Evaluation of Fat Accumulation and Adipokine Production During The

cells

Article Evaluation of Fat Accumulation and Adipokine Production during the Long-Term Adipogenic Diﬀerentiation of Porcine Intramuscular Preadipocytes and Study of the Inﬂuence of Immunobiotics

1,2, 1,2,3, , 1,2,4, , 1,2 Asuka Tada †, AKM Humayun Kober † ‡, Md. Aminul Islam † ‡ , Manami Igata , Michihiro Takagi 1,2, Masahiko Suzuki 1,2, Hisashi Aso 2,5 , Wakako Ikeda-Ohtsubo 1,2, Kazutoyo Yoda 6, Kenji Miyazawa 6, Fang He 6, Hideki Takahashi 7,8 , Julio Villena 1,9,* and Haruki Kitazawa 1,2,* 1 Food and Feed Immunology Group, Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan; [email protected] (A.T.); [email protected] (A.H.K.); [email protected] (M.A.I.); [email protected] (M.I.); [email protected] (M.T.); [email protected] (M.S.); [email protected] (W.I.-O.) 2 Livestock Immunology Unit, International Education and Research Center for Food and Agricultural Immunology (CFAI), Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan; [email protected] 3 Department of Dairy and Poultry Science, Chittagong Veterinary and Animal Sciences University, Chittagong 4225, Bangladesh 4 Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh 5 Cell Biology Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan 6 Technical Research Laboratory, Takanashi Milk Products Co., Ltd., Yokohama Kanagawa 241-0023, Japan; [email protected] (K.Y.); [email protected] (K.M.); [email protected] (F.H.) 7 Laboratory of Plant Pathology, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan; [email protected] 8 Plant Immunology Unit, International Education and Research Center for Food Agricultural Immunology, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan 9 Laboratory of Immunobiotechnology, Reference Centre for Lactobacilli, (CERELA-CONICET), Tucuman 4000, Argentina * Correspondence: [email protected] (J.V.); [email protected] (H.K.); Tel.: +54-381-4310465 (J.V.); +81-22-757-4372 (H.K.) These authors contributed equally to this work. † JSPS Postdoctoral research fellow. ‡ Received: 19 February 2020; Accepted: 15 July 2020; Published: 17 July 2020

Abstract: The degree of fat accumulation and adipokine production are two major indicators of obesity that are correlated with increased adipose tissue mass and chronic inflammatory responses. Adipocytes have been considered effector cells for the inflammatory responses due to their capacity to express Toll-like receptors (TLRs). In this study, we evaluated the degree of fat accumulation and adipokine production in porcine intramuscular preadipocyte (PIP) cells maintained for in vitro differentiation over a long period without or with stimulation of either TNF-α or TLR2-, TLR3-, or TLR4-ligands. The cytosolic fat accumulation was measured by liquid chromatography and the expression of adipokines (CCL2, IL-6, IL-8 and IL-10) were quantified by RT-qPCR and ELISA at several time points (0 to 20 days) of PIP cells differentiation. Long-term adipogenic differentiation (LTAD) induced a progressive fat accumulation in the adipocytes over time. Activation of TLR3 and TLR4

Cells 2020, 9, 1715; doi:10.3390/cells9071715 www.mdpi.com/journal/cells Cells 2020, 9, 1715 2 of 22

resulted in an increased rate of fat accumulation into the adipocytes over the LTAD. The production of CCL2, IL-8 and IL-6 were significantly increased in unstimulated adipocytes during the LTAD, while IL-10 expression remained stable over the studied period. An increasing trend of adiponectin and leptin production was also observed during the LTAD. On the other hand, the stimulation of adipocytes with TLRs agonists or TNF-α resulted in an increasing trend of CCL2, IL-6 and IL-8 production while IL-10 remained stable in all four treatments during the LTAD. We also examined the influences of several immunoregulatory probiotic strains (immunobiotics) on the modulation of the fat accumulation and adipokine production using supernatants of immunobiotic-treated intestinal immune cells and the LTAD of PIP cells. Immunobiotics have shown a strain-specific ability to modulate the fat accumulation and adipokine production, and differentiation of adipocytes. Here, we expanded the utility and potential application of our in vitro PIP cells model by evaluating an LTAD period (20 days) in order to elucidate further insights of chronic inflammatory pathobiology of adipocytes associated with obesity as well as to explore the prospects of immunomodulatory intervention for obesity such as immunobiotics.

Keywords: porcine intramuscular adipocyte; long-term adipogenic differentiation (LTAD); inflammation; adipokines; fat accumulation; immunobiotics

1. Introduction Obesity is a global public health concern having a high socio-economic impact. According to the World Health Organization, globally there are about 1.9 billion overweight adults, 650 million of whom are obese, and worldwide obesity has nearly tripled since 1975 until 2016 [1]. Obesity is characterized by increased adipose tissue mass in the whole body, which is associated with abnormal or excessive fat accumulation in the adipocytes along with subclinical chronic inflammatory responses [2]. However, the consequence of obesity-associated metabolic disorders mostly depends on whether adipose depot is expanded by the increase of adipocyte size (hypertrophy), or by the formation of new adipocytes from precursors (hyperplasia) [3]. The balance of hypertrophic expansion of existing adipocytes and the development of new adipocytes through adipogenesis within the individual has a profound impact on metabolism. Healthy white adipose tissue expansion is achieved by the recruitment and differentiation of adipose precursor cells rather than the increment of fat in mature adipocytes [4]. However, in obesity, the excessive hypertrophied adipocytes have been associated with metabolic abnormalities such as type 2 diabetes, hypertension, cardiovascular diseases, hepatic steatosis, musculoskeletal disorders, metabolic disorders and cancer [5,6]. Adipocyte hypertrophy has also been associated with elevated lipolysis [7], increased inflammatory cytokines secretion [8], and reduced production of anti-inflammatory adipokines such as leptin [8] and adiponectin [9]. On the other hand, adipogenic expansion through preadipocytes differentiation can offset the negative impact of metabolic disorders [3]. Modulation of adipogenic differentiation by the immune system has been considered as an adaptive response that is essential for healthy remodeling of adipose tissue and its normal function [10]. Therefore, a deep understanding of the cellular and molecular changes of fat accumulation and inflammatory responses over the long-term adipogenic differentiation (LTAD) of preadipocytes is of great interest. There are two major classes of adipose tissue in the mammalian body: the white adipose tissue that is specialized to store excess energy as fat, and the brown adipose tissue, which has a remarkable ability to dissipate excess energy from substrate (lipids and glucose) oxidation directly as heat [11]. On the other hand, adipocytes can be found in different anatomical locations (subcutaneous, visceral, intermuscular, intramuscular or bone depots) having different physiological and pathological properties [12]. Adipocytes interjecting themselves between and among skeletal muscle fibers are called intramuscular adipocytes and in meat animals, they are termed overall as a marbling fat [13]. Cells 2020, 9, 1715 3 of 22

There is considerable knowledge on preadipocyte proliferation, differentiation into mature cells, adipocyte ability to conduct lipid metabolism or produce adipokines in normal conditions and in the context of abnormal metabolism. Most of these studies have been performed in visceral adipocytes while less is known about the cellular and molecular changes of intramuscular adipocytes in both physiological and pathological conditions [14]. In order to study the immunobiology and adipogenesis of porcine intramuscular white adipose tissue in vitro, we have previously established a clonal porcine intramuscular preadipocyte (PIP) line from the Musculus longissimus thoracis muscle tissue of Duroc pigs [15], which have bred to produce marbling muscle [16]. We reported that adipogenesis in the PIP cell line can be induced by a combination of octanoate and oleate in the presence of dexamethasone and insulin, which results in the production of functionally mature adipocytes [15]. Therefore, this cell line has been used for the study of adipogenesis. Moreover, we also demonstrated that PIP cells and mature adipocytes derived from them after a short differentiation period are able to respond to acute challenges with cytokines or microbial products by triggering inflammatory responses [17]. Therefore, the PIP cell model is considered relevant for studying adipogenesis and inflammation of adipocytes in the porcine host and its impact on high-quality meat production. On the other hand, attention has focused on the composition of fat within human skeletal muscle as determinants of insulin resistance and involvement in the metabolic syndrome [18,19]. Then, considering the anatomical, physiological and immunological similarities between pigs and humans [14], it is tempting to speculate that the PIP cell line could also be used as a human model. We speculated that the characterization of the immunobiology and adipogenesis of PIP cells under a sustained rather than an acute inflammatory environment could help to position this cell line as a useful laboratory tool for metabolic and immunological studies of adipocytes in the context of obesity, as well as therapeutic alternatives to mitigate its adverse effects. Innate immune receptors, in particular the Toll-like receptors (TLRs) family, are likely to be involved in obesity-associated inflammatory signaling as the over-expression of TLR2 and TLR4 has been observed in the adipose tissue of obese individuals [20,21]. Activation of TLRs stimulate adipocytes to secrete adipokines, which contribute to the development of local and systemic inflammatory responses [22,23]. Expressions of TLRs including TLR-2, -3 and -4, and their ligand responsiveness have been demonstrated in adipocytes of human [24] and mouse [25] origins. In this regard, we previously demonstrated that nine members of TLR family (TLR-1-9) are expressed in PIP cells as well as in differentiated mature porcine adipocytes [17]. In a recent study, we also demonstrated the acute activation of TLR-2, -3 and -4 in porcine adipocytes by their ligand stimulation resulted in overexpression of several cytokines and chemokines including interleukin 8 (IL-8), IL-6 and the C-C motif chemokine ligand 2 (CCL2) [26], which were reported to be linked with obesity-related inflammatory responses in adipocytes [27]. In addition, TNF-α has been implicated as a mediator in the induction of insulin resistance and adipose tissue inflammation [28]. In vitro studies suggested that TNF-α affects glucose homeostasis and fat accumulation in adipocytes, as well as their differentiation (reviewed in ref [29]). We have also demonstrated that TNF-α stimulation significantly affect the biology of PIP cells during in vitro differentiation for a period of four days [17,30]. Immunoregulatory probiotic bacteria or immunobiotics are known to be involved in the beneficial modulation of the host immune responses [31,32]. As adipocytes do not come in direct contact with orally administered immunobiotic bacteria, the cross-talk between immunobiotics and gut immune cells is believed to influence the adipocyte’s immunity [32,33]. In a previous study, our research group demonstrated that probiotic strains including Lactobacillus rhamnosus GG, L. gasseri TMC0356, and L. rhamnosus LA-2 were able to exert immunobiotic effects with significant reduction in the expression of proinflammatory cytokines and chemokines in adipocytes after an acute challenge with TNF-α [17]. Exploring the trend of immunobiotic-mediated changes in adipocytes over a longer period of differentiation and under a sustained inflammation would provide a better understanding of their potential benefits on the progressive fat accumulation and the chronic inflammatory responses of adipocytes. Cells 2020, 9, 1715 4 of 22

The elucidation of the immunological regulators and the cellular and molecular mechanisms involved in the process of adipogenesis are of great interest in order to improve our understanding of the adipose tissue physiology and pathology as well as to develop new strategies to reduce their negative consequences in the obese host. In the present work, we investigated the effects of LTAD on the progressive fat accumulation and adipokines production in the porcine intramuscular adipocytes. We also studied whether immunobiotic strains are able to influence fat accumulation and/or inflammation during LTAD. This work constitutes a step forward to establish an in vitro model that could allow the study of the effects of sustained inflammation on the biology of adipocytes as well as the beneficial effect of immunobiotics in this context.

2. Materials and Methods

2.1. Cells and Culture Conditions The PIP cell line, originally established by our group [15] was used in the present study. The culture condition and adipogenesis induction were performed according to the method described previously [17,33]. Briefly, the PIP cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Paiseley, Scotland, UK) with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 mg/mL streptomycin as a growth medium by using 75 cm2 flask (BD Japan, Tokyo, Japan). The 4-day post-confluent PIP cells were washed with phosphate buffer saline (PBS), and stimulated with PBS containing 0.04% EDTA and kept in a CO2 incubator for 5 min with Trypsin buffer (0.04% EDTA, 0.02% trypsin in PBS). Cells were prepared at a density of 2.5 104/cm2 and were induced to long-term × adipogenesis (20 days) by adding a differentiation medium: DMEM containing 10% FBS, 50 ng/mL insulin (swine, Sigma), 0.25 µM dexamethasone (Sigma), 2 mM octanoate (Wako), 200 µM oleate (Ardorich, Milwaukee, WI, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin. The medium was changed at every second day. The cells and culture supernatants were collected at day 0, 1, 2, 4, 8, 12, 16 and day 20 of differentiation for performing studies. Antigen presenting cells (APCs) were isolated from porcine Peyer’s patches according to the method described in our previous publication [34]. Briefly, porcine Peyer´s patches were cut into small pieces and gently pressed through nylon mesh to prepare single immune cell suspensions. After several washes in complete RPMI medium, residual erythrocytes were lysed in 0.2% NaCl followed by a hypertonic rescue in 1.5% NaCl. Then, immune cells were fractionated by density gradient centrifugation using Lymphocyte Mammal (Cedarlane, Corbyville, ON, Canada) and the mononuclear cell suspension containing a mixed population of T, B and APCs was suspended in complete DMEM supplemented with 10% FCS, 50 U/mL penicillin and 50 µg/mL streptomycin (Nacalai Tseque, Kyoto, Japan). Finally, the APCs including macrophage and dendritic cells were separated by their ability to adhere to a glass plate.

