The Effects And Mechanisms Of Soluble Dietary Fiber, Apple-derived Pectin, On Metabolism And Gut Barrier Function In Rats With Diet-induced Obesity

In recent years, obesity and related metabolic disorders have emerged as major health concerns. As a largest epidemic disease, obesity has become an independent risk factor which influences clinical results of surgical patients. Obesity is associated with increased risks for developing type 2 diabetes mellitus (T2DM), hyperlipidemia, hypertension and so on.Obesity and related metabolic disorders are attributable to a combination of genetics, unhealthy diet and lifestyle. Recent studies have demonstrated that disturbance of gut microbiota, especially the ratio of Bacteriodetes to Firmicutes phylum, is closely related to obesity and metabolic disorders. In addition, obese subjects exhibit systemic chronic inflammation and a high level of serum endotoxins (lipopolysaccharides (LPS), a key component of the cell wall of gram-negative bacteria), termed "metabolic endotoxemia", which associates with gut barrier dysfunction. Gut microbiota in the lumen is normally isolated by the intestinal epithelium from lamina propria and deeper layers, and LPS derived from gut microbiota is confined to the gut lumen and does not penetrate healthy intestinal epithelium. However, a damaged intestinal epithelium or other gut barrier dysfunction can lead to disturbance of gut microbiota, allow for LPS permeation and cause metabolic endotoxemia. Recognition of LPS by Toll-like receptor 4 (TLR4) of host cells triggers downstream inflammatory events that contributes to the development of obesity and metabolic disorders such as insulin resistance. Tight junctions are key components for maintaining gut barrier integrity. Another important protein for gut barrier function is intestinal alkaline phosphatase (IAP) that is a type of glycoprotein anchored in the apical membrane of enterocytes. IAP has multiple roles in maintenance of gut barrier, including detoxification of LPS via its dephosphorylation. remission of systemic inflammation, protection of gut barrier function and modulation of gut microbiota.Although various approaches are recommended for obesity management, such as dieting, behavior therapy, exercise, pharmacotherapy, and bariatric surgery, they are often defective. Here, we consider that modification of gut microbiota, protection of gut barrier, remission of metabolic endotoxemia, and relief of systemic inflammation may provide a novel strategy for the treatment of obesity and related metabolic disorders.Dietary fiber is a polymer extracted or synthetized from plants; it cannot be absorbed or digested by the human intestine. Recent animal experiments and clinical trials have shown that dietary fiber has hypolipidemic and hypoglycemic effects and may contribute to weight loss. Apple-derived pectin is the main soluble fiber in apples and can be fermented by gut microbiota in the colon to produce metabolites with local intestinal and systemic effects. Apple-derived pectin may also help to maintain the balance of gut microbiota.The aim of the present study was to assess potential effects of apple-derived pectin on diet-induced obesity in rats. We found that apple-derived pectin could modulate gut microbiota, preserve gut barrier function, and alleviate metabolic endotoxemia and inflammation in diet-induced obese rats. Our findings suggest that apple-derived pectin may be useful for the clinical management of obesity.PARTⅠEstablishment of rat models of diet-induced obesityObjective:To establish a standard, stable and repeatable rat models of diet-induced obesity.Method:Male Sprague-Dawley rats weighing 90±10 g (aged 4 weeks, n= 40) were used to establish rat models of diet-induced obesity. Rats were randomized into two groups and received either a standard chow diet (Chow group, n= 8) as a control or a high fat diet (HFD) (n= 32) to induce obesity for 8 weeks. Body weight was recorded every week. At the end of the 8th week, if the weight of rats fed on a HFD is significantly greater than those in the Chow group, rat models of diet-induced obesity were built successfully.Results:After high fat feeding, the weight of the rats fed on a HFD was significantly greater than those in the Chow group.Conclusion:Establishment of rat models of diet-induced obesity by a high fat diet is stable and repeatable, which is suitable for follow-up study.PART ⅡThe effects of soluble dietary fiber, apple-derived pectin, on metabolism and gut barrier function in rats with diet-induced obesityObjective:To study the effect of soluble dietary fiber, apple-derived pectin, on metabolism and gut barrier function in rats with diet-induced obesity.Method:On the basis of rat models of diet-induced obesity,16 diet induced obese rats were selected randomly for the following interventions. They were randomized to receive either a HFD (HF group, n= 8) or a HFD supplemented with pectin (5% wt/wt) (HF-P group, n=8) for 6 weeks. The Chow group continued a standard chow diet for 6 weeks. Body weight was recorded every week. At the end of the 6th week, rats were fasted for 12 hours, and then anesthetized by intraperitoneal administration of chloral hydrate (10%,0.3 mL/100 g body weight). Portal venous blood (about 3 mL) was immediately collected for blood chemistry. Abdominal subcutaneous adipose tissue and epididymal adipose tissue were removed, dried with filter