Unacylated Ghrelin Regulates Glycolipid Metabolism And Its Intervention In Non-alcoholic Fatty Liver Disease

Objective: Ghrelin is a gastrointestinal hormone known to stimulate appetite.It exists in two forms: acylated ghrelin(AG)and unacylated ghrelin(UAG).AG induces fat accumulation and reduces insulin sensitivity by binding to the growth hormone secretagogue receptor,UAG acts as a separate hormone that antagonizes the effects of AG,but its receptor has yet not been identified.Therefore,there are few studies on the activity of UAG which is mainly focused on the research of feeding regulation.The regulation of glycolipid metabolism by UAG and its possible targets was investigated in the present study.Furthermore,the effects of peripheral and central administration of UAG on nonalcoholic fatty liver disease(NAFLD)and its possible mechanism were explored.Methods: Immunofluorescent staining was used to observe the expression of c-fos in orexin-A immunopositive neurons in the lateral hypothalamic area(LHA)after LHA and peripheral administration of UAG.Neuronal firing records were used to detect the effects of UAG in the LHA on the discharge of glucose-sensitive(GS)neurons.After peripheral and LHA administration of UAG,enzyme-linked immunosorbent assay(Elisa)was used to determine the content of glucose,insulin,total cholesterol(TC),triglyceride(TG),and low density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)in serum;Real-time polymerase chain reaction(RT-PCR)was used to detect the mRNA expression of carnitine palmitoyl transferase-1(CPT-1)and acetyl Coenzyme A carboxylase-1(ACC-1);anti orxin-A antibody was pre-administered to observe its effect on glucose and lipid metabolism regulated by LHA-administered UAG.Rats were fed with high-sugar,high-fat,and high-cholesterol diets to establish a pathological model of NAFLD,and UAG was administered peripherally and centrally for intervention;Elisa was used to determine changes in serum lipid biochemical indicators,alanine aminotransferase(ALT),aspartate aminotransferase(AST)and Homeostatic model assessment of insulin resistance(HOMA-IR).RT-PCR was used to detect the mRNA expression of liver lipase;Colorimetric determination was used to detect changes in hepatic TG;Hematoxylin-eosin(HE)staining was used to observe pathological changes in liver tissue;Western Blot(WB)was used to measure the protein expression of insulin signaling pathway factors phosphoinositide 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(PKB/Akt),phosphorylated Akt(p-Akt),mammalian target of rapamycin(mTOR)and phosphorylated mTOR(p-mTOR)in the liver and hypothalamus,respectively.Results: Compared with the control group,administration of UAG into LHA activated orexin-A immunopositive neurons in LHA,and stimulated the discharge of glucose-inhibited(GI)neurons in LHA,which was partially blocked by pre-administration of anti orexn-A antibody;however,peripheral administration of UAG did not activate orexin-A immunopositive neurons in LHA.Compared with the control group,peripheral and LHA administration of UAG significantly reduced the levels of glucose,insulin,TC,TG,and LDL-C levels in serum(p < 0.05-0.01),and significantly increased serum HDL-C levels(p < 0.05-0.01);increased CPT-1 mRNA expression(p < 0.05-0.01),and decreased ACC-1 mRNA expression(p < 0.05-0.01)in the liver.These series of physiological changes caused by LHA administration of UAG is blacked partially by pre-administration of anti orexin-A antibody(p < 0.05).Compared with the control group,the levels of blood lipid,transaminase,and HOMA-IR in the NAFLD model group were significantly increased(p < 0.01),hepatic TG were significantly increased(p < 0.01),hepatic CPT-1 mRNA expression was reduced(p < 0.05-0.01),while ACC-1 m RNA expression was increased.(p < 0.05-0.01),the liver tissue structure was disordered with steatosis in liver cells,indicating that the NAFLD pathological model was successfully established.Compared with the NAFLD model group,the levels of serum TC,TG,LDL-C,ALT,AST,and HOMA-IR decreased significantly after peripheral and central administration of UAG(p < 0.01),while the levels of serum HDL-C increased significantly.(p < 0.05);hepatic TG were significantly decreased(p < 0.01);the mRNA expression of hepatic CPT-1 was enhanced(p < 0.01),while ACC-1 was weakened(p < 0.01);liver tissue damage was significantly restored.Compared with the NAFLD model group,the protein expression of p-PI3 K,p-Akt and p-mTOR in liver and hypothalamus was enhanced by peripheral and central administration of UAG,respectively(p < 0.01).Conclusions: Peripheral and LHA administration of UAG participates in the regulation of glucose and lipid metabolism in normal rats.The role of UAG in LHA may be related to its activation of orexin-A immunopositive neurons,while the mechanism of peripheral UAG is more complicated and needs to be further explored.Peripheral and central administration of UAG has a certain intervention effect on diet-induced NAFLD,and this effect may be related to its stimulation of hepatic and hypothalamic insulin signaling pathway factor phosphorylation and restoration of pathway activity.This topic provides experimental and theoretical basis for improving the mechanism of UAG participating in the regulation of glucose and lipid metabolism and discovering UAG as a potential therapeutic target for NAFLD.