Obesity has become a public health dilemma that has steadily increased in the past few decades. It is a risk factor for various metabolic disturbances, including type 2 diabetes, dyslipidemia and cardiovascular disease [1
]. It is associated with an expansion of adipose tissue due to excessive energy intake and lack of exercise, resulting in an abnormal accumulation of body fat [2
Mammals were previously believed to have two major types of adipose tissue, called white adipose tissue (WAT) and brown adipose tissue (BAT), which showed difference in function and morphology [3
]. WAT stores excessive energy as triglycerides, whereas BAT dissipates chemical energy as heat [4
]. Uncoupling protein 1 (UCP1), which is specifically expressed in BAT, plays an important role in energy expenditure, and promoting its expression could reduce obesity and metabolic dysfunction [5
]. Recently, “brown-like” adipocytes were identified in WAT depots in response to certain stimuli, and these adipocytes were referred to as beige or ‘brite’ (brown in white) adipocytes [6
]. Similar to classic brown adipocytes, beige adipocytes in WAT are composed of multilocular/small lipid droplets and high amounts of mitochondria that contain UCP1 and can ‘burn’ fat [7
]. Browning of WAT is inducible and promoted by dietary supplements such as olive oil, resveratrol and capsaicin [8
]. Thus, the induction of browning in WAT represents an attractive strategy to combat obesity and its complications.
Adipogenesis is a differentiation process during which the mesenchymal precursor cells develop into mature adipocytes [9
]. The differentiation of preadipocytes into adipocytes requires the sequential expression of adipogenic transcription factors, including peroxisome proliferators-activator receptor-γ (PPARγ), CCAAT/enhancer-binding protein-α (C/EBPα) and sterol regulatory element-binding protein-1c (SREBP-1c) [10
]. AMP-activated kinase (AMPK) is a master regulator in control of energy homeostasis and lipid metabolism, and its activation inhibits the expression of PPARγ and C/EBPα and adipogenesis [11
]. Phosphorylation of AMPK suppresses lipid synthesis by inactivating lipogenic genes in adipose tissue, such as acetyl-CoA carboxylase (ACC), a key enzyme of lipogenesis that synthesizes malonyl-coenzyme A (malonyl-CoA) from acetyl-CoA [12
]. Therefore, inhibition of preadipocyte proliferation and differentiation might be an effective strategy for preventing obesity and its associated metabolic disorders.
Milk fat globule membrane (MFGM), a tri-layer membrane consisting of proteins and lipids, is derived from the apical surface of mammary epithelial cells, which surrounds the fat globules in milk [13
]. Several benefits of MFGM components, such as anti-cancer, antibacterial and anti-inflammatory, have been reported [14
]. Recently, MFGM has been attracting much attention in protection against obesity and related metabolic disorders. Milk sphingomyelin, one of the major polar lipids in MFGM, significantly inhibited intestinal lipid absorption and reduced serum and liver lipid levels in obese/diabetic KK-Ay mice [15
]. Milk sphingomyelin also improved lipid metabolism by reducing hepatic triglycerides and inducing gene expression associated with cholesterol biosynthesis in high-fat diet-fed mice [16
]. In addition to functions attributed to its individual components, clinical trials in overweight and obese individuals also showed that the addition of a dairy fraction rich in MFGM reduced postprandial cholesterol levels, improved insulin response and increased the production of anti-inflammatory markers, which attenuated the negative effects of a high-saturated-fat meal [17
]. In addition, milk fat globule membrane in the diet of young mice reduced body fat accumulation against obesity in later life [18
]. However, whether MFGM could alleviate obesity by altering adipogenesis or brown remodeling of WAT is not fully understood.
The objective of the present study was to investigate the effect of MFGM on adipogenesis and WAT browning in high-fat diet-induced mice.
2. Materials and Methods
Lacprodan® MFGM-10 was obtained from Arla Co. (Sønderhøj, Viby J, Denmark). Antibodies against C/EBPα, SREBP-1c and β-actin were purchased from Bioss Inc. (Bios, Beijing, China) and antibodies against PPARγ, AMPK, p-AMPK, ACC and p-ACC were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody against UCP1 was obtained from Abcam (Cambridge, UK).
