The Study On The Effects Of TSHR—knockout On Hepatic Gluconeogenesis In Mice

Background:Subclinical hypothyroidism (SCH) is characterized by an increased level of plasma thyroid-stimulating hormone (TSH) and normal levels of free thyroxin (FT4) and free triiodothyronine (FT3). With increasing evidence from clinical studies, the association between SCH and other diseases, such as hypercholesterolemia and coronary heart disease, is receiving increasing attention. Some studies have also found associations between SCH and insulin resistance and fasting hyperinsulinemia. Amati reported that SCH patients had significantly lower improvements in insulin sensitivity relative to the euthyroid group under similar regular exercise and body weight loss. And some studies observed an improvement of insulin sensitivity when the level of serum TSH was reduced within normal range in SCH patients. Moreover, in the population of nondiabetic elderly men, it was reported that the TSHR-Asp727Glu polymorphism was associated with insulin resistance, suggesting that TSHR (TSH receptor) plays a role in glucose metabolism. Taken together, these clinical studies strongly suggest a possible role for TSH in glucose metabolism.TSH is synthesized and secreted by the pituitary gland and directly regulates thyroid function via binding to TSHR located in the membrane of thyrocytes. Recently, multiple studies have found that extrathyroidic tissues express TSHR. Our previous studies demonstrated that functional TSHR was expressed in hepatocytes, and the hepatic cAMP/PKA/CREB pathway was involved in the TSH-induced up-regulation of cholesterol synthesis. The hepatic cAMP/PKA/CREB pathway is also vital for regulating glucose metabolism in response to fasting. In the fasted state, hepatic glucose production is promoted (including gluconeogenesis and glycogenolysis) to maintain fasting glucose homeostasis via the cAMP/PKA/CREB pathway. Therefore, abnormal regulation of the cAMP/PKA/CREB pathway could lead to variations in hepatic glucose production, which is vital for fasting blood glucose.AMP-activated protein kinase (AMPK) is considered to be a cellular monitor of energy charge that can regulate glucose and lipid metabolism in liver. Once hepatic AMPK is activated, it can phosphorylate downstream substrates, such as glycogen synthase kinase (GSK)3β, to regulate hepatic glucose metabolism, including decreasing hepatic gluconeogenesis and glycogenolysis and enhancing glycogen synthesis. Phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatese (G6P) are the rate-limiting enzymes in hepatic gluconeogenesis. The evidence from Andrade’s study showed that TSH could suppress AMPK activation in the thyroid gland. Based on the above findings, we hypothesized that TSH may be involved in the regulation of hepatic glucose metabolism.Objective:To discuss the possible role of TSHR on the hepatic glucose production, we design the experiments as follows:1. Establish TSHR-knockout mice modle; observe the effects of TSHR deletion on hepatic gluconeogenesis.2. Observe the effects of TSH on PEPCK and G6P mRNA in HepG2cell line after treated with0.2μm TSH; we want to know whether TSH takes part in the modulation of hepatic gluconeogenesis directly or not.Methods:1. Development of TSHR-knockout mice:We got the wild-type mice and TSHR-knockout mice by breeding heterozygote mice and determined the genotype by PCR. And all mice lived in SPF environment of the experimental animal center.2. Detection of metabolic index:at the end of the experiment, body length, body weight and organ index of all mice were measured. And we assayed the levels of serum lipid profiles, total T4, TSH, insulin, glucagon, insulin like growth factor-1(IGF-1) and fasting glucose and then calculated the insulin sensitivity index (ISI).3. Tolerance tests:Glucose tolerance tests (OGTT), insulin tolerance tests (ITT) and pyruvate tolerance tests (PTT) were performed to evaluate the glucose metabolism.4. Effects of TSHR-knockout on hepatic structure and hepatic glycogen:we observed the histological characteristics of liver cells by HE staining; and we also observe the distribution of glycogen in liver using PAS staining.5. Effects of TSHR-knockout on the content of hepatic glycogen:we got the standard curve by detecting the absorbance of standard glucose solution using anthrone colorimetry method; and then we calculated the content of hepatic glycogen in mice.6. Effects