| Cold temperature environment is a physical phenomenon ubiquitous in nature,and it is linked to human life and health.An exposure to cold temperatures diminishes the body’s efficiency in functioning and even leads to frostbite,death,among other health consequences.Therefore,preventing the damage of the body and improving the body’s cold resistance or heat production capability in cold temperature environments are critical.Peripheral tissues are essential metabolic effectors when the body is in a cold temperature environment.After an exposure to cold temperature,skeletal muscle trembles and produces heat,liver glycogen metabolism increases,and adipose tissue changes;Adipose tissue,as an important thermogenic organ in peripheral tissues,can regulate and promote the body’s thermogenesis through the browning of white fat and the activation of brown fat.This change is essential for regulating cold resistance or heat production after cold temperature exposure.When white fat experiences browning,the neuroendocrine system,particularly the hypothalamus-pituitary-thyroid axis,plays a key role.The release level of its related hormones is closely related to white fat browning and brown fat activation.Estrogen receptors（ER）have three subtypes:ERα,ERβ,and G protein-coupled ER（GPER）.They are found throughout the brain,including the hypothalamus,hippocampus,amygdala,cerebral cortex,and other areas,and serve a variety of physiological functions.The estrogen receptor may be engaged in the process of energy metabolism,and ERαis considered to be involved in the control of adipose tissue function,according to studies.The increased expression of ERαin the hypothalamus following exposure to cold temperatures can stimulate the browning of white fat and play a major role in the heat production of peripheral fat;Therefore,this study took ERαas the research entry point to clarify the molecular mechanism of ERαin the regulation of brown fat phenotypic transformation by activation of hypothalamus-pituitary-thyroid axis after exposure to cold temperature,providing a new research target for exploring the mechanism of adipose tissue transformation after cold temperature exposure as well as heat production and cold acclimatization of the body in a cold temperature environment.Objective:In this study,the characteristics of phenotypic transformation of adipose tissue after exposure to cold temperature were investigated with an animal model in cold temperature exposure.This study also aimed to clarify the changes of hypothalamus-pituitary-thyroid axis and its role in fat transformation after exposure to cold temperature,and to reveal the role of ERαin the changes of hormones related to hypothalamus-pituitary-thyroid axis induced by cold temperature,so as to provide theoretical basis for the improvement of hypothalamus regulating thermogenesis in adipose tissue.Methods:1.The animal model of C57BL/6 mice exposed to cold temperature（4°C）for 14 days and 28 days was established,and the physiological indexes such as body weight and anal temperature of mice were recorded during the establishment of the model.2.The expression quantity of UCP1 and PGC1αin scapular brown fat activation and inguinal white fat browning was determined by RT-PCR;The changes of UCP1 and PGC1αin protein level and the altered expression of ERαin hypothalamus were detected by Western Blotting;3.The morphological changes of scapular brown fat and inguinal white fat were observed by hematoxylin-eosin（HE）staining;4.The changes of hypothalamus-pituitary-thyroid axis related hormones in serum of mice exposed to cold temperature were detected by enzyme-linked immunosorbent assay（ELISA）;5.Pretreatment of hemithyroidectomy was conducted on C57BL/6 mice to observe the changes of adipose tissue phenotype after hypothalamic-pituitary-thyroid axis-related hormone reduction.Modeling with hemithyroidectomy and recording body weight and rectal temperature.Hypothalamus-pituitary-thyroid axis correlated hormones were determined by ELISA;RT-PCR and Western Blotting were applied to detect the changes in m RNA levels of Ucp1 and Pgc1αand UCP1 protein levels;The morphological changes in scapular brown fat and inguinal white fat were observed by HE staining in mice undergoing hemithyroidectomy;Methimazole suppress thyroid hormones,and adipose tissue heat production is observed;6.The thyroid function was inhibited by methimazole,and the changes of adipose tissue phenotype were observed after the decrease of thyroid hormone.RT-PCR and Western Blotting were used to detect the changes of Ucp1 and Pgc1αat the m RNA level and UCP1at the protein level,respectively;HE staining was used to observe the morphological changes of adipose tissue in mice with hypothyroidism;7.ERα-/-mice were constructed.The cold temperature exposure model was established with ERα-/-mice,and the body weight and anal temperature were recorded;The changes in related hormones of hypothalamus-pituitary-thyroid