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Using dynamic gene module map analysis to identify targets that modulate free fatty acid induced cytotoxicity
Zheng Li1, Shireesh Srivastava, Robert Findlan
1Cellular and Molecular Biology Lab, Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan 48824, USA.
Abstract:
The objective of this study was to identify pathways that regulate the cytotoxicity induced by free fatty acids (FFAs) in human hepatoblastoma cells (HepG2/C3A). Gene expression profiles of HepG2/C3A cells were obtained at three time points, after 24, 48, and 72 h of exposure to different types of FFA. Saturated fatty acid (palmitate) was found to be cytotoxic. The pathways activated by the different FFAs at the different time points were identified using global gene module map analysis. Unsaturated FFAs exerted transcriptional regulation mainly within the first 24 h, whereas saturated FFA, palmitate, regulated energy production pathways, such as the electron transport chain (ETC) and tricarboxylic acid cycle, within the first 24 h. In the next 24 h, palmitate up-regulated 36 cell death relevant pathways and down-regulated several protective pathways, such as the pentose phosphate pathway and glutathione-related pathways. In the final 24 h, the FFAs did not induce significant transcriptional regulation. We hypothesized that palmitate induced cytotoxicity by first perturbing metabolic pathways in the initial 24 h, resulting in changes to factors, such as metabolites or signaling molecules, which subsequently triggered cell death relevant pathways in the next 24 h. The uptake and release of 27 metabolites were measured to further elucidate the metabolic changes in the first 24 h. It was determined that ketone bodies such as beta-hydroxybutyrate and acetoacetate were important in separating the toxic from the nontoxic phenotypes. A regression model was used to identify the genes relevant to these metabolites. Some of the genes identified to be important were experimentally validated. It was found that ETC genes such as NADH dehydrogenase and succinate dehydrogenase were involved in palmitate induced cytotoxicity.
Insights
Saturated fatty acids like palmitate induce cell death in liver cells by disrupting energy pathways and triggering cell death pathways. Ketone bodies and electron transport chain genes are key factors in this toxicity.
Area of Science:
- Cell Biology
- Metabolomics
- Molecular Biology
Background:
- Free fatty acids (FFAs) play crucial roles in cellular metabolism and signaling.
- Dysregulation of FFA metabolism is implicated in various pathologies, including liver diseases.
- Understanding FFA-induced cytotoxicity is vital for developing therapeutic strategies.
Purpose of the Study:
- To identify molecular pathways regulating cytotoxicity induced by free fatty acids (FFAs) in human hepatoblastoma cells (HepG2/C3A).
- To elucidate the temporal dynamics of gene expression changes in response to different FFAs.
- To investigate the role of metabolic perturbations in FFA-induced cell death.
Main Methods:
- Gene expression profiling of HepG2/C3A cells exposed to FFAs over 72 hours.
- Global gene module map analysis to identify activated pathways.
- Metabolite profiling (27 metabolites) and regression modeling to identify key genes.
- Experimental validation of identified genes, including electron transport chain (ETC) genes.
Main Results:
- Saturated fatty acid (palmitate) was cytotoxic, while unsaturated FFAs showed transient effects.
- Palmitate initially regulated energy production pathways (e.g., electron transport chain, tricarboxylic acid cycle) within 24 hours.
- Subsequently, palmitate upregulated 36 cell death pathways and downregulated protective pathways (e.g., pentose phosphate pathway) within 48 hours.
- Ketone bodies (beta-hydroxybutyrate, acetoacetate) differentiated toxic from non-toxic phenotypes.
- Electron transport chain genes (NADH dehydrogenase, succinate dehydrogenase) were validated as involved in palmitate-induced cytotoxicity.
Conclusions:
- Palmitate induces cytotoxicity by first perturbing metabolic pathways, leading to downstream activation of cell death pathways.
- Metabolic alterations, particularly involving ketone bodies, are critical early events in FFA-induced liver cell toxicity.
- Electron transport chain function is a key target in understanding and potentially mitigating palmitate-induced hepatotoxicity.
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