Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells

Abstract

Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells. Am J Physiol Gastrointest Liver Physiol 307: G66-G76, 2014. First published April 24, 2014 doi:10.1152 doi:10. /ajpgi.00335.2013 was recently associated with the susceptibility to nonalcoholic fatty liver disease, a common cause of chronic liver disease characterized by abnormal triglyceride accumulation. Although it is established that PNPLA3 has both triacylglycerol lipase and acylglycerol O-acyltransferase activities, is still unknown whether the gene has any additional role in the modulation of the human liver metabolome. To uncover the functional role of PNPLA3 on liver metabolism, we performed high-throughput metabolic profiling of PNPLA3 siRNA-silencing and overexpression of wild-type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells. Metabolomic analysis was performed by using GC/MS and LC/MS platforms. Silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown. A significant decrease in amino-and ␥-glutamyl-amino acids and dipeptides and a significant increase in cysteine sulfinic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed. Overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing, but in the opposite direction. These findings were replicated by the exploration of canonical pathways associated with PNPLA3 silencing and Met148 overexpression. Overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling.PNPLA3; metabolome; nonalcoholic fatty liver disease; Huh-7 cells; Ile148Met variant; systems biology THE PROTEIN ENCODED BY THE patatin-like phospholipase domain containing 3 (PNPLA3) gene, also known as adiponutrin, is a multifunctional enzyme that belongs to the IPLA2/lipase (calcium-independent phospholipase A2-epsilon) family, which has both triacylglycerol lipase and acylglycerol O-acyltransferase activities (7).

PNPLA3 gene in liver diseases

“…These liposoluble micronutrients are known to influence cell proliferation and differentiation [93], and have been shown to be effective in liver cancer prevention trials [94]. In addition, in vitro overexpression of the 148M mutation in Huh-7 cell lines was associated with a 1.75 fold increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction [95], a hallmark observed in many cancer cells [96]. Furthermore, another study in humans observed that the lactate-to-pyruvate ratio was increased in rs738409 [G] homozygotes with NAFLD compared to healthy controls.…”

Genetic Basis of Alcoholic and Nonalcoholic Fatty Liver Disease

“…Interestingly, overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid in comparison with the empty vector, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling [ 31 ] and could explain the role of PNPLA3 in disease progression. …”

“…Of note, the silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown; a signifi cant decrease in amino-and gamma-glutamyl amino acids and dipeptides and a signifi cant increase in cysteine sulfi nic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed [ 31 ]. On the other hand, overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing but in the opposing direction.…”

“…Figure 7.3 depicts the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway of glycerolipid metabolism , in which PNPLA3 involvement is highlighted. Nevertheless, the role of PNPLA3 in the liver seems to be broader than adipose tissue, involving hepatocyte triacylglycerol remodeling [ 18 , 30 ] and global metabolic changes [ 31 ].…”

“…Finally, a recent study uncovered the functional role of PNPLA3 on liver metabolism by performing high-throughput metabolic profi ling of PNPLA3 siRNA silencing and overexpression of wild-type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells [ 31 ]. Of note, the silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown; a signifi cant decrease in amino-and gamma-glutamyl amino acids and dipeptides and a signifi cant increase in cysteine sulfi nic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed [ 31 ].…”