A dramatic increase in obesity percentage is becoming not only a serious public health problem, but also a global scale economic problem. Obesity contributes to a high risk of CVD, type-2 diabetes, and hypertension. Moreover, emerging data show that obesity is associated with immune dysfunction and increased susceptibility to bacterial and viral infections( Reference Huttunen and Syrjanen 1 ). Accordingly, the rise in medical costs associated with obesity will likely result in serious economic impact in many countries.
Diet plays an important role in the control of obesity without significant economic costs. Among the many dietary approaches for controlling obesity, nutritional energy restriction is effective for short-term body weight control and body fat loss. However, this approach carries a risk of relapse( Reference Elfhag and Rossner 2 ). An alternative approach, which produces a long-term effect, is the increased intake of Ca and dairy products( Reference Zemel 3 ). In fact, many human observational studies and clinical trials have so far suggested an inverse relationship between intake of dietary Ca or dairy products and body fat( Reference Lin, Lyle and McCabe 4 – Reference Zemel, Richards and Milstead 9 ). The underlying theory is that dietary Ca down-regulates intracellular Ca2+ in adipocytes by reducing the level of parathyroid hormone and vitamin D (1,25(OH)2D), thereby modulating lipid metabolism and TAG accumulation in adipocytes( Reference Zemel 10 – Reference Shi, Norman and Okamura 12 ). In addition, dairy products act as an efficient source of dietary Ca as well as other bioactive substances such as angiotensin-converting enzyme inhibitors and branched-chain amino acids, which are mainly present in whey proteins. Thus, dairy consumption may increase the intake of dietary Ca and other active ingredients, thereby modulating energy metabolism, and attenuating the risk of obesity.
In the aforementioned studies, non-fat dried milk (NFDM) and/or normal yogurt were utilised as dairy products to examine the anti-obesity effects. A previous study showed that there was no remarkable difference in the anti-obesity effects of NFDM and normal yogurt( Reference Sun and Zemel 13 ). A recent development is the addition of probiotics to fermented milk products, such as yogurt. Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host( Reference Hill, Guarner and Reid 14 ). There are a wide variety of health-promoting effects of probiotics, such as the prevention and/or treatment of diarrhoea, infectious diseases and allergic diseases, in a strain-dependent manner( Reference Szajewska and Mrukowicz 15 – Reference Yoda, Miyazawa, Harata, Kitazawa, Villena and Alvarez 18 ). However, emerging data suggest that probiotics may also confer anti-obesity effects( Reference Mekkes, Weenen and Brummer 19 ). Given the fact that probiotic dairy products are commonly consumed, the anti-obesity effects of probiotic dairy products should be compared with those of normal dairy products.
The present study was designed to evaluate the influence of fermentation of milk with probiotics and supplementation of milk with whey proteins on the anti-obesity effects of dietary Ca and dairy products, using aP2-agouti transgenic mice as the test model. Mice of the aP2-agouti transgenic type are susceptible to diet-induced obesity, and exhibit a human pattern of expression of obesity-related genes( Reference Bruckbauer and Zemel 20 ). These mice can be maintained at a normal weight on a standard AIN-93G diet( Reference Reeves 21 ), but can characteristically develop mild to moderate obesity (25–40 % increase in adipose tissue mass), when fed high sucrose/fat diets for 6 to 12 weeks. In previous studies, aP2-agouti transgenic mice were commonly used to examine the short-term effects of Ca on obesity and oxidative and inflammatory stress( Reference Shi, Dirienzo and Zemel 11 – Reference Sun and Zemel 13 ). Fermented milk supplemented with whey proteins was prepared using probiotic strains of Lactobacillus rhamnosus GG, Lactobacillus paracasei TMC0409 (TMC0409) and Streptococcus thermophilus TMC1543. These probiotic strains and whey proteins demonstrated considerable anti-obesity effects in previous studies( Reference Tabuchi, Tamura and Yamada 22 – Reference Kawase, Hashimoto and Hosoda 27 ). The fermented milk product was tested for its anti-obesity effects, using the aP2-agouti murine model.
