GP73 Regulates Systemic Glucose Homeostasis By Stimulating Hepatic Gluconeogenesis

Objective:Hyperglycemia is caused by defective insulin secretion,impaired biological effects of insulin,or excessive gluconeogenesis.Long-term hyperglycemia leads to muti-organ damage and functional failure,especially in eyes,kidneys,heart,blood vessels,and nerves.Here we aimed to explore the contribution of Golgi Protein 73（GP73）to systemic glucose homeostasis and its underlying mechanistic.Methods:1.Clinical data from healthy and diabetic subjects were collected,the plasma levels of GP73 in these two groups were compared and analyzed;In patients with diabetics,the correlation of plasma glycosylated hemoglobin（Hb A1c）levels and plasma GP73 levels were analyzed.2.C57/BL6N mouse were intraperitoneally injected with citric acid buffer containing streptozotocin（STZ）along with a high-sugar and high-fat diet.Fasting blood glucose was monitored and the expressions of GP73 in the plasma and muti-organs in this model was analyzed by ELISA and RT-PCR.3.Two T2DM mice models including db/db mice and HFD+STZ mice and one STZ-induced T1DM mice were used.GP73monoclonal antibody（m Ab）and an equal volume of mouse immunoglobulin G（Ig G）were injected into the tail vein of the above mice.Fasting blood glucose,body weight.,plasma Hb A1c,ALT,AST,TG,CHO levels,and the m RNA level of key gluconeogenesis enzymes in the liver were analyzed and compared between GP73 m Ab and Ig G–treated mice.4.By using CRISPR/Cas9 technology,we generated whole body GP73-knockout（GP73 KO）mice.Fasting blood glucose,body weight,specific gravity of organs,pyruvate tolerance（PTT）,glucose tolerance（GTT）,insulin tolerance（ITT）,m RNA expression levels of key enzymes of gluconeogenesis and glycolysis in liver and kidney were analyzed in GP73 KO and WT mice.Results:1.Diabetic patients had significantly higher plasma GP73 levels than age-and sex-matched healthy subjects.Specifically,the plasma concentrations of GP73 were strikingly correlated with average blood glucose during the months preceding the demise（glycated A1c,Hb A1c）.2.The HFD-STZ T2DM mice displayed significant rise in plasma GP73 levels.We found strong upregulation of the GP73 m RNA in white adipose tissue and liver.3.HFD-STZ induced diabetic mice and db/db mice were used to mimic the human disease of type 2 diabetes.Animals with fasting glucose over 199.8 mg/d L were treated with Ig G isotype or m Ab against GP73.As we can see,GP73 m Ab caused a significant decrease in fasting blood glucose levels compared to controls treated with a preimmune Ig G at 6 h after administration.We then injected STZ-induced diabetic T1DM mice with GP73 m Ab twice a day continuing for 4 weeks to examine long-term metabolic outcome.Antibody administration did not cause liver damage and alter the body weight,but caused a significant decrease in blood glucose level and Hb A1c level within 4 weeks of treatment compared to controls treated with a preimmune Ig G.Reduced plasma TG and CHO levels was detected in GP73m Ab-treated groups.Notably,mice receiving the GP73 m Ab exhibited significantly reduced glucogenic genes expressions（G6pc,Pck1,Fbp1 and Pcx）compared to control animals.4.GP73^-/-homozygous showed undetectable circulating and multiorgan GP73 levels.GP73^-/-male mice showed lower glucose levels in the fed and fasted state when compared with littermate controls.The pyruvate tolerance test（PTT）also showed abnormalities in GP73^-/-mice,but the glucose tolerance test（GTT）and insulin tolerance test（ITT）were not abnormal.Accordingly,fasting induced gluconeogenic genes（G6pc,Pck1,Fbp1 and Pcx）in liver and kidney were significantly lower in GP73^-/-mice.Conclusion:Based on the above results,we found that the hyperglycemic environment can up-regulate the high expression of GP73 in plasma,and GP73m Ab could cause a significant decrease glucose of diabetic model mice without significant side effects.Further studies showed that the gluconeogenesis ability of GP73^-/-mice was decreased,and the fasting induced gluconeogenic genes in liver and kidney were significantly lower,suggesting that GP73 regulates glucose metabolism by promoting gluconeogenesis.