Study On The Function And Mechanism Of LKB1 Regulating White Fat Browning

At present,nearly 2.2 billion people worldwide are overweight,accounting for about one-third of the world's total population,of which obesity accounts for 10%.The number of obese people in China has reached 95 million,ranking first in the world.Obesity is a high risk factor for diseases such as cardiovascular disease,type 2 diabetes and cancer.Prevention and treatment of obesity is an important issue in the field of public health.Abnormal increase and accumulation of white fat cells is an important indicator of obesity.Studies have shown that cold stimulation,adrenergic receptor activation can stimulate white fat browning.White fat browning,the body's stored energy is uncoupled by uncoupling protein(UCP1)to release heat,increasing energy metabolism.Therefore,study of the molecular mechanism of white fat browning,providing new ideas for combating obesity and related metabolic diseases.Liver kinase B1 is a serine/threonine kinase that regulates phosphorylation of the AMP-dependent protein kinase(AMPK)family and is involved in cell metabolism,proliferation,differentiation,and the occurrence of tumors,but the role of LKB1 in regulating browning of white fat has not been clarified.In the early stage of the study,the LKB1/AMPK signaling pathway in the hypothalamus of diet induced obese rats was found to be impaired.The hypothalamus is a high level center that regulates energy metabolism and regulates the production of peripheral fat.Therefore,this study intends to explore the effect of LKB1 on white fat browning and its mechanism through both central and peripheral aspects.Methods:1.The LKB1 adeno-associated virus was constructed and synthesized,which was injected into the third ventricle of Sprague-Dawley(SD)rats.One week after the injection,the rats were fed with standard feed(chow fat,CF)or high fat(HF).The rats were randomly divided into four groups,namely CF-AAV group: standard feed,intracerebroventricular injection of AAV;HF-AAV group: high fat diet,intracerebroventricular injection of AAV;HF-LKB1-L group: high fat diet,intraventricular injection of low titer LKB1-AAV;HF-LKB1-H group: high fat diet,Intraventricular injection of high titer LKB1-AAV.Changes in food intake were monitored daily and changes in body weight were measured weekly.2.After 9 weeks of high-fat intervention,the dual-energy X-ray absorptiometry was used to detect changes in body fat content.The expression of LKB1 AAV in the hypothalamus was observed by Western Blot.The expression of LKB1/AMPK signaling pathway was detected by Western Blot.The morphology of white fat cells was observed by HE staining.The number of fat cells was observed by DNA detection of fat cells.The changes of lipid accumulation in liver tissue were observed by oil red O staining;the presence and quality of brown fat in the interscapular region of SD rats were observed;the changes of blood lipid metabolism were analyzed;the lipogenic factors,fat breakdown genes,and fat-producing genes were detected by RT-PCR.And mRNA expression of fatty acid oxidation factor;Western blot was used to detect the expression of brown fat marker protein UCP1 in white adipose tissue;immunohistochemical staining was used to observe the expression of NPY and POMC neuropeptides in hypothalamus and mRNA level expression by RT-PCR;Western Blot method was used to detect the expression of melanocortin receptor MC3R/MC4 R protein in hypothalamus.The content of serum epinephrine was detected by ELISA,and the mechanism of hypothalamic overexpression of LKB1 to regulate food-induced obesity was further explored.3.The browning induction solution was induced to differentiate into beige fat cells by constructing a 3T3-L1 monoclonal cell line overexpressing or silencing LKB1.Firstly,MTT assay was used to detect the effect of LKB1 expression on the proliferation of 3T3-L1 cells.Oil red O staining was used to observe the effect of LKB1 expression on the differentiation morphology of 3T3-L1 cells and lipid droplet formation.The changes of lipid content were detected by microplate reader.Immunofluorescence assay was used to detect the expression of UCP1 in differentiated and mature cells.RT-PCR was used to detect the expression of brown fat-specific gene,beige fat marker gene,lipogenesis-related gene,lipolytic gene and fatty acid oxidation factor.Western Blot detection The expression of brown fat-specific genes,lipogenic factors and LKB1/AMPK signaling pathway proteins was investigated,and the effect and mechanism of peripheral LKB1 on browning of 3T3-L1 adipose precursor cells were investigated.Results:1.The rats in the hypothalamic overexpressed LKB1 AAV group had reduced food intake,decreased body fat content and body weight.2.High-fat diet can significantly increase blood cholesterol,low-density lipoprotein levels,and lower high-density lipoprotein levels.Overexpression of LKB1 in the central region can lower cholesterol and low-density lipoprotein and increase high-density lipoprotein,suggesting that overexpression of LKB1 in the hypothalamus can ameliorate dyslipidemia caused by high fat.3.A high-fat diet can increase the volume and number of fat cells.When the hypothalamic overexpresses LKB1,the volume of adipocytes becomes smaller and the number increases.In addition,the expression of UCP1 protein in white adipose tissue is up-regulated,suggesting that white fat is brown-like.Oil red O staining showed that the central intervention of LKB1 liver cells decreased lipid droplets and hepatic steatosis was alleviated.Secondly,central overexpression of LKB1 promotes brown fat production in the interscapular region.4.Overexpression of LKB1 in the hypothalamus can promote POMC expression and inhibit NPY expression.The ELISA test showed an increase in adrenaline content,suggesting an increase in sympathetic nerve activity.Western Blot results showed that central overexpression of LKB1 activates hypothalamic AMPK activity and enhances POMC neuronal function,possibly activating the hypothalamic AMPK-POMC-sympathetic axis,thereby inhibiting the formation of obesity.5.The expression of LKB1 does not affect the proliferation of 3T3-L1 adipose precursor cells;overexpression of LKB1 can promote the differentiation of 3T3-L1 adipose precursor into multiple small lipid droplets,and the expression of brown fat-specific gene UCP1 is up-regulated,beige fat Specific genes(Tmem26 and CD137)were up-regulated,suggesting browning.After LKB1 was silenced,the 3T3-L1 adipose precursor differentiated into a large fat droplet of a single compartment,and the brown fat-specific gene expression was down-regulated,and the beige fat-specific gene was also down-regulated.6.Western Blot found that overexpression of LKB1 promoted PPAR? expression,while silencing LKB1 expression inhibited PPAR? expression.PPAR? plays a crucial role in fat metabolism.Therefore,in order to verify the role of LKB1 in regulating browning of 3T3-L1 cells,PPAR? antagonist GW9662 was added to a cell line overexpressing LKB1,and the change in browning effect of LKB1 was observed.Validation of LKB1 from both positive and negative by overexpression and silencing of LKB1 expression may promote white fat browning by modulating the expression of PPAR?.Conclusions:1.Overexpression of LKB1 in the central region may promote browning of white fat and increase energy consumption of the body by activating the AMPK-POMC-SNS axis in the hypothalamus,thereby inhibiting white fat production,reducing body weight and inhibiting the occurrence of diet induced obesity.2.LKB1 promotes browning of 3T3-L1 adipose precursor cells and may promote browning of white fat by regulating the expression of PPAR?.