulation is mediated by the inhibiting of DNA binding activity of the HNF-4a with CYP7A1 promoter r, we designed a plasmid encoding the human CYP7A1 reporter containing the HNF-4a binding site (human CYP7Al/luc), which was transfected into HepG2 cells, and the luciferase activity was mearsured after bTSH stimulation.6. In order to investigate whether the cAMP/PKA signalingpathway was involved in the regulation of TSH on CYP7A1 expression, liver cells were pre-incubated with the PKA inhibitor, H89 and the adenylyl cyclase inhibitor SQ22536 before subsequent stimulation with bTSH, and then the cAMP and CYP7A1 levies were tested.7. Our study found that blocking cAMP/PKA pathway cannot completely eliminate the influence of TSH on CYP7A1, so we further explore the role of PI3K/Akt signal pathway. HepG2 cells were pre-incubated with the PI3K inhibitor, LY294002 before subsequent stimulation with bTSH, and then Akt and CYP7A1 levies were tested. And we further explore the mechanism of the regulating of CYP7A1 by TSH via PI3K/Akt pathway.Results:1. TSH represses hepatic CYP7A1 activity leading to decreased total BA level in vivo and in vitro(1) the effect of bTSH on CYP7A1 expressionRelative to the control, CYP7A1 protein levels were reduced by 23% and 52%, respectively, with 1 and 4 uM bTSH treatment, and this effect was accompanied by an increase in HMG-CoA reductase (HMGCR), the rate limiting enzyme of cholesterol synthesis.(2) the effect of bTSH on CYP7A1 mRNARelative to the control, CYP7A1 mRNA expression was reduced by 33%and 61%, respectively (p<0.01)。(3) Male Wistar rats were surgically thyroidectomized (Tx) or sham-operated (Sh). Tx rats were injected with thyroxine (T4), and serum total thyroxine (TT4) levels were not significantly different between the Tx group and the sham group (P>.05). After different doses of bovine TSH (bTSH) were injected into Tx rats, relative to the rats receiving only T4, Tx rats receiving TSH and T4 exhibited significantly higher serum TSH levels. Liver and serum total BA levels decreased dose-dependently in Tx rats following TSH administration, and serum TC levels correspondingly increased. Simultaneously, the expression and activity of liver CYP7A1 protein were also decreased.2. TSH-mediated repression of BA synthesis is dependent on TSHRs in the liver(1) Relative to littermate TSHR(+/+) mice, the levels of serum total BAs, the total BA pool size and levels of intrahepatic total BAs and gallbladder total BAs were increased in TSHR(-/-) mice, with a concurrent increase in the gallbladder volume. All the serum TC, LDL-cholesterol levels and liverTC levels were decreased. CYP7A1 protein levels increased in TSHR(-/-) mice. Similar changes were observed in CYP7A1 mRNA and serum C4 levels, which represent the activity of CYP7A1.(2) The TSH-induced decreases in CYP7A1 expression and total BA levels were markedly reversed after TSHR expression was silenced by lentivirus-based RNAi in hepatocytes.(3) An RNAi system to silence the expression of liver TSHR was successfully achieved in mice. Increased serum total BAs and decreased TC levels in the sera and liver of RNAi mice were observed. Additionally, CYP7A1 protein expression was up-regulated and HMGCR expression was down-regulated. 3. HNF-4α is essential for the TSH-mediated regulation of CYP7A1(1) Compared to their respective Tshr (+/+) littermates, the expression of Fxr, Lrh-1, Pxr and Fgfr4 mRNA in liver and fgf15 mRNA in intestine remained unchanged in Tshr (-/-) mice supplemented with TH(2) HNF-4α is the most abundant orphan nuclear receptor expressed in the liver, which plays an critical role in the regulation of CYP7A. We revealed that both the mRNA and protein levels of HNF-4α were markedly increased in Tshr(-/-) mice liver(3) When a plasmid encoding the human CYP7A1 reporter containing the HNF-4α binding site (human CYP7A1/luc) was transfected into HepG2 cells, bTSH treatment significantly decreased the CYP7A1 reporter activity. However, when the HNF-4α binding site in the CYP7A1 promoter was mutated (mHNF4α-ph1887/luc), bTSH did not affect reporter activity.4. TSH increases the phosphorylation of HNF-4α via cAMP/PKA pathway(1) Our previous study showed that TSH increases cAMP production via TSHR, and cAMP represses CYP7A1 via the PKA-mediated phosphorylation of HNF-4α. The system used in this study showed that TSH caused an evident increase in the phosphorylation of HNF-4α relative to the control; indeed, this phenomenon was disrupted using the PKA inhibitor H89 in HepG2 cells.(2) The phosphorylation of HNF-4α impairs its nuclear localization and DNA binding activity, which represses CYP7A1 expression. The inhibiting effect of TSH on CYP7A1 was partially but not completely reversed using H89 or the adenylate cyclase (AC) inhibitor (SQ22536), suggesting that pathways other than the cAMP/PKA pathway might also be involved in the TSH-mediated repression of CYP7A1.5. TSH reduces the expression of nuclear HNF-4a by increasing the levels of mature SREBP-2(1) bTSH-treated cells exhibited a dose-dependent increase in Akt phosphorylation。(2) The ability of TSH to reduce HNF-4a expression was prevented when PI3K activity was inhibited via treatment with the PI3K inhibitor, LY294002, but not by treatment with the inactive analog, LY303511。(3) bTSH increases both cytoplasmic (premature) and nuclear (mature) SREBP-2 via the PI3K/Akt pathway.(4) When cells were transfected with a plasmid expressing SREBP-2, the increase in SREBP-2 expression led to a decrease in the protein levels of HNF-4a and CYP7A1. Similarly, the human CYP7A1/luc reporter activity was strongly inhibited by SREBP-2, but the mHNF4α-ph1887/luc reporter activity was not affected.(5) When SREBP-2 was silenced,the TSH-induced changes in HNF-4a and CYP7A1 expression were largely blocked.Conclusion:1.We here highlighted the novel effect of TSH on cholesterol conversion through suppression of the CYP7A1 activity in the liver. This effect directly contributed to the elevated cholesterol level and the decreased bile acid contents. The mechanism involved inthe TSH-TSHR binding was mediated by both the cAMP/PKA and the PI3K/Akt signaling pathways and their downstream molecules, SREBP-2 and HNF-4a2. Our data define a previously unidentified direct action and mechanism for TSH in the down-regulation of cholesterol conversion in the liver via TSHR through cAMP/PKA/HNF-4a phosphorylation and PI3K/Akt/SREBP-2/HNF-4a signaling pathways. This is a novel effect of TSH on cholesterol metabolism in liver and might partially explain the pathogenesis of hypercholesterolemia in hypothyroidism. The new information prompts us reconsidering the physiological and pathological effects of TSH on extra-thyroidal tissues.