of TSHR-knockout on the expression of G6P, PEPCK and glucokinase (GK) mRNA:We detected the expression levels of G6P, PEPCK and GK mRNA to assess the effects of TSHR-knockout on hepatic gluconeogenesis using real-time PCR.7. Effects of TSHR-knockout on the expression of hepatic CREB, AMPK and GSK3β:we detected the expression levels of hepatic CREB, AMPK, GSK3β and its phosphorylated protein levels using Western Blot method.8. Effects of TSH stimulation on the expression of G6P and PEPCK mRNA in HepG2cells:added0.2μm TSH in cultured HepG2cells and then detected the expression levels of G6P and PEPCK mRNA to observe the effects of TSH on hepatic gluconeogenesis in vitro.Results:1. TSHR-knockout mice exhibit decreased fasting blood glucose but normal plasma insulin level.6weeks-old and8weeks-old TSHR-knockout mice fed the supplemented diet exhibited equal levels of serum TSH and TT4compared with the same age of wild-type mice (all p>0.05). The body weight of TSHR-knockout mice was significantly lower than wild-type mice (p<0.01); however, the ratio of organ weight to body weight (including the liver, heart and kidney) in TSHR-knockout mice did not show significant differences compared with wild-type mice (all p>0.05). The results showed that liver function was normal in TSHR-knockout mice relative to wild-type mice based on the findings that liver HE staining did not show obvious changes. Moreover, no significant differences were observed in serum insulin, glucagon and IGF-1concentrations between TSHR-knockout mice and wild-type mice (all p>0.05), but the level of fasting glucose was reduced to69%in TSHR-knockout mice (p<0.01). In line with decreased fasting glucose, TSHR-knockout mice had increased insulin sensitivity index relative to wild-type mice (p<0.01).2. TSHR-knockout improves glucose tolerance and reduces gluconeogenesis.In OGTT, TSHR-knockout mice displayed lower levels of blood glucose throughout the test compared with wild-type mice (all p<0.05). At30min after glucose loading, the blood glucose level in TSHR-knockout mice was62%that of wild-type mice (p<0.05). In ITT, neither time point showed significant difference in blood glucose throughout the test when compared the two types of mice (all p>0.05). In PTT, TSHR-knockout mice showed significantly lower glucose levels than wild-type mice at all time points after the i.p. pyruvate injection (all p<0.01). The AUC results also supported that TSHR deletion improved pyruvate tolerance, as represented by a significant reduction in the AUC of TSHR-knockout mice compared with wild-type mice (approximately25%, p<0.01).3. Increased hepatic glycogen contents in TSHR-knockout mice.The hepatic glycogen assay showed a significant increase (approximately70%) in TSHR-knockout mice compared with wild-type mice (p<0.05). Prunosus particles representing glycogen were observed in the hepatocytic cytoplasm. Consistent with the glycogen content assay result, hepatic glycogen staining also showed a significant augmentation of glycogen storage in TSHR-knockout mice.4. Effects of TSH on gluconeogenic gene expression.Compared to wild-type mice, the expression levels of PEPCK and G6P mRNA were reduced by36%and47%, respectively, in TSHR-knockout mice (all p<0.05). And the expression level of GK mRNA was1.38-fold increase in TSHR-knockout mice (p<0.05). In HepG2cells, the abundance of PEPCK and G6P mRNAs were significantly increased to268%and159%after20-hours exposure to TSH, respectively, suggesting a direct action of TSH on gluconeogenesis gene expression (all p<0.05).5. Effects of TSHR-knockout on proteins involved in regulating hepatic glucose metabolism.The expression of phospho-CREB was diminished in TSHR-knockout mice (p<0.05), while the expression of total CREB was unaltered (p>0.05). The expression of total AMPK showed no significant difference between TSHR-knockout mice and wild-type mice (p>0.05). However, the expression level of phospho-AMPK was significantly increased in the liver of TSHR-knockout mice (p<0.05). Moreover, as one of the downstream target proteins of AMPK, the expression level of phospho-GSK3β was also enhanced in TSHR-knockout mice (p<0.05).Conclusions:1. TSHR-knockout could decrease fasting glucose and inhibit hepatic glucose production, and the possible mechanisms were related to phospho-CREB inhibition and AMPK activation.2. TSH maybe directly increases the expression of PEPCK, G6P mRNA in HepG2cells.