Oleoylethanolamide improves energy disposal in a cellular model of Alzheimer’s disease

ABSTRACT

Oleoylethanolamide (OEA) is an endogenous lipid mediator which is being discussed as a weight-loss drug for obesity. In addition to its homoeostatic functions, OEA has neuroprotective and anti-inflammatory capabilities. To further investigate the properties of OEA against neurodegenerative diseases, we studied the influence of OEA on mitochondrial function with a focus on mitochondrial energy metabolism in a cellular model of Alzheimer’s disease (AD). SH-SY5Y-APP₆₉₅ cells were used as a model for an early stage of AD. Vector-transfected SH-SY5Y-MOCK cells served as controls. Using these cells, we investigated adenosine triphosphate (ATP) production, various glucose- and fat-metabolising genes as well as fatty acid oxidation (FAO) and lactate/pyruvate levels in cells treated with OEA. Incubation with OEA showed a significant increase in ATP levels in both cell lines. Pyruvate dehydrogenase 1 gene expression was significantly decreased in SH-SY5Y-MOCK cells, whereas FAO and lactate/pyruvate ratio significantly increased in SH-SY5Y-APP₆₉₅ cells. Based on the increased ATP concentration, we conclude that incubation with OEA leads to a disease-specific higher energy availability in the cells. In SH-SY5Y-MOCK cells, this seems to result from the elevated conversion of pyruvate to acetyl-CoA, whilst in SH-SY5Y-APP₆₉₅ cells it may be caused by an increased lactate level and more FAO.

KEYWORDS:

• Oleoylethanolamide
• Alzheimer disease
• mitochondrial dysfunction
• lipid metabolism
• energy supply

Introduction

Obesity and type 2 diabetes mellitus (T2DM) are two risk factors for the development of neurological diseases such as Alzheimer’s disease (AD) (Pugazhenthi et al. 2017; Silva et al. 2019). The combination of an increasingly ageing society and an unfavourable lifestyle thus leads to a major burden of these diseases on healthcare systems. Since AD research still has several failures in developing disease modifying therapies (Gao et al. 2016; Breijyeh and Karaman 2020), the prevention of T2DM and obesity thus represent modifiable risk factors in the prevention of AD (Xu et al. 2015; Yen et al. 2022).

Oleoylethanolamide (OEA), an endogenous lipid mediator with modulating effects on eating behaviour, is produced in the intestine by enterocytes after the ingestion of dietary fat (Laleh et al. 2018, 2019). The mediation does not appear to be by OEA via the endocannabinoid receptors, but is has been shown that OEA is a potent agonist of peroxisome proliferator-activated receptor-alpha (PPARα) (Laleh et al. 2018; Lin et al. 2022). The PPARα mediated activation is thought to be responsible for the anti-inflammatory and radical scavenging effects (González-Aparicio and Moratalla 2014; Payahoo et al. 2018). Additionally, targets such as transient receptor potential vanilloid 1 (TRPV1) and G protein-coupled receptor 119 (GPR119) have been investigated for the appetite suppressing effect of OEA (Bowen et al. 2017). OEA has already been shown to be able to suppress the transcription factor NF-κB, which can influence various inflammatory processes (Orio et al. 2018). In addition to its endogenous production, OEA is an FDA-approved supplement for weight control in type 2 diabetes mellitus. It has been shown that supplementation with 250 mg OEA over 8 weeks in obese test subjects led to a significant reduction of IL-6 and TNF-α concentrations in plasma, two activators of NF-κB which makes OEA interesting as a potential antioxidant acting compound on mitochondria (Payahoo et al. 2018).

However, the influence of OEA on mitochondrial energy metabolism in neuronal cell models has not been investigated. Since mitochondria are essential for providing cellular energy by the oxidative phosphorylation system (OXPHOS) (Grimm and Eckert 2017), preventing a dysfunction is an essential early starting point for the avoidance of diseases such as T2DM and AD in the long term as mitochondrial function seems to be impaired in both (Kelley et al. 2002; Esselun et al. 2022; Reutzel et al. 2022). Increasing mitochondrial energy supply is an essential aspect of healthy cellular energy metabolism. Therefore, in the present work, we investigated the effect of OEA in SH-SY5Y-MOCK and SH-SY5Y-APP₆₉₅ cells on the cellular bioenenergetic metabolites ATP, lactate, and pyruvate.