Materials and methods
Fermented milk
Fermented milk was supplemented with 1 % whey protein concentrate 80, and was fermented with probiotic strains of L. rhamnosus GG, L. paracasei TMC0409 and S. thermophilus TMC1543 (Takanashi Milk Products Company Limited). There were more than 10 million viable cells of each strain in 1 ml of this product. After fermentation, probiotic-fermented milk (PFM) was lyophilised and stored at − 80°C until use. The lyophilised PFM was composed of 36·8 % protein, 0·8 % fat and 49·3 % carbohydrates, and contained 1469 kJ (351 kcal) of total energy/100 g of the powder.
Mice and diets
The present study was subdivided into two phases. Phase I was designed to induce weight gain and fat accretion, and Phase II involved assessment of weight and fat loss as described below. The study utilised aP2-agouti transgenic mice from our colony. Three to four animals were housed together in polypropylene cages at a room temperature of 22 ± 2°C and a 12 h light–12 h dark cycle throughout the experimental period. The mice had free access to water. The present study was approved by the Institutional Care and Use Committee of The University of Tennessee.
Phase I
Male aP2-agouti transgenic mice of 6 weeks of age (n 45) were subjected to a basal (standard) diet for 6 weeks, consisting of a modified AIN-93G diet with suboptimal Ca intake (0·4 %), a soy protein isolate as the protein source, sucrose as the sole carbohydrate source providing 62 % of energy and fat supplying 24 % of energy intake with lard serving as the supplemental fat source (Table 1). The mice were monitored for 6 weeks, during which food intake and spillage were measured daily, and body weight, fasting blood glucose and food consumption were assessed weekly.
Table 1 Mouse diet composition
NFDM, non-fat dried milk; PFM, probiotic-fermented milk.
* Mineral and vitamin mix from AIN-93G diet( Reference Reeves 21 ).
Phase II
At the end of the Phase I duration of 6 weeks, five randomly selected representative mice were killed under isofluorane anaesthesia for baseline measurements. The remaining forty mice, weighing an average of 29·85 (se 0·39) g, were randomised into four groups (ten per group). Each group followed one of four different diets: (1) the basal diet, ad libitum feeding; (2) the basal diet, restricted to 70 % of the energy intake of the ad libitum group by using the pair-fed protocol; (3) the basal diet, with NFDM substituted for soy as a protein source, Ca increased to 1·2 % and energy restricted to 70 % of the energy intake of the ad libitum group and (4) the basal diet, with the PFM substituted for soy as the protein source, Ca increased to 1·2 % and energy restricted to 70 % of the energy intake of the ad libitum group. Macronutrient levels in these diets were adjusted to ensure that the protein, carbohydrate and fat contents were identical to those of the basal diet used as a control (Table 1). At the end of the 6 weeks, all the forty mice were killed under isofluorane anaesthesia. The fat pads, skeletal muscles (soleus and gastrocnemius) and livers were excised, weighed and used for further study as described below.
Lipolysis assay
Perirenal adipose tissue was immediately dissected, washed several times with Hank's Balanced Salt Solution, minced into small pieces and digested with 0·8 g/l type I collagenase in a shaking Water-bath at 37°C for 30 min. Adipocytes were then filtered through sterile 500-μm nylon mesh, and cultured in Dulbecco's Modified Eagle's Medium supplemented with 1 % foetal bovine serum for 4 h. Glycerol released into the culture medium was used to measure lipolysis, as described previously( Reference Xue, Moustaid and Wilkison 28 ). Glycerol was measured using the 1-step enzymatic fluorometric method( Reference Boobis and Maughan 29 ).
