Prenylcysteine Oxidase 1 Is a Key Regulator of Adipogenesis

The process of adipogenesis involves the differentiation of preadipocytes into mature adipocytes. Excessive adipogenesis promotes obesity, a condition that increasingly threatens global health and contributes to the rapid rise of obesity-related diseases. We have recently shown that prenylcysteine oxidase 1 (PCYOX1) is a regulator of atherosclerosis-disease mechanisms, which acts through mechanisms not exclusively related to its pro-oxidant activity. To address the role of PCYOX1 in the adipogenic process, we extended our previous observations confirming that Pcyox1−/−/Apoe−/− mice fed a high-fat diet for 8 or 12 weeks showed significantly lower body weight, when compared to Pcyox1+/+/Apoe−/− mice, due to an evident reduction in visceral adipose content. We herein assessed the role of PCYOX1 in adipogenesis. Here, we found that PCYOX1 is expressed in adipose tissue, and, independently from its pro-oxidant enzymatic activity, is critical for adipogenesis. Pcyox1 gene silencing completely prevented the differentiation of 3T3-L1 preadipocytes, by acting as an upstream regulator of several key players, such as FABP4, PPARγ, C/EBPα. Proteomic analysis, performed by quantitative label-free mass spectrometry, further strengthened the role of PCYOX1 in adipogenesis by expanding the list of its downstream targets. Finally, the absence of Pcyox1 reduces the inflammatory markers in adipose tissue. These findings render PCYOX1 a novel adipogenic factor with possible pathophysiological or therapeutic potential.


Introduction
Adipose tissue plays an important role in energy homeostasis through its capacity to store energy, mobilize stored lipids for fuel, and secrete hormones and cytokines [1]. Cardiovascular diseases, type 2 diabetes, and metabolic syndrome are all associated with excess fat accumulation in white adipose tissue (WAT) [2]. Understanding the molecular events that regulate adipocyte differentiation is an essential step towards preventing obesity and metabolic diseases because adipocyte differentiation determines adipose tissue mass.
Adipocyte differentiation involves hormonal stimulators and the induction of a network of transcription factors that induce changes in gene expression and cell morphology. At the top of this transcriptional hierarchy are the basic region/leucine zipper proteins CCAAT-enhancer binding protein (C/EBP)δ, C/EBPβ, C/EBPα, and the nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ [3]. These transcription factors are induced in a sequential and tightly regulated manner, and all are essential for normal adipogenesis. However, several important unanswered questions remain, and a more complete understanding of the developmental origin as well as the cellular and molecular components of adipose tissue is necessary to further illuminate how excess adiposity contributes to the onset and/or progression of metabolic disorders.
We have recently shown that prenylcysteine oxidase 1 (PCYOX1) deficiency in apolipoprotein E deficient mice (Apoe −/− ) is associated with reduced body weight and adipose tissue deposits [4], but the role of PCYOX1 in adipogenesis has never been addressed since its discovery in 1997 by Casey's group in studies related to the metabolism of prenylated proteins [5,6].
To substantiate the contribution of PCYOX1 in the adipogenic process, we investigated its role in vitro and in vivo, taking advantage of a complementary approach based on gene silencing and label-free quantitative proteomics.
Here we describe for the first time the role of PCYOX1 as a novel regulator of adipogenesis. Employing proteomics and gene expression analysis in Pcyox1 silenced cells, we show that it acts upstream of the key factors that control the adipogenic process, while the in vivo absence of the Pcyox1 gene in mice is associated with reduced adipose deposits, and related adipose tissue inflammation. Overall, these data identify PCYOX1 as a master regulator and potential therapeutic target in obesity-related diseases.

Stable Transfection with Short Hairpin RNA (shRNA)
Stable gene silencing of Pcyox1 and Cebpb in 3T3-L1 cells was achieved using shRNA plasmids (Santa Cruz Biotechnology, Dallas, TX, USA), a pool of 3 target-specific lentiviral vector plasmids each encoding 19-25 nt (plus hairpin) shRNAs designed to knock down Pcyox1 and Cebpb gene expression. A shRNA plasmid encoding a scrambled shRNA sequence that does not induce the degradation of any cellular message was taken as negative control (control cells, shNEG). Cells at 50-70% confluency were transfected for 24 h with a multiplicity of infection (MOI) of 5 for Pcyox1 shRNA and 7.5 for Cebpb shRNA lentiviral particles, and 5 µg/mL of Polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for each well of a 12 well/plate and cultured in complete medium containing 2 µg/mL puromycin to allow selection.