Material and methods

Chemicals

The chemicals used for this research were purchased from either Merck, Sigma Aldrich, Cayman Chemical or Thermo Fisher Scientific in the highest purity available. Oleoylethanolamide (OEA) (purity ≥98%) specifically, was ordered from Cayman Chemical (90265). A mixture of DMSO and cell culture medium in a 1:1 ratio served as solvent for OEA and therefore as a control in the experiments.

Cell lines

For all experiments, human neuronal SH-SY5Y-MOCK cells or SH-SY5Y-APP₆₉₅ cells were used. SH-SY5Y-MOCK cells contain the empty vector pCEP4 (Invitrogen, Europe) and served as a control group, SH-SY5Y-APP₆₉₅ cells are transfected with the amyloid precursor protein 695 which is considered an established model for early stages of Alzheimer’s disease (Stockburger et al. 2014). Cells were maintained at 37°C and cultured at a CO₂ concentration of 5%. The medium used was Dulbecco’s modified Eagle medium (DMEM) (Gibco, Thermo Scientific, Waltham, MA, USA) containing 10% (v/v) heat-inactivated foetal bovine serum (FBS), 60 units/mL penicillin, 60 μg/mL streptomycin, 5 mL vitamins, 5 mL non-essential amino acids (NEAA), 1 mM sodium pyruvate and 0.3 mg/mL hygromycin. DMEM with only 2% FBS was used to conduct experiments, the remaining components were identical. Cells were passaged every 3 days and were used for the experiments when a confluency of 70–80% was reached. Two days before incubation, cells were harvested and either seeded into 6-well plates (qPCR, 5 × 105 cells/well) or 96-well plates (ATP, Fatty Acid Oxidation (FAO) Assay, 2 × 104 cells/well) in DMEM. After 48 hours, cells were treated with OAE at different concentrations for 24 hours (resp. again after 48 hours for the FAO assay) or with a 1:1 mixture of DMEM and DMSO as a control. To investigate the effect of OEA on cell damage, a subset of cells was additionally exposed to 25 µM rotenone 1 h after OEA treatment.

Measurement of ATP concentrations

ATP concentrations were determined using the ATPlite Luminescence Assay System (#6016947; Perkin Elmer, Rodgau-Jügesheim, Germany). This assay utilises the light emission that occurs when ATP is combined with luciferin. The emitted light was evaluated with a ClarioStar plate reader (BMG Labtech, Ortenberg, Germany). Plates were removed from the incubator to cool down to room temperature for 15 minutes. Subsequently, lysis buffer was added and after 5 minutes cells were incubated with monitoring reagent for additional 40 minutes in the dark. The results were normalised to the cell count.

Quantitative real-time PCR (qRT-PCR)

RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Quantification was performed on the Nanodrop 2000c spectrometer (Thermo Fischer Scientific, Waltham, MA, USA). To remove residual genomic DNA, samples were treated with the TURBO DNA-free Kit according to the manufacturer’s instructions (Thermo Fischer Scientific, Waltham, MA, USA). The RNA was transcribed into complementary DNA using the iScript cDNA Synthesis Kit (Bio-Rad, Munich, Germany). For rRT-PCR, the CfX96 Connect System (Biorad, Munich, Germany) was used. A list of the primers used (Biomol, Hamburg, Germany) can be found in Table 1. The synthesised cDNA was diluted 1:10 with RNA-free water (Qiagen, Hilden, Germany). Samples were analysed in triplicate at 10 μL. The following PCR conditions were chosen: initial Denaturation (3 min; 95°C) followed by 45 cycles of denaturation (10 s; 95°C), annealing (30 s; 58°C) and elongation (29 s; 72°C). Actin-β (ACTB), glyceraldehyde-3- phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase 1 (PGK1) were used as reference genes. Expression was analysed with −(2∆∆Cq) using Bio-Rad CfX manager software.

Table 1. Oligonucleotide primer sequences, product sizes, and primer concentrations for quantitative real-time PCR.

