The Mechanism Of Chronic Transient Hypoxia Ameliorate Obesity And Fatty Liver In High Fat Diet-induced Mice

Chapter 1 IntroductionIn the past 30 years,with the continuous improvement of the economy and living quality,overweight in our country has become a more and more serious issue.Obesity impaires glucose uptake,leads to insulin and leptin resistance and inflammation,and reduces the sympathetic nerve activity innervated by noradrenergic nerves.It is an important risk factor for hypertension,type 2 diabetes(T2DM),non-alcoholic fatty liver(NAFLD),and other chronic diseases.At present,the main treatment methods for obesity and non-alcoholic fatty liver disease are from three aspects: energy absorption and energy expenditure,including diet contral and living habits,exercise,surgery,and medication.However,obesity intervention is very difficult and needs to be implemented almost throughout life.Therefore,to identify an effective method with low side effects and good patient compliance has become the focus of research and attention.In recent years,the use of hypoxia for the treatment of obesity and non-alcoholic fatty liver has aroused researcher’s attention.When the body is exposed to low-oxygen environment,which is different from hypoxia in adipose tissue,the oxygen concentration in the air decreases,leading to reduced partial pressure of oxygen in the alveoli.However,due to different hypoxia methods used in studies(time,oxygen concentration,hypoxia mode,etc.),the conclusions are not consistent.This study explores whether long-term transient hypoxia environment(using 10% oxygen concentration for 1 hour every day)has an effect on reducing obesity and fatty liver induced by high fat diet,which can provide new basis for the treatment of obesity and fatty liver.Chapter 2 The high fat diet-induced mice are reduces body weight and fatty liver when their subjected to long-term transient hypoxic environmentObjective: To establish a mouse model of obesity and fatty liver with 60% high-fat diet and to investigate the role of long-term transient hypoxic in controlling high-fat-induced obesity and fatty liver.Methods: Mice were 5 weeks old and randomly divided into five groups:(1)chow diet under normoxia;(2)chow diet,1 hour per day at 10% oxygen concentration;(3)high-fat diet under normoxia;(4)after four weeks feeding with high-fat diet,mice were exposed to 10% oxygen for 1 hour per day;(5)high-fat diet and mice were exposed to 10% oxygen for 1 hour per day.1.Body weight and food intake were measured weekly,glucose tolerance test(GTT),and insulin tolerance test(ITT)were performed.H&E staining of lungs and heart was conducted to evaluate adverse effects of the hypoxic treatment.2.Liver appearance of mice were observed,subcutaneous fat,perirenal fat,brown fat and liver were measured.H&E staining was performed to observe changes in fat and liver structure;oil red O staining to assess liver lipid droplets;serum levels of ALT and AST were assayed to assess liver function.3.Immunohistochemistry was carried out to assess expression of PPARα and UCP1 in brown fat or liver;expression of Fasn(fatty acid synthase)and UCP1 in liver was examined by Western blot;m RNAs of UCP1,PGC1α,CPT1 A,SCD1,PPARα and ATGL for lipid metabolism,and M2 macrophage marker CD206 and Arginase in the liver were quantified by real-time PCR.4.Elisa assay on serum epinephrine in mice at different time points following transient hypoxic stimulation.Results:1.Long-term transient hypoxia did not affect the appetite of mice and had no effect on the body weight of mice fed with chow diet,and cardiopulmonary H&E staining showed no adverse effect on mice;however,the hypoxic environment reduced body weight of obese mice induced by high-fat diet and improve GTT,but had little effect on ITT.2.Long-term transient hypoxia reduced the obese mice weight of subcutaneous and perirenal fat and liver,and reduced the size of fat vacuoles,the accumulation of fat and lipid droplet content in obese mice liver;it also inhibited the increase of AST and ALT induced by high fat diet.3.Long-term transient hypoxia up-regulated UCP1 and PPARγ in brown fat;up-regulated UCP1 and PPARα and down-regulated Fasn(fatty acid synthase)in liver tissues;Real-time PCR results showed that UCP1,PGC1α,PPARα,CPT1 A and ATGL were increased in the liver,while SCD1 was down-regulated.CD206 and Arginase(M2 type macrophage marker)were increased.4.ELISA assays showed serum epinephrine was increased after hypoxic stimulation.Conclusions: Long term transient