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. 2021 Jul 26;13(8):2552. doi: 10.3390/nu13082552

Figure 3.

Figure 3

Effects of exendin-4 and PASK deficiency on the response of hepatic Gck, Gckr mRNAs and GCK, GCKR protein levels to fasting/refeeding and glucokinase activity. Quantitative real-time PCR was used to analyze the expression of Gck (A) and Gckr (B) mRNA levels in the liver from wild-type (WT) and PASK-deficient mice (Pask−/−). mRNA levels were measured in 48-h fasted (FASTED) and 3-h refed (REFED) specimens in the absence or presence of exendin-4 treatment and normalized by Rn 18s. Data are shown as mean ± SEM of 3–5 animals per group and expressed as fold-over the fasted condition mean. A Gck mRNA: ‡ p < 0.05, ‡‡ p < 0.01 vehicle vs. exendin-4 by two-way ANOVA followed by Fisher’s LSD test. Immunoblot analysis of GCK (C,E) and GCKR (D,E) in the liver from wild-type (WT) and PASK-deficient mice (Pask−/−). Liver lysates from non-fasted (NON-FASTED), 48-h fasted (FASTED) and 3-h refed (REFED) livers in the absence or presence of exendin-4 (Ex4) treatment were processed for Western blot analysis. A representative blot of 2 prepared blots is also shown (E), from which the data shown in the graph were collected (C,D). The values were normalized by total protein detected by Stain-Free Biorad© and expressed as fold-over the non-fasted wild-type mice. Data are shown as mean ± SEM of 3–5 animals per condition; * p < 0.05 WT vs. Pask−/−, ‡ p < 0.05, ‡‡‡ p < 0.001 vehicle vs. exendin-4 by two-way ANOVA followed by Tukey post-hoc test. Total glucose-phosphorylating activity assays (F) were performed at 30 mM, with low-Km hexokinase (HK) activities at 0.3 mM glucose. GCK activity was obtained by the difference between individual values at high and low glucose (G). Liver homogenates from 3-h refeeding after prolonged fasting (REFED) in the absence or presence of exendin-4 (Ex4) treatment from wild-type (WT) and PASK-deficient mice (Pask−/−). (F) Total glucose-phosphorylating activity and (G) glucokinase (GCK) activities are shown as mean ± SEM of 3–5 animals per group and expressed as a percentage of the control mean; * p < 0.05, *** p < 0.001 WT vs. Pask−/− and ‡ p < 0.05, ‡‡‡ p < 0.001 vehicle vs. exendin-4 by two-way ANOVA followed by Tukey post-hoc test.