Obesity is a metabolic disease caused by the increment of adipose tissue mass due to hyperplastic and hypertrophic growth of adipocytes. Obesity is closely associated with others diseases, such as insulin resistance, type 2 diabetes, hypertension, atherosclerosis, cardiovascular disorders, metabolic syndrome and some type of cancers. The main cause of obesity is the excessive intake of dietary carbohydrate and fat that stimulate the adipogenic process, which in turn causes changes in adipokine secretion, activation of pro-inflammatory pathways and increase of oxidative stress (Baret et al., Reference Baret, Septembre-Malaterre, Rigoulet, d'Hellencourt, Priault, Gonthier and Devin2013; Kusunoki et al., Reference Kusunoki, Yang, Yoshizaki, Nakagawa, Ishikado, Kondo, Morino, Sekine, Ugi, Nishio, Kashiwagi and Maegawa2013). Childhood obesity is increasingly an important concern in pediatric research. The relationship between breast-feeding in the first six months and childhood obesity (Han et al., Reference Han, Lawlor and Kimm2010), as well as milk nutritional factors that influence newborn health, have been studied (Fujisawa et al., Reference Fujisawa, Yamaguchi, Nagata, Satake, Sano, Matsushita, Kitsuta, Nakashima, Nakanishi, Nakagawa and Ogata2013; Romacho et al., Reference Romacho, Glosse, Richter, Elsen, Schoemaker, van Tol and Eckel2015). Preadipocyte cells are transformed under hormonal and nutritional stimulation into adipose tissue during the late-fetal and post natal period, therefore, the nutritional factors in the diet of newborn babies influence the future development of adipose tissue (Spalding et al., Reference Spalding, Arner, Westermark, Bernard, Buchholz, Bergmann, Blomqvist, Hoffstedt, Näslund, Britton, Concha, Hassan, Rydén, Frisén and Arner2008; Tang et al., Reference Tang, Zeve, Suh, Bosnakovski, Kyba, Hammer, Tallquist and Graff2008). Milk is undoubtedly a complete food for the newborn with high nutritional value and several bioactive compounds. The main high energy value compounds in human milk are lactose and fat, and in the latter case the fatty acid profile can exert a complex effect on adipose tissue both promoting and controlling inflammation (Guenther Boden, Reference Guenther Boden2011; Romacho et al., Reference Romacho, Glosse, Richter, Elsen, Schoemaker, van Tol and Eckel2015).
During gastrointestinal digestion pH and enzymes alter food composition to a very considerable extent, and in particular this process impacts both the quality and the quantity of fatty acids through physical and biochemical mechanisms. Milk of various animal species is different for structure and size of fat globules, composition in triglycerides and also enzymatic activity and these features can influence the milk fat digestion (Devle et al., Reference Devle, Ulleberg, Naess-Andresen, Rukke, Vegarud and Ekeberg2014). Recently Santillo et al. (Reference Santillo, Figliola, Ciliberti, Caroprese, Marino and Albenzio2018) submitted milk from different sources (human, formula, donkey, bovine, ovine and caprine) to in vitro digestion to gain information on the fatty acids pattern liberated upon simulated enzymatic hydrolysis. The present study aimed to evaluate the effect of digested milk, with particular regard to its fatty acid composition, from human, donkey, bovine, ovine, caprine and formula milk on mature adipocytes 3T3-L1. In particular cellular viability, apoptosis, oxidative response and gene expression levels of P65 NF-κB, HMGB1, SREBP-1c and FAS were evaluated.
