Nonalcoholic fatty liver disease (NAFLD) is a condition characterized by the accumulation of fat in the liver, which can progress to more severe liver injury [1]. The liver is a central organ in lipid metabolism, responsible for processing dietary fats, synthesizing lipoproteins, and regulating lipid storage and release. Despite numerous clinical trials exploring responses to various medications and supplements, no approved treatment has yet been suggested for managing NAFLD.

Lipoic acid, also known as alpha-lipoic acid (ALA), is a natural compound that plays a crucial role in lipid metabolism and various other biological processes [2]. It has been investigated for its potential to improve NAFLD by promoting the breakdown of triglycerides (a type of lipid) in liver cells [3]. Additionally, lipoic acid exhibits antioxidant properties that can reduce oxidative stress in the liver, a factor in the development and progression of NAFLD [4, 5, 6]. The pharmacological effects of ALA are multifaceted, as it influences several aspects of lipid metabolism, including energy production, antioxidant defense, fatty acid oxidation, and insulin sensitivity regulation [7].

Previous research showed that ALA serves as a cofactor for various enzymes involved in mitochondrial energy production, particularly in the citric acid cycle (Krebs cycle) and oxidative phosphorylation [2]. These processes are critical for breaking down macronutrients, including lipids. Furthermore, ALA has demonstrated potential benefits in improving insulin sensitivity, a factor associated with abnormal lipid metabolism and elevated circulating lipids (hyperlipidemia) in conditions such as type 2 diabetes and obesity [7]. In particular, previous studies showed that ALA exhibited similar effects to metformin, significantly upregulating the glycolytic enzymes glucokinase (GCK), hesokinase-1 (HK-1) and pyruvate kinase (PK), and the glycogen synthesis enzyme GS, and downregulating the gluconeogenic enzymes PEPCK and G6Pase, thus decreased glucose production, and promoted glycogen synthesis and glucose utilization in livers. Moreover, ALA markedly increased PKB/Akt and GSK3$\beta$ phosphorylation, and nuclear carbohydrate response element binding protein (ChREBP) expression in livers [8]. Research also suggests that ALA may directly modulate cholesterol metabolism by regulating the expression of genes involved in cholesterol synthesis and transport, contributing to lipid homeostasis [9]. Although the molecular mechanisms responsible for the effects of ALA on lipid metabolism are not fully understood, previous reports indicated its interactions with peroxisome proliferator-activated receptors (PPARs) [10]. It was demonstrated that in rats and mice, activation of PPAR-$\gamma$ and PPAR-$\alpha$ prevented hyperinsulinemia, hypertriglyceridemia, high fat diet-induced insulin resistance and hepatic steatosis [11, 12, 13]. These nuclear receptor proteins play a significant role in regulating various aspects of lipid metabolism. Specifically, lipoic acid may activate PPAR-alpha and PPAR-gamma, responsible for regulating fatty acid metabolism and insulin sensitivity, respectively [10]. This interaction may enhance fatty acid oxidation, leading to improved lipid profiles and reduced lipid accumulation in the liver. However, it is worth noting that the effects of ALA on PPARs can vary based on factors such as dosage, duration of supplementation, and specific tissue or cell types, potentially accounting for inconsistencies in published results under various experimental conditions. Given the increasing global prevalence of NAFLD and the absence of approved treatment options, coupled with the potential benefits of ALA supplementation and its positive metabolic effects, this study aims to investigate the impact of ALA on lipid content in an in vitro model of NAFLD and its impact on intracellular fatty acid content and metabolism.

NAFLD is a persistent disorder associated with liver damage and closely linked to obesity, insulin resistance, and metabolic syndrome. We recently showed a significant association between steatosis and alterations in mitochondrial dynamics, impacting the metabolic function of liver cells and contributing to intracellular lipid accumulation [4]. However, the direct effects of ALA on hepatic liver metabolism have not been investigated. This study investigates the impact of ALA on gene expression within the primary lipid regulatory pathways using a model of hepatic steatosis induced by exogenous fatty acids [15]. In our experimental model, we observed an accumulation of lipid droplets in cells exposed to the PA:OA mixture. There was an increase in free palmitic acid levels, while oleic acid concentrations decreased. This is consistent with previous studies indicating that oleic acid induces more effective steatogenic effects and reduces cytoplasmic oleic acid concentrations [17]. The accumulation of cytoplasmic palmitic acid has been linked to increased apoptosis through PPAR$\alpha$ and PPAR$\delta$ activation, resulting in enhanced beta-oxidation and oxidative stress [18]. PPAR$\alpha$ and PPAR$\delta$ belong to the superfamily of nuclear receptors. PPARs are transcription factors activated by various fatty acids and their derivatives [19]. Three isotypes of PPAR exist, PPAR$\alpha$, PPAR$\gamma$ (expressed principally in adipose tissue and regulate adipogenesis and insulin sensitivity) and PPAR$\delta$. PPAR$\delta$ stimulates temporary or more severe fatty acid accumulation in mice and potentially in the human liver [20]. The lipogenic activity of PPAR$\delta$ increases the production of monounsaturated fatty acid (MUFAs), which are activators of PPAR$\delta$ and may protect the liver from FFA-mediated lipotoxicity and inflammatory response [21]. In addition, it was demonstrated that human PPAR$\delta$ in the mouse liver promoted hepatic steatosis that was associated with a significant loss of fat mass, suggesting extensive adipose tissue lipolysis and consequently an influx of fatty acids into the liver. This effect required PPAR$\alpha$ signaling, with PPAR$\alpha$ working downstream of PPAR$\delta$ [22]. Our study, through the administration of free fatty acids to liver cells, mimics prolonged food deprivation, including the release of large quantities of fatty acids from adipose tissue, followed by their oxidation in the liver. It has been demonstrated that PPAR$\alpha$ is involved in the transcriptional response to fasting, it is induced during fasting in wild-type mice, indicating that PPAR$\alpha$ plays a critical role in managing energy stores during fasting. By modulating gene expression, PPAR$\alpha$ stimulates the oxidation of hepatic fatty acids to provide substrates that can be metabolized by other tissues [23].

