Thyrotropin Increases Hepatic Triglyceride Content Through Upregulation Of SREBP-1c Activity

Background:Non-alcoholic fatty liver disease (NAFLD) is the non-alcohol-induced accumulation of extra fat in liver cells, resulting in diseases ranging from benign steatosis to advanced cirrhosis and cancer. Importantly, people with NAFLD have an increased risk of developing cardiovascular problems, such as heart attack and stroke. The prevalence of NAFLD ranges from9to36.9%of the population in different regions of the world. Approximately20%of the United States population suffers from NAFLD, and the prevalence of this condition is increasing. The exact cause of NAFLD remains unknown. However, endocrine disorders such as obesity, insulin resistance, and hypothyroidism likely play a strong role in the pathology of this disease.Subclinical hypothyroidism (SCH), characterized by an elevated thyrotrophin (TSH) level and a normal free thyroxine (T4) level, has recently been demonstrated as a risk factor for NAFLD. The prevalence of NAFLD gradually increased with increasing TSH levels. Moreover, a prospective case-control study showed that SCH is an independent factor that predicts the development of NAFLD, with a hazard ratio (95%CI) of2.21(1.42-3.44) after adjustment for the indicators of the metabolic syndrome. Moreover, clinical studies indicated a positive association between TSH and serum TG level. As TSH is the only thyroid function component affected in SCH, we endeavor to investigate whether TSH induces the accumulation of extra fat in liver cells in the development of NAFLD and determine the underlying molecular mechanism. Notably, no related studies have been reported to date.Accumulation of triglycerides within hepatocytes is the hallmark of NAFLD and the liver is a central organ in the regulation of triglyceride metabolism. Moreover, sterol regulatory element binding protein lc (SREBP-lc) is a key lipogenic transcription factor, which directly activates the expression of more than30genes, dedicated to the synthesis and uptake of fatty acids and triglyceride. Increased SREBP-lc levels were found in patients with histologically diagnosed NAFLD. SREBP-lc was regulated at multiple levels, such as the proteolytic cleavage of SREBP-lc precursors and post-translational modification of mature SREBP-lc. Studies have reported that SREBP-lc expression were decreased in peroxisome proliferator-activated receptor a (PPARa)-null mice compared with the wild type, suggesting the PPARa-dependent induction of hepatic fatty acid synthesis and SREBP-1c activation. Moreover, it is known that PPARa agonists enhance the activity of the Srebp-1c promoter through direct binding with the DR1motif.We hypothesized that TSH might play a novel role in the regulation of triglyceride content in liver, which, at least partially, involves the development of NAFLD. Objectives:We have previously demonstrated that functional TSHR exists in liver cells. The main purpose of this study was to demonstrate if TSH increases hepatic triglyceride content through TSHR, in which SREBP-lc activation induced by cAMP/PKA/PPARa signaling pathway played an indispensible role, suggesting an essential role for TSH in the pathogenesis of disorders associated with lipid metabolism, particularly NAFLD.Methods:1. Cell culture1) HepG2cells were routinely maintained in MEM/EBSS and RPMI1640, respectively, supplemented with10%fetal bovine serum2) Hepatocytes were isolated from C57BL/6J male mice using the two-step collagenase perfusion protocol. The isolated mouse hepatocytes were then cultured at80%-90%confluence in DMEM media containing10%FBS in rat-tail collagen type I coated plates.2. Animal model1) Tshr-/-mice (stain name:B6;129S1-Tshrtm1Rmar/J) were purchased from Jackson laboratory. When grown to8weeks, male mice were subjected to high-fat diet, forskolin or H89treatment2) Fenofibrate treated mouse model:eight week-old C57BL/6J male mice were administered fenofirate through daily gavage.3. Oil Red O Staining, H&E Staining and electron microscopy analysis for detecting lipid droplet accumulation in liver cells.4. Intracellular triglyceride content was measured using a colorimetric assay.5. RT-PCR was adopted to determine the expression of PPARa, SREBP-1c mRNA and the mRNA expression of genes involved in triglyceride metabolism.6. Protein extraction and western blotting analysis was adopted to determine the expression of PPARa, p-SREBP-1and n-SREBP-1rotein.7. Immunohistochemistry and Immunofluorescence were adopted to determine the expression and intracellular distribution of PPARa and SREBP-1protein.8. Silencing of genes using siRNA:small interfering RNAs (siRNAs) targeting the human SREBP-1and PPARa gene was designed and transfected into cells. Expression of genes involved in triglyceride metabolism and intracellular TG content were detected.9. Plasmid construction and transfection:pSV-SPORT plasmids