Materials and methods
Milk treatments
Commercial brands of bovine milk (BM), caprine milk (CM), and liquid formula milk (FM) were purchased at a local store. Ovine milk (OM) and donkey milk (DM) were taken at a dairy farm located in Foggia (Apulian region, Italy) and pasteurized (63 °C for 30 min). Five lactating women (1–3 months after delivery) were recruited for human milk (HM) samples collection. Gross composition of the milk samples is reported in online Supplementary Table 1S. Milk sources were previously subjected to in vitro digestion according to Minekus et al. (Reference Minekus, Alminger, Alvito, Ballance, Bohn, Bourlieu, Carriere, Boutrou, Corredig, Dupont, Dufour, Egger, Golding, Karakaya, Kirkhus, Le Feunteun, Lesmes, Macierzanka, Mackie, Marze, McClements, Menard, Recio, Santos, Singh, Vegarud, Wickham, Weitschies and Brodkorb2014). Free fatty acids in digested milk sources were analyzed as described in Santillo et al. (Reference Santillo, Figliola, Ciliberti, Caroprese, Marino and Albenzio2018) and are reported in online Supplementary Table 2S.
Cell culture conditions
Murine 3T3-L1 fibroblasts were propagated and differentiated as described by Lin et al. (Reference Lin, Berg, Iyengar, Lam, Giacca, Combs, Rajala, Du, Rollman, Li and Hawkins2005). Briefly, the cells were propagated in DM0 (DMEM containing 5 mm glucose, 10% FBS -fetal bovine serum-, and penicillin/streptomycin [100 units/ml each]) and allowed to reach confluence. After 2 d (day 0), the medium was changed to DM1 (DMEM containing 10% FBS and 160 nm insulin, 250 µm dexamethasone, and 0.5 mm 3-isobutyl-1-methylxanthine). Two days later (day 2), the medium was switched to DM2 (DMEM containing FBS 10% and 160 nm insulin). After another 2 d, the cells were switched backed to DM0 for 2 d. Finally 3T3-L1 adipocytes were treated with BSA medium (DMEM medium add BSA 500 µm) conjugated with 2% of different sources digested milk, previously filtered through 0.2 mm filter. Control group (CTR) was treated only with BSA medium. Time of contact was 24 h for XTT and ROS assay and 48 h for gene expression analysis.
Cell viability
Cell viability was determined with XTT Cell Proliferation Assay Kit (ATCC, Manassas, VA). Briefly, the cells were maintained in BSA medium added with digested milk from different sources for 24 h, then washed with PBS and incubated for 4 h with activated-XTT solution. The absorbance was read using a spectrophotometer at 630 nm and subtracted from the 450 nm values to eliminate non-specific readings. The viability was expressed as the percentage relative to absorbance of BSA.
Caspase 3–7 assay
Apoptotic assay was performed using CellEvent™ Caspase-3–7 Green Detection Reagent (Molecular Probes-Life Technology) a fluorogenic substrate for activated caspase-3–7. Briefly, 3T3-L1 adipocytes were incubated with 5 µm of CellEvent™ Caspase-3–7 Green Detection Reagent at 37 °C for 30 min. The excitation/emission was 502/530 nm, measured using a Wallac 1420 Fluorescent Plate Reader.
Measurement of ROS generation
Treated cells seeded in a 96-well plate were incubated with 10 µmol/l CM-H2DCFDA (Molecular Probes-Life Technology, Brooklyn, NY) for 45 min at 37 °C, and the intracellular formation of ROS was measured at excitation/emission wavelengths of 485/530 nm using a Wallac 1420 Fluorescent Plate Reader (D'Apolito et al., Reference D'Apolito, Colia, Lasalvia, Capozzi, Falcone, Pettoello-Mantovani, Brownlee, Maffione and Giardino2017).
RT reaction and real-time quantitative PCR
Total RNA from treated cells was extracted using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). Total RNA was then isolated following the manufacturer's instructions. The mRNA was reverse transcribed by SuperScript III First Strand Synthesis System (Invitrogen, Carlsbad, USA). Experiments were performed in quadruplicate in optical 96-well reaction plates on a CFX96 Touch Real-Time PCR detection System using iQ SYBR green supermix (Bio-Rad Laboratories Inc., California, USA). Experiment setup and data analysis were performed using CFX manager software. Expression levels of NF-κB p65, HMGB1, SREBP-1c and FAS were normalized to β-actin and GAPDH levels in the same sample. Melting curves were analyzed to ensure that fluorescence signals solely reflected specific amplicons. PCR conditions were as follows: 7 min at 95 °C and 45 cycles of 30 s at 95 °C and 30 s at 60 °C (D'Apolito et al., Reference D'Apolito, Du, Zong, Catucci, Maiuri, Trivisano, Pettoello-Mantovani, Campanozzi, Raia, Pessin, Brownlee and Giardino2010).
