Fig 2: Palmitoleic acid reduces enzymatic activities of MDH2, PC, and PEPCK under HFD. C57BL/6N male mice of 5–6 weeks of age were fed with LFD or HFD for 12 weeks. At the 13th week, BSA, oleic acid, or palmitoleic acid was administered intragastrically. At the 18th week, mice were sacrificed, and liver tissue was collected. The enzyme activities of MDH2, PC, and PEPCK in the liver were measured. (A) Enzymatic activity of MDH2 in the liver of HFD-fed mice. (B) Enzymatic activity of PC in the liver of HFD-fed mice. (C) Enzymatic activity of PEPCK in the liver of HFD-fed mice. The data are mean ± s.e. (error bars). n = 4–7 mice per group. *, p < 0.05, **, p < 0.01, HFD BSA vs. HFD palmitoleic acid; ††, p < 0.01, †††, p < 0.001, HFD oleic acid vs. HFD palmitoleic acid.

Fig 3: Palmitoleic acid reduced the expression of SIRT3 and MDH2 but did not change the protein levels of PC, PEPCK, and SIRT1 in the liver of mice under HFD. C57BL/6N male mice of 5–6 weeks of age were fed with HFD for 12 weeks. At the 13th week, BSA, oleic acid, or palmitoleic acid were administered intragastrically. At the 18th week, mice were euthanized, and liver tissues were collected. (A) Expression of SIRT3, SIRT1, and gluconeogenic enzymes in the liver of mice under HFD. Protein expression was detected using Western blot analysis. (B) Quantification of SIRT3 protein level. (C) Quantification of PC protein level. (D) Quantification of PEPCK protein level. (E) Quantification of MDH2 protein level. (F) Quantification of SIRT1 protein level. AU, arbitrary units. The data are mean ± s.e. (error bars). n = 4–6 mice per group. *, p < 0.05, **, p < 0.01, HFD BSA vs. HFD palmitoleic acid; †, p < 0.05, oleic acid vs. palmitoleic acid.

Fig 4: OXGR1 is not required for AKG-induced gluconeogenesis suppression.(A to D) Body weight (A), body composition (B), gWAT index (C), and gastrocnemius (Gastro.) muscle index (D) of male wild-type (WT) control (littermates) or OXGR1KO mice. At 12 weeks of age, both control and knockout (KO) mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 11 weeks (n = 8). (E to G) Representative images (E) and quantification (F and G) of hematoxylin and eosin (H&E) staining in gWAT and gastrocnemius (n = 8). H&E staining shows the size of gastrocnemius fibers (F) and adipocytes (G). gWAT bar is 50 μm; gastrocnemius bar is 25 μm. (H to L) Blood glucose (H), serum HbA1c level (I), insulin level (J), and PTT (K and L) (n = 8). (M to O) Immunoblots (M) and quantification (N), and enzyme activity (O) of PEPCK, G6Pase, and FBP (n = 4 to 8). Results are presented as means ± SEM. (A, H, J, and K) *P ≤ 0.05 by two-way ANOVA followed by post hoc Bonferroni tests. In (B) to (D), (F), (G), (I), (L), (N), and (O), different letters between bars indicate P ≤ 0.05 by one-way ANOVA followed by post hoc Tukey’s tests.

Fig 5: 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.