Mitochondrial General Control of Amino Acid Synthesis 5 Like 1 Regulates Glutaminolysis, Mammalian Target of Rapamycin Complex 1 Activity, and Murine Liver Regeneration

Abstract

Background and Aims The regenerative capacity of the liver plays a protective role against hepatotoxins and impaired regeneration exacerbates liver dysfunction in nonalcoholic fatty liver disease (NAFLD). Mitochondrial bioenergetic and ‐synthetic functions are important contributory factors in hepatic regeneration, and the control of mitochondrial protein acetylation is implicated in the mitochondrial susceptibility to liver stressors. Here, we evaluated the role of general control of amino acid synthesis 5 like 1 (GCN5L1), a mediator of mitochondrial metabolism and acetylation, in modulating murine liver regeneration (LR) in response to acute CCl4‐induced hepatotoxicity. Approach and Results Initial metabolomic screening found that liver GCN5L1 knockout (LKO) mice have augmented glutaminolysis. Absence of GCN5L1 modified enzyme activity of liver‐enriched glutaminase enzyme (glutaminase 2; GLS2), and GCN5L1 levels modulated GLS2 oligomerization and acetylation. This metabolic remodeling resulted in the elevation of α‐ketoglutarate levels, which are known to activate mammalian target of rapamycin complex 1 (mTORC1). This signaling pathway was induced with increased phosphorylation of S6 kinase in LKO hepatocytes, and inhibition of glutaminolysis reversed aberrant mTORC1 signaling. At the same time, glutaminolysis, activity of GLS2, and activation of mTORC1 signaling were reversed by the genetic reintroduction of the mitochondrial isoform of GCN5L1 into LKO primary hepatocytes. Finally, LKO mice had a more robust regenerative capacity in response to CCl4 hepatoxicity, and this response was blunted by both the mTORC1 inhibitor, rapamycin, and by pharmacological blunting of glutaminolysis. Conclusions These data point to a central role of glutaminolysis in modulating the regenerative capacity in the liver. Furthermore, inhibition of mitochondrial GCN5L1 to augment LR may be a useful strategy in disease states linked to hepatotoxicity.

Mitochondrial GCN5L1 regulates glutaminase acetylation and hepatocellular carcinoma

“… 21 Here, we tested whether ablation of GCN5L1 had the same metabolic profile in human HepG2 cells following GCN5L1 KO versus control cells cultured in Earle's Balanced Salt Solution (EBSS) with glutamine supplementation for 2 or 4 hours. Consistent with GCN5L1 LKO primary hepatocytes, 21 mTORC1 signalling was highly activated by glutamine in GCN5L1 KO HepG2 cells (Figure 3F ). Given that glutamine metabolism is critical to maintain GSH levels and cellular redox control, 26 which regulates apoptosis, we analysed cell apoptosis in HepG2 cells following GCN5L1 depletion.…”

“…The levels of GCN5L1 and mitochondrial protein acetylation are significantly decreased during mouse liver regeneration. 21 To examine the expression levels of GCN5L1 in HCC, we analysed HCC tumours and adjacent liver tissues from the combined DEN and CCl 4 model (Figure S1A ). We performed immunoblot analyses of tumours and their adjacent normal liver tissues and found that GCN5L1 protein levels were decreased in DEN‐induced HCC tumours in comparison with adjacent liver tissues (Figure S1B ).…”

“…Loss of GCN5L1 has been reported to activate mTORC1 by enhancing glutaminolysis during murine liver regeneration. 21 Here, we tested whether ablation of GCN5L1 had the same metabolic profile in human HepG2 cells following GCN5L1 KO versus control cells cultured in Earle's Balanced Salt Solution (EBSS) with glutamine supplementation for 2 or 4 hours. Consistent with GCN5L1 LKO primary hepatocytes, 21 mTORC1 signalling was highly activated by glutamine in GCN5L1 KO HepG2 cells (Figure 3F ).…”

“…Glutaminase activity assays were previously described. 21 Briefly, mitochondria from HepG2 cells or HCC samples were used for endogenous glutaminase activity assays. To test the efficiency of glutaminase inhibitors, a plasmid encoding human GLS1 or GLS2 was transfected into 293T cells, and 2 h before cell harvest, inhibitors were incubated with the cells.…”

“…Loss of GCN5L1 activates GLS2 activity by reducing its acetylation to promote glutaminolysis, followed by mTORC1 activation to support hepatocyte growth. 21 GLS1 and GLS2 share sequence homology, especially in their catalytic domains. Thus, we hypothesised that GCN5L1 may regulate both GLS1 and GLS2 acetylation and activity to modulate glutaminolysis and thereby modulate the development of HCC.…”