Fig 2: Effects of H19 inhibition on genes of fatty acid and cholesterol metabolism and gluconeogenesis in Hepa 1–6 cells. (A) Hepa 1–6 cells were transfected with either the scramble or H19 siRNA (5 nM, 48 h) and total cellular lipid content was evaluated using Bodipy. Total RNA isolated from Hepa 1–6 cells transfected as in “A” was reverse transcribed and assessed for the relative transcript expression of genes of fatty acid and cholesterol metabolism, namely Fasn, Dgat2, Hmgcr, Hmgcs, Acadl, Hadha (B) and gluconeogenesis, namely G6Pase, Pcx, Fbp1 and Pck1 (C). Cells transfected as in “A” were lysed and lysates (20–40 μg) were resolved on SDS-PAGE, transferred to nitrocellulose membranes and probed with antibodies against G6Pase (D), FBP1 (E), PCK1 (F) and PCx (G). β-actin or Vinculin were used as the loading controls and densitometric analyses are given in the panels below. All experiments were done at least three times and data are presented as means ± SEM.**p < 0.01, *p < 0.05, ns: non-significant.

Fig 3: Overexpression of VDAC1 increases gluconeogenic genes and impairs mitochondrial function in Hepa 1–6 cells. Hepa 1–6 cells were transfected with either the empty vector or the VDAC1 overexpression clone (0.5 and 1 μg). At 48h, cells were lysed and lysates (20 μg) were probed for the levels of VDAC1 (A). (B) Hepa 1–6 cells were transfected as in “A” and on completion of incubation, cells were washed and labelled with annexin V and Propidium Iodide (PE) and analyzed for apoptosis by flow cytometry. Annexin V (FITC-A) positive cells are represented. Hepa 1–6 cells transfected with either the empty vector or the VDAC1 overexpression clone (0.5 and 1 μg) were lysed and lysates (20–40 μg) were probed to evaluate G6Pase (C), PCx (D), PCK1 (E) and FBP1 (F) protein levels by Western Blot analysis. β-actin or Vinculin were used as loading controls. Representative blots are shown and densitometric analyses (n = 3) are given in the panels below. (G) Hepa 1–6 cells transfected with either the empty vector or the VDAC1 clone (1 μg) were incubated in the presence of sodium lactate (20 mM) and sodium pyruvate (2 mM) and glucose output into the media was measured. Data was normalized to the protein content. Cells transfected as in “C” were analyzed for mitochondrial Ca2+ levels using Rhod-2 AM (1 μM) (H), cytosolic Ca2+ levels using Fluo-3 AM (1 μM) (I) and mitochondrial ROS (J). (K) Representative OCR of Hepa 1–6 cells after transfection with either the empty vector or the VDAC1 clone (1 μg) for 48h in the presence of 2 μM oligomycin, 1.25 μM FCCP and 2 μM rotenone and 2 μM antimycin and quantitative analysis of basal respiration, maximal respiration, proton leak, spare respiratory capacity and ATP production. Experiments were performed thrice and data are presented as means ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05, ns: non-significant.

Fig 4: P2RX4/SLC25A11 mediates the intracellular transport of AKG.(A and B) Intracellular and medium 13C-AKG level (A) and intracellular total AKG level (B) (n = 3). (C and D) Cytosol (mitochondrial-free) (C) and mitochondrial (D) AKG level (n = 6). (E) Relative changes of dicarboxylate carriers in response to AKG treatment (n = 3). (F) SLC25A11 protein expression of mitochondrial membrane protein in AKG-treated primary hepatocytes (PA treatment) (n = 6). (G) Immunoblots of SLC25A11 in si-SLC25A11–treated primary hepatocyte (n = 3). (H to J) Immunoblots (H) and quantification (I), and enzyme activity (J) of PEPCK, G6Pase, and FBP in primary hepatocyte (PA treatment) (n = 4 to 8). (K) Relative changes of purine receptors in response to AKG treatment (n = 3). (L) Schematic representation of Biacore. (M) Immunoblots of protein expression after AKG pull-down in mice liver. (N) Immunoblots and quantification of P2RX4 in the liver. Twelve-week-old male mice were treated with vehicle or 5-BDBD (P2RX4 antagonist, 5 mg/kg) (n = 3). (O) Cytosol (mitochondrial-free) and mitochondrial AKG level (n = 6 per group). (P) Immunoblots and quantification of P2RX4 in si-P2RX4–treated primary hepatocyte (n = 3). (Q to S) Immunoblots (Q) and quantification (R), and enzyme activity (S) of PEPCK, G6Pase, and FBP in si-P2RX4–treated primary hepatocyte (PA treatment) (n = 4 to 8). (T) Graphic abstract mechanism of AKG on gluconeogenesis. Results are presented as means ± SEM. (B, F, G, N, and P) *P ≤ 0.05 by nonpaired Student’s t test. In (C), (D), (I), (J), (O), (R), and (S), different letters between bars indicate P ≤ 0.05 by one-way ANOVA followed by post hoc Tukey’s tests.

Fig 5: Effect of CK2 downregulation on blood glucose, hormone secretion and FBP1 expression in mice. (a) Schematic illustration of the experimental setting. On day 0, αTC1 cells (WT or KO) were transplanted under the left kidney capsule of mice. Sham-transplanted mice served as negative control. Fasting blood glucose levels and body weights were measured over 28 days twice a week. On day 28, IPGTT was performed. (b) Body weight of mice transplanted with WT or KO cells from day 0 to day 28 (n=6 each). Sham-transplanted mice served as negative control (n=5 each). (c) AUC of the body weights shown in (b). (d) Blood glucose levels of mice transplanted with WT or KO cells from day 0 to day 28 (n=6 each). Sham-transplanted mice served as negative control (n=5 each). (e) AUC of the blood glucose levels shown in (d). (f) Quantitative analysis of blood glucose levels on day 28 according to the IPGTT of mice transplanted with WT or KO cells (n=6 each). Sham-transplanted mice served as negative control (n=5 each). (g) AUC of IPGTT results shown in (f). (h) Quantitative analysis of GCG secretion of mice transplanted with WT or KO cells (n=8 each). Sham-transplanted mice served as negative control (n=8 each). (i) Quantitative analysis of insulin secretion of mice transplanted with WT or KO cells (n=3 each). Sham-transplanted mice served as negative control (n=3 each). (j) Schematic illustration of the experimental setting. Mice were treated for 3 days with CX-4945 or DMSO (control) and blood, kidney and liver samples were collected to study GCG and insulin secretion and FBP1 expression. (k) Quantitative analysis of GCG secretion of mice treated with CX-4945 or DMSO (control) (n=3 each). (l) Quantitative analysis of insulin secretion of mice treated with CX-4945 or DMSO (control) (n=3 each). (m) Representative western blots of FBP1 and α-tubulin expression from liver and kidney tissue extracts. (n, o) Quantitative analysis of FBP1 from data shown in (m) (n=2 or 3 each). Data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. (a, j) Generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. Ctrl, control (DMSO); CX, CX-4945; INS, insulin