Measurements of Diacylglycerols in Skeletal Muscle by Atmospheric Pressure Chemical Ionization Mass Spectrometry
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METHODS
Measurements of Diacylglycerols in Skeletal Muscle by Atmospheric Pressure Chemical Ionization Mass Spectrometry Su-Yeon Lee • Jung Ran Kim • Mi-Young Ha Soon-Mi Shim • Tae-Sik Park
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Received: 28 August 2012 / Accepted: 20 January 2013 / Published online: 8 February 2013 Ó AOCS 2013
Abstract Diacylglycerol (DAG) is an intermediate lipid involved in the synthesis of phospholipids and triglycerides. As signaling regulators, DAG activate novel protein kinase C leading to decreased response to insulin in skeletal muscle. Alteration of DAG contents correlates with development of metabolic dysregulation in obese and diabetic conditions. Recent advances in lipidomics using mass spectrometry allow expanded measurements of various lipid species. This study describes a rapid measurement of DAG species using the triple quadrupole mass spectrometry using atmospheric pressure chemical ionization in a positive ion mode. DAG in the cells and muscle tissues were separated depending on differences in chain lengths and degree of unsaturation. The limit of detection and quantification for DAG was 0.2 to 17 pmol for this method. When C2C12 cells were treated with palmitate or oleate, we found a 12-fold and 2-fold DAG increase respectively compared to the no-treatment control. In the muscles of obese db/db mice, DAG levels were elevated by 6-fold compared to those of wild-type skeletal muscles. The
present analytical method provides a rapid and sensitive quantification of DAG molecular species from various biological samples and can be used to correlate the degree of metabolic dysregulation with lipotoxic metabolites. Keywords Fatty acid
Diacylglycerol Obesity Protein Kinase C
Abbreviations APCI Atmospheric pressure chemical ionization DAG Diacylglycerol(s) ELSD Evaporative light scattering detector FFA Free fatty acid(s) HPLC High performance liquid chromatography MS/MS Tandem mass spectrometry PBS Phosphate-buffered saline PKC Protein kinase C
Introduction S.-Y. Lee and J. R. Kim contributed equally to this work. S.-Y. Lee J. R. Kim T.-S. Park (&) Department of Life Science, Gachon University, Jinrikwan 304B, Bokjung-dong, Sujung-gu, Sungnam-Si, Gyeonggi-do 461-701, Republic of Korea e-mail: [email protected] M.-Y. Ha Nonghyup Food Safety Research Institute, Seoul 137-130, Republic of Korea S.-M. Shim Department of Food Science and Technology and Carbohydrate Bioproduct Research Center, Sejong University, Seoul 143-747, Republic of Korea
Obesity is a worldwide epidemic and a major cause of type 2 diabetes. Overnutrition due to a high fat diet and increased caloric intake causes metabolic imbalance and develops metabolic disorder [1, 2]. Since the majority of the patients with type 2 diabetes are obese, a positive correlation between obesity and insulin resistance has been suggested [3]. In humans, plasma free fatty acid (FFA) and triacylglycerol (TAG) levels are elevated and show a linear correlation with blood glucose and hepatic glucose production [4–6]. From these reasons, a mechanism has gained credibi
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