Metabolic Phenotype Study Of ATP Synthase Inhibitory Factor 1 Gene Knockout Mice

Background With the improvement of living standards,obesity becomes more common and the incidence of obesity-induced illnesses like type II diabetes is rising,a trend that has placed a huge burden on health care and the government.The occurrence of type II diabetes mainly because of energy metabolic disorder.The energy-supplying substances are mainly glucose and fatty acids,which are oxidized to form water and carbon dioxide in the mitochondria,releasing energy,and driving ATP synthase to produce ATP for all kinds of physiological activities.Mitochondrial complex V(ATP synthase)is the molecular force that produces ATP and also determines the main function of mitochondria.The ATP synthase inhibitory factor 1(ATPIF1)plays an important role in regulating ATP synthase activity.Upon ATPIF1 interaction with the ATP synthase,both the synthetic and hydrolytic activities of the ATP synthase are inhibited,which is important in the maintenance of the normal level of ATP.Previous studies have shown that elevated intracellular ATP levels cause insulin resistance.We assume that the elevated ATP may lead to insulin resistance on the basis of previous work.The objective of this experiment is to verify the causal relationship between elevated ATP level and insulin resistance.Objective CRISPR/Cas9 technique is used to construct ATPIF1 gene knockout mouse model,then studying the effects of ATPIF1 gene knockout on intracellular ATP level and insulin resistance under the condition of high fat diet.Methods 1.The establishment of animal model: the 12-weeks old ATPIF1 gene knockout mice(KO for short)and C57BL/6 mice(WT for short),which is reared in humidity 40%~50% and temperature(24±1.0)?,within the independent ventilation cage system.The rate of day and night is 12h/12 h.Both groups of mice were given high fat diet.2.The measurement of body weight: the body weight of mice was measured once a week.3.Measurement of food intake: food intake were measured on the first week and 7th week of high fat diet.4.Measurement of insulin level: after 5 weeks and 26 weeks of high fat diet,insulin levels were tested with the insulin ELISA kit.5.Detection of insulin resistance: after 12 weeks and 13 weeks of high fat diet,respectively,then fasting blood glucose,insulin tolerance test,glucose tolerance test,homeostasis model assessment of insulin resistance and C-peptide levels were tested.6.Detection of energy metabolism: after 19 weeks of high fat diet,the metabolic indexes were detected with metabolic cages.7.Histochemical determination of lipid accumulation in liver: after 26 weeks of high fat diet,the liver was stained with hematoxylin and eosin.8.Primary cell culture of mice: to simulate the feed condition of high fat diet,primary cells were induced by high glucose(33m M glucose)medium,then detect the ATP level and TG level.9.Western Blot Technology: verify the results of ATPIF1 gene knockout and the expression of AKT phosphorylation modification after ATPIF1 gene knockout.10.Detection the function of cellular mitochondrial respiratory chain in tissue by seahorse: after 12 weeks of normal diet,detecting the function of complex?,?,?and ?,respectively.Results 1.Changes in body weight and tissues showed that KO mice were sensitive to high fat diet,and the weight of subcutaneous fat tissue,gonadal fat tissue and liver in KO mice,respectively,was higher than that of WT mice.2.Metabolic Cage Indexes showed that the metabolic rate of KO mice were reduced.And the weight of fat tissue account for body weight in KO mice was higher than that of WT mice.3.Insulin secretion test showed that the secretion of insulin and C peptide in KO mice are higher than WT mice.4.Detection of insulin resistance showed that the phenomenon of insulin resistance is more obvious in KO mice.5.The results of primary cell culture showed that with the development of induction,the high level ATP in the primary cells of KO mice decreased,but the accumulation of triglyceride was more than WT,which indicated that high level ATP in primary cell of the KO mice promoted the storage of triglyceride.6.The function of mitochondrial complex ?,?,? and ?in the liver and muscle in KO mice and WT mice was detected by Seahorse and the results showed no differences.This showed that under the condition of normal diet,the mitochondrial function of KO mice did not change significantly compared with WT mice.Conclusion The phenotype of the ATPIF1 gene knockout mice suggested that in the obese condition,elevated intracellular ATP promoted insulin resistance.Before becoming obesity,decreased intracellular ATP levels increase the risk of obesity.