Oxidized Low Density Lipoprotein (oxLDL) Assay
The quantitation of oxLDL in the cell culture medium was performed by a quantitative immunoenzymatic assay according to the manufacturer's instructions (MyBioSource, Inc., San Diego, CA, USA). Briefly, shNEG and shPcyox1 silenced cells were induced to differentiate for 9 days. After this period, medium was collected and oxLDL levels were measured.

PCOYX1 Activity Assay
PCYOX1 activity was assessed as previously described [4]. Briefly, H 2 O 2 produced in cells by the PCYOX1 reaction was measured using the Amplex Red Kit (Life Technologies, Milan, Italy). In the presence of peroxidase, the Amplex Red reagent is converted by H 2 O 2 into the red-fluorescent oxidation product resorufin. The resorufin produced was measured following the fluorescence emission in a microplate reader Infinite 200 (TECAN, Mannedorf, Switzerland), equipped for excitation at 530 nm and fluorescence emission detection at 590 nm. Results are expressed as picomoles of H 2 O 2 /µg protein.

Label-Free Mass Spectrometry (LC-MS E ) Analysis
Cell pellets were dissolved in 25 mmol/L NH 4 HCO 3 containing 0.1% RapiGest (Waters Corporation, Milford, MA, USA), sonicated, and centrifuged at 13,000× g for 10 min. After 15 min of incubation at 80 • C, proteins were reduced with 5 mmol/L dithiothreitol (DTT) at 60 • C for 15 min, and carbamidomethylated with 10 mmol/L iodoacetamide for 30 min at room temperature in darkness. Digestion was performed with sequencing grade trypsin (Promega, Milan, Italy) (1 µg every 20 µg of proteins) overnight at 37 • C. After digestion, 2% trifluoroacetic acid (TFA) was added to hydrolyze RapiGest and inactivated trypsin. Tryptic peptides were used for label-free mass spectrometry analysis, LC-MS E , performed on a hybrid quadrupole-time of flight mass spectrometer (SYNAPT-XS, Waters Corporation, Milford, MA, USA) coupled with a UPLC Mclass system and equipped with a nano-source (Waters Corporation, Milford, MA, USA), as previously described [8,9]. Statistical analysis was performed by means of Progenesis QIP v 4.1 (Nonlinear Dynamics) using a Uniprot mouse protein sequence database (v2020). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [10] partner repository with the dataset identifier PXD039943 and 10.6019/PXD039943.

GO Analysis
The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING 10.5) database [11] was used to identify enriched Gene Ontology (GO) terms in the biological process, molecular function, or cellular component categories, as previously described [12]. The enrichment function of STRING, which calculates an enrichment p value based on a Antioxidants 2023, 12, 542 4 of 17 hypergeometric test using the method of Benjamini and Hochberg for correction of multiple testing (p value cut-off < 0.05), was used.

Mass Spectrometry-Based Quantification of PCYOX1
Tryptic peptides (0.5 µg/uL), obtained as described above, were added with the stable isotope labelled proteotipic PCYOX1 peptide (CPSIILHD(R) from Thermofisher Scientific (Milan, Italy), desalted with ZipTip C18 (Millipore, Burlington, MA, USA) according to the manufacturer's instruction, and then dissolved in water with 0.1% formic acid before mass spectrometry analysis. Two microliters of each sample, containing 10 fmol/µL of labelled heavy peptide, were injected into a Xevo TQ-S micro triple quadrupole mass spectrometer coupled to a Waters ACQUITY ultra-performance liquid chromatography (UPLC) M-Class system through an ionKey source (Waters Corporation, Milford, MA, USA), and analysed as previously described [13].

Mice and Diets
Pcyox1 −/− mice were bred with Apoe −/− mice (B6.129P2-Apoetm1Unc/J, stock 002052, JAX™ Mice Strain) as previously described [4]. Intercrosses of resulting Apoe −/− / Pcyox1 +/− mice generated offspring that entered the study. All procedures were approved by the Institutional Animal Care and Ethics Committee of the University of Milan and by the Ministry of Health DGSAF (N. 782-2020; approval 10 August 2020). Mice were housed in an air-conditioned room at 22 ± 0.5 • C with a 12-h lighting cycle and free access to food and water. For experiments, double knockout mice (Pcyox1 −/− /Apoe −/− ) and control mice (Pcyox1 +/+ /Apoe −/− ) were fed ad libitum with a high fat diet (HFD) containing 0.2% cholesterol, 21.2% fat (42% kcal), and 17.5% protein by weight (Teklad diet TD.88137; Envigo, Milan, Italy), starting at 11 weeks of age and continuing for 8 or 12 weeks. After the indicated period of HFD, mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (75 mg/kg) and medetomidine (1 mg/kg) prior to visceral fat harvest. Abdominal visceral adipose tissue (VAT) was rapidly removed, weighed, and snap frozen for RNA and mass spectrometry analyses. Each experimental session included animals of both genotypes (Pcyox +/+ /Apoe −/− n = 23; Pcyox −/− /Apoe −/− n = 19), with animals being assigned to the experimental groups according to genotype, and with the investigators blinded to group assignment during all experimental stages and when evaluating outcome measures.

