The Effect Of TSH On HMG-CoA Reductase Phosphorylation In The Liver

Background:Hypercholesterolemia is a dominant risk factor for atherosclerosis and cardiovascular diseases. HMG-CoA reductase (HMGCR) is the rate-limiting enzyme in cholesterol biosynthesis, so its activity is instrumental in controlling de novo cholesterol synthesis. To maintain the cholesterol homeostasis, HMGCR could be regulated by multiple mechanisms such as transcription, translation, enzyme degradation rate, phosphorylation-dephosphorylation, and feedback inhibition. Hormones could regulate the expression of HMGCR by acting at different levels. For example, glucocorticoids act at a post-translational level, whereas insulin reportedly affects both the transcriptional and post-translational processes. Recently, Wu et al found that the changes of the phosphorylated HMGCR played an important role in regulating of the hepatic cholesterol biosynthesis process. The HMGCR is physiologically present in the cell in unphosphorylated active form and phosphorylated inactive form. In general, phosphorylation of HMG-CoA reductase leads to inactivation of the enzyme, while dephosphorylation activates it. The ratio of the phosphorylated form to total form indicates an inactivation state of HMGCR. The AMPK seems to be the major kinase that targets the HMGCR in the liver.Subclinical hypothyroidism (SCH) is a type of thyroid function abnormality, its characteristic is the elevated serum TSH and normal serum thyroid hormone levels, as well as an increased serum cholesterol level. Our study and other clinical studies have addressed a positive correlation between the high serum TSH and increased cholesterol levels. Studies find that: the TSH receptor also expresses in a lot of tissues such as lymphocytes, adipose cells, retroocular fibroblasts, erythrocytes, osteocytes, neurons and astrocytes et al besides the thyroid tissue. And TSH can take part in the function modulation of these tissues through binding with the TSH receptor. Our laboratory data have shown that the functionalTSHR was expressed in hepatocytes and the TSH could enhance the expression of HMGCR by activating the cAMP/PKA pathway, leading to an elevated cholesterol level via the TSHR in the hepatocytes.Objective:To discuss whether TSH decreases the HMGCR phosphorylation via PKA/AMPK signal pathway in the liver.Methods:1. To identify whether the TSH could regulate the phosphorylation of the HMGCR, we chose the primary mouse hepatocytes and HepG2 cells, and treated the cells with different TSH concentrations (0,1,4 μM).2. To assess whether the TSH could affect the activation of AMPK, we stimulated the hepatocytes and HepG2 cells with different bTSH concentrations (0,1,4 μM), then tested the expression of the p-AMPK (T172) and AMPK.3. To test whether AMPK is involved in a TSH-induced inhibition of the hepatic HMGCR phosphorylation. The HepG2 cells were nucleofected with recombinant pcDNA3 plasmids coding for constitutively active AMPK (CA-AMPK) in prior to 4 μM bTSH treatment for 48 hours. The protein levels of p-ACC, phosphorylated HMGCR and total HMGCR were determined respectively by western immunoblotting.4. To investigate whether the TSH affects the AMPK activity associated the PKA. HepG2 cells were pretreated with H89 (10 μM) for 1 hours followed by incubation with bTSH (4 μM) for 48 hours in the presence or absence of H89. The protein levels of phosphorylated AMPK, total AMPK, phosphorylated HMGCR and total HMGCR were determined by western immunoblotting.5. To realize the change of the HMGCR phosphorylation in subclinical hypothyroidism, we set up the subclinical hypothyroidism mouse model. The expression of p-HMGCR, HMGCR, p-AMPK and AMPK was analyzed using western immunoblotting analysis in subclinical hypothyroidism mice and the control mice.6. To test whether the TSH decreases the liver HMGCR phosphorylation by AMPK via the TSH receptor, we set up the Tshr-KO mouse model. The expression of p-HMGCR, HMGCR, p-AMPK, AMPK and p-ACC was analyzed using western immunoblotting analysis in Tshr-KO and wild-type littermate mice.Results:1. TSH decreases the hepatic HMGCR phosphorylation levels.After the stimulation of TSH in the primary mouse hepatocytes, the expression of the phosphorylated HMGCR decreased and