2.2. Triglyceride Assay Cytosolic triglyceride (TG) content of porcine adipocytes was analyzed using the TG assay kit (Wako Chemicals Inc., Virginia, USA) according to the manufacturer’s instruction. Briefly, cells were harvested by using a cell scraper and crushed by ultrasonic fragmentation followed by the treatment with 600 µL of lysis buffer (20 mm Tris, 1 mM EDTA). Then, 100 µL of lysate were used for protein assay, and the remaining volume was used for TG assay. Lipid extraction was performed according to Folch’s method [35]. In brief, 500 µL of Chloroform–methanol (2:1, v/v) were added to the lysate sample. The lysate samples were subjected for ultrasonic fragmentation then centrifuged at 15,000 rpm for 5 min at 4 ◦C. Then cellular protein content was measured by BCA (bicinchoninic acid) protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the TG data was normalized by protein concentration (µg/mg protein). Cells 2020, 9, 1715 5 of 22

2.3. Oil red O and Hematoxylin Staining For quantitative analysis of differentiation and cytosolic fat accumulation in the adipocytes, cultured cells were stained with Oil red O staining kit (GENMED Scientifics INC., Wilmington, DE, USA) according to the protocol described in previous publications [17,36]. In brief, cells were rinsed three times in PBS and then fixed in 10% (v/v) formalin for 30 min, then cells were dissolved in isopropanol for 1 min and then stained with Oil red O (Sigma-Aldrich, Tokyo, Japan) for 30 min at 37 ◦C. Thereafter, cells were washed twice with deionized water and stained with Mayer’s Hematoxylin solution (Merck, Darmstadt, Germany). The stained cells were then washed with deionized water and immersed in aqueous ammonia. Then, the cells were dried to examine and snapped the lipid droplet accumulation under the inverted tissue culture microscope (Olympus IX70). Photographs of stained cells were further analyzed for the size and number of cells by using the ImageJ software (National Institute of Health, Bethesda, MD, USA) according to the method described by Parlee et al. [36].

2.4. Fatty acid Analysis by Gas–Liquid Chromatography (GLC) GLC-based fatty acid analysis of the adipocytes culture-supernatant was performed according to our previous publication [17]. In brief, the processed samples were converted to fatty acid methyl esters and analyzed using a Hitachi G-6000 gas chromatograph equipped with ﬂame ionization detectors and a TC-70 column (0.25 mm 60 m, GL Sciences Inc., Tokyo, Japan) in Helium carrier gas. × The concentrations of three free fatty acids: palmitic acid, stearic acid and oleic acid released by the adipocyte were measured by GLC.

2.5. Induction of Fat Accumulation by SFAs Stimulation Adipocytes were cultured at a density of 2.5 104/cm2 in 6-well plates (BD Falcon, Tokyo, Japan). × The 4-day-old confluent cells maintained in differentiation medium were stimulated by exogenous SFAs: palmitic acid, myristic acid and stearic acid (Sigma-Aldrich, Osaka, Japan) each with four different doses (0 as control, and 250, 25, and 2.5 µmol/L) and kept for the next 4 days. Both SFA treated and untreated cells were washed with PBS three times to remove the oleic acid left in the differentiation medium, and the samples were harvested for TG assay. The degree of fat accumulation into the adipocytes was quantified by TG assay kit as described earlier.

2.6. Induction of Inflammatory Responses during the Long-Term Adipogenic Differentiation (LTAD) Inflammatory responses were induced in differentiated adipocytes by the stimulation with TNF-α and three chemical ligands: Pam3CSK4, poly(I:C) and LPS for the activation of TLR2, TLR3 and TLR4, respectively. Pam3CSK4 is a synthetic triacylated lipopeptide, poly(I:C) is a synthetic double-stranded RNA while LPS is purified from the cell-wall of Gram-negative bacteria. Preadipocytes were cultured at a density of 2.5 104/cm2 in a six-well plate (BD Falcon, Tokyo, Japan) and 4-day post confluent PIP × cells treated to induce their differentiation as described previously. The differentiated adipocytes were stimulated either by TNF-α (2.5 ng/mL), Pam3CSK4 (10 ng/mL), poly(I:C) (0.1 µg/mL) or LPS (0.1 µg/mL) for 12 h and kept for 20 days with a regular changing of culture media. In order to determine the dose of each stimulant, CCL2 expression was verified. The concentration of TLR ligands were chosen according to their ability to induce 5-fold increase of CCL2 expression. TNF-α concentration was chosen according to our previous study [17]. TNF-α (2.5 ng/mL) stimulation resulted in a 10-fold increase in the expression of CCL2 when compared to control. The mRNA and protein of four adipokines: CCL2, IL-6, IL-8, and IL-10 were determined by RT-qPCR and ELISA, respectively, as described below in Sections 2.9 and 2.10. Cells 2020, 9, 1715 6 of 22

2.7. Lactic Acid Bacteria Strains Bacterial isolates were provided by the Takanashi Milk Product Company (Yokohama, Japan). Seven strains of lactic acid bacteria (LAB) were used in this study: L. gasseri TMC0356, L. rhamnosus GG, L. rhamnosus LA-2, L. paracasei TMC0409, Streptococcus thermophilus TMC1543, Bifidobacterium bifidum TMC3108 and B. bifidum TMC3115. Lactobacilli and bifidobacteria strains were grown in MRS (deMan-Rogosa-Sharp) medium (Difco, Detroit, MI, USA) for 16 h at 37 ◦C. S. thermophilus was grown in Elliker medium (Difco, Detroit, MI, USA) for 16 h at 37 ◦C. The cultured bacteria were washed with PBS and heated at 60 ◦C for 30 min. After washing twice with PBS, resuspended in DMEM (10% FCS, 1% streptomycin/penicillin) and adjusted 2.5 109 cells/mL. × 2.8. Effect of Immunobiotic Stimulations on the Fat Accumulation and Adipokines Production in Adipocytes For the assessment of immunobiotic effect in terms of their potential to regulate fat accumulation and inflammatory responses of adipocytes, we accomplished two types of in vitro experiments. First, mononuclear cells isolated from porcine Peyer’s patches were stimulated with the different LAB strains. The immunocompetent cells were seeded in a 6-well cell culture plate (BD Falcon, 1 106 cells/well) and stimulated with LAB (5 107 cells/well) in 5% CO humidified atmosphere × × 2 at 37 ◦C, for 24 h, in a complete RPMI 1640 medium (Sigma) supplemented with 2% fetal bovine serum (FCS). After stimulation, cell free supernatant (CFS) was collected for further experiments. Twenty-day-old differentiated mature porcine adipocytes were pre-stimulated for 48 h with CFS. In a second set of experiments, adipocytes (1 106 cells/well) were directly stimulated with LAB strains × (5 107 cells/well) for 48 h. × 2.9. Quantitative Real Time PCR Total RNA was isolated from treated and untreated cells using TRIzol reagent (Invitrogen) according to our previous study [17]. The cDNAs were synthesized using a Quantitect reverse transcription (RT) kit (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. The RT-qPCR was performed with an Applied Biosystems Real-time PCR System 7300 (Applied Biosystems, Warrington, UK), using the Platinum SYBR Green qPCR SuperMix-UDG (uracil-DNA glycosylase) with ROX (6-carboxyl-X-rhodamine) (Invitrogen). The sequences of primers for CCL2, IL-6, IL-8, IL-10 and β-actin used for this study are available in our previous publications [17,34]. The PCR thermal cycling conditions were 5 min at 50 ◦C, followed by 5 min at 95 ◦C, and then 40 cycles of 15 s at 95 ◦C, 30 s at 60 ◦C, and 30 s at 72 ◦C. The reaction mixtures contained 2.5 µL of sample cDNA and 7.5 µL of master mix including the sense and antisense primers. Each reaction ran in duplicate and the expression of β-actin in each sample was used as a reference to normalize differences between samples and to calculate the normalized fold expression of target genes.

2.10. ELISA Assay The protein concentration of IL-6, IL-8, IL-10, CCL2, leptin and adiponectin in the cell-free supernatant (100 µL) of adipocytes culture were measured by using commercially available ELISA kits: porcine IL-6, IL-8, and IL-10 ELISA kits (R&D Systems, Minneapolis, MN, USA); porcine CCL2 ELISA kit (Kingﬁsher Biotech, Inc. ST. Paul, MN, USA); pig leptin ELISA Kit (Wuhan Huamei Biotech Co., Ltd., Wuhan, China); and pig adiponectin ELISA Kit (Wuhan Huamei Biotech Co., Ltd., Wuhan, China). Estimation of the concentration of each protein was performed according to the manufacturers’ instructions.

2.11. Statistical Analysis All results were expressed as mean SD and are the average of three independent experiments. ± Relative indices were calculated as the ratio of fat deposition in adipocyte cells, and the values are normalized by common logarithmic transformation and confirmed as approximate values included Cells 2020, 9, 1715 7 of 22 significantly into normal distribution by the Kolmogorov–Smirnov test. Then, they were adjusted similarly to the mean of control groups. One-way analysis of variance (ANOVA) followed by Fisher’s least significant difference test was performed by the GLM procedure of the SAS program (Version 9.1) for the pairwise mean comparisons. Mean comparison of mRNA expression and protein concentrations between control (day 0) and several time points were performed by a repeated measures ANOVA procedure by using the PROC GLM model implemented in the SAS program considering the ‘day 0 measurement’ as a fixed factor. Cells 2020, 9, x 7 of 22 3. Results 3. Results 3.1. Fat Accumulation and Fatty Acid Production in Porcine Adipocytes during LTAD 3.1. Fat Accumulation and Fatty Acid Production in Porcine Adipocytes during LTAD In order to investigate the effect of LTAD on the fat accumulation of porcine adipocytes, quantitative analysisIn oforder TG to was investigate performed. the Continuouseffect of LTAD fat accumulationon the fat accumulation was observed of porcine over the adipocytes, 20 days of diquantitativefferentiation analysis (Figure of1A). TG Thewas performed. cytosolic TG Continuous content wasfat accumulation remarkably was increased observed when over PIP the cells20 (beforedays of di differentiationfferentiation) were (Figure compared 1A). The with cytosolic adipocytes. TG content Fat content was remarkably was increased increased about when 10-fold PIP after 20cells days (before of culture differentiation) with the di werefferentiation compared medium. with adipocytes. Oil red O Fat staining content also was confirmed increased about the progressive 10-fold after 20 days of culture with the differentiation medium. Oil red O staining also confirmed the accumulation of fat in the cytoplasm of adipocytes (Figure1B). The lipid droplets within the adipocytes progressive accumulation of fat in the cytoplasm of adipocytes (Figure 1B). The lipid droplets within were increased in number and size in a time dependent manner (Figure1B). the adipocytes were increased in number and size in a time dependent manner (Figure 1B).

FigureFigure 1. 1.Long-term Long-term adipogenic adipogenic differentiation differentiation mediated mediated fat accumulation fat accumulation into the porcine into the intramuscular porcine preadipocyteintramuscular (PIP) preadipocytein vitro culture. (PIP) in The vitro PIP culture cells (2.5. The10 PIP4/cm cells2) were (2.5 kept× 104/cmin vitro2) wereculture kept for in 20vitro days × withculture differentiation for 20 days medium,with differentiation and cells were medium, harvested and cells at different were harvested time points at different to estimate time the points degree to of cytosolicestimate fat the accumulation degree of cytosolic by using fat a accumulationtriglyceride (TG) by assayusing kita triglyceride (A). Data shown (TG) assay are the kit mean (A). DataSD of ± threeshown independent are the mean experiments ± SD of three performed independent in triplicates. experiments Different performed letters in triplicates. (a, b, c, and Different d) labeled letters upon the(a, timeb, c, pointsand d) indicatelabeled upon significant the time differences points indicate (p < 0.05), significant while differences the same letter (p < indicates0.05), while no the significant same differenceletter indicate betweens no thesignificant contrast difference pairs. Oil between red O staining the contrast of adipocytes pairs. Oil demonstrated red O staining the of fat adipocytes droplets red colordemonstrated while the nucleusthe fat droplets takes a bluered color whenwhile observedthe nucleus under takes microscope a blue color (B ).when observed under microscope (B). 3.2. Size and Number of Adipocyte Counts during LTAD 3.2. Size and Number of Adipocyte Counts during LTAD The size of the adipocytes was also progressively increased over the longer period of diﬀerentiation (FigureThe2A). size The averageof the adipocytes number of livewas adipocytes also progressively (as estimated incre inased a stained over areathe longer of 10,000 periodµm2 ) of was signiﬁcantlydifferentiation decreased (Figure over2A). The time average (Figure number2B). of live adipocytes (as estimated in a stained area of 10,000 μm2) was significantly decreased over time (Figure 2B).