Statistical Analysis
Data analysis was performed with GraphPad Prism 9.3 software (GraphPad Software Inc., San Diego, CA, USA). All data sets were tested for normality of distribution and analysed using the Student t test or analysis of variance (ANOVA) for multiple comparison followed by Dunnett's or Tukey's post hoc test as indicated. Statistical significance level was accepted at p < 0.05.

Pcyox1 −/− Mice Have Decreased Adiposity
Pcyox1 −/− /Apoe −/− mice fed an HFD for 8 or 12 weeks showed 16.3% and 20.2% lower body weight, respectively, when compared to Pcyox1 +/+ /Apoe −/− mice ( Figure 1A), due to a 40% and 52.5% reduction in visceral adipose content ( Figure 1B), thus confirming preliminary observations [4]. Pcyox1 mRNA and protein, measured by quantitative mass spectrometry, were both present in the VAT of Pcyox1 +/+ /Apoe −/− mice fed an HFD for 8 weeks ( Figure 1C,D). As previously demonstrated, serum analysis in Pcyox1 −/− /Apoe −/− mice showed significantly decreased triglycerides, free fatty acid, and cholesterol concentrations, without any effects attributable to differences in food intake [4]. Overall, these data suggest that PCYOX1 may play a role in promoting in vivo adipogenesis. To address this, we explored the role of PCYOX1 in adipogenesis in in vitro differentiating cells. spectrometry, were both present in the VAT of Pcyox1 +/+ /Apoe −/− mice fed an HF weeks ( Figure 1C,D). As previously demonstrated, serum analysis in Pcyox1 −/− / mice showed significantly decreased triglycerides, free fatty acid, and cholesterol c trations, without any effects attributable to differences in food intake [4]. Overal data suggest that PCYOX1 may play a role in promoting in vivo adipogenesis. To a this, we explored the role of PCYOX1 in adipogenesis in in vitro differentiating ce

PCYOX1 Expression Is Induced during Adipocyte Differentiation In Vitro
Pcyox1 mRNA expression is significantly increased when 3T3-L1 are fully differentiated to adipocyte-like cells after 9 days from the beginning of the differentiation protocol ( Figure 2A). This occurs concomitantly with a rising expression of Pparg, a nuclear receptor critically involved in adipocyte differentiation, and Fabp4, a downstream target of PPARγ, which is a terminal marker of adipocyte differentiation ( Figure 2B,C).

PCYOX1 Expression Is Induced during Adipocyte Differentiation In Vitro
Pcyox1 mRNA expression is significantly increased when 3T3-L1 are fully differenti ated to adipocyte-like cells after 9 days from the beginning of the differentiation protoco ( Figure 2A). This occurs concomitantly with a rising expression of Pparg, a nuclear recep tor critically involved in adipocyte differentiation, and Fabp4, a downstream target o PPARγ, which is a terminal marker of adipocyte differentiation ( Figure 2B,C). To explore the determinants of PCYOX1 induction during adipogenesis, we analysed the effects of the three components of the cocktail used to induce differentiation: insulin dexamethasone (DEX), and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Insulin minimally perturbed Pcyox1 expression ( Figure 3A), whereas IBMX and dexamethasone strongly increased Pcyox1 gene expression ( Figure 3B,C). Because IBMX and DEX are each necessary for maximal and sustained expression of the pro-adipogenic transcription factor CCAAT/enhancer binding protein β (C/EBPβ) [14], we evaluate its mRNA modulation in the initial stages of differentiation ( Figure 3D).

