mTORC1/NF-κB axis controls amino acid catabolism by regulating the expression of the key enzymes in human hepatocytes

In addition to serving as building blocks for protein synthesis, amino acids also provide energy and precursors that are used by cells through catabolism. Mechanistic target of rapamycin complex 1 (mTORC1) is a central coordinator of cellular metabolism. However, little is known regarding the function of mTORC1 in amino acid catabolism. The aims of this study were to explore the mechanism by which mTORC1 controls the conversion of glutamate to α-ketoglutarate and ornithine to putrescine, and mTORC1 regulates the expression amino acid catabolism-related genes in hepatocyte. HL-7702 were treated with ornithine, rapamycin or SC75741, alone or in combination; the plasmids pRNAT-U6.1/Neo-shRaptor and pIRES2-EGFP-Rheb were transfected into HL-7702 cells to silencing Raptor or overexpressing Rheb . The intracellular content of glutamate, oxaloacetate, α-ketoglutaric acid, and aspartic acid, and the intracellular level of aspartate aminotransferase (AST), ornithine decarboxylase (ODC), glutamate dehydrogenase (GDH), and glutamic acid decarboxylase (GAD) were measured by ELISA. The concentrations of intracellular ornithine and putrescine were measured by HPLC. The mRNA level of amino acid catabolism-related genes was detected by qRT-PCR, and the protein level of mTORC1 and NF-κB was investigated by western blot. Our results demonstrate that mTORC1 regulates amino acid catabolism by inducing the expression of AST , ODC , GDH , and GAD , which is mediated by NF-κB. This finding constitutes a novel mechanism by which amino acid catabolism is regulated in hepatocytes. amino acid catabolic gene expression. We found that rapamycin or Raptor silencing inhibited the expression of amino acid catabolic genes and the activation of NF-κB in HL-7702 cells. The serum and amino acid starvation significantly decreased the activation of NF-κB, but the intracellular agonist glutamate and ornithine, or Rheb ovexpression, significantly enhanced its activation. The data from hepatocytes HL-7702, which were based on active and inactive forms of mTORC1 and phosphorylation or dephosphorylation of transcription factor NF-κB, indicate that the expression of AST, GDH, GAD, and ODC is regulated by

cells were pretreated with 100 nM rapamycin for 8 hours. Three groups-control, ornithine, and ornithine with rapamycin-were established. HL-7702 cells were collected and dissolved in 1 mL of lysis buffer, and protein concentration was determined.
For the analysis of putrescine, the protein samples were treated with n-hexane to remove lipids, and the mixture was extracted with N-butanol/trichloromethane (1:1 v/v ratio), which the extracting agent was removed by aspiration and evaporation to dryness under a stream of nitrogen at 40°C. Samples were then dissolved in 0.1 mM HCl for derivatization. For derivatization, the mixture was combined with dansyl chloride, which was then aspirated and evaporated to dryness under a stream of nitrogen at 40°C. Samples were then dissolved in 1 mL methanol for HPLC analysis with a C18 column (150 mm × 4.6 mm, 5 µm) at 30°C. The mobile phase contained A (methanol) and B (water), which was used according to the following program: 0 min, 55% A; 7 min, 65% A; 14 min, 70% A; 20 min, 70% A; 27 min, 90% A; 30 min, 100% A. The flow rate was 1.5 ml/min, and the injection volume was 20 μL.
Putrescine was tentatively identified by comparing its retention time with that of authentic standards under identical analysis conditions at 254 nm.
For the detection of ornithine, 200 μL of each protein sample was mixed with 10 μL of 1.0 mg/mL norleucine, 100 μL of 1 mM triethylamine-acetonitrile solution, and 100 μL of 0.1 mM phenyl isothiocyanate-acetonitrile solution and let stand at room temperature for 1 hour. Next, 400 μL nhexane was added, and the mixture was left standing for 10 min, and the lower clear solution was passed through a 0.45-μm filter. Next, 2.0 μL of each sample was injected into the HPLC system with a DIONEX Acclaim 120 C18 column (250 mm × 4.6 mm, 5 µm) at 40°C (Thermo Fisher Scientific Inc.,

