Glucose promotes cell growth and casein synthesis via ATF4/Nrf2-Sestrin2- AMPK-mTORC1 pathway in dairy cow mammary epithelial cells

Abstract In the dairy industry, glucose (Glu) is used as bioactive substance to increase milk yield. However, the molecular regulation underneath needs further clarification. Here, the regulation and its molecular mechanism of Glu on cell growth and casein synthesis of dairy cow mammary epithelial cells (DCMECs) were investigated. When Glu was added from DCMECs, both cell growth, β-casein expression and the mechanistic target of rapamycin complex 1 (mTORC1) pathway were increased. Overexpression and silencing of mTOR revealed that Glu promoted cell growth and β-casein expression through the mTORC1 pathway. When Glu was added from DCMECs, both Adenosine 5'-monophosphate-activated protein kinase α (AMPKα) and Sestrin2 (SESN2) expression were decreased. Overexpression and silencing of AMPKα or SESN2 uncovered that AMPKα suppressed cell growth and β-casein synthesis through inhibiting mTORC1 pathway, and SESN2 suppressed cell growth and β-casein synthesis through activating AMPK pathway. When Glu was depleted from DCMECs, both activating transcription factor 4 (ATF4) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression were increased. Overexpression or silencing of ATF4 or Nrf2 demonstrated that Glu depletion promoted SESN2 expression through ATF4 and Nrf2. Together, these results indicate that in DCMECs, Glu promoted cell growth and casein synthesis via ATF4/Nrf2-SESN2-AMPK-mTORC1 pathway. Graphical Abstract


Introduction
Glucose (Glu) is one of the most effective nutrients for the rapid response to the metabolic activity of animals.It is the main source of energy for the body's metabolism. 1 In animal cells, Glu provides energy for cell metabolism, and in dairy cow mammary epithelial cells (DCMECs), Glu, as a crucial nutrient, is an important substrate for milk protein synthesis. 2Glu provides the material source and the main energy source for the milk protein synthesis in dairy cow mammary gland. 3][6] In dairy cow mammary gland, the milk protein synthesis is a complex biological process, and it is mainly regulated by hormones, nutrients and environmental factors, 4,7 and involves many signaling pathways. 8,9he Adenosine 5 0 -monophosphate (AMP)-activated protein kinase (AMPK) pathway and the mammalian target of rapamycin complex 1 (mTORC1) pathway are the two main signaling pathways that regulate protein synthesis in response to the changes of extracellular nutrients, such as amino acids and Glu. 10 Studies have shown that AMPK is a key energy sensor for the changes in Glu concentration in cells and it is necessary for the body to maintain Glu balance. 11In cells, the AMPK is phosphorylated and activated by Glu starvation, and then the phosphorylated AMPK inhibits the mTORC1 pathway and subsequent various cell physiological activities, including cell growth, protein synthesis, fat synthesis. 12Recent studies have shown that Glu, as a nutritional factor, regulates milk protein synthesis through the AMPK-mTORC1 pathways, 13,14 but the specific signaling pathways and molecular mechanisms remain unclear.
Sestrins are a family of highly conserved proteins.Sestrin2(SESN2) is a member of proteins family and it is induced upon various conditions of stress, including DNA damage, oxidative stress and glucose or amino acid starvation. 15SESN2 plays an important role in reducing the accumulation of reactive oxygen species (Ros), maintaining energy balance, enhancing autophagy, decreasing protein synthesis, and regulating cell growth. 16n this study, the regulation of Glu on cell growth and casein synthesis and its underlying molecular mechanism and pathway were explored.Our results revealed that Glu is an important nutritional factor for cell growth and casein synthesis.In DCMECs, Glu promoted cell growth and casein synthesis via ATF4/Nrf2-SESN2-AMPK-mTORC1 pathway.This study enhanced our understanding about the regulation of Glu on cell growth and casein synthesis and its molecular mechanism, and provided scientific experimental data for the artificial regulation of casein synthesis and application of Glu in the dairy industry.