2.2. Animals and Treatments
Male C57BL/6J mice (5-week-old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were housed under 12 h light/dark cycle and a constant temperature (24 °C). After one-week acclimation, mice were subsequently divided into two groups randomly. The mice in the different groups were fed, respectively, a normal chow diet (ND group, KeAoXieLi Feed Co., Ltd., Beijing, China) (n = 10) or a high-fat diet (KeAoXieLi Feed Co., Ltd., Beijing, China) (n = 40) for 8 weeks. The normal chow diet provided 3.01 kcal/g of energy (25.75% calories from protein, 13.40% calories from fat and 60.85% calories from carbohydrate), whereas the high-fat diet provided 5.24 kcal/g of energy (20.05% calories from protein, 60.08% calories from fat and 20.04% calories from carbohydrate). Then mice fed high-fat diet were randomly divided into 4 groups (n = 10/group) and fed with high-fat diet (HFD group, n = 10), high-fat diet with MFGM at 100 mg/kg BW (M100 group, n = 10), 200 mg/kg BW (M200 group, n = 10) and 400 mg/kg BW (M400 group, n = 10) for another 8-week. Body weight and food intake were monitored every other day. At the end of the experimental period, mice were anesthetized after fasting and blood was taken to collect serum. The adipose tissues were removed, rinsed with physiological saline solution, weighed and stored at −80 °C until analysis. The animal protocols (Approval No. KY160018) were approved by the Animal Experimentation Committee of China Agricultural University (Beijing, China).
2.3. Western Blot Analysis
Adipose tissues were homogenized in an ice cold lysis buffer (Beyotime Biotech, Haimen, Jiangsu, China) containing 1% NP-40, 0.5% deoxycholate, 0.1% SDS and 1% protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The homogenate was centrifuged at 12,000× g for 10 min at 4 °C and the supernatant was collected. Protein concentration in samples was determined using BCA Protein Assay Reagent (Beyotime Biotech, Haimen, Jiangsu, China). The 20 µg of protein of each sample was electrophoretically separated in 7.5% to 12.5% sodium dodecyl sulfate polyacrylamide gel and then transferred to polyvinylidene diflouride membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% (w/v) skimmed milk powder in tris-buffered saline supplemented with 0.05% (v/v) Tween 20 (TBST) for 2 h and then incubated with primary antibodies overnight at 4 °C. The membranes were next incubated with horseradish-peroxidase-conjugated secondary antibodies at room temperature for 1 h. The bands were visualized with enhanced chemiluminescence (ECL) reagents (Millipore, Billerica, MA, USA), and quantified densitometrically using the software ImageJ 1.47v (Wayne Rasband, Bethesda, MD, USA).
2.4. Total RNA Isolation and Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA from epididymal WAT was extracted with Trizol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The cDNA was synthesized from total RNA (3 µg) in a 25 μL reaction volume using the TIANScript RT Kit (Tiangen Biotech, Beijing, China). Real-time PCR was performed using 1 μL cDNA with the SYBR Premix Ex Taq RT-PCR kit (Takara, Otsu, Shiga, Japan) on Techne Quantica real-time PCR detection system (Techne, Staffordshire, UK). The thermal profile settings were starting with a denaturation at 95 °C for 180 s followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s. The SYBR green fluorescence was read at the end of each extension step (72 °C). Relative expression levels of the mRNA of the target genes were normalized to 18S mRNA levels. PCR sequences of various genes are presented in Table 1
2.5. Measurement of Serum Biochemical Parameters
Serum was separated after blood sampling by centrifugation at 1500× g for 15 min. The concentrations of serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C) cholesterol and free fatty acids (FFA) were determined using commercially diagnostic kits supplied by R&D Systems (Minneapolis, MN, USA) according to the manufacturer’s instructions.
2.6. Histological Analysis
Epididymal WAT was dissected, washed in saline and immediately fixed in 4% paraformaldehyde. Fixed tissues were embedded in paraffin and 4 μm sections were prepared, stained with hematoxylin and eosin (H&E) for general morphological observations. Images were taken at 200× magnification. The adipocyte sizes of white adipose tissue were quantitated using Photoshop CS6 (Adobe Systems Inc., San Jose, CA, USA). Adipocyte volume (v) was calculated as the formulation v = πd3
/6 and the adipocyte weight equal volume multiplied by density (about 0.915 g/cm3
]. Adipocyte number was determined by dividing the epididymal WAT weight by the average adipocyte weight.
2.7. Statistical Analysis
All data were presented as means ± S.E.M. (the standard error of the mean). Significance for multiple comparisons was determined using one-way ANOVA followed by Duncan corrections. Statistical significance was established at p < 0.05 level. All statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA).
Obesity and metabolic diseases are rapidly becoming major global health problems. Consequently, research to identify natural products for the prevention and treatment of obesity is gaining more and more attention [20
]. MFGM is a bioactive component of milk which possesses various health benefits but its anti-obesity effect remains unclear. In the current study, we found that MFGM suppressed HFD-induced obesity in mice, which is attributable to the inhibition of adipogenic differentiation by the activation of AMPK pathway. MFGM also upregulated the UCP1 protein expression in both BAT and inguinal WAT. These findings suggest that MFGM supplementation could mitigate obesity by inhibiting adipogenesis, improving browning of WAT and increasing BAT activity.
