High Fat Diet Induces Hypothyroidism Through Endoplasmic Reticulum Stress,with The Disturbance Of Thyroid Lipid Profile

Background:Hypothyroidism is a common and potentially serious disease of deficient secretion of thyroid hormones by the thyroid gland,characterized with increased serum thyrotropin(TSH)level and decreased serum thyroxine(T4)level.The commonest cause of hypothyroidism is the dysfunction of thyroid gland itself,known as primary hypothyroidism,often due to auto-immune thyroid disorders,thyroid irradiation or surgery,environmental factors and anti-thyroidal drugs.The onset of hypothyroidism is insidious,with no specific symptoms,while the deficiency of thyroxine could influence almost every system of the body.Severe untreated hypothyroidism can lead to heart failure,psychosis,and coma.Thyroid gland is one of the critical endocrine organs,functioning in synthesizing and secreting thyroid hormone.The functional units are thyroid follicles,composed of the follicle lumen and its thyroid epithelial cells.Normal biosynthesis of thyroid hormone relies on several major components:thyroglobulin(Tg),thyroperoxidase(TPO),and iodide.The precursor protein of thyroid hormone,Tg,is produced by thyrocytes and is the major secretory glycoprotein of thyrocytes.Normal thyrocytes synthesize and secret large amounts of Tg to meet the need for hormone production.Deficiency of Tg has been proved to be pathogenic in many Tg mutant cases,resulting in congenital goiter or hypothyroidism.TPO catalyzes the iodination of Tg and the coupling of tyrosyl residues to produce T4 and triiodothyronine(T3).The sodium iodide symporter(NIS)mediates the iodide uptake into thyroid follicular cells,these three molecules are critical to the maintenance of thyroid function.The thyroid epithelial cells are abundant of endoplasmic reticulum(ER).Early studies revealed that hypothyroidism was one of the ER storage diseases,suggesting the relationship between ER function and hypothyroidism.Endoplasmic reticulum provides as the principle site for the folding and maturation of membrane and secretory protein.The maintenance of the ER homeostasis is critical to the normal function of ER.Any physiological or pathological process that disturbs the ER homeostasis would result in the misfolding and aggregation of protein,which triggers a serious of responses to attenuate the ER load,termed the unfolded protein response(UPR).The process involves three ER transmembrane sensors:PKR-like ER kinase(PERK),activating transcription factor 6(ATF6),and Inositol-requiring enzyme 1(IRE1).The UPR could inhibit global protein synthesis,induce the transcription of chaperones assisting in protein folding and trigger ER-associated protein degradation(ERAD)of misfolded proteins.Severe and persistent UPR leads to cell apoptosis.Lipids are small hydrophobic molecules that carry out a multitude of crucial roles.For example,they constitute the biological membranes,store energy,potentially regulate gene expression and particulate in cell signalling pathways.Lipid metabolism disturbance is a key component in developing metabolic syndrome,characterized by dyslipidemia,insulin resistance,abdominal obesity and hypertension.The prevalence of metabolic syndrome is strongly associated with the severity of obesity;its physiopathology is related to both genetics and food intake habits,especially the consumption of a highcaloric,high-fat and high-carbohydrate diet.Excess intake of dietary fat has been shown to lead to dysfunction and disease in many organs,for instance,adipose tissue metabolism,hepatic fat storage,pancreatic islets function and many more other organs to be studied.Recent studies found that excess dietary fat intake and obesity related lipid metabolism dysfunction might make a possible contribution to the pathogenesis of hypothyroidism,revealing the intriguing regulating role of lipids in the maintenance of thyroid function.Dietary high-fat lard intake in rats induced decreased serum T4 concentration in parallel with elevated serum TSH concentration,as well as abnormal morphology change of thyroid gland.Diet-induced obese,ob/ob,and db/db mice displayed a variable degree of primary thyroid hypofunction,expanded intrathyroidal adipose depot and steatosis in thyroid follicular cells.However,the precise mechanism that lipid metabolism