| Background:Reduced production and secretion, or insufficient physiological effect of thyroid hormones, is the central feature of the clinical state termed hypothyroidism. Overt hypothyroidism is characterized by lower levels of free thyroxine (FT4) and free triiodothyronine (FT3), while subclinical hypothyroidism is defined as serum FT4 and FT3 levels within their respective reference ranges in the presence of mildly elevated serum thyroid stimulating hormone (TSH) concentrations.In recent years, hypothyroidism has reached epidemic proportions worldwide. Some researches indicated that hypothyroidism, closely associated with various risk factors of cardiovascular diseases, was the independent risk factor for the development and progression of coronary heart diseases. Hypothyroidism has also been proved to be an indispensable reason for secondary hypertension and dyslipidemia. It is essential to investigate the uncertain cause of hypothyroidism. Previous studies have pointed out that autoimmune thyroid disorders, abnormal iodine intake and inflammation may result in hypothyroidism. However, these reasons are still insufficient to explain its high incidence.Dietary fat is an essential component of the human diet and serves many important functions. It is necessary to provide essential fatty acids (EFA), facilitate absorption of fat-soluble vitamins (A, D, E and K), prevent heat loss, and protect vital organs from shock during normal activities. However, excess dietary fat intake promotes ectopic accumulation of triglycerides and free fatty acids in non-adipose depots, known as lipotoxicity, which contributes to chronic cellular dysfunction and injury. In recent years, lipotoxicity has been well documented in the pathogenesis of many metabolic-related diseases, such as diabetes, non-alcoholic fatty liver disease and metabolic syndrome.The thyroid gland, a crucial endocrine organ that synthesizes and secretes thyroid hormones, plays an important role in organic metabolism and in the development, differentiation and maintenance of the central nervous system, skeletal system, and cardiovascular system. Thus, maintaining thyroid homeostasis is essential for human health. The production and secretion of thyroid hormones by the thyroid are directly regulated by the hypothalamus-pituitary-thyroid axis. The pituitary gland serves as a sophisticated biosensor of thyroid hormone levels and regulates TSH levels according to the feedback of FT4 and FT3. A decrease in circulating thyroid hormone stimulates more secretion of TSH by the pituitary. TSH then acts via specific receptors on the membrane of thyroid follicular cells to facilitate compensatory stimulation of thyroid hormone production and secretion, which involves a number of thyroid hormone synthesis-related molecules, such as sodium/iodide symporter (NIS), thyroglobulin (Tg), and thyroperoxidase(TPO).Limited studies have demonstrated altered thyroid hormone levels in animals fed a high-fat diet. A mixed high-fat diet resulted in elevated serum TSH with normal serum T3 and T4 levels in a study conducted by Araujo RL, whereas timedependent decreases in serum TT3 and TT4 were observed according to Han et al. The different high-fat compositions and different duration of feeding in the two studies may underlie the inconsistent results.Rats were fed a high-fat lard diet in this study, we focused on not only serologic but also morphological evidence to address the harm of dietary fat lard overload on thyroid in an animal model. Our new view might be helpful in providing ways to protect against hypothyroidism.Objectives:The main purpose of the study was to observe the effects of lipotoxicity on thyroid, from the aspects of not only serologic, morphological and molecular changes in vivo, but also the molecular expression and function of Tg, NIS and TPO in vitro, aid in the development of novel methods to protect against hypothyroidism and perfect the theory of lipotoxicity.Methods:1. Animal feeding:Forty male SD rats (6-week-old, weighting 170-190 g) were acclimatized to the housing condition for 1 week, then randomly assigned to one of the two diets (n=20 each group) for 24 weeks:(1) normal control diet:100% standard rodent chow (NC group,3.49 kcal/g); (2) high-fat lard diet:85% standard rodent chow supplemented with 15% lard (HF group,4.14 kcal/g). Fasting blood samples were collected by subclavian venous puncture at the 12th and 18th weeks, respectively. All rats were sacrificed for fasting blood and thyroid at the 24th week.2. Primary human thyrocytes culture:human thyroid tissues were cut into pieces and digested in a mixture of type I collagenase and trypsin for 40-60 min at 37 ℃, then cultured in DMEM/F12 with newborn fetal serum and TSH.3. Serum lipid parameters analysis:Serum levels of triglycerides, total cholesterol (TC) and LDL-cholesterol (LDL-C) were measured using automated spectrophotometry.4. Serum TT4, FT4 and TSH analysis:Serum TT4, FT4 and TSH were measured by ELISA kits.5. Thyroid morphological evaluation:High-frequency ultrasound scanning, light microscopic analysis and transmission electron microscopic observation of rat thyroid were conducted. The thyroid volume was also calculated under ultrasound scanning.6. Thyroid lipid