Low concentrations of α-lipoic acid reduce palmitic acid-induced alterations in murine hypertrophic adipocytes

Abstract Obesity is a metabolic disorder with excessive body fat accumulation, increasing incidence of chronic metabolic diseases. Hypertrophic obesity is associated with local oxidative stress and inflammation. Herein, we evaluated the in vitro activity of micromolar concentrations of α-lipoic acid (ALA) on palmitic acid (PA)-exposed murine hypertrophic 3T3-L1 adipocytes, focussing on the main molecular pathways involved in adipogenesis, inflammation, and insulin resistance. ALA, starting from 1 µM, decreased adipocytes hypertrophy, reducing PA-triggered intracellular lipid accumulation, PPAR-γ levels, and FABP4 gene expression, and counteracted PA-induced intracellular ROS levels and NF-κB activation. ALA reverted PA-induced insulin resistance, restoring PI3K/Akt axis and inducing GLUT-1 and glucose uptake, showing insulin sensitizing properties since it increased their basal levels. In conclusion, this study supports the potential effects of low micromolar ALA against hypertrophy, inflammation, and insulin resistance in adipose tissue, suggesting its important role as pharmacological supplement in the prevention of conditions linked to obesity and metabolic syndrome. Graphical Abstract


Introduction
Obesity is a condition, characterized by an excessive accumulation of body fat, able to negatively affect the health status by increasing the incidence of several chronic metabolic diseases, such as insulin resistance and diabetes mellitus (engin 2017).In particular, in people with obesity, changes in the flow of free fatty acids (FFAs) and in oxidized cholesterol levels occur in adipose tissue, activating the endoplasmic reticulum (eR) stress response and determining the onset of a lipotoxicity condition, with consequent alterations in cellular homeostasis and activation of proinflammatory pathways (Paniagua 2016).Among these, the most important is the nuclear factor κB (NF-κB) transcription factor, accountable for the activation of a wide range of genes that code for proinflammatory proteins (Nandipati et al. 2017).evidence supports a key role for obesity in the development of low-grade inflammation also characterised by enhanced infiltration and activation of innate and adaptive immune cells into adipose tissue.These in turn secrete proinflammatory cytokines affecting the development of complications related to obesity, such as local and systemic insulin resistance (Cooke et al. 2016).
In this regard, several in vitro and in vivo studies have shown that variations in the cellular redox state, and the activation of inflammatory and stress-signaling pathways, such as c-Jun N-terminal kinase (JNK) and NF-κB pathways, appear to be the main responsible for the alteration of the main insulin signaling pathways and for the reduction of glucose uptake in adipose tissue (Strauss 1999;Spiller et al. 2018;Zhou et al. 2020).In the presence of an inflammatory condition an impairment on insulin receptor substrate-1 (IRS-1) function and downstream insulin/PI3K pathway occur, resulting in glucose uptake inhibition and leak of insulin signal transduction (Guria et al. 2023).
Recent years have seen rising interest in α-lipoic acid (ALA), a cofactor of vital importance for the cells, thanks to its high antioxidant capacity and its protective effect against various pathological chronic conditions (Salehi et al. 2019).The disulfide bond in the molecule gives a reductive potential necessary for the catalysis of mitochondrial 2-ketoacid dehydrogenases and is involved in the redox-dependent regulation of several multienzyme complexes.These functions make ALA essential for cell growth, carbohydrates oxidation, and mitochondrial redox balance regulation (Packer and Cadenas 2011).ALA is normally produced by the cells of our body, but it is also present in nature in plants and animals, especially in red meats, but also in some vegetables such as potatoes, broccoli, and spinach.ALA is known especially for its strong antioxidant activities able to scavenge free radicals, to restore endogenous antioxidants (e.g.glutathione [GSH]) and to upregulate intracellular antioxidant capacity through activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-signaling (McCarty et al. 2022).Nrf2 regulates the expression of essential components of GSH and thioredoxin (Trx) antioxidant systems, plus enzymes implicated in NAdPH renewal and ROS detoxification, so performing a vital role in preserving cellular redox homeostasis (Speciale et al. 2020).Previous study from our group demonstrated that nanomolar concentrations of ALA inhibited TNF-α-induced proinflammatory NF-κB pathway in human umbilical vein endothelial cells following the activation of Nrf2 transcription factor (Fratantonio et al. 2018).Additionally, short treatment with ALA in obese rats reversed lipoapoptosis in the liver, through the inhibition of the kinases linked to oxidative and eR stress, and, secondly, it increased the Nrf2-mediated antioxidant responses (Valdecantos et al. 2015).Prevention of oxidative stress may then be part of the therapeutic benefit of ALA, directly or undirectly regulating the transcription of genes related to antioxidant and anti-inflammatory pathways.