Animal ethics
All the experimental procedures applied in this study were conducted according to the principles of Northeast Agricultural University (Harbin, China) and Shihezi University (Shihezi, China) Animal Care and Use Committee, which approved the study protocols.

Cell culture and treated
The primary DCMECs were obtained from our lab. 17riefly, the mammary gland tissue from lactating healthy Holstein was obtained by surgery.The mammary gland tissue was digested with 1 mg/mL collagenase, and the DCMECs were obtained.DCMECs cultured with Dulbecco's Modified Eagle's Medium (11960044, Gibco, California, USA) and Ham's F 12 nutrient medium (11765054, Gibco) (v/v ¼ 1:1, DMEM/F12) containing 10% fetal bovine serum (FBS; 10082147, Gibco), 100 U of penicillin and 100 U of streptomycin (C0222, Beyotime, Shanghai, China).The DCMECs used in current study were 10-15 generations.To identify the purified cells, the expression of cytokeratin 18 (CK18, a marker of epithelial cell) and b-casein (CSN2, one of the representative components of milk protein), and the secretion of TG were tested.
For the experiments of Glu supply, DCMECs were cultured in 6 well plates with DMEM/12 containing 10% FBS, 100 U of penicillin and 100 U of streptomycin.When the cells reached 80% confluence, they were starved with Glu (cells cultured with DMEM/F12 media without Glu) for 6 h, and then treated with Glu starvation (DMEM/F12 media without all of Glu, GluÀ), middle concentration Glu (DMEM/F12 media with 1,000 mg/L) and high concentration Glu (DMEM/F12 media with 4,500 mg/L, Gluþ) for 0, 6, 12, 18 and 24 h.
For the experiments of genes function, DEMECs treated with genes overexpression or silencing, and then cultured with GluÀ (0 mg/L) or Gluþ (4,500 mg/L) for 12 h.

Western blotting (WB)
The WB experiment was carried out according to the conventional method. 18Cells were tremated with GluÀ, Gluþ, or/and gene overexpression or silencing.The protein samples were collected with western and IP cell lysate (P0013, Beyotime) containing protease phosphatase inhibitor mixture (P1045, Beyotime) and quantitative analyzed with BCA protein assay kit (P0011, Beyotime) follow the operating instructions.About 25 lg of total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (FFP20, Beyotime).The PVDF membrane was blocked with Western blocking buffer (P0023B, Beyotime) and then incubated with primary antibody (diluted with blocking buffer).The membrane was washed three times with TBSTw (ST671, Beyotime) and then incubated with HRPconjugated secondary antibody (diluted with blocking buffer).Then, the membrane was washed 3 times with TBSTw and visualized with Super ECL Plus (P0018M, Beyotime Biotechnology Co., Ltd).The proteins bands were analyzed with ImageJ software.
DCMECs were plated in 96 well plates with a density of 5.0 Â 10 4 cells/ml and normal cultured with DMEM/12 containing 10% FBS.After 12 h, cells were treated with GluÀ, Gluþ, or/and gene overexpression or silencing and then prepared as previously reported. 19The absorbance of samples at 450 nm was determined using an automated enzyme-linked immunosorbent assay (ELISA) reader (MD, SpectraMAX M3, USA).
The gene overexpression experiment was carried out using Lipo8000 Transfection Reagent (C0533, Beyotime) follow the operating instructions.Cells were plated into 6 well plates and treated according to the experimental requirements.The medium was changed to OPTI-MEM medium (11058021, Thermo Fisher Scientific Co., Ltd, USA) before the transfection experiment.The amounts of overexpression plasmids and Lipo8000 Transfection Reagent applied were 2.5 lg and 5 lL per well, respectively.The cells that were not transfected were blank controls (B), and the cells transfected with empty plasmids pCMV-C-myc were empty plasmid controls (EV).