disturbance effect the thyroid function and whether it could directly interfere in thyroid lipid profile remains to be studied.In this study,we demonstrated the impaired thyroid function in high fat diet SD rats,with significant decreased Tg level.We also observed the high fat diet induced ER stress in thyrocytes,activating UPR signaling pathways.Meanwhile,we investigated the thyroid lipid profile in rats fed with HFD,measured by Ultra-high performance liquid chromatography coupled with mass spectrometry(UPLC-MS).We found that excess intake of dietary fat induced disturbance of thyroid lipid profile,which may provide the evidence of the correlation of lipid profile and organ function and also a new prospect in understanding the pathogenesis of hypothyroidism.Objective:1.In vivo,rats were fed either with control diet(CD)or HFD diet.The serum thyroid hormones and key molecules for thyroid hormone synthesis were observed.The effect of HFD on endoplasmic reticulum stress was studied in thyroid tissue and in palmitic acid treated human primary thyroid cells.The Tg protein synthesis and degradation were also studied to clarify the effects of lipid metabolism disturbance on the thyroid,and explore the possible mechanism of pathogenesis.2.UPLC-MS/MS was used to study the change of lipid profile in thyroid tissue between the rats fed with HFD and CD,to identify the key lipid molecules causing hypothyroidism,and to provide a new prospect for hypothyroidism.MethodsPart 1 High fat diet induces hypothyroidism through endoplasmic reticulum stress1.Animals1.1 Male SD rats were fed with eihter CD or HFD.Blood was collected by subclavian vein at the 8th weeks and 18th weeks respectively.Serum thyroid hormones were anlyzed.1.2 Male SD rats were randomly divided into 4 groups:CD + PBS group,CD +4-PBA group,HFD + PBS group,HFD + 4-PBA group.Rats were fed with either CD or HFD for 18 weeks.From the 9th week,4-PBA or PBS was given daily at a dose of 100 mg/kg/d i.p.till the end of the experiment.1.3 SD rats rats were randomly divided into 3 groups:CD group,HFD group and HC group.Rats of HC group were fed with HFD for the first 8 weeks of the experiment.From the 9th week,HC rats were fed with CD till the end of the experiment(18 weeks).2.Thyroid function was evaluated by 24-hour radioactive iodine uptake test at the 18th weeks.1?Ci[1311]was injected intraperitoneally.After 24 hours,rats were sacrificed and thyroid tissue radioactivity was measured.The percentage of iodine uptaken per unit weight of thyroid tissue were expressed to evaluate the thyroid function.3.Thyroid morphological observation:the thyroid general pathological changes and ultrastructure morphology were observed by H&E staining and transmission electron microscopy.4.Assessing the mRNA levels of the key protein for thyroid hormone synthesis:detection of Tg,Nis,Tpo,Ttfl,Pax8,Hex mRNA levels by real time-PCR.5.Assessing the protein levels of the key protein for thyroid hormone synthesis:protein levels of Tg,NIS,TPO in the thyroid tissue were observed by Western Blotting.6.Assessing the activation level of endoplasmic reticulum stress:the activation levels of eIF2a,IRE1,ATF6 in thyroid tissue and palmitate treated human thyroid primary cell were observed by western blotting.7.Assessing the glycosylation of Tg:changes in the glycosylation of Tg in palmitate treated human thyroid primary cell were observed by using endo H and PNGase F.8.Assessing the degradation level of Tg:by inhibiting the function of proteasome,we observed whether the decrease of Tg treated with palmitate could be improved or not;by inhibiting the newly synthesized protein,the degradation of Tg was studied.9.Evaluation of the therapeutic effect of improving endoplasmic reticulum stress:by using endoplasmic reticulum stress inhibitor 4-PBA(beginning at the 9th weeks to 18th weeks)or withdrawal of HFD(beginning at the 9th weeks to 18th weeks),the endoplasmic reticulum stress markers,Tg and thyroid function were studied.Part 2 HFD induces disturbance of thyroid lipid profile1.Sample collection:rats were fed with either CD or HFD for 18 weeks,and then were sacrificed.The thyroid tissues were collected and immediately frozen in liquid nitrogen.2.Lipidomics:lipid profile of the thyroid tissue was detected and analyzed by ultra performance liquid chromatography-mass