Fatty acid synthase activity assay
Fatty acid synthase (FAS) activity in crude cytosolic extracts of mouse adipose tissue was determined spectrophotometrically. Mouse abdominal fat pads were homogenised in a 250 mm-sucrose solution containing 1 mm-EDTA, 1 mm-dithiothreitol and 100 μm-phenylmethylsulfonyl fluoride (pH 7·4). The homogenate was centrifuged at 18 500 g for 1 h, and the infranatant was used for measuring the oxidation rate of NADPH. The final FAS activity was normalised to DNA, which was measured using a CyQUANT cell proliferation assay kit, according to the manufacturer's instructions (Packard Instrument Company).
Total RNA extraction
A total cellular RNA isolation kit (Ambion, Inc.) was used to extract total RNA from murine abdominal adipose tissue and soleus muscle, according to the manufacturer's instruction.
Quantitative real-time PCR
Quantitative real-time PCR was performed to measure the expression levels of genes related to lipid metabolism and insulin sensitivity in adipocytes and skeletal muscle, as described previously( Reference Sun and Zemel 13 , Reference Bruckbauer and Zemel 20 , Reference Sun and Zemel 30 ). Briefly, murine abdominal adipose tissue FAS, 11β-hydroxysteroid dehydrogenase type I (11β-HSD1), glucocorticoid receptor 1 (GR1) mRNA, and mouse soleus muscle PPARα, PPARδ, mitochondrial uncoupling protein 3 (UCP3) and GLUT type 4 (GLUT4) mRNA were quantified using an ABI 7300 Real-Time PCR System (Applied Biosystems) with a TaqMan 1000 Core Reagent Kit (Applied Biosystems). The primers and probe sets were ordered from Applied Biosystems TaqMan Assays-on-Demand Gene Expression Collection of primers and probes, and used according to the manufacturer's instructions. Pooled total RNA from adipocytes was serially diluted in the range of 1·6–25·0 ng, and used to establish a standard curve; total RNA from unknown samples were also diluted in this range. Reactions of quantitative real-time PCR for standards and unknown samples were performed according to the instructions of the ABI 7300 Real-Time PCR System and the TaqMan 1000 Core Reagent Kit. The mRNA quantification data from each sample were normalised to 18S levels.
Measurement of blood chemistry markers
Immediately after killing the mice, blood was collected via cardiac puncture and was centrifuged forthwith under refrigeration to obtain plasma for the measurement of insulin, malondialdehyde (MDA), TNF-α, adiponectin, monocyte chemotactic protein-1 and C-reactive protein. Insulin and adiponectin levels were assessed using the Mouse Insulin and the Adiponectin ELISA Kits, respectively (Linco Research). MDA was assessed using the TBARS Assay Kit (ZeptoMetrix Corporation). The level of TNF-α was measured using the specific Mouse TiterZyme ELISA Kits from Assay Designs, Inc. Plasma C-reactive protein levels were measured using the Mouse C-reactive protein ELISA Kit (Life Diagnostics, Inc.).
Statistical analysis
All data were expressed as means with their standard errors. The data were evaluated for statistical significance using one-way ANOVA, and the identification of a significantly different group depended upon separation in the least-significant-difference test, conducted with the SPSS software (SPSS, Inc.). Differences with P< 0·05 were considered statistically significant.
Results
Mice that were fed the basal (0·4 % Ca/high-sucrose/high-fat) diet exhibited a 49 % weight gain during phase II, and energy restriction in those mice resulted in a reduction of >3 % of body weight (Fig. 1(a)). The mice that were fed the PFM diet showed the most significant weight reduction among the mice subjected to energy restriction; furthermore, the PFM diet resulted in the greatest suppression of fat accumulation (Fig. 1(b)).
Fig. 1 Body weight change (a) and fat mass (b) in mice fed each diet after phase II. Values are means, with their standard errors represented by vertical bars (n 10). Mean value was significantly different from that of the energy-restricted group fed the 0·4 % calcium diet: † P< 0·05, †† P< 0·01. Mean value was significantly different from that of the energy-restricted group fed the 1·2 % calcium non-fat dried milk (NFDM) diet: ‡ P< 0·05, ‡‡ P< 0·01.