Gene silencing
The specific small interfering RNAs of mTOR, AMPKa, SESN2, ATF4, Nrf2 and negative control (NC) were synthesized by GenePharma (Shanghai, China).The gene silencing experiment was carried out using Lipo8000 Transfection Reagent (C0533, Beyotime) follow the operating instructions.The amounts of siRNA and Lipo8000 Transfection Reagent applied were 100 pmol and 5 lL per well, respectively.The untransfected cells were blank controls (B), and the cells transfected with negative control siRNA were negative controls.The siRNA sequences used in current study were shown in Table 2.

Statistical analysis
Results were reported as mean ± SD (n ¼ 3).Statistical significance between groups were analyzed with oneway ANOVA.p > 0.05 was considered no significant difference, 0.01 < p < 0.05 was considered to be statistically significant and p < 0.01 was considered highly significant.All data of this study were averaged from three independent experiments.

Glu promotes cell growth and casein synthesis
To evaluate the effect of Glu on cell growth and casein synthesis, DCMECs were treated with different concentrations of Glu (0, 1,000 and 4,500 mg/L) for 12 h.The cell growth and the expression of b-casein (CSN2) were tested.Results showed that the cell growth (Fig. 1A) and the expression of CSN2 (Fig. 1B,  C) were significantly increased in cells treated with 1,000 and 4,500 mg/L of Glu.DCMECs were treated with 4,500 mg/L of Glu for 0, 6, 12, 18 and 24 h.The cell growth and the expression of CSN2 were tested.
Results showed that the cell growth (Fig. 1D) and the expression of CSN2 (Fig. 1E, F) were significantly increased in cells treated with 6, 12, 18 and 24 h, and the increase was time dependent within 0-12 h.The result suggested that Glu was an important nutritional factor for cell growth and casein synthesis of DCMECs.It significantly enhanced cell growth and casein synthesis in DCMECs.In follow up experiments, DCMECs were treated with 4,500 mg/L of Glu for 12 h was defined as Gluþ.

Glu activates mTORC1 and promotes cell growth and casein synthesis via mTORC1 pathway
The mTORC1 pathway is one of the key pathways regulating cell growth and milk protein synthesis. 22To investigate whether mTORC1 pathway was regulated by Glu and the effect of mTORC1 on the regulation of the cell growth and casein synthesis activated by Glu, DCMECs were treated with GluÀ, Gluþ, Glu À and mTOR overexpression (GluÀ/mTOR GO), or Glu þ and mTOR silencing (Gluþ/si-mTOR).The the expression of mTOR, p-mTOR, ribosome S6 protein kinase 1 (S6K1), p-S6K1 and CSN2, and the rate of p-mTOR/mTOR and p-S6K1/S6K1 were tested.Results showed that the expression of mTOR, p-mTOR, S6K1, p-S6K1 and CSN2 (Fig. 2A, B), the rate of p-mTOR/mTOR and p-S6K1/S6K1 (Fig. 2C) and the cell growth (Fig. 2D) were significantly increased in cells responded to Gluþ, but these increases were blocked by mTOR silencing.Conversely, the expression of mTOR, p-mTOR, S6K1, p-S6K1 and CSN2 (Fig. 2E, F), the rate of p-mTOR/mTOR and p-S6K1/S6K1 (Fig. 2G) and the cell growth (Fig. 2H) were significantly decreased in cells responded to GluÀ, but these decreases were restored by mTOR overexpression.

