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Dietary cellulose nanofiber modulates obesity and gut microbiota in high-fat-fed mice
Bioactive Carbohydrates and Dietary Fibre 22 (2020) 100214
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Dietary cellulose nanofiber modulates obesity and gut microbiota in high-fat-fed mice Takao Nagano a, *, Hiromi Yano b a
Department of Food Science, Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa, 921-8836, Japan b Department of Health & Sports Science, Faculty of Health Science and Technology, Kawasaki University of Medical Welfare, 288 Matsushima, Kurashiki, Okayama, 719-1197, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Cellulose nanofiber Gut microbiota High-fat diet Lactobacillaceae Obesity 16S ribosomal RNA genes
Cellulose nanofiber (CN) has a unique feature of dispersing in water and producing a dispersion of high viscosity, similar to highly viscous soluble dietary fibers (DFs). The beneficial effects of soluble DFs on glycemic control are associated with viscosity, thus making CN a potential option to attenuate obesity and related diseases. Here, we studied the effects of CN intake on obesity and gut microbiota in high-fat diet (HFD)-fed mice. The CN-treated mice were provided 0.1% or 0.2% CN dispersions via drinking water for seven weeks. HFD-fed mice weighed more than mice fed a normal-fat diet (NFD). The weight gain in HFD-fed mice was suppressed in 0.2% CN-treated group, but not in 0.1% CN-treated group, when compared with CN-untreated group. Also, the accumulation of epididymal and subcutaneous fat in HFD-fed mice was lower in 0.2% CN treated group than in the CN-untreated group. Moreover, the fecal gut microbiota was analyzed by sequencing bacterial 16S ribosomal RNA genes. Oral administration of 0.2% CN, but not 0.1% CN, increased bacterial diversity and induced changes in the gut microbiota composition of HFD-fed mice. The principal component analysis (PCA) at the phylum level showed a shift in the microbiota composition resulting from feeding HFD. This shift in the PC1 axis was reversed by 0.2% CN intake. In addition, the relative abundance of Streptococcaceae and Rikenellaceae decreased while that of Lactobacillaceae increased, upon higher intake of CN. These results suggest that CN consumption has inhibitory effects on obesity mediated via control of gut microbiota balance.
1. Introduction In recent decades, obesity is a growing epidemic in developed countries and induces related metabolic diseases, such as type 2 diabetes and cardiovascular disease. Diets comprising high fat and low dietary fiber are the most common causes of obesity. In addition, the develop ment of obesity has been correlated with gut microbiota structures that may play a significant role in energy homeostasis (Cani & Everard, 2016; Dahiya et al., 2017; Lynch & Pedersen, 2016; Subramanian et al., 2015). For these reasons, many researchers have been investigating anti-obesity food components using high-fat diets (HFD)-induced obesity animal models to find solutions to obesity and other metabolic diseases and thereby provide potential health benefits for humans (Genda et al., 2018; Miyamoto et al., 2018; Weitkunat et al., 2017; Zhai, Lin, Zhao, Li, & Yang, 2018).
Dietary fibers are one of the most important food components and their beneficial effects on our health dependent on their physicochem ical properties, such as solubility, viscosity, and fermentability by the gut microbiota (Gidley & Yakubov, 2019; Kaczmarczyk, Miller, & Freund, 2012; Wu, Shi, Wang, & Wang, 2016). Cellulose is the main component of plants and the most consumed dietary fiber. Dhital, Gid ley, and Warren (2015) investigated the inhibition of α-amylase activity by cellulose. They found that cellulose reduced α-amylase-mediated starch hydrolysis in a concentration dependent manner, and its inhibi tory activity was exerted by non-specific adsorption of α-amylase to cellulose. However, the inhibition of enzyme activity by cellulose is much lower than that by a polysaccharide thickener, guar gal actomannan, as cellulose is not soluble in water and shows low viscosity at 0.1–3% concentration (Slaughter, Ellis, Jackson, & Butterworth, 2002). Sasaki and Kohyama (2012) studied starch digestibility by
* Corresponding author. Department of Food Science, Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, 1-308, Suematsu, Nonoich, Ishikawa, 921-8836, Japan. E-mail address: [email protected] (T. Nagano). https://doi.org/10.1016/j.bcdf.2020.100214 Received 16 June 2019; Received in revised form 29 January 2020; Accepted 17 February 2020 Available online 20 February 2020 2212-6198/© 2020 Published by Elsevier Ltd.
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adding non-starch polysaccharides, such as pectin, konjac glucomannan, guar gum, and xanthan gum. They found that increased viscosity due to addition of the non-starch polysaccharides reduced starch digestibility in an in vitro assay. These results indicate that viscosity is a key factor in inhibition of α-amylase activity. The Isogai group developed a method to prepare cellulose nanofiber (CN) from plant cellulose by catalytic oxidation with 2,2,6,6-tetramethl piperidine-1-oxyl radical (TEMPO). This CN has unique features of dispersing in water and high viscosity (Saito, Kimura, Nishiyama, & Isogai, 2007). Since CN dispersions showed high viscosity, they also investigated inhibitory effects of CN on postprandial increases in blood glucose and insulin in mice. The postprandial increase in blood insulin was suppressed by CN in a concentration dependent manner. These re sults suggest that CN is a potential option to attenuate obesity and related disorders (Shimotoyodome, Suzuki, Kumamoto, Hase, & Isogai, 2011). However, the beneficial effects of CN intake on obesity and gut microbiota remain unclear. In the present study, we investigated the effect of CN supplementa tion on obesity in a HFD-fed mouse model of obesity. In addition, the gut microbiota composition was analyzed by sequencing bacterial 16S ri bosomal RNA genes (16S rDNA) to elucidate the effect of CN intake on the intestinal microbiota in HFD-fed mice.
