Energy metabolism disorders in rare and common diseases. Toward bioenergetic modulation therapy and the training of a new generation of European scientists

Energy metabolism alterations are found in a large number of rare and common diseases of genetic or environmental origin. The number of patients that could benefit from bioenergetic modulation therapy (BIOMET) is therefore very important and includes individuals with pathologies as diverse as mitochondrial diseases, acute coronary syndrome, chronic kidney disease, asthma or even cancer. Although, the alteration of energy metabolism is disease specific and sometimes patient specific, the strategies for BIOMET could be common and target a series of bioenergetic regulatory mechanisms discussed in this article. An excellent training of scientists in the field of energy metabolism, related human diseases and drug discovery is also crucial to form a young generation of MDs, PHDs and Pharma or CRO-group leaders who will discover novel personalized bioenergetic medicines, through pharmacology, genetics, nutrition or adapted exercise training. The Mitochondrial European Educational Training (MEET) consortium was created to pursue this goal, and we dedicated here a special issue of Organelle in Focus (OiF) to highlight their objectives. A total of 10 OiFs articles constitute this Directed Issue on Mitochondrial Medicine. As part of this editorial article, we asked timely questions to the PR. Jan W. Smeitink, professor of Mitochondrial Medicine and CEO of Khondrion, a mitochondrial medicine company. He shared with us his objectives and strategies for the study of mitochondrial diseases and the identification of future treatments.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Molecular and functional characterization of the plastid-localized Phosphoenolpyruvate enolase (ENO1) from Arabidopsis thaliana

The Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non-root-hair cells, but not in the mesophyll of leaves. Two T-DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.     

Effects of coumestrol on lipid and glucose metabolism as a farnesoid X receptor ligand

In the course of an effort to identify novel agonists of the farnesoid X receptor (FXR), coumestrol was determined to be one such ligand. Reporter and in vitro coactivator interaction assays revealed that coumestrol bound and activated FXR. Treatment of Hep G2 cells with coumestrol stimulated the expression of FXR target genes, thereby regulating the expression of target genes of the liver X receptor and hepatocyte nuclear factor-4α. Through these actions, coumestrol is expected to exert beneficial effects on lipid and glucose metabolism.     

Born to run; the story of the PEPCK-Cmus mouse

In order to study the role of the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-C) in skeletal muscle, PEPCK-Cmus mice were created by introducing the cDNA for the enzyme, linked to the human alpha-skeletal actin gene promoter, into their germ line. Two founder lines generated by this procedure were bred together, creating a line of mice that have 9.0 units/g skeletal muscle of PEPCK-C, as compared to 0.080 units/g in muscle from control animals. The mice were more active than controls in their cages and could run for up to 5 km, at a speed of 20 m/min without stopping (control mice run for 0.2 km at the same speed). Male PEPCK-Cmus mice are extremely aggressive, as well as hyperactive. During strenuous exercise, they use fatty acids as a fuel more efficiently than do controls and produce far less lactate than do control animals, perhaps due to the greatly increased number of mitochondria in their skeletal muscle. PEPCK-Cmus mice also store up to five-times more triglyceride in their skeletal muscle, but have only marginal amounts of triglyceride in their adipose tissue depots, despite eating 60% more than controls. The concentration of leptin and insulin the blood of 8-12 months of PEPCK-Cmus mice is far lower than noted in the blood of control animals of the same age. These mice live longer than controls and the females remain reproductively active for as long as 35 months. The possible reasons for the profound alteration in activity and longevity caused the introduction of a simple metabolic enzyme into the skeletal muscle of the mice will be discussed.     

Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.     

Studies of regulatory metabolism in Moniezia expansa: Effects of cambendazole and mebendazole

Effects of cambendazole and mebendazole on the respiratory metabolism of the anterior portion of Moniezia expansa were investigated in vitro. Anaerobically and in the presence of glucose, both drugs inhibited glucose uptake and increased glycogen utilisation. They reduced succinate production, by inhibiting fumarate reductase and (in the case of cambendazole) phosphoenolypyruvate carboxykinase activities, and increased lactate production. The additional lactate formed was accumulated in the worms. The drugs diminished ATP synthesis and/or turnover of adenine nucleotides. Aerobically, the drugs exerted similar effects on glucose uptake, glycogen utilisation and adenine nucleotides but the formation of end products was unaffected. Hexokinase and phosphofructokinase activities were inhibited by the drugs in vitro, but were not inhibited in extracts of parasites preincubated with the drugs.     

Leishmania mexicana: molecular cloning and characterization of enolase

The gene of Leishmania mexicana enolase was cloned and overexpressed in Escherichia coli as an active enzyme; the protein was biochemically analyzed. This enolase shares with enolases from other trypanosomatids the presence of three atypical residues, each with a reactive side group, near the active site, already described for the enzyme from Trypanosoma brucei. The natural enzyme was purified, using a three-step procedure, from a cytosolic fraction of L. mexicana promastigotes. The kinetic properties of the purified recombinant enzyme were similar to those of the natural enzyme. Both the recombinant and natural enzyme were inhibited by inorganic pyrophosphate. Subcellular localization analysis after differential centrifugation showed that the enzyme activity is only associated with the cytosolic fraction. However, an apparently inactive form of enolase was detected by Western blots in the microsomal fraction. Digitonin treatment of parasites and immunofluorescence studies with permeabilized and non-permeabilized parasites showed that enolase is also associated with membranes and it was found at the external face of the plasma membrane.