Statistical analysis
Data on the effect of different digested milk treatments on mature adipocytes 3T3-L1 cellular viability, apoptosis, oxidative response and gene expression levels of P65 NF-κB, HMGB1, SREBP-1c and FAS were analyzed using ANOVA for repeated measures (SAS Institute, 2011). Where significant effects were found (P < 0.05), the Student's t-test was used to locate significant differences between means.
Results and discussion
3T3-L1 adipocytes viability and apoptosis
The cytotoxic effect of digested human, bovine, caprine, ovine and donkey milk and of liquid commercial formula on mature 3T3-L1 adipocytes, detected by using XTT assay, is reported in Table 1 (and graphically in online Supplementary Figure S2). Treatments with digested milk samples were compared to each other as percentages, keeping the control group as 100% of viability. Digested milk samples reduced cell viability compared with the control group; however, no significant differences were observed among different sources of digested milk. Cell death may be ascribed to different mechanisms such as necrosis or apoptosis. Apoptosis is an important biological process by which the body removes aged cells during physiological or pathological conditions (Sergeev, Reference Sergeev2009) and it is described as an active, programed process of autonomous cellular dismantling that avoids eliciting inflammation. Necrosis has been characterized as passive, accidental cell death resulting from environmental perturbations with uncontrolled release of inflammatory cellular contents. In order to evaluate the mechanism by which digested milk reduced cell viability the caspase 3–7 activity was evaluated. Caspase-3–7 are effector proteins that, activated by caspase initiators, induce apoptosis in cells (Fink and Cookson, Reference Fink and Cookson2005). Table 1 shows the effect of digested milk treatments on caspase 3–7 activity in 3T3-L1 mature adipocytes (graphical representation is given in online Supplementary Figure S3). All of the treatments significantly reduced the caspase 3–7 activity compared to that found in the control. However, no significant differences in caspase activity were found among digested milk treatments. Results on caspase activity were in accordance with cell viability and indicate a late stage of apoptotic events in the control group where the major presence of live cells was able to produce caspase.
Table 1. Effect of digested milk treatments on viability, caspase 3–7 activity and ROS concentration in 3T3-L1 mature adipocytes
CTR, control treatment; FM, digested formula milk; HM, digested human milk; DM, digested donkey milk; BM, digested bovine milk; OM, digested ovine milk; CM, digested caprine milk.
a NS, ***P < 0.001.
b Cell viability was expressed as % of control (BSA).
c Caspase 3–7 activity and ROS concentration were expressed as relative fluorescence units (RFU).
Level of ROS concentration
Since cell death is induced by oxidative stress (Navarro-Yepes et al., Reference Navarro-Yepes, Burns, Anandhan, Khalimonchuk, del Razo, Quintanilla-Vega, Pappa, Panayiotidis and Franco2014), we next investigated the effect of digested milk samples on ROS production in 3T3-L1 mature adipocytes, as shown in Table 1 (and graphically in online Supplementary Figure S4). In all digested milk treatments ROS level was higher than the control, however, the digested human and formula milk showed lower levels of ROS than DM, BM, OM and CM digested samples. Mitochondria are the main source of ROS due to cellular respiration; the respiratory process converts metabolic compounds such as carbohydrate, fat and protein to CO2 and H2O releasing various types of ROS at the same time. This event is opposed by endogenous cellular antioxidant mechanisms such as superoxide dismutase, catalase and glutathione peroxidase but when ROS output exceeds antioxidant defenses, the cell enters a state of oxidative stress (Rigoulet et al., Reference Rigoulet, Yoboue and Devin2011). The role of ROS in adipose tissue is complex. In preadipocytes