| Background:Thyroid-stimulating hormone (TSH) is secreted by the pituitary gland and regulates thyroid growth and differentiation. However, recent studies have discovered that TSH is a tropic hormone that has multiple effects on metabolism, including cholesterol synthesis and glucose metabolism. Several epidemiological studies have indicated positive correlations between TSH and total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-c). Subclinical hypothyroidism (SCH), which is characterized by normal thyroid hormone levels and elevated TSH levels, is often accompanied by hypercholesterolemia and associated with cardiovascular disease. Therefore, more studies are focusing on the relationship between TSH and hypercholesterolemia to explore novel approaches for preventing cardiovascular disease.The liver is the major organ involved in cholesterol metabolism. Sterol regulatory element-binding protein-2 (SREBP-2) is synthesized as a precursor protein in the endoplasmic reticulum and subsequently undergoes sequential proteolytic cleavage to attain the N-terminal transcriptionally active form; the active form translocates into the nucleus to promote the expression of target genes involved in cholesterol biosynthesis and uptake in the liver, such as 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS),3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and low-density lipoprotein receptor (LDLR). Liver-specific SREBP-2 transgenic mice exhibit a significant increase in the rate of cholesterol synthesis and elevated expression of HMGCS (13-fold), HMGCR (75-fold) and LDLR (5.8-fold). Our previous research demonstrated that TSH upregulates the expression of HMGCR, a rate-limiting enzyme in liver cholesterol synthesis, via the cAMP/PKA/CREB pathway. Furthermore, Min HK et al. showed that the accumulation of triglycerides and free cholesterol in nonalcoholic fatty liver disease (NAFLD) was accompanied by the inhibition of AMPK activity and increased expression of SREBP-2 and HMGCR. Recently, evidence has suggested that TSH levels are closely associated with NAFLD. Therefore, these findings suggest a possible new role for TSH in hepatic steatosis and dyslipidemia. We found that TSH increases SREBP-2 expression in liver cells, but the precise molecular pathway remains unclear.AMP-activated protein kinase (AMPK) is a crucial cellular energy sensor that is activated by phosphorylation at Thr172 in the alpha subunit in response to various metabolic stressors, and AMPK is a major regulator of glucose and lipid metabolism that phosphorylates and inactivates numerous metabolic enzymes, including glycogen synthase, acetyl-CoA carboxylase (ACC), HMGCR, and CREB-regulated transcription coactivator 2 (CRTC2). Recently, AMPK has attracted more interest as a pharmacological target for therapeutic intervention in metabolic disorders. Chronic activation of AMPK leads to a compensatory increase in SREBP-2 and HMGCR expression in the liver of a new transgenic mouse model with liver-specific expression of constitutively active (CA)-AMPK-1, but in adipose tissue, SREBP-2 and HMGCR mRNA expression was down-regulated. A recent study demonstrated that AMPK interacts with and phosphorylates SREBP-1 and SREBP-2, thereby inhibiting SREBP activation by impeding its proteolytic maturation and nuclear translocationBut it remains unclear whether there is a direct link between TSH, AMPK and SREBP-2.Objectives:We aimed to investigate whether AMPK plays an active role in regulating hepatic cholesterol metabolism by modulating SREBP-2 and whether AMPK affects the TSH-mediated upregulation of SREBP-2 in the liver; these data may provide a basis for the future clinical treatment of hypercholesterolemia associated with TSH.Methods:1. In Vitro, the HepG2 cells were treated with AMPK activator (AICAR) or TSH,AMPK activity was measured using the SAMS peptide phosphorylation assay, SREBP-2 cytoplasmic precursor and nuclear active forms and its target gene, HMGCR and HMGCS, were observed.2. In Vivo, we established Tshr+/+ and Tshr-/- mouse models, Tshr+/+ and Tshr-/-mice were injected intraperitoneally with AICAR, SREBP-2 cytoplasmic precursor and nuclear active forms and its target gene, HMGCR and HMGCS, were observed.3. HepG2 cells were transfected with pc-DNA3.1 encoding 2×flag-tagged human nuclear SREBP-2 and then were treated with AICAR, immunoprecipitation of total protein with SREBP-2 antibodies and immunoblot analysis with p-Thr antibodies were to demonstrated that the SREBP-2 nuclear forms were phosphorylated by AMPK on threonine residues.4. In the HepG2 cells and L02 cells treated with AICAR, and in the livers of Tshr-/- mice and Tshr+/+ mice, immunoprecipitation of total protein with SREBP-2 antibodies and immunoblot analysis with p-Thr antibodies were to demonstrated that the SREBP-2 cytoplasmic precursor were phosphorylated by AMPK on threonine residues.5. We utilized double immunofluorescence staining to test TSH repressed the AMPK-mediated phosphorylation of SREBP-2.6. The main methods:Real-Time PCR, immunoprecipitation, immunoblot analysis immunofluorescence, plasmid transient transfection and mouse models.Results:1. TSH up-regulated