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.

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