Fig 2: Insulin stimulates metabolic flux into the methionine cycle and transsulfuration pathway through ATF4. (A) Steady state metabolomic profiling from primary mouse hepatocytes transfected with control (siCT) or Atf4-targeting siRNAs followed by 100 nM insulin stimulation for 6 h performed in quadruplicate. The heat map displays a rank order of metabolites significantly (p < 0.05) induced with insulin in the control cells, but not those with Atf4 knockdown. (B) Protein normalized peak areas of the top 6 metabolites (A) plotted as mean ± SD. (C) Schematic of the interconnections between serine and tetrahydrofolate metabolism, the methionine cycle, and the transsulfuration pathway. Metabolites found to be significantly induced by insulin in an ATF4-dependent manner in (A) are highlighted in red. (D) Gene expression analysis of primary mouse hepatocytes transfected with control (siCT) or Atf4-targeting siRNAs followed by overnight serum starvation and treatment with 100 nM insulin for 8 h. Data are plotted as mean ± SEM relative to siCT-transfected serum-starved cells (n = 3 independent experiments). (E) Protein normalized peak areas of 3-13C-serine (400 µM, last 1 h) tracing into labeled SAM and cystathionine in primary mouse hepatocytes serum-starved overnight and subsequently treated with 100 nM insulin following a 30-min pretreatment with vehicle (DMSO), 20 nM rapamycin, or 500 nM Torin1 for 8 h. Data are plotted as mean ± SD and are representative of two independent experiments performed in triplicate. (F) Protein normalized peak areas of 3-13C-serine tracing (400 µM, last 1h) in primary mouse hepatocytes transfected with control (siCT) or Atf4-targeting siRNAs followed by overnight serum starvation and treatment with 100 nM insulin for 8 h. Data are plotted as mean ± SD and are representative of two independent experiments performed in triplicate. (G) Protein normalized peak areas of labeled SAM from 13C5-methionine (200 µM, last 1h) tracing of cells treated as in (F). (H) Fractional enrichment of SAM isotopologues from 13C5-methionine tracing as in (G). (I) Protein normalized peak areas of labeled cystathionine from 13C5-methionine tracing as in (G). Data are plotted as mean ± SD for tracing studies and are representative of two independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-way ANOVA).

Fig 3: hnRNPK regulates the expression of genes coding for aminoacyl-tRNA synthetases via the eIF2a/Atf4 pathway. (A) Western blot showing the increased Atf4 expression 48 h after hnRNPK KD, and ISRIB treatment suppressed the increase. Dimethyl sulfoxide (DMSO) was used as the control. The blots shown are representative of three experiments. (B–F) qRT-PCR to detect the expression of Aars (B), Gars (C), Iars (D), Nars (E), Sars (F) 48 h after hnRNPK KD with or without ISRIB treatment. n = 3, mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05.

Fig 4: ATF4 is both necessary and sufficient for insulin-mTORC1 signaling to induce select gene targets. (A) Immunoblot analysis of primary mouse hepatocytes transfected with control (siCT) or Atf4-targeting siRNAs followed by overnight serum starvation and treatment with 100 nM insulin for 8 h. (B) Gene expression in cells treated as in (A) plotted as mean ± SEM relative to the siCT-transfected serum-starved cells (n = 3 independent experiments). (C) Immunoblot analysis of Atf4fl/fl and LAtf4KO primary mouse hepatocytes serum-starved overnight, and subsequently, treated with 100 nM insulin for 6 h following a 30-min pretreatment with vehicle (DMSO) or 20 nM rapamycin. (D) Gene expression in cells treated as in (C) plotted as mean ± SEM relative to the Atf4fl/fl serum-starved cells (n = 3). (E) Immunoblot analysis of Atf4fl/fl and LAtf4KO primary mouse hepatocytes infected with AdGFP or AdATF4 (MOI = 10) for 24 h. (F) Gene expression from a representative experiment of cells treated as in (E) plotted as mean ± SEM relative to the Atf4fl/fl AdGFP sample (n = 3). (G) Immunoblot analysis of WT primary mouse hepatocytes infected with AdGFP or AdATF4 (MOI = 10) for 24 h followed by treatment with vehicle (DMSO) or 20 nM rapamycin for 8 h. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-way ANOVA (B,D) or one-way ANOVA (F)).

Fig 5: Feeding and insulin induce hepatic ATF4 and established gene targets by mTORC1 signaling. (A and B) Eight-week-old male mice were fasted for 12 h and refed with a high carbohydrate diet for 6 h following pretreatment with vehicle or 10 mg/kg rapamycin (n = 6/group). (A) Liver lysates were immunoblotted to assess mTORC1 signaling and ATF4, with mean ATF4 protein to tubulin quantification plotted ± SEM and normalized to the fasted group (right). (B) Liver gene expression plotted as mean ± SEM relative to the fasted group. (C) Immunoblot analysis of primary mouse hepatocytes serum-starved overnight and treated with 100 nM insulin following a 30-min pretreatment with vehicle (DMSO), 20 nM rapamycin, or 750 nM Torin1 for the indicated time points. (D) Immunoblot analysis of primary mouse hepatocytes transfected with control (siCT) or a combination of Eif4ebp1 (4ebp1) and Eif4ebp2 (4ebp2) siRNAs, followed by overnight serum starvation and treatment for 8 h with 100 nM insulin following a 30-min pretreatment with vehicle, 20 nM rapamycin or 500 nm Torin1. (E) Immunoblot analysis of primary mouse hepatocytes treated for the indicated time points as in (C). (F) Gene expression in cells treated as in (E) plotted as mean ± SEM relative to serum-starved cells (n = 4 independent experiments). (G) Immunoblot analysis of primary human hepatocytes serum-starved overnight, and subsequently, treated with 100 nM insulin following a 30-min pretreatment with vehicle (DMSO), 20 nM rapamycin, or 500 nM Torin1 for the indicated time points. (H) Gene expression in cells treated as in (G) plotted as mean ± SEM (n = 3) relative to the serum-starved cells. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (one-way ANOVA).