Figure 2. Measurement of proliferation and size of porcine adipocyte during long-term adipogenic differentiation. (A) The average area (size) of the adipocytes. (B) Average count of Oil red O stained (live) adipocytes. Images of stained adipocytes were analyzed for size and number of cells using

Cells 2020, 9, x 7 of 22

3. Results

3.1. Fat Accumulation and Fatty Acid Production in Porcine Adipocytes during LTAD In order to investigate the effect of LTAD on the fat accumulation of porcine adipocytes, quantitative analysis of TG was performed. Continuous fat accumulation was observed over the 20 days of differentiation (Figure 1A). The cytosolic TG content was remarkably increased when PIP cells (before differentiation) were compared with adipocytes. Fat content was increased about 10-fold after 20 days of culture with the differentiation medium. Oil red O staining also confirmed the progressive accumulation of fat in the cytoplasm of adipocytes (Figure 1B). The lipid droplets within the adipocytes were increased in number and size in a time dependent manner (Figure 1B).

Figure 1. Long-term adipogenic differentiation mediated fat accumulation into the porcine intramuscular preadipocyte (PIP) in vitro culture. The PIP cells (2.5 × 104/cm2) were kept in vitro culture for 20 days with differentiation medium, and cells were harvested at different time points to estimate the degree of cytosolic fat accumulation by using a triglyceride (TG) assay kit (A). Data shown are the mean ± SD of three independent experiments performed in triplicates. Different letters (a, b, c, and d) labeled upon the time points indicate significant differences (p < 0.05), while the same letter indicates no significant difference between the contrast pairs. Oil red O staining of adipocytes demonstrated the fat droplets red color while the nucleus takes a blue color when observed under microscope (B).

3.2. Size and Number of Adipocyte Counts during LTAD The size of the adipocytes was also progressively increased over the longer period of Cells 2020, 9, 1715 8 of 22 differentiation (Figure 2A). The average number of live adipocytes (as estimated in a stained area of 10,000 μm2) was significantly decreased over time (Figure 2B).

FigureFigure 2. 2. MeasurementMeasurement of of proliferation proliferation and and size size of of porcine porcine adipocyte adipocyte during during long long-term-term adipogenic adipogenic Cells 2020, 9, x 8 of 22 differentiationdifferentiation.. (A (A) )The The average average area area (size) (size) of of the the adipocytes. adipocytes. (B (B) )Average Average count count of of Oil Oil red red O O stained stained (live)(live) adipocytes. Images Images of of stained stained adipocytes adipocytes were were analyzed analyzed for sizefor sizeand numberand number of cells of using cells ImageJusing ImageJsoftware. software. Data shown Data shownare the meanare the meanSD of three± SD independentof three independent experiments experiments performed performed in triplicates. in ± triplicates.The asterisks The (**) asterisks indicated (**) statistical indicated di ffstatisticalerences of differences adipocyte numberof adipocyte and size number when and compared size when with comparedDay 4 at the with significance Day 4 at the level significan of p < 0.01.ce level of p < 0.01.

3.3.3.3. Release Release of of Free Free Fatty Fatty Acids Acids from from Porcine Porcine Adipocytes Adipocytes during during LTAD LTAD TheThe release release of of free free fatty fatty acids acids by by adipocytes adipocytes during during LTAD LTAD was was also also investigated investigated (Figure (Figure 33AA).). SinceSince the the fat fat accumulation accumulation reached reached its its relative relative stationary stationary phase phase at at day day 8 8and and continued continued until until day day 20, 20, thethe FFA FFA contents contents of of adipocytes adipocytes were were measured measured at at day day 8 8 and and 20 20 of of differentiation. diﬀerentiation. There There were were no no remarkableremarkable differences diﬀerences of palmitic acid (C 16:0),16:0), stearicstearic acidacid (C(C 18:0)18:0) andand oleic oleic acid acid (C (C 18:1) 18:1) contents contents in inthe the culture culture supernatant supernatant measured measured between between days days 8 and 8 and 20 (Figure20 (Figure3A). 3 However,A). However, the stearic the stearic acid: acid: oleic oleicacid acid ratio ratio was was changed changed by the by LTAD.the LTAD.

Figure 3. Long-term adipogenic differentiation (LTAD) mediated release of free fatty acids (FFA) from Figureadipocytes, 3. Long and-term the adipogenic influence of differentiation saturated fatty (LTAD acids (SFA)) mediated in fat release accumulation. of free fatty The acids concentrations (FFA) from of adipocytes,three FFA: Palmiticand the influence acid, Stearic of saturated acid and Oleic fatty acidacids were (SFA measured) in fat accumulation. in the differentiated The concentrations adipocyte by ofgas–liquid three FFA: chromatography Palmitic acid, Stearic (A). The acid 4-day-old and Oleic confluent acid were adipocytes measured (2.5 in the10 differentiated4 /cm2) were challengedadipocyte × bywith gas three–liquid di ff chromatographyerent concentrations (A). (asThe of 4250,-day 25,-old and confluent 2.5 µmol adipocytes/l) of each of (2.5 three × 10 SFA4 /cm for2) 4 were days challengedfollowed by with quantifying three different the fat concentrations accumulation by (as a of TG 250, assay 25, kit and (B 2.5). The µmol/l) asterisk of each (*) indicated of three statisticalSFA for 4di daysfferences followed of fat by accumulation quantifying the of SFAfat accumulation treated adipocyte by a TG when assay compared kit (B). The to untreated asterisk (*) control indicate withd statisticalsignificance differences level of p of< 0.05. fat accumulation of SFA treated adipocyte when compared to untreated control with significance level of p < 0.05. We also evaluated the ability of FFA to modify fat accumulation in adipocytes. For this purpose, adipocytesWe also were evaluated stimulated the ability with diofff FFAerent to concentrations modify fat accumulation of palmitic acid,in adipocytes. myristic acidFor this or stearic purpose, acid adipocytes(0.1, 0.01 or were 0.001 sµtimulatedg/mL) and with the fat different accumulation concentrations was evaluated of palmitic (Figure acid,3B). Palmitic myristic acid acid stimulation or stearic acidsignificantly (0.1, 0.01 increased or 0.001 µg/m the fatL) accumulation and the fat accumulation in adipocytes was at a evaluated concentration (Figure lower 3B than). Palmitic 0.01 µ g acid/mL stimulationwhile myristic significantly acid significantly increased reduced the fat fataccumulation accumulation in whenadipocytes used atat aa concentrationconcentration oflower 0.1 µ thang/mL 0.01(Figure µg/m3B).L while However, myristic no e acidffect significantly was observed reduced for any fat dose accumulation for the stearic when acid used (Figure at a3 concentrationB). of 0.1 µg/mL (Figure 3B). However, no effect was observed for any dose for the stearic acid (Figure 3B).

3.4. Adipokine Expression Dynamics in Adipocytes during LTAD To examine the effects of LTAD in production of immune mediators, mRNA expression and protein concentration of four adipokines IL-6, IL-8, IL-10 and CCL2 were examined during the 20 days of adipogenesis (Figure 4). The mRNA expression of CCL2 increased progressively during LTDA while the expression of IL-6 and IL-8 were significantly increased only at the end of the studied period. On the other hand, no significant changes were observed for IL-10 mRNA (Figure 4A). The protein concentration of the adipokines evaluated changed in a similar fashion as observed for the mRNA levels (Figure 4B). Furthermore, the secretion of leptin and adiponectin by the adipocytes was measured by ELISA during the 20 days of differentiation (Figure 5). Significantly augmented levels were observed at days 4 and 16 for adiponectin, while leptin production was significantly increased after day 12 of adipogenesis.

Cells 2020, 9, 1715 9 of 22

3.4. Adipokine Expression Dynamics in Adipocytes during LTAD To examine the effects of LTAD in production of immune mediators, mRNA expression and protein concentration of four adipokines IL-6, IL-8, IL-10 and CCL2 were examined during the 20 days of adipogenesis (Figure4). The mRNA expression of CCL2 increased progressively during LTDA while the expression of IL-6 and IL-8 were significantly increased only at the end of the studied period. On the other hand, no significant changes were observed for IL-10 mRNA (Figure4A). The protein concentration of the adipokines evaluated changed in a similar fashion as observed for the mRNA levels (Figure4B). Furthermore, the secretion of leptin and adiponectin by the adipocytes was measured by ELISA during the 20 days of differentiation (Figure5). Significantly augmented levels were observed at days 4 and 16 forCells adiponectin, 2020, 9, x while leptin production was significantly increased after day 12 of adipogenesis.9 of 22

FigureFigure 4. 4.Long-term Long-term adipogenic adipogenic didifferentiationfferentiation mediated expression expression dynamics dynamics of ofadipokines adipokines in inthe the adipocytesadipocytes without without any any exogenous exogenous inflammatory inflammatory triggers. triggers. The The mRNA mRNA expression expression (A ()A and) and protein protein concentrationconcentration (B ()B of) of CCL2, CCL2, IL-6, IL-6, IL-8 IL-8 and and IL-10 IL-10 in in adipocytes adipocytes over over thethe 2020 daysdays ofof cultureculture asas measuredmeasured by RT-qPCRby RT-q andPCR ELISA, and ELISA, respectively. respectively. Data shown Data areshown the mean are the SDmean of three ± SD independent of three independent experiments ± performedexperiments in triplicates. performed The in triplicates. asterisks: The (*) and asterisk (**) indicateds: (*) and ( statistical**) indicate didff erencesstatistical when differences compared when with daycompared 0 at the significancewith day 0 at levels the significan of p < 0.05ce levels and p of< p0.01, < 0.05 respectively. and p < 0.01, respectively.

Cells 2020, 9, 1715 10 of 22 Cells 2020, 9, x 10 of 22

Cells 2020, 9, x 10 of 22

FigureFigure 5. 5. Long-termLong-term adipogenic adipogenic di differentiationfferentiation mediatedmediated secretion secretion of of adiponectin adiponectin and and leptin leptin in in adipocytes.adipocytes.Figure 5. TheL ongThe ELISA-based- termELISA adipogenic-based quantification quantification differentiation of of adiponectin adiponectin mediated and secretion and leptin leptin of concentration concentration adiponectin in andin adipocytes adipocytes leptin in as as measuredmeasuredadipocytes. over over The the ELISA the20 days 20-based daysof culture. quantification of culture. Data shown Dataof adiponectin are shown the mean are and the leptinSD mean of concentration three ± independentSD of threein adipocytes experimentsindependent as ± performedexperimentsmeasured in over triplicates.performed the 20 The daysin triplicates. asterisk of culture. (*) The indicated Data asterisk shown the (*) statistical areindicate the dmean di thefferences statistical± SD ofof protein threedifferences independent concentration of protein experiments performed in triplicates. The asterisk (*) indicated the statistical differences of protein whenconcentration compared when with daycompared 0, at the with significance day 0, at levelthe significan of p < 0.05.ce level of p < 0.05. concentration when compared with day 0, at the significance level of p < 0.05. 3.5.3.5. E Effectffect of of TNF- TNFα-αand and TLR TLR Ligands Ligands on on Fat Fat Accumulation Accumulation in in Adipocytes Adipocytes during during LTAD LTAD 3.5. Effect of TNF-α and TLR Ligands on Fat Accumulation in Adipocytes during LTAD α InIn order order to to evaluate evaluate the the e ff effectect of of TNF- TNF-αand and TLR TLR ligands ligands on on the the fat fat accumulation accumulation of of porcine porcine In order to evaluate the effect of TNF-α and TLR ligands on the fat accumulation of porcine adipocytes,adipocytes, the the cytosolic cytosolic TGTG contentcontent waswas estimated.estimated. Progressive Progressive fat fat accumulation accumulation was was observed observed in adipocytes,α the cytosolic TG content was estimated. Progressive fat accumulation was observed in inTNF TNF--α andand TLR TLR-ligands-ligands st stimulatedimulated adipocytes adipocytes as as well well as in unstimulated unstimulated cells cells (Figure (Figure6 ).6). TNF-α and TLR-ligands stimulated adipocytes as well as in unstimulated cells (Figure 6). Interestingly,Interestingly, fat fat deposition deposition rate rate was was accelerated accelerated in in adipocytes adipocytes challenged challenged with with TLR3 TLR3 or or TLR4 TLR4 ligands ligands sinceInterestingly, significantly fat deposition higher levels rate was of TG accelerated were found in adipocytes from day challenged 8 until the with end TLR3 of the or studiedTLR4 ligands period sincesince significantlysignificantly higher levelslevels ofof TG TG were were found found from from day day 8 until8 until the the end end of ofthe the studie studied periodd period when compared to controls. On the other hand, TNF-α and TLR2 ligand stimulation did not result in whenwhen comparedcompared to controls. On On the the other other hand, hand, TNF TNF-α- αand and TLR2 TLR2 ligand ligand stimulation stimulation did did not not result result in in significant changes in fat deposition as compared to control adipocytes at the end of the study period significantsignificant changes inin fatfat depositiondeposition as as compared compared to to control control adipocytes adipocytes at atthe the end end of theof the study study period period (Figure6). ((FigureFigure 66).).