PCYOX1 Is Critical for Adipogenesis In Vitro
To examine the role of PCYOX1 in adipogenesis, short-hairpin shRNA retroviral con structs targeting three regions of the Pcyox1 mRNA were generated, and stably transfected into 3T3-L1 preadipocytes. The control and knockdown cell lines were then induced to differentiate for 9 days. Before the induction of differentiation, Pcyox1 mRNA levels were To explore the determinants of PCYOX1 induction during adipogenesis, we analysed the effects of the three components of the cocktail used to induce differentiation: insulin, dexamethasone (DEX), and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Insulin minimally perturbed Pcyox1 expression ( Figure 3A), whereas IBMX and dexamethasone strongly increased Pcyox1 gene expression ( Figure 3B,C). Because IBMX and DEX are each necessary for maximal and sustained expression of the pro-adipogenic transcription factor CCAAT/enhancer binding protein β (C/EBPβ) [14], we evaluate its mRNA modulation in the initial stages of differentiation ( Figure 3D). ated to adipocyte-like cells after 9 days from the beginning of the differentiation protocol ( Figure 2A). This occurs concomitantly with a rising expression of Pparg, a nuclear receptor critically involved in adipocyte differentiation, and Fabp4, a downstream target of PPARγ, which is a terminal marker of adipocyte differentiation ( Figure 2B,C). To explore the determinants of PCYOX1 induction during adipogenesis, we analysed the effects of the three components of the cocktail used to induce differentiation: insulin, dexamethasone (DEX), and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Insulin minimally perturbed Pcyox1 expression ( Figure 3A), whereas IBMX and dexamethasone strongly increased Pcyox1 gene expression ( Figure 3B,C). Because IBMX and DEX are each necessary for maximal and sustained expression of the pro-adipogenic transcription factor CCAAT/enhancer binding protein β (C/EBPβ) [14], we evaluate its mRNA modulation in the initial stages of differentiation ( Figure 3D).

PCYOX1 Is Critical for Adipogenesis In Vitro
To examine the role of PCYOX1 in adipogenesis, short-hairpin shRNA retroviral constructs targeting three regions of the Pcyox1 mRNA were generated, and stably transfected into 3T3-L1 preadipocytes. The control and knockdown cell lines were then induced to differentiate for 9 days. Before the induction of differentiation, Pcyox1 mRNA levels were

PCYOX1 Is Critical for Adipogenesis In Vitro
To examine the role of PCYOX1 in adipogenesis, short-hairpin shRNA retroviral constructs targeting three regions of the Pcyox1 mRNA were generated, and stably transfected into 3T3-L1 preadipocytes. The control and knockdown cell lines were then induced to differentiate for 9 days. Before the induction of differentiation, Pcyox1 mRNA levels were reduced in silenced cells (−92.8 ± 1.1% with respect to control cells, n = 5, p < 0.001), and remained significantly reduced until day 9 (−86.4 ± 2.5%, n = 8, p < 0.001 versus control cells). Similarly, treatment of 3T3-L1 cells with the Pcyox1 shRNA reduced endogenous Antioxidants 2023, 12, 542 7 of 17 PCYOX1 protein levels by 79.1 ± 6%, as assessed by mass spectrometry (n = 3; p < 0.0002). The shPcyox1-treated 3T3-L1 were also examined for their ability to differentiate into adipocytes compared to control cells. In response to adipogenic inducers, control cells underwent efficient morphological differentiation into lipid droplet-containing adipocytes. By contrast, Pcyox1-depleted 3T3-L1 preadipocytes accumulated fewer lipid droplets when induced to undergo differentiation ( Figure 4A). reduced in silenced cells (−92.8 ± 1.1% with respect to control cells, n = 5, p < 0.001), and remained significantly reduced until day 9 (−86.4 ± 2.5%, n = 8, p < 0.001 versus control cells). Similarly, treatment of 3T3-L1 cells with the Pcyox1 shRNA reduced endogenous PCYOX1 protein levels by 79.1 ± 6%, as assessed by mass spectrometry (n = 3; p < 0.0002). The shPcyox1-treated 3T3-L1 were also examined for their ability to differentiate into adipocytes compared to control cells. In response to adipogenic inducers, control cells underwent efficient morphological differentiation into lipid droplet-containing adipocytes. By contrast, Pcyox1-depleted 3T3-L1 preadipocytes accumulated fewer lipid droplets when induced to undergo differentiation ( Figure 4A). The mRNA expression of Fabp4, a terminal marker of adipogenesis ( Figure 4B), was found to be significantly reduced in Pcyox1-silenced cells compared to control cells at the end of differentiation.
To identify the developmental stage regulated by PCYOX1, we harvested control and Pcyox1-depleted 3T3-L1 cells at 0, 1, and 9 days after the addition of adipogenic inducers in order to evaluate the expression of known transcriptional factors involved in adipogenesis. Pparg ( Figure 4C) and Cebpa ( Figure 4D) were significantly lower in Pcyox1-silenced cells compared to controls at day 9. Importantly, the earlier induction of Cebpb was unaffected by the loss of PCYOX1 in 3T3-L1 cells, being induced to the same extent in control and Pcyox1-silenced cells 24 h after the addition of the adipogenic cocktail ( Figure 4E), suggesting that PCYOX1 acts downstream of C/EBPβ to promote the adipogenic program.
To assess whether C/EBPβ can directly regulate PCYOX1, we silenced cells for Cebpb. Although there was a significantly lower expression of Cebpb (−54 ± 6% in silenced cells with respect to control cells treated with a negative construct, n = 3, p < 0.002) and the The mRNA expression of Fabp4, a terminal marker of adipogenesis ( Figure 4B), was found to be significantly reduced in Pcyox1-silenced cells compared to control cells at the end of differentiation.
To identify the developmental stage regulated by PCYOX1, we harvested control and Pcyox1-depleted 3T3-L1 cells at 0, 1, and 9 days after the addition of adipogenic inducers in order to evaluate the expression of known transcriptional factors involved in adipogenesis. Pparg ( Figure 4C) and Cebpa ( Figure 4D) were significantly lower in Pcyox1-silenced cells compared to controls at day 9. Importantly, the earlier induction of Cebpb was unaffected by the loss of PCYOX1 in 3T3-L1 cells, being induced to the same extent in control and Pcyox1silenced cells 24 h after the addition of the adipogenic cocktail ( Figure 4E), suggesting that PCYOX1 acts downstream of C/EBPβ to promote the adipogenic program.
To assess whether C/EBPβ can directly regulate PCYOX1, we silenced cells for Cebpb. Although there was a significantly lower expression of Cebpb (−54 ± 6% in silenced cells with respect to control cells treated with a negative construct, n = 3, p < 0.002) and the known C/EBPβ downstream targets Pparg and Cebpa, Pcyox1 expression was unaffected ( Figure 5).