Thus, ALA can potentially constrain inflammation-mediated sequelae linked to obesity and progression to metabolic syndrome or other chronic disease.In vivo data confirmed anti-inflammatory activity of ALA since dietary supplementation for ten weeks significantly reduced systemic inflammation and cardiovascular disease-related risk factors in healthy overweight women (Huerta et al. 2015).In addition, ALA improved insulin sensitivity in animal models of insulin resistance and obesity, and also in patients with Type 2 diabetes (Akbari et al. 2018).A multitude of mechanisms have been hypothesized to explain the molecular basis of ALA protective effects on insulin resistance, Type 2 diabetes, and metabolic syndrome (Tibullo et al. 2017).However, up until now, the basic mechanism is still not clear.Mechanistic studies on insulin responsive cells shown that ALA quickly promotes glucose uptake by activating components of the insulin signaling pathway by changing intracellular redox status (Moini et al. 2002).However, many of these findings have been obtained using very high concentrations of ALA (ranging 0.1 − 2.5 mM), not only nonrelevant from the physiological and clinical perspective, but also probably altering the thiols redox intracellular equilibrium.
For this purpose, the present study had the objective of evaluating in vitro activities of ALA on murine hypertrophic adipocytes using physiological concentrations.In particular, concentration range used (1-100 µM) mimicks oral or intravenous pharmacological ALA supplementation (Hermann et al. 1996), even if it cannot be excluded that the regular consumption of foods rich in ALA could provide similar plasma concentrations.Adipocytes hypertrophy was induced by high PA concentrations using a previously validated experimental model able also to affect inflammatory and insulin pathways (Muscarà et al. 2019;Molonia et al. 2020).In particular, we focussed on the main molecular pathways involved in adipogenesis (PPAR-γ pathway), inflammatory process (NF-κB pathway), and insulin resistance (IRS-1/PI3K/Akt pathway).At our knowledge, our study is the first to report the anti-adipogenic, anti-inflammatory, and insulin-sensitizing effects of micromolar physiological ALA doses (Hermann et al. 1996) by means of an in vitro experimental model of PA-induced hypertrophic adipocytes.

Antihypertrophic effects of ALA
Adipocyte hypertrophy is a known feature of dysfunctional adipose tissue that appears characterized by excessive fat accumulation involving overexpression of crucial transcription factors of the adipogenesis process, such as Liver X receptor alpha (LXRα), CCAAT-enhancer-binding protein alpha (C/eBPα), peroxisome proliferator-activated receptor-γ (PPAR-γ), and sterol regulatory element-binding protein 1c (SReBP-1c), in turn responsible for the activation of lipogenic genes, such as fatty acid binding protein 4 (FABP4), adiponectin, and fatty acid synthase (FAS) (Longo et al. 2019).
To evaluate the impact of ALA on PA-induced hypertrophy in adipocytes, Oil Red O staining histological technique was performed (Figure S1A) (see Supplementary Material for experimental details).The results obtained show that treatment with 1 mM PA for 24 h caused a sharp increase in the amount of lipid deposits compared to control cells (1.28 ± 0.06 fold increase vs. CTR; p < 0.05), whereas pretreatment with ALA, at all the tested concentrations (1-25-100 µM), dose-dependently reduced hypertrophic condition induced by PA [1.10 ± 0.05 (p < 0.05 vs CTR and vs PA), 1.02 ± 0.05 (p < 0.05 vs PA), 0.97 ± 0.04 (p < 0.05 vs PA), respectively, for ALA 1 μM, ALA 25 μM, and ALA 100 μM].Moreover, ALA dose-dependently reduced lipid accumulation even in adipocytes not exposed to PA [0.98 ± 0.05, 0.92 ± 0.05 (p < 0.05 vs CTR) and 0.85 ± 0.06 (p < 0.05 vs CTR and vs ALA 1 µM), respectively, for ALA 1 μM, ALA 25 μM and ALA 100 μM] suggesting a potential lipolytic effects.However, these data cannot exclude or confirm the potential lipolytic effect of ALA, although short-term treatment with ALA demonstrated the involvement of this mechanism in 3T3-L1 (Fernández-Galilea et al. 2012).
