Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on Hepatic Steatosis in Zucker Rats

Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on Hepatic Steatosis in Zucker Rats. PLoS ONE 9(5): e98401. doi:10.1371/journal. pone.0098401 Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on Hepatic Steatosis in Zucker Rats Julio Plaza-Diaz 0 Carolina Gomez-Llorente 0 Francisco Abadia-Molina 0 Maria Jose Saez-Lara 0 Laura Campan a-Martin 0 Sergio Mun oz-Quezada 0 Fernando Romero 0 Angel Gil 0 Luis Fontana 0 Jose Luis Balcazar, Catalan Institute for Water Research (ICRA), Spain 0 1 Department of Biochemistry & Molecular Biology II, School of Pharmacy, University of Granada , Granada , Spain , 2 Institute of Nutrition & Food Technology ''Jose Mataix'', Biomedical Research Center, University of Granada , Granada , Spain , 3 Department of Cell Biology, School of Sciences, University of Granada , Granada , Spain , 4 Department of Biochemistry & Molecular Biology I, School of Sciences, University of Granada , Granada , Spain , 5 Hero Global Technology Center , Hero Spain, S.A., Alcantarilla, Murcia , Spain We have previously described the safety and immunomodulatory effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 in healthy volunteers. The scope of this work was to evaluate the effects of these probiotic strains on the hepatic steatosis of obese rats. We used the Zucker rat as a genetic model of obesity. Zucker-Leprfa/fa rats received one of three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo for 30 days. An additional group of Zucker-lean+/fa rats received a placebo for 30 days. No alterations in intestinal histology, in the epithelial, lamina propria, muscular layers of the ileal or colonic mucosa, or the submucosae, were observed in any of the experimental groups. Triacylglycerol content decreased in the liver of ZuckerLeprfa/fa rats that were fed L. rhamnosus, B. breve, or the mixture of B. breve and L. paracasei. Likewise, the area corresponding to neutral lipids was significantly smaller in the liver of all four groups of Zucker-Leprfa/fa rats that received probiotics than in rats fed the placebo. Zucker-Leprfa/fa rats exhibited significantly greater serum LPS levels than Zuckerlean+/fa rats upon administration of placebo for 30 days. In contrast, all four groups of obese Zucker-Leprfa/fa rats that received LAB strains exhibited serum LPS concentrations similar to those of Zucker-lean+/fa rats. Serum TNF-a levels decreased in the Zucker-Leprfa/fa rats that received B. breve, L. rhamnosus, or the mixture, whereas L. paracasei feeding decreased IL-6 levels in the serum of Zucker-Leprfa/fa rats. In conclusion, the probiotic strains reduced hepatic steatosis in part by lowering serum LPS, and had an anti-inflammatory effect in obese Zucker rats. - Funding: Part of the research currently in progress in the authors laboratory is funded by the company Hero Spain, S. A. through the grant #3545 managed by the Fundacion General Empresa-Universidad de Granada. No additional external funding was received for this study. The funders played a role in the study design, but had no role in the decision to publish or preparation of the manuscript. Competing Interests: Part of the research currently in progress in the authors laboratory is funded by the company Hero Spain, S. A. through the grant #3545 managed by the Fundacion General Empresa-Universidad de Granada. Fernando Romero is employed by Hero Global Technology Center, Hero Spain, S.A. This center is part of the food company HERO, headquartered in Switzerland. There are no patents, products in development or marketed products to declare. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. Obesity has reached pandemic levels and is becoming a serious health problem worldwide. In developing countries, the prevalence of obesity has tripled over the last 20 years owing to the adoption of a Western lifestyle (i.e., low physical activity levels, sedentariness, and excessive eating) [1,2]. The increasing prevalence of type 2 diabetes (T2D), cardiovascular disease, and certain types of cancer is linked to obesity. Thus, approximately 90% of T2D cases are attributable to excess body weight, and 200 million people worldwide are estimated to have glucose intolerance and insulin resistance syndrome (IRS), a figure that is expected to rise to 420 million by the year 2025 [3]. Recent evidence indicates that the intestinal microbiota plays a crucial role in body weight and fat mass [46], and, accordingly, suggests an association between the gut microbiota and T2D [7 9]. Probiotics are live microorganisms that confer a health benefit on the host when administered in adequate amounts [10], although dead bacteria and bacterial molecular components may also exhibit probiotic properties. Strains belonging to Bifidobacterium and Lactobacillus are the most widely used probiotic bacteria and are included in many functional foods and dietary supplements [1113]. Probiotics can modulate the gut microbiota and the mucosal immune system [1416]. For probiotics to be successful, they must exhibit certain characteristics: i.e., tolerance to gastrointestinal conditions (gastric acid and bile), ability to adhere to the gastrointestinal mucosa, and competitive exclusion of pathogens [17,18]. We have previously described the isolation of three lactic acid bacterial (LAB) strains from the feces of exclusively breast-fed newborn infants. These strains were selected based on their probiotic properties, such as adhesion to intestinal mucus, sensitivity to antibiotics and resistance to biliary salts and low pH. We identified these strains as Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035, and Lactobacillus rhamnosus CNCM I-4036 [19]. Recently, we demonstrated the tolerance and safety of these three strains in a multi-centre, randomized, double-blind, placebocontrolled trial with healthy volunteers [20]. Oral administration of these LAB strains modified the bacterial populations in the feces of the volunteers, and all three strains exerted varying degrees of immunomodulatory effects [20]. Thus, administration of B. breve CNCM I-4035 resulted in a significant increase in fecal secretory IgA content. In addition, IL-4 and IL-10 was increased, whereas IL-12 was decreased, in the serum of volunteers treated with any of the three strains. A large body of evidence has highlighted the concept that putative intestinal bacteriaderived compounds may affect liver metabolism and, therefore, cause systemic diseases [6,21,22]. Serum LPS levels have been proposed to increase upon obesity and steatosis, leading to a metabolic endotoxemia capable of modulating proinflammatory cytokines, as well as glucose and lipid metabolism in the (...truncated)