The Effects of PCYOX1 in Adipogenesis Are Independent of Its Pro-Oxidant Activity
We previously demonstrated that PCYOX1 is able to generate H2O2, which in turn induces the formation of oxLDL [4], a known activator of PPARγ because a variety of oxLDL components act as PPARγ ligands [15]. Therefore, we assessed whether PCYOX1 could induce the formation of oxLDL during adipogenesis. First, we measured PCYOX1 activity in control and Pcyox1-silenced cells, in comparison with CHO cells overexpressing PCYOX1. The results obtained indicated that, in differentiated adipocytes, PCYOX1 is expressed but is not active (1.4 pmol H2O2/µ g protein in CHO cells overexpressing PCYOX1, undetectable in differentiated adipocytes, n = 3). Furthermore, the levels of oxLDL, as assessed by immunoenzymatic assay, were not different between control cells and Pcyox1silenced cells (1.1 ± 0.3 ng/mL and 1.0 ± 0.2 ng/mL, respectively, n = 6).

PCYOX1 Significantly Affects the Cell Proteome
The impact of PCYOX1 deletion on adipogenesis was further investigated by proteomics. Label-free quantitative mass spectrometry revealed that 43 proteins were significantly less abundant in the Pcyox1-deficient cells, and only two were more abundant in the Pcyox1-deficient cells compared to control cells (Table 1).

