Metabolome response to glucose in the ß-cell line INS-1 832/13.

Glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 to 10 mM glucose and in response to increasing concentrations of glucose in the INS-1 832/13 ß-cell line. U-[(13)C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.

Analysis of fatty acid composition in insulin secreting cells by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry.

A comprehensive two-dimensional gas chromatography (GC×GC) time-of-flight mass spectrometry method was developed for determination of fatty acids (irrespective of origin, i.e., both free fatty acids and fatty acids bound in sources such as triglycerides) in cultured mammalian cells. The method was applied to INS-1 cells, an insulin-secreting cell line commonly used as a model in diabetes studies. In the method, lipids were extracted and transformed to fatty acid methyl esters for analysis. GC×GC analysis revealed the presence of 30 identifiable fatty acids in the extract. This result doubles the number of fatty acids previously identified in these cells. The method yielded linear calibrations and an average relative standard deviation of 8.4% for replicate injections of samples and 12.4% for replicate analysis of different samples. The method was used to demonstrate changes in fatty acid content as a function of glucose concentration on the cells. These results demonstrate the utility of this method for analysis of fatty acids in mammalian cell cultures.

Reducing time and increasing sensitivity in sample preparation for adherent mammalian cell metabolomics.

A simple, fast, and reproducible sample preparation procedure was developed for relative quantification of metabolites in adherent mammalian cells using the clonal ß-cell line INS-1 as a model sample. The method was developed by evaluating the effect of different sample preparation procedures on high performance liquid chromatography- mass spectrometry quantification of 27 metabolites involved in glycolysis and the tricarboxylic acid cycle on a directed basis as well as for all detectable chromatographic features on an undirected basis. We demonstrate that a rapid water rinse step prior to quenching of metabolism reduces components that suppress electrospray ionization thereby increasing signal for 26 of 27 targeted metabolites and increasing total number of detected features from 237 to 452 with no detectable change of metabolite content. A novel quenching technique is employed which involves addition of liquid nitrogen directly to the culture dish and allows for samples to be stored at -80 °C for at least 7 d before extraction. Separation of quenching and extraction steps provides the benefit of increased experimental convenience and sample stability while maintaining metabolite content similar to techniques that employ simultaneous quenching and extraction with cold organic solvent. The extraction solvent 9:1 methanol: chloroform was found to provide superior performance over acetonitrile, ethanol, and methanol with respect to metabolite recovery and extract stability. Maximal recovery was achieved using a single rapid (∼1 min) extraction step. The utility of this rapid preparation method (∼5 min) was demonstrated through precise metabolite measurements (11% average relative standard deviation without internal standards) associated with step changes in glucose concentration that evoke insulin secretion in the clonal ß-cell line INS-1.