AMP-regulated protein kinase activity in the hearts of mice treated with low- or high-fat diet measured using novel LC-MS method.

I M Rybakowska,K Kaletha,E M Slominska,M Olkowicz,P Romaszko,R T Smolenski

doi:10.1007/s11010-015-2360-z

Abstract

AMP-regulated protein kinase (AMPK) is involved in regulation of energy-generating pathways in response to the metabolic needs in different organs including the heart. The activity of AMPK is mainly controlled by AMP concentration that in turn could be affected by nucleotide metabolic pathways. This study aimed to develop a procedure for measurement of AMPK activity together with nucleotide metabolic enzymes and its application for studies of mice treated with high-fat diet. The method developed was based on analysis of conversion of AMARA peptide to pAMARA by partially purified heart homogenate by liquid chromatography/mass spectrometry (LC/MS). Activities of the enzymes of nucleotide metabolism were evaluated by analysis of conversion of substrates into products by HPLC. The method was applied for analysis of hearts of mice fed 12 weeks with low- (LFD) or high-fat diet (HFD). The optimized method for AMPK activity analysis (measured in presence of AMP) revealed change of activity from 0.089 ± 0.035 pmol/min/mg protein in LFD to 0.024 ± 0.002 in HFD. This coincided with increase of adenosine deaminase (ADA) activity from 0.11 ± 0.02 to 0.19 ± 0.06 nmol/mg tissue/min and decrease of AMP-deaminase (AMPD) activity from 1.26 ± 0.35 to 0.56 ± 0.15 nmol/mg tissue/min for LFD and HFD, respectively. We have proven quality of our LC/MS method for analysis of AMPK activity. We observed decrease in AMPK activity in the heart of mice treated with high-fat diet. However, physiological consequences of this change could be modulated by decrease in AMPD activity.

Highlights

AMP-regulated protein kinase (AMPK) is involved in the regulation of energy consumption and fuel generation paths in response to the need of organs such as the liver, central nervous system, adipose tissue, skeletal muscle, and cardiac muscle [1]
This study aimed to develop a procedure for measurement of AMPK activity together with nucleotide metabolic enzymes and its application for studies of mice treated with high-fat diet
The method developed was based on analysis of conversion of AMARA peptide to phosphorylated AMARA (pAMARA) by partially purified heart homogenate by liquid chromatography/mass spectrometry (LC/MS)

Summary

Introduction

AMP-regulated protein kinase (AMPK) is involved in the regulation of energy consumption and fuel generation paths in response to the need of organs such as the liver, central nervous system, adipose tissue, skeletal muscle, and cardiac muscle [1]. AMPK could be activated by AMP by several mechanisms. One of them is allosteric activation of the phosphorylated enzyme; the second is promotion of phosphorylation of Thr-172 by the upstream kinase and the third is the inhibition of dephosphorylation of Thr-172 by protein phosphatases [2]. Pharmacological activation of AMPK in the hypothalamus increases food intake and so AMPK is identified as a target for anti-obesity and antidiabetic drugs [4, 5]. Some commonly used drugs such as metformin activate AMPK in cardiomyocytes [6]. Another activator of AMPK is a precursor of nucleotide synthesis AICAR (5-aminoimidazole-4-carboxyamide riboside).

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Results