Western blot analysis
Cells were harvested with trypsin, washed with cold phosphate-buffered saline, and lysed in cell lysis buffer. The cells were then placed on ice for 10 min and centrifuged at 10625 g RCF at 4°C for 10 min.
Lysate protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, USA).
Equal amounts (40 μg) of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% gels (w/v)), transferred to polyvinylidene fluoride (PVDF) membranes, and incubated with the primary antibody overnight at 4°C. Membranes were then incubated with the peroxidase-conjugated secondary antibody for 1 hour at room temperature.
Enhanced chemiluminescence (ECL) reagent (Amersham) was used with the Western Blotting System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) to detect proteins of interest. Protein bands were quantified on a Gel-Pro Analyzer 4.0 (Media Cybernetics, USA).

RT-qPCR analysis
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to determine mRNA levels of AST, GDH, ODC, and GAD and the Raptor, Rheb in HL-7702 cells of treatment and control groups. Total RNA from the untreated and treated cells was reverse-transcribed with an oligo (dT)12-18 primer using the AMV 1st Strand cDNA Synthesis Kit (Takara Co. Ltd., China).
cDNA sequences were amplified with the primers shown in Table S1. The reactions were run using the KAPA SYBP® FAST qPCR Kit optimized for LightCycler® 480 (KAPA BIOSYSTEMS, Inc, Boston, Massachusetts, USA) according to the manufacturer's instructions. One microliter of cDNA was amplified in a 25-μL reaction that contained 10 μM forward primer (0.5 μL), 10 μM reverse primer (0.5 μL), SYBR Premix Ex Taq (12.5 μL), and nuclease-free water (10.5 μL). Cycling conditions consisted of an initial denaturation step at 95°C for 5 min, then 40 cycles at 95°C for 5 sec, 54°C for 30 sec, and 72°C for 20 sec, followed by a final extension at 72°C for 10 min. Three technical replicates were performed per sample. 2 -ΔΔCT values were calculated to determine expression levels, and the qPCR results were analyzed by student's t-test to compare expression levels between untreated and treated groups. 3 independent experiments were performed.

In vitro transfection
The plasmids pRNAT-U6.1/Neo-shRaptor and pIRES2-EGFP-Rheb were transfected into HL-7702 cells using Lipofectamine TM2000 (Invitrogen, Carlsbad, New Mexico, USA) per the manufacturer's instructions. Transfectants were selected by culturing cells in the presence of G418 (Hyclone Laboratories, Inc. Logan, Utah, USA) for 48 hours and were imaged using a ZEISS AX10 fluorescence microscope (Carl Zeiss Microscopy, Thornwood, NY, USA), and then cells were collected. For ELISA assay, cell lysates were prepared by 5 freeze-thaw cycles; for western blot analysis, cells lysates were prepared by lysed in cell lysis buffer.

Statistical Analyses
Statistical analyses were conducted using SPSS PASW Statistics for Windows, v18.0 (SPSS Inc., Chicago, IL, USA). Data were analyzed using standard parametric statistics, one-way ANOVA, followed by Tukey's method. Data are expressed as mean ± SD. Results are presented as the average of at least 3 independent experiments unless stated otherwise. Statistical significance was accepted when p≤0.05.