Although energy restriction treatment did not influence lipolysis in the adipose tissue, mice that were fed a high-Ca/dairy-based (NFDM or PFM) diet exhibited a significant increase in lipolysis compared with mice that were fed low-Ca diets. The PFM diet showed a greater increase in adipose tissue lipolysis than did the NFDM diet (Fig. 2(a)). FAS activity in the adipose tissue was significantly decreased by energy restriction, Ca supplementation and the change in protein sources (Fig. 2(b)). The expression of the FAS gene was also decreased by Ca supplementation and by replacement of protein sources (data not shown). Furthermore, the PFM diet suppressed expression of the FAS gene more than did the NFDM diet.
Fig. 2 Effect of each diet on adipose tissue lipolysis (a) and fatty acid synthase (FAS) activity (b). Values are means, with their standard errors represented by vertical bars (n 10). Mean value was significantly different from that of the basal diet-fed group: * P< 0·05, ** P< 0·01. †† Mean value was significantly different from that of the energy-restricted group fed the 0·4 % calcium diet (P< 0·01). Mean value was significantly different from that of the energy-restricted group fed the 1·2 % calcium non-fat dried milk (NFDM) diet: ‡ P< 0·05, ‡‡ P< 0·01.
Dairy-based diets produced a significant decrease in the expression of a gene encoding 11β-HSD1, an enzyme related to the glucocorticoid pathway and glucocorticoid receptor (Fig. 3). The PFM diet down-regulated the 11β-HSD1 gene expression in adipose tissue to a greater degree than did the NFDM diet (Fig. 3(a)). Both diets triggered comparable effects on GR1 expression (Fig. 3(b)).
Fig. 3 Effect of each diet on mRNA expression of genes encoding 11β-HSD1 (11β-hydroxysteroid dehydrogenase type I) (a) and GR1 (glucocorticoid receptor 1) (b) in skeletal muscle of mice fed each diet. Values are means, with their standard errors represented by vertical bars (n 10). Mean value was significantly different from that of the energy-restricted group fed the 0·4 % calcium diet: † P< 0·05, †† P< 0·01. ‡ Mean value was significantly different from that of the energy-restricted group fed the 1·2 % calcium non-fat dried milk (NFDM) diet (P< 0·05).
In the skeletal muscle, the expressions of genes encoding PPARα, PPARδ and UCP3, which regulate fat oxidation, were significantly increased in mice that were fed the PFM diet as compared with other mice (Fig. 4).
Fig. 4 mRNA expression of genes encoding PPAR-α (a), PPAR-δ (b) and UCP3 (uncoupling protein 3) (c) in skeletal muscle of mice fed each diet. Values are means, with their standard errors represented by vertical bars (n 10). Mean value was significantly different from that of the energy-restricted group fed the 1·2 % calcium non-fat dried milk (NFDM) diet: ‡ P< 0·05, ‡‡ P< 0·01.
To examine the indices of insulin sensitivity, expression of the gene encoding GLUT4 in skeletal muscle (Fig. 5(a)) and the circulating insulin level (Fig. 5(b)) were measured. In mice that were fed the PFM diet, GLUT4 expression was significantly increased compared with the other mice. The PFM diet also caused a greater decrease in the plasma insulin level than did the NFDM diet.
Fig. 5 Effect of each diet on GLUT4 expression (a) and plasma insulin level (b) in skeletal muscle. Values are means, with their standard errors represented by vertical bars (n 10). † Mean value was significantly different from that of the energy-restricted group fed the 0·4 % calcium diet (P< 0·05). ‡ Mean value was significantly different from that of the energy-restricted group fed the 1·2 % calcium non-fat dried milk (NFDM) diet (P< 0·05). To convert insulin in μU/ml to pmol/l, multiply by 6·945.
