Research Article
Alterations to the microbiota–colon–brain axis in high-fat-diet-induced obese mice compared to diet-resistant mice

https://doi.org/10.1016/j.jnutbio.2018.08.016Get rights and content

Abstract

Obesity is underpinned by both genetic and environmental factors, including a high-saturated-fat diet. Some mice develop diet-induced obesity (DIO), but others remain diet resistant (DR) despite intake of the same high-saturated-fat diet, a phenomenon that mimics characteristics of the human obese phenotype. Microbiota–colon–brain axis regulation is important for energy metabolism and cognition. Using DIO and DR mouse models, this study aimed to examine gut microbiota, colonic inflammation and cognitive function to elucidate the role of microbiota–gut–brain regulation in DIO. C57Bl6/J mice fed a chronic saturated-palmitic-acid diet for 22 weeks showed significant body weight gain differences, with the top one third gaining 48% heavier body weight than the lower one third. There was significant reduction in gut microbiota richness and diversity in DIO mice but not in DR mice. At the phylum level, DIO mice had increased abundance of Firmicutes and Antinobacteria, and decreased abundance of Bacterioides and Proteobacteria in gut microbiota. DIO mice exhibited reduced tight junction proteins, increased plasma endotoxin lipopolysaccharide (LPS) and increased inflammation in the colon and liver. Recognition memory and spatial memory were impaired in DIO mice, associated with decreased Bacteroidetes. Further examination showed that hippocampal brain-derived neurotrophic factor was significantly decreased in DIO mice (vs. DR). Conversely, DR mice showed no changes in the above parameters measured. Therefore, gut microbiota, colon inflammation and circulating LPS may play a major role in the development of the obese phenotype and cognitive decline associated with a chronic high-saturated-palmitic-acid diet.

Introduction

Diets rich in saturated fat can induce obesity, associated with insulin resistance and cognitive decline. Palmitic acid (PA, C16:0) is the most common saturated fatty acid in human diets in the modern society, accounting for approximately 65% of saturated fatty acids and 32% of total fatty acids in human serum [1]. Patients with metabolic syndrome have a significantly higher level of PA but not other saturated fatty acids, such as myristic acid or stearic acid, within erythrocytes [2]. Obesity is a polygenic disorder and results from a complex interaction with an obesogenic environment, including saturated fat intake [3]. For example, mice or rats fed a high-fat diet develop either obesity or resistance to obesity but have comparable body weight when fed a lab chow diet [4], [5], [6], [7]. Interestingly, there are differences in gene expression between diet-induced obesity (DIO) mice and their diet-resistant (DR) counterparts [8], [9], showing clear genetic distinction between resistant and nonresistant rodents. Therefore, DIO and DR models mimic some characteristics of the human obese phenotype, including a polygenic mode of inheritance and the fact that some (but not all) individuals are susceptible to weight gain when exposed to an obesogenic environment [10].

The role of gut microbiota in contributing to obesity has been recognized. For example, an imbalance of Firmicutes and Bacteroidetes, the primary bacterial phyla comprising the gastrointestinal microbiota, has been reported in the leptin-deficient ob/ob mouse model [10], [11]. Consistent with animal studies, similar differences with an increase in the ratio of Firmicutes/Bacteroidetes have been found in the distal gut microbiota in obese individuals [12]. However, alterations in the gut microbiota in obese individuals have not been reported by all investigators [13]. It has even been reported that ratios of Firmicutes to Bacteroidetes are lower in some obese adults compared to lean controls [14], whereas a study employing an 8-week high-saturated-fat diet altered gut microbiota with increased Firmicutes and reduced Bacteriodetes in mice [15]. However, rare studies have examined microbiota in obesity in a manner that encompasses the interaction between environmental and genetic factors using DIO compared to DR phenotypes.

Obesity has been associated with a chronic low-grade level of inflammation. Alterations in the gut microbiota may play a role in intestinal inflammation and epithelial functions in the development of obesity. High-fat dietary intake increases circulating plasma levels of lipopolysaccharide (LPS, endotoxin), a breakdown product of outer layer of Gram-negative bacteria [16]. LPS and saturated fatty acids exert effects on the toll-like receptor 4 (TLR4) [17], which is expressed at high levels in the colon but lower levels in the small intestine [18], [19]. TLR4 and its adaptor protein, myeloid differentiation primary-response protein 88 (MyD88), activate the inflammatory signaling pathway, including inhibitor kappa B alpha (IκBα) and nuclear factor-kappa B (NFκB) [20]. In addition, intestinal macrophages are essential for intestinal homeostasis and play critical roles in protective immunity and inflammation [21], [22]. The colon has a high density of macrophages compared to other parts of the gastrointestinal tract [23]. In the healthy mouse colon, most resident macrophages are resistant to TLR stimulation and express anti-inflammatory markers, such as CD206, to prevent inflammation [24]. On the other hand, experimental colitis results in the accumulation of TLR-responsive proinflammatory macrophages, most of which become CD11c+ and F4/80+, indicating upregulated proinflammatory actions [24]. A recent report showed that colonic proinflammatory macrophages play a causal role in the development of insulin resistance, as decreased infiltration of colonic proinflammatory macrophages improved high-fat-diet-induced insulin resistance in macrophage-specific chemokine (C-CMotif) receptor 2 (Ccr2) knockout (M-Ccr2KO) mice [25]. Another study found that the switch from anti-inflammatory to proinflammatory macrophages occurred in adipose and muscle tissue, as well as the liver and pancreas, contributing to insulin resistance in obesity [26]. However, the status of macrophages in the colon of DIO and DR mice requires further investigation. Furthermore, the role of colonic inflammation in generating obesity induced by a high-saturated-fat diet and altered gut microbiota is unknown.

