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 synthase (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 and is released as an acyl-CoA ester by carnitine palmitoyl transferase 2 (CPT2), where it can then enter the fatty acid β-oxidation pathway, a series of four enzymatic steps that results in the production of a two carbon acetyl-CoA, one NADH, and one FADH2, regenerating an acyl-CoA that is now two carbons shorter [1–3]. Very long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the a,b-dehydrogenation of long chain acyl-CoA substrates with various carbon chain length and maximal activity to C14-CoA, to its enoyl-CoA product utilizing the electron transfer flavoprotein (ETF), a mitochondrial matrix electron shuttle protein, as an electron acceptor [1, 2, 4]. Reduced ETF transfers its’ reducing equivalents to its redox partner, the ETF dehydrogenase (ETFDH), which in turn delivers the reducing equivalents to the ubiquinone pool and complex III of the electrons transport chain (ETC) [1, 2, 4].
VLCAD deficiency (VLCADD) is an autosomal recessive disorder caused by biallelic mutations in ACADVL gene . The frequency of VLCADD in various populations is about 1:30,000 to 1:100,000 live births [6, 7]. Symptoms of VLCADD include hypoglycemia, recurrent rhabdomyolysis, myopathy, hepatopathy, and cardiomyopathy. Symptoms can present in infancy, later in childhood, or in adolescence or early adulthood . Treatment for VLCADD patients involves a low-fat diet consisting mainly of medium chain triglyceride (MCT) or triheptanoin supplementation with smaller more frequent meals [9–12]. However, many patients still have episodes of rhabdomyolysis and cardiomyopathy that can lead to hospitalization suggesting the need for additional treatment options. Episodes of metabolic decompensation are typically triggered by physiologic stress such as illness or excess exercise, but the cause often remains unidentified . Ultimate outcome is improved by identification of the disorder through newborn screening .
Peroxisome proliferator-activated receptors (PPAR) are nuclear receptors that play key roles in the regulation of fatty acid β-oxidation, lipid metabolism, inflammation, and cellular growth and differentiation [15–19]. They are divided into several categories based on the specific promotors that they stimulate. PPARδ is a major activator of oxidative metabolism and is ubiquitously expressed [15, 20, 21]. It is activated by polyunsaturated fatty acids such as arachidonic acid, oleic acid, dexamethasone, and eicosanoids such as prostaglandin 1 (PGA1), carbaprostacyclin (cPGI), and 15-deoxy-∆12,14-prostaglandin J2 (15d-J2) [22, 23]. In vivo experiments with PPARδ agonists have examined their effects in a variety of diseases and cellular processes including diabetes, obesity, and lipid metabolism. In a two week clinical study, treatment of moderately obese men with dyslipidemia with GW501516, a PPARδ agonist, resulted in a decrease in fasting and postprandial plasma triglycerides, low-density lipoprotein (LDL) cholesterol, apoB compared to placebo, as well as reduction in liver fat content and urinary isoprostanes (a marker of whole-body oxidative stress) . Four weeks of treatment of insulin-resistant middle-aged obese rhesus monkeys with GW501516 induced a dose-dependent rise in serum high density lipoprotein cholesterol while lowering the levels of small-dense LDL, fasting triglycerides, and fasting insulin . Genetically obese ob/ob mice had reduced plasma glucose and blood insulin levels after treatment GW501516 . Genetically predisposed obese Leprdb/dbmice treated with GW501516 demonstrated a decrease in lipid accumulation, while PPARδ-deficient mice were prone to obesity on a high-fat diet . Recent studies with herbal supplements such as bavachinin (a pan-PPAR agonist) from the glucose-lowering malaytea scurfpea herb and ginger (a PPARδ agonist) reduced obesity in obese db/db mice, and diet induced obesity in C57BL6J mice, respectively [26, 27].
REN001 (formerly known as HPP593), a PPARδ agonist (Reneo Pharmaceuticals), has been shown to reduce oxidative stress and inflammation in renovascular hypertensive Goldbatt’s 2 kidney 1 clip (2KIC) rats . 2KIC mice treated with this REN001 for 30 days had no necrosis in kidneys, reduced oxidative stress-responsive proteins, and decreased pro-death protein BNIP3 in kidney tubules . REN001’s proposed mechanism is the inhibition of BNIP3 activation resulting in preserved mitochondrial function and oxidative stress control.
Bezafibrate is a pan-PPAR agonist used to treat hyperlipidemia as it increases high density (HDL) cholesterol levels, decreasing total and LDL cholesterol levels . Since PPARs can increase fatty acid β-oxidation there has been interest in repurposing bezafibrate as a treatment for fatty acid oxidation disorders. In an in vitro study, VLCADD patient derived fibroblast cell lines treated with two versions of bezafibrate demonstrated a 3-fold increase in palmitate oxidation with an increase in VLCAD mRNA, protein, and enzyme activity. RT-PCR showed an increase in other genes encoding proteins in the β-oxidation pathway . Similarly, treatment of CPT2 deficient human myoblast cells with bezafibrate, and the PPARα agonist GWδ 0742 led to an increase in CPT1-B and CPT2 mRNA levels with increased CPT2 activity, while GWα 7647, another PPARα agonist, had minimal effect . Treatment with bezafibrate of fibroblasts from 26 patients with mitochondrial fatty acid oxidation trifunctional protein (MTP) deficiency with various mutations led to improved cellular palmitate oxidation in 6 of 26 cell lines . In an open label trial treating patients with CPT2 deficiency, patients showed an increased or no change in incidence of rhabdomyolysis episodes, but an improvement in quality of life scores [33, 34]. However, in a randomized double blind placebo control clinical trial in patients with VLCAD or CPT2 deficiency, bezafibrate failed to improve cardiac function or whole-body fatty acid oxidation . One possibility for this dichotomy is the limited PPARδ effect of bezafibrate.
Finally, in a clinical study examining the effect of resveratrol, a mitochondrial antioxidant, had no effect on exercise tolerance or whole body fatty acid oxidation in patients with VLCAD or CPT2 deficiency. Thus, a clinical need for additional therapies for this group of disorders remains.
In this study, we examined the effects of REN001 in VLCADD patient derived fibroblast cell lines in advance of clinical trials with this agent.