Inhibition of energy metabolism by 2‐methylacetoacetate and 2‐methyl‐3‐hydroxybutyrate in cerebral cortex of developing rats

Abstract

Mitochondrial beta-ketothiolase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiencies are inherited neurometabolic disorders affecting isoleucine catabolism. Biochemically, beta-ketothiolase deficiency is characterized by intermittent ketoacidosis and urinary excretion of 2-methyl-acetoacetate (MAA), 2-methyl-3-hydroxybutyrate (MHB) and tiglylglycine (TG), whereas in MHBD deficiency only MHB and tiglylglycine accumulate. Lactic acid accumulation and excretion are also observed in these patients, being more pronounced in MHBD-deficient individuals, particularly during acute episodes of decompensation. Patients affected by MHBD deficiency usually manifest severe mental retardation and convulsions, whereas beta-ketothiolase-deficient patients present encephalopathic crises characterized by metabolic acidosis, vomiting and coma. Considering that the pathophysiological mechanisms responsible for the neurological alterations of these disorders are unknown and that lactic acidosis suggests an impairment of energy production, the objective of the present work was to investigate the in vitro effect of MAA and MHB, at concentrations varying from 0.01 to 1.0 mmol/L, on several parameters of energy metabolism in cerebral cortex from young rats. We observed that MAA markedly inhibited CO2 production from glucose, acetate and citrate at concentrations as low as 0.01 mmol/L. In addition, the activities of the respiratory chain complex II and succinate dehydrogenase were mildly inhibited by MAA. MHB, at 0.01 mmol/L and higher concentrations, strongly inhibited CO2 production from all tested substrates, as well as the respiratory chain complex IV activity. The other activities of the respiratory chain were not affected by these metabolites. The data indicate a marked blockage in the Krebs cycle and a mild inhibition of the respiratory chain caused by MAA and MHB. Furthermore, MHB inhibited total and mitochondrial creatine kinase activities, which was prevented by the use of the nitric-oxide synthase inhibitor L-NAME and glutathione (GSH). These data indicate that the effect of MHB on creatine kinase was probably mediated by oxidation or other modification of essential thiol groups of the enzyme by nitric oxide and other by-products derived from this organic acid. In contrast, MAA did not affect creatine kinase activity. Taken together, these observations indicate that aerobic energy metabolism is inhibited by MAA and to a greater extent by MHB, a fact that may be related to lactic acidaemia occurring in patients affected by MHBD and beta-ketothiolase deficiencies. If the in vitro effects detected in the present study also occur in vivo, it is tempting to speculate that they may contribute, at least in part, to the neurological dysfunction found in these disorders.

Evidence for a synergistic action of glutaric and 3‐hydroxyglutaric acids disturbing rat brain energy metabolism

“…We investigated the in vitro effect of these metabolites added separately or combined on glucose utilization, lactate generation, glucose and acetate oxidation, as well as on key enzyme activities involved in energy metabolism in brain of developing rats. The control values obtained in the various parameters analyzed in rat cerebral cortex were similar to those found by other investigators (Leighton et al, 1985;Toyomizu et al, 1999;Rasia-Filho et al, 2002;Sgaravatti et al, 2003;Reis de Assis et al, 2004;Schweigert et al, 2004;Rosa et al, 2005;Schuck et al, 2007a,b).…”

Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl‐CoA thiolase (T2) deficiency

“…Several patients with T2 deficiency developed chronic neurological impairment, mainly extrapyramidal, independent of frank ketoacidosis (Buhaş et al, ; Fukao et al, ; Paquay et al, ). In vitro studies indicate that 2MAA and 2M3HB exert neurotoxic effects (Leipnitz et al, ; Rosa et al, ). Therefore, accumulated isoleucine‐catabolic metabolites may contribute to neurological impairment in patients with T2 deficiency (Fukao et al, ; Paquay et al, ).…”

“…However, an increasing body of evidence indicates that chronic neurological impairment, mainly extrapyramidal manifestations, can exist independent of frank ketoacidosis even in patients with T2 deficiency confirmed at the molecular level (Buhaş et al, 2013;Fukao et al, 2018;Paquay et al, 2017). In vitro studies indicate that 2MAA and 2M3HB exert neurotoxic effects (Leipnitz et al, 2010;Rosa et al, 2005).…”

Myoglobin-H2O2 catalyzes the oxidation of β-ketoacids to α-dicarbonyls: Mechanism and implications in ketosis

“…Another β-ketoacid, 2-methylacetoacetate (MAA), accumulates in isoleucinemic patients, whose 2-methylacetoacetyl-CoA thiolase is defective [4], leading to massive urinary excretion of MAA, triglylglycine and 2-methyl-3-hydroxybutyrate [5]. Isoleucinemic patients are characterized by recurrent ketoacidosis, lethargy, vomiting, and usually manifest mental retardation, convulsions and coma [6]. …”