Diisopropylamine dichloroacetate, a novel pyruvate dehydrogenase kinase 4 inhibitor, as a potential therapeutic agent for metabolic disorders and multiorgan failure in severe influenza

Abstract

Severe influenza is characterized by cytokine storm and multiorgan failure with metabolic energy disorders and vascular hyperpermeability. In the regulation of energy homeostasis, the pyruvate dehydrogenase (PDH) complex plays an important role by catalyzing oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle and fatty acid synthesis, and thus its activity is linked to energy homeostasis. The present study tested the effects of diisopropylamine dichloroacetate (DADA), a new PDH kinase 4 (PDK4) inhibitor, in mice with severe influenza. Infection of mice with influenza A PR/8/34(H1N1) virus resulted in marked down-regulation of PDH activity and ATP level, with selective up-regulation of PDK4 in the skeletal muscles, heart, liver and lungs. Oral administration of DADA at 12-h intervals for 14 days starting immediately after infection significantly restored PDH activity and ATP level in various organs, and ameliorated disorders of glucose and lipid metabolism in the blood, together with marked improvement of survival and suppression of cytokine storm, trypsin up-regulation and viral replication. These results indicate that through PDK4 inhibition, DADA effectively suppresses the host metabolic disorder-cytokine cycle, which is closely linked to the influenza virus-cytokine-trypsin cycle, resulting in prevention of multiorgan failure in severe influenza.

Figure 1. Time course of changes in PDH activity and ATP levels in the skeletal muscles, heart, lungs, liver and brain of IAV-infected mice.

Mice were infected with IAV/PR/8/34(H1N1) at 120 pfu intranasally and the levels of PDH activity (A) and ATP (B) in the skeletal muscles, heart, lungs, liver and brain were analyzed at days 0 (d0), 3 (d3) and 7 (d7) post-infection. PDH activity levels after IAV infection relative to the values at day 0. Data are mean ± SD of 5 mice per group. ^# P<0.05, ^## P<0.01 vs. day 0, by one-way analysis of variance (ANOVA) with Tukey post hoc test.

Figure 2. Time course of changes in PDK4 protein expression levels in the skeletal muscles, heart, lungs and liver of IAV-infected mice.

Mice were infected with 120 pfu of IAV, and PDK4 protein expression levels in the skeletal muscles, heart, lungs and liver were measured by western immunoblotting at day 0 (d0), day 3 (d3) and day 7 (d7) post-infection. β-Actin was used as internal control. PDK4 expression levels after IAV infection relative to the values at day 0. Data are mean ± SD of three experiments from 5 mice per band. *P<0.05, **P<0.01, vs. day 0, by one-way analysis of variance (ANOVA) with Tukey post hoc test.

Figure 3. Treatment with DADA restores suppressed PDH activity and ATP levels in the skeletal muscles, heart, lungs and liver of IAV-infected mice.

Mice infected with 120 pfu of IAV were treated orally with DADA at 50 mg/kg or vehicle at 12-h intervals for 14 days, and the levels of PDH activity (A) and ATP (B) in the skeletal muscles, heart, lungs, liver and brain of mice were analyzed at day 7 post-infection. PDH activity levels relative to the values of the control (no-infection). Values are mean ± SD of 5 mice per group. ^# P<0.05, ^## P<0.01, vs. no-infection, *P<0.05, **P<0.01, vs. infected group treated with vehicle, by one-way analysis of variance (ANOVA) and Tukey post hoc test.

Figure 4. DADA improves blood glucose, lactate, β-hydroxybutyric acid, free fatty acids and ATP levels.

Mice infected with 120 and 200 pfu of IAV were treated with oral DADA at 50 mg/kg or vehicle at 12-h intervals and the levels of glucose, lactate, free fatty acids, β-hydroxybutyric acid, and ATP in the blood were analyzed at day 7 post-infection. Values are mean ± SD of 5 mice per group. ^# P<0.05 and^## P<0.01 vs. no-infection. *P<0.05, **P<0.01, vs. infected group treated with vehicle, by one-way analysis of variance (ANOVA) and Tukey post hoc test.

Figure 5. DADA suppresses induction of various cytokines in the lungs of IAV-infected mice.

Mice infected with 120 pfu of IAV were treated with oral DADA at 50 mg/kg or vehicle at 12-h intervals and the levels of various cytokines in the lungs at day 2 post-infection were analyzed by ELISA. Data are mean ± SD of 5 mice per group. ^# P<0.05, ^## P<0.01, vs. no-infection, *P<0.05, **P<0.01, vs. infected group treated with vehicle, by one-way analysis of variance (ANOVA) with Tukey post hoc test.

Figure 6. Effects of DADA on viral replication.

Mice infected with 120 pfu of IAV were treated with oral DADA at 50 mg/kg or vehicle at 12-h intervals and viral NS1 replication in the lungs was analyzed quantitatively by real-time PCR at days 2 (d2), 4 (d4) and 6 (d6) post-infection. Data were normalized relative to GAPDH expression, which was used as the internal control. Open columns: control (no-infection) group [values are below the detection level (ND)]. Data are mean ± SD of three experiments in 3 mice per group. *P<0.05, **P<0.01, vs. infected group treated with vehicle, by one-way analysis of variance (ANOVA) and Tukey post hoc test.

Figure 7. Effects of DADA on trypsin up-regulation.

Mice infected with 120 pfu of IAV were treated with oral DADA at 50 mg/kg or vehicle at 12-h intervals and trypsin mRNA levels in the skeletal muscles, heart, liver and brain were measured by real-time PCR at day 4 post-infection. Trypsin mRNA levels in the lungs were also measured at days 2 (d2), 4 (d4) and 6 (d6) post-infection. Trypsin mRNA expression levels after IAV infection relative to that of no-infection. Values are mean ± SD of three experiments in 5 mice per each group. ^# P<0.05, ^## P<0.01, vs. no-infection, *P<0.05, **P<0.01, vs. infected mice treated with vehicle, by one-way analysis of variance (ANOVA) and Tukey post hoc test.

Figure 8. Effects of DADA on survival rate, body weight, and food and water intake.

Mice infected with 60 pfu of IAV, representing the 50% lethal dose, were treated with oral DADA at 50 mg/kg, vehicle, or DCA administered peritoneally at 28 mg/kg at 12-h intervals for 14 days. The survival rate (A), body weight (B), food intake (C), and water intake (D) by infected mice were monitored. Differences in survival rate were analyzed by Kaplan-Meier and log-rank tests. Data are mean ± SD of 15 mice per group. *P<0.05, **P<0.01, vs. infected group treated with vehicle, by two-way ANOVA.

Figure 9. Effects of DADA on pathological changes in the lungs.

Mice infected with 60 pfu of IAV were treated orally with DADA at 50 mg/kg or vehicle at 12-h intervals for 6 days. The lungs were isolated at days 0 (d0), 2 (d2), 4 (d4) or 6 (d6) post-infection, processed and stained with hematoxylin and eosin for histopathological evaluation. Each result is representative of 5 animals in each group. Bar, 300 µm.

Figure 10. Diagram illustrating the proposed pathogenic mechanism of MOF in severe influenza involving the host metabolic disorders–cytokine cycle linked to the influenza virus–cytokine–trypsin cycle.

PPARs, peroxisome proliferator-activated receptors; PDK4, pyruvate dehydrogenase kinase 4; DADA, diisopropylamine dichloroacetate.