spectrometry.Results:Part 1 High fat diet induces hypothyroidism through endoplasmic reticulum stress.1.The weight of HFD rats was significantly higher than that of the control group.At the 8th weeks,HFD group exhibited elevated level of TSH.Whereas at 18th weeks,HFD group exhibited elevated level of TSH in parallel with decreased level of T4.2.HFD group showed significant decrease in 24h iodide uptake,indicating impaired thyroid function.3.The thyroid gland of HFD rats showed enlarged follicle cavities and flattened follicular epithelial cells.The transmission EM showed the luminal swelling ER in thyroid epithelial cell of HFD rats,indicating the pathological changes of ER function.4.HFD did not lead to the decreased expression of Tg,Nis,Tpo,suggesting that HFD-induced hypothyroidism is not due to the decrease of the transcription level of the three molecules critical for hormone synthesis.Consistently,there was no significant difference in up-regulating transcription factor Ttf1 and Pax8 between two groups,while the down-regulating transcription factor Hex decreased.5.Western blotting results showed that HFD mainly caused a significant decrease of Tg protein.6.HFD induced ER stress in the thyroid tissue.Western blotting results showed that P-eIF2a level increased in the thyroid of HFD group and in the palmitate treated human primary thyroid cells,indicating the activation of ER stress.7.The glycosylation of Tg was also studied using endo H and PNGase F.Results showed that the larger proportion of Tg was endo H-sensitive in palmitate treated cells,indicating the retention of Tg protein in the ER and the decrease of Tg protein reaching the Golgi,strongly suggesting the degradation of these Tg proteins through the ERAD pathway.8.Palmitate treatment resulted in the decrease of Tg protein in primary cells,whereas inhibition of proteasome improved the palmitate-induced downregulation of Tg.Inhibiting the newly synthesized Tg protein by cycloheximide,the result showed that under the treatment of palmitate,Tg degraded more rapidly than the control.9.Alleviating ER stress by 4-PBA or by the withdrawal of HFD could improve the decrease of Tg and thyroid dysfunction.Part 2 HFD induces disturbance of thyroid lipid profile1.Total FFA content was significantly higher in HFD group.Taking the saturation degree into consideration,the total content of MUFAs showed increased concentration in HFD group.FFA 18:1 and 22:3 were significantly higher in HFD rats.2.The total TG content between CD and HFD group showed no significant difference.Then we analyzed the constitute difference unsaturation indices of of TG by the ratio of saturated,mono-unsaturated or poly-unsaturated TGs to the total content of TGs.the unsaturation index for the HFD group was significantly decreased when considering poly-unsaturated fatty acids,showing a tendency of the decrease of poly-unsaturated fatty acids,Mono-unsaturated TGs 52:1,54:1,54:2,54:3,56:2 and 56:3 significantly increased in HFD group.Besides,significant increase of saturated TGs 52:0 was observed in HFD rats.3.The total content of DAGs showed no significant difference between CD and HFD group,though some specific individual DAG molecules increased[DAG(36:1),DAG(38:2)]in HFD group4.The CD and HFD group showed no significant difference in the total content of phospholipids,as well as in the contents of PC,PE,PI,PG,PS and CL.The concentration of PG(36:2),PG(40:6),and PI(36:3)significantly increased in HFD group,however a relatively more variety of phospholipids decreased in HFD group,including PC(34:2),PC(40:6),PE(34:2),PE(38:6),PE(40:7),PE(40:8),PG(36:4),PG(38:6),PG(40:8),PG(42:10),PG(44:12),PI(34:2),PI(37:4),PI(38:8),and PI(39:4).5.The concentration of lysophospholipids(LPC,LPE)was evaluated.HFD group showed a significant increase of total LPC concentration as well as LPC 18:0 and 18:1.6.Cer(d1 8:1/18:0)and Cer(d18:2/20:0)increased in HFD group.SM(d16:0/20:1),SM(d16:0/20:2)SM(d16:0/22:2)increased in HFD group,and SM(d16:0/17:1),SM(d16:0/24:2),SM(d16:0/26:1),SM(d16:0/26:3),SM(d20:0/19:1),SM(d22:0/19:1)decreased.Conclusion:1.ER stress mediated HFD induced decrease of thyroglobulin and hypothyroidism.Alleviating ER stress helped inprove the decrease of thyroglobulin and hypothyroidism.2.Excess intake of dietary fat induced disturbance of thyroid lipid profile.