The PFM diet significantly increased the plasma level of adiponectin (Fig. 6(a)), and down-regulated inflammatory cytokines TNF-α, monocyte chemotactic protein-1 and C-reactive protein (Fig. 6(b)–(d)). Plasma MDA, a marker of oxidative stress, was significantly decreased in mice subjected to energy restriction compared with mice that were fed the basal diet (Fig. 6(d)). The PFM diet caused a greater decrease of the plasma MDA level than did the other diets.
Fig. 6 Effects of each diet on indices of inflammatory and oxidative stress in plasma. The production levels of adiponectin (a), TNF-α (b), monocyte chemotactic protein-1 (MCP-1) (c), C-reactive protein (CRP) (d) and malondialdehyde (MDA) (e) in plasma. Values are means, with their standard errors represented by vertical bars (n 10). † Mean value was significantly different from that of the energy-restricted group fed the 0·4 % calcium diet (P< 0·05). ‡ Mean value was significantly different from that of the energy-restricted group fed the 1·2 % calcium non-fat dried milk (NFDM) diet (P< 0·05).
Discussion
The present study demonstrated that milk fermented by three selected probiotic strains and supplemented with whey proteins significantly augmented the anti-obesity effects of Ca and dairy products in energy-restricted aP2-agouti transgenic mice. The PFM diet reduced body weight and body fat, and modulated adipose tissue lipid metabolism, such as the promotion of lipolysis and the suppression of FAS activity, to a greater degree than did the NFDM diet. In contrast, energy restriction affected just the reduction of body weight and body fat, but not lipolysis and FAS gene expression (data not shown) in adipose tissue. Similar results were observed in a previous study( Reference Shi, Dirienzo and Zemel 11 ). Considering the fact that obesity is associated with abnormal lipid metabolism( Reference Hu, Zhang, Song and Baez 31 ), it is important to formulate methods of counteracting abnormal lipid metabolism, so as to intrinsically control obesity. The previous study additionally demonstrated that dietary Ca and NFDM played a key role in the modulation of lipid metabolism in adipose tissue under energy restriction( Reference Shi, Dirienzo and Zemel 11 ). Since both the PFM diet and the NFDM diet had the same composition of Ca in the present study, it is observed that fermentation of milk with probiotics and/or supplementation of milk with whey proteins, promote the modulatory effect of dietary Ca and dairy products on lipid metabolism.
To assess in clear terms the modulatory effect of the PFM diet on lipid metabolism, the adipocyte glucocorticoid pathway was analysed. 11β-HSD1 is an enzyme that converts inactive cortisone to cortisol, the principal active glucocorticoid, resulting in activation of the glucocorticoid pathway. In adipose tissue, glucocorticoids promote the differentiation of pre-adipocytes into adipocytes, which could lead to increased body fat mass( Reference Wang 32 ). Moreover, the elevation of adipose 11β-HSD1 activity is observed in obese subjects( Reference Rask, Walker and Soderberg 33 ). In the present study, the PFM diet significantly suppressed the gene expression of 11β-HSD1 compared with the NFDM diet. On the other hand, there was no difference between the two diets in the expression level of the gene encoding GR, which works downstream of 11β-HSD1 in the glucocorticoid pathway. The PFM diet may inhibit the activated glucocorticoid pathway in adipose tissue through the suppression of 11β-HSD1 gene expression, resulting in a greater reduction of body fat than that observed with the NFDM diet.
The most impressive outcomes of the present study were that the PFM diet up-regulated the expression of genes related to fatty acid oxidation, such as PPAR and UCP3 and up-regulated glucose uptake via GLUT4 in skeletal muscle in contrast to the NFDM diet. Inefficient muscle fatty acid utilisation promotes fat storage above oxidation, leading to increased adipose tissue and TAG storage within skeletal muscle, thereby promoting obesity and insulin resistance( Reference Blaak 34 ). In obese subjects, abnormality of GLUT4 expression inhibits the transport of plasma glucose into skeletal muscle cells, resulting in an increased plasma insulin level. In fact, the present study showed that the PFM diet decreased plasma insulin level significantly as compared with the NFM diet. Therefore, the PFM diet may increase energy consumption in skeletal muscle and enhance the anti-obesity effects of dietary Ca and dairy products.