Primer	Sequence	Manufacturer	Product Size	Concentration (nM)
ß-Actin (ACTB) NM_001101.2	5´-ggacttcgagcaagagatgg-3´ 5´-agcactgtgttggcgtacag-3´	Biomol, Hamburg, Germany	234	200
Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) NM_002046.2	5´-gagtcaacggatttggtcgt-3´ 5´-ttgattttggagggatctcg-3´	Biomol, Hamburg, Germany	238	200
Phosphoglycerate kinase 1 (PGK1) NM_000291.2	5´-ctgtgggggtatttgaatgg-3´ 5´-cttccaggagctccaaa-3´	Biomol, Hamburg, Germany	198	200
Laktatdehydrogenase (LDHA2) NM_033195	5´-ggtgtccctttgaaggatct-3‘ 5´-tgcagtcacttctttgtgga-3´	Biomol, Hamburg, Germany	189	200
Pyruvat-Dehydrogenase-Kinase (PDK1) NM_001278549	5´-atacggatcagaaaacagaca-3´ 5´-cagacgcctagcattttcat-3´	Biomol, Hamburg, Germany	291	200
Acyl-Coenzym-A-Oxidase 1 (ACOX1) NM_004035	5´-ctgaaggctttcacctcctg-3´ 5´-catgccacacaccaactttc-3´	Biomol, Hamburg, Germany	225	200
Carnitin-Palmitoyltransferase 1 (CPT1) NM_001136052	5´-ggcgtccaattatgtcagtg-3´ 5´-ctcgtactgggcagagcata-3´	Biomol, Hamburg, Germany	211	200

Lactate and pyruvate measurement

The cellular pyruvate and L(+)-lactate was measured using a Pyruvate Assay Kit resp. Lactate Assay Kit by following the manufacturer’s instructions (#MAK071& MAK064; Sigma-Aldrich, St. Louis, USA). Before the measurement the cells were treated with OEA concentrations between 5 and 0,05 µM for 24 or 48 hours. The enzymatic assays result in a colorimetric product, which is determined using a ClarioStar plate reader (BMG Labtech, Ortenberg, Germany) at a wave length of 570 nm. The determined colorimetric product is proportional to the pyruvate/lactate present in the probe.

Fatty acid oxidation assay

Fatty acid oxidation of the cells was determined using a quantitative colorimetric assay by following the manufacturer’s instructions (#BR00001; Assay Genie, Dublin, Ireland). The cells were incubated for 24 or 48 hours with OEA concentrations of 5 to 0.05 µM. The incubation time of the enzyme assay lasted 90 minutes. Fluorescence measurements were performed using a ClarioStar plate reader (BMG Labtech, Ortenberg, Germany) at a chosen excitation of 492 nm.

Statistics

Data were tested for normal distribution using the D’Agostino & Pearson test or the Shapiro-Wilk test. The ROUT test (Q = 1%) was used to identify outliers. Two-tailed t-tests were performed to test significance between two measurement groups. A one-way Analysis of Variance (ANOVA) was used to test for statistically significant differences between multiple means, and the Kruskal-Wallis test was used for non-normally distributed values (Prism 9.4.1 GraphPad Software, San Diego, CA, USA). A significance level α = 0.05was assumed for the test statistics. The results of all experiments are presented as mean values ± standard deviation (SEM).

Results

Changes in energy metabolism in SH-SY5Y-APP695 cells.

SH-SY5Y-MOCK and SH-SY5Y-APP₆₉₅ cells were used as a model to study the effect of OEA on the early phase of Alzheimer’s disease. The established SH-SY5Y-APP₆₉₅ model is characterised by increased amyloid-β production due to mutations in the APP gene (Stockburger et al. 2014). Our research group has already shown that SH-SY5Y-APP₆₉₅ cells also exhibit differences in energy metabolism consequently. A study by Grewal et al. (2020) showed a significant reduction in mitochondrial respiration in the APP model due to reduced complex activity of the respiratory chain. In line with this, ATP production was also reduced in SH-SY5Y-APP₆₉₅ cells compared to SH-SY5Y-MOCK cells (Grewal et al. 2020).

Effect of oleoylethanolamide on adenosine triphosphate levels

Incubation with OEA concentrations of 5 µM, 0.5 µM, 0.05 µM and 0.025 µM for 24 h resulted in a significant increase in ATP levels in SH-SY5Y-MOCK cells (p = 0.01; p = 0.014; p = 0.027; p = 0.014) and in SH-SY5Y-APP₆₉₅ cells at OEA concentrations of 5 µM, 1 µM, 0.5 µM, 0.1 µM, 0.05 µM and 0.025 µM (p = 0.004; p = 0.001; p = 0.009; p = 0.0002; p = 0.003; p = 0.006) (Figure 1). The mean increase of ATP concentration was 26.7% in SH-SY5Y-MOCK cells and 31.2% in SH-SY5Y-APP₆₉₅ cells. The highest OEA concentration of 100 μM led to a decrease in ATP concentration. A drop of the OEA-induced ATP increase was recorded starting at an OEA concentration of 0.001 μM.