The Effects of PCYOX1 in Adipogenesis Are Independent of Its Pro-Oxidant Activity
We previously demonstrated that PCYOX1 is able to generate H 2 O 2 , which in turn induces the formation of oxLDL [4], a known activator of PPARγ because a variety of oxLDL components act as PPARγ ligands [15]. Therefore, we assessed whether PCYOX1 could induce the formation of oxLDL during adipogenesis. First, we measured PCYOX1 activity in control and Pcyox1-silenced cells, in comparison with CHO cells overexpressing PCYOX1. The results obtained indicated that, in differentiated adipocytes, PCYOX1 is expressed but is not active (1.4 pmol H 2 O 2 /µg protein in CHO cells overexpressing PCYOX1, undetectable in differentiated adipocytes, n = 3). Furthermore, the levels of oxLDL, as assessed by immunoenzymatic assay, were not different between control cells and Pcyox1-silenced cells (1.1 ± 0.3 ng/mL and 1.0 ± 0.2 ng/mL, respectively, n = 6).
We then evaluated gene expression in the VAT of mice fed an HFD for 8 weeks. As shown in Figure 8, Pcyox1 deficiency was associated with a significant reduction in the expression of the genes involved in adipogenesis, such as Cd36, Pparg, Lpl, and Ldlr ( Figure 8).
xidants 2023, 12, x FOR PEER REVIEW 11 of Figure 6. GO analysis of proteins modulated by Pcyox1 silencing. Enriched GO terms in the biolo ical process category are highlighted in different colors: red, lipid metabolic processes; violet, ox dation-reduction processes; blue, fatty acid β-oxidation; green, tricarboxylic acid cycle; yellow, gl coneogenesis.
Furthermore, the results from the proteomic analysis were validated in independe experiments by assessing the mRNA levels of the less abundant proteins: hormone-sens tive lipase (Lipe), carbonic anhydrase 3 (Car3), perilipin-1 (Plin1), 1-acyl-sn-glycerol- We then evaluated gene expression in the VAT of mice fed an HFD for 8 weeks. As shown in Figure 8, Pcyox1 deficiency was associated with a significant reduction in the expression of the genes involved in adipogenesis, such as Cd36, Pparg, Lpl, and Ldlr ( Figure 8).   We then evaluated gene expression in the VAT of mice fed an HFD for 8 weeks. As shown in Figure 8, Pcyox1 deficiency was associated with a significant reduction in the expression of the genes involved in adipogenesis, such as Cd36, Pparg, Lpl, and Ldlr ( Figure 8).  To determine the effect of the ablation of Pcyox1 on adipose tissue inflammation, we examined the expression of inflammatory markers serum amiloyd A3 (SAA3), MAC2, CD11b, CD11c, and EMR1. All of them were decreased in the VAT of Pcyox1 −/− /Apoe −/− mice versus Pcyox1 +/+ /Apoe −/− mice after 8 weeks on HFD (Figure 9). To determine the effect of the ablation of Pcyox1 on adipose tissue inflammation, we examined the expression of inflammatory markers serum amiloyd A3 (SAA3), MAC2, CD11b, CD11c, and EMR1. All of them were decreased in the VAT of Pcyox1 −/− /Apoe −/− mice versus Pcyox1 +/+ /Apoe −/− mice after 8 weeks on HFD (Figure 9).

Discussion
New treatments that directly target adipose tissue accumulation may be possible if we understand all the factors necessary for normal adipogenesis. To the list of these factors, we add PCYOX1 as a novel regulator of adipogenesis. Our results demonstrate that PCYOX1 is expressed in adipose tissue, and that its deficiency results in reduced fat deposits. In agreement with these findings, in vitro experiments revealed that PCYOX1 is necessary for adipogenesis. PCYOX1 first emerged at the end of the 1990s as a lysosomal enzyme involved in the catabolism of prenylated proteins acting as a flavin adenine dinucleotide (FAD)-dependent thioether oxidase able to generate a stoichiometric amount of H2O2 [5,16]. Afterwards, thanks to the advent of proteomics, the knowledge of PCYOX1 was extended to the finding that this protein belongs to the proteome of lipoproteins, in which it contributes to their oxidative modifications [17]. Furthermore, Pcyox1 silencing in vitro was found to affect the cellular proteome by influencing multiple functions related