FigureFigure 6. 6.Regulation Regulation of of fat fat accumulationaccumulation in adipocytes by by the the stimulation stimulation of of immune immune receptors. receptors. AdipocytesFigureAdipocytes 6. Regulation were were stimulatedstimulated of fat either accumulation by by TNF TNF-- α in α(2.5 adipocytes(2.5 ng ng/m/LmL)) or by Pam3csk4 or the Pam3csk4 stimulation (10 ng/m (10 ofngL) / immuneormL) poly(I:C) or poly(I:C) receptors. (0.1 (0.1Adipocytesμg/mµg/LmL)) or or LPSwere LPS (0.1 stimulated (0.1 μg/mµg/LmL)) foreither for 12 12 hby h and TNF and kept- keptα (2.5 for for ng 20 20 /m days daysL) or with withPam3csk4 regular regular (10 changing changing ng/mL culture) or culture poly(I:C) media. media. (0.1 Pam3CSK4,μPam3g/mLCSK) or4, poly(I:C), LPSpoly(I:C (0.1 and)μ, andg/m LPS LLPS)represent for represent 12 h the and the ligands kept ligands for TLR2,for 20 daysTLR2, TLR3 with TLR3 and regular TLR4,and TLR4, respectively. changing respectively. culture Data shownData media. arePam3shown the averageCSK are4, the poly(I:C averageSD of), 3 and± independent SD LPS of 3 representindependent experiments the experimentsligands performed for performedTLR2, in triplicates. TLR3 in andtriplicates. The TLR4, asterisk Therespectively. asterisk (*) indicated (* Data) ± theshownindicate statistical ared the the di ﬀ statisticalaverageerence between ±difference SD of each3 independent between treated each group experiments treated cells and group performed the cells untreated and in thetriplicates. control untreated of correspondingThe control asterisk of (*) timeindicatecorresponding points,d the at the statisticaltime signiﬁcance points difference, at levelsthe significance ofbetweenp < 0.05. levels each treatedof p < 0.05. group cells and the untreated control of corresponding time points, at the significance levels of p < 0.05.

Cells 2020, 9, 1715 11 of 22

Cells 2020, 9, x 11 of 22 3.6. Effect of TNF-α on the Adipokine Production in Adipocytes during LTAD 3.6. Effect of TNF-α on the Adipokine Production in Adipocytes during LTAD We next aimed to evaluate whether the stimulation with TNF-α was able to change the expressions We next aimed to evaluate whether the stimulation with TNF-α was able to change the of four adipokines in porcine adipocytes during LTAD. As observed in non-stimulated control expressions of four adipokines in porcine adipocytes during LTAD. As observed in non-stimulated adipocytes, TNF-α-challenged adipocytes showed a progressive augment in CCL2 expression and control adipocytes, TNF-α-challenged adipocytes showed a progressive augment in CCL2 expression protein concentration (Figure7). However, adipocytes under TNF- α stimulation had significantly and protein concentration (Figure 7). However, adipocytes under TNF-α stimulation had higher levels of CCL2 mRNA (Figure7A) and protein (Figure7B) when compared to unchallenged significantly higher levels of CCL2 mRNA (Figure 7A) and protein (Figure 7B) when compared to controls. In fact, CCL2 expression in controls was up-regulated four-fold at day 2 while in TNF-α unchallenged controls. In fact, CCL2 expression in controls was up-regulated four-fold at day 2 while stimulated adipocytes this adipokine increased more than 20-fold when compared to basal levels. in TNF-α stimulated adipocytes this adipokine increased more than 20-fold when compared to basal Similarly, both mRNA and protein levels of IL-8 and IL-6 were increased from day 8 (Figure7) and levels. Similarly, both mRNA and protein levels of IL-8 and IL-6 were increased from day 8 (Figure their values were significantly higher when compared to unchallenged adipocytes. Interestingly, 7) and their values were significantly higher when compared to unchallenged adipocytes. mRNA levels for IL-10 were significantly reduced in TNF-α-challenged adipocytes already from day 8 Interestingly, mRNA levels for IL-10 were significantly reduced in TNF-α-challenged adipocytes onwards. In addition, a slight but not significant decrease in IL-10 protein concentration was observed already from day 8 onwards. In addition, a slight but not significant decrease in IL-10 protein after 20 days of LTAD (Figure7B). concentration was observed after 20 days of LTAD (Figure 7B).

FigureFigure 7. TNFTNF--αα mediatedmediated adipokine adipokine production production in in adipocytes. The The mRNA mRNA expressions expressions ( (AA)) and and proteinprotein concentration ((BB)) ofof CCL2, CCL2, IL-6, IL-6, IL-8, IL-8, IL-10 IL-10 in in the the adipocytes adipocytes at diatff erentdifferent time time points points of 20 of days 20 daysof di ffoferentiation. differentiation. Data Data shown shown are are the the mean mean ±SD SD of of three three independentindependent experimentsexperiments performed ± inin triplicates. triplicates. The The ‘* ‘*’’ indicated indicated statistical statistical difference differencess when when compared compared with with day day 0 0,, and and ‘ ‘†’’ indicated † statisticalstatistical di differencesfferences when when compared compared with with control control group group at the sameat the time same point, time both point, at the both significance at the significancelevel of p < 0.05.level of p < 0.05.

3.7.3.7. Effects Effects of of TLR2 TLR2 on on the the Adipokine Adipokine Production Production in in Adipocytes during LTAD InIn order order to to investigate investigate the the effects effects of of TLR2 TLR2 activation activation during during LTAD, LTAD, adipocytes were were challenged challenged withwith Pam3CSK4 Pam3CSK4 or LPS, LPS, respectively respectively ( (FigureFigure 88).). TLR2 activation resulted inin a fluctuantfluctuant but significantsignificant increaseincrease of of CCL2, CCL2, IL IL-8-8 and IL IL-6-6 mRNA expression and protein concentrations during during the the period of adipogenesisadipogenesis studied (Figure(Figure8 8AA,B).,B). TLR2-activated TLR2-activated adipocytes adipocytes also also showed showed a significanta significant reduction reduction of ofIL-10 IL-10 mRNA mRNA (Figure (Figure8A) 8A and) and protein protein (Figure (Figure8B) 8 levelsB) levels from from day day 12 onward. 12 onward.

Figure 8. TLR2 mediated adipokine production in adipocytes. The figure displayed the TLR2 mediated mRNA expression (A) and protein concentration (B) of CCL2, IL-6, IL-8, IL-10 in the adipocytes at different time points of 20 days of adipogenesis. Data shown are the mean ± SD of three independent experiments performed in triplicates. The ‘*’ indicated statistical differences when compared with day 0, and ‘†’ indicated statistical differences when compared with control group at the same time point, both at the significance level of p < 0.05.

Cells 2020, 9, x 11 of 22

3.6. Effect of TNF-α on the Adipokine Production in Adipocytes during LTAD We next aimed to evaluate whether the stimulation with TNF-α was able to change the expressions of four adipokines in porcine adipocytes during LTAD. As observed in non-stimulated control adipocytes, TNF-α-challenged adipocytes showed a progressive augment in CCL2 expression and protein concentration (Figure 7). However, adipocytes under TNF-α stimulation had significantly higher levels of CCL2 mRNA (Figure 7A) and protein (Figure 7B) when compared to unchallenged controls. In fact, CCL2 expression in controls was up-regulated four-fold at day 2 while in TNF-α stimulated adipocytes this adipokine increased more than 20-fold when compared to basal levels. Similarly, both mRNA and protein levels of IL-8 and IL-6 were increased from day 8 (Figure 7) and their values were significantly higher when compared to unchallenged adipocytes. Interestingly, mRNA levels for IL-10 were significantly reduced in TNF-α-challenged adipocytes already from day 8 onwards. In addition, a slight but not significant decrease in IL-10 protein concentration was observed after 20 days of LTAD (Figure 7B).

Figure 7. TNF-α mediated adipokine production in adipocytes. The mRNA expressions (A) and protein concentration (B) of CCL2, IL-6, IL-8, IL-10 in the adipocytes at different time points of 20 days of differentiation. Data shown are the mean ± SD of three independent experiments performed in triplicates. The ‘*’ indicated statistical differences when compared with day 0, and ‘†’ indicated statistical differences when compared with control group at the same time point, both at the significance level of p < 0.05.

3.7. Effects of TLR2 on the Adipokine Production in Adipocytes during LTAD In order to investigate the effects of TLR2 activation during LTAD, adipocytes were challenged with Pam3CSK4 or LPS, respectively (Figure 8). TLR2 activation resulted in a fluctuant but significant increase of CCL2, IL-8 and IL-6 mRNA expression and protein concentrations during the period of Cells 2020, 9, 1715 12 of 22 adipogenesis studied (Figure 8A,B). TLR2-activated adipocytes also showed a significant reduction of IL-10 mRNA (Figure 8A) and protein (Figure 8B) levels from day 12 onward.

Figure 8. 8.TLR2 TLR2 mediated mediated adipokine adipokine production production in adipocytes. in adipocytes. The figure The displayedfigure displayed the TLR2 the mediated TLR2 mRNAmediated expression mRNA expression (A) and protein (A) and concentration protein concentration (B) of CCL2, (B IL-6,) of IL-8, CCL2, IL-10 IL-6, in IL the-8, adipocytes IL-10 in the at diadipocytesfferent time at different points of time 20 days points of adipogenesis. of 20 days of adipogenesis. Data shown are Data the shown mean areSD the of mean three independent± SD of three ± experimentsindependent performed experiments in triplicates. performed The in ‘*’ triplicates. indicated The statistical ‘*’ indicate differencesd statistical when compared differences with when day 0,compared and ‘ ’ indicated with day statistical 0, and ‘†’ diindicatefferencesd statistical when compared differences with when control compared group at with the samecontrol time group point, at † the same time point, both at thep significance level of p < 0.05. Cells 20both20, 9, at x the significance level of < 0.05. 12 of 22 3.8. Effects of TLR4 on the Adipokine Production in Adipocytes during LTAD 3.8. Effects of TLR4 on the Adipokine Production in Adipocytes during LTAD In order to investigate the effects of TLR4 activation during LTAD, adipocytes were challenged with In order to investigate the effects of TLR4 activation during LTAD, adipocytes were challenged Pam3CSK4 or LPS, respectively (Figure9). TLR4 activation also resulted in a significant up-regulation with Pam3CSK4 or LPS, respectively (Figure 9). TLR4 activation also resulted in a significant up- of CCL2, IL-6 and IL-8 (Figure9A,B). Particularly, the TLR4-activated adipocytes showed a sustained regulation of CCL2, IL-6 and IL-8 (Figure 9A,B). Particularly, the TLR4-activated adipocytes showed increase in CCL2 mRNA (Figure9A) and protein concentration (Figure9B) from day 1 of LTAD, a sustained increase in CCL2 mRNA (Figure 9A) and protein concentration (Figure 9B) from day 1 of while IL-8 and IL-6 were up-regulated from days 8 and 12, respectively (Figure9A,B). On the contrary, LTAD, while IL-8 and IL-6 were up-regulated from days 8 and 12, respectively (Figure 9A,B). On the both mRNA expression (Figure9A) and protein concentration (Figure9B) of IL-10 were significantly contrary, both mRNA expression (Figure 9A) and protein concentration (Figure 9B) of IL-10 were reduced after TLR4 activation. significantly reduced after TLR4 activation.

Figure 9. 9. TLR4TLR4 mediated mediated adipokine adipokine production production in adipocytes. in adipocytes. The figure The figure displayed displayed the TLR4 the mediated TLR4 mediatedmRNA expression mRNA expression (A) and protein (A) and concentration protein concentration (B) of CCL2, (B) IL-6, of CCL2, IL-8, IL-10 IL-6, in IL- the8, IL adipocytes-10 in the at adipocytesdifferent time at different points of time 20 days points of adipogenesis.of 20 days of adipogenesis. Data shown Data are the shown mean are SDthe ofmean three ± SD independent of three ± independentexperiments performed experiments in triplicates. performed The in triplicates. ‘*’ indicated The statistical ‘*’ indicate differencesd statistical when differencescomparedwith when day compared0, and ‘ ’ indicated with day statistical0, and ‘†’ indicate differencesd statistical when compared differences with when control compared group with at the control same timegroup point, at † theboth same at the time significance point, both level at the of psignificance< 0.05. level of p < 0.05.