axis in ERα-/-mice were determined by ELISA;RT-PCR was applied to detect the changes in m RNA levels of Ucp1 and Pgc1α;The morphological changes in scapular brown fat and inguinal white fat were observed by HE staining in mice;Results:1.Effects of phenotypic transformation of adipose tissue after exposure to cold temperature（1）Changes of adipose tissue in mice exposed to cold temperature for 14 daysWestern Blotting and RT-PCR were applied to determine the protein and m RNA expressions of Ucp1 and Pgc1αin the browning of inguinal white adipose tissue and scapular brown fat activation.The results showed that the protein and m RNA expressions of Ucp1 and Pgc1αwere significantly increased after 14 days of cold temperature exposure compared with the control group;（2）Changes of physiological indexes and adipose tissue of mice exposed to cold temperature for 28 daysAfter 28 days of exposure to cold temperature,the results of physiological indexes showed that compared with the control group,the anal temperature and body weight of mice in the cold temperature exposure group decreased significantly in the first three days of exposure,and with the increase of exposure time,the anal temperature and body weight were adjusted back,without significant difference after 14 days compared with the control group;According to the results of HE staining,"brown fat-like"multi-cavity lipid droplets appeared in the single-cavity bullous structure of inguinal white fat,and the volume of lipid droplets of scapular brown fat decreased significantly but the number increased.In other words,the inguinal white fat experienced browning and the scapular brown fat became activated;The results of Western Blotting and RT-PCR revealed that the protein and m RNA expressions of Ucp1 and Pgc1αin the browning of inguinal white fat and scapular brown fat activation increased significantly.2.Changes of hypothalamus-pituitary-thyroid axis-related hormones after exposure to cold temperature and its role in fat transformation（1）Changes of hormones related to hypothalamus-pituitary-thyroid axis after 14 and28 days of exposure to cold temperatureAfter 14 days and 28 days of exposure to cold temperature,the serum of mice was collected and the changes of related hormones were determined by ELISA;The results showed that the expression quantity of TRH,TSH and T4 in exposure group was significantly higher than that in control group,suggesting that exposure to cold temperature for 14 days and 28 days promoted the activation of hypothalamus-pituitary-thyroid axis in mice;（2）Changes of hormones related to hypothalamus-pituitary-thyroid axis and adipose tissue of mice undergoing hemithyroidectomy after 14 and 28 days of exposure to cold temperatureThe changes in hypothalamus-pituitary-thyroid axis related hormones were determined by ELISA at 14 and 28 days after exposure to cold temperature in the animal model with mice undergoing hemithyroidectomy.The results showed that the secretion of TRH,TSH and T4 in mice which underwent hemithyroidectomy decreased,and the secretion of TRH and TSH increased in those mice after exposure to cold temperature;After 28 days of exposure to cold temperature,the anal temperature of mice undergoing hemithyroidectomy and C57BL/6 mice decreased significantly in the first four days after exposure,and then gradually recovered;Under room temperature exposure,mice undergoing hemithyroidectomy had lower anal temperature than C57BL/6 mice.The body weight of both mice undergoing hemithyroidectomy and C57BL/6 mice decreased significantly in the first three days of cold temperature exposure,and then gradually increased to a level without significant difference with room temperature exposure group;The morphology of inguinal white adipose tissue and scapular brown fat of mice undergoing hemithyroidectomy was observed by HE staining after exposure to cold temperature.The results showed that the volume of white fat droplets increased but the number decreased in mice undergoing hemithyroidectomy.However,after 28 days of exposure to cold temperature,the volume of white fat droplets decreased but the number increased,and a"brown fat-like"multi-cavity lipid droplet structure appeared in mice undergoing hemithyroidectomy;The lipid droplets of scapular brown fat were consistent with those of white fat.Western Blotting and RT-PCR showed that the expression of Ucp1 decreased significantly in mice undergoing hemithyroidectomy.After 14 and 28 days of exposure to cold temperature,the expression of related proteins and genes in mice undergoing hemithyroidectomy increased significantly.It is suggested that white fat browning and brown fat activation occurred in mice undergoing hemithyroidectomy after exposure to cold temperature.