Metabolic Outcomes of Anaplerotic Dodecanedioic Acid Supplementation in Very Long Chain Acyl-Coa Dehydrogenase (VLCAD) Deficient Fibroblasts
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H OH metabolites OH Article Metabolic Outcomes of Anaplerotic Dodecanedioic Acid Supplementation in Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Deficient Fibroblasts Igor Radzikh, Erica Fatica †, Jillian Kodger, Rohan Shah, Ryan Pearce and Yana I. Sandlers * Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA; [email protected] (I.R.); [email protected] (E.F.); [email protected] (J.K.); [email protected] (R.S.); [email protected] (R.P.) * Correspondence: [email protected] † Current address: Department of Laboratory Medicine and Pathology, Rochester, MN 55904, USA. Abstract: Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD, OMIM 609575) is associ- ated with energy deficiency and mitochondrial dysfunction and may lead to rhabdomyolysis and cardiomyopathy. Under physiological conditions, there is a fine balance between the utilization of different carbon nutrients to maintain the Krebs cycle. The maintenance of steady pools of Krebs cycle intermediates is critical formitochondrial energy homeostasis especially in high-energy demand- ing organs such as muscle and heart. Even-chain dicarboxylic acids are established as alternative energy carbon sources that replenish the Krebs cycle by bypassing a defective β-oxidation path- way. Despite this, even-chain dicarboxylic acids are eliminated in the urine of VLCAD-affected individuals. In this study, we explore dodecanedioic acid (C12; DODA) supplementation and inves- Citation: Radzikh, I.; Fatica, E.; tigate its metabolic effect on Krebs cycle intermediates, glucose uptake, and acylcarnitine profiles Kodger, J.; Shah, R.; Pearce, R.; in VLCAD-deficient fibroblasts. Our findings indicate that DODA supplementation replenishes the Sandlers, Y.I. Metabolic Outcomes of Krebs cycle by increasing the succinate pool, attenuates glycolytic flux, and reduces levels of toxic Anaplerotic Dodecanedioic Acid very long-chain acylcarnitines. Supplementation in Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Keywords: VLCAD deficiency; dodecandioic acid; acylcarnitines; Krebs cycle Deficient Fibroblasts. Metabolites 2021, 11, 538. https://doi.org/10.3390/ metabo11080538 Academic Editor: Maria Fuller 1. Introduction Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD, OMIM 609575) is Received: 16 April 2021 manifested by a wide range of clinical phenotypes including hypertrophic and dilated Accepted: 9 August 2021 cardiomyopathy, rhabdomyolysis, myopathy, and hypoglycemia [1–4]. Some of these Published: 13 August 2021 symptoms can be ameliorated by nutritional restrictions. Current dietary strategies are primarily focused on eating frequent meals and avoiding consumption of very long-chain Publisher’s Note: MDPI stays neutral fatty acids, instead offering carbohydrates and medium-chain triglycerides as the energy with regard to jurisdictional claims in source [5]. Despite the special diet, nutritional management has limited success to treat published maps and institutional affil- some symptoms and metabolic derangements in VLCADD affected individuals. iations. VLCAD deficiency leads to a defect in long-chain fatty acids catabolic pathway with subsequent disruptions in energy production [6,7] and mitochondrial function [8,9]. In high energy demanding organs such as muscle and heart, carbon energy substrate utilization and the maintenance of adequate pools of Krebs cycle intermediates in mitochondria Copyright: © 2021 by the authors. are especially critical. Disruptions in adequate energy production and mitochondrial Licensee MDPI, Basel, Switzerland. bioenergetic pathways are implicated in the risk of development rhabdomyolysis [10] This article is an open access article and cardiomyopathy [11]. To target impaired mitochondrial bioenergetics in VLCADD, distributed under the terms and anaplerotic therapy was proposed as a new treatment strategy [12,13]. This therapeutic conditions of the Creative Commons approach is directed towards the restoration of energy production by replenishing pools of Attribution (CC BY) license (https:// Krebs cycle intermediates [14] while bypassing defective β-oxidation of long-chain fatty creativecommons.org/licenses/by/ acids (LCFA). Nutritional therapy with a recently FDA-approved anaplerotic triglyceride 4.0/). Metabolites 2021, 11, 538. https://doi.org/10.3390/metabo11080538 https://www.mdpi.com/journal/metabolites Metabolites 2021, 11, x FOR PEER REVIEW 2 of 13 Metabolites 2021, 11, 538 2 of 12 acids (LCFA). Nutritional therapy with a recently FDA-approved anaplerotic triglyceride triheptanoin in VLCADD demonstrated long-term positive effects, such as improvement triheptanoinof some clinical in VLCADDsymptoms