3.9. Effect Effect of of TLR3 TLR3 on on the the Adipokine Adipokine Production Production in in Adipocytes Adipocytes during during LTAD LTAD We also investigated the the effect effect of of the the a activationctivation of of TLR3 TLR3 during during LTAD. LTAD. The The stimulation stimulation of of adipocytes with the the TLR3 TLR3 ligand ligand poly(I:C) poly(I:C) significantly significantly up up-regulated-regulated the the mRNA mRNA expression expression and and the the protein concentration ofof the chemokine CCL2 when compared with before treatment timetime pointpoint (day(day 0) as0) wellas well as untreatedas untreated unchallenged unchallenged control control cells atcells corresponding at corresponding time pointstime points (Figure ( Figure10A,B). 10 InA addition,,B). In poly(I:C)addition, inducedpoly(I:C) an induced increase an in increase IL-8 mRNA in IL levels-8 mRNA (Figure levels 10 A)(Figure and protein 10A) and concentration protein concentration (Figure 10B). The(Figure mRNA 10B). expression The mRNA of expression IL-6 was increased of IL-6 was at 12increased h post poly(I:C)at 12 h post treated poly(I:C) adipocytes treated (Figureadipocytes 10A), while(Figure no 10 significantA), while no variations significant in IL-6variations protein in level IL-6 wasprotein observed level was when observed compared when to compared the basal level to (Figurethe basal 10 levelB). The (Figure expression 10B). The of IL-10 expression was significantly of IL-10 was reduced significantly from dayreduced 8 onwards from day when 8 onwards compared when compared to before treatment (day 0), as well as corresponding control at the same time point (Figure 10A), while IL-10 protein concentration showed no differences when compared with before treatment time point (day 0) but significant difference as compared to the unchallenged control cells at day 8, 12, 16 and 20 (Figure 10B).

Cells 2020, 9, 1715 13 of 22 to before treatment (day 0), as well as corresponding control at the same time point (Figure 10A), while IL-10 protein concentration showed no differences when compared with before treatment time point (day 0) but significant difference as compared to the unchallenged control cells at day 8, 12, 16 andCells 202020 (Figure, 9, x 10B). 13 of 22

FigureFigure 10. 10.TLR3 TLR3 mediated mediated adipokine adipokine production production in adipocytes.in adipocytes. The mRNAThe mRNA expressions expressions (A) and (A protein) and concentrationprotein concentration (B) of CCL2, (B) of IL-6, CCL2, IL-8, IL- IL-106, IL-8, in IL the-10 adipocytes in the adipocytes at different at different time points time ofpoints 20 days of 20 of differentiation. Data shown are the mean SD of 3 independent experiments performed in triplicates. days of differentiation. Data shown are the± mean ±SD of 3 independent experiments performed in The ‘*’ indicated statistical differences when compared with day 0, and ‘ ’ indicated statistical differences triplicates. The ‘*’ indicated statistical differences when compared † with day 0, and ‘†’ indicated whenstatistical compared differences withthe when control compared group atwith the samethe control time point, group both at the at the same significance time point, level both of p at< 0.05. the 3.10. Immunobioticsignificance level Mediated of p < Regulation 0.05. of Fat Accumulation and Adipokine Production in Adipocytes during LTAD

3.10.Previously, Immunobiotic we Mediated demonstrated Regulation that someof Fat immunobioticAccumulation and strains Adipokine are able Production to differentially in Adipocytes modulate fat accumulationduring LTAD and proinflammatory mediators’ expression during short term adipogenic differentiation of PIP cells [16]. Then, we aimed to evaluate whether the same lactobacilli and bifidobacteria strains Previously, we demonstrated that some immunobiotic strains are able to differentially modulate were able to modulate fat accumulation and CCL2 expression during LTAD. For this purpose, two sets fat accumulation and proinflammatory mediators’ expression during short term adipogenic of experiments were performed. First, porcine adipocytes were treated directly with bacterial strains. differentiation of PIP cells [16]. Then, we aimed to evaluate whether the same lactobacilli and As shown in Figure 11 (Panel-a) no effect on fat accumulation was observed for the evaluated strains bifidobacteria strains were able to modulate fat accumulation and CCL2 expression during LTAD. withFor this the exception purpose, two of L. sets paracasei of experimentsTCM0409, were which performed. significantly First, reduced porcine this adipocytes parameter. were In addition, treated onlydirectlyB. bifidum with bacterialTCM3108 strains. induced As ashown significant in Figure up-regulation 11 (Panel- ofa) CCL2no effect expression. on fat accumulation In a second setwas of experiments,observed for wethe performed evaluated severalstrains inwith vitro thesteps exception to simulate of L. LAB-immuneparacasei TCM cells-adipocytes0409, which significantly interaction. Antigenreduced presenting this parameter. cells isolatedIn addition, from o porcinenly B. bifidum Peyer’s TCM3108 patches wereinduced stimulated a significant with up the-regulation different LAB of strainsCCL2 andexpression. the cell freeIn a supernatants second set of (CFS) experiments, were collected. we performed Adipocytes several werestimulated in vitro steps with to the simulate different CFSLAB obtained-immune in cells the- previousadipocytes step, interacti and faton. accumulation Antigen presenting and CCL2 cells expression isolated were from evaluated. porcine Peyer’s The CFS frompatches immune were stimulated cells stimulated with withthe different TCM3108, LAB LA-2 strains and and TCM0409 the cell strains free supernatants significantly (CFS) reduced were fat accumulationcollected. Adipocytes while only were CFS stimulated from TCM3108 with treatmentthe different improved CFS obtained CCL2 expressionin the previous (Figure step, 11: and Panel-b). fat accumulationTo gain further and CCL2 insight expression into the CFS were mediated evaluated regulation. The CFS of from adipogenesis, immune cells the mRNA stimulated expression with ofTCM3108, PPARγ and LA- GLUT42 and TCM0409 were quantified. strains significantly As shown inreduced Figure fat12, accumulation only the CFS fromwhileL. only rhamnosus CFS fromGG significantlyTCM3108 treatment down-regulated improved theCCL2 expression expression of ( PPARFigureγ ,11 while: Panel TCM3108-b). and TCM0409 treatments up-regulated GLUT4 expression. Finally, we aimed to characterize the cytokine profile induced by LAB strain on porcine antigen presenting cells that would be responsible for the effects on porcine adipocytes. A strain-dependent ability in the modulation of cytokine expression in porcine intestinal immune cells was observed (Figure 13). The most remarkable effect was found for L. paracasei TMC0409, which was able to significantly increase the expressions of IL-1β, IL-2, IL-12, IFN-β and IFN-γ. L. rhamnosus GG increased the mRNA expression levels of IL-10, IL-12, IL-6 and IFN-γ (Figure 13). On the other hand, L. rhamnosus LA-2 significantly reduced the expression of IL-1β while it improved the levels of IL-6 and IFN-γ (Figure 13). Lactobacillus gasseri TMC0356 stimulation increased the expression of IL-2, IL-6, IL-10 and IFN-γ when compared with untreated control cells. B. bifidum TCM3115 enhanced the expression of IL-6 and IL-10 while S. thermophilus TCM1543 increased IFN-β and IFN-γ expression. B. bifidum TMC3108 only increased the expression of IL-6 when compared to control cells (Figure 13).

Figure 11. Immunobiotic mediated regulation of fat accumulation and inflammatory responses in an immune cell-adipocyte co-culture model. A total of 1 × 106 immune cells/well of a 6-well plate were cultured on adipocytes 4-days differentiated. Bacterial samples (5 × 108 cells/well) were also added

Cells 2020, 9, x 13 of 22

Figure 10. TLR3 mediated adipokine production in adipocytes. The mRNA expressions (A) and protein concentration (B) of CCL2, IL-6, IL-8, IL-10 in the adipocytes at different time points of 20 days of differentiation. Data shown are the mean ±SD of 3 independent experiments performed in triplicates. The ‘*’ indicated statistical differences when compared with day 0, and ‘†’ indicated statistical differences when compared with the control group at the same time point, both at the significance level of p < 0.05.

3.10. Immunobiotic Mediated Regulation of Fat Accumulation and Adipokine Production in Adipocytes during LTAD Previously, we demonstrated that some immunobiotic strains are able to differentially modulate fat accumulation and proinflammatory mediators’ expression during short term adipogenic differentiation of PIP cells [16]. Then, we aimed to evaluate whether the same lactobacilli and bifidobacteria strains were able to modulate fat accumulation and CCL2 expression during LTAD. For this purpose, two sets of experiments were performed. First, porcine adipocytes were treated directly with bacterial strains. As shown in Figure 11 (Panel-a) no effect on fat accumulation was observed for the evaluated strains with the exception of L. paracasei TCM0409, which significantly reduced this parameter. In addition, only B. bifidum TCM3108 induced a significant up-regulation of CCL2 expression. In a second set of experiments, we performed several in vitro steps to simulate LAB-immune cells-adipocytes interaction. Antigen presenting cells isolated from porcine Peyer’s patches were stimulated with the different LAB strains and the cell free supernatants (CFS) were collected. Adipocytes were stimulated with the different CFS obtained in the previous step, and fat accumulation and CCL2 expression were evaluated. The CFS from immune cells stimulated with CellsTCM3108,2020, 9, 1715 LA-2 and TCM0409 strains significantly reduced fat accumulation while only CFS from14 of 22 TCM3108 treatment improved CCL2 expression (Figure 11: Panel-b).

Cells 2020, 9, x 14 of 22

and cultured for 2 or 4 days. Adipocyte cells were stained with Oil red O stained and fat accumulation in the adipocytes was quantitatively analyzed by image J software. The ELISA-based concentration FigureoFiguf CCL2re 11. 11 . proteinImmunobiotic Immunobiotic in supernatant mediated of regulation regulation adipocyte of- ofculture fat fat accumulation accumulation stimulated and by and inflammatory immune inﬂammatory cells responses and responses lactic in an acid in 6 anbacteriaimmune immune (LAB)cell cell-adipocyte-adipocyte. All data co shown- co-cultureculture are model. the model. mean A totalA ± totalSD of 1of of× three10 1 6 immune 10independentimmune cells/well cellsexperiments of/well a 6-well of aperformed plate 6-well were plate in × 8 weretriplicate. cultured The on asterisk adipocytess: ‘*’ and 4-days ‘**’ indicate diﬀerentiated.d statistical Bacterial differences samples either8 (5 between10 cells bars/well) of panel were-a alsoand cultured on adipocytes 4-days differentiated. Bacterial samples (5 × 10 cells× /well) were also added addedcontrol and or culturedbetween for bars 2 or of 4 panel days.-b Adipocyte and IMU, cellswith were significant stained levels with of Oil p red< 0.05 O stained and p and< 0.0 fat1,

accumulationrespectively. inControl the adipocytes, untreated was adipocytes; quantitatively IMU, cell analyzed free supernatant by image (CFS) J software. medium The obtained ELISA-based from concentrationimmunocompetent of CCL2 cell protein (antigen in presenting supernatant cell) of culture adipocyte-culture; Panel-a, adipocytes stimulated directly by immune exposed cells to LAB and; lacticPanel acid-b, adipocytes bacteria (LAB). exposed All to data CFS shown obtained are from the mean differentSD LAB of treatedthree independent immunocompetent experiments cells; ± performedTMC3108, in Bifidobacterium triplicate. The bifidum asterisks: TMC3108; ‘*’ and ‘**’ LGG, indicated Lactobacillus statistical rhamnosus differences GG; either LA-2, between Lactobacillus bars ofrhamnosus panel-a and LA- control2; TMC0409, or between Lactobacillus bars ofparacasei panel-b TMC0409. and IMU, with significant levels of p < 0.05 and p < 0.01, respectively. Control, untreated adipocytes; IMU, cell free supernatant (CFS) medium obtained fromTo gain immunocompetent further insight cell into (antigen the CFS presenting mediated cell) regulation culture; of Panel-a, adipogenesis, adipocytes the directly mRNA exposed expression of PPARγto LAB; and Panel-b, GLUT4 adipocytes were quantified. exposed to CFSAs shown obtained in fromFigure diff 1erent2, only LAB the treated CFS immunocompetentfrom L. rhamnosus GG significantlycells; TMC3108, downBifidobacterium-regulated the bifidum expressionTMC3108; of PPARγ, LGG, Lactobacillus while TCM3108 rhamnosus and GG;TCM0409 LA-2, Lactobacillus treatments up- regulatedrhamnosus GLUT4LA-2; expression. TMC0409, Lactobacillus paracasei TMC0409.

Figure 12. Immunobiotic mediated regulation of adipocyte differentiation in an immune cell-adipocyte Figure 12. Immunobiotic mediated regulation of adipocyte differentiation in an immune cell- co-culture model. The mRNA expressions of PPARγ and GLUT4 resulted from various immunobiotic adipocyte co-culture model. The mRNA expressions of PPARγ and GLUT4 resulted from various lactic acid bacteria (LAB) stimulation into the co-culture model. A total of 1 106 immune cells/well immunobiotic lactic acid bacteria (LAB) stimulation into the co-culture model.× A total of 1 × 106 of 6-well plate were cultured on adipocytes 4-days differentiated. LAB samples (5 108 cells/well) immune cells/well of 6-well plate were cultured on adipocytes 4-days differentiated. ×LAB samples (5 also added into the adipocyte plate and maintained for further 2 or 4 Days. All data shown are the × 108 cells/well) also added into the adipocyte plate and maintained for further 2 or 4 Days. All data average SD of 3 independent experiments performed in triplicate. The asterisk (*) indicated statistical shown± are the average ±SD of 3 independent experiments performed in triplicate. The asterisk (*) differences when compared with control at the significance level of p < 0.05. TMC3108, Bifidobacterium indicated statistical differences when compared with control at the significance level of p < 0.05. bifidum TMC3108; LGG, Lactobacillus rhamnosus GG; LA-2, Lactobacillus rhamnosus LA-2; TMC0409, TMC3108, Bifidobacterium bifidum TMC3108; LGG, Lactobacillus rhamnosus GG; LA-2, Lactobacillus Lactobacillus paracasei TMC0409. rhamnosus LA-2; TMC0409, Lactobacillus paracasei TMC0409.