Figure 1. Effect of oleoylethanolamide (OEA) on ATP level in SH-SY5Y-MOCK and SH-SY5Y-APP₆₉₅ cells.

(A) SH-SY5Y-MOCK cells were incubated with OEA at different concentrations for 24 h and measured against cells treated with a 1:1 mixture of DMSO and cell culture media. (B) ATP level in rotenone [25 µM] treated SH-SY5Y-MOCK cells. Before rotenone impairment, SH-SY5Y-MOCK cells were incubated with different concentrations of OEA or a 1:1 mixture of DMSO and cell culture media for 1 h before exposure to 25 μM rotenone for another 24 h. SH-SY5Y-APP₆₉₅ were treated likewise. Likewise treatment with OEA in SH-SY5Y-APP₆₉₅ cells. Data are displayed as means ± SEM. n = 19. Statistical significance was tested via one-way ANOVA (*p < 0.05; ** p < 0.01; ***p < 0.001).

An additional treatment with 25 μM rotenone (respiratory chain complex-I inhibitor) before OEA incubation resulted in a significant decrease of ATP levels in both cell models (p < 0.0001). This tended to be counteracted by OEA, especially in SH-SY5Y-APP₆₉₅ cells, however the results were not significant (data not shown).

Effect of oleoylethanolamide on gene expression

To investigate the OEA-dependent increase in ATP concentration, relevant mitochondrial genes were examined in SH-SY5Y cells after incubation with OEA at concentrations of 5 µM, 0.5 µM and 0.05 µM for 24 h.

In SH-SY5Y-MOCK cells treated with OEA, relative PDK1 expression decreased compared to the control group. At the 5 µM and 0.5 µM concentrations, the results were significant (p = 0.0431; p = 0.0450). Analysis of LDHA2 expression showed no significant effect in SH-SY5Y-MOCK cells, as did expression of the CPT1 gene (Figure 2(a–c)).

Figure 2. Effect of oleoylethanolamide (OEA) on the relative gene expression in SH-SY5Y cells after incubation with OEA for 24 h, determined using quantitative real-time PCR compared to SH-SY5Y cells incubated with a 1:1 mixture of DMSO and cell culture media. (A) Gene expression of phosphoinositide-dependent kinase-1 (PDK1) in SH-SY5Y-MOCK cells. (B) Gene expression lactate dehydrogenase a (LDHA2) in SH-SY5Y-MOCK cells. (c) Gene expression of carnitine palmitoyltransferase 1 (CPT1) in SH-SY5Y-MOCK cells. (D) – (F) Are equal to (A) – (C) in SH-SY5Y-APP₆₉₅ cells. Calculation of normalization factor based on geometric mean of multiple control genes levels of ß-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and phosphoglycerate kinase 1 (PGK1). Data are displayed as means ± SEM. n = 14. Statistical significance was tested via one-way ANOVA (*p < 0.05).

Examination of these three cellular metabolism key enzymes in SH-SY5Y-APP₆₉₅ cells revealed that PDK1 gene expression also tended to decrease (ns) upon treatment with OEA, whereas LDHA2 expression showed a modest trend to increase (ns). The expression of the CPT1 gene was equally unaffected as in SH-SY5Y-MOCK cells (Figure 2(d–f)).

Effect of oleoylethanolamide on cellular lactate and pyruvate levels

To further investigate the effect of OEA on the energy availability of the cell, both lactate and pyruvate levels were assessed in SH-SY5Y-MOCK and SH-SY5Y APP₆₉₅ cells. Incubation of SH-SY5Y-MOCK cells with OEA at concentrations of 5 µM and 0.05 µM for 24 hours, or twice 0.05 µM over 48 hours, showed a tendency for lactate and pyruvate levels to decrease compared to the control group, but this was not significant at any concentration (Figure 3a,b). Overall, the lactate/pyruvate ratio increased by 13.2% on average as a result of OEA treatment (Figure 3c).