hypoxic reduces the severity of high-fat diet induced obesity and fatty liver in mice,and did not affect their appetite or health.Hypoxic stimulation also upregulated serum levels of epinephrine and increased macrophage M2-type gene expression in the liver.Chapter 3 Long-term transient hypoxic environment reduces body weight and fatty liver in high fat diet-induced mice by increasing adrenaline levelsObjective: To investigate whether the effect of long-term transient hypoxic on high-fat diet induced obesity and fatty liver is mediated by epinephrine.Methods: Mice were 5 weeks old and randomly divided into five groups:(1)chow diet under normoxia;(2)high-fat diet under normoxia;(3)after four weeks of high-fat diet,daily intraperitoneal injection of epinephrine(0.1mg/kg);(4)after four weeks of high-fat diet,treatment with 10% oxygen for1 hour per day;(5)after four weeks of high-fat diet,treatment with 10% oxygen for1 hour per day and simultaneously with daily intraperitoneal injection of propranolol(2mg/kg).1.Body weight and body size monitored weekly,glucose tolerance test(GTT),and insulin tolerance test(ITT)were performed;the weight of liver and fat were measured,H&E staining was conducted to observe the structure of liver and fat;oil red O,PAS and Masson staining to assess the lipid droplets,glycogen and fibrosis in liver,triglyceride and cholesterol content in liver were performed,the levels of ALT,AST and epinephrine in serum were assayed.2.Real-time PCR and Western blot were carried out to assess expression of UCP1,PGC1α,CPT1 A,PPARα,ATGL,ADRβ3 and M2 macrophage markers CD206 and Arginase in liver;expression of CPT1 A,FASN,CD206 in liver were exaimed by immunohistochemistry.3.H&E staining of lung,kidney and heart were performed to evaluate adverse effects of hypoxic or intraperitoneal injection of epinephrine.Results:1.Epinephrine and long-term transient hypoxic reduced high-fat diet induced obese mices’ body weight,improved GTT,reduced the weight of subcutaneous,inguinal,perirenal and abdominal fat and fat vacuole size,as well as reduced triglyceride and cholesterol in liver,ALT and AST in serum;both intraperitoneal injection of epinephrine and hypoxic increased the level of epinephrine.2.Intraperitoneal injection of epinephrine and hypoxic upregulated Adiponectin,UCP1,PGC1α,p-AMPK,CPT1 A,PPARα,ATGL,ADRβ3,CD206 and downregulated SCD1,FASN,ACC,while propranolol would partially counteract the effect of long-term transient hypoxic.3,H&E staining of lung,kidney and heart showed that hypoxic or intraperitoneal injection of epinephrine have not adverse effect.Conclusions: Chronic transient hypoxic attenuates high-fat diet induced obesity and fatty liver through upregulation of epinephrine.Chapter 4 Effects of adrenaline on macrophages,liver cells induced by high fatmediumObjective: To establish an in vitro fatty liver-induced cell model and a macrophage model in obese condition and to investigate the effect of epinephrine on lipid droplet formation and macrophage chemotaxis at the cellular level.Methods:1.A mixture of palmitic acid and oleic acid(2:1)was used as the "high-fat" treatment for human normal hepatocytes LO2 to replicate the fatty liver-induced cell model,and the cells were divided into five groups:(1)normal control group;(2)high-fat induced group;(3)high-fat epinephrine group;(4)high-fat propranolol group;(5)high-fat epinephrine+propranolol group.The expression of PGC1α,CPT1 A,PPARα,ATGL,ADRβ3,IR,FASN and ACC were detected by Western blot and Real-time PCR.2.Human monocyte macrophage THP-1 was induced by PMA to adhere to the wall,and then epinephrine or propanolol in palmitic and oleic acid mixed medium for 48 h.Oil red O staining was performed to detect lipid production;Western blot and Real-time PCR were performed to detect the expression of CD206 and Arginase.Results:1.Human normal hepatocytes LO2 were induced by palmitic and oleic acid mixture,epinephrine reduced their glucose consumption as well as cellular triglyceride production,decreased their lipid droplet,upregulated PGC1α,CPT1 A,PPARα,ATGL,ADRβ3 and IR,and down-regulated Fasn and ACC,while propranolol antagonized these effects.2,Human monocyte macrophage THP-1 was induced by PMA and cultured in mixed palmitic and oleic acid medium,epinephrine reduced their lipid production and upregulated CD206 and Arginase,while propranolol has the opposite effect.Conclusions: Epinephrine reduced lipid accumulation in macrophages or hepatocytes and induced THP1 cells chemotaxis to macrophage M2 type.