the accumulation of mitochondrial ROS could inhibit cell proliferation (Carrière et al., Reference Carrière, Fernandez, Rigoulet, Pénicaud and Casteilla2003, Reference Carrière, Carmona, Fernandez, Rigoulet, Wenger, Pénicaud and Casteilla2004; Wang and Hai, Reference Wang and Hai2015), whereas in mature adipocytes from obese rats high level of ROS were observed (Furukawa et al., Reference Furukawa, Fujita, Shimabukuro, Iwaki, Yamada, Nakajima, Nakayama, Makishima, Matsuda and Shimomura2017) and protection of adipocytes from oxidative stress is recognized as a potential clinical strategy in obesity treatment (Kusunoki et al., Reference Kusunoki, Yang, Yoshizaki, Nakagawa, Ishikado, Kondo, Morino, Sekine, Ugi, Nishio, Kashiwagi and Maegawa2013). In this study the lower capacity of HM and FM to induce oxidative stress in mature adipocyte could be ascribed to the peculiar free fatty acid profile of digested milk. The activity of PPARγ, involved in adipogenic pathways, can be influenced by fatty acids transported into the adipocytes (Fernyhough et al., Reference Fernyhough, Okine, Hausman, Vierck and Dodson2007). Caprilic acid has been shown to induce ROS generation and might also modulate PPARγ activity indirectly via the ROS signaling pathways (Guo et al., Reference Guo, Xie and Han2006). Accordingly, digested milk from ruminant species showed a mean content of free caprilic acid higher than FM and HM (6.69, 12.22, 5.17 µg/ml for bovine, ovine and caprine respectively, vs. 4.85, 3.5 µg/ml of extract for formula and human milk respectively), and this could partly explain the lower ROS content in the former samples. Palmitic acid also represents an inflammatory mediator in the adipose tissue inducing white adipose tissue expansion and increasing inflammation through oxidative stress (Kennedy et al., Reference Kennedy, Martinez, Chuang, LaPoint and McIntosh2009), palmitic acid being found in high levels in digested donkey milk. On the other hand, many natural lipid compounds with anti-inflammatory and antioxidant effects have been used to treat obesity such as n-3 PUFA, EPA and DHA, MUFA and CLA (Kusunoki et al., Reference Kusunoki, Yang, Yoshizaki, Nakagawa, Ishikado, Kondo, Morino, Sekine, Ugi, Nishio, Kashiwagi and Maegawa2013; Chang et al., Reference Chang, Wu and Hsu2015). Here we observed higher free oleic and linoleic acid levels in digested human and formula milk; oleic acid 1.5- and 6-folds higher and linoleic acid 2- and 5.5-folds higher than that found in milk from ruminant species, respectively.
Gene expression of NF-κB p65 and HMGB1
Since ROS are key signaling molecules that play an important role in the progression of inflammatory disorders (Mittal et al., Reference Mittal, Siddiqui, Tran, Reddy and Malik2014), NF-κB p65 and HMGB1 were evaluated. The effect of digested milk source on the expression of NF-κB p65 and HMGB1 in 3T3-L1 mature adipocyte are shown in Fig. 1a and 1b, respectively. All digested milk elicited a significant over-expression of NF-κB p65 in 3T3-L1 adipocytes compared to the control. Among treatments, the lowest gene expression was found in HM, BM, OM and CM, the highest in FM and an intermediate behavior was shown in DM. The p65 subunit of NF-κB is readily activated when cells are stimulated with various agents such as inflammatory cytokines, LPS, oxidative or shear stress (Yang et al., Reference Yang, Hori, Takahashi, Kawabe, Kato and Okamoto1999), and exogenous administration of SFA was reported to exert a pro-inflammatory effect that plays a major role in the activation of NF-κB (Suganami et al., Reference Suganami, Tanimoto-Koyama, Nishida, Itoh, Yuan, Mizuarai, Kotani, Yamaoka, Miyake, Aoe, Kamei and Ogawa2007). Inflammation induced in adipocytes by fatty acids is rather specific, in particular palmitate activates the NF-κB transcription factor in 3T3-L1 adipocyte and DHA and linoleate disrupt and prevent the NF-κB activation by palmitate (Ajuwon and Spurlock, Reference Ajuwon and Spurlock2005; Kennedy et al., Reference Kennedy, Martinez, Chuang, LaPoint