Zhx2 Accelerates Obesity Through Inhibiting Adipogenesis And Thermogenesis Of Adipocytes

BackgroundThe World Obesity Federation’s World Obesity Atlas 2023 indicates that global prevalence of obesity increases year by year.Global study predicts that more than half the global population will be living with overweight and obesity by 2035.Currently,the prevalence of overweight in Chinese adults（≥ 18 years old）is 34.3%,and the prevalence of obesity is 16.4%,which has become a major challenge to the health of the Chinese population.It has been reported that obesity is closely related to the development of multiple diseases.Compared with healthy individuals,patients with obesity have abnormal immune functions and serious metabolic disorders,which greatly increases the risk of cardiovascular diseases,diabetes,osteoarthritis,cancer and other diseases.There are no effective measures for obesity up to date.Therefore,it is urgent to explore new strategies to intervene obesity.As one of the primary organs of energy metabolism,adipose tissue plays an important role in obesity progression.The adipose tissue expands through adipocyte hypertrophy charaterized as the increase of adipocyte size and adipogenesis as the increase of adipocyte number,which is closely correlated with the systemic metabolism.When the energy is excessive,adipocytes are differentiated to generate a large amount of new functional adipocytes,which strengthen energy metabolism efficiency,thus significantly suppressing the development of obesity.Adipose tissues are divided into white adipose tissues（WAT）and thermogenic adipose tissues.White adipose tissues,containing single-locular large lipid droplets and few mitochondria,mainly function as energy storage.Thermogenic adipose tissues including beige adipose tissues and brown adipose tissues,contain multi-locular lipid droplets,high abundance of mitochondria and expressing thermogenic marker uncoupling protein 1（UCP1）.UCP1 releases potential energy in the form of heat by dissipating proton gradient in mitochondrial intima,thus promoting trophic catabolism and ATP synthesis uncoupling.Thermogenic adipose tissues could accelerate energy metabolism,inhibit the development of obesity,improve the metabolism index.Thermogenic adipocytes can be activated by cold exposure or adrenergic receptor stimulation.The UCP1 dependent thermogenesis is the main pathway to accelerate energy metabolism.In addition,there are a variety of synergistic thermogenic pathways such as Ca2+cycle,creatine cycle and tag-fatty acid cycle.Zinc fingers and homeoboxes 2（Zhx2）belongs to zinc fingers and homeoboxes（ZHX）family.Zhx2 is a novel transcription factor widely expressed in liver and other tissues.It has been reported that Zhx2 regulates lipid metabolism in liver and inhibits the progression of fatty hepatitis,liver cancer and other diseases.In addition,Zhx2 deficiency promotes macrophages apoptosis and reduces atherosclerosis pathogenesis.Zhx2 enhances glycolysis of macrophages,leading to increased production of lactic acid and further aggravation of sepsis.Relevant studies reveal the important role of Zhx2 in glycolipid metabolism and the diversity of biological functions.However,the role of Zhx2 in adipose tissues remains completely unknown so far.In this study,we used the adipocyte conditional Zhx2-knockout mice(Adipoq-Cre+/-,Zhx2^fl/fl;Zhx2^AKO)and control mice(Adipoq-Cre^-/-,Zhx2^fl/fl;Zhx2^fl/fl)to preliminarily explore the effects of Zhx2 on the development of obesity and adipocyte metabolism.Objectives1.To identify the role of Zhx2 in the development of obesity.2.To explore the effect of Zhx2 on adipocyte metabolism.Methods and results1.Zhx2 is highly expressed in brown adipose tissuesAdipose tissues are divided into white adipose tissues,beige adipose tissues and brown adipose tissues,and brown adipose tissues are responsible for heat generation and accelerated energy consumption.Western blotting was used to detect Zhx2 expression in adipose tissues,and the results showed that Zhx2 was highly expressed in brown adipose tissues,suggesting that Zhx2 may be related to the energy metabolism of adipose tissues.2.Adipose Zhx2 levels decreases in response to adipogenesis and thermogenesisWestern blotting was performed to detect Zhx2 and thermogenic index UCP1 in thermogenic adipocyte after stimulation,and the results showed that the Zhx2 protein level in adipocyte was significantly decreased during adipogenesis and thermogenesis.Meanwhile,we found that Zhx2 protein level was also reduced in the adipose tissues after stimulating thermogenesis.These results indicate that Zhx2 was highly expressed in brown adipose tissues or decreased during the adipogensis and enhancing thermomgensis.3.Adipocyte conditional knockout Zhx2 significantly inhibits high fat diet-induced obesityIn order to clarify the role of Zhx2 in the development of obesity and its effect on energy metabolism of adipocytes,Zhx2^AKO and Zhx2^fl/fl mice were constructed and given normal diet and high fat diet（HFD）for 24 w.Then the follow experiments were performed.