Obesity, defined as an excess abnormal accumulation of body fat mass, leads to a series of metabolic complications [21
]. C57BL/6 mice fed with high-fat diet are widely used to assess the development of obesity [22
]. Chronic administration of a high-fat diet to C57BL/6 mice resulted in obesity development [23
]. In the present study, mice fed with HFD induced obesity while MFGM supplement mitigated the body weight gain without influencing the food intake.
Adipose tissue is an important contributor to energy storage and energy utilization [24
]. However, excess adipose tissue can have many negative effects on an individual’s health [25
]. Over-nutrition leads to the expansion of adipose tissue which is associated to the increase in the size and number of adipocytes, and eventually causes adipocyte hypoxia, inflammation and insulin resistance [26
]. In our study in mice, high-fat diet increased fat pad weights and the size of adipocytes with the elevation of serum TG levels. After administration of MFGM, the mice had smaller adipocytes in epididymal WAT and the TG levels in serum was closer to that in the ND group. These observations are consistent with previous reports that high-fat diet-induced obese mice exhibited a modest hypertriglyceridaemia characterized by increases in TC, LDL-C and HDL-C levels and reduction in the ratio of HDL-C/LDL-C. MFGM treatment also lowered the levels of TC, HDL-C and LDL-C in serum and up-regulated the ratio of HDL-C/LDL-C. These observations suggested that MFGM reduces weight gain is partially due to the alleviation of lipid accumulation in WAT and the improvement of lipid profiles.
Adipogenesis can be separated into adipogenic commitment and differentiation [27
]. Several transcription factors, including PPARγ, C/EBPa and SREBP-1c, play roles during the differentiation of pre-adipocytes [28
]. Among these factors, PPARγ has been recognized as a key nuclear receptor transcription factor in the control of adipogenesis. It regulates the expressions of the genes related to fatty acid synthesis, oxidation and adipogenesis, such as ACC, FAS and aP2 [29
]. PPARγ mediates high-fat diet-induced adipocyte differentiation, whereas PPARγ-deficient mice were protected from adipocyte hypertrophy under a high-fat diet [30
]. C/EBPα is another transcription factor and promotes adipogenesis in the early phase of preadipocyte differentiation. PPARγ and C/EBPα reinforce each other to drive adipogenic differentiation [31
]. Besides, SREBP-1c can activate PPARγ by increasing its expression and by stimulating the production of its ligands [32
]. We found that the mRNA expression levels of PPARγ, C/EBPα, SREBP-1c, FAS, aP2 and ACC were remarkably increased in mice fed high-fat diet, but these genes were significantly decreased when treated with different concentrations of MFGM, showing its inhibitory effects on adipogenic differentiation.
AMP-activated protein kinase (AMPK) is a key signaling protein in the regulation of cellular energy homeostasis, lipid metabolism and mitochondrial biogenesis [33
]. AMPK activation in adipose tissue suppresses PPARγ and inhibits adipogenesis, which consequently reduces fat accumulation [34
]. The phosphorylation of AMPK also enhances fatty acid oxidation by the inactivation of lipogenic enzymes such as ACC [35
]. Dietary bioactive compounds such as sulforaphane [11
], oxyresveratrol [36
] and green tomato extract [37
] prevent high-fat diet-induced obesity and reduce lipid accumulation by stimulating AMPK activity. Consistent with these reports, MFGM administration induced a statistically significant increase in the phosphorylation of AMPK and ACC accompanied by the upregulation of PPARγ, C/EBPα and SREBP-1c protein expression.
Browning of WAT is an attractive strategy for combating obesity and associated metabolic diseases [7
]. Beige adipocytes express UCP1 and other protein machinery involved in thermogenesis. UCP1, which dissipates the proton gradient across the mitochondrial inner membrane to produce heat, is generally considered as a crucial marker of browning of WAT and BAT activity. The browning of WAT is inducible and promoted by many factors including adipokines, myokines, cytokines, and dietary factors [8
]. Curcumin, a naturally occurring curcuminoid of turmeric with anti-obesity effect, induces a brown-like phenotype in both 3T3-L1 and primary white adipocytes [38
]. Berberine increased the expression of UCP1 and other thermogenic genes in white and brown adipose tissue of mice [6
]. Consistent with these findings, MFGM also significantly upregulated the protein expression of UCP1 in BAT and inguinal WAT, suggesting the potential role of MFGM on BAT activity and browning of inguinal WAT. However, the exact mechanism of MFGM on the browning of WAT needs further investigation.