Fig 2: Glutamine supplementation exacerbates the lipotoxic effects of palmitate in HER2/neu-positive SKBR3 cells(A) SKBR3 cells were transfected with the transcriptional reporter construct pCAX-XBP1-?DBD-venus which results in the expression of a functional fluorescent XBP1-venus fusion protein after mRNA splicing by the ER-stress regulator IRE1 as described in [14, 35]. The cells were treated with 250 µM palmitate 24 h post-transfection, either in full growth medium, drop-out medium without glutamine or drop-out medium with glutamate instead of glutamine. Green fluorescent cells, that are indicative of XBP1-splicing and ER-stress activation, were counted using the InCell Analyzer 2200. (B) SKBR3 cells were treated with 250 µM palmitate or vehicle for 24 h either in full growth medium or drop-out medium without glutamine. Total protein was harvested and subjected to western blot analysis to evaluate the protein expression changes for the ER-stress related marker DDIT3/CHOP and the growth factor receptors HER2 and HER3. Each experiment was performed in triplicate, representative images are shown. Signal was quantified using ImageJ. Intensities were normalized to loading control and vehicle-control. The data is presented as mean ± SD. * = p = 0.05, ** = p = 0.01, *** = p = 0.001, one-way ANOVA with Bonferroni post-test (A), Student’s t-test (B). n = 3. (C) Overview of palmitate induced lipotoxicity in SKBR3 cells. Exogenous palmitate application results in increased activity in glycine production and decreased fatty acid synthesis. Glycine production is accomplished by transamination reactions. Fatty acid synthesis down regulation occurs through activation of AMPK. Metabolites and enzymes increased abundance are depicted in orange. Decreases in ACACA activity and early glycolytic pathway metabolites are depicted in blue.

Fig 3: AMPK phosphorylates KHK-A and promotes the association between KHK-A and p62.Immunoprecipitation or immunoblot analyses were performed with the indicated antibodies. (A) Huh7 cells transfected with Flag–KHK-A were pretreated with SB203580 (10 µM), SP600125 (25 µM), U0126 (20 µM), or compound C (5 µM) before hypoxia stimulation for 6 hours in the presence of CQ (10 µM). (B) Huh7 cells expressing Flag–KHK-C or Flag–KHK-A were cotransfected with HA-AMPKa and treated with or without hypoxia for 6 hours or H2O2 (0.5 mM) for 1 hour in the presence of the lysosome inhibitor CQ (10 µM). (C) Huh7 cells transfected with Flag–KHK-A were pretreated with or without compound C (5 µM) for 30 min before hypoxia stimulation for 6 hours in the presence of the lysosome inhibitor CQ (10 µM). ACC1, acetyl-CoA carboxylase 1. (D) WT and AMPKa1/2 double-knockout (DKO) mouse embryonic fibroblasts (MEFs) infected with lentivirus expressing Flag–KHK-A were treated with or without A769662 (0.5 mM) for 6 hours in the presence of the lysosome inhibitor CQ (10 µM). (E and F) Bacterially purified GST–KHK-A, GST–KHK-C (E), or GST–KHK-A S80A (F) was incubated with or without bacterially purified active AMPK in the presence of [?-32P]ATP. Autoradiography and immunoblot analyses were performed. (G) Huh7 cells expressing WT Flag–KHK-A, Flag–KHK-C, or Flag–KHK-A S80A were treated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 µM). (H) WT and AMPKa1/2 DKO MEFs were cultured with or without hypoxia for 6 hours. (I) An in vitro AMPK kinase assay was performed by mixing bacterially purified WT GST–KHK-A, GST–KHK-A S80A, or GST–KHK-C on glutathione agarose beads with or without purified active AMPK in the presence of AMP and ATP for 1 hour. The glutathione agarose beads were then washed and incubated with purified His-p62 for a GST pulldown analysis. (J) Huh7 cells expressing Flag–KHK-A, Flag–KHK-C, or Flag–KHK-A S80A were treated with or without hypoxia for 4 hours in the presence of the lysosome inhibitor CQ (10 µM). (K) Huh7 cells expressing Flag–KHK-A and HA-AMPKa were pretreated with N-acetyl-l-cysteine (NAC) (5 mM) or Trolox (100 µM) for 30 min before hypoxia stimulation for 6 hours. (L) Huh7 cells expressing Flag–KHK-A were pretreated with NAC (5 mM) or Trolox (100 µM) for 30 min before hypoxia stimulation for 6 hours in the presence of the lysosome inhibitor CQ (10 µM).

Fig 4: Insulin Induces SREBP1/ACC1 Expression through TDG-Mediated DNA Demethylation(A–C and E) HepG2 or MCF7 cells stably infected with nontargeting control or TDG#1 shRNA were cultured in the absence or presence of 200 nM insulin for 72 h. (D and F) HepG2 or MCF7 cells were treated with 200 nM demethylase inhibitor gemcitabine (Gem) for 30 min prior to 200 nM insulin treatment for 48 h. Relative TDG, SREBP1, and ACC1 mRNA, determined via qRT-PCR, was shown as the mean plus SEM of a representative experiment performed in triplicates. ***p < 0.001 (Student’s t test). Cell lysates were analyzed by western blot with specified antibodies. flSREBP1, full-length SREBP1. (G and H) HepG2 (G) or MCF7 (H) cells stably infected with nontargeting control or TDG#1 shRNA were cultured in the absence or presence of 200 nM insulin for 48 h. Relative 5caC abundance in the SREBP1 promoter was detected by the DIP assay. *p < 0.05 and ***p < 0.001 (ANOVA). (I–K) HepG2 cells were stably transfected with vector or pBabe-TDG. Cell lysates were analyzed by western blot with specified antibodies (I). Relative 5caC abundance in the SREBP1 promoter was detected by the DIP assay (J). The relative amount of TDG binding to the SREBP1 promoter was analyzed by the ChIP assay (K). (L and M) SREBP1 expression values were shown in breast (L; TCGA) and liver (M; GSE87630) cancer tissues compared to adjacent noncancerous tissues (normal) from database analysis. **p < 0.01 and ***p < 0.001 (Welch’s t test). (N and O) Correlations of HCC tumor/peri-tumor tissue (N) and HCC tumor/normal liver tissue (O) mixture ratios of SREBP1 mRNA and promoter 5caC abundance on a logarithmic scale were depicted.

Fig 5: Expression of FNDC5 is lower in muscle tissue from AMPK DMKO mice compared to wild-type mice but is unaltered after exhaustive treadmill exercise. (A) The maximum speed attained during a progressive treadmill running exercise capacity test in wild-type and AMPK DMKO mice. AMPK alpha pThr172 (B) and ACC2 pSer212 (C) phosphorylation levels and FNDC5 protein content (D) at rest (Rest) or immediately following exercise (Post Ex) in tibialis anterior muscle tissue (N = 3–5). Error bars represent standard error of the mean. ND, not detectable. *Significantly different from wild-type, P < 0.05. **Significantly different from wild-type Rest.