Citation: D’Annibale, O.M.; Phua, Y.L.; Van’t Land, C.; Karunanidhi, A.; Dorenbaum, A.; Mohsen, A.-W.; Vockley, J. Treatment of VLCAD-Deficient Patient Fibroblasts with Peroxisome Proliferator-Activated Receptor δ Agonist Improves Cellular Bioenergetics. Cells 2022, 11, 2635. https://doi.org/10.3390/ cells11172635 Academic Editors: Louise Torp Dalgaard, Anandwardhan A. Hardikar and Mugdha Joglekar Received: 7 July 2022 Accepted: 20 August 2022 Published: 24 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Treatment of VLCAD-Deficient Patient Fibroblasts with Peroxisome Proliferator-Activated Receptor δ Agonist Improves Cellular Bioenergetics Olivia M. D’Annibale 1,2 , Yu Leng Phua 1 , Clinton Van’t Land 1 , Anuradha Karunanidhi 1 , Alejandro Dorenbaum 3 , Al-Walid Mohsen 1,2 and Jerry Vockley 1,2,4, * 1 Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA 2 Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15261, USA 3 Reneo Pharmaceuticals, Inc., 18575 Jamboree Road Suite 275-S, Irvine, CA 92612, USA 4 UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA 15224, USA * Correspondence: gerard.vockley@chp.edu Abstract: Background: Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an au- tosomal recessive disease that prevents the body from utilizing long-chain fatty acids for energy, most needed during stress and fasting. Symptoms can appear from infancy through childhood and adolescence or early adulthood, and include hypoglycemia, recurrent rhabdomyolysis, myopathy, hepatopathy, and cardiomyopathy. REN001 is a peroxisome-proliferator-activated receptor delta (PPARδ) agonist that modulates the expression of the genes coding for fatty acid β-oxidation enzymes and proteins involved in oxidative phosphorylation. Here, we assessed the effect of REN001 on VLCAD-deficient patient fibroblasts. Methods: VLCAD-deficient patient and control fibroblasts were treated with REN001. Cells were harvested for gene expression analysis, protein content, VLCAD enzyme activity, cellular bioenergetics, and ATP production. Results: VLCAD-deficient cell lines responded differently to REN001 based on genotype. All cells had statistically significant increases in ACADVL gene expression. Small increases in VLCAD protein and enzyme activity were observed and were cell-line- and dose-dependent. Even with these small increases, cellular bioenergetics improved in all cell lines in the presence of REN001, as demonstrated by the oxygen consumption rate and ATP production. VLCAD-deficient cell lines containing missense mutations responded better to REN001 treatment than one containing a duplication mutation in ACADVL. Discussion: Treating VLCAD- deficient fibroblasts with the REN001 PPARδ agonist results in an increase in VLCAD protein and enzyme activity, and a decrease in cellular stress. These results establish REN001 as a potential ther- apy for VLCADD as enhanced expression may provide a therapeutic increase in total VLCAD activity, but suggest the need for mutation-specific treatment augmented by other treatment measures. Keywords: fatty acid oxidation; VLCAD deficiency; ACADs; PPARs; acyl-CoA dehydrogenases; REN001; cellular bioenergetics 1. Introduction Long-chain fatty acids enter cells via protein fatty acid transporters on the cell surface concurrent with or followed by conjugation to a CoA group by a fatty acyl-CoA synthases (FACS) [1,2]. Long-chain fats are activated in the cytoplasm and require a series of three enzymatic steps that constitute what is known as the carnitine cycle [1,2]. Carnitine palmitoyl transferase 1 (CPT1) replaces the CoA moiety of the long-chain acyl-CoA with carnitine (acylcarnitine), which is transported by carnitine-acylcarnitine translocase (CAT) across the inner mitochondrial membrane in exchange for a free carnitine molecule from the mitochondrial matrix [1,2]. The carnitine of the acylcarnitine is replaced with a CoA Cells 2022, 11, 2635. https://doi.org/10.3390/cells11172635 https://www.mdpi.com/journal/cells