<i>Lactobacillus rhamnosus GG</i> Protects against Non-Alcoholic Fatty Liver Disease in Mice

<div><p>Objective</p><p>Experimental evidence revealed that obesity-associated non-alcoholic fatty liver disease (NAFLD) is linked to changes in intestinal permeability and translocation of bacterial products to the liver. Hitherto, no reliable therapy is available except for weight reduction. Within this study, we examined the possible effect of the probiotic bacterial strain <i>Lactobacillus rhamnosus GG</i> (LGG) as protective agent against experimental NAFLD in a mouse model.</p><p>Methods</p><p>Experimental NAFLD was induced by a high-fructose diet over eight weeks in C57BL/J6 mice. Fructose was administered via the drinking water containing 30% fructose with or without LGG at a concentration resulting in approximately 5×10⁷ colony forming units/g body weight. Mice were examined for changes in small intestinal microbiota, gut barrier function, lipopolysaccharide (LPS) concentrations in the portal vein, liver inflammation and fat accumulation in the liver.</p><p>Results</p><p>LGG increased beneficial bacteria in the distal small intestine. Moreover, LGG reduced duodenal IκB protein levels and restored the duodenal tight junction protein concentration. Portal LPS (P≤0.05) was reduced and tended to attenuate TNF-α, IL-8R and IL-1β mRNA expression in the liver feeding a high-fructose diet supplemented with LGG. Furthermore liver fat accumulation and portal alanine-aminotransferase concentrations (P≤0.05) were attenuated in mice fed the high-fructose diet and LGG.</p><p>Conclusions</p><p>We show for the first time that LGG protects mice from NAFLD induced by a high-fructose diet. The underlying mechanisms of protection likely involve an increase of beneficial bacteria, restoration of gut barrier function and subsequent attenuation of liver inflammation and steatosis.</p></div

<i>Lactobacillus rhamnosus GG</i> elevates bacterial numbers in the distal small intestine.

<p>Total bacterial numbers (A), LGG numbers (B), the phyla Firmicutes (C), and Bacteriodetes (D) were measured via qPCR. Data are shown as means ± SEM (**P<0.01, ***P<0.001; <i>n</i> = 5–6). Abbreviations: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080169#pone-0080169-g001" target="_blank">Figure 1</a>.</p

Nutritional and weight parameters.

<p>Animal groups: C, control diet; F, high-fructose diet; CLGG control diet supplemented with <i>Lactobacillus rhamnosus GG</i> (LGG); FLGG, F supplemented with LGG; ALT, alanine-aminotransferase. The detailed feeding protocols of the four animal groups are described in material and methods. Data are shown as means ± SEM (<i>n</i> = 6).</p>a<p>P<0.05 compared to C;</p>b<p>P<0.05 compared to F.</p

<i>Lactobacillus rhamnosus GG</i> improves diet-induced NAFLD.

<p>LGG ameliorates high-fructose diet-induced NAFLD via modulation of the intestinal microbiota. LGG products e.g. lactate may increase butyrate producing Firmicutes leading to an improved intestinal barrier and reduced portal plasma LPS concentrations as well as a decreased inflammation and fatty acid accumulation in the liver. Abbreviations: NAFLD, non-alcoholic fatty liver disease; LPS, lipopolysaccharides.</p

Effect of High Sugar Intake on Glucose Transporter and Weight Regulating Hormones in Mice and Humans

<div><p>Objective</p><p>Sugar consumption has increased dramatically over the last decades in Western societies. Especially the intake of sugar-sweetened beverages seems to be a major risk for the development of obesity. Thus, we compared liquid versus solid high-sugar diets with regard to dietary intake, intestinal uptake and metabolic parameters in mice and partly in humans.</p><p>Methods</p><p>Five iso-caloric diets, enriched with liquid (in water 30% vol/vol) or solid (in diet 65% g/g) fructose or sucrose or a control diet were fed for eight weeks to C57bl/6 mice. Sugar, liquid and caloric intake, small intestinal sugar transporters (GLUT2/5) and weight regulating hormone mRNA expression, as well as hepatic fat accumulation were measured. In obese versus lean humans that underwent either bariatric surgery or small bowel resection, we analyzed small intestinal GLUT2, GLUT5, and cholecystokinin expression.</p><p>Results</p><p>In mice, the liquid high-sucrose diet caused an enhancement of total caloric intake compared to the solid high-sucrose diet and the control diet. In addition, the liquid high-sucrose diet increased expression of GLUT2, GLUT5, and cholecystokinin expression in the ileum (P<0.001). Enhanced liver triglyceride accumulation was observed in mice being fed the liquid high-sucrose or -fructose, and the solid high-sucrose diet compared to controls. In obese, GLUT2 and GLUT5 mRNA expression was enhanced in comparison to lean individuals.</p><p>Conclusions</p><p>We show that the form of sugar intake (liquid versus solid) is presumably more important than the type of sugar, with regard to feeding behavior, intestinal sugar uptake and liver fat accumulation in mice. Interestingly, in obese individuals, an intestinal sugar transporter modulation also occurred when compared to lean individuals.</p></div

Primers used for mRNA detection.

<p>TNF-α, tumor necrosis factor alpha; IL-1β, interleukin 1 beta; h, human; IL-8R, interleukin 8 receptor; ChREBP, carbohydrate response-element binding protein; ACC1, acetyl-CoA carboxylase 1; FAS, fatty acid synthase.</p

<i>Lactobacillus rhamnosus GG</i> improves markers of intestinal barrier function.

<p>The tight junction molecules occludin (A), and claudin-1 (B), as well as the inflammatory marker pIκB kinase (C) were analysed in the proximal intestine. Representative western blots and quantitative analyses of the blots are shown (A–C). LPS in portal plasma was measured (D). Data are shown as means ± SEM (*P<0.05, **P<0.01; <i>n</i> = 4–6). Abbreviations: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080169#pone-0080169-g001" target="_blank">Figure 1</a>; LPS; lipopolysaccharides.</p

<i>Lactobacillus rhamnosus GG</i> ameliorates fructose-induced hepatic fatty acid accumulation.

<p>Representative Oil-Red-O (A) and hematoxilin & eosin (B) stainings showing fat accumulation in the liver. Abbreviations: C, control diet; F, high-fructose diet; CLGG, control diet with <i>Lactobacillus rhamnosus GG</i> (LGG) supplementation; FLGG, F with LGG supplementation.</p

<i>Lactobacillus rhamnosus GG</i> modulates fructose-induced hepatic fatty acid metabolism.

<p>Hepatic ChREBP (A), ACC1 (B) and FAS (C) mRNA expression was measured. Concentrations of triglycerides in the liver were analysed (D). Data are shown as means ± SEM (**P<0.01; ***P<0.001; <i>n</i> = 4–6). Abbreviations: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080169#pone-0080169-g001" target="_blank">Figure 1</a>; ChREBP, carbohydrate response element-binding protein; ACC1, acetyl-CoA carboxylase 1; FAS, fatty acid synthase.</p

Obese humans showed an increased small intestinal sugar transporter expression compared to normal weight humans.

<p>Small intestinal hGLUT2, hGLUT5 and hCCK mRNA expression was detected (A/B/C). Data are shown as means ± SEM (*P<0.05; **P<0.01; n = 12–20). hGLUT2/5, human glucose transporter 2/5; hCCK, human cholecystokinin; Ob, obese; Nw, normal weight.</p