（1）Adipocyte conditional knockout Zhx2 has no significant effect on mice fed with normal diet4 w male Zhx2^AKO and Zhx2^fl/fl mice were administered normal diet for 24 w,and the body weight,adipose tissues weight,insulin sensitivity,glucose tolerance and serological metabolism indexes of mice in two groups were monitored.The results showed that there were no significant differences in the above parameters between the two groups,and no obvious fatty liver disease was observed in these mice.The results of qRT-PCR showed that there was no significant difference in expression of adipogenesis genes（Ppary,Fabp4.Adipoq,Plin1）and thermogenic genes（Pparα,Ucp1,Pgc1α,Dio2,Cidea,Cox8b,Elovl3）between Zhx2^AKO and Zhx2^fl/fl mice.These results indicate that adipocyte conditional knockout Zhx2 has no significant effect on metabolism of mice under normal diet.（2）Adipocyte conditional knockout Zhx2 protects mice from obesityTo clarify the role of Zhx2 in obesity,4 w male Zhx2^AKO and Zhx2^fl/fl mice were fed with HFD for 24 w.Compared with Zhx2^fl/fl mice,Zhx2^AKO mice had significantly decreased body weight,less fat content,improved insulin sensitivity,increased glucose tolerance,improved serological metabolic indexes and hepatic steatosis as well as upregulated UCP1 expression.These results suggest that adipocyte conditional knockout Zhx2 significantly protects mice from obesity.4.Zhx2^AKO mice display increased cold tolerance and energy expenditureThe cause of obesity is that calories intake is in excess of calories expenditure,leading to the accumulation of energy in adipocytes in the form of lipids.In order to clarify the role of Zhx2 in regulating adipocytes in HFD induced obesity,Zhx2^AKO mice were used to investigate the effect of Zhx2 on energy consumption in mice.Combining with the cell model,the role of Zhx2 in adipocyte thermogenesis was charaterized.The results of metabolic cage showed that Zhx2-knockout did not affect the food intake and water intake under HFD,but the VO2,VCO2 and RER of Zhx2^AKO mice were higher than those of Zhx2^fl/fl mice.The energy metabolism of Zhx2-knockout was notably increased,suggesting that Zhx2^AKO mice could enhance the energy consumption of obese mice.It is shown that brown adipose tissues promote energy consumption and inhibit obesity mainly by thermogenesis.The results of metabolic cage and infrared imaging showed that the heat production was enhanced in Zhx2^AKO mice.Adipose tissue can provide a large amount of heat for the body to maintain core body temperature and enhance the cold tolerance in mice.To assess their cold tolerance,we monitored the rectal temperature of individual mice placed into a 4℃ cold chamber.The rectal temperature test indicated that the body temperature of Zhx2^AKO mice was significantly higher than that of Zhx2^fl/fl mice.Collectively,these data demonstrate that Zhx2^AKO mice display increased cold tolerance and energy expenditure.5.Zhx2 inhibits adipocytes adipogenesis and thermogenesisIn order to further clarify the effect of Zhx2 on adipocytes adipogenesis and thermogenesis,brown adipose tissues in Zhx2^AKO obese mice and primary brown mature adipocytes were used to detect the expression of adipogenesis genes（Pparγ,Fabp4,Adipoq,Plin1）and thermogenic genes（Pparα,Prdm16,Ucp1,Pgc1α,Dio2,Cidea,Cox8b,Elovl3）.qRT-PCR and Western blotting results showed that the expressions of adipogenesis genes such as Pparγ and thermogenic genes such as Ucpl were significantly enhanced in adipose tissues or primary brown mature adipocytes from Zhx2^AKO mice.Meanwhile,overexpression of Zhx2 in primary adipocytes from Zhx2^AKO mice significantly decreased Ucp1 and Pgc1α expression.These results suggest that Zhx2 can inhibit adipocyte thermogenesis.Meanwhile,RNA-seq was performed in the brown adipose tissues from Zhx2^AKO or Zhx2^fl/fl mice fed with HFD for 24 w.KEGG pathway and GSEA enrichment analysis showed that the thermogenesis pathway was significantly enriched in adipose tissues from Zhx2^AKO mice,which further indicates that Zhx2 could inhibit adipocytes thermogenesis.Conclusions and significanceThis study indicates that Zhx2 is highly expressed in brown adipose tissues and decreased during the adipogensis and thermomgensis.Adipocyte conditional Zhx2-knockout significantly improves the progression of obesity induced by HFD.We demonstrate that Zhx2-knockout promotes energy consumption in mice,and Zhx2 inhibits adipogenesis and thermogenesis in brown adipocytes.In conclusion,this study reveals that Zhx2 promotes HFD-induced obesity by regulating brown adipocytes thermogenesis.Thus this study further clarifies the novel biological function of Zhx2 and provides a new weight loss target for obesity treatment in clinical,which has important theoretical research value and potential application prospect in clinical.