and McIntosh2009). The gene expression of HMGB1 was significantly up-regulated in HM, FM, CM and in DM with the highest gene expression, while it was comparable to the control in BM and OM. HMGB1 is a group of non-histone DNA-binding proteins with the role of stabilizing the nucleosomes, to help DNA binding and then to take part in DNA replication, transcription and repair (Wang et al., Reference Wang, Xu, Xie, Yao, Shen, Wan, Zhang, Fu, Chen, Zou, Li and Zhang2016). Nevertheless, under signals like stress, cell death, infection or inflammation, HMGB1 is found to be released from adipose tissue especially from obese persons (Gunasekaran et al., Reference Gunasekaran, Viranaicken, Girard, Festy, Cesari, Roche and Hoareau2013). Excessive consumption of SFA and in particular palmitate expands adipose tissue and contributes to inflammation and weight gain (Kennedy et al., Reference Kennedy, Martinez, Chuang, LaPoint and McIntosh2009). HMGB1 promotes inflammation and its receptors interact with NF-κB p65 forming a positive feedback loop to sustain inflammatory conditions (Wang et al., Reference Wang, Xu, Xie, Yao, Shen, Wan, Zhang, Fu, Chen, Zou, Li and Zhang2016). Both NF-κB p65 and HMGB1 expression in 3T3-L1 mature adipocyte treated with digested donkey milk could be an outcome of the free fatty acid profile characterized by the highest content of SFA and palmitic acid, the absence of DHA, and low content of linoleic acid.
Fig. 1. Effect of digested milk treatments on the expression of NF-κB p65, HMGB1, SREBP-1c and FAS in 3T3-L1 mature adipocyte. The mRNA levels of (a) NF-κB p65, (b) HMGB1, (c) SREBP-1c and (d) FAS were measured by reverse transcription quantitative polymerase chain reaction. The mRNA expression levels of target genes were normalized using β-actin and GAPDH. Treatments not sharing a common letter differ significantly from one another (P < 0.05). CTR, control treatment; FM, digested formula milk; HM, digested human milk; DM, digested donkey milk; BM, digested bovine milk; OM, digested ovine milk; CM, digested caprine milk.
Gene expression of SRBP-1c and FAS
The effect of digested milk treatments on the expression of SRBP-1c and FAS in 3T3-L1 mature adipocyte are shown in Fig. 1c and 1d, respectively. All digested milk treatments influenced the gene over-expression of SRBP-1c with FM and HM showing the highest levels. For FAS expression, BM showed the highest level, OM and CM the intermediate and FM, HM and DM the lowest levels, however HM and DM had comparable levels to the control. These two genes are closely related although their expression is sequential. SREBP-1c is one of the transcription factors that are implicated in adipose tissue differentiation, also involved in lipid metabolism and regulation of lipid homeostasis by controlling the expression of genes required for fatty acids and lipids production such as FAS (Shimomura et al., Reference Shimomura, Hammer, Richardson, Ikemoto, Bashmakov, Goldstein and Brown1998; Jang et al., Reference Jang, Jung, Seo, Kim, Shim, Cho, Chung and Yoon2017).
In conclusion, all digested milk treatments decreased cell viability in mature adipocytes and induced cell death, partially due to apoptotic event, although no differences were observed among milk sources. In all digested milk treatments ROS level was increased relative to the control, however, 3T3-L1 mature adipocytes treated with digested human and formula milk showed lower levels of ROS probably due to the peculiar free fatty acid profile of these milks, yielding lower free caprilic acid after digestion. All digested milk treatments exerted a pro-inflammatory effect in mature adipocytes through over expression of HMGB1 and NF-κB p65 although lower gene expression was found in human milk and milk from ruminant species treatments. Finally, the study of SRBP-1c and FAS genes demonstrated a slight expression of the adipogenic pathway in adipocytes stimulated with different digested milk sources.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029919000104.