Figure 3. Effect of oleoylethanolamide (OEA) on lactate and pyruvate level in SH-SY5Y cells.

Cells were incubated with 5 µM or 0,05 µM for 24 h, resp. again with 0,05 µM after 24 h. A 1:1 mixture of DMSO and cell culture media served as a control. (A) Lactate Levels of SH-SY5Y-MOCK cells after OEA incubation. (B) Pyruvate Levels of SH-SY5Y-MOCK cells after OEA incubation. (C) Lactate to pyruvate ratio in SH-SY5Y-MOCK cells after OEA incubation. (D) Lactate Levels of SH-SY5Y-APP₆₉₅ cells after OEA incubation. (E) Pyruvate Levels of SH-SY5Y-APP₆₉₅ cells after OEA incubation. (F) Lactate to pyruvate ratio in SH-SY5Y-APP₆₉₅ cells after OEA incubation. Data are displayed as means ± SEM. n = 8. Statistical significance was tested via one-way ANOVA (*p < 0.05; ***p < 0,001).

Incubation of SH-SY5Y-APP₆₉₅ cells with OEA at concentrations of 5 µM and 0.05 µM for 24 hours, resp. 48 hours, showed a tendency for increased lactate levels and decreased pyruvate levels compared to the control group (Figure 3d,e). This was significant at a concentration of 0.05 µM over 48 hours (p = 0.0215) and was 61.7%. Overall, the OEA treatment increased the lactate/pyruvate ratio by 30,9% (p = 0.0008) over 48 hours at a concentration of 0.05 µM (Figure 3f).

Effect of oleoylethanolamide on the fatty acid oxidation

To determine what caused the increase in ATP levels after incubation with OEA in SH-SY5Y-APP₆₉₅ cells, fatty acid oxidation (FAO) was measured after incubation with OEA at concentrations of 5 and 0.05 µM after 24 h, resp. after 48 h.

FAO activity in SH-SY5Y-MOCK cells did not change after treatment with different concentrations of OEA, neither over the 24-hour period, nor over 48 h compared to the control group (Figure 4a).

Figure 4. Effect of oleoylethanolamide (OEA) on the fatty acid oxidation (FAO) activity in SH-SY5Y-MOCK cells and SH-SY5Y-APP₆₉₅ cells. Cells were incubated with 5 µM or 0,05 µM for 24 h, resp. again with 0,05 µM after 24 h. A 1:1 mixture of DMSO and cell culture media served as a control. Data are displayed as means ± SEM. n = 9. Statistical significance was tested via one-way ANOVA (*p < 0.05).

FAO activity in SH-SY5Y-APP₆₉₅ cells was significantly increased by incubation with two times 0.05 µM OEA (after 24 and 48 h) compared to the control group (Figure 4b). The increase was 18.4%. Incubation with 5 µM and 0,05 µM OEA for 24 h resulted in an increase in FAO as well, however the effect was not significant.

Discussion

OEA increases cellular ATP levels in SH-SY5Y-MOCK and SH-SY5Y-APP₆₉₅ cells without radical scavenging function

The provision of energy in the form of ATP is essential for the cell to maintain a normal cell metabolism. The largest amount is produced via mitochondrial respiration and a lack of ATP is associated with a variety of neurological diseases, such as Alzheimer’s disease (Atlante et al. 2022). Thus, determination of ATP levels may provide a marker of mitochondrial functionality. Moreover, ATP is the universal energy carrier in cells and thus also maps the general viability of cells. Improved cell viability has already been shown in isolated substantia nigra cells treated with OEA concentrations between 1 and 0.5 μM (Calabrese et al. 2007). As far as we know, there is no investigation in the current literature regarding the effect of OEA on cellular ATP levels. In both cell lines, a concentration range of 10 μM to 0.01 μM OEA showed an increase in ATP levels compared to the DMSO control group. These results were significant at 5 μM, 0.5 μM, 0.05 μM and 0.025 μM OEA, respectively. The observed increase can possibly be explained by the antioxidant properties of OEA (Giudetti et al. 2021; Sabahi et al. 2022). Mitochondria are a major target site for reactive oxygen species (ROS), as the electron transport chain produces a large proportion of ROS. Inadequate detoxification or increased ROS production results in oxidative stress within the mitochondrion. Increased levels of radicals in the cell can thus lead to extensive damage to DNA, lipids and enzymes, which would also affect cellular ATP synthesis (Onyango et al. 2016). It has already been shown that incubation of HUVEC cells with OEA increased the activity of antioxidant enzymes such as catalase, superoxide dismutase and glutathione peroxidase and reduced cellular ROS levels (Ma et al. 2015).