Finally, we aimed to characterize the cytokine profile induced by LAB strain on porcine antigen presenting cells that would be responsible for the effects on porcine adipocytes. A strain-dependent ability in the modulation of cytokine expression in porcine intestinal immune cells was observed (Figure 13). The most remarkable effect was found for L. paracasei TMC0409, which was able to significantly increase the expressions of IL-1β, IL-2, IL-12, IFN-β and IFN-γ. L. rhamnosus GG increased the mRNA expression levels of IL-10, IL-12, IL-6 and IFN-γ (Figure 13). On the other hand, L. rhamnosus LA-2 significantly reduced the expression of IL-1β while it improved the levels of IL-6 and IFN-γ (Figure 13). Lactobacillus gasseri TMC0356 stimulation increased the expression of IL-2, IL-6, IL- 10 and IFN-γ when compared with untreated control cells. B. bifidum TCM3115 enhanced the expression of IL-6 and IL-10 while S. thermophilus TCM1543 increased IFN-β and IFN-γ expression. B. bifidum TMC3108 only increased the expression of IL-6 when compared to control cells (Figure 13).

Cells 2020, 9, 1715 15 of 22 Cells 2020, 9, x 15 of 22

Figure 13. Immunobiotic mediated inflammatory responses in the antigen presenting cells (APCs) Figure 13. Immunobiotic mediated inflammatory responses in the antigen presenting cells (APCs) of of Payer’s patches. The APCs were seeded at 1 106 cells/well of 6-well plate and cultured with Payer’s patches. The APCs were seeded at 1 × 106× cells/well of 6-well plate and cultured with each each bacterial strain (5 108 cells/well) separately for 24 h and the mRNA expressions of IL-1β, IL-2, bacterial strain (5 × 108 ×cells/well) separately for 24 h and the mRNA expressions of IL-1β, IL-2, IL-4, β γ IL-4,IL-6, IL-6,IL-10, IL-10, IL-12, IL-12, IFN-β IFN-and IFNand-γ) IFN- were )analyzed were analyzed with RT with-qPCR. RT-qPCR. Data shown Data are shown the mean are the ±SD mean of SD of 3 independent experiments performed in triplicates. The asterisks: (*) and (**) indicated ±3 independent experiments performed in triplicates. The asterisks: (*) and (**) indicated statistical statisticaldifferences di ffwitherences significant with significant levels of levelsp < 0.05 of andp < 0.05p < 0.01, and prespectively.< 0.01, respectively. TMC0356, TMC0356, LactobacillusLactobacillus gasseri gasseriTMC0356;TMC0356; LGG, LGG,L. rhamnosusL. rhamnosus GG; GG; LA-2, LA-2, L. rhamnosusL. rhamnosus LALA-2;-2; TMC0409, TMC0409, L. L. paracasei paracasei TMC0409;TMC0409; TMC1543,TMC1543, StreptococcusStreptococcus thermophilus thermophilusTMC1543; TMC1543; TMC3108, TMC3108,Bifidobacterium Bifidobacterium bifidum TMC3108; bifidum TMC3108; TMC3115, B.TMC3115, bifidum TMC3115. B. bifidum TMC3115. 4. Discussion 4. Discussion Adipocyte inflammation is a common feature of obesity and obesity-related complications. Adipocyte inflammation is a common feature of obesity and obesity-related complications. The The inflammatory reactions in adipocytes initiate through their sensing of either microbial associated inflammatory reactions in adipocytes initiate through their sensing of either microbial associated molecular patterns (MAMPs) or damage associated molecular patterns (DAMPs). Unlike the molecular patterns (MAMPs) or damage associated molecular patterns (DAMPs). Unlike the microorganism derived MAPMs which induce a rapid onset of proinflammatory reaction, DAMPs are microorganism derived MAPMs which induce a rapid onset of proinflammatory reaction, DAMPs endogenous molecules (e.g., free fatty acids, cholesterol, ceramides) derived from dead adipocytes, are endogenous molecules (e.g., free fatty acids, cholesterol, ceramides) derived from dead which can lead to a non-infectious chronic inflammation [37–39]. In our previous study, we have adipocytes, which can lead to a non-infectious chronic inflammation [37–39]. In our previous study, demonstrated the MAMPs-mediated acute inflammatory responses in adipocytes using a short-term we have demonstrated the MAMPs-mediated acute inflammatory responses in adipocytes using a differentiation period (4 days) of PIP cells [17,26,30]. However, like other standard in vitro adipocytes short-term differentiation period (4 days) of PIP cells [17,26,30]. However, like other standard in vitro models,adipocytes our previous models, approaches our previous have approaches the limitation have of not the able limitation to address of thenot mechanisms able to address of sustained the inflammationmechanisms of because sustained of theinflammation short period because of adipocytes of the short culture. period Therefore,of adipocyte heres culture. we expanded Therefore, the utilityhere we and expanded potential the application utility and of potential our in vitro applicationPIP cells modelof our byin vitro evaluating PIP cells an LTADmodel periodby evaluating (20 days) inan order LTAD to period elucidate (20 furtherdays) i insightsn order ofto sustainedelucidate further inflammation insights on of the sustained pathobiology inflammation of adipocytes on the as wellpathobiology as to explore of ad theipocytes prospects as of well beneficial as to explore immunomodulatory the prospects interventionsof beneficial immunomodulatory for obesity. interventionThe results for of obesity. the present study indicated that cytosolic TG content of porcine mature adipocyte progressivelyThe result increased of the present over study the period indicate ofd LTAD that cytosolic evaluated TG here.content This of porcine finding mature is consistent adipocyte with previousprogressively studies increase demonstratingd over the that period in the of mouse LTAD 3T3 evaluated adipocyte here. cell This line, finding fat accumulation is consistent resulted with fromprevious the increasestudies demonstrating of TG synthesis that while in the cholesterol mouse 3T3 or itsadipocyte derivatives cell line, did notfat accumulation necessarily accumulate resulted duringfrom the the increase adipocyte of TG diff synthesiserentiation while [40 ].cholesterol The enlargement or its derivatives of lipid droplets did not andnecessarily the enhancement accumulate of TGduring content the adipocyte are associated differentiation with an increase [40]. The in enlargement lipolysis. High of lipid rate droplets of lipolysis and causes the enhance the increasement of of circulatingTG content FFA are levelsassociated and their with delivery an increase to the in liver lipolysis. increasing High TG rate synthesis, of lipolysis which causes could the lead increase to hepatic of steatosiscirculating [41 FFA]. Along levels with and TG their synthesis delivery in to the the liver, liver the increasing increased TG delivery synthesis, of FFA which exacerbates could lead insulin to resistance,hepatic steatosis which [ promotes41]. Alongdyslipidemia. with TG synthesis Saturated in the fattyliver, acids,the increased particularly delivery lauric of FFA acid exacerbates and palmitic acids,insulin are resistance, capable of which stimulating promotes an inflammatory dyslipidemia. response Saturated through fatty acids, the TLR4 particularly signaling lauric pathway acid [42 and,43 ]. Leepalmitic et al. [ acids,44] evaluated are capable the e ff ofect stimulating of different an fatty inflammatory acids on the response TLR4 signaling through and the reported TLR4 signaling that lauric, palmitic,pathway and[42,4 stearic3]. Leeacids et al. could[44] evaluated induce COX-2 the effect expression of different through fatty anacids NF-kB-dependent on the TLR4 signaling mechanism and inreported a macrophage that lauric, cell line.palmitic, These and results stearic suggest acids thecould existence induce of COX a correlation-2 expression between through fat accumulation an NF-kB- anddependent inflammatory mechanism responses in a macrophage in the adipocytes cell line. that These experienced results suggest an LTAD. the existence of a correlation between fat accumulation and inflammatory responses in the adipocytes that experienced an LTAD.