Glu inhibits AMPK and activates mTORC1 pathway through inhibiting AMPK
AMPK is reported to be a sensor for Glu levels in cells. 23To measure the effect of Glu on the expression and phosphorylation of AMPK and whether AMPK was involved in the regulation of Glu on mTORC1 pathway, DCMECs were treated with GluÀ, GluÀ/si-AMPKa, Glu þ or Gluþ/AMPKa GO.The expression of AMPKa, p-AMPKa, mTOR, p-mTOR, S6K1 and p-S6K1, and the rate of p-AMPKa/AMPKa, p-mTOR/mTOR and p-S6K1/S6K1 were tested.Results showed that the expression of AMPKa and p-AMPKa   (Fig. 3A, B), and the rate of p-AMPKa/AMPKa (Fig. 3C) were significantly decreased in cells response to Gluþ, but these decreases were restored by AMPKa overexpression.Conversely, the expression of AMPKa and p-AMPKa (Fig. 3D, E), and the rate of p-AMPKa/AMPKa (Fig. 3F) were significantly increased in cells response to Glu-, but these increases were blocked by AMPKa silencing.The expression of mTOR, p-mTOR, S6K1 and p-S6K1 (Fig. 3A, B), and the rate of p-mTOR/mTOR and p-S6K1/S6K1 (Fig. 3C) were significantly increased in cells response to Gluþ, but these increases were blocked by AMPKa overexpression.Conversely, the expression of mTOR, p-mTOR, S6K1 and p-S6K1 (Fig. 3D, E), and the rate of p-mTOR/mTOR and p-S6K1/S6K1 (Fig. 3F) were significantly decreased in cells response to Glu-, but these decreases were restored by AMPKa silencing.The effect of AMPKa on Glu mediated cell growth and casein synthesis in DCMECs was tested.The result showed that AMPKa suppressed Glu-mediated cell growth and casein synthesis (Fig. S1).

Glu inhibits SESN2 and suppresses AMPK pathway through SESN2
Previous studies have shown that SESN2 is one of the upstream regulator of AMPK. 16To investigate the effect of Glu on the expression of SESN2 and whether SESN2 was involved in the regulation of Glu on AMPK, DCMECs were treated with GluÀ, Gluþ, GluÀ/si-SESN2 or Gluþ/SESN2 GO.The expression of SESN2, AMPKa and p-AMPKa, and the rate of p-AMPKa/AMPKa were tested.The result showed that the expression of SESN2, AMPKa and p-AMPKa (Fig. 4A, B), and the rate of p-AMPKa/AMPKa (Fig. 4C) were significantly decreased in cells response to Gluþ, but these decreases were restored by SESN2 overexpression.Conversely, both the expression of SESN2, AMPKa and p-AMPKa (Fig. 4D, E), and the rate of p-AMPKa/AMPKa (Fig. 4F) were significantly increased in cells responded to GluÀ, but these increases were blocked by SESN2 silencing.
The effect of SESN2 on AMPKa and mTORC1 pathway in DCMECs was tested.The result showed that SESN2 activated AMPKa and suppressed mTORC1 pathway by AMPKa (Fig. S2).The effect of SESN2 on Glu-mediated cell growth and casein synthesis in DCMECs was tested.The result showed that SESN2 suppresses Glu-mediated cell growth and casein synthesis (Fig. S3).
DCMECs were treated with GluÀ, Gluþ, Gluþ/ATF4 GO or Gluþ/Nrf1 GO, the expression of SESN2 was tested.The result showed that the expression of SESN2 was significantly decreased in cells treated with Gluþ, and this decrease was restored by ATF4 or Nrf2 overexpression (Fig. 5C, D).Conversely, the expression of SESN2 was increased in cells treated with GluÀ, and this increase was blocked by ATF4 or Nrf2 silencing (Fig. 5E, F).

