Inhibition of the oxygen sensor PHD2 in the liver improves survival in lactic acidosis by activating the Cori cycle

Abstract

Loss of prolyl hydroxylase 2 (PHD2) activates the hypoxia-inducible factor-dependent hypoxic response, including anaerobic glycolysis, which causes large amounts of lactate to be released from cells into the circulation. We found that Phd2-null mouse embryonic fibroblasts (MEFs) produced more lactate than wild-type MEFs, as expected, whereas systemic inactivation of PHD2 in mice did not cause hyperlacticacidemia. This unexpected observation led us to hypothesize that the hypoxic response activated in the liver enhances the Cori cycle, a lactate-glucose carbon recycling system between muscle and liver, and thereby decreases circulating lactate. Consistent with this hypothesis, blood lactate levels measured after a treadmill or lactate tolerance test were significantly lower in Phd2-liver-specific knockout (Phd2-LKO) mice than in control mice. An in vivo (13)C-labeled lactate incorporation assay revealed that the livers of Phd2-LKO mice produce significantly more glucose derived from (13)C-labeled lactate than control mice, suggesting that blockade of PHD2 in the liver ameliorates lactic acidosis by activating gluconeogenesis from lactate. Phd2-LKO mice were resistant to lactic acidosis induced by injection of a lethal dose of lactate, displaying a significant elongation of survival. Moreover, oral administration of a PHD inhibitor improved survival in an endotoxin shock mice model. These data suggest that PHD2 is a potentially novel drug target for the treatment of lactic acidosis, which is a serious and often fatal complication observed in some critically ill patients.

Keywords: PHD inhibitor; gluconeogenesis; hyperlactatemia; hypoxic response; sepsis.

Fig. 1.

Inactivation of Phd2 in the liver reduces blood lactate levels. (A) Lactate efflux analysis in Phd2^+/+ and Phd2^−/− MEFs. Values were from triplicated dishes. Error bars indicate 1 SEM. (B) The blood lactate level in Control and Phd2-SKO mice. Error bars, SEM. (C) The blood lactate level 50 min after the treadmill in Control and Phd2-SKO mice. Error bars, SEM. (D) Urinary lactate measurement. Lactate levels in urine were measured before (Left) and 20 min after (Right) the lactate injection in Control and Phd2-SKO mice. Error bars, SEM. (E) Real-time RT-PCR analysis of Phd2 mRNA in the livers of Control and Phd2-LKO mice. Error bars, SEM. (F) Blood lactate levels in Control and Phd2-LKO mice. Error bars, SEM. (G) Treadmill experiment in Control and Phd2-LKO mice. The blood lactate levels 50 min after the exercise (Left) and time to exhaustion (Right) are shown. Error bars, SEM. (H) Lactate tolerance test. 0.5 mg per gram body weight of lactate was intraperitoneally injected, and the blood lactate levels were measured at the indicated time (Left). Area under the curve (AUC) was also analyzed (Right). Error bars, SEM. *P = 0.0448; †P = 0.000342; ‡P = 0.000118; §P = 2.53 × 10⁻⁷ versus Control.

Fig. S1.

Systemic or liver-specific inactivation of Phd2. (A) Real-time RT-PCR analysis of transporters or enzymes involved in the skeletal muscles of Control and Phd2-SKO mice: glucose transporter GLUT1 (Slc2a1), phosphofructokinase, muscle (Pfkm), phosphoglycerate kinase 1 (Pgk1), LDHA (Ldha), and PDH kinase 1 (Pdk1). Error bars, SEM. (B) Phd2 intact allele frequency was analyzed in the indicated tissues of Phd2-LKO mice (n = 3) by real-time PCR. The copy number of Phd2 locus and Rn18s locus was compared. All of the values were normalized with the control DNA (genomic DNA from livers of Phd2^F/F mice carrying no Cre-recombinase transgene; n = 3). The unpaired Student t-test was performed versus control DNA. Note that gene disruption in Phd2-LKO mice is liver-specific. Error bars, SEM. (C) Blood glucose levels under the ad libitum (Left) or fasted (Right) conditions in Control and Phd2-LKO mice. Error bars, SEM.

Fig. 2.

Inactivation of Phd2 in the liver activates gluconeogenesis from lactate. (A) Real-time RT-PCR analysis of transporters or enzymes involved in gluconeogenesis in the livers of Control and Phd2-LKO mice. Error bars, SEM. (B) LC/MS-based quantification of total ¹³C atoms in glucose that were newly synthesized from ¹³C₃-lactate. Data were not corrected for the natural abundances of ¹³C_x-glucose (Materials and Methods). Error bars, SEM.

Fig. S2.

Gluconeogenesis in Phd2-LKO liver. (A) Real-time RT-PCR analysis of transporters or enzymes involved in gluconeogenesis in the livers of Control and Phd2-LKO mice: MCT1 (Slc16a1), monocarboxylate transporter 4 (MCT4) (Slc16a3), lactate dehydrogenase B (LDHB) (Ldhb), pyruvate carboxylase (PC) (Pcx), MDH2 (Mdh2), malate dehydrogenase 1 (MDH1) (Mdh1), fructose-1,6-bisphosphatase 1 (FBP1) (Fbp1), and glucose-6-phosphate dehydrogenase (G6PD) (G6pd). Error bars, SEM. (B) LC/MS-based quantification of nonlabeled (¹²C₆) and ¹³C-labeled (¹³C₁–¹³C₆) glucose. Note that the shaded bars indicate the estimated natural abundances of ¹³C_x-glucose. Error bars, SEM.

Fig. 3.

Quantification of total ¹³C atoms in ¹³C₃-lactate-derived metabolites. Cultured hepatocytes from Control and Phd2-LKO mice were labeled with ¹³C₃-lactate. Total ¹³C atoms in indicated metabolites were quantified (µmol/g protein), using CE/MS. Data were not corrected for the natural abundances of ¹³C_x-glucose (Materials and Methods). Error bars, SEM.

Fig. S3.

Expression of glycolytic genes in Phd2-LKO liver. The expression levels of Hk2, Pfkl, and Pklr in the livers of Control or Phd2-LKO mice were analyzed by real-time RT-PCR. Error bars, SEM.

Fig. 4.

Inactivation of Phd2 in the liver causes resistance to lactic acidosis. (A) Venous blood gas analysis. pH (Left) and base excess (Right) were analyzed in the venous sample from the retro-orbital sinus 20 min after the injection of 0.5 mg/g body weight of lactate. Error bars, SEM. (B) Kaplan–Meier survival analysis after the injection of 0.5 mg/g body weight of lactate. MS indicates median survival. (C) The blood lactate analysis in indicated points after LPS injection in vehicle or PHD inhibitor GSK360A-treated wild-type mice (Left). AUC was also calculated (Right). Error bars, SEM. *P = 0.00598; †P = 0.000641; ‡P = 0.00742 versus Control. (D) Survival analysis in LPS-induced endotoxin shock model. Wild-type mice were orally treated with vehicle or PHD inhibitor GSK360A immediately after the LPS injection. U.D., undefined. (E) Scheme of how the Cori cycle is activated in the liver upon PHD2-inactivation.

Fig. S4.

GSK360A-induced hypoxic response. Real-time RT-PCR analysis of Ldha, Pdk1, Pgk1, and L-type amino acid transporter 1 (LAT1) (Slc7a5), known as direct HIF-target mRNAs, in the livers of vehicle or GSK360A treated wild-type male mice (n = 3). Total RNA from the liver was isolated 6 h after the oral administration of vehicle (1% methyl cellulose) or GSK360A (30 mg/kg body weight). Error bars, SEM.