The Regulation Mechanism Of Heparin On Mammalian Food Intake And Glucose Homeostasis

Energy homeostasis is a dynamic balance process of energy intake,storage and consumption during animal growth and development,which is the foundation for animal to maintain growth and development.Improving animal energy positive balance can not only improve feed efficiency rate and production efficiency,but also reduce feeding costs and excretion.Therefore,the regulation of energy homeostasis plays an important role in livestock and poultry production.Energy homeostasis is mainly regulated by the nervous system and the endocrine system.The hypothalamic arcuate nucleus Ag RP neurons can change the metabolic rate of the body while regulating the animal energy intake by feeling the body's nutritional level,insulin regulated glucose homeostasis not only affect energy production,skeletal muscle and other tissue nutrient deposition,but also inhibit central energy intake.Heparin as a glycosaminoglycan that exists in extracellular matrix and circulating blood,it is mainly secreted by mast and basophilic cells.It has anticoagulant effect and participates in the body's immune regulation.It is the first time to find that heparin plays an important role in the regulation of food intake and glucose homeostasis.Part 1: The central mechanism of heparin promoting animal food intakeMice were used as objects to detect the serum heparin content in different energy states at first,and the results showed that the serum heparin content was significantly increased by starvation of 24 h or 60% energy restriction for one week.Acute injection of heparin significantly improved mice food intake,while long-term treatment of heparin not only promoted the food intake,but also increased body weight gain,feed efficiency rate,and reduced the energy expenditure.Further through c-fos staining in hypothalamus of NPY-GFP mice and patch clamp test of Ag RP neurons in Ag RP-td Tomato mice showed that heparin could promote hypothalamic Ag RP neuron activity.Western Blot,q PCR and Ch IP have found that heparin may promote the expression and secretion of neurotransmitters in Ag RP neurons by activating Fox O1.It has been proved that heparin can increase the food intake mainly by activating the Ag RP neurons in the hypothalamus after the orexigenic effects of heparin was blocked by a DREADD induced specific activation or inhibition of Ag RP neurons,or intracerebroventricular anti-Ag RP antibody.Part 2: The molecular mechanism of heparin promoting the excitability of Ag RP neuronsN38 cells as the object of research found that heparin can significantly promote inflammatory pathways and reduced insulin pathway.However,the effect of heparin on expression and secretion of Ag RP was not blocked by heparin co-treatment with agonist or antagonist of inflammatory pathway.Using agarose gel chromatography found that heparin could be directly binding to the insulin receptor on Ag RP neurons.Meanwhile,the results of insulin and insulin receptor Co-IP and immunocytochemistry in N38 cells confirm that heparin can inhibit the binding of insulin to its receptor in Ag RP neurons.What's more,high doses of insulin can inhibit the effect of heparin on the c-fos expression of Ag RP neurons,Ag RP promoter activity and promoting the food intake.Western Blot,Ch IP,immunostaining of hypothalamic arcuate nucleus and N38 cells also showed that heparin significantly inhibited Fox O1 phosphorylation and promoted Fox O1 to nucleus translocation,which combined with Ag RP promoter and activated Ag RP expression.The effect of heparin on the activity of Ag RP promoter,Ag RP expression and food intake was reversed by Fox O1 antagonist.These results indicate that heparin inhibits the insulin pathway by blocking the binding of insulin and its receptor in Ag RP neurons,and promotes the translocation of Fox O1 into the nucleus,thereby enhancing the expression of Ag PR and the excitability of Ag PR neurons,which eventually leads to an increase in food intake.Part 3: The effect of heparin on glucose metabolism and insulin receptor sensitivityBased on the results of the mechanism of heparin on Ag PR neurons,we hypothesized that heparin may also regulate insulin sensitivity and affect glucose metabolism.To verify this assumption,the content of serum heparin in mice with different insulin sensitivity was compared at first and found that obese or diabetic mice(ob/ob)mice were higher than normal mice.After long-term intraperitoneal injection of heparin,the Oral Glucose Tolerance Test(OGTT)and insulin tolerance test(Insulin resistance test,ITT)showed that heparin could significantly inhibit insulin sensitivity.In contrast,ob/ob mice were long-term injected with heparinase to degrade endogenous heparin can reduce the body weight and alleviate the symptoms of diabetes mellitus.In addition,heparin have no effect on glucose in STZ induced type I diabetes mice.What's more,exogenous addition insulin could or could not restore the glucose level in type I diabetes mice treated by saline or heparin.Finally,in order to reveal the target tissue regulated by heparin,we used fluorescent labeling glucose 2-NBDG to trace glucose uptake and found that heparin significantly inhibited skeletal muscle glucose uptake,but had no effect on liver and adipose tissue.It also has similar effects in C2C12 cells.Therefore,heparin can induce insulin resistance and inhibit glucose uptake in skeletal muscle by reducing the body's insulin sensitivity,which leads to the increase of blood glucose.Part 4: The molecular mechanism of heparin regulating the skeletal muscle insulin sensitivityIn order to further clarify the signaling pathway of heparin regulating insulin sensitivity in skeletal muscle,Western Blot found that heparin could significantly inhibit the inflammatory and insulin pathway in C2C12 cells.The results of agarose gel chromatography showed that heparin also could be directly binding with insulin receptor.Subsequently,293 T cells were transfected with a stable expression of IR-GFP,FRET detection showed that heparin could significantly inhibit the binding of insulin(Cy3 tag)with insulin receptor,which is further confirmed by the CO-IP in skeletal muscle tissue.Cells and in vivo test showed that high concentrations of insulin could block the inhibitory effect of heparin on glucose uptake.In addition,293 T cells and muscle immunofluorescence assay showed that heparin significantly inhibited GLUT4 translocation,however this effect was blocked after adding insulin,continuously activating Akt or transfection of GLUT4 glycosylation mutant plasmid N57-GLUT4-GFP.Finally,the fluorescent labeled sialic acid was added to the overexpressed GLUT4-GFP 293 T cells,FRET was used to detect GLUT4 glycosylation and found that heparin could inhibit its translocation to cell membrane by decreasing GLUT4 glycosylation and this effect disappeared after the GLUT4 glycosylation mutation.Therefore,heparin can inhibit the insulin pathway,GLUT4 glycosylation and translocation to the cell membrane by blocking the binding of insulin and its receptor in skeletal muscle cells,and ultimately leads to a high blood glucose and insulin resistance.In summary,this study found that heparin can inhibit insulin pathway by blocking the binding of insulin and its receptor,thus the Ag RP neurons excitability was activated by promoting the translocation of Fox O1 to the nucleus and combining the Ag RP promoter;on the other hand,the translocation of GLUT4 to the cell membrane and glucose absorption in skeletal muscle were inhibited,eventually leaded to animal food intake and body weight increased,elevated blood glucose,reduce energy consumption and improve feed conversion rate.