Discussion
New treatments that directly target adipose tissue accumulation may be possible if we understand all the factors necessary for normal adipogenesis. To the list of these factors, we add PCYOX1 as a novel regulator of adipogenesis. Our results demonstrate that PCYOX1 is expressed in adipose tissue, and that its deficiency results in reduced fat deposits. In agreement with these findings, in vitro experiments revealed that PCYOX1 is necessary for adipogenesis. PCYOX1 first emerged at the end of the 1990s as a lysosomal enzyme involved in the catabolism of prenylated proteins acting as a flavin adenine dinucleotide (FAD)-dependent thioether oxidase able to generate a stoichiometric amount of H 2 O 2 [5,16]. Afterwards, thanks to the advent of proteomics, the knowledge of PCYOX1 was extended to the finding that this protein belongs to the proteome of lipoproteins, in which it contributes to their oxidative modifications [17]. Furthermore, Pcyox1 silencing in vitro was found to affect the cellular proteome by influencing multiple functions related to inflammation, oxidative stress, and platelet adhesion [4,13]. We also showed that Pcyox1 deficiency in Apoe −/− mice on C57/BL6J background mice (B6.129P2-Apoetm1Unc/J) retards atheroprogression, is asso-ciated with decreased features of lesion vulnerability and lower levels of lipid peroxidation, and reduces plasma lipid levels and inflammation, thus highlighting for the first time the role of PCYOX1 in vivo. Indeed, the pioneering study of Beigneux et al. [18] showed the absence of any histologic abnormalities in a survey of >30 tissues from Pcyox1-deficient mice on a mixed C57BL/6-129/SvJae genetic background. The observation that Pcyox1 deficiency in Apoe −/− mice fed with HFD, a model of obesity-accelerated atherosclerosis accompanied by development of a metabolic syndrome phenotype [19], have less adipose tissue depots, led us to further investigate the role of PCYOX1 in adipogenesis.
The differentiation of preadipocytes into adipocytes is regulated by an elaborate network of transcription factors that coordinate the expression of hundreds of proteins responsible for establishing the mature fat-cell phenotype. At the center of this network are the two principal adipogenic factors, PPARγ and C/EBPα, which oversee the entire terminal differentiation process. PPARγ in particular is considered the master regulator of adipogenesis; without it, precursor cells are incapable of expressing any known aspect of the adipocyte phenotype [20].
We found that the knockdown of Pcyox1 in 3T3-L1 preadipocytes renders them defective in inducing markers of terminal differentiation such as C/EBPα, PPARγ, and FABP4 and in accumulating fat, suggesting that PCYOX1 plays a pivotal role during the early events of the process. In this process, we ruled out the reciprocal regulation between PCYOX1 and C/EBPβ, suggesting that PCYOX1 acts independently from this crucial transcription factor. In an attempt to define the sequence of events leading to terminal adipogenesis, it was proposed that C/EBPβ and C/EBPδ simultaneously control the expression of both PPARγ and C/EBPα. Alternatively, some investigators have suggested that C/EBPβ induces C/EBPα and that, together, these factors regulate PPARγ expression [21]. The precise role of C/EBPβ and C/EBPδ in regulating this cascade of factors has been questioned, however, in knockout mice. Specifically, Tanaka et al. [22] demonstrated that adipocyte differentiation in vitro proceeds according to the proposed transcriptional regulatory cascade in which adipogenic transcription factors such as C/EBP family members and PPARs are activated sequentially. However, in vivo, C/EBPα and PPARγ can be induced without expression of C/EBPβ and C/EBPδ. These data suggest that there is some redundancy in the early steps of adipogenesis in vivo where alternative pathways operate to ensure the expression of PPARγ and C/EBPα. Over the last few years, many studies suggested that many additional transcription factors are potential components of this complex network of factors responsible for inducing adipogenic gene expression [23][24][25]. It is likely that additional factors of parallel pathways are induced early and converge on PPARγ at a stage downstream of C/EBPβ and C/EBPδ [21,25].
The evidence that PCYOX1 generates H 2 O 2 , which in turn leads to the oxidative modifications of lipoproteins [4], suggested that it might be involved in the synthesis of PPARγ ligands. Indeed, it has been reported that oxLDL induces PPARγ activation, and that 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), and oxidized phospholipids, which are components of oxLDL, are involved in oxLDL-induced PPARγ activation [15]. However, our findings excluded that PCYOX1 might contribute to the adipogenic process by providing a ligand for PPARγ, as far as oxLDL is concerned.
Importantly, Perilipin-1 is abundantly expressed in mature adipocytes, phosphorylated in a cAMP-dependent manner, and localized to lipid droplet surfaces during differentiation of 3T3-L1 adipocytes into lipid-accumulating mature adipocytes. Plin1-knockout (KO) mice exhibited striking phenotypes [26,27], being lean, with microscopically reduced lipid droplet sizes in adipose tissues, increased glucose tolerance and resistance to diet-induced obesity [28]. In addition, the high lipolytic activities in the WAT of Plin1-KO mice prevent the accumulation of triglycerides, suggesting that Perilipin-1 exerts essential roles in lipid droplet formation and triglyceride metabolism in vivo.
Additionally, isoform 2 of the 1-acyl-sn-glycerol-3-phosphate acyltransferases (Agpat2) is a key enzyme involved in lipid synthesis, which is highly expressed in adipose tissue. It catalyses the acylation of lyso-phospatidic acid (LPA) to produce phosphatidic acid (PA), which will subsequently enter triglyceride or phospholipid synthesis. Cellular studies have supported that Agpat2 is necessary for adipocyte differentiation, suggesting that the absence of WAT in Agpat2 KO was the result of altered adipogenesis [29,30]. Furthermore, decreased levels of LPC in Agpat2 KO could lead to reduced synthesis of LPA, which has been previously shown to be a physiological PPARγ ligand [31].
Recent advances in research have shown the relevance of carboxylesterases to metabolic diseases such as obesity and fatty liver disease [32]. Expression of Ces1 was induced during 3T3-L1 adipocyte differentiation [33], and administration of Ces1 inhibitors to HFD fed mice or db/db mice protected from weight gain reduced plasma lipids, ameliorated liver steatosis, and improved glucose tolerance [34]. Furthermore, in the adipose tissue of obese and type 2 diabetic patients, the activity of Ces1 is elevated, which is consistent with other studies showing that Ces1 expression is higher in adipose tissue from obese patients compared to lean subjects [35,36].
Hormone-sensitive lipase (Lipe) is rate-limiting for diacylglycerol and cholesteryl ester hydrolysis in adipose tissue and essential for complete hormone-stimulated lipolysis [37]. Gene expression profiling in Lipe −/− mice suggests that it is important for modulating adipogenesis and adipose metabolism. In vitro studies showed that Lipe increases during differentiation [38], likely providing ligands for the activation of PPARγ.
PCYOX1 also modulates Car3, which belongs to the family of carbonic anhydrases, enzymes involved in the promotion of fatty acid synthesis in adipocytes and the liver [39]. Car3 is highly abundant in tissues that can store lipids [40,41], and increases in rodents fed Western-type high-fat diets [42], becoming one of the most abundant transcripts in both human [43] and rodent [44] adipose tissues, accounting for up to 2% of the total mRNA. Moreover, Car3 constitutes the most abundant protein in mature adipocytes, comprising up to 24% of the total soluble protein fraction [40].
Overall, the modulation of a plethora of genes involved in adipogenesis supports the hypothesis that PCYOX1 not only acts at early stages of adipogenesis, but also may account for the persistent expression of genes relevant for terminal adipogenic differentiation.
Importantly, PCYOX1 deficiency abrogated, in vitro and in vivo, the expression of CD36, a multifunctional immuno-metabolic receptor that is involved in many physiological and pathological processes [45]. Compared to wild-type mice, CD36 deficient mice with an HFD exhibited reduced adipose tissue inflammation, as evidenced by decreased pro-inflammatory cytokine levels in adipose tissue, and less macrophage and T-cell accumulation in adipose tissue [46,47].
Consistent with the role of PCYOX1 in the regulation of adipose tissue inflammation is the finding that SAA3, one of the members of the acute-phase proteins serum amyloid A family, is also downregulated in Pcyox1 −/− /Apoe −/− mice. Furthermore, SAA3 has been recently utilized for monitoring the adipose inflammatory state, possibly serving as an index of the number of infiltrated macrophages in adipose tissue [48]. In support of this hypothesis, we also found that the macrophage markers Mac2, Cd11b, Cd11c, and Emr1 were all decreased in the adipose tissue of Pcyox1 deficient mice, thus substantiating the hypothesis of a crosstalk between adipocytes and infiltrated macrophages as an important pathological phenomenon leading to adipose tissue inflammation.

