| Background:Thyrotrophin (TSH) is synthesized and secreted by the pituitary gland and develops ability by acting on TSH receptor (TSHR). Several studies have demonstrated that TSHR expression is not confined to thyrocytes but can be detected in a number of non-thyroidal cells, such as brain, lymphocytes, adipocytes, osteocytes and fibroblasts. And it expression and activity in these non-thyroid tissues may have physiopathological relevance. We have previously demonstrated the expression of functional TSHR protein in hepatocytes. Therefore, the role of TSH binding to TSHR need to be deeply explored in liver, In recent years, more and more evidences have demonstrated that TSH is positively associated with high triglyceride levels; and the studies have revealed a positive correlation between serum triglyceride levels and TSH, independent of thyroid hormone, even when the TSH levels are within normal range, but there is no clear mechanism directly linking the two.Triglycerides are mainly synthesized in liver, which are regulated by sterol regulatory element binding protein-lc (SREBP-lc), which is one of SREBPs isoforms (the membrane-bound transcription factors) belong to the basic helix-loop-helix leucine zipper (bHLH-Zip) family. SREBPs could be modified by phosphorylation to regulate their activity. Moreover, AMP-activated protein kinase (AMPK)-induced SREBP-1c phosphorylation at Ser372suppressed the accumulation of nuclear SREBP-1c. Therefore, AMPK-regulated SREBP-1c phosphorylation is vital for hepatic triglyceride synthesis.AMPK is known as a cellular energy sensor. ACC is its downstream substrate and the key enzyme of triglyceride synthesis, and ACC plays an important role in triglyceride metabolism. AMPK has many phosphorylation sites and its a subunit Thr-172is the major and necessary AMPK activating site. AMPKa at Ser-173was phosphorylated to impede Thr-172phosphorylation and thus affect the role of AMPK activation in adipocytes. But there is no report whether TSH affects AMPK Ser173and how TSH activates AMPK to act on lipid synthesis.We have previously demonstrated that TSH, by binding to TSHR on hepatocytes, plays an important role in cholesterol synthesis by cAMP/PKA/CREB in the liver. In this project, we intended to explore whether TSH, via binding to TSHR, regulated hepatic triglyceride synthesis through AMPKa-induced SREBP-lc phosphorylation. It would provide new ideas and therapeutic targets for the clinical treatment of hypertriglyceridemia, obesity, and metabolic disorders.Objectives:The clinical investigation has showed that elevation of serum triglyceride (TG) has also been observed in patients with subclinical hypothyroidism (SCH), but it was not clear in the mechanism linking the two. Our epidemiological surveys have found that serum TSH level was positively associated with serum TG levels in patients with SCH, and the previous studies have confirmed the functional TSH receptors were present in hepatocytes. As there is no report about the regulation of TSH on liver TG metabolism abroad, we will explore the novel hypothesis that TSH plays an important role in hepatic TG synthesis based on TSH as the research object, in order to provide the new perspective and theories for TSH-regulated lipid metabolism in non-thyroidal cells.Methods:1. TSH receptor knockout (Tshr-/-) and wild-type (Tshr+/+) mice with or without intraperitoneal injection of AMPK activator (AICAR), and C57mice with or without intraperitoneal injection of PKA inhibitor (H89) were built to assay liver triglyceride content, the phosphorylation or nuclear protein expression levels of hepatic lipid-related key molecule SREBP-lc, or the transcriptional levels of its downstream molecules, such as ACC1, fatty acid synthase (FASN), and stearoyl CoA desaturase1(SCD1) in liver.2. In the HepG2cells stimulated with or without TSH, the triglyceride content, the phosphorylation or nuclear protein expression levels of lipid-related key molecule SREBP-1c, and the transcriptional levels of its downstream molecules, such as ACC1, FASN, and SCD1in hepatocytes were determained. With the treatment of the flag-tagged wild-type SREBP-lc and the S372A mutant plasmids to hepatocytes, our study showed the transcriptional activity of downstream lipid-related molecules including FASN and SCD1.3. With AICAR, a constitutively active AMPK (CA-AMPK) plasmid or or PKA inhibitor (H89) used to pretreat HepG2cells, TSH-induced expression levels of SREBP-1c and its downstream molecules related to TG metabolism, the secondary TG content were tested.4. The main methods:triglyceride content assay, Real-time PCR, immunoprecipitation, immunofluorescence, plasmid transfection and mice models.Results:1. The hepatic triglyceride content was decreased by approximately25%in the Tshr-/-mice relative to their littermate Tshr+/+mice (P<0.05). The transcriptional levels of regulating triglyceride synthesis key protein such as SREBP-1c, ACC1, FASN, and SCD1were significantly decreased approximately20-80%in the Tshr-/-mice (P<0.05). Compared with Tshr+/+mice, nuclear SREBP-lc expression decreased, while SREBP-lc Ser372, ACC Ser79and AMPK Thr172not Serl73phosphorylation increased in Tshr-/-mice. The transcriptional level of AMPK showed no significant change. The phosphorylation of SREBP-lc, the mRNA levels and TG content appears corresponding changes in Tshr+/+and Tshr-/-mice received intraperitoneal injections of AICAR for2weeks. Nuclear SREBP-lc expression was decreased in C57mice received intraperitoneal injections of H89. It showed the effects of TSH, rely on TSHR, on TG synthesis in liver in vivo.2. When TSH stimulation, the amount of triglyceride increased approximately30%(P<0.05). Immunofluorescent staining showed that very strong positive signaling for SREBP-1located in the nucleus of hepatocyte by TSH. Western blotting showed the expression of SREBP-1c phosphorylation was decreased in HepG2cells exposed to TSH. A almost2-fold increase in transcriptional activity of SREBP-lc targeting downstream enzymes including ACC1, FASN and SCD1were detected(P <0.05). Compared to wild-type SREBP-lc, the S372A mutant strongly promoted the transcriptional activity of downstream lipid-related molecules including FASN and SCD1with treatment to hepatocytes.It showed the S372A mutant play an important role in enhancing basal transcription of lipid-related genes in HepG2cells. These results showed TSH affected SREBP-1c expression to promote hepatic TG synthesis.3. In HepG2cells, AMPK Ser173phosphorylation level was unchanged while AMPK Thr172phosphorylation and activity were reduced by TSH stimulation. When AICAR or CA-AMPK was employed to stimulate AMPK activity, the variances of TSH-induced SREBP-lc phosphorylation and TG content were partly reversed. When H89was employed to inhibit PKA activity, the decrease of TSH-induced AMPK phosphorylation was partly reversed. Therefore, it showed that AMPK played an indispensable role in TSH-regulated SREBP-lc phosphorylation and TG content.Conclusions:1. TSH increase hepatic TG accumulation.1. SREBP-lc phosphorylation plays an important role in TSH-regulated hepatic TG accumulation2. AMPK is involved in the regulation of hepatic SREBP-lc phosphorylation and TG content by TSH. |
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