Role of Modied Diet and Gut Microbiota in Metabolic Endotoxemia in Mice Iram Liaqat ( [email protected] ) GC University, Lahore https://orcid.org/0000-0002-4638-8253 Arjumand Iqbal Durrani University of Engineering and Technology Urooj Zafar University of Karachi Saima Rubab Lahore Pharmacy College Mehwish Faheem GC University, Lahore Muhammad Mubin University of Agriculture Faisalabad Chand Raza GC University, Lahore Nauman Aftab GC University, Lahore Research Article Keywords: Gut microbiota, High fat diet, Obesity, Metabolic endotoxemia, Mice Posted Date: June 22nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-603542/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Archives of Microbiology on July 24th, 2021. See the published version at https://doi.org/10.1007/s00203-021-02491-4. Page 1/15 Abstract This study was aimed to investigate the effect of cultured gut microbiota (GM) from obese humans coupled HFD in inducing metabolic endotoxemia in humanized mice. In total, 30 strains were isolated from 10 stool samples of obese patients. Following morphological and biochemical characterization, 16S rRNA gene sequencing of six abundant isolates identied these as Klebsiella aerogenes, Levilactobacillus brevis, Escherichia coli, Staphylococcus aureus, Bacillus cereus and Bacillus subtilis (MZ052089- MZ052094). In vivo trial using above six isolates, known as human gut microbiota (HGM), was performed for six months. Sixteen mice were distributed into four groups i.e., G1 (control) mice fed with chow diet, group 2 (G2) mice with HFD, group 3 (G3) mice with HFD + HGM and group 4 (G4) mice with chow diet + HGM. Body mass index (BMI) and plasma endotoxins were measured pre and post experiment. In vivo study revealed that HFD + HGM caused signicant increase (3.9 g/cm at 20 weeks) in the body weight and BMI (0.4g/cm post experiment) of G3 mice compared to the other groups. One way ANOVA showed signicantly higher level of endotoxins (2.41, 4.08 and 3.7 mmol/l) in mice groups G2, G3 and G4, respectively, indicating onset of metabolic endotoxemia. Cecal contents of experimental mice groups showed more diversied microbiota in mice groups G1 and G4 compared to G2 and G3 where high fat feeding alone and combined with obese gut microbiota caused a shift in microbial diversity as observed by all ve strains belonging to either Firmicutes or Bacteriodetes phyla, respectively. In conclusion, current study reported that minor alteration in GM composition through HFD feeding and cultured GM transfer has signicant impact in development of metabolic endotoxemia, possibly via modied intestinal permeability. Introduction Metabolic endotoxemia is a state of increased level of lipopolysaccharides (LPS) or endotoxins in the blood (Wang et al. 2010). During this condition, proinammatory molecules such as interleukin-1, interleukin-6, tumor necrosis factors (TNF- α) are increasingly expressed. LPS are large, heat stable endotoxins, present in outer membrane of Gram -ive bacterial cell wall. It creates a permeability barrier at the bacterial surface and is a mainly responsible to confer innate resistance that Gram-negative bacteria display against various antimicrobials (Boutagy et al. 2016; Dalby et al. 2018). Human intestine is colonized by copious amount of different bacterial phyla, and these bacteria are associated with food metabolism, energy harvesting and development of innate immunity. Among bacterial phyla as inhabitant of human gut, abundant ones include Firmicutes (64%), Bacteroidetes (23%), Proteobacteria (8%), Fusobacteria, Verrucomicrobia and Actinobacteria (3%). Firmicutes is the most common with 200 genera including Mycoplasma, Bacillus and Clostridium (Arumugam et al. 2014). Gram- ive bacteria makes approximately 70% of total microbes and any imbalance is central to development of metabolic diseases e.g., obesity and type 2 diabetes mellitus (DM) (Bailey and Holscher, 2018), due to continuous low grade inammation also known as metabolic endotoxemia. Regular chow diet is the normally used high ber diet, composed of agricultural byproducts (wheat, corn, alfalfa, soybeans and supplemented with minerals, vitamins) and is palatable to mice. On the other hand, HFD contains cornstarch, amino acid supplemented casein, ber from cellulose, sucrose or lord. Two ingredients differ between two diets. One is phytoestrogen content from soy, that is high in chow diet only and other is sucrose that is present only in HFD. Phytoestrogen content of chow diet controls anxiety, locomotion, memory, insulin-thyroid levels, lipogenesis and lipolysis etc. (Hooper et al. 2018). High ber content of chow diet make it equivalent to the prebiotic, thus suppresses adiposity and other