Additionally, damage to cells with rotenone, a specific complex I inhibitor, results in additional deficits in mitochondrial respiration and increased oxidative stress in cellular models. Furthermore, rotenone is a popular model for simulating neurodegenerative diseases, as this respiratory chain complex shows deficits in Parkinson`s Disease and in early AD (Friedland-Leuner et al. 2014). Incubation with rotenone resulted in a decrease of ATP levels in both cellular models, as already shown by Stockburger et al. (2014). Incubation of OEA resulted in a tendency towards protection. This observation again supports the assumption that OEA acts as an antioxidant. To verify whether OEA also acts as a ROS scavenger in our cell model, ROS formation was measured in both cellular models. None of the investigated concentrations showed a significant effect on the formation of ROS, which led us to the assumption that in our cell model the increase of ATP levels is not triggered by a radical scavenging function of OEA, but via other energy production pathways such as fatty acid oxidation or glycolysis, which will be discussed in more detail in 4.2. and 4.3. Another promising target appears to be the transcription factor NF-E2-related factor 2 (NRF2). It plays a central role in maintaining cell homoeostasis by regulating various enzymes that regulate the redox system. Recent studies have shown that Nrf2-regulated genes can increase stress resilience in many pathologies, including neurodegenerative diseases (Calabrese et al. 2007, 2010, 2016). Understanding the ability of neuronal cells to cope with chronic redox stress is of fundamental interest and should therefore be investigated in future work on OEA. Furthermore, for the SH-SY5Y-APP₆₉₅ cells an influence on the APP metabolism would be conceivable, since OEA acts as a PPAR-α agonist and is thus able to modulate the degradation of the amyloid precursor protein positively via the α-secretase cleavage (Corbett et al. 2015). Thus, OEA treatment could lead to lower Aβ levels, which has a positive effect on cell viability. This would explain why the ATP levels in SY5Y-APP₆₉₅ cells showed a higher proportional increase due to OEA treatment than in SY5Y-MOCK cells. Improved cell viability has already been shown in isolated substantia nigra cells treated with OEA concentrations between 1 and 0.5 μM (Gonzalez-Aparicio et al. 2014). As far as we know, there is no investigation in the current literature regarding the effect of OEA on cellular ATP levels.

OEA improves cellular glucose metabolism in SH-SY5Y-MOCK and SH-SY5Y-APP₆₉₅ cells

In order to determine whether the ATP increase is due to an increased cellular glucose metabolism, the two possible diversion pathways for pyruvate were investigated in both cell models. In the presence of oxygen, acetyl-CoA is generated from pyruvate by pyruvate dehydrogenase (PDH), which can enter the citrate cycle and thus contribute to the electron transport chain . Under low-oxygen conditions, pyruvate is oxidised to lactate by the LDHA. Therefore, the expression of PDK1 was investigated. The kinase causes the inactivation of PDH and thus inhibits energy production. Therefore, PDK1 functions as a molecular switch between glycolysis and aerobic respiration to cover the cellular ATP demand. In this context, PDK1 showed a significant decrease in SH-SY5Y-MOCK cells after incubation with OEA, indicating a lower inhibition of PDH by PDK1. Thus, there could be an improved energy supply by NADH and FADH2 in the further course and subsequently an increase in the ATP level by treatment with OEA. This effect also tended to be observed in the transfected cell model. The effect on mRNA expression of LDHA2 was not significant in either cell model, although there was a tendency for an increase by OEA in the SH-SY5Y cells. In the context of decreased PDK1 expression, the increased consumption of pyruvate by PDH could result in less pyruvate available for anaerobic lactate production. Treatment with OEA therefore appears to induce increased mitochondrial metabolism of pyruvate, particularly in SH-SY5Y-MOCK cells, and thus provide increased energy supply. However, in SH-SY5Y-APP₆₉₅ cells, OEA also appears to have effects on glucose metabolism, as PDK1 was not downregulated to the same extent, but there was an increase in LDHA2 expression. This would be plausible because it could lead to greater PDH inhibition, making more pyruvate available, which would then be degraded to lactate by LDHA. This would be conceivable, as it could lead to a stronger PDH inhibition, making more pyruvate available, which would then be degraded to lactate via LDHA. This pathway of energy provision could also explain the higher ATP levels found in SH-SY5Y-APP₆₉₅ cells as well as in SH-SY5Y-MOCK cells. However, these assumptions still have to be clarified in future studies.

