Fig 2: miR-214 inhibition and ATF4 overexpression reverse MEG3 knockdown-mediated decreases in FoxO1, G6pc and Pepck protein expression. Cells were transfected with the indicated plasmids, followed by treatment with 0.5 mM palmitate for 4 h. (A–D) Western blot analysis revealed that MEG3 knockdown significantly suppressed the palmitate-mediated increase in (B) FoxO1, (C) Pepck, and (D) G6pc protein expression. (E–H) By contrast, the miR-214 inhibitor additionally induced the palmitate-mediated increase in (F) FoxO1, (G) Pepck and (H) G6pc protein expression. (I–L) ATF4 overexpression also induced the palmitate-mediated increase in (J) FoxO1, (K) Pepck and (L) G6pc protein expression. ß-tubulin served as the loading control in the western blot analyses. *P<0.05 vs. group 1; #P<0.05 vs. group 3;$P<0.05 vs. group 4. si, small interfering RNA; ctrl, control; MEG3, maternally expressed gene 3; miR, microRNA; FoxO1, forkhead box protein O1; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase catalytic subunit; ATF4, activating transcription factor 4.

Fig 3: Expression of pck1 transiently increases in response to low-pH stress. Changes in expression levels of g6pca.1 (A), g6pca.2 (B), pck1 (C), and pck2 (D) and body glucose content (E) were assessed in the zebrafish larvae after low-pH exposure. g6pca.1 (A) and pck1 (C) were up-regulated until 12 h after the onset of low-pH stress. Data are expressed as means ± SE. *time and *int indicate significant main effect of time and interaction of two factors (time x low-pH treatment), respectively. Different letters indicate significant differences at P < 0.05 between time points within each treatment group (a, b, c, … for control, and x, y, z, … for low-pH; in D, the two treatment groups are combined), and asterisks indicate the significant differences between treatments at the same time points.

Fig 4: pck1 knockdown had no effect on acid-induced decrease in glutamine and glutamate levels, although Pck1-mediated cataplerosis is arrested. pck1 knockdown increased body contents of serine, threonine, valine, and isoleucine, but decreased those of methionine and phenylalanine (A). Low-pH stress decreased glutamine, glycine, glutamate, threonine, alanine, proline, and phenylalanine (A). Body content of malate, a Krebs cycle intermediate, was accumulated in the pck1 morphants (B). Data are expressed as means ± SE. *MO and *pH indicate significant main effects of pck1 MO and low-pH treatment, respectively. NS, no significance.

Fig 5: pck1 knockdown eliminates acid-induced H+ excretion. Western-blot analysis of the protein samples obtained from control MO-injected (1st lane) and pck1 MO-injected (2nd lane) larvae (A). A band immunoreactive to human PEPCK antibody was detectable at ~69 kDa in the control, but not in the pck1 morphant. The effects of pck1 knockdown and 6-h exposure to pH 7 or pH 4 environments on Pck1 protein (B), glucose content (C), V-ATPase protein (D), and H+ secretion from the yolk-sac skin (E) were assessed. pck1 knockdown decreases not only Pck1 protein level, but also glucose content, V-ATPase protein, and acid-induced excretion of H+. Open and closed column indicate control MO- and pck1 MO-treated samples, respectively. Data are expressed as means ± SE. Asterisks indicate significant differences between control and morphant in the same pH, and daggers indicate significant differences between pH 7 and pH 4 in the same treatment groups (P < 0.05). N.D., not detectable.