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α-Ketoglutaric acid ameliorates hyperglycemia in diabetes by inhibiting hepatic gluconeogenesis via serpina1e signaling - PubMed

  • ️Sat Jan 01 2022

. 2022 May 6;8(18):eabn2879.

doi: 10.1126/sciadv.abn2879. Epub 2022 May 4.

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α-Ketoglutaric acid ameliorates hyperglycemia in diabetes by inhibiting hepatic gluconeogenesis via serpina1e signaling

Yexian Yuan et al. Sci Adv. 2022.

Abstract

Previously, we found that α-ketoglutaric acid (AKG) stimulates muscle hypertrophy and fat loss through 2-oxoglutarate receptor 1 (OXGR1). Here, we demonstrated the beneficial effects of AKG on glucose homeostasis in a diet-induced obesity (DIO) mouse model, which are independent of OXGR1. We also showed that AKG effectively decreased blood glucose and hepatic gluconeogenesis in DIO mice. By using transcriptomic and liver-specific serpina1e deletion mouse model, we further demonstrated that liver serpina1e is required for the inhibitory effects of AKG on hepatic gluconeogenesis. Mechanistically, we supported that extracellular AKG binds with a purinergic receptor, P2RX4, to initiate the solute carrier family 25 member 11 (SLC25A11)-dependent nucleus translocation of intracellular AKG and subsequently induces demethylation of lysine 27 on histone 3 (H3K27) in the seprina1e promoter region to decrease hepatic gluconeogenesis. Collectively, these findings reveal an unexpected mechanism for control of hepatic gluconeogenesis using circulating AKG as a signal molecule.

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Figures

Fig. 1.
Fig. 1.. Serum AKG level is negatively correlated with HbA1c.

(A) Two-tailed Pearson’s correlation coefficient analysis of plasma AKG level and HbA1c in mice. (B) Plasma AKG level in chow, DIO, and DB mice. Chow male mice were fed a chow diet at 8 weeks of age (n = 22). For DIO mice, 8-week-old male C57BL/6 mice were fed an HFD for 12 weeks (n = 30). DB (db/db diabetes) male mice were fed a chow diet at 10 weeks of age (n = 9). (C) Two-tailed Pearson’s correlation coefficient analysis of plasma AKG level with HbA1c in Chinese adults (36 males and 6 females). (D) Two-tailed Pearson’s correlation coefficient analysis of plasma AKG and related metabolite level with HbA1c in Chinese adults (36 males and 6 females). *P < 0.05 indicates significant correlation between human plasma AKG and related metabolite level with HbA1c. Gln, glutamine; Glu, glutamic acid; α-keval, α-ketoisovaleric acid; SUA, succinic acid; α-kehex, α-ketoleucine. Results are presented as means ± SEM. (A, C, and D) Two-tailed Pearson’s correlation coefficient analysis. In (B), different letters between bars indicate P ≤ 0.05 by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s tests.

Fig. 2.
Fig. 2.. Chronic AKG supplementation prevents diet-induced hyperglycemia.

(A) Blood glucose from chow-fed mice supplemented with 2% AKG for 11 weeks (n = 8). (B to E) Blood glucose (B), serum HbA1c level (C), insulin level (D), and glucagon level (E) from HFD-fed mice supplemented with 2% AKG for 11 weeks (n = 8). (F to H) GTT (1 g/kg) (F), ITT (1 U/kg) (G), and PTT (1 g/kg) (H) in HFD-fed mice (n = 8). (I) Serum insulin level during PTT (1 g/kg) (n = 8). (J to L) Time course of blood glucose and glucose infusion rate (GIR) during hyperinsulinemic-euglycemic clamping (Clamp) (J), glucose disposal rate (GDR) (K), and endogenous glucose production (EGP) (L) from HFD-fed mice supplemented with 2% AKG for 11 weeks (n = 8 to 9). (M to P) mRNA expression (M), immunoblots (N) and quantification (O), and enzyme activity (P) of PEPCK, G6Pase, and FBP in the mice liver (n = 8). Results are presented as means ± SEM. (A, B, D, and F to J) *P ≤ 0.05 by two-way ANOVA followed by post hoc Bonferroni tests. (C, E, K to M, O, and P) *P ≤ 0.05 by nonpaired Student’s t test.

Fig. 3.
Fig. 3.. 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. 4.
Fig. 4.. 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.

Fig. 5.
Fig. 5.. 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. 6.
Fig. 6.. 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. 7.
Fig. 7.. 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.

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