Phenylbutyrate therapy for pyruvate dehydrogenase complex deficiency and lactic acidosis

Abstract

Lactic acidosis is a buildup of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1α subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57BL/6 wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1α in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1α through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noa(m631) zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis.

Conflict of interest statement

Competing interests: the authors declare that they have no competing interests.

Figures

Fig. 1. PDHC phosphorylation and activity after drug treatment of human wild-type (WT) fibroblasts

A. Western blotting of BA1054 fibroblasts treated with 0.25, 0.5, or 1 mM of phenylbutyrate for 24 hours, or untreated. The images are representative of two independent experiments. B. The average band intensities of phosphorylated E1 normalized for total E1 from two independent experiments. C. Two independent fibroblast cell lines (BA1054 and BA1020) were treated with phenylbutyrate at 1 mM for 24 hours before measurement of PDH enzyme activity. *: p<0.05; **: p<0.01.

Fig. 2. PDHC phosphorylation status in mouse tissues

Western blot analysis of brain, muscle, and liver mitochondrial extract was performed with antibodies against the phosphorylated form of E1α (P-E1α). Each lane corresponds to brain, muscle, or liver mitochondrial extracts from an independent, representative mouse. Total E1α protein and cytochrome c oxidase (COX) are shown as controls. The graphs present densitometric quantification of n=5 mice from each treatment group. Means ± SDs are shown. *p

Fig. 3. Analysis of PDHC activity in mouse brain

PDHC activity was measured in brain mitochondrial extracts of mice treated with phenylbutyrate [250 mg/kg/day (n=10) or 500 mg/kg/day (n=8)] and in saline controls (n=16). **p

Fig. 4. PDK inhibition by phenylbutyrate

A. Lineweaver-Burk plot of WT recombinant E1α in the absence (■) or in the presence of 0.25 mM (●), 0.5 mM (□), and 1 mM (○) of phenylbutyrate. B. Replot of slopes measured from (A) against the concentration of phenylbutyrate. The intersection of the line with the x axis gives the value of Ki. C and E. Phenylbutyrate (PB, stick representation) in the two identified pockets on the protein surface of PDK2. D and F. Specific interactions of phenylbutyrate with amino acid residues (stick representation) at the binding sites. Van der Waals interaction spheres of the amino acid residues (stick representation) in contact with the inhibitor have been removed for clarity.

Fig. 5. PDHC activity in PDHC-deficient cells

PDHC activity is shown for WT control (white bar) and PDHC-deficient cells incubated with phenylbutyrate. The molecular defects of the corresponding patient cell lines are shown in Table 1. Patients 1-12 (dark gray bars) have mutations in PDHA1, patient 13 (light gray bar) has PDHC deficiency due to abnormal ubiquitination and degradation of E1β (66), and patients 14 and 15 (black bars) have mutations in PDHX. *p<0.05.

Fig. 6. Position of responsive and nonresponsive PDHA1 mutations

A. Picture of heterotetrameric human E1 [Protein Data Bank (PDB: 3EXE)] with the four chains highlighted (α, green; β, cyan; α′, magenta; β′, yellow), and the positions (arrows) of non-responsive mutations Met181-α and Arg349-α shown as ball and stick colored in blue. The positions of Met181-α and Arg349-α are part of the α subunit, and are underneath the β subunit. The square indicates the region with phenylbutyrate-responsive mutations, and is enlarged in (B). B. Orientation is the same as in (A). The following elements are shown: Asn135-α, Val138-α, Pro221-α, Tyr214-α, and Arg234-α (red), phosphorylation sites Ser203-α, Ser264-α, Ser271-α (yellow), and coenzyme ThDP (blue). The ribbon of the N-terminus (N-ter) of the E1α protein (residues 1 to 35) is depicted in gray; the structural elements containing Pro221-α, Tyr214-α, and Ser203-α are shown in cyan. The loop containing Ser264-α and Ser271-α is shown in magenta. The loop containing the residues Asn135-α and Val138-α is shown in green.

Fig. 7. Effect of phenylbutyrate on zebrafish noam631 mutants

A. Pigmentation phenotype of untreated noam631, treated mutant noam631 and untreated wild-type larvae at 8 days after fertilization (n=14 per group). The bar graph shows quantification of zebrafish pigmentation, calculated with ImageJ software reporting mean gray value for a given area. The binary representation assumes that 0 is black and the maximum value (255 at 8 bit) is white. B. Movements of the larvae are plotted as the distance travelled by the larvae relative to time in 48-well plate, as previously described (31). C and D. Lactate and pyruvate concentrations in untreated or treated mutant noam631 larvae and in wild-type larvae (n=6 per group). *: p<0.05.

Fig. 8. Effect of phenylbutyrate on blood lactate on hepatectomized mice

Blood lactate concentration at specified time intervals after partial hepatectomy in mice treated with saline or phenylbutyrate (n=5 per group). *: p