Alters Gene Expression in Obese Mice
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International Dairy Journal 90 (2019) 15e22 Contents lists available at ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj Milk fermented with Lactobacillus casei NCDC19 improves high fat and sucrose diet alters gene expression in obese mice * S. Jangra , 1, R.K. Sharma, R. Pothuraju, G. Bhakri Department of Animal Biochemistry, ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India article info abstract Article history: Dietary incorporation of milk fermented with indigenous probiotic Lactobacillus casei NCDC19 (Lc19) was Received 22 May 2018 investigated in mice fed a high-fat and sucrose diet (HFS diet). Epididymal fat mass and adipocyte size Received in revised form were significantly reduced in the group fed HFS diet supplemented with Lc19 compared with the group 9 November 2018 fed HFS diet only. However, body weight differences were not statistically significant. Fasting blood Accepted 11 November 2018 glucose, serum triglyceride, insulin and homeostatic model assessment of insulin resistance score were Available online 1 December 2018 found to be significantly decreased in the Lc19 treatment group; liver triglyceride levels remained un- affected. Epididymal fat adiponectin and liver carnitine palmitoyl-transferase 1, peroxisome proliferator- activated receptor a, forkhead box protein A2 and peroxisome proliferator-activated receptor gamma coactivator 1b were significantly up-regulated in the Lc19 group. The influence of Lc19 on the expression of genes related to energy expenditure suggests a possible role of L. casei in gut physiology modulation, which might be affecting the host metabolism. © 2018 Elsevier Ltd. All rights reserved. 1. Introduction lactobacilli and Streptococcus thermophilus) was shown to attenuate diet-induced obesity, hepatic steatosis and insulin resistance by Probiotics are defined as live microorganisms that confer increasing hepatic natural killer T cells and reducing inflammatory health-benefits upon the host when administered in an adequate signalling (Ma et al., 2008). Lactobacillus gasseri SBT2055 reduced amount (FAO/WHO, 2002). They are an important class of func- adipocyte size by inhibiting dietary fat absorption (Hamad et al., tional foods that have attracted consumers’ attention because of 2009) and anti-obesity effects through improvement of inflam- their potential effectiveness in prevention/treatment of hyper- matory state in adipose tissue (Miyoshi, Ogawa, Higurashi, & cholesterolaemia (Shin et al., 2010), certain cancers (Kumar et al., Kadooka, 2014). L. gasseri BNR17 has also been reported to atten- 2010) and immune diseases (Borchers, Selmi, Meyers, Keen, & uate fat accumulation in body by increasing energy expenditure Gershwin, 2009). In addition, certain reports have also demon- through up-regulating the expression of genes involved in fatty strated the beneficial effects of some bacterial strains on obesity acid b-oxidation (Kang et al., 2013). Lactobacillus casei NCDC19 (Kang et al., 2013; Park et al., 2013; Pothuraju, Sharma, exerts anti-obesity effects by up-regulating the expression of adi- Chagalamarri, Kavadi, & Jangra, 2015; Rather et al., 2014) and in- ponectin in epididymal fat, and bifidobacterial counts in the colon sulin resistance (Ma, Hua, & Li, 2008; Okubo, Takemura, Yoshida, & (Rather et al., 2014). However, the exact mechanism of action of Sonoyama, 2013). probiotics has not been completely elucidated as yet. Different probiotics have different capacities to modulate Moreover, beneficial effects of probiotics have been reported to metabolic phenotypes. Lactobacillus rhamnosus PL60 and Lactoba- be strain dependent (Fak & Backhed, 2012; Qiao et al., 2015; Yin, Yu, cillus plantarum PL62 have been reported to exert beneficial effects Fu, Liu, & Lu, 2010). There are also reports of probiotics indicating on diet-induced obesity through production of CLA (Lee et al., 2006, no effects on body weight gain (Arora et al., 2012), and also in some 2007). VSL#3 (mixture of viable lyophilised bifidobacteria, cases probiotics have been reported to cause weight gain in rodents (Andersson et al., 2010; Yin et al., 2010). Evidently, the contradic- tory reports suggest that more studies are necessary to delineate * Corresponding author. Tel.: þ91 94 67094 023. the effects of probiotics belonging to different species/strains. E-mail address: [email protected] (S. Jangra). L. casei NCDC19 is an indigenous strain that has been reported to 1 Present address: School of Bioengineering and Biosciences, Lovely Professional exhibit potential probiotic attributes (Kapila, Vibha, & Sinha, 2006) University, Phagwara, Punjab, India. https://doi.org/10.1016/j.idairyj.2018.11.002 0958-6946/© 2018 Elsevier Ltd. All rights reserved. 