Obesity - Inflammation - Metabolic Disease: Effect of Lactobacillus Casei Shirota

Obesity and metabolic syndrome are linked by inflammation. Gut flora seems to play an important role in the development of inflammation and metabolic syndrome in obesity. Modulation of gut flora by probiotics has been shown in animal studies to positively influence inflammation and metabolic disturbances. Lactobacillus casei Shirota is able to decrease metabolic endotoxemia by altering gut flora composition and gut permeability which leads to an improvement in neutrophil function and insulin resistance in obesity. The aim of the current study is to investigate the effect of Lactobacillus casei Shirota supplementation over 12 weeks on neutrophil function (phagocytosis, oxidative burst and TLR expression) in patients with metabolic syndrome. Furthermore the investigators aim to investigate the effect of Lactobacillus casei Shirota supplementation over 12 weeks on glucose tolerance, insulin resistance, inflammation, gut flora composition, gut permeability, and endotoxemia in metabolic syndrome

Obesity and metabolic disorders (type 2 diabetes and insulin resistance) are tightly linked to inflammation. Obesity, a pandemic affecting 30-50% of the adult population, is mediated by a variety of genetic and environmental factors. It is well described that cytokines cause insulin resistance which causes hyperinsulinemia and excessive fat storage in adipose tissue and the liver. However, the triggering factor, linking inflammation to metabolic syndrome has not been fully elucidated yet. Recently it has been hypothesized that the gut flora is an important factor in this vicious cycle of obesity, metabolic disease and inflammation. Firstly, metabolic activities of the gut microbiota facilitates the extraction of calories from ingested dietary substances and helps to store these calories in host adipose tissue for later use. Second, the gut bacterial flora of obese mice and humans include fewer Bacteroidetes and correspondingly more Firmicutes than that of their lean counterparts, suggesting that differences in caloric extraction of ingested food substances may be due to the composition of the gut microbiota. Furthermore, bacterial lipopolysaccharide derived from the intestinal microbiota may trigger inflammation, linking it to high-fat diet-induced metabolic syndrome. High-fat diet induces insulin resistance and oxidative stress in mice and is associated with increased gut permeability. high fat diet induces a low-grade endotoxemia in mice ("metabolic endotoxemia) and infusing endotoxin causes weight gain and insulin resistance. This has also been shown in humans, where patients with fatty liver had a susceptibility to higher gut permeability, possibly causing increased endotoxin levels. Endotoxin and Lipopolysaccharide-binding protein (LBP) is elevated in obese patients, patients with type 2 diabetes and patients with liver steatosis. Endotoxin causes a significant increase in proinflammatory cytokine production in adipocytes via a TLR mediated pathway, contribution to the proinflammatory state in obesity. Endotoxin levels correlate with adiponectin and insulin suggesting a pathophysiological link between obesity, inflammation and metabolic disease. As described above, endotoxin is related to increased inflammation and oxidative stress, causing insulin resistance. Adipocytes have been shown to play a dynamic role in regulation of inflammation by producing cytokines via a Toll-like receptor (TLR)/Nuclear Factor kappa B (NFkB) mediated pathway.But not only adipocytes are in a proinflammatory state - also circulating mononuclear cells have been described to be activated. Clinical evidence suggests immune dysfunction in obesity, since obese patients are more prone to infections after surgery, higher incidence of lower respiratory infection which is also underlined by impairment of cell-mediated immune responses in vivo and in vitro and a reduced intracellular killing by neutrophils. A similar situation has been recently described in alcoholic cirrhosis and alcoholic hepatitis, which is also a proinflammatory condition with impaired innate immunity, leading to infection. Endotoxin has been described as a key mediator and inadequate activation of neutrophils leading to high oxidative burst and energy depletion of the cells with consecutive impaired phagocytic capacity has been described. The most effective therapy of obesity - weight loss - leads to significant improvement of mononuclear cell activation. However, there is no data available on the effect of weight loss on gut flora, gut permeability and endotoxin. Since weight loss is usually very hard to achieve, other therapeutic strategies have been tested. Since gut flora seems to be crucial in the development of the vicious cycle of obesity, inflammation and metabolic disease, several studies tried to modify the composition of gut microbiota. In mice treatment with antibiotics improved glucose tolerance by altering expression of genes involved in inflammation and metabolism. A similar result was found in mice treated with a probiotic that increases the number of Bifidobacterium spp., which leads to improved glucose tolerance, insulin secretion and a decrease in inflammatory tone. Finally treatment of mice with a probiotic decreased hepatic insulin resistance via a C-Jun N-terminal Kinase (JNK) and NFkB pathway, supporting the concept that intestinal bacteria induce endogenous signals that play a pathogenic role in hepatic insulin resistance. Among the vast amount of bacteria described to alter gut flora and exert positive effects on the host, we have chosen to study Lactobacillus casei Shirota several reasons: Firstly this commercially available preparation delivers a high bacterial number in a relatively small volume and is available as a palatable milk drink. Furthermore Lactobacillus casei Shirota has been proven to survive the passage through the stomach and is present in the lower intestinal tract. It has also been shown that this bacterial strain can increases the amount of Lactobacilli and decreases the number of gram-negative organisms in the bacterial flora. This bacterial strain has been shown to be effective in modulating natural killer cell function and neutrophil function. We hypothesize that Lactobacillus casei Shirota is able to decrease metabolic endotoxemia by altering gut flora composition and gut permeability which leads to an improvement in neutrophil function and insulin resistance in obesity Specific Aims: To investigate the effect of Lactobacillus casei Shirota supplementation over 12 weeks on neutrophil function (phagocytosis, oxidative burst and TLR expression) in patients with metabolic syndrome. To investigate the effect of Lactobacillus casei Shirota supplementation over 12 weeks on glucose tolerance, insulin resistance, inflammation, gut flora composition, gut permeability, and endotoxemia in metabolic syndrome Plan of investigations: Patients: 30 Patients with metabolic syndrome and increased gut permeability will be randomized to either receive food supplementation with a milk drink containing Lactobacillus casei Shirota (3 bottles a day, 65 ml each, containing Lactobacillus casei Shirota at a concentration of 10^8 colony forming units/ml) for twelve weeks or standard medical therapy.

