Adenine nucleotide-mediated regulation of hepatic PTP1B activity in mouse models of type 2 diabetes.

Abstract

Plasma 5'-AMP (pAMP) is elevated in mouse models of type 2 diabetes. However, the metabolic regulatory role of adenine nucleotides in type 2 diabetes remains unclear. Adenine nucleotides and their metabolites in plasma and liver were examined by HPLC. 1H NMR-based metabolomics analysis was performed to explore the changes of metabolites in mouse models of type 2 diabetes. Na+/K+ ATPase and Na+/H+ exchanger activity were measured in response to adenine nucleotide metabolites. Human recombinant protein tyrosine phosphatase 1B (PTP1B) was used for enzyme kinetic assays. Protein binding assays were performed with microscale thermophoresis. The intracellular pH of hepatocyte AML12 cell lines was measured using the BCECF-AM method. We also analysed pAMP levels in participants with type 2 diabetes. Elevation of pAMP was a universal phenomenon in all mouse models of type 2 diabetes including db/db vs lean mice (13.9 ± 2.3μmol/l vs 3.7 ± 0.9μmol/l; p < 0.01), ob/ob vs lean mice (9.1 ± 2.0μmol/l vs 3.9 ± 1.2μmol/l; p < 0.01) and high-fat diet/streptozotocin-induced vs wild-type mice (6.6 ± 1.5μmol/l vs 4.1 ± 0.9μmol/l; p < 0.05); this elevation was required for the occurrence of hyperglycaemia in obese mice. 1H NMR-based metabolomics study following HPLC analysis revealed that the metabolite profile in wild-type mice treated with 5'-AMP was similar to that in db/db diabetic mice, especially the accumulation of a large quantity of ATP and its metabolites. The glucose-lowering drug metformin reduced the severity of hyperglycaemia both in 5'-AMP-induced wild-type mice and db/db mice. Metformin decreased the accumulation of liver ATP but not its metabolites in these hyperglycaemic mice. ATP and metformin reciprocally change cellular pH homeostasis in liver, causing opposite shifts in liver activity of PTP1B, a key negative regulator of insulin signalling. Furthermore, pAMP levels were also elevated in individuals with type 2 diabetes (45.2 ± 22.7nmol/l vs 3.1 ± 1.9nmol/l; p < 0.01). These results reveal an emerging role for adenine nucleotide in the regulation of hyperglycaemia and provide a potential therapeutic target in obesity and type 2 diabetes.