In 2015, the National Center for Health Statistics (NCHS) published a report that found the prevalence of obesity in U.S. adults (20+ years old) increased from 30.5% in 1999-2000 to 37.7% in 2013-2014, and 13.9% to 17.2% for US youth (age 2-19) (1). Another NCHS report released in October 2017 found obesity rates for those same groups in 2015-2016 at 39.8% and 18.5%, respectively (2). This increasing obesity rate, and the associated increase in obesity-related diseases, exerts a significant (and increasing) financial strain on both private and state provided healthcare (3).
Obesity increases the risk for a variety of diseases, including type 2 diabetes, neurodegenerative disorders, and some cancers. The mechanisms underlying this phenomenon are not clear, however chronic low-grade inflammation observed in obese patients has been suggested as a contributing factor (4). Obesity derived low-grade inflammation utilizes a similar pathway to tissue damage-induced inflammation, however it is induced by continuous metabolic surplus. Unlike traditional inflammation, the chronic low-grade inflammation in obesity usually remains unresolved. This chronic inflammation is characterized by increased immune cell infiltration of adipose tissue and increased levels of inflammatory cytokines, such as Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6) and IL-1β (4).
Research has indicated that obesity and other metabolic disorders may be associated with intestinal microbiota population (5). Additionally, disruption of the intestinal epithelial barrier has been implicated in the pathogenesis of chronic low-grade inflammation. Decreased barrier function allows for increased translocation of lipopolysaccharide (LPS) from the intestines to the circulatory system. The systemic circulation of LPS (and presumably other bacterial toxins) stimulates pro-inflammatory reactions that remain unresolved during continuous metabolic surplus, leading to insulin resistance and other metabolic disorders (reviewed in 4).
Given the links between systemic LPS circulation and obesity-related inflammation, scientists at the Milk Science Research Institute in Japan questioned if oral feeding of a probiotic bacteria, Lactobacillus gasseri SBT2055 (LG2055), would improve intestinal barrier function in mice whose intestinal barrier was compromised by a high fat diet. The researchers hypothesized that the improved intestinal barrier as a result of LG2055 treatment would limit systemic LPS exposure, therefore decreasing pro-inflammatory reactions in mouse adipose tissue.
The researchers established three groups of C57BL/6 mice: a normal-fat diet (NFD) group, a high-fat diet (HFD) group, and a high-fat diet containing LG2055 (HFD-LG) group. The mice were kept on these diets for 21 weeks, and during that time several tests were performed. The specifics of each diet and the entire experimental protocol can be found here.
At 18 weeks, mice were fasted for 12 hours and fed 4 kDa FITC-Dextran to evaluate intestinal permeability. Blood was then collected from the orbital sinus and plasma was analyzed for FITC-Dextran concentration 1-4 hours after feeding, as plasma FITC-Dextran levels indicate intestinal barrier (paracellular) permeability. At 1 hour after feeding, mice in the HFD group showed higher FITC-dextran plasma concentration than the NFD group. This indicated that the HFD group had increased paracellular permeability of the intestinal epithelial barrier. The HFD-LG group had lower FITC-dextran plasma levels than the HFD group and approximately equal levels to the NFD group.
Next, at 21 weeks, sera from mice were analyzed for anti-LPS antibodies using Chondrex, Inc.’s Mouse Anti-LPS IgG Antibody Assay Kit. The researchers used the presence of anti-LPS antibodies as a measure of LPS translocation from the intestines, indicating relative levels of intestinal permeability. Consistent with the FITC-dextran experiments, the HFD group showed the highest anti-LPS IgG antibody levels, followed by the HFD-LG group, with the NFD group showing the lowest levels.
The intestinal permeability test results were confirmed by in vitro tests of paracellular permeability of Caco-2 cell monolayers. This test showed that apical treatment of Caco-2 cell monolayers with LG2055 prevented the increased paracellular permeability of Caco-2 cells that were basally treated with IFN-γ and TNF-α.
Post-mortem, epididymal adipose tissue was analyzed via flow cytometry for immune cell recruitment. Specifically, the ratio of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages and the presence of CD4-positive and CD8-positive cells were measured. The HFD group showed increased M1 levels compared to the NFD group, while no change in the M2 population was observed between these groups. Therefore, the M1:M2 ratio in the HFD group was higher than the NFD group. The HFD-LG group, meanwhile, showed higher M2 levels than both the HFD and NFD groups, while the M1 macrophage numbers were between the NFD and HFD groups (NFD < HFD-LG < HFD). This resulted in a lower M1:M2 ratio in HFD-LG epididymal tissue, which is generally considered as less pro-inflammatory. The HFD-LG group also showed decreased CD8-positive T-cell levels as compared to the HFD group, indicating that LG2055 can modify T-cell responses in adipose tissue.
The researchers also analyzed the body weight, adipose tissue weight, and liver weight of experimental animals at 21 weeks. The HFD group had higher body weight, adipose tissue weight, and liver weight than the NFD group. The HFD-LG group, which had the same caloric intake as the HFD (both more than the NFD group), had significantly lower body and adipose tissue weights than the HFD group.
It has been suggested that LPS translocation plays a significant role in obesity-related inflammation (4), and this study performed at the Milk Science Research Institute supports those findings. The FITC-dextran experiment confirmed that a HFD can disrupt intestinal epithelial barrier function. The analysis of serum anti-LPS antibody levels showed that the increased intestinal permeability caused by a HFD can lead to increased systemic exposure to LPS. Furthermore, increased exposure to LPS was correlated with pro-inflammatory immune cell recruitment in the HFD group. Meanwhile, the HFD-LG group had decreased systemic LPS exposure and exhibited a lower M1:M2 ratio than the HFD group, indicating that the HFD-LG group immune cell profile skews away from pro-inflammatory reactions found in the HFD group.
Overall, this data supports a few key observations. First, increased intestinal permeability associated with a HFD can be mitigated by oral feeding of LG2055. Additionally, oral feeding of LG2055 can modify immune cell recruitment in adipose tissues towards an M2 macrophage favored response, which is generally less pro-inflammatory than M1 favored responses. This shift in the M1:M2 ratio was positively correlated with the anti-LPS antibody levels, suggesting that reduced LPS exposure could contribute to the shift in M1:M2 ratio. This is consistent with hypothesis that translocation of LPS from the gut and systemic exposure to LPS contributes to chronic low-grade inflammation in obesity. Considering that the body weight and adipose tissue weight in the HFD-LG group was lower than in the HFD group, this study also shows that manipulation of intestinal flora could be a potential treatment for obesity and obesity-related diseases. While this link is tenuous, further research on the complex relationship between diet, intestinal flora, and disease could lead to novel strategies for treating metabolic disorders like obesity.
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