demonstratedand hospitalization long-term frequency positive [15–18]. effects, Nevertheless, such as improvement VLCADD ofpresents some clinicalwith a symptomsvery heterogenic and hospitalization phenotype frequencyand clinical [15 outcomes–18]. Nevertheless, under triheptanoin VLCADD presentssupplementation with a very are also heterogenic heterogenic phenotype [16,17]. Or andal triheptanoin clinical outcomes has a limited under triheptanoineffect on the supplementationperipheral tissues are [19] also and heterogenic may induce [16 gastro,17].intestinal Oral triheptanoin symptoms has leading a limited to some effect degree on the peripheralof lipotoxicity. tissues Triheptanoin [19] and may also induce does not gastrointestinal significantly symptomsreduce episodes leading of to rhabdomyoly- some degree ofsis, lipotoxicity. thus the search Triheptanoin for the new also therapeutic does not significantly agents is still reduce ongoing. episodes of rhabdomyolysis, thusMore the search recently, for the to newaddress therapeutic exercise agentsintolerance is still and ongoing. rhabdomyolysis another anaple- rotic Moremolecule recently, was investigated. to address exercise Bleeker intolerance et al. demonstrated and rhabdomyolysis that ketone another ester-induced anaplerotic nu- moleculetritional ketosis was investigated. has beneficial Bleeker outcomes et al.demonstrated and improves that exercise ketone performance ester-induced in nutri- five tionalVLCAD-affected ketosis has subjects beneficial [20]. outcomes The data and presen improvested in exercise this study performance suggest that in five anaplerotic VLCAD- affectedketone ester subjects increases [20]. The ATP data levels presented in muscles. in this study suggest that anaplerotic ketone ester increasesEven-chain ATP levels dicarboxylic in muscles. acids (DAs) also exhibit anaplerotic characteristics and have been Even-chainproposed as dicarboxylic an alternative acids energy (DAs) su alsobstrate exhibit [21]. anaplerotic DAs are produced characteristics endogenously and have beenthrough proposed the corresponding as an alternative fatty acid energy ω-oxidation substrate or [21 from]. DAs the are β-oxidation produced of endogenously longer dicar- throughboxylic acids. the corresponding In normal circumstances, fatty acid ! th-oxidationese pathways or from are theconsideredβ-oxidation minor of fates longer how- di- carboxylicever, when acids.β-oxidation In normal becomes circumstances, impaired, fatty these acids pathways are channeled are considered to produce minor DAs. fatesDAs β however,(C6-C12) metabolism when -oxidation in mitochondria becomes impaired, and peroxisomes, fatty acids yields are channeled a Krebs cycle to produce intermediate DAs. DAsand gluconeogenic (C6-C12) metabolism precursor in mitochondria succinyl-CoA. and Due peroxisomes, to their anaplerotic yields a Krebs effect cycleand high interme- en- diateergy density and gluconeogenic [22], DAs were precursor proposed succinyl-CoA. as an alternative Due to fuel their energy anaplerotic substrate effect in andparental high energy density [22], DAs were proposed as an alternative fuel energy substrate in parental nutrition [21–23]. It is evident however that overproduction and accumulation of hepatic nutrition [21–23]. It is evident however that overproduction and accumulation of hepatic DA are toxic [24]. Dicarboxylic aciduria is strongly associated with fatty acids oxidation DA are toxic [24]. Dicarboxylic aciduria is strongly associated with fatty acids oxidation disorders while C8-C12 DAs are eliminated in urine in FAOD-affected individuals [25,26] disorders while C8-C12 DAs are eliminated in urine in FAOD-affected individuals [25,26] especially during metabolic instability and fasting episodes. especially during metabolic instability and fasting episodes. Dodecanedioic acid (DODA), a twelve carbon/medium-chain water-soluble DA de- Dodecanedioic acid (DODA), a twelve carbon/medium-chain water-soluble DA de- rives from β-oxidation of longer chain DAs and by the direct ω-oxidation of lauric acid rives from β-oxidation of longer chain DAs and by the direct !-oxidation of lauric acid (C14). DODA enters mitochondria through the mitochondrial transport system similar to (C14). DODA enters mitochondria through the mitochondrial transport system similar the corresponding monocarboxylic fatty acids, but does not require a carnitine shuttle to the corresponding monocarboxylic fatty acids, but does not require a carnitine shut- [27,28]. Mitochondria (Figure 1) and peroxisomes are major sites of DODA oxidative me- tle [27,28]. Mitochondria (Figure1) and peroxisomes are major sites of DODA oxidative metabolismtabolism [29,30]. [29,30 ]. Figure 1. DODA oxidative metabolism in mitochondria and peroxisomes yields acetyl-CoA and succinyl-CoA that can anaplerotically