Tracking biochemical changes induced by iron loading in AML12 cells with synchrotron live cell, time-lapse infrared microscopy

Abstract

Hepatocytes are essential for maintaining homeostasis of iron and lipid metabolism in mammals. Dysregulation of either iron or lipids has been linked with serious health consequences, including non- alcoholic fatty liver disease (NAFLD). Considered the hepatic manifestation of metabolic syndrome, NAFLD is characterised by dysregulated lipid metabolism leading to a lipid storage phenotype. Mild to moderate increases in hepatic iron have been observed in approximately 30% of individuals with NAFLD; however, direct observation of the mechanism behind this increase has remained elusive. To address this issue, we sought to determine the metabolic consequences of iron loading on cellular metabolism using live cell, time-lapse Fourier transform infrared (FTIR) microscopy utilising a synchrotron radiation source to track biochemical changes. Use of Synchrotron FTIR is non-destructive and label-free, and allowed observation of spatially-resolved, sub-cellular biochemical changes over a period of 8 hours. Using this approach, we have demonstrated that iron loading in AML12 cells induced perturbation of lipid metabolism congruent with steatosis development. Iron loaded cells had approximately three times higher relative ester carbonyl concentration compared to controls, indicating accumulation of triglycerides. The methylene/methyl ratio qualitatively suggests the acyl chain length of fatty acids in iron loaded cells increased over the 8 hour period of monitoring compared to a reduction observed in the control cells. Our findings provide direct evidence that mild to moderate iron loading in hepatocytes drives de novo lipid synthesis, consistent with a role for iron in the initial hepatic lipid accumulation that leads to development of hepatic steatosis.

Selenotranscriptome Network in Non-alcoholic Fatty Liver Disease

“…ACSL6 is more highly expressed in NASH than HOC, although it has previously been reported in several cell lines, including the hepatoma cell line HepG2, not to be associated with ferroptosis ( 67 ). Increases in acyl-CoA synthetases are consistent with our previous findings in AML12 mouse hepatocytes that lipid accumulation occurs within 16 hours of iron loading and continues to increase over the subsequent 8 hours ( 70 ). However, the mechanism behind differential regulation of three ACSL isoforms, all of which are associated with cell death pathways, remains unclear.…”

The Interplay of Iron and Lipid Homeostasis in Non-Alcoholic Fatty Liver Disease

“…Iron has been shown to have a role in altering hepatic equilibrium between lipid uptake, synthesis, oxidation, and export, leading to lipid accumulation due to impairment of mitochondrial β-oxidation gene and protein expression (35,36), and dysregulated lipid synthesis (37)(38)(39). In mice, we demonstrated that iron loading increased the expression of genes involved in the biosynthesis of cholesterol (40) and increased lipid polyunsaturation in a hepatic cell line, potentially increasing susceptibility to oxidative stress (41). This review focuses on the role of iron loading in the dysregulation of lipid metabolism and the consequences for the development and progression of NAFLD.…”

“…ELOVL2 elongates C20:4 to C22:4 and C20:5 to C22:5 and protein levels were increased in rats fed with a high fat diet plus ferric citrate, although no change in activity was evident (112). Nevertheless, increases in the ELOVL family of proteins were consistent with increases in synthesis of longer-chain fatty acids and may explain the increase in fatty acid chain length observed in AML12 cells incubated with ferric ammonium citrate (41). Elovl3 and Elovl5, which are believed to be involved in the elongation of multiple length fatty acids, exhibited reduced gene expression in mice fed with 2% carbonyl iron (38,71).…”

Synchrotron Infrared Microspectroscopy for Stem Cell Research

“…In recent decades, SR-FTIR-based microspectroscopy and imaging have been widely applied in stem cell-related research. A phenotypic screening approach revealed that the chemical information in single cells could be further studied [ 13 , 53 ]. SR-FTIR was applied here to identify changes related to molecular biomarkers in cancer stem cells.…”