encoding a dominant negative mutant of rat SREBP-lc (DN-SREBP-lc) was transfected into cells. mRNA expression of related genes and intracellular TG content were detected.10. Dual luciferase activity assays:a DNA fragment corresponding to^the SREBP-lc promoter was fused to a luciferase gene and the resultant luciferase reporter construct was transfected into cells. Dual luciferase activity assays were adopted to detect SREBP-lc promoter activity upon PPARa or TSH treatment.11. Co-IP assays were performed to measure the serine phosphorylation of PPARa in response to PKA stimulation.12. Blood samples from the animals were obtained for analyses of blood lipid and liver function using Olympus AU5400autoanalyzer in Clinical Laboratory of Provincial Hospital Affiliated to Shandong University.13. Blood samples from the animals were obtained for analyses of serum levels of TT4, TT3as well as TSH were detected using radioimmunoassay according to the manufacturer’s protocol.14. For statistical analysis, values were presented as the mean±standard deviation, median (interquartile range) or number (percentage). Statistical differences were determined by unpaired Student’s t-test or one-way analysis of variance (ANOVA).Results:1. TSH promotes triglyceride accumulation in liverTSH caused a concentration-and time-dependent effect on intracellular triglyceride contents in HepG2cells. After fed with a high-fat diet for20weeks, Tshr+/+mice exhibited a uniformly pale yellow fatty liver, while Tshr-/-mice had a relatively normal liver. Importantly, the hepatic triglyceride content was decreased in the Tshr-/-mice. Correspondingly, the Tshr-/-mice showed reduced fat accumulation in the hepatic intracellular vacuoles. Tshr-/-mice fed with a chow diet also showed less lipid droplet accumulation and a reduction in the triglyceride content compaired with Tshr+/+mice.2. The expression profile of molecules related to triglyceride metabolism in Tshr-/-miceThe expression of the key genes associated with lipogenesis and fatty acid oxidation as well as the genes responsible for the rate-limiting step of fatty acid uptake and export were almost entirely reduced in the Tshr-/-mouse liver, especially for the genes related to de novo lipogenesis. The mRNA expression of SREBP-lc, a key regulatory molecule involved in lipogenesis, was significantly decreased.3. Indispensible role of SREBP-lc in TSH-induced lipid accumulationA prominent decrease in the precursor and mature forms of SREBP-1proteins was observed in both high-fat diet and chow fed Tshr-/-mouse livers. Immunohistochemical staining of HepG2cells showed that TSH stimulated a stronger fluorescence signal, showing SREBP-1protein expression in the cells, particularly in the nuclei. Transfecting cells with SREBP-1siRNA resulted in a disappearance of the TSH-mediated increase in intracellular triglyceride. Similarly, when the plasmid encoding DN-SREBP-lc was used, the TSH-mediated increase in the hepatic triglyceride content was completely prevented.4. PPARa activation triggered SREBP-lc gene expression and liver triglyceride homeostasis are disrupted through pharmacological treatment with fenofib ratesThe triglyceride content and the expression of SREBP-1of mice liver increased dose-dependently after fenofibrate gavage. The physiological and pharmacological effects of PPARa activation through fenofribrate on the induction of SREBP-lc expression and the elevated intracellular triglyceride content were achieved in HepG2cells incubated with different doses of fenofibrate. When PPARα-siRNA was used, the expression of SREBP1declined.After fenofibrate treatment, the luciferase activity of the plasmid (SREBP-lc-1564/+1-luc) containing the PPARa binding site (DR1) increased in a dose-dependent manner.5. PPARa mediates the regulation of SREBP-lc by TSH and PKA mediates phosphorylation of PPARaPPARa protein expression was remarkably decreased in both chow and high-fat diet Tshr-/-mouse livers. In either the mice primary hepatocytes of Tshr+/+or HepG2cells, TSH induced the expression of PPARa. The expression levels of SREBP1c and PPARa and their downstream genes were induced after forskolin treatment in the livers of Tshr-/-and Tshr+/+mice. When mice were treated with H89, a selective and potent inhibitor of PKA, the expression of SREBP1in the liver exhibited an opposite trend. Pretreatment with MK886, a specific PPARa inhibitor, reversed the effects of TSH on the activation of PPARa, SREBP-1. Forskolin increased serine phosphorylation of PPARa.Conclusions:TSH increased hepatic triglyceride content via TSHR/cAMP/PKA/PPARa/SREBP-lc signaling pathway, in which PKA, PPARa and SREBP-lc all played an indispensible role. These findings highlight a novel physio-pathological role for TSH in the regulation of triglyceride metabolism in the liver and suggest that TSH or hepatic TSHR might have key therapeutic importance in preventing fatty liver disease.