Furthermore, previous studies have indicated that PPAR$\alpha$ activation up-regulates UCP2 expression in hepatocytes by increased transcription involving unknown proteins [24]. Our data show PPAR$\alpha$ and PPAR$\delta$ activation pathways due to PA/OA. In particular, we highlighted the correlation with UCPs. PPARs, together with UCPs, stimulate thermogenesis as well as beta oxidation to dissipate the energy contained in the reduced carbon chains of fatty acids. The administration of ALA after treatment with PA:OA was able to decrease the lipid content, the free palmitic acid and the expression of PPAR$\alpha$ and PPAR$\delta$ by regulating lipid metabolism.

To this regard, we found that ALA mitigates the overexpression of UCPs induced by PA:OA treatment. While UCPs activate thermogenesis pathways to catabolize excess fatty acids, overexpression, particularly of UCP2, may contribute to compromised mitochondrial physiology and liver diseases [25, 26]. Finally, in order to analyze the genetic pathway of cholesterol biosynthesis, our results suggest that the PA:OA model was able to upregulate the gene expression of the main target enzymes of the biosynthetic pathway such as SREBF2, HMGCR, DHCR7 and CYP51A1 and that ALA restores this upregulation confirming a regression in cholesterol biosynthesis.

Furthermore, the expression of the LDL receptor and PCSK9 genes were analyzed. LDLR mediate LDL clearance and increased LDLR expression improves LDL hepatic absorption and decreases plasma LDL. Conversely, PCSK9 functions as a chaperone, guiding LDLR to internal degradation and preventing its recycling to the cell surface [27]. Our results showed an overexpression of both genes in PA:OA treated cells and a decrease mediated by ALA treatment. Elevated HMGCR activity and PCSK9 are associated with diminished LDLR expression and LDL uptake in the liver. Although the expected expression of PCSK9 and LDLR should be inversely proportional, the increase in LDLR could be the effect mediated by PPARs which allows the influx of fatty acids into the hepatocyte or a secondary mechanism affected by the consequence of feedback.

Our data showed other free fatty acid profiles present in the cytoplasm such as linoleic acid and arachidonic acid. Both fatty acids are responsible for activating the prostaglandin pathway through the action of cyclooxygenases. PA:OA treatment highlighted an accumulation of linoleic acid while arachidonic acid concentration was significantly downregulated compared to control cells. Interestingly, 1 µm ALA showed a reduction in free linoleic acid concentration. Therefore, in order to investigate cyclooxygenases, we measured the gene expression of COX1 and COX2. Both genes were overexpressed following PA:PO treatment and ALA administration significantly downregulated their expression. To this regard, COX-1 and COX-2 are heme proteins, and such a prosthetic group is crucial for the expression of catalytic activity [28]. Recent studies demonstrated that ALA reduces lipotoxicity and induces the heme oxygenase system, the enzymes responsible for regulating intracellular heme content, in acute lung injury and an in vitro model of steatosis [29, 30]. Consistently with this hypothesis, previous reports showed that prostaglandin synthesis reduces in response to treatments that upregulate the expression of heme oxygenase (HO)-1 [31, 32]. Our results reveal that ALA acts by inhibiting cyclooxygenases, playing a role in the inflammation exhibited in hepatic steatosis.

In conclusion, our results demonstrated a direct effect of ALA on hepatic liver metabolism and lipid profile that combined with the other systemic effects of improved insulin resistance, oxidative stress and systemic inflammation may represent a strong rationale for the use of ALA in metabolic syndrome treatment or NAFLD. Since ALA is commercially available for several clinical purposes, its use could be easily and highly recommended for patients with NAFLD.

The datasets used and/or analysed in this study are reported within the manuscript and are available from the corresponding authors.

LL, DT, TZ, GLV and IAB designed the research study. LL, ELS, TZ, GZ and SR performed the research. AN, AMA, AS, ET and SR contributed to formal analysis, data interpretation and in editing the final version of the manuscript. LL, FMS, MA, WC and FG analyzed the data. GLV and IAB wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

We thank all the present and past members of the Giovanni Li Volti’s lab for their technical support.

This research was funded by the researchers supporting project number (RSP2024R261, to AMA) King Saud University, Riyadh, Saudi Arabia. This work was supported by Piano di Incentivi per la Ricerca di Ateneo 2020-2022, Linea di Intervento 2, “IMYTRA” to GLV.

Given their role as Guest Editor, GLV had no involvement in the peer-review of this article and have no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Margaret O. James. The authors declare no other conflict of interest.