Rapamycin inhibits catabolism of glutamate and ornithine in HL-7702 cells
To determine whether mTORC1 regulates the conversion between substrate and product in amino acid catabolism, the inhibitory effect of rapamycin on glutamate or ornithine catabolism was examined in HL-7702 cells. HL-7702 cells were starved, which were then divided into 3 groups: control, glutamate, and glutamate with rapamycin. The concentration of intracellular glutamate, oxaloacetate, α-ketoglutaric acid, and aspartic acid was measured by ELISA. As shown in Fig. 1a, the intracellular glutamate concentration in the glutamate group was significantly higher than that in the control group (p < 0.01). The intracellular glutamate concentration of the glutamate with rapamycin group was significantly higher versus the glutamate group (p < 0.05). These data indicate that exogenous glutamate was absorbed by starved cells and that rapamycin reduced their utilization of glutamate.
Furthermore, the intracellular oxaloacetate concentration of the glutamate group was significantly lower than that in the control group (p < 0.01). The intracellular oxaloacetate concentration of the glutamate with rapamycin group was significantly higher compared with the glutamate group (p < 0.01). These data indicate that intracellular oxaloacetate was utilized and that rapamycin blocked this utilization. The intracellular concentration of α-ketoglutaric acid and aspartic acid was higher in the glutamate group compared with the control group (p < 0.01) and no change in the glutamate with rapamycin group (p > 0.05). These results suggest that rapamycin prevents the accumulation of αketoglutaric acid and aspartic acid.
Ornithine is converted to polyamine by ODC in cancer cells [21]. To characterize the inhibitory effects of rapamycin on ornithine catabolism, HL-7702 cells were starved, which were then divided into 3 groups: control, ornithine, and ornithine with rapamycin. The concentrations of intracellular ornithine and putrescine were measured by HPLC. The intracellular ornithine concentration of the ornithine group was significantly higher than that in the control group (Fig. 1b) (p < 0.05) and the ornithine content of the ornithine with rapamycin group was significantly higher than that in the ornithine group (p < 0.05). These data indicate that exogenously added ornithine was absorbed by starved cells and that rapamycin blocked ornithine utilization.
Glutamate and ornithine promote AST and ODC expression via activation of mTORC1 and NF-κB in HL-

cells
Transamination between glutamate and α-ketoglutaric acid can be catalyzed by AST to produce oxaloacetate and aspartic acid, and the decarboxylation of ornithine to putrescine can be catalyzed by ODC; moreover, NF-κB has recently attracted attention as functioning in metabolic disorders [16,17], thus, we speculated that AST and ODC expression is regulated by mTORC1 via NF-κB. To evaluate whether glutamate or ornithine can promote AST and ODC expression by mTORC1 via NF-κB, we first examined the effect of glutamate or ornithine on the activation of mTORC1 signaling and on NF-κB. Glutamate or ornithine was added to starve HL-7702 cells, and phosphorylation of S6 and 4EBP1 was assessed, which are phosphorylated in an mTORC1-dependent manner. Phosphorylation of NF-κB was also measured. The results showed that glutamate significantly increased the phosphorylation of S6, 4EBP1, and NF-κB p65 ( Fig. 2a and 2b) compared with the starved group.
Ornithine increased the phosphorylation of mTOR, 4EBP1, and NF-κB ( Fig. 2c and 2d). As a result, expression of AST and ODC was increased by glutamate or ornithine in mRNA and protein levels The results showed that rapamycin significantly inhibited activation of mTORC1 and NF-κB ( Fig. 3a and 3b) (p < 0.05), and the mRNA level of AST, GDH, GAD, and ODC and the corresponding intracellular enzyme levels were significantly decreased by rapamycin ( Fig. 3c and 3d) (p < 0.01).
These data suggest that mTORC1 and NF-κB are associated with the expression of these catabolic genes.
To further evaluate the effect of rapamycin on AST, GDH, GAD, and ODC expression, the degree of mTORC1 activation was reduced by knocking down Raptor, a critical component of mTORC1, using targeting shRNA (Fig. S1) in HL-7702 cells, and then phosphorylation of S6, 4EBP1, and NF-κB was measured. The phosphorylation of all targets was inhibited by Raptor silencing (Fig. 4a, 4b). The levels of metabolic gene expression and intracellular enzymes were also measured. The expression pattern of the genes was similar to that in the rapamycin-treated group (Fig. 4c, 4d) (p < 0.01). These results further demonstrate that mTORC1 and NF-κB are involved in catabolic gene expression.
To conform the function NF-κB in the expression of AST, GDH, GAD, and ODC, we used SC75741, a specific inhibitor of NF-κB, to inhibit NF-κB activation in HL-7702 cells, and then the levels of metabolic gene expression and intracellular enzymes were measured. The results showed that NF-κB activation was inhibited by the inhibitor (Fig. 5a), and the levels of AST, GDH, GAD, and ODC mRNA, and the corresponding intracellular enzyme were significantly decreased ( Fig. 5b and 5c, respectively) (p < 0.01), suggesting that NF-κB directs the expression of AST, GDH, GAD, and ODC.

