Fig 2: Increase in glucose metabolism induced by GLP-1R signalling is mediated by HIF-1a. (A) BRIN-BD11 cells were treated as indicated for 18 hours and Hif1a protein levels were determined by immunoblot analysis. (B) After treatment with Exendin-4 for 18 hours HIF-1a mRNA expression was evaluated by qRT-PCR. (C) BRIN-BD11 cells were pre-incubated with either vehicle, 1 µM Torin 1 or 50 µM LY294002 for 30 min as indicated, followed by addition (or not) of 50 nM Exendin-4 for 18 hours. Immunoblot shows Hif1a protein expression and phosphorylation levels of mTOR at serine 2448. (D–J) BRIN-BD11 cells were transfected with either HIF-1a specific siRNAs (siHIF1a) or non-targeting siRNAs (siControl) for 24 h, and subsequently exposed to 50 nM Exendin-4 (or not) for 18 h. (D) Exendin-4’s ability to promote protein levels of Pdh and Pfkp was significantly blunted in cells transfected with siHIF1a as compared to siControl transfected cells. (E) At the mRNA level, siHIF1a transfection caused a reduction of approximately 80% in HIF-1a expression. (F) mRNA expression of glycolytic enzymes was determined by qRT-PCR analysis. (G–J) Determination of glucose consumption, 2-NBDG uptake, SSC-A and FSC-A following HIF-1a knockdown. Data are mean ± SEM, n = 3; n.s. = non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001.

Fig 3: 18 h of GLP-1R stimulation induces mRNA and protein expression of glycolytic enzymes. (A) mRNA expression of the glycolytic enzymes Pyruvate Kinase (Pklr), Lactate dehydrogenase A (Ldha), Aldolase A (Aldoa), Phosphofructokinase (Pfkp), Glucose-6-phosphate isomerase (Gpi) and Phosphoglycerate mutase 2 (Pgam2) was assessed by qRT-PCR in BRIN-BD11 cells exposed (or not) to 50 nM Exendin-4 for 18 hours. (B) Protein levels of glycolytic enzymes were analysed by immunoblot following 0, 2, 4, 8, 12 and 18 hours of exposure to 50 nM Exendin-4. Expression levels of (C) Pfkp, (D and E) Pyruvate Kinase M1 and M2 (Pkm1, Pkm2), (F) Hexokinase 1, (G) Hexokinase 2 and (H) Pyruvate dehydrogenase a1 subunit (Pdh) were quantified by band densitometry analysis. Data represent mean ± SEM, n = 3; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 4: Isoflurane induces miR-21 expression and phosphorylation of AKT(A) SKOV3 and TOV21G cells were treated without or with isoflurane for 1 or 2 h, and the expressions of miR-21 were assessed by qRT-PCR. U6 was used as an internal control. (B) SKOV3 and TOV21G cells were treated with isoflurane for 1 or 2 h, the phosphorylation of AKT was measured by Western blot. ß-actin was used as the loading control. (C) SKOV3 and TOV21G cells were transfected with control miRNAs or pre-miR-21 for 72 h. The expression of miR-21 was assessed by qRT-PCR. U6 was used as the internal control. (D) The proteins and (E) mRNAs of HK2, PKM2, and LDHA were measured in SKOV3 and TOV21G cells without or with miR-21 overexpression. (F) SKOV3 and TOV21G cells without or with isoflurane treatment were transfected with control antisense or anti-miR-21 for 72 h. The expression of miR-21 was assessed by qRT-PCR. U6 was used as an internal control. (G) The mRNAs of GLUT1, HK2, PKM2, and LDHA were measured by qRT-PCR. All experiments were performed at least three times independently and the data shown are mean ± S.D.; *: P<0.05; **: P<0.01.

Fig 5: Glycolytic enzymes were activated by isoflurane treatmentsSKOV3 and TOV21G cells were treated without or with isoflurane for 1 or 2 h, cells were then cultured in new medium for 24 h. (A) The protein expressions of GLUT1, HK2, PKM2, and LDHA were measured by Western blot. ß-actin was used as a loading control. (B) The mRNA expressions of glycolytic enzymes were measured by qRT-PCR. (C) SKOV3 cells were treated without or with isoflurane for 2 h, cells were incubated in fresh medium for 24 h. Cells were fixed and stained with antibody against GLUT1. DAPI was used for the nuclei staining. (D) Quantitation of the membrane localization of GLUT1 based on the results of Figure 2C using ImageJ software. All experiments were performed at least three times independently and the data shown are mean ± S.D.; *: P<0.05; **: P<0.01; ***: P<0.001.