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A comparison between different human hepatocyte models reveals profound differences in net glucose production, lipid composition and metabolism in vitro.

Abstract (Expand)

Hepatocytes are responsible for maintaining a stable blood glucose concentration during periods of nutrient scarcity. The breakdown of glycogen and de novo synthesis of glucose are crucial metabolic … pathways deeply interlinked with lipid metabolism. Alterations in these pathways are often associated with metabolic diseases with serious clinical implications. Studying energy metabolism in human cells is challenging. Primary hepatocytes are still considered the golden standard for in vitro studies and have been instrumental in elucidating key aspects of energy metabolism found in vivo. As a result of several limitations posed by using primary cells, a multitude of alternative hepatocyte cellular models emerged as potential substitutes. Yet, there remains a lack of clarity regarding the precise applications for which these models accurately reflect the metabolic competence of primary hepatocytes. In this study, we compared primary hepatocytes, stem cell-derived hepatocytes, adult donor-derived liver organoids, immortalized Upcyte-hepatocytes and the hepatoma cell line HepG2s in their response to a glucose production challenge. We observed the highest net glucose production in primary hepatocytes, followed by organoids, stem-cell derived hepatocytes, Upcyte-hepatocytes and HepG2s. Glucogenic gene induction was observed in all tested models, as indicated by an increase in G6PC and PCK1 expression. Lipidomic analysis revealed considerable differences across the models, with organoids showing the closest similarity to primary hepatocytes in the common lipidome, comprising 347 lipid species across 19 classes. Changes in lipid profiles as a result of the glucose production challenge showed a variety of, and in some cases opposite, trends when compared to primary hepatocytes.

Authors: F. Bonanini, M. Singh, H. Yang, D. Kurek, A. C. Harms, A. Mardinoglu, T. Hankemeier

Date Published: 1st Apr 2024

Publication Type: Journal

Development of HILIC-MS/MS method for acyl-CoAs covering short- to long-chain species in a single analytical run

Abstract (Expand)

Acyl-CoAs play a significant role in numerous physiological and metabolic processes making it important to assess their concentration levels for evaluating metabolic health. Considering the important …he important role of acyl-CoAs, it is crucial to develop an analytical method that can analyze these compounds. Due to the structural variations of acyl-CoAs, multiple analytical methods are often required for comprehensive analysis of these compounds, which increases complexity and the analysis time. In this study, we have developed a method using a zwitterionic HILIC column that enables the coverage of free CoA and short- to long-chain acyl-CoA species in one analytical run. Initially, we developed the method on a QTOF instrument for the identification of acyl-CoA species, optimizing their chromatography and retention times. Later, a targeted HILIC-MS/MS method was created in scheduled multiple reaction monitoring mode on a QTRAP instrument. The performance of the method was evaluated based on various parameters such as linearity, precision, recovery and matrix effect. This method was applied to identify the difference in acyl-CoA profiles in HepG2 cells cultured in different conditions. Our findings revealed an increase in levels of acetyl-CoA, medium- and long-chain acyl-CoA while a decrease in the profiles of free CoA in the starved state, indicating a clear alteration in the fatty acid oxidation process.

Authors: Madhulika Singh, Ligia Akemi Kiyuna, Christoff Odendaal, Barbara M. Bakker, Amy C Harms, Thomas Hankemeier

Date Published: 11th Sep 2023

Publication Type: Journal

Development of a targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry based lipidomics platform applied to a coronavirus disease severity study.

Abstract (Expand)

The importance of lipids seen in studies of metabolism, cancer, the recent COVID-19 pandemic and other diseases has brought the field of lipidomics to the forefront of clinical research. Quantitative … and comprehensive analysis is required to understand biological interactions among lipid species. However, lipidomic analysis is often challenging due to the various compositional structures, diverse physicochemical properties, and wide dynamic range of concentrations of lipids in biological systems. To study the comprehensive lipidome, a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)-based screening method with 1200 lipid features across 19 (sub)classes, including both nonpolar and polar lipids, has been developed. HILIC-MS/MS was selected due to its class separation property and fatty acyl chain level information. 3D models of class chromatographic retention behavior were established and evaluations of cross-class and within-class interferences were performed to avoid over-reporting these features. This targeted HILIC-MS/MS method was fully validated, with acceptable analytical parameters in terms of linearity, precision, reproducibility, and recovery. The accurate quantitation of 608 lipid species in the SRM 1950 NIST plasma was achieved using multi-internal standards per class and post-hoc correction, extending current databases by providing lipid concentrations resolved at fatty acyl chain level. The overall correlation coefficients (R(2)) of measured concentrations with values from literature range from 0.64 to 0.84. The applicability of the developed targeted lipidomics method was demonstrated by discovering 520 differential lipid features related to COVID-19 severity. This high coverage and targeted approach will aid in future investigations of the lipidome in various disease contexts.

Authors: Z. Zhang, M. Singh, A. Kindt, A. B. Wegrzyn, M. J. Pearson, A. Ali, A. C. Harms, P. Baker, T. Hankemeier

Date Published: 31st Aug 2023

Publication Type: Journal

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