the total HMGCR increased in a TSH concentration-dependent manner (p< 0.05). The ratio of the phosphorylated form to total form of HMGCR, demonstrating an inactivation state of the HMGCR, showed a decline after the TSH-stimulating increase, suggesting that TSH could directly decrease the phosphorylated HMGCR, which might lead to an increase in the activity of the hepatocytic HMGCR. Similar results were also found in HepG2 cells,2. Inhibition of HMG-CoA reductase phosphorylation by TSH via decreasedAMPK activity(1) The treatment of the primary mouse hepatocytes with TSH decreased the expression of the phosphorylated AMPK (active form) in a concentration-dependent manner, compared to the control group. The amount of total AMPK was unaltered. Similar results were also found in the HepG2 cells.(2) when the cells were nucleofected with recombinant pcDNA3 plasmids coding for constitutively active AMPK (CA-AMPK), the phosphorylated ACC, the best-characterized activation of AMPK, increased, and the phosphorylated HMGCR also elevated compared with the cells without any stimulation (p< 0.05). When TSH was supplemented, the change was dramatically blocked. The ratio of p-HMGCR/HMGCR decreased by 20.1% in the cells co-treated with CA-AMPK and TSH compared with the cells with CA-AMPK alone (p< 0.05).3. The PKA inhibitor attenuates the inhibiting effect of TSH on AMPK activityThe expression of p-AMPK, AMPK, p-HMGCR and HMGCR were measured (Fig, 3A). The TSH alone decreased the ratio of p-AMPK/AMPK and p-HMGCR/HMGCR relative to the control (p< 0.05), but the treatment with H89 down-regulated this action by TSH, whereas the expression of the phosphorylated AMPK and the phosphorylated HMGCR increased compared with the cells stimulated by TSH lonely (both p< 0.05).4. The HMGCR phosphorylation decreases in subclinical thyroidism mice.(1) After two months, compared with the control group, in the MMI-treated mice, the circulating TSH levels increased (p< 0.05), but the serum FT3 level (p> 0.05) did not change. The cholesterol levels in the SCH mice increased relative to the control mice.(2) The expression of the hepatic HMGCR protein was increased while the p-HMGCR expression decreased in the SCH mice than that in the control group (p< 0.05). The ratio of the phosphorylated HMGCR to the total HMGCR decreased in SCH mice.(3) In the liver of the SCH mice in vivo, western blotting showed that the expression of total AMPK did not significant change (p> 0.05), whereas the expression of the phosphorylated AMPK (activated) was decreased compared with the control mice (p< 0.05).5. TSH decrease the liver HMGCR phosphorylation by AMPK via TSH receptors(1) The serum T4, FT4 and TSH levels showed no differences (all p > 0.05) between the wild-type and the Tshr-KO littermate mice. The body weight of Tshr-KO mice were significantly lower than that of wild-type mice (p< 0.05), however, the ratio of liver weight to body weight in Tshr-KO mice did not show significant differences compared with the wild-type mice (p> 0.05). Compared with the littermate wild-type mice, the serum cholesterol levels in the Tshr-KO mice decreased by 35% (p< 0.05).(2) the HMGCR expression in the livers of the Tshr-KO mice, compared with the wild-type mice, showed a decrease by 42.1%, and the phosphorylated HMGCR increased by 21.9% (both groups,p <0.05). Ratio of the phosphorylated HMGCR and HMGCR increased by 94.3% (p< 0.05). We found consistently that, compared with the wild-type mice, the activity of AMPK was elevated by 3- fold and the expression of p-ACC was increased by 100.5% (both groups, p< 0.05) in the liver of the Tshr-KO mice.Conclusion:In the present study, we demonstrated a significant decrease in the ratios of p-HMGCR/HMGCR and p-AMPK/AMPK,which indicating the unactivated state of HMGCR and the activated state of AMPK respectively, in the primary mouse hepatocytes and liver cell line in a dose-dependent manner following bTSH stimulation for 48 hours. The changes above were inversed when the cells were treated with CA-AMPK plasmid or H89. In the Tshr-KO mice, the ratios of liver p-HMGCR/HMGCR and p-AMPK/AMPK were increased relative to the littermate wild-type mice. Our main finding in this study is that the TSH decreases the phosphorylation of HMGCR via AMPK in the liver.