Conclusions
In summary, we report here that PCYOX1 is a novel regulator of adipogenesis, which acts upstream of known transcription factors and genes involved in lipogenesis, lipid droplet formation as well as lipid binding, and inflammation. These novel findings expand our knowledge on PCYOX1 biology and functions beyond its role in the metabolism of prenylated proteins [5,6,18,49]. Indeed, PCYOX1 is emerging as a multifunctional protein with a relevant role in atherogenesis by modulating lipid metabolism, inflammation and lipid peroxidation [4,17], in thrombosis by likely regulating circulating coagulant and inflammatory factors [13], and lastly, in adipogenesis. How PCYOX1 can modulate such different processes, which might not be restricted to its enzymatic pro-oxidant activity, is under investigation. Understanding the biology and mechanisms of all functions of this unique enzyme will help to provide additional therapeutic opportunities in addressing such conditions. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12030542/s1, Table S1: Primers used for qRT-PCR; Table S2: Primers purchased from Qiagen; Table S3: List of eEnriched GO terms in the biological process category considering all the proteins modulated by Pcyox1 silencing.  Data Availability Statement: Data collected in the study will be made available using the data repository Zenodo (https://zenodo.org accessed on 22 February 2023) with restricted access upon request to direzione.scientifica@ccfm.it. Proteomic data are available via ProeomeXchange with identifier PXD039943. Any remaining information can be obtained from the corresponding Author upon reasonable request.