Cells 2020, 9, 1715 16 of 22

In addition to fat accumulation, it was observed that LTAD led to an increased production of IL-6, IL-8 and specially CCL2 that was enhanced more than ten-fold in porcine adipocytes when compared to PIP cells. Both the mRNA and protein expression of CCL2 were significantly increased in adipocytes over the time of differentiation. The over-expression of CCL2 in adipose tissue increases macrophage recruitment [45], whereas a reduction of CCL2 or its receptor CCR2 lowered proinflammatory macrophages accumulation in the adipose tissue and provides protection from insulin resistance as well as hepatic steatosis [45]. Activated macrophages, both immigrant and adipose tissue residents, further trigger inflammatory responses through the release of various proinflammatory cytokines such as TNF-α and IL-6, via NF-kB activation and signaling (reviewed in [43]). In our hands, LTAD led to the production of new mature adipocytes, which will be able to secrete proinflammatory adipokines in response to the progressive stress of fat accumulation. These results suggest the existence of a correlation between fat accumulation and inflammatory responses in porcine adipocytes during LTAD. This is in line with previous studies reporting the correlation of fat accumulation and the increased expression of cytokines and chemokines including CCL2, IL-6 and IL-8 in the obese individuals [46]. Adiponectin and leptin are two physiologically important adipokines released from white adipocytes [46,47]. In addition to fat accumulation dynamics, we observed here that adiponectin and leptin production followed a moderate increasing trend over LTAD. The secretion of adiponectin and leptin is known to involve distinct intracellular trafficking pathways [47], suggesting that adipocytes rely on different avenues for the constitutive and regulated secretion of these adipokines. It has been reported that adipocyte size correlated with the production of adiponectin and leptin [3,8]. Then, it is tempting to speculate that the continuous differentiation of PIP cells during LTAD led to the development of a higher number of matured adipocytes that upon reaching a critical mass of fat accumulation increased their production of adiponectin and leptin. In addition to metabolic and endocrine functions, adipocytes derived from the differentiation of porcine intramuscular preadipocytes could be a source for adult stem cells. It was reported that human adipocytes generated through adipogenic differentiation in vitro exhibit similar physiological properties of bone marrow-derived mesenchymal stem cells [48] because of the more abundant cell number and the high purity as compared to adipose-derived stem cells [49]. A recent study demonstrated that porcine-derived differentiated fat cells exhibit efficient proliferative activity, multipotent differentiation to adipocytes, osteoblasts, and myocytes, normal diploid karyotypes during the long-term in vitro culture [50]. Therefore, pigs being considered as the closest approximate animal model for studying human disease, porcine derived differentiated adipocytes maintained in long-term in vitro culture can be an excellent stem cell model applied to tissue engineering and regenerative biomedicine. Of note, the changes in the levels of adipokines observed during the LTAD evaluated here can be explained by other processes that are not mutually exclusive. On the one hand, the increased synthesis of CCL2, IL-6, IL-8, adiponectin and leptin during LTAD of PIP cells could be associated to the release of DAMPs. The progressive death of adipocytes may lead to the release of DAMPs, which may subsequently induce a low-grade inflammatory response. On the other hand, the long-term culture of adipocyte progenitors such as preadipocytes can induce senescence of the growing cells, which function as mediators of inflammation associated with obesity [51,52]. It was reported that in addition to no longer undertaking their original function effectively, senescent cells can induce the secretion of proinflammatory factors in the tissue in which they reside [53]. Cellular senescence in adipocytes attenuates the physiological resolution of inflammation leading to a low-grade chronic inflammation, insulin resistance, type-2 diabetes, and companion senescence-associated secretory phenotype in the obese individual [51–53], indicating that these phenotypes are associated with impaired adipokine production. A detailed evaluation of senescence, cell death and DAMPs-mediated signaling pathway in PIP cells during LTAD would be of great value to further characterize this process in vitro and establish in parallel with the in vivo physiology. We also evaluated the influence of TLRs activation on the degree of fat accumulation and cytokines production by porcine adipocytes during LTAD. Results demonstrated that fat accumulation in Cells 2020, 9, 1715 17 of 22 adipocytes was increased after stimulation with poly(I:C) or LPS which induced the activation of TLR3 and TLR4, respectively. We previously reported the presence of TLR1-9 in PIP cells [17], and in a follow up study, we demonstrated the activation of TLR2, TLR3 and TLR4 in differentiated adipocytes after specific ligand stimulation [26]. Studies have demonstrated that TLR2 and TLR4 agonists activate macrophages and adipocytes in the adipose tissue, resulting in increased release of various adipokines [54,55]. Fat accumulation and obesity-induced activation of TLR-mediated proinflammatory signaling cascades were demonstrated in the adipose tissue of mice [56]. Kim et al. [56] provided evidence of increased expression of TLR 1-9 and TLR 11-13 in murine adipose tissue in response to obesity induced by high-fat diet administration, causing activation of both MyD88- and non-MyD88 signaling cascades as well as downstream up-regulation of NF-kB activity and the subsequent release of adipokines. The expression patterns of adipokines varied significantly with the timing of TLR-ligands stimulation. The expression of CCL2, IL-6 and IL-8 were increased in the later time points of stimulation when a decreased IL-10 expression was noticed. Interestingly, cytokine expression dynamics varied between ligands. Our results are in accordance with a study conducted by Kopp et al. [24] who have demonstrated that the specific ligands of TLRs can influence IL-6 and CCL2 release in human adipocytes in a ligand-specific manner. The stimulation with poly(I:C) resulted in CCL2 production at an early stage of the study while the other ligands triggered the synthesis of this chemokine at later stages. TLR3 is expressed in the porcine adipocytes [26] as well as human adipocytes [57]. TLR3 can recognize mRNA from dying cells, a process frequently observed in obese adipose tissue [55,57]. It was reported that the in vitro stimulation of human and mouse adipocytes with poly(I:C) led to an increased expression of proinflammatory factors such as IL-6, IL-8 and CCL2. However, TLR3 signaling had no influence on the obesity-induced inflammation in adipose tissue in vivo [57]. On the other hand, LPS and TNF-α stimulation led to a strong induction of CCL2 protein at a later stage of the differentiation period. The TNF-α-mediated expression of IL-6 and IL-8 was stronger than the TLRs ligand stimulations. In addition, we evaluated here the ability of LAB to modify the fat accumulation and CCL2 expression of 20-day-old porcine adipocytes through host’ intestinal immune-competent cells. For this purpose, we stimulated porcine APCs from Peyer’s patches with different LAB strains, collected the CFS and treated porcine adipocytes with them. Results indicated that the rate of cytosolic fat accumulation was significantly reduced in a strain dependent manner. Remarkably, a reduced fat accumulation was recorded when the CFS from L. rhamnosus LA-2 and L. casei TMC0409-treated immune cells were used. In addition, no significant effect in the expression of CCL2 was observed after the treatment of adipocytes with CFS with the exception of CFS from the TMC3108 strain that up-regulated this chemokine. These results contrast significantly with our previous studies on four-day-old porcine adipocytes. When the CFS from L. rhamnosus LA-2 and L. rhamnosus GG were used to stimulate four-day-old porcine adipocytes, a significant reduction in the expression of CCL2 was observed, even after the stimulation with TNF-α [17]. Those previous findings were in agreement with studies performed in the murine macrophage-like cell line J774.1 stimulated with L. rhamnosus GG followed by a co-culture with the mouse preadipocyte cell line 3T3-L1, which resulted in a decreased fat accumulation [58]. The effect of the GG strain was associated to its ability to improve IFN-γ in immune cells and reduced PPAR-γ mRNA expression in 3T3-L1 cells [59,60]. Here, we demonstrated that L. rhamnosus GG was capable of upregulating the expression of IFN-γ in porcine APCs as well as down regulating PPAR-γ in adipocytes. However, the GG strain was not capable of reducing fat accumulation or CCL2 expression. These results show an important difference between the 4- and 20-day-old porcine adipocytes in terms of the most efficient immunobiotic strain capable of regulating their response. Interestingly, L. rhamnosus GG, similarly to the strains able to reduce fat accumulation in 20-day-old porcine adipocytes, increased the expression of IFN-γ, IL-6, IL-12 and IL-10 in APCs. Then, it is tempting to speculate that the presence of these cytokines in CFS would not be responsible for Cells 2020, 9, 1715 18 of 22 modulating fat accumulation in adipocytes. On the contrary, the GG strain was not capable of modulating IL-1β or IFN-β expression in immune cells as the strains with the ability to reduce fat accumulation. It has been shown that IL-1β impairs insulin sensitivity in adipose tissue by affecting insulin signal transduction [61]. Paradoxically, it was reported that adipogenesis and the expression of / the markers PPARγ, CEPBA, FABP4, and GLUT4 were significantly elevated in IL-1β− − mice under a / high-fat diet treatment [62]. Moreover, it was shown that IL-1Ra− − mice have a lean phenotype due to decreased fat mass, related to a defect in adipogenesis and increased energy expenditure [63]. On the other hand, it was demonstrated that the overexpression of IFN-β blocked body weight gain and adipose tissue expansion in mice under high-fat diet treatment. Furthermore, IFN-β protected animals against adipose tissue inflammation [64]. Then, our results indicate that the cytokines able to regulate fat accumulation and inflammation in porcine adipocytes are different when 4- and 20-day-old cells are considered. Evaluating the effects of IFN-γ, IL-1β and IFN-β signaling pathways in PIP cells and in 4- and 20-day-old porcine adipocytes in terms of their fat accumulation abilities and the inflammatory cytokines expression will significantly enhance our understanding of the biology of these porcine cells. In addition, those studies could improve our knowledge of the effect of immunobiotics and will enhance our capacity to select the most appropriate strains for their application in the prevention of obesity and its related inflammatory complications.

5. Conclusions We presented here a long-term adipogenic differentiation model using a porcine intramuscular preadipocytes cell line (PIP cells) and evaluated the effects of the sustained inflammation induced by TNF-α and TLRs ligands on the degree of fat accumulation and adipokine production. In our hands, activation of TNF, TLR3 and TLR4 pathways resulted in an increased fat accumulation and inflammatory factors expression of the adipocytes. Since in obese subjects, adipocytes are subject to sustained inflammatory responses, the characterization of the immunobiology and adipogenesis of PIP cells under a sustained rather than an acute inflammatory environment performed in this work indicate that this cell line could be used for the study of metabolic and immunological alterations in adipocytes in the context of obesity and the potential therapeutic alternatives to mitigate those effects. Despite the need for more in-depth investigations of immune signaling pathways, metabolic activities, senescence and cell death to establish conclusively a physiological relevance of LTAD in PIP cells to in vivo human adult intramuscular adipocytes, this is a first step in the positioning of this cell line as a useful laboratory tool for obesity studies.

Author Contributions: Conceptualization, A.T., A.H.K., M.A.I., J.V. and H.K.; methodology, A.T., A.H.K., M.A.I., M.I., M.T. and M.S.; software, M.A.; validation, M.A.I. and J.V.; formal analysis, A.T., A.H.K., M.A.I.; investigation, J.V., H.K.; resources, K.M., K.Y., F.H.; data curation, A.T., writing—original draft preparation, A.H.K. and M.A.I.; writing—review and editing, J.V., W.I.-O., H.T. and H.A.; visualization, M.A.I.; supervision, J.V., H.K.; project administration, H.K; funding acquisition, H.K. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by a Grant-in-Aid for Scientific Research (A) (19H00965), (B) (16H05019), Challenging Exploratory Research (26660216, 16K15028), Open Partnership Joint Projects of JSPS Bilateral Joint Research Projects from the Japan Society for the Promotion of Science (JSPS) to HK, and the Research Project on Development of Agricultural Products and Foods with Health-promoting benefits (NARO), and the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry, Japan to HA, and the grants for “Scientific Research on Innovative Areas” from the Ministry of Education, Culture, Science, Sports and Technology (MEXT) of Japan (Grant numbers: 16H06429, 16K21723, and 16H06435) to HT. MAI and AHK were supported by JSPS (Postdoctoral Fellowship for Foreign Researchers, Program No. 18F18081 and 15F15401, respectively). This work was also supported by the JSPS Core-to-Core Program, A. Advanced Research Networks entitled Establishment of international agricultural immunology research-core for a quantum improvement in food safety. Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be constructed as a potential conflict of interest. Cells 2020, 9, 1715 19 of 22

References

1. World Health Organization. Obesity and Overweight. Available online: https://www.who.int/news-room/ fact-sheets/detail/obesity-and-overweight. (accessed on 28 December 2019). 2. Shoelson, S.E.; Herrero, L.; Naaz, A. Obesity, Inflammation, and Insulin Resistance. Gastroenterology 2007, 132, 2169–2180. [CrossRef][PubMed] 3. Ghaben, A.L.; Scherer, P.E. Adipogenesis and metabolic health. Nat. Rev. Mol. Cell Boil. 2019, 20, 242–258. [CrossRef][PubMed] 4. Longo, M.; Zatterale, F.; Naderi, J.; Parrillo, L.; Formisano, P.; Raciti, G.; Beguinot, F.; Miele, C. Adipose Tissue Dysfunction as Determinant of Obesity-Associated Metabolic Complications. Int. J. Mol. Sci. 2019, 20, 2358. [CrossRef][PubMed] 5. Blüher, M. Adipose tissue inflammation: A cause or consequence of obesity-related insulin resistance? Clin. Sci. 2016, 130, 1603–1614. [CrossRef][PubMed] 6. Reilly, S.M.; Saltiel, A.R. Adapting to obesity with adipose tissue inflammation. Nat. Rev. Endocrinol. 2017, 13, 633–643. [CrossRef] 7. Laurencikiene, J.; Skurk, T.; Kulyté, A.; Hedén, P.; Åström, G.; Sjölin, E.; Rydén, M.; Hauner, H.; Arner, P. Regulation of Lipolysis in Small and Large Fat Cells of the Same Subject. J. Clin. Endocrinol. Metab. 2011, 96, E2045–E2049. [CrossRef][PubMed] 8. Skurk, T.; Alberti-Huber, C.; Herder, C.; Hauner, H. Relationship between Adipocyte Size and Adipokine Expression and Secretion. J. Clin. Endocrinol. Metab. 2007, 92, 1023–1033. [CrossRef][PubMed] 9. Meyer, L.K.; Ciaraldi, T.P.; Henry, R.R.; Wittgrove, A.C.; Phillips, S.A. Adipose tissue depot and cell size dependency of adiponectin synthesis and secretion in human obesity. Adipocyte 2013, 2, 217–226. [CrossRef] 10. Asterholm, I.W.; Tao, C.; Morley, T.S.; Wang, Q.A.; Delgado-López, F.; Wang, Z.V.; Scherer, P.E. Adipocyte Inflammation Is Essential for Healthy Adipose Tissue Expansion and Remodeling. Cell Metab. 2014, 20, 103–118. [CrossRef] 11. Contreras, C.; Gonzalez, F.; Fernø, J.; Diéguez, C.; Rahmouni, K.; Nogueiras, R.; López, M. The brain and brown fat. Ann. Med. 2014, 47, 150–168. [CrossRef][PubMed] 12. Wueest, S.; Schoenle, E.J.; Konrad, D. Depot-specific differences in adipocyte insulin sensitivity in mice are diet- and function-dependent. Adipocyte 2012, 1, 153–156. [CrossRef] 13. Hausman, G.J.; Basu, U.; Du, M.; Fernyhough-Culver, M.; Dodson, M.V. Intermuscular and intramuscular adipose tissues: Bad vs. good adipose tissues. Adipocyte 2014, 3, 242–255. [CrossRef][PubMed] 14. Hausman, N.L.; Borrero, J.C.; Fisher, A.; Kahng, S. Improving accuracy of portion-size estimations through a stimulus equivalence paradigm. J. Appl. Behav. Anal. 2014, 47, 485–499. [CrossRef][PubMed] 15. Sanosaka, M.; Minashima, T.; Suzuki, K.; Watanabe, K.; Ohwada, S.; Hagino, A.; Rose, M.; Yamaguchi, T.; Aso, H. A combination of octanoate and oleate promotes in vitro differentiation of porcine intramuscular adipocytes. Comp. Biochem. Physiol. Part B Biochem. Mol. Boil. 2008, 149, 285–292. [CrossRef][PubMed] 16. Suzuki, K.; Kadowaki, H.; Shibata, T.; Uchida, H.; Nishida, A. Selection for daily gain, loin-eye area, backfat thickness and intramuscular fat based on desired gains over seven generations of Duroc pigs. Livest. Prod. Sci. 2005, 97, 193–202. [CrossRef] 17. Suzuki, M.; Tada, A.; Kanmani, P.; Watanabe, H.; Aso, H.; Suda, Y.; Nochi, T.; Miyazawa, K.; Yoda, K.; He, F.; et al. Advanced Application of Porcine Intramuscular Adipocytes for Evaluating Anti-Adipogenic and Anti-Inflammatory Activities of Immunobiotics. PLoS ONE 2015, 10, e0119644. [CrossRef] 18. Vettor, R.; Milan, G.; Franzin, C.; Sanna, M.; De Coppi, P.; Rizzuto, R.; Federspil, G. The origin of intermuscular adipose tissue and its pathophysiological implications. Am. J. Physiol. Metab. 2009, 297, E987–E998. [CrossRef] 19. Coen, P.M.; Goodpaster, B.H. Role of intramyocelluar lipids in human health. Trends Endocrinol. Metab. 2012, 23, 391–398. [CrossRef] 20. Ghanim, H.; Mohanty, P.; Deopurkar, R.; Sia, C.L.; Korzeniewski, K.; Abuaysheh, S.; Chaudhuri, A.; Dandona, P. Acute Modulation of Toll-Like Receptors by Insulin. Diabetes Care 2008, 31, 1827–1831. [CrossRef][PubMed] 21. Vitseva, O.I.; Tanriverdi, K.; Tchkonia, T.T.; Kirkland, J.L.; McDonnell, M.E.; Apovian, C.M.; Freedman, J.; Gokce, N. Inducible Toll-like receptor and NF kappaB regulatory pathway expression in human adipose tissue. Obesity 2008, 16, 932–937. [CrossRef] Cells 2020, 9, 1715 20 of 22