（3）Methimazole was used to inhibit the level of thyroid hormone,and the effect of cold temperature exposure on the transformation of adipose tissue in mice was observed.After the model of hypothyroidism was established by the intervention of methimazole,the changes of hormones related to the hypothalamic-pituitary-thyroid axis were detected by ELISA.The results showed that the secretion of T4-T3 in mice with hypothyroidism decreased;the secretion of T4-T3 in mice with hypothyroidism decreased after low temperature exposure;On the first day of hypothermia exposure,the rectal temperature of the methimazole group and C57BL/6 mice decreased significantly,and then gradually recovered.The body weight of the methimazole group and C57BL/6 mice decreased significantly 3 days before low temperature exposure,and then gradually returned to normal,with no significant difference from the control group.The morphology of inguinal white fat and brown fat in the scapular region of mice after low temperature exposure was observed by HE staining.The results showed that the volume of white fat lipid droplets in the methimazole group increased and the number decreased;after 28 days of low temperature exposure,the volume of white fat lipid droplets in methimazole group increased and the number decreased;the results of brown fat in the scapular region were consistent with that of white fat.Western Blotting and RT-PCR showed that the expression of UCP1 in the methimazole group was significantly decreased.After 28 days,the expressions of UCP1 and PGC1αwere also decreased in the methimazole group.（4）Molecular mechanism of thyroid-related hormones regulating white fat browningWestern Blotting detected the expression of JAK2/STAT3 signaling pathway;after 28days of cold temperature exposure,P-JAK2 and P-STAT3 were significantly increased,indicating that cold temperature exposure promoted the activation of JAK2/STAT3signaling pathway.After inhibiting the thyroid function of mice,the expression of JAK2/STAT3 signaling pathway in the methimazole group was decreased;the JAK2/STAT3 signaling pathway in the methimazole group was also decreased after cold temperature exposure.The above results suggest that reducing thyroid hormone inhibits the JAK2/STAT3 signaling pathway.3.Role of ERαin hypothalamus-pituitary-thyroid related hormone changes induced by cold temperature（1）Changes of ERαin hypothalamus after 28 days of exposure to cold temperatureThe expression quantity of ERαprotein in hypothalamus was determined by Western Blotting.After 28 days of cold temperature exposure,the protein expression of ERαin hypothalamus was significantly higher than that in control group;（2）Effects of ERα-/-mice on physiological indexes and hypothalamus-pituitary-thyroid related hormones after cold temperature inductionIn the first three days after exposure to cold temperature,the anal temperature of ERα-/-mice and C57BL/6 mice decreased significantly.Compared with the control group,the anal temperature of ERα-/-mice decreased more significantly.The body weight of ERα-/-mice and C57BL/6 mice decreased in the first three days after exposure to cold temperature.The body weight of ERα-/-mice decreased more significantly than that of the control group.ELISA was applied to determine the expression quantity of hormones related to hypothalamus-pituitary-thyroid axis in serum of ERα-/-mice.The results indicated that after 28 days of exposure to cold temperature,the secretion of TRH,TSH,T4 and T3 in ERα-/-mice was lower than that of the control group.（3）Role of hypothalamic ERα-/-mice in fat transformation after 28 days of exposure to cold temperatureHE staining was employed to observe the morphology of inguinal white adipose tissue and scapular brown fat of ERα-/-mice.The results showed that the volume of lipid droplets in ERα-/-mice increased significantly and the density decreased.Exposure to cold temperature could induce"brown fat-like"multi-cavity lipid droplets in inguinal white fat of ERα-/-mice,with reduced volume and increased density of brown fat droplets.However,the browning and activation degree of increased white fat were lower than those of exposure group;RT-PCR was performed to determine the expression quantity of proteins related to inguinal white fat browning and scapular brown fat activation after exposure to cold temperature;The results showed that the expression quantity of Ucp1 and m RNA expression quantity of Ucp1 and Pgc1αin ERα-/-mice decreased significantly after 28 days of exposure to cold temperature.It is suggested that ERαmolecule is involved in the white fat browning and brown fat activation in mice exposed to cold temperature.Conclusion:1.Exposure to cold temperature can promote white fat browning and brown fat activation.2.Hypothalamus-pituitary-thyroid axis is involved in cold temperature induced fat thermogenesis:hormones related to hypothalamus-pituitary-thyroid axis promote the browning of white fat and the activation of brown fat;Thyroid hormone promotes white fat browning through JAK2/STAT3 signaling pathway;3.Hypothalamic ERαcan regulate TRH-TSH-T4/T3 secretion and control adipose tissue thermogenesis. |
PDF Full Text Request