content assay:Thyroid triglycerides and free fatty acids were extracted and assayed by enzymatic methods.7. The mRNA level of thyroid hormone synthesis-related molecules assay:Real time-PCR was applied to determine the mRNA expression of NIS, Tg, TPO, Deiodinase 1, TSHR, Pendrin and DUOX2 in rat thyroid, and the mRNA expression of NIS, Tg, TPO in primary human thyrocytes.8. The protein level of thyroid hormone synthesis-related molecules assay:Western blotting, immunohistochemistry and immunoflurescence were used to determine the protein expression of NIS, Tg, TPO in thyroid.9. Function of Tg, NIS, TPO assay:Iodide uptake, secretory Tg content and TPO activity assay were adopted.10. For statistical analysis, values were presented as the mean ± standard deviation. Statistical differences were determined by unpaired Student’s t-test, one way analysis of variance(ANOVA) or Pearson correlation analysis.Results:1. A high-fat lard diet increased the body weight and serum lipid profiles in rats The two groups of rats had similar body weights at the baseline. However, the rats in the high-fat lard diet (HF) group gained significantly more weight than those in the normal control diet (NC) group from the 18th week to the end of the study (P<0.05).At the 24th week, the rats in the HF group exhibited a significant increase of approximately 79% in the serum triglyceride levels compared with those in the NC group (P =0.002). There was no significant difference in either TC or LDL-C between the two groups.2. A high-fat lard diet altered rat serum levels of thyroid hormones and TSH Rats in the HF group exhibited an obvious decrease in the serum TT4 and FT4 levels from exposure to a high-fat lard diet for 18 weeks when compared with the NC group, while serum TSH concentrations were considerably higher at the corresponding time points.3. A negative correlation between serum levels of triglycerides and TT4 in ratsA Pearson’s correlation analysis indicates that serum TT4 levels were negatively associated with serum triglyceride concentrations (r=-0.35; P=0.027). This correlation showed the concentration-dependent effects of serum triglyceride levels on thyroid function.4. A high-fat lard diet increased rat thyroid triglyceride contentThyroid triglyceride content of the rats in the HF group was approximately 2.4-fold (P =0.032) higher than that of the NC group, suggesting that a high-fat lard diet could increase the triglyceride content in the thyroid gland, not just in the serum.5. A high-fat lard diet altered thyroid morphological ultrasonic features in ratsThe thyroid glands in the NC group showed a homogeneous appearance, while those in the HF group presented an enlarged thyroid with lower echotexture and relatively heterogeneous features. Additionally, the thyroid volumes of either each or the total lobe measured by the scanning exhibited an obvious enlargement, with an increase of approximately 50% over that in the NC group.6. A high-fat lard diet disturbed rat thyroid histomorphology and ultrastructureThyroid tissue from the HF group was characterized by different degrees of focal colloid goiter, in which follicular epithelial cells became flattened and the follicles distended with colloid. Electron micrographs showed that in the follicular epithelial cells of the HF group, fewer microvilli and wider perinuclear gaps were observed. There were rare secretory vesicles at the apical pole, while dilated ER cisternae were present in the basal pole. Nuclear twist and chromatin condensation were also noted.7. A high-fat lard diet decreased the expression of thyroid hormone synthesis-related proteinsReal time-PCR revealed the down-regulated mRNA expression of NIS. Immunohistochemistry confirmed the same variation trend of the protein expression of NIS in rat thyroid.8. Palmitic acid treating increases intracellular triglyceride and free fatty acid contents in human primary thyrocytes.Intracellular accumulations of triglyceride and free fatty acid in cells increased in a dose- and time-dependent manner when exposed to palmitic acid treatment.9. Palmitic acid decreases the expression and activity of thyroglobulin (Tg), sodium iodide symporter (NIS) and thyroperoxidase (TPO) in human primary thyrocytes.Real time-PCR revealed the down-regulated mRNA expression of Tg, NIS and TPO in cells exposed to palmitic acid treatment, while western blotting and immunoflurescence confirmed the same variation trend of the protein expression of Tg, NIS and TPO. Functional assay revealed the downregulation of secretory Tg content, iodide uptake and TPO activity.Conclusions:1. Long term high-fat lard feeding could increase the triglyceride content in the thyroid gland, not just in the serum of rats. Palmitic acid overloading could trigger intracellular accumulation of triglycerides and free fatty acids as well.2. Long term high-fat lard feeding could dramatically decrease the levels of TT4 and FT4 and increase the concentration of TSH in the serum, affect the structure of the thyroid gland, and down-regulate the protein expression of thyroid hormone synthesis-related molecule NIS in rats. Palmitic acid overloading could downregulate the expression and function of Tg, NIS and TPO. |
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