Enhanced mTORC1 activation upregulates amino acid metabolic genes expression in HL-7702 cells
To complement the results that Raptor silencing decreases mTORC1 activation and expression of AST, GDH, GAD and ODC, we cloned and overexpressed Rheb, an upstream positive effector of mTORC1, in HL-7702 cells to enhance mTORC1 activation (Fig. S2). We also measured AST, GDH, GAD and ODC expression and the concentration of the corresponding enzymes. Rheb overexpression upregulated mTORC1 signaling (Fig. 6a) and NF-κB phosphorylation (Fig. 6b), and the expression of these catabolic genes was enhanced both in mRNA level (Fig. 6c) (p < 0.01) and in protein level (Fig. 6d) (p < 0.01).
These results indicate that the expression of these catabolic genes and NF-κB activation are increased by greater activation of mTORC1.
To further verify mTORC1 regulates the expression of AST, GDH, GAD and ODC through NF-κB, the Rheb-over expressed HL-7702 cells were treated with 10 µM SC75741 for 12 h, and then NF-κB phosphorylation and expression of the amino acid catabolic genes were determined. The results showed that NF-κB activation was enhanced by Rheb over expression and inhibited by SC75741 (Fig. 7a), and the expression pattern of the genes was similar to phosphorylation of transcription factor NF-κB (Fig. 7b). These results indicate that mTORC1 controls the expression of these catabolic genes via NF-κB in HL-7702.

Discussion
Amino acid catabolism supplies energy and precursors for the synthesis of macromolecules and to support cellular function, for which key enzymes play an important role. In this study, we demonstrated that mTORC1 regulates the expression of amino acid-catabolic genes in HL-7702 cells.
Two recent reports showed that plasma AST levels are regulated by mTORC1 in rats [13,22]. GDH activity is related to mTORC1 activity in ovarian cancer cells [14], and prolactin induces ODC expression via mTOR signaling in mink uterine epithelial cells [23]; however, the relationship between GAD and mTOR signaling has not been reported. In our study, the expression of AST, GDH, GAD, and ODC was inhibited by rapamycin and Raptor silencing. Additionally, intracellular levels of AST, GDH, GAD, and ODC were regulated by mTORC1. Further, the conversion of glutamate to α-ketoglutarate and ornithine to putrescine were controlled by mTORC1.
A recent report showed that cooperative NF-κB/STAT3 signaling functions in lymphoma metabolic reprogramming and aspartate transaminase (GOT2) gene expression [19]. In our previous study, we found that mTORC1 regulates peptidoglycan-induced inflammation via NF-κB in murine macrophages [24]. In the present study, we focused on the regulation of NF-κB in amino acid catabolic gene expression. We found that rapamycin or Raptor silencing inhibited the expression of amino acid catabolic genes and the activation of NF-κB in HL-7702 cells. The serum and amino acid starvation significantly decreased the activation of NF-κB, but the intracellular agonist glutamate and ornithine, or Rheb ovexpression, significantly enhanced its activation. The data from hepatocytes HL-7702, which were based on active and inactive forms of mTORC1 and phosphorylation or dephosphorylation of transcription factor NF-κB, indicate that the expression of AST, GDH, GAD, and ODC is regulated by mTORC1/NF-κB axis. Thus, we conclude that mTORC1 regulates the expression of amino acid catabolic genes is via NF-κB.
In the present study, ornithine induced ODC expression, which was regulated by mTORC1, and the utilization of ornithine in HL-7702 cells was significantly blocked by rapamycin. However, the content of putrescine has no significant difference between ornithine group and glutamate with rapamycin group, meaning rapamycin has no effect on putrescine accumulation. In fact, putrescine can stimulate