The Phagotest® (Orpegen Pharma, Heidelberg, Germany) is used to measure phagocytosis by using Fluorescein isothiocyanate (FITC)-labelled opsonized E. coli bacteria.

The Phagotest® (Orpegen Pharma, Heidelberg, Germany) is used to measure phagocytosis by using FITC-labelled opsonized E. coli bacteria. The Phagoburst® kit (Orpegen Pharma, Heidelberg, Germany) is used to determine the percentage of neutrophils that produce reactive oxidants with or without stimulation.

change in indices of glucose tolerance and insulin resistance (frequently sampled in an oral glucose tolerance test) at baseline and after 3 months Homeostasis model assessment (HOMA)- Insulin Resistance (IR): HOMA is calculated by [fasting glucose*fasting insulin/22.5] insulin (U/L), glucose (mmol/l) - higher values indicating more severe insulin resistance Quantitativer Insulin Sensitivitäts-Check Index (QUCIKI): QUICKI is calculated by [1/log (insulin0)+log(glucose0)] insulin (mU/L), glucose (mg/dL) - lower values indicating a improvement of insulin sensitivity Insulin Sensitivity Index (ISI): 0.222-00333*BMI - 0.0000779*Ins120 -0.0004222*age insulin (mU/L) lower values indicating a improvement of insulin sensitivity

Inclusion Criteria: Age >18 Informed consent Fasting blood glucose >95mg/dL Metabolic syndrome defined by the National Cholesterol Education Program (NCEP) Adult Treatment Panel-III (ATP-III) -ATPIII criteria (3 out of 5) Abdominal obesity (waist circumference >102 in men or >88 in women) Elevated blood pressure (>135/>85) or drug treatment for elevated blood pressure Fasting blood glucose >100mg/dL or previously known type 2 diabetes mellitus, High Density Lipoprotein (HDL) cholesterol <40 mg/dL (men) or <50 mg/dL (women) or drug treatment for low HDL cholesterol Triglycerides >150 mg/dL or drug treatment for elevated for high triglycerides HbA1C ≤7.0% Exclusion Criteria: Drug treatment for diabetes mellitus Liver cirrhosis (biopsy proven) or elevated transaminases (>2x Upper Limit of Normla (ULN)) Inflammatory bowel disease (Crohns disease, ulcerative colitis) Celiac disease Alcohol abuse (more than 40g alcohol per day in the history) Clinical evidence of active infection Antibiotic treatment within 7 days prior to enrolment Use of immunomodulating agents within previous month (steroids etc.) Concomitant use of supplements (pre-, pro-, or synbiotics) likely to influence the study Any severe illness unrelated to metabolic syndrome Malignancy Pregnancy

Stadlbauer V, Leber B, Lemesch S, Trajanoski S, Bashir M, Horvath A, Tawdrous M, Stojakovic T, Fauler G, Fickert P, Hogenauer C, Klymiuk I, Stiegler P, Lamprecht M, Pieber TR, Tripolt NJ, Sourij H. Lactobacillus casei Shirota Supplementation Does Not Restore Gut Microbiota Composition and Gut Barrier in Metabolic Syndrome: A Randomized Pilot Study. PLoS One. 2015 Oct 28;10(10):e0141399. doi: 10.1371/journal.pone.0141399. eCollection 2015.

Leber B, Tripolt NJ, Blattl D, Eder M, Wascher TC, Pieber TR, Stauber R, Sourij H, Oettl K, Stadlbauer V. The influence of probiotic supplementation on gut permeability in patients with metabolic syndrome: an open label, randomized pilot study. Eur J Clin Nutr. 2012 Oct;66(10):1110-5. doi: 10.1038/ejcn.2012.103. Epub 2012 Aug 8.