SREBP-2 precursors and the nuclear active forms, HMGCR and HMGCS mRNA expression in HepG2 cells.SREBP-2 preferentially regulates genes that are responsible for cholesterol synthesis and uptake in the liver such as HMGCR and HMGCS. The protein levels of the SREBP-2 precursor and the nuclear active form increased in a time-dependent manner in HepG2 cells after treatment with TSH (4μM). TSH treatment up-regulated HMGCS mRNA expression at 48 h and HMGCR mRNA expression at both 24 and 48 h (p<0.05).2. AMPK activation by AICAR directly inhibited SREBP-2 expression in HepG2 cells.SREBP-2 is regulated in multiple ways, including sterol feedback mechanisms and modulation of mRNA expression. SREBP-2 is a direct target of AMPK, which inhibits SREBP-2 transcriptional activity. To explore whether AMPK activation regulates the expression of the SREBP-2 precursor and nuclear active forms, we treated HepG2 cells with AICAR (an AMPK activator) for 12 or 24 h. Phospho-AMPK (p-AMPK) and phospho-ACC (p-ACC), indicators of AMPK activation, were markedly up-regulated in a time-dependent manner. The SREBP-2 precursor levels slightly decreased in response to AICAR at 12 h and were significantly decreased by 24 h. However, the nuclear active form of SREBP-2 exhibited a significant decrease during the AICAR treatment period.To confirm the data above, SREBP-2 mRNA expression was determined by real-time PCR. AICAR-mediated AMPK activation decreased SREBP-2 mRNA expression in a time-dependent manner. The immunofluorescent staining intensity of nuclear SREBP-2 was obviously reduced by AICAR treatment in HepG2 cells.3. AMPK activation reduced SREBP-2 protein levels and target gene expression in the livers of AICAR-treated Tshr-/- and Tshr+/+ mice.It has been previously reported that TSH suppresses AMPK activity in the thyroid gland and the liver. To further investigate the effects of TSH and AMPK on SREBP-2, we established Tshr+/+ and Tshr-/- mouse models. The SREBP-2 precursor and nuclear active forms were decreased in the livers of Tshr-/- mice compared with Tshr+/+ mice, although the exact mechanisms by which this occurs remain unclear. The mRNA expression of HMGCR was also significantly reduced, and HMGCS mRNA expression exhibited a decreasing trend.AMPK activity increased in the livers of PBS-treated Tshr-/- mice compared with Tshr+/+ mice. Furthermore, the SREBP-2 precursor and nuclear active forms were significantly down-regulated in Tshr-/- mice. AICAR treatment enhanced AMPK activity and reduced SREBP-2 protein levels in the livers of both Tshr-/- and Tshr+ mice. AICAR significantly decreased HMGCR mRNA expression.4. AICAR stimulated AMPK activity and suppressed the TSH-induced up-regulation of SREBP-2 in HepG2 cells.To further confirm the effects of TSH on AMPK activity, we performed a SAMS peptide phosphorylation assay in vitro. In HepG2 cells, AMPK activity was markedly decreased by TSH compared with control and increased by AICAR treatment. We next examined whether the TSH-induced up-regulation of SREBP-2 occurred via AMPK activation. The TSH-induced up-regulation of the SREBP-2 precursor and nuclear active forms was ameliorated by AICAR.To determine the functional consequences of the AMPK-mediated suppression of SREBP-2, the gene expression of SREBP-2 target genes was assessed by RT-PCR. Consistent with the suppressed SREBP-2 protein levels, the TSH-mediated up-regulation of HMGCR and HMGCS mRNA expression was attenuated by AICAR in HepG2 cells.5. Threonine phosphorylation of SREBP-2 was observed in AICAR-treated cells with activated AMPK and in mouse liversAMPK, an evolutionally conserved serine/threonine kinase, plays an important role in regulating key metabolic processes by phosphorylating multiple enzymes involved in fatty acid oxidation and cholesterol synthesis, leading to their activation or inhibition. A recent study demonstrated that SREBP-2 is a direct target of AMPK in HEK293T cells and in the LDLR’1’mouse liver. To further investigate whether AMPK is involved in the phosphorylation of SREBP-2, we detected phosphorylation at threonine (p-Thr) and serine (p-Ser) residues with specific antibodies. Immunoprecipitation of total protein with SREBP-2 antibodies and immunoblot analysis with p-Ser or p-Thr antibodies demonstrated that the SREBP-2 precursor and nuclear forms were phosphorylated on threonine residues, not on serine residues, in HepG2 cells. AMPK activity was higher in the livers of Tshr-/- mice than in Tshr+/+ mice, and consistent with the above observations, the level of threonine phosphorylation of the endogenous SREBP-2 precursor was higher in Tshr-/- mice than in Tshr+/+ mice. Similar results are obtained using liver L02 cells.6. AMPK could phosphorylate SREBP-2, but TSH repressed the AMPK-mediated phosphorylation of SREBP-2.Conclusions:1. AMPK inhibite the up-regulation of SREBP-2 and its target genes HMGCR and HMGCS mediated by TSH in liver.2. SREBP-2 precursor and nuclear forms can be directly phosphorylated by AMPK on threonine residues,3. TSH interacts with AMPK to influence the phosphorylation of SREBP-2 and regulate its activity. |
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