Evidence shows a link between gut microbiota, central levels of brain-derived neurotrophic factor (BDNF) and cognitive behavior. For example, one study showed that administration of gut bacteria Citrobacter rodentium by oral gavage caused memory dysfunction in mice exposed to stress conditions [27], while another reported that oral antimicrobial treatment transiently altered the composition of the microbiota and increased hippocampal BDNF expression [28]. On the other hand, reduced BDNF expression in the hippocampus contributes to working-memory impairment in germ-free mice [27]. These studies suggest that BDNF may be influenced by gut microbiota, causing effects on cognitive function. However, it is not known whether cognition and altered BDNF levels are affected by a diet high in saturated PA or by the presence of obesity, and whether alterations in these factors occur concurrently with altered gut microbiota.

A useful tool in preclinical obesity research to study the interaction between diet and genes is the C57 male mouse model, incorporating mice genetically prone to DIO and their DR counterparts. When exposed to a high-fat diet, the DIO mice gain weight and become obese, while the DR mice remain as lean as the control mice. Therefore, the DIO and DR mouse model can provide insight into whether changes in the gut microbiota, colon inflammation and cognition are driven by the high-saturated-PA diet or by the ensuing obesity. In the current study, by using both chow- and PA-diet-fed DIO and DR mice, we determined whether shifts in the microbiota profile result solely from the consumption of an obesogenic PA diet or are a manifestation of the obese phenotype. Furthermore, we examined levels of LPS (an endotoxin) in plasma, colonic tight junction protein levels (reflecting the permeability of colon epithelia), macrophage shifting toward a proinflammatory phenotype and markers of inflammation in the colon, as well as hippocampal BDNF and cognitive behavior.

Section snippets

Animals and treatments

Seventy-two C57Bl/6J male mice (6 weeks old) were obtained from the Animal Resources Center (Perth, WA, Australia). They were housed in environmentally controlled conditions (temperature 22°C, 12-h light/dark cycle) and allowed ad libitum access to food and water throughout the study. Mice were fed standard laboratory chow [i.e. low-fat (LF) diet, AIN93M, Speciality Feeds, WA, Australia] for the first week to facilitate habituation to the new environment and then randomized into dietary

Chronic PA diet increased body weight, adiposity, food intake and hepatic injury in DIO mice

On a chronic high-PA diet, the DIO mice gained significantly more body weight than the DR and LF mice from 5 weeks, and this persisted throughout the rest of the dietary intervention (Fig. 1A). The final body weight of DIO mice was 48% and 54% heavier than that of DR and LF mice (both P<.01) (Fig. 1B). Moreover, DIO mice consumed significantly more calories compared to the DR (P<.05) and LF mice (P<.05) (Fig. 1C). The DIO mice had higher inguinal, epididymal, perirenal and mesenteric fat masses

Discussion

In this study, we found that some mice developed obesity while others were DR and remained lean when fed the same chronic high-PA diet. There was a significant reduction in gut microbiota richness and diversity in the DIO mice but not the DR mice. DIO mice had reduced tight junction protein expression, increased plasma LPS and increased inflammation in colon and liver. The gut microbiota (e.g., Bacteroidetes) is important for cognitive function [46]; in this study, we found that recognition and

Acknowledgments

We wish to acknowledge the University of Wollongong and Illawarra Health and Medical Research Institute for financially supporting the experimental works. This study was also supported by a Diabetes Australia Research Trust Research Project (Y12G-YUYI) awarded to Dr. YH Yu. We are grateful for support for this study from the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Dean Special Foundation of Xuzhou Medical University (2012KJZ07), General Financial

Disclosure statement

The authors have no actual or potential conflicts of interest to report.

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    Grants, sponsors and funding sources: This study was supported by Diabetes Australia Research Trust Research Project (Y12G-YUYI), The Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Dean Special Foundation of Xuzhou Medical University (2012KJZ07), General Financial Grant from the Chinese Postdoctoral Science Foundation (2014M561713) and The Program for Youth Science and Technology Innovative Research Team of Xuzhou Medical University.

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    Contributed equally to this paper.

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