22. Maurizi, G.; Della Guardia, L.; Maurizi, A.; Poloni, A. Adipocytes properties and crosstalk with immune system in obesity-related inflammation. J. Cell. Physiol. 2017, 233, 88–97. [CrossRef][PubMed] 23. Desruisseaux, M.; Nagajyothi; Trujillo, M.E.; Tanowitz, H.B.; Scherer, P.E. Adipocyte, Adipose Tissue, and Infectious Disease. Infect. Immun. 2006, 75, 1066–1078. [CrossRef] 24. Kopp, A.; Buechler, C.; Neumeier, M.; Weigert, J.; Aslanidis, C.; Schölmerich, J.; Schäffler, A. Innate Immunity and Adipocyte Function: Ligand-specific Activation of Multiple Toll-like Receptors Modulates Cytokine, Adipokine, and Chemokine Secretion in Adipocytes. Obesity 2009, 17, 648–656. [CrossRef][PubMed] 25. Schäffler, A.; Scholmerich, J.; Salzberger, B. Adipose tissue as an immunological organ: Toll-like receptors, C1q/TNFs and CTRPs. Trends Immunol. 2007, 28, 393–399. [CrossRef][PubMed] 26. Igata, M.; Islam, A.; Tada, A.; Takagi, M.; Kober, A.K.M.H.; Albarracin, L.; Aso, H.; Ikeda-Ohtsubo, W.; Miyazawa, K.; Yoda, K.; et al. Transcriptome Modifications in Porcine Adipocytes via Toll-Like Receptors Activation. Front. Immunol. 2019, 10, 1180. [CrossRef] 27. Caër, C.; Rouault, C.; Le Roy, T.; Poitou, C.; Aron-Wisnewsky, J.; Torcivia, A.; Bichet, J.-C.; Clément, K.; Guerre-Millo, M.; Andre, S. Immune cell-derived cytokines contribute to obesity-related inflammation, fibrogenesis and metabolic deregulation in human adipose tissue. Sci. Rep. 2017, 7, 3000. [CrossRef] 28. Oliver, E.; McGillicuddy, F.C.; Phillips, C.M.; Toomey, S.; Roche, H.M. The role of inflammation and macrophage accumulation in the development of obesity-induced type 2 diabetes mellitus and the possible therapeutic effects of long-chainn-3 PUFA. Proc. Nutr. Soc. 2010, 69, 232–243. [CrossRef] 29. Cawthorn, W.P.; Sethi, J.K. TNF-alpha and adipocyte biology. FEBS Lett. 2007, 582, 117–131. [CrossRef] 30. Tada, A.; Islam, A.; Kober, A.H.; Fukuyama, K.; Takagi, M.; Igata, M.; Albarracin, L.; Ikeda-Ohtsubo, W.; Miyazawa, K.; Yoda, K.; et al. Transcriptome Modifications in the Porcine Intramuscular Adipocytes during Differentiation and Exogenous Stimulation with TNF-α and Serotonin. Int. J. Mol. Sci. 2020, 21, 638. [CrossRef] 31. Clancy, R.L. Immunobiotics and the probiotic evolution. FEMS Immunol. Med. Microbiol. 2003, 38, 9–12. [CrossRef] 32. Villena, J.; Kitazawa, H. Editorial: Immunobiotics—Interactions of Beneficial Microbes with the Immune System. Front. Immunol. 2017, 8.[CrossRef][PubMed] 33. Fabersani, E.; Abeijon-Mukdsi, M.C.; Ross, R.; Medina, R.B.; González, S.; Gauffin-Cano, P. Specific Strains of Lactic Acid Bacteria Differentially Modulate the Profile of Adipokines In Vitro. Front. Immunol. 2017, 8, 425. [CrossRef][PubMed] 34. Villena, J.; Chiba, E.; Vizoso-Pinto, M.G.; Tomosada, Y.; Takahashi, T.; Ishizuka, T.; Aso, H.; Salva, S.; Alvarez, S.; Kitazawa, H. Immunobiotic Lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells. BMC Microbiol. 2014, 14, 126. [CrossRef][PubMed] 35. Folch, J.; Lees, M.; Stanley, G.H.S. A simple method for the isolation and purification of total lipides from animal tissues. J. Boil. Chem. 1957, 226, 497–509. 36. Parlee, S.D.; Lentz, S.I.; Mori, H.; MacDougald, O. Quantifying size and number of adipocytes in adipose tissue. Methods Enzym. 2014, 537, 93–122. [CrossRef] 37. Chen, G.Y.; Nuñez, G. Sterile inflammation: Sensing and reacting to damage. Nat. Rev. Immunol. 2010, 10, 826–837. [CrossRef] 38. Grant, R.W.; Dixit, V.D. Adipose tissue as an immunological organ. Obesity 2015, 23, 512–518. [CrossRef] 39. Kuroda, M.; Sakaue, H. Adipocyte Death and Chronic Inflammation in Obesity. J. Med. Investig. 2017, 64, 193–196. [CrossRef] 40. Green, H.; Kehinde, O. Sublines of mouse 3T3 cells that accumulate lipid. Cell 1974, 1, 113–116. [CrossRef] 41. Caspar-Bauguil, S.; Kolditz, C.-I.; Lefort, C.; Vila, I.; Mouisel, E.; Beuzelin, D.; Tavernier, G.; Marques, M.-A.; Zakaroff-Girard, A.; Pécher, C.; et al. Fatty acids from fat cell lipolysis do not activate an inflammatory response but are stored as triacylglycerols in adipose tissue macrophages. Diabetologia 2015, 58, 2627–2636. [CrossRef] 42. Hwang, D.H.; Kim, J.A.; Lee, J.Y. Mechanisms for the activation of Toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid. Eur. J. Pharmacol. 2016, 15, 24–35. [CrossRef] 43. Rocha, D.M.U.P.; Caldas, A.P.S.; De Oliveira, L.L.; Bressan, J.; Hermsdorff, H.H.M. Saturated fatty acids trigger TLR4-mediated inflammatory response. Atherosclerosis 2016, 244, 211–215. [CrossRef][PubMed] Cells 2020, 9, 1715 21 of 22

44. Lee, J.Y.; Sohn, K.H.; Rhee, S.H.; Hwang, D. Saturated Fatty Acids, but Not Unsaturated Fatty Acids, Induce the Expression of Cyclooxygenase-2 Mediated through Toll-like Receptor 4. J. Boil. Chem. 2001, 276, 16683–16689. [CrossRef][PubMed] 45. Kanda, H.; Tateya, S.; Tamori, Y.; Kotani, K.; Hiasa, K.-I.; Kitazawa, R.; Kitazawa, S.; Miyachi, H.; Maeda, S.; Egashira, K.; et al. MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity. J. Clin. Investig. 2006, 116, 1494–1505. [CrossRef] 46. Gerhardt, C.; Romero, I.; Cancello, R.; Camoin, L.; Strosberg, A. Chemokines control fat accumulation and leptin secretion by cultured human adipocytes. Mol. Cell. Endocrinol. 2001, 175, 81–92. [CrossRef] 47. Xie, L.; O’Reilly, C.P.; Chapes, S.K.; Mora, S. Adiponectin and leptin are secreted through distinct trafficking pathways in adipocytes. Biochim. Biophys. Acta (BBA)—Mol. Basis Dis. 2008, 1782, 99–108. [CrossRef] [PubMed] 48. Poloni, A.; Maurizi, G.; Leoni, P.; Serrani, F.; Mancini, S.; Frontini, A.; Zingaretti, M.C.; Siquini, W.; Sarzani, R.; Cinti, S. Human Dedifferentiated Adipocytes Show Similar Properties to Bone Marrow-Derived Mesenchymal Stem Cells. Stem Cells 2012, 30, 965–974. [CrossRef] 49. Jumabay, M.; Zhang, R.; Yao, Y.; Goldhaber, J.I.; Boström, K.I. Spontaneously beating cardiomyocytes derived from white mature adipocytes. Cardiovasc. Res. 2009, 85, 17–27. [CrossRef] 50. Peng, X.; Song, T.; Hu, X.; Zhou, Y.; Wei, H.; Peng, J.; Jiang, S. Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro. BioMed Res. Int. 2015, 2015, 1–10. [CrossRef] 51. Eckel-Mahan, K.; Latre, A.R.; Kolonin, M.G. Adipose Stromal Cell Expansion and Exhaustion: Mechanisms and Consequences. Cells 2020, 9, 863. [CrossRef] 52. Pokrywczynska, M.; Maj, M.; Kloskowski, T.; Buhl, M.; Balcerczyk, D.; Jundziłł, A.; Szeliski, K.; Rasmus, M.; Drewa, T. Molecular Aspects of Adipose-Derived Stromal Cell Senescence in a Long-Term Culture: A Potential Role of Inflammatory Pathways. Cell Transplant. 2020, 29.[CrossRef][PubMed] 53. Burton, D.; Faragher, R. Obesity and type-2 diabetes as inducers of premature cellular senescence and ageing. Biogerontology 2018, 19, 447–459. [CrossRef][PubMed] 54. Fresno, M.; Alvarez, R.; Cuesta, N.; Escudero, M.F. Toll-like receptors, inflammation, metabolism and obesity. Arch. Physiol. Biochem. 2011, 117, 151–164. [CrossRef] 55. Ballak, D.B.; Van Asseldonk, E.J.P.; Van Diepen, J.A.; Jansen, H.; Hijmans, A.; Joosten, L.A.B.; Tack, C.J.; Netea, M.G.; Stienstra, R. TLR-3 is Present in Human Adipocytes, but Its Signalling is Not Required for Obesity-Induced Inflammation in Adipose Tissue In Vivo. PLoS ONE 2015, 10, e0123152. [CrossRef] [PubMed] 56. Kim, S.J.; Choi, Y.; Choi, Y.H.; Park, T. Obesity activates toll-like receptor-mediated proinflammatory signaling cascades in the adipose tissue of mice. J. Nutr. Biochem. 2012, 23, 113–122. [CrossRef] 57. Brenner, C.; Simmonds, R.; Wood, S.; Rose, V.; Feldmann, M.O.; Turner, J.J. TLR Signalling and Adapter Utilization in Primary Human In vitro Differentiated Adipocytes. Scand. J. Immunol. 2012, 76, 359–370. [CrossRef] 58. Miyazawa, K.; Yoda, K.; Hiramatsu, M.; He, F. Potent effects of, and mechanisms for, modification of crosstalk between macrophages and adipocytes by lactobacilli. Microbiol. Immunol. 2012, 56, 847–854. [CrossRef] [PubMed] 59. Marion-Letellier, R.; Déchelotte, P.; Iacucci, M.; Ghosh, S. Dietary modulation of peroxisome proliferator-activated receptor gamma. Gut 2008, 58, 586–593. [CrossRef] 60. Harata, G.; He, F.; Kawase, M.; Hosono, A.; Takahashi, K.; Kaminogawa, S. Differentiated implication of Lactobacillus GG and L. gasseri TMC0356 to immune responses of murine Peyer’s patch. Microbiol. Immunol. 2009, 53, 475–480. [CrossRef] 61. Bing, C. Is interleukin-1β a culprit in macrophage-adipocyte crosstalk in obesity? Adipocyte 2015, 4, 149–152. [CrossRef] 62. Nov, O.; Shapiro, H.; Ovadia, H.; Tarnovscki, T.; Dvir, I.; Shemesh, E.; Kovsan, J.; Shelef, I.; Carmi, Y.; Voronov, E.; et al. Interleukin-1β Regulates Fat-Liver Crosstalk in Obesity by Auto-Paracrine Modulation of Adipose Tissue Inflammation and Expandability. PLoS ONE 2013, 8, e53626. [CrossRef][PubMed] Cells 2020, 9, 1715 22 of 22

63. Alsaggar, M.; Mills, M.; Liu, D. Interferon beta overexpression attenuates adipose tissue inﬂammation and high-fat diet-induced obesity and maintains glucose homeostasis. Gene Ther. 2016, 24, 60–66. [CrossRef] [PubMed] 64. Somm, E.; Henrichot, E.; Pernin, A.; Juge-Aubry, C.E.; Muzzin, P.; Dayer, J.-M.; Nicklin, M.J.; Meier, C.A. Decreased Fat Mass in Interleukin-1 Receptor Antagonist-Deﬁcient Mice: Impact on Adipogenesis, Food Intake, and Energy Expenditure. Diabetes 2005, 54, 3503–3509. [CrossRef][PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).