Discussion
Glu is one of the most widely distributed and important monosaccharides in nature. 25It plays an important role in organism, and it is the most important energy material and energy source of the cell. 26In dairy industry, the supply of Glu significantly affects the milk yield of lactating animals. 27,28If the glucose supply is insufficient, the milk yield of lactating cows will be significantly reduced. 29Studies from Zhang show that, in mammary epithelial cells of dairy cows, Glu and amino acid deficiency inhibits casein synthesis. 14In the result of our study, the cell growth and casein synthesis were promoted by Glu addition in DCMECs.Our results were consistent with the previous study and these results on cellular and molecular levels revealed the basis underlying milk production increased by Glu addition in dairy industry.
The mTORC1 pathway is the key signaling hub that regulates cellular protein homeostasis, growth, and proliferation in health and disease. 30,31It is also the key pathway that regulates milk protein synthesis in response to the hormones and nutrients in DCMECs. 32Study has shown, the mTORC1 pathway is involved in the regulation of milk protein synthesis at the level of translation in response to amino acids signals, in particular, leucine, histidine and arginine signals. 33In our research, the mTORC1 pathway was involved in the regulatory process of Glu on cell growth and casein synthesis and Glu promotes cell growth and casein synthesis via mTORC1 pathway.A, B) and (D, E) expression of SESN2, AMPKa and p-AMPKa; (C) and (F) rate of p-AMPKa/AMPKa.DCMEC were cultured with DMEM/F12 without all of Glu (GluÀ), DMEM/F12 þ 4,500 mg/L Glu (Gluþ) for 12 h, Glu À and transfected with negative control siRNA (GluÀ/NC), Glu À and transfected with SESN2 siRNA (GluÀ/si-SESN2), Glu þ and transfected with empty vector (Gluþ/EV) and Glu þ and transfected with SESN2 expression vector (Gluþ/SESN2 GO).Data were expressed as the mean values and standard deviations (n ¼ 3); Ã p < 0.05; ÃÃ p < 0.01; ÃÃÃ p < 0.001.Glu: glucose; SESN2: sestrin2; AMPKa: adenosine 5'-monophosphate-activated protein kinase a; p-AMPKa: phosphorylated AMPKa.(B, C, E, F) The value in "Glu À group" was set to "1".
AMPK is an important kinase regulating energy homeostasis, and it is one of the central regulators of eukaryote cell and organism metabolism, in particular, lipid and Glu metabolism. 34At the same time, AMPK is also a key protein involved in a variety of signaling pathways, such as AMPK-mTORC1 pathway. 35In our study, AMPK was involved in the Glu mediated casein synthesis.In the process of this regulation, the expression and phosphorylation of AMPK was activated by Glu starvation, and then inhibited cell growth and casein synthesis by suppressing mTORC1 pathway.
SESN2 is a highly conserved stress-induced protein, which is widely distributed in various animal cells. 15,16ESN2 is induced to express when cells are exposed to gene toxin and hypoxia, and the expressed SESN2 is widely involved in the regulation of various metabolic pathways in cells.36 Previous studies have shown that SESN2 can respond to Glu or amino acid starvation, and then inhibit the mTORC1 signaling pathway through different pathways.37,38 Our previous researches have shown that, SESN2 responds to amino acid starvation and then inhibits the mTORC1 pathway by binding to SH3BP4.39,40 In our research, the expression of SESN2 was activated by Glu starvation, and then the SESN2 inhibited the mTORC1 pathway and subsequent cell growth and casein synthesis by activating the AMPK pathway.

Table 1 .
Primer sequences used for plasmid construction.
mTORAACTGCAGAACAGCCTCCCACAAAAC (the PstI site is underlined) GCGTCGACAGCCATAGCCTCCTTCAC (the Sal I site is underlined) AMPKa CGGAATTCATGGTGATGGAATATGTCTCA (the EcoR I site is underlined) GAAGATCTTCACATGGAGGCCCCGGCCGA (the Bgl II site is underlined) SESN2 CGGAATTCCACACCATGATCGTTGCGG (the EcoR I site is underlined) GAAGATCTCAGGTGAGTAAATGGGCTTCC (the Bgl II site is underlined) ATF4 CGGAATTCATGGCCGAGATGAGCTTTC (the EcoR I site is underlined) GAAGATCTGAGGACCCTTTTCTTCTCCCT (the Bgl II site is underlined) Nrf2 CCCAAGCTTATGATGGACTTGGAGCTGCCGC (the Hind III site is underlined) GCTCTAGAGTTTTTCTTAATATCTGGCCTC (the Xba I site is underlined)