To verify the increase in the hypertrophic condition of fully differentiated 3T3-L1 adipocytes following PA exposure, and the molecular targets by which the ALA determines reduction of lipid deposits, the expression of PPAR-γ was then evaluated.This transcriptional factor is, indeed, the master modulator of the adipogenesis process and principal responsible of lipid accumulation in adipose tissue (Ambele et al. 2020).
The results obtained confirm that PA causes a marked increase in PPAR-γ levels with respect to unexposed control cells (Figure S1B).Conversely, pretreatment with ALA dose-dependently reduced PA effect restoring PPAR-γ levels to control cells.In addition, cells pretreated with 25 and 100 µM ALA showed PPAR-γ levels lower than control cells.Interestingly, ALA pretreatment significantly diminished the levels of this protein even in adipocytes not exposed to PA, confirming the modulatory effects on basal PPAR-γ levels.
In addition, gene expression of FABP4, a downstream gene of PPAR-γ signaling pathway and a transporter of fatty acids involved in the control of lipid droplets storage (Moreno-Vedia et al. 2022) (Furuhashi et al. 2014), was estimated.Figure S1C shows that PA induced a strong increase in FABP4 mRNA levels, which was, in contrast, significantly and dose dependently reduced by pretreatment with ALA starting form 1 µM.On the other hand, treatment with ALA alone had no effect on FABP4 expression as previously observed for PPAR-γ.Although FABP4 is transcriptionally regulated by PPAR-γ agonists, these results let us hypothesize that FABP4 expression can be modulated also by other factors than PPAR-γ, such as AMPK and C/eBP (Kajita et al. 2008;Furuhashi et al. 2014;Moreno-Vedia et al. 2022), during PA exposure.
Similar effects were also shown by other authors, but at significantly higher ALA doses.In fact, ALA (250 and 500 µM) was able to reduce PPAR-γ and FABP4 during 3T3-L1 adipocytes differentiation (Cho et al. 2003), and to improve lipid profile in adipose tissue of high-fat diet-fed rats affecting fatty acids transporters (Wołosowicz et al. 2022).These findings suggest that the effects of low concentrations of ALA to reduce adipocytes hypertrophy induced by high PA concentrations might be associated with PPAR-γ pathway inhibition.

Protective effects of ALA against PA-induced oxidative stress and NF-κB pathway activation
Several studies have shown that obesity can induce local and systemic oxidative stress that in turn induce inflammation (Martin et al. 2015).Since ALA showed strong antioxidant properties (Rochette et al. 2015;Fratantonio et al. 2018;Salehi et al. 2019), its protective effects on intracellular ROS levels were evaluated (see Supplementary Material for experimental details).Our data show that ROS levels were significantly increased in fully differentiated adipocytes following the treatment with PA compared to control cells (Figure S2).Conversely the pretreatment with ALA significantly reduced intracellular ROS levels induced by PA starting from 1 µM.In addition, ALA pretreatment did not affect basal ROS levels in PA-unexposed fully differentiated cells with values comparable to control cells (Figure S2).This data, therefore, confirms the possible protective role of ALA against changes in the intracellular redox status.
It has been shown that oxidative stress into adipose tissue induces the activation of redox-sensitive kinases and transcription factors, such as NF-κB, which trigger macrophage infiltration and determine the expression of pro-inflammatory cytokines (Pérez-Torres et al. 2021).
In our experimental conditions, PA determined an inflammatory state in fully differentiated adipocytes inducing the nuclear translocation of NF-ᴋB, as demonstrated by increased p65 nuclear levels compared to control (Figure S3A).Conversely, ALA pretreatment significantly and dose-dependently inhibited NF-κB activation induced by PA starting from 1 µM (Figure S3A).Furthermore, ALA 25 and 100 μM reduced p65 nuclear basal levels, compared to controls, also in cells not exposed to PA and this effect might be associated with a redox modulation of NF-κB exerted by ALA.
