Differential Regulation of Fatty Acid Composition by AKT Isoforms in Hepatocellular Carcinoma Cell Lines

Background/Objectives: AKT signaling is a central regulator of cancer metabolism; however, the isoform-specific roles of AKT1, AKT2, and AKT3 in fatty acid metabolism remain incompletely understood in hepatocellular carcinoma (HCC). This study aimed to investigate the contributions of individual AKT isoforms to fatty acid regulation in an HCC cells model. Methods: Stable knockdown models of AKT1, AKT2, and AKT3 were established in Mahlavu cells, whereas stable knockdown models of AKT1 and AKT2 were established in Huh7 cells because endogenous AKT3 expression was very low. Targeted fatty acid profiling was performed using gas chromatography–mass spectrometry (GC–MS) to evaluate alterations in fatty acid composition following AKT isoform knockdown. Results: Knockdown of AKT1, AKT2, and AKT3 in Mahlavu cells, and AKT1 and AKT2 in Huh7 cells, was associated with reduced relative abundance of several common fatty acids, including myristic acid (C14:0), myristoleic acid (C14:1), 7-hexadecenoic acid (C16:1), arachidic acid (C20:0), eicosenoic acid (C20:1), and erucic acid (C22:1). In addition, isoform-specific differences in fatty acids composition were also observed in both HCC cell lines. Compared with scrambled control cells, knockdown of AKT1 or AKT3, but not AKT2, was associated with significant reductions in palmitic acid (C16:0) and stearic acid (C18:0) in Mahlavu cells. In contrast, Huh7 cells exhibited distinct changes in fatty acid composition, showing unchanged or increased relative abundance of palmitic acid (C16:0) and stearic acid (C18:0) following AKT1 or AKT2 knockdown. Moreover, in Huh7 cells very-long-chain fatty acids (VLCFAs), behenic acid (C22:0) and nervonic acid (C24:1) were significantly reduced following AKT2 knockdown, but not AKT1 knockdown. Conclusions: These findings suggest that AKT isoforms play a role regarding fatty acid composition in HCC cells, with both shared and isoform-associated patterns which should be investigated in further studies.