ANGPTL4 regulate glutamine metabolism and fatty acid oxidation in nonsmall cell lung cancer cells

Figures

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  FIGURE 1. ANGPTL4 knockdown reduces the proliferation and ATP content of A549 and H1299 cells. (A) The viability of normal NSCLC and ANGPTL4 knockdown cells was analysed by CCK in A549 cells and H1299 cell. (B) The expression of ANGPTL4 in normal NSCLC, ANGPTL4 vector and ANGPTL4 knockdown cells were determined by Western blot analysis of protein level. **P < 0.01. (C) The ATP content in normal NSCLC (ANGPTL4 vector transfection cell) and ANGPTL4 knockdown cells was analysed. *P < 0.05

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  FIGURE 2. ANGPTL4 perform OXPHOS but not aerobic glycolysis. The OXPHOS and aerobic glycolysis in normal NSCLC (ANGPTL4 vector transfection cell) and ANGPTL4 knockdown cells were determined (A) Representative trace (left) and mean data (centre) of glycolytic ECAR when assayed in minimal medium and treated with glucose in Seahorse. Glycolytic reserve (right) is the difference between glycolytic capacity and glycolysis rate and was measured as the difference between resulting ECAR values after in‐Seahorse exposure to glucose and maximal ECAR values obtained after oligomycin treatment. (B) Representative trace (left) and mean data (centre) of oxygen consumption rate (OCR) from cells as in a. Values are averages of 5–8 basal OCR measurements minus averages of 3–5 OCR readings after rot/AA treatment of individual wells from indicated cell types. Spare respiratory capacity is the difference between basal OCR values and maximal OCR values obtained after FCCP uncoupling (right). *p < 0.05, **p < 0.01, 2DG, 2‐deoxy‐d‐glucose; FCCP, carbonyl cyanide‐4 (trifluoromethoxy) phenylhydrazone; rot/AA, rotenone and antimycin A

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  FIGURE 3. ANGPTL4 promotes glutamine metabolism and fatty acid oxidation. (A) (A) Glutamine consumption determined in NSCLC cells, isotope abundance of α‐KG (M+5), malate (M+4), aspartate (M+4) in normal NSCLC, ANGPTL4 vector and ANGPTL4 knockdown cells were traced by 13C5‐glutamine. *P < 0.05. (B) Fatty acid oxidation determined in NSCLC cells, the content of [13C16]‐acetyl‐CoA and [13C16]‐acetylcarnitine isotope in normal NSCLC, ANGPTL4 vector and ANGPTL4 knockdown cells were traced by 13C16‐palmitate. *P < 0.05. (C) The expression of GLS and CPT1 in normal NSCLC(ANGPTL4 vector transfection cell) and ANGPTL4 knockdown cells were determined by Western blot analysis of protein level. **P < 0.01. *P < 0.05

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  FIGURE 4. ANGPTL4 knockdown inhibited the tumour growth, glutamine metabolism and fatty acid oxidation in subcutaneous xenograft model. (A). ANGPTL4 in tumour and the tumour volume of subcutaneous xenograft model for NSCLC cells. (B) Glutamic acid, acetyl coa and lactate content were determined in subcutaneous xenograft model. **P < 0.01

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  FIGURE 5. ANGPTL4 overexpression promoted NSCLC proliferation and OXPHOS of A549 cells, but inhibited by CPT1 and GLS inhibitors. (A) ANGPTL4 overexpression promoted NSCLC proliferation. (A) The viability of ANGPTL4 overexpression cells in normal environmental and without glutamine was analysed by CCK in A549 cells. (B) Representative trace and mean data (centre) of oxygen consumption rate (OCR) from ANGPTL4 overexpression cells with CPT1 inhibitor (Etomoxir sodium salt, ETO, 15 μM) or GLS inhibitor (BPTES, 20 μM) were determined. Results are representative of (for sample data) or represent the mean of 3–6 independent experiments. *P < 0.05, **P < 0.01, 2DG, 2‐deoxy‐d‐glucose; FCCP, carbonyl cyanide‐4 (trifluoromethoxy) phenylhydrazone; rot/AA, rotenone and antimycin A

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  FIGURE 6. ANGPTL4 overexpression promotes glutamine metabolism and fatty acid oxidation, but inhibited by CPT1 (ETO, 15 μM) and (BPTES, 20 μM). (A) Glutamine consumption determined in NSCLC cells, isotope abundance of α‐KG (M+5), malate (M+4) and aspartate (M+4) in NSCLC cells were traced by 13C5‐glutamine. *P < 0.05. (B) Fatty acid oxidation determined in NSCLC cells, the content of [13C16]‐acetyl‐CoA and [13C16]‐acetylcarnitine isotope were traced by 13C16‐palmitate. *P < 0.05. NSCLC cell which pretreatment with GLS inhibitor BPTES (20 μM,Selleck) or CPT1 inhibitor ETO (15 μM,Selleck)for 24h and ANGPTL4 overexpression NSCLC cell combined with BPTES or ETO. *P < 0.05