Mechanism Of Adenine Nucleotide-mediated Insulin Resistance And Hyperglycemia

Type 2 diabetes is a complex chronic disease that has attracted widespread attention worldwide.The incidence of diabetes has increased every year and become one of the major public health problems.The rise in free fatty acids(FFA)caused by obesity is a major risk factor in the development of type 2 diabetes.Our previous study has demonstrated that the increase of plasma adenine nucleotide 5'-AMP is an upstream signal for insulin resistance and hyperglycemia in type 2 diabetic mice;enhanced extracellular 5'-AMP signaling in wild-type mice causes insulin resistance and hyperglycemia;cell damage caused by FFA is a source of elevated plasma 5'-AMP in diabetes;strengthening 5'-AMP signal can elevate adenosine content in liver and muscle,inhibiting the phosphorylation of insulin receptor induced by insulin,leading to insulin resistance.However,the mechanism by which adenine nucleotides induce hyperglycemia needs further investigation.Firstly,we found that hepatic ATP and adenine nucleotide metabolites increased significantly in type 2 diabetic mice.Using metabolomics method,we found that the changes of metabolites in the liver of db/db and 5'-AMP treated wild type mice were similar.ATP,adenosine,hypoxanthine,xanthine,and uric acid were significantly elevated in the liver of db/db and 5'-AMP treated mice;similar results were observed in the liver of other mice models of type 2 diabetes.However,in the liver of obese mice without hyperglycemia,we only found an increase in adenosine.These results indicate that elevated levels of ATP and adenine nucleotide metabolites in the liver are markers of type 2 diabetes.Plasma adenine nucleotides metabolites were increased in type 2 diabetic mice.Compared with control group,plasma 5'-AMP was increased in db/db and high-fat-diet/streptozotocin induced diabetic mice(db/db vs lean:13.9±2.3?mol/L vs 3.7±0.9?mol/L,p<0.01;HF+STZ vs CK:6.6±1.5?mol/L vs 4.1±0.9?mol/L,p<0.05);hypoxanthine,xanthine and uric acid were also elevated in plasma.Only uric acid was increased in plasma of non-diabetic obese mice.Elevated levels of adenine nucleotide metabolites in plasma were observed in mice with increased level of FFA.Injection of 5'-AMP can induce glucose intolerance in wild type mice.The content of adenine nucleotide metabolites in blood glucose and plasma was positively correlated with the dose of 5'-AMP injected:at lower dose,compared with control group,only glucose intolerance and an increase in plasma uric acid content were observed;at higher dose,hyperglycemia was observed(20.7±2.8 mmol/L vs 7.7±0.8 mmol/L,p<0.01),and plasma levels of adenine nucleotides and metabolites increased significantly.FFA is an important cause of the increased extracellular adenine nucleotides.FFA induced release of nucleotides from vascular endothelial cells in a short time,prior to apoptosis.Glibenclamide can inhibit this effect.Glibenclamide is an inhibitor of transmembrane conduction regulators and volume-regulated anion channels in cystic fibrosis,indicating that these two channels may be involved in FFA-induced nucleotides release.In addition,FFA can inhibit RBCs'ability to degrade hydrogen peroxide.The accumulation of hydrogen peroxide can induce the release adenine nucleotides from RBCs.5'-AMP decreased intracellular pH of cultured hepatocytes,increasing the activity of the protein-tyrosine phosphatase PTP1B.5'-AMP and adenosine increased levels of ATP and adenine nucleotide metabolites in cultured hepatocytes.The adenine nucleotide metabolites hypoxanthine,xanthine and uric acid could inhibit the activity of Na~+/K~+-ATPase,increasing intracellular ATP.In vitro experiments showed that ATP can increase the activity of PTP1B.PTP1B has a role in the negative regulation of insulin signaling,and the enhancement of its activity can inhibit insulin signaling.The decrease in pH of the assaying buffer caused by ATP was the reason for the increase of PTP1B activity.Exogenous 5'-AMP decreased the intracellular pH of cultured hepatocytes.5'-AMP,hypoxanthine,xanthine,and uric acid inhibited the activity of the Na~+/H~+exchanger,impairing regulation of intracellular pH.Metformin can moderate hyperglycemia by increasing intracellular pH and inhibiting the activity of PTP1B.Metformin could moderate 5'-AMP induced insulin resistance.HPLC results showed that metformin reduced the levels of ATP and adenosine in the liver of db/db and 5'-AMP treated mice,whereas the levels of hypoxanthine,xanthine and uric acid remained unchanged.In vitro experiments showed that cationic metformin can inhibit the activity of PTP1B by increasing the pH of the assaying buffer.Metformin increased the pH of cultured hepatocytes in a short period.This presents a new potential mechanism for the hypoglycemic effect of metformin.Adenine nucleotides were involved in obesity induced metabolic disorders of testis.Compared with control mice,adenosine was elevated in testes of ob/ob,db/db and high fat diet induced obese mice(ob/ob vs lean:36.8±12.4 nmol/g protein vs 20.8±3.9 nmol/g protein,p<0.05;db/db vs lean:36.1±19.5 nmol/g protein vs 16.7±2.3 nmol/g protein,p<0.05;HF vs CK:26.9±5.17 nmol/g protein vs 20.2±3.5 nmol/g protein,p<0.05).Compared with control group,mice treated with 5'-AMP had similar results(49.8±23.6 nmol/g protein vs 20.9±2.7 nmol/g protein;p<0.05).Injection of 5'-AMP decreased the expression of genes related to hormone metabolism,and a significant decrease of serum testosterone was observed(328±44 ng/d L vs 235±30 ng/d L;p<0.01).Metabolomics analysis showed that the testes injected with 5'-AMP mice had similar metabolic profiles to the testes of infertile ob/ob mice.The changes of metabolites content were also similar.AKT/mTOR affect spermatogenesis in male mice,and phosphorylation levels of AKT/mTOR were decreased in the testes of ob/ob and 5'-AMP treated mice.The adenosine accumulation induced metabolic disorder in testis is a reason for the decline of reproductive potential in obese mice.