Fig 2: Serpina1e is required for the inhibitory effects of AKG on hepatic gluconeogenesis.(A and B) Principal coordinate analysis plot (A) and volcano plot of AKG-induced transcriptome signature (B) (n = 3). (C) mRNA expression of serpina1e in si-serpina1e–treated primary hepatocyte (n = 6). (D to F) Immunoblots (D) and quantification (E), and enzyme activity (F) of PEPCK, G6Pase, and FBP in primary hepatocyte (PA treatment) (n = 4 to 8). (G) mRNA expression of serpina1e in the liver (n = 8). (H and I) Immunoblots (H) and quantification (I) of p-FAK and p-Akt protein expression in the liver (n = 3). (J) mRNA expression of serpina1e in Alb-serpina1e-/- mice liver (n = 8). (K to M) Blood glucose (K), PTT (L), and ITT (M) in Alb-serpina1e-/- mice (n = 8). (N to P) Immunoblots (N) and quantification (O), and enzyme activity (P) of PEPCK, G6Pase, and FBP in the liver (n = 4 to 8). (Q) Serum insulin level (n = 8). (R) mRNA expression of JMJD3, LSD1, and UTX in AKG-treated primary hepatocyte (PA treatment) (n = 8). (S and T) mRNA (S) and protein (T) expression of JMJD3 in si-JMJD3–treated primary hepatocyte (n = 3 to 6). (U) Chromatin immunoprecipitation analysis of H3K27me3 in promoter of serpina1e in si-JMJD3–treated primary hepatocyte (PA treatment) (n = 8). (V) mRNA expression of serpina1e in si-JMJD3–treated primary hepatocyte (PA treatment) (n = 8). (W to Y) Immunoblots (W) and quantification (X), and enzyme activity (Y) of PEPCK, G6Pase, and FBP in si-JMJD3–treated primary hepatocyte (PA treatment) (n = 4 to 8). Results are presented as means ± SEM. In (E) to (G), (O) to (Q), (U), (X), and (Y), different letters between bars indicate P = 0.05 by one-way ANOVA followed by post hoc Tukey’s tests. (C, I, J, R to T, and V) *P = 0.05 by nonpaired Student’s t test. (K to M) *P = 0.05, **P = 0.01 by two-way ANOVA followed by post hoc Bonferroni tests.

Fig 3: 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 4: Acute AKG administration prevents diet-induced hyperglycemia in HFD mice and T1D diabetic mice.(A to C) Serum AKG level (A), blood glucose level (B), and insulin level (C) in male C57BL/6 mice (12 weeks old) fed with HFD intraperitoneal saline or AKG (10 mg/kg body weight) for 3 hours (n = 6 to 8). (D) PEPCK, G6Pase, and FBP mRNA expression in mice liver (n = 6). (E) PEPCK, G6Pase, and FBP mRNA expression in the liver. Mice (12 weeks old) fed with HFD intraperitoneal actinomycin D (1 mg/kg) or actinomycin D (1 mg/kg) + AKG (10 mg/kg) for 1, 2, and 3 hours (n = 6). (F to H) Immunoblots (F) and quantification (G), and enzyme activity (H) of PEPCK, G6Pase, and FBP in mice liver (n = 3 to 8). (I and J) Serum insulin level (I) and blood glucose (J) in T1D male mice (12 weeks old) fed with chow diet intraperitoneal saline or AKG for 6 hours (n = 8). (K to M) Immunoblots (K) and quantification (L), and enzyme activity (M) of PEPCK, G6Pase, and FBP in mice liver (n = 3 to 8). Results are presented as means ± SEM. (B, C, E, and J) *P = 0.05 by two-way ANOVA followed by post hoc Bonferroni tests. (A, D, G to I, L, and M) *P = 0.05 by nonpaired Student’s t test.

Fig 5: AKG suppresses hepatic gluconeogenesis in vitro.(A) Schematic representation of primary hepatocyte treated with AKG. (B to E) Basal glucose production (B), immunoblots (C) and quantification (D), and enzyme activity (E) of PEPCK, G6Pase, and FBP from 12-week-old male C57BL/6 mouse primary hepatocyte cultured with vehicle or 0.25 mM PA for 24 hours and then treated with vehicle or 100 µM AKG for 6 hours (n = 4 to 12). (F) Schematic representation of liver slice treated with AKG. (G to I) Immunoblots (G) and quantification (H), and enzyme activity (I) of PEPCK, G6Pase, and FBP. Liver slices were cultured with 0.25 mM PA for 24 hours and then treated with vehicle or 100 µM AKG for 6 hours (n = 3 to 8). (J) Schematic representation of primary hepatocyte treated with AKG. (K to M) Immunoblots (K) and quantification (L), and enzyme activity (M) of PEPCK, G6Pase, and FBP. Twelve-week-old male C57BL/6 mice were fed an HFD for 11 weeks. Primary hepatocytes were treated with vehicle or 100 µM AKG for 6 hours (n = 3 to 8). Results are presented as means ± SEM. In (B), (D), and (E), different letters between bars indicate P = 0.05 by one-way ANOVA followed by post hoc Tukey’s tests. (H, I, L, and M) *P = 0.05 by nonpaired Student’s t test.