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

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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.

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AMP-activated protein kinase--development of the energy sensor concept.

D Hardie, Simon A. Hawley, John W. Scott (2006)

The LKB1-->AMPK cascade is switched on by metabolic stresses that either inhibit ATP production (e.g. hypoxia, hypoglycaemia) or that accelerate ATP consumption (e.g. muscle contraction). Any decline in cellular energy status is accompanied by a rise in the cellular AMP: ATP ratio, and this activates AMPK by a complex and sensitive mechanism involving antagonistic binding of the nucleotides to two sites on the regulatory gamma subunits of AMPK. Once activated by metabolic stress, AMPK activates catabolic pathways that generate ATP, while inhibiting cell growth and biosynthesis and other processes that consume ATP. While the AMPK system probably evolved in single-celled eukaryotes to maintain energy balance at the cellular level, in multicellular organisms its role has become adapted so that it is also involved in maintaining whole body energy balance. Thus, it is regulated by hormones and cytokines, especially the adipokines leptin and adiponectin, increasing whole body energy expenditure while regulating food intake. Some hormones may activate AMPK by an LKB1-independent mechanism involving Ca2+/calmodulin dependent protein kinase kinases. Low levels of activation of AMPK are likely to play a role in the current global rise in obesity and Type 2 diabetes, and AMPK is the target for the widely used antidiabetic drug metformin.

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AMP-activated protein kinase plays a role in the control of food intake.

Ulrika Andersson, Karin Filipsson, Caroline Abbott … (2004)

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that acts as an intracellular energy sensor maintaining the energy balance within the cell. The finding that leptin and adiponectin activate AMPK to alter metabolic pathways in muscle and liver provides direct evidence for this role in peripheral tissues. The hypothalamus is a key regulator of food intake and energy balance, coordinating body adiposity and nutritional state in response to peripheral hormones, such as leptin, peptide YY-(3-36), and ghrelin. To date the hormonal regulation of AMPK in the hypothalamus, or its potential role in the control of food intake, have not been reported. Here we demonstrate that counter-regulatory hormones involved in appetite control regulate AMPK activity and that pharmacological activation of AMPK in the hypothalamus increases food intake. In vivo administration of leptin, which leads to a reduction in food intake, decreases hypothalamic AMPK activity. By contrast, injection of ghrelin in vivo, which increases food intake, stimulates AMPK activity in the hypothalamus. Consistent with the effect of ghrelin, injection of 5-amino-4-imidazole carboxamide riboside, a pharmacological activator of AMPK, into either the third cerebral ventricle or directly into the paraventricular nucleus of the hypothalamus significantly increased food intake. These results suggest that AMPK is regulated in the hypothalamus by hormones which regulate food intake. Furthermore, direct pharmacological activation of AMPK in the hypothalamus is sufficient to increase food intake. These findings demonstrate that AMPK plays a role in the regulation of feeding and identify AMPK as a novel target for anti-obesity drugs.

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Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR.

C. Young, Helen R. Russell, G Shulman … (1999)

Insulin increases glucose uptake through the translocation of GLUT-4 via a pathway mediated by phosphatidylinositol 3-kinase (PI3K). In contrast, myocardial glucose uptake during ischemia and hypoxia is stimulated by the translocation of GLUT-4 to the surface of cardiac myocytes through a PI3K-independent pathway that has not been characterized. AMP-activated protein kinase (AMPK) activity is also increased by myocardial ischemia, and we examined whether AMPK stimulates glucose uptake and GLUT-4 translocation. In isolated rat ventricular papillary muscles, 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, as well as cyanide-induced chemical hypoxia and insulin, increased 2-[(3)H]deoxyglucose uptake two- to threefold. Wortmannin, a PI3K inhibitor, did not affect either the AICAR- or the cyanide-stimulated increase in deoxyglucose uptake but eliminated the insulin-stimulated increase in deoxyglucose uptake. Immunofluorescence studies demonstrated translocation of GLUT-4 to the myocyte sarcolemma in response to stimulation with AICAR, cyanide, or insulin. Preincubation of papillary muscles with the kinase inhibitor iodotubercidin or adenine 9-beta-D-arabinofuranoside (araA), a precursor of araATP (a competitive inhibitor of AMPK), decreased AICAR- and cyanide-stimulated glucose uptake but did not affect basal or insulin-stimulated glucose uptake. In vivo infusion of AICAR caused myocardial AMPK activation and GLUT-4 translocation in the rat. We conclude that AMPK activation increases cardiac muscle glucose uptake through translocation of GLUT-4 via a pathway that is independent of PI3K. These findings suggest that AMPK activation may be important in ischemia-induced translocation of GLUT-4 in the heart.

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Author and article information

Contributors

I. M. Rybakowska: +48 58 349 14 65 , iwonar@gumed.edu.pl

Journal

Journal ID (nlm-ta): Mol Cell Biochem

Journal ID (iso-abbrev): Mol. Cell. Biochem

Title: Molecular and Cellular Biochemistry

Publisher: Springer US (New York )

ISSN (Print): 0300-8177

ISSN (Electronic): 1573-4919

Publication date (Electronic): 25 February 2015

Publication date PMC-release: 25 February 2015

Publication date (Print): 2015

Volume: 404

Issue: 1-2

Pages: 5-10

Affiliations

[ ]Department of Biochemistry and Clinical Physiology, Medical University of Gdansk, Debinki 1, 80-211 Gdańsk, Poland

[ ]Department of Biochemistry, Medical University of Gdansk, Gdańsk, Poland

[ ]Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poznan, Poland

Article

Publisher ID: 2360

DOI: 10.1007/s11010-015-2360-z

PMC ID: 4544673

PubMed ID: 25711403

SO-VID: e9fd5134-debb-4944-958f-f0835d9551f3

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

History

Date received : 23 October 2014

Date accepted : 14 February 2015

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ScienceOpen disciplines: Biochemistry

Keywords: amp-regulated protein kinase,amp-deaminase,adenosine deaminase,high-fat diet

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ScienceOpen disciplines: Biochemistry

Keywords: amp-regulated protein kinase, amp-deaminase, adenosine deaminase, high-fat diet

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

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GLUT4 biology

Most cited references 12

AMP-activated protein kinase--development of the energy sensor concept.

AMP-activated protein kinase plays a role in the control of food intake.

Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR.

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