Obesity is associated with chronic inflammation and oxidative stress( Reference Fantuzzi 35 , Reference Furukawa, Fujita and Shimabukuro 36 ). Adipose tissue normally releases into circulation anti-inflammatory cytokines (which also regulate glucose levels and fatty acid breakdown) such as adiponectin( Reference Jaziri, Aubert and Roussel 37 ), and pro-inflammatory cytokines such as TNF-α, and maintains the balance between these anti- and pro-inflammatory cytokines. Nonetheless, the enlarged adipocytes observed in obese subjects produce more of TNF-α and monocyte chemotactic protein-1, which migrate and infiltrate monocytes/macrophages( Reference Deshmane, Kremlev and Amini 38 ), leading to chronic inflammation and oxidative stress. The significant increase in plasma adiponectin levels in mice that were fed the PFM diet indicated that the PFM diet induced an anti-inflammatory response. This anti-inflammatory response was consistent with the up-regulation of the expression of genes related to fatty acid oxidation and glucose uptake in skeletal muscle. The PFM diet also markedly reduced plasma C-reactive protein and MDA, which are suitable markers for indicating the level of inflammation and oxidative stress, respectively. These outcomes were consistent with the significant reduction of plasma TNF-α and monocyte chemotactic protein-1 observed in mice that were fed the PFM diet. Therefore, the results of the present study suggest that the PFM diet regulates the balance between anti-inflammatory and inflammatory response and suppresses oxidative stress in obese mice.
The present study did not assess the contribution in isolation of each of the probiotic strains and whey proteins to the above-mentioned effects of the PFM diet. For instance, L. rhamnosus GG, is known to increase the expression of genes related to fatty acid oxidation and glucose uptake in skeletal muscle, and reduce plasma insulin level in murine models( Reference Kim, Park and Kim 39 , Reference Tabuchi, Ozaki and Tamura 40 ). Whey proteins, which are enriched in branched-chain amino acids, are, in addition, considered as potent up-regulators of fatty acid utilisation in skeletal muscle( Reference Sun and Zemel 30 , Reference Sun and Zemel 41 , Reference Arakawa, Masaki and Nishimura 42 ). Therefore, further studies are needed to clarify precisely the contribution of each of these factors as a potent enhancer of the anti-obesity effects of dietary Ca and dairy products.
In summary, the present study demonstrated the new finding that fermentation of milk with selected probiotics and supplementation of milk with whey proteins may promote the anti-obesity effects of dietary Ca and dairy products during nutritional energy restriction in aP2-agouti mice; and this may be done via regulation of lipid and glucose metabolisms and via reduction of oxidative and inflammatory stress. Data from this study also provide the possibility of developing an effective strategy for the control of weight and obesity without high medical costs.
Acknowledgements
The authors would like to thank Enago (www.enago.jp) for the English language review.
This work was supported by Takanashi Milk Products Company Limited.
K. Y., M. K., A. K., K. M., G. H., M. Hosoda, M. Hiramatsu and F. H. are employees of Takanashi Milk Products Company Limited. X. S. and M. B. Z. have no competing interests. The authors have no conflicts of interest. The funding agency had no role in the design, conduct, findings or the writing of this research report.
M. B. Z., M. K., M. Hiramatsu, and F. H. designed this study. X. S. and M. B. Z. conducted the animal study. K. Y., M. K., A. K., K. M., G. H., M. Hosoda and F. H. provided technical support and prepared the fermented milk. K. Y., X. S., M. K., K. M., and F. H. analysed the data. K. Y. and F. H. wrote and revised the manuscript.