paper and weighted. The liver samples were removed to determine lipid profile and used for hematoxylin-eosin staining. The intestine samples were collected and used for hematoxylin-eosin staining and western blot.Result:In contrast with the Chow group, rats in the HF group gained more body weight, developed adipose tissue more rapidly and exhibited higher levels of serum total cholesterol, triglycerides, glucose, insulin, as well as liver total cholesterol and triglycerides. Moreover, the expression levels of gut tight junction proteins like claudinl, occludin and zonula occludens 1 (ZO1) in rats of the HF group were significantly lower than those in the Chow group.Compared with the HF group, after 6 weeks of apple-derived pectin treatment, rats in HF-P group had a significantly reduced weight gain, a significantly reduced adipose tissue and exhibited a lower level of serum total cholesterol and liver total cholesterol and triglycerides. There was a downward trend in serum triglycerides, glucose and insulin. The expression level of claudinl improved significantly, while there was only a downward trend in occludin and ZO1.Conclusion:Apple-derived pectin can alleviate HFD-induced body weight gain, fat mass development, dyslipidemia, hyperglycemia, hyperinsulinism and improve liver lipid profile in obese rats. Moreover it can also improve the expression of gut tight junction proteins and protect gut barrier.PART IIIThe mechanisms of effects of soluble dietary fiber, apple-derived pectin, on metabolism and gut barrier function in rats with diet-induced obesityObjective:To study the underlying mechanisms of effects of soluble dietary fiber, apple-derived pectin, on metabolism and gut barrier function in rats with diet-induced obesityMethods:On the basis of Part II of this study, portal venous blood was immediately collected in a dry tube without heat source and was used to determine the level of serum LPS by Chromogenic End-point Tachypleus Amebocyte Lysate (CE TAL) assay. Serum inflammatory factors tumor necrosis factor a (TNFa) and interleukin 6 (IL6) were determined by ELISA. The intestine samples were collected to deterimine the expression level of TNFa, IL6, interleukin 10 (IL10), toll like receptor 4 (TLR4) by quantitative real-time polymerase chain reaction (qPCR), and intestinal alkaline phosphatase (IAP) by western blot and immunohistochemistry. Cecum content was collected to analyze gut microbiota by 16s rRNA pyrosequencing analysis.Results:1. Soluble dietary fiber, apple-derived pectin, alleviated metabolic endotoxemia.In contrast with the Chow group, rats in the HF group showed a significantly higher level of serum LPS and TLR4 mRNA in the intestinal samples. However, supplemtation of apple-derived pectin could reduce the level of serum LPS and TLR4 mRNA level in the intestinal samples.2. Soluble dietary fiber, apple-derived pectin, remitted systemic and intestinal inflammation.In contrast with the Chow group, rats in the HF group showed a significantly higher level of serum TNFa, IL6, as well as a higher level of TNFa mRNA, IL6 mRNA and a lower level of IL10 mRNA in intestinal samples. However, rats in the HF-P group exhibited similar results to the Chow group.3. Soluble dietary fiber, apple-derived pectin, improved expression of intestinal IAP.Compared with the Chow group, rats in HF group showed a significantly lower expression of IAP. However supplementation of apple-derived pectin could improve the expression of IAP to a normal level.4. Soluble dietary fiber, apple-derived pectin, modulated gut microbiota.At the phylum level, rats in the HF group had a significantly lower level of Bacteroidetes and a higher level of Firmicutes than the Chow group.At the class level, rats in the HF group presented a significantly higher level of Bacilli (a class of Firmicutes phylum) and Gammaproteobacteria (a class of Proteobacteria phylum) as well as a significantly lower level of Bacteroidia (a class of Bacteroidetes phylum) and Deltaproteobacteria (a class of Proteobacteria phylum).At the order level, we observed a significantly lower level of Bacteroidales (an order of Bacteroidia class. Bacteroidetes phylum) and a higher level of Lactobacillales (an order of Bacilli class. Firmicutes phylum) in the HF group than in the Chow group.At the family level, we observed a significantly lower level of Bacteroidaceae (a family of Bacteroidales order. Bacteroidia class. Bacteroidetes phylum) in the HF group than in the Chow group.At the genus level, we observed a dramatically lower level of Bacteroides (a genus of Bacteroidaceae family, Bacteroidales order, Bacteroidia class, Bacteroidetes phylum) and a higher level of Lactococcus (a genus of Streptococcaceae Family, Lactobacillales Order, Bacilli Class, Firmicutes Phylum) in the HF group than in the Chow group.At the species level, Clostridium ruminantium (a species of Clostridium Genus, Clostridiaceae Family, Clostridiales Order, Clostridia Class, Firmicutes Phylum) was the only species that displayed a significant increase upon high fat diet.After pectin supplementation for six weeks, gut microbiota in different levels in the HF-P group was similar to that in the Chow group.Conclusion:Apple-derived pectin could modulate gut microbiota, and alleviate metabolic endotoxemia and systemic inflammation in obese rats. In addition, expression of IAP in intestinal samples was improved with pectin supplementation.