In order to determine the mechanisms involved in ALA effects on NF-κB inhibition, cytoplasmic levels of phosphorylated IKK α/β, the main NF-κB activators, were also evaluated.In presence of an extracellular stimulus, IKKα/β, in fact, is activated, resulting in phosphorylation of the IκB inhibitor and translocation of NF-κB into the nucleus (Hayden and Ghosh 2014).PA determined the activation (phosphorylation) of IKK α/β (Figure S3B) in fully differentiated adipocytes.Conversely, ALA pretreatment dose-dependently inhibited IKK α/β phosphorylation induced by PA, thus confirming NF-κB pathway inhibition (Figure S3B), although the statistical significance was reached only at 25 and 100 µM.
Additionally, IL-6 gene expression was evaluated by real time-PCR to confirm the transcriptional activity of NF-κB.IL-6, in fact, is the most representative cytokine modulated by NF-κB and released by adipose tissue following a proinflammatory stimulus.The results demonstrate that PA activated a significant overexpression of IL-6 gene expression in fully differentiated adipocytes when compared to control, whereas the pretreatment with ALA was able to reduce the mRNA levels of this pro-inflammatory cytokine (Figure S3C).However, even if ALA 1 µM was able to affect NF-κB nuclear levels, it did not significantly modulate IL-6 gene expression in our experimental conditions.
Therefore, ALA can be considered an antioxidant capable of neutralizing ROS and determining, presumably in part by the modulation of intracellular oxidative stress, the inhibition of an inflammatory state in adipose tissue.However, other authors demonstrated that ALA, but not other tested antioxidants, inhibited TNF-α-induced NF-κB-dependent gene expression, implying that ALA inhibited NF-κB activation independent of its antioxidant function (Zhang and Frei 2001;Ying et al. 2011).In our study, in PA-exposed cells we observed both a reduction in ROS levels and an inhibition of NF-κB pathway by ALA, so supporting the modulation of oxidative stress induced by PA.However, in cells treated only with ALA, the reduction of basal NF-κB levels was not associated with a similar behaviour of ROS concentrations that were similar to control cells.A possible justification of these effects is that, more than a merely direct scavenger of ROS, ALA could produce a weak and transient modification of the cellular thiol redox environment, inducing an hormetic protective response affecting signaling cascades (Shay et al. 2008).Moini and coworkers demonstrated that ALA (250 µM) increases the intracellular GSH content in 3T3-L1 adipocytes, shifting the intracellular environment toward more reducing conditions (Moini et al. 2002), and this effect can be mediated by an adaptive cellular response.In addition, ALA ameliorated ROS production and endoplasmic reticulum stress in human hepatocytes exposed PA and this effect was associated to Nrf2 pathway and activated antioxidant enzymes.Nrf2 regulates the expression of important components of the GSH and Trx antioxidant system, as well as enzymes involved in NAdPH regeneration and ROS detoxification, so performing an essential role in preserving cellular redox homeostasis.In this regard, a previous study from our group showed that ALA (0.25 and 1 µM) was able to modulate the cellular adaptive protective response through the activation of the Nrf2 pathway and inhibition of the NF-κB one, thus protecting endothelial cells from TNF-α-induced dysfunction (Fratantonio et al. 2018).Growing evidence have demonstrated, indeed, the existence of a close crosstalk between Nrf2 and NF-κB, where the two pathways reciprocally inhibit the transcription and/or activity of downstream proteins (Wardyn et al. 2015;Hennig et al. 2018).

Effects of ALA on PA-induced insulin resistance
Growing evidence suggest that oxidative stress and the activation of inflammatory signaling pathways lead to inhibition of insulin signal transduction in adipose tissue of people with obesity (Pérez-Torres et al. 2021).Adipocytes represent the main sites of insulin action and, therefore, play an important role in glucose metabolism as well as in the regulation of glucose homeostasis and glucose uptake (Herman and Kahn 2006).
