Inhibition of the mTORC1/NF-κB Axis Alters Amino Acid Metabolism in Human Hepatocytes

Meng Zhang ,Yuting Fu,Zhigang Wang,Yanfeng Wang,Yuze Ma,Hao Huang ,Yuhao Chen ,Zhixin Guo ,Qihua Fu

doi:10.1155/2021/8621464

Abstract

In addition to serving as the building blocks for protein synthesis, amino acids can be used as an energy source, through catabolism. The transamination, oxidative deamination, and decarboxylation processes that occur during amino acid catabolism are catalyzed by specific enzymes, including aspartate aminotransferase (AST), glutamate dehydrogenase (GDH), glutamic acid decarboxylase (GAD), and ornithine decarboxylase (ODC); however, the overall molecular mechanisms through which amino acid catabolism occurs remain largely unknown. To examine the role of mechanistic target of rapamycin complex 1 (mTORC1) on amino acid catabolism, mTORC1 was inactivated by rapamycin or shRNA targeting Raptor, versus activated by overexpressing Rheb or amino acids in human hepatocytes. The expression of amino acid catabolic genes and related transcription factor was investigated by RT/real-time PCR and western blot analysis. A few types of amino acid metabolite were examined by ELISA and HPLC analysis. The data showed that inactivated mTORC1 resulted in inhibition of NF-κB and the expression of AST, GDH, GAD, and ODC, whereas activated mTORC1 enhanced NF-κB activation and the expression levels of the catabolism-associated genes. Further, inhibition of NF-κB reduced the expression levels of AST, GDH, GAD, and ODC. mTORC1 upregulated NF-κB activation and the expression of AST and ODC in response to glutamate and ornithine treatments, whereas rapamycin inhibited the utilization of glutamate and ornithine in hepatocytes. Taken together, these results indicated that the mTORC1/NF-κB axis modulates the rate of amino acid catabolism by regulating the expression of key catabolic enzymes in hepatocytes.

Highlights

The availability of nutrients and energy is critical for cellular growth and proliferation
To verify nuclear factor (NF-)κB directs the expression of amino acid catabolismassociated genes, we predicted the transcription factor binding sites (TFBS) and the transcription factor binding motifs (TFBM) of NF-κB by bioinformatics analysis, and the TFBS and TFBM of NF-κB in the promoter sequence of the AST, glutamate dehydrogenase (GDH), glutamic acid decarboxylase (GAD), and ornithine decarboxylase (ODC) were found (Figure S3, Figure S4)
The results showed that the inhibition of mechanistic target of rapamycin complex 1 (mTORC1) signal pathway by rapamycin reduces the phosphorylation level of IKKα, reducing the phosphorylation level of NF-κB and attenuated degradation of IκBα (Figures 1(a) and 1(b)) (p < 0:05), and the mRNA levels of AST, GDH, GAD, and ODC and the corresponding intracellular enzyme levels were significantly decreased by rapamycin treatment (Figures 1(c) and 1(d)) (p < 0:01)

Summary

Introduction

The availability of nutrients and energy is critical for cellular growth and proliferation. The primary amino acid catabolic pathways include transamination, oxidative deamination, and decarboxylation, which are catalyzed by specific enzymes, including aspartate aminotransferase (AST), glutamate dehydrogenase (GDH), glutamic acid decarboxylase (GAD), and ornithine decarboxylase (ODC), in liver cells. Glutamate can be converted into α-ketoglutarate via oxidative deamination, which is catalyzed by GDH [5]. The liver is rich in GDH, which catalyzes the reversible oxidative deamination of glutamate into α-ketoglutarate and ammonia, bridging the amino acid-to-glucose pathway [6,7,8]. A nonproteinogenic amino acid that is not utilized during de novo protein synthesis, is derived from arginine, via citrulline, and the decarboxylation of ornithine by ODC results in the generation of putrescine in mammalian cells [10, 11]. Amino acid catabolism enzymes have been associated with liver diseases

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