metabolic syndromes. Previous human intervention studies have performed using prebiotics such as oligofructose, inulin, galacto-oligosaccharides, resistant dextrin, insoluble dietary ber, and whole grains. Oligofructose is found in many fruits and vegetables and is an oligosaccharide having one glucose molecule combined with several molecules of fructose. Inulin is a fructose-polymerized polysaccharide, abundantly present in vegetables (burdock and onion) and. In these studies, subjects with obesity, overweight subjects, and subjects with type 2 diabetes showed signicant decrease in LPS levels in blood (Dehghan et al. 2014; Parnell et al. 2017). Likewise, other studies showed Page 2/15 decreased plasminogen activator inhibitor-1 (PAI-1), and improved glucose metabolism. One study concluded that galacto-oligosaccharides reduced LPS levels, and improved obesity by suppressing appetite. In mice, chronic administration of LPS resulted in hyperphagia by decreasing leptin sensitivity of afferent vagal nerves. Following galacto- oligosaccharide administration, reduced LPS levels and appetite suppression supports association between LPS and appetite (de La Serre et al. 2010). HFD acts as strong trigger for the systemic inammation and increased BMI. Previously, it demonstrated that increase in LPS is related to HFD consumption (Li et al. 2011). Unaware of true mechanism of increased plasma LPS linkage with HFD, causative factor was thought to be changes in intestinal microbiota, possibly a switch from Gram + ive ones to Gram –ive bacteria. Though still there is no understanding of direct effect of GM on metabolic endotoxemia, but evidently it was thought that microbial dysbiosis make the gut leaky, induce inammation and led to endotoxemia (Li et al. 2011; Bailey and Holscher, 2018). Increased HFD consumption modies GM, results in increased amount of systemic level of bacterial products and increases the gut permeability for these bacterial products. A meal intake of excess HFD provokes the excess formation of LPS from the Gram -ive bacteria’s cell wall, increased the excess formation of chylomicron resulting in LPS inltration into the blood circulation (Ghoshal et al. 2009; Caesar et al. 2012). Bacterial endotoxins translocation and increased intestinal permeability are the two most important contributing factors that play an important role in the progress of metabolic endotoxemia (Fei and Zhao, 2013). Both dietary modulations and gut microbiota are the most signicant contributions to human health. Turnbaugh et al. (2006) transferred the human fecal microbiota into germ free mice to create humanized mice, which were successfully colonized with donor’s microbiota. Switching this humanized mice to HFD for a single day resulted in changed metabolic pathways and microbiome. The authors observed that though colonization establishes the initial microbial community, however diet can signicantly alter this primary community. Further, humanized mice showed high adiposity index following feeding with HFD. Their study established an animal model to study the effect of genetic and environmental factors on the GM and host metabolism. However, Turnbaugh and colleagues (2006) used fecal microbiota from normal healthy individuals and there is difference in the gut microbiota and metabotypes of lean vs obese individuals (Waldram et al. 2009). Additionally, variation in gut microbiota has been reported across populations of different ethnicity. For example, Jain et al. (2018) revealed microbial community with dominant Firmicutes, Actinobacteria and underrepresented Bacteroides in Indian and Chinese population. While, Brooks et al. (2018) documented 12 microbial genera and families from two US-based 1,673 individuals. Majority of these include the family Christensenellaceae, overlap with closely related taxa and form clusters showing similar metabolic processes. Hence, with all this background the current study seeks to establish whether a chow diet administered therapeutically to mice inoculated with fecal microbiota from obese individuals, is able to change the gut microbiota to a composition associated with healthy individuals using an in vivo approach (mice). The study also aims to clarify whether a chow diet reduces adiposity and metabolic endotoxemia in such mice possibly by acting as prebiotic. Materials And Methods In total, clinically diagnosed obese (n = 10) volunteer subjects were selected from the Mayo Hospital Lahore and general population. All subjects gave fully written informed consent about participation in study which was approved by Institutional Bioethics Committee (IBC) vide letter no. IBC 2431, GC University, Lahore. Sample collection and inoculum preparation