Thus, in order to investigate the ALA protective effect on insulin resistance induced by PA, intracellular glucose uptake in fully differentiated 3T3-L1 adipocytes was examined (see Supplementary Material for experimental details).The results show that PA exposure determined, in our experimental conditions, a sharp reduction in insulin-induced glucose uptake in 3T3-L1 cells, with value below the control insulin treatment (Figure S4A), thus confirming the establishment of an insulin resistance state.data obtained with ALA pretreatment confirm, instead, the protective effect of this molecule, that significantly and dose-dependently improved glucose uptake altered by PA.Interestingly, ALA pretreatment alone increased glucose uptake levels to values above those observed in cells exposed to insulin alone resulting in an insulin-sensitizing effect starting from 1 µM (Figure S4A).ALA effects were also confirmed evaluating protein levels of GLUT-1, as representative insulin-dependent glucose transporter.GLUT-1 was preferred to other transporters, since its expression is less affected by chronic treatments during adipocytes differentiation (Kozka et al. 1991).Our data showed that, in our experimental conditions, GLUT-1 is induced by insulin treatment and deeply reduced after PA exposure, demonstrating that this transporter is involved in insulin sensitivity/resistance in fully differentiated 3T3-L1 (Figure S4B) as also confirmed by previous data on glucose uptake (Figure S4A).The pretreatment with ALA determined, instead, at all tested concentrations and in a dose-dependent and statistically significant manner, an increase in GLUT-1 protein levels altered by PA.Interestingly, ALA treatment induced GLUT-1 protein also without PA exposure with values above those observed in cells exposed to insulin alone (Figure S4B).These results, together with data on cellular glucose uptake, let us hypothesize that ALA induces not only GLUT-1 expression but also its translocation to the plasma membrane so improving glucose entry into the cells (Figure S4B).evidence suggests that ALA (30 mg/kg for 4 weeks) improves glucose metabolism in high fat diet-fed mice (dajnowicz et al. 2022) and attenuates insulin resistance, increasing PI3K/Akt phosphorylation at both 100 and 200 mg/kg •pro die in high fat diet-fed mice (Yang et al. 2014).In our experimental conditions, fully differentiated adipocytes exposure to PA resulted in a statistically significant reduction in the levels of PI3K (Figure S5A and B) and pAkt (Figure S5A and C) compared to control cells exposed only to insulin, thus confirming that PA induces an alteration of the insulin signaling pathway.In contrast, the pretreatment with ALA, starting from 1 µM, was able to significantly and dose-dependently increase the levels of PI3K and pAkt altered by PA.Also in this case, ALA was able to induce pAkt levels with or without PA exposure, to levels higher than cells exposed to insulin alone (Figure S5A and C).
These findings agree with those reported by other authors in 3T3-L1 adipocytes using different experimental models, but also in this case were obtained at very low and physiological concentations of ALA (< 100 µM) starting from 1 µM.In fact, R(+) ALA (2.5 mM) stimulated basal glucose uptake and augmented GLUT-1 in the plasma membrane fraction similarly to the action of insulin and these effects were associated to IRS-1 tyrosine phosphorylation and PI3K/Akt activation in 3T3-L1 adipocytes (estrada et al. 1996;Yaworsky et al. 2000).Moreover, Rudich and coll.(Rudich et al. 1999) reported that ALA (200 µM) prevented glucose oxidase-induced insulin-resistance in 3T3-L1 adipocytes improving glucose uptake and GLUT plasma membrane levels.Moini and coll.(Moini et al. 2002), instead, demonstrated the insulin-sensitizing activity of 250 µM ALA in 3T3-L1, reporting also that wortmannin, a PI3K inhibitor, repressed ALA-stimulated glucose uptake.

Conclusions
While being aware that the results were obtained using a model of murine adipocytes and that the translation into the human clinical setting suffers from considerable limitations, this study has clearly highlighted the protective effects of ALA against hypertrophy, inflammation, and insulin resistance induced by PA in adipose tissue.Additionally, the effects of ALA on basal lipid levels let us hypothesize the activation of adaptive mechanisms such as thermogenic process by increasing energy expenditure instead of accumulation as previously reported (Kim et al. 2004).Our data, compared to the existing literature using high concentrations of ALA, confirmed that ALA exerts its protective effects at low micromolar concentrations, starting from 1 µM, and suggest an important role for this product as pharmacological supplement in the prevention of pathological conditions linked to obesity and metabolic syndrome.