Incubation of OEA in SH-SY5Y-MOCK cells tended to result in lower levels of lactate and pyruvate, while the ratio of both metabolites remained largely unchanged. Here, it would be conceivable that more pyruvate is converted to acetyl-CoA due to the significant decrease of PDK1 expression. Since acetyl-CoA is efficiently consumed in the respiratory chain of the non-APP₆₉₅-transfected control group, this is measured in lowered levels. In contrast, it was observed that in SH-SY5Y-APP₆₉₅ cells, a significant decrease in pyruvate was observed after incubation with OEA, while the ratio of lactate to pyruvate was increased. Here, we hypothesise that mitochondrial respiration, which is impaired in SH-SY5Y-APP₆₉₅ cells compared to SH-SY5Y-MOCK cells (Ma et al. 2015; Dieter et al. 2022) should be compensated by an increased glycolytic rate by OEA. This could explain the higher percentage increase in ATP in SH-SY5Y-APP₆₉₅ cells.

The effect of OEA and other amides on glucose metabolism is not reported in the literature. Only the effect of other PPAR-α agonists such as fenofibrate on PDK1 activity is known. Hann et al. (2013) showed a significant decrease in PDK1 activity by fenofibrate, which is in line with our data (Hann et al. 2013).

OEA increases fatty acid oxidation in SH-SY5Y-APP₆₉₅ cells but not in control cells

Subsequently, we checked whether the increase in ATP level is due to changes in lipid metabolism. For this purpose, the expression of CPT1 as an integral outer mitochondrial membrane protein, which catalyses the conversion of acyl-CoA into acylcarnitine, was examined. Lamichane et al. (2018) reported that PPAR-α increases the expression of genes encoding mitochondrial enzymes related to lipid metabolism, including CPT1 (Lamichane et al. 2018). Since OEA is discussed as a PPAR-α agonist, we assumed an increase in expression of this enzyme, which, did not prove true in either cell model. In contrast, Poornima et al. (2022) observed a PPAR-α-dependent upregulation of CPT1 in HepG2 cells with concomitant changes in fatty acid oxidation (Poornima et al. 2022). Fatty acid oxidation was significantly increased in SH-SY5Y-APP₆₉₅ cells, especially after 48 hours of incubation. Again, this suggests that the effect could be PPAR-α-mediated, leading to increased FAO in the neuronal cell model. Genes activated by PPAR-α encode proteins involved in processes such as lipid transport, mitochondrial β-oxidation, lipogenesis and lipoprotein uptake. Consistent with our results, Guzman et al. (2004) were able to record a significant increase in fatty acid release in isolated adipocytes and a significant increase in fatty acid oxidation in isolated muscle and liver cells through treatment with OEA [10 μM] (Guzmán et al. 2004). In addition, it has already been shown in vivo that the effect on FAO appears to be via PPAR-α activation. An increase in FAO was observed in wild-type mice, whereas the effect was absent in PPAR-α knock-out animals (Li et al. 2015). In SH-SY5Y-MOCK cells, OEA does not appear to exert any effect on fatty acid oxidation. Consequently, the relatively higher increase in ATP levels in SH-SY5Y-APP₆₉₅ cells could be due to the additional energy provided by the FAO.

Conclusion

In this work, we describe for the first time the effect of OEA in the neuronal cell model SH-SY5Y-MOCK and SH-SY5Y-APP₆₉₅ cells on cellular energy processes. We observed an increase in cellular ATP levels in both investigated cell models, which seems to occur via a positive effect on glucose metabolism in SH-SY5Y-MOCK cells. Interestingly, in contrast, the increase in ATP levels in SH-SY5Y-APP₆₉₅ cells seems to be based primarily on a combination of increased FAO and enhanced glycolysis to compensate for inefficient mitochondrial respiration.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data presented in this study are available on request from the corresponding author.