16 S. Jangra et al. / International Dairy Journal 90 (2019) 15e22 and has not been fully explored especially in relation to adiposity, Table 1 a insulin resistance and related gene expression. Fermented milk is Composition of experimental high-fat and sucrose diet. an important vehicle for delivery of probiotics. Various studies are Ingredient Content (%) available on animals and humans involving the administration of Casein 19.5 probiotic through fermented milk (Hamad et al., 2009; Kadooka, L-Methionine 0.3 2010; 2013; Sato et al., 2008). Therefore, the present investigation Sucrose 28.78 was carried out to evaluate the effects of dietary supplementation Corn starch 15.0 of milk fermented with probiotic L. casei NCDC19 (Lc19) on Milk fat 21.0 Soybean oil e adiposity, insulin resistance and expression of genes related to lipid Cellulose 10.52 metabolism in C57BL/6 mice. Mineral mixture (AIN-76) 3.5 Vitamin mixture (Teklad) 1.0 2. Materials and methods CaCO3 0.4 Ethoxyquin 0.004 Fat (% kcal) 42.63 À 2.1. Preparation of Lactobacillus casei NCDC19 fermented milk Total energy (kcal 100 g 1 diet) 443.3 a Casein was a product of M/s Modern Dairies, Karnal (India); cow milk fat L. casei NCDC19 was obtained from the National Collection of (ghee) prepared by experimental dairy, NDRI, Karnal (India). Dairy Cultures, Dairy Microbiology Division, National Dairy Research Institute, Karnal, India. Before preparation of probiotic fermented milk, the culture was activated by sub-culturing three times in skim milk. Thereafter, fermented milk was prepared by 2.3. Analysis of blood parameters inoculating (1%, v/v) the sterile skim milk with activated culture, and incubation at 37 C for 20e22 h. Fermented milk was prepared At the end of study, blood obtained by cardiac puncture was on alternate days and stored at 4 C. Viable counts in fermented transferred into non-heparinized tubes. Tubes were kept undis- milk were determined by pour plating method as per formula: turbed at room temperature for 30 min, centrifuged at 2000 Âg for À cfu g 1 ¼ (no. of colonies  dilution factor)/volume of sample 15 min at 4 C, and serum was collected. Serum total cholesterol plated. (TC), HDL-C and triglyceride (TG) levels were determined using Fermented skim milk (Lc19) contained viable counts in the commercial kits (Span Diagnostics Pvt. Ltd., Surat, India). LDL-C À range of 8.5e9.0 log cfu g 1. concentration was calculated by using Friedewald's equation (Friedewald, Levy, & Fredrickson, 1972), and VLDL-C calculated by 2.2. Experimental animals and diets dividing triglycerides value by 5. Serum insulin level was measured by using ultrasensitive mouse insulin ELISA kit (Crystal Chem Inc., All animal experiments were conducted at the small animal Elk Grove Village, IL, USA). Serum PYY was measured by using house, National Dairy Research Institute, India, in accordance with Human/Mouse/Rat enzyme immunoassay kit (RayBiotech Inc., the guidelines of Institutional Animal Ethics Committee. Eighteen Norcross, GA, USA). Homeostatic model assessment of insulin C57BL/6 male mice (10e12 weeks old) were purchased from Indian resistance (HOMA-IR) was calculated using the following equation Institute of Integrative Medicine, Jammu, India. Mice were kept in (Haffner, Miettinen, & Stern, 1997): À ventilated plastic cages with soft husk bedding at controlled room HOMA-IR ¼ fasting insulin (mUmL1)  fasting glucose À temperature (22 ± 2 C). The animals were acclimatised for 2 weeks (mmol L 1)/22.5. during which they were fed on normal chow ad libitum and had free access to water. After the acclimatisation period animals were 2.4. Analysis of liver lipids randomised according to their body weights, divided into 3 groups ¼ (n 6 animals per group; 2 mice per cage), and assigned on Analysis of liver lipids was done following the method described experimental diets as follows: (i) HFS group, received high-fat and by Morton et al. (2005). Briefly, liver tissue was homogenised in sucrose diet (HFS diet) ad libitum; (ii) HFS-SM group, received skim isopropanol (1:10), and then shaken for 45 min. The supernatant milk along with HFS diet; (iii) HFS-FM group, received Lc19 along obtained by centrifugation at 3000 Âg for 10 min was used for with HFS diet. The composition of HFS diet is shown in Table 1. Skim À analysis with a commercial kit (Span diagnostics Pvt. Ltd.). milk comprised (g 100 mL 1): protein, 3.6, carbohydrate 4.9, and À fat, 0.3; energy content was 35 kcal 100 mL 1. Skim milk/Lc19 was provided around 9:30 a.m. and removed 2.5. Histological examination of epididymal fat tissue around 4:30 p.m. During this period, HFS diet and water was fi removed so that the animals consumed the skim milk/Lc19. HFS diet Epididymal fat tissue was xed in 10% (v/v) formalin and sent to and water