Illumina Miseq platform (Illumina, San Diego, CA, USA), as described previously (Nagano, Katase, & Tsumura, 2019). All sequence reads were analyzed by the TechnoSuruga laboratory (Shizuoka, Japan). The paired reads were joined together with the fastq-join program (http://code.goo gle.com/p/ea-utils/). Only reads that had quality value (QV) scores of �20 for more than 99% of the sequence were extracted. Chimeras were detected and omitted by USEARCH 6.1.544_i86 (Edgar, Haas, Clemente, Quince, & Knight, 2011). Operational taxonomic units (OTUs) were determined by clustering sequences with a similarity threshold of 97%, and α- and β-diversities of the gut microbiota were analyzed using Quantitative Insight into Microbial Ecology (QIIME ver1.8.0) (Caporaso, Kuczynski, Stombaugh et al., 2010). Taxonomic analysis of the 16S rDNA sequences was also conducted based on the Ribosomal Database Project, using the Metagenome@KIN analysis software (World Fusion, Tokyo, Japan) (Takahashi, Tomita, Nishioka, Hisada, & Nishijima, 2014). 2.4. Statistical analysis
2. Methods
The statistical significance of differences was analyzed using nonparametric Kruskal-Wallis test followed by Mann-Whitney’s post-hoc tests, and the principal component analysis (PCA) was conducted using Origin 2019 software (OriginLab, Northampton, MA, USA). The data were considered significant at P < 0.05.
2.1. Materials
3. Results
The normal fat diet (NFD, 10% kcal fat; D12450J) and HFD (60% kcal fat; D12492G) with no added cellulose were purchased from Research Diets, New Brunswick, NJ, US. The CN (BiNFi-s™, WFo10002) was obtained from Sugino Machine (Uozu, Japan).
3.1. Feed, water, and CN intake Feed intake was 11.7 � 0.4 kcal/day, 11.4 � 0.6 kcal/day, 11.4 � 0.7 kcal/day, and 11.3 � 6.0 kcal/day for NFD-fed and CN-untreated, 0.1% and 0.2% CN-treated HFD-fed mice groups, respectively. Feed intake was significantly higher in the NFD group compared with that in HFD groups based on weight (P < 0.01), but not calories. Water intake was 3.65 � 0.75 mL/day, 3.66 � 0.64 mL/day, 4.63 � 0.57 mL/day, and 4.69 � 0.85 mL/day for NFD-fed and CN-untreated, 0.1% and 0.2% CNtreated HFD-fed mice groups, respectively. There was no difference in water intake between NFD-fed and CN-untreated HFD-fed mice groups. However, mice from these groups consumed lower amounts of water than the mice in the 0.1% and 0.2% CN-treated HFD-fed groups (P < 0.01). As mice were provided CN via drinking water, CN intake was 4.63 � 0.57 mg/day and 9.34 � 1.07 mg/day for 0.1% and 0.2% CN-treated HFD-fed mice, respectively.
2.2. Animal experiments Eight-week-old male C57BL/6N mice were obtained from Clea Japan (Tokyo, Japan). Four mice were housed per cage in a temperaturecontrolled room (22 � 1� C, 12-h light/dark cycle) and conventional conditions. The experiment involving the mice including the procedures performed, were approved by the Institutional Animal Care and Use Committee of Kawasaki University of Medical Welfare (#18–015). After three-day acclimatization, the mice were randomly divided into NFDfed (NFD, n ¼ 8), CN-untreated HFD-fed (CN0%, n ¼ 7), 0.1% CNtreated HFD-fed (CN0.1%, n ¼ 8), and 0.2% CN-treated HFD-fed (CN0.2%, n ¼ 8) groups. The NFD group was fed with NFD only, while CN0%, CN0.1%, and CN0.2% groups were fed with HFD. CN0.1% and CN0.2% groups were orally administered 0.1 wt% and 0.2 wt% CN dispersions, respectively, via drinking water provided through a water bottle, throughout the experiment (seven weeks). For glucose tolerance test (GTT) conducted at seventh week, each mouse was lightly anes thetized by the inhalation of Isoflurane prior to glucose administration (2 g/kg, i.p.). Blood samples were collected from the tail vein, and blood glucose levels were measured using the glucose monitoring device AccuChek, (Roche, Basel, Schweiz) immediately prior to, and at 15, 30, 60 and 120 min after glucose administration (Bradley, Jeon, Liu, & Maratos-Flier, 2008). On the final day of the experiment, mice were rendered unconscious by Isoflurane exposure and sacrificed by cervical dislocation for euthanasia. Epididymal, visceral, and subcutaneous fat were taken and weighed. The cecum was collected and pH levels of the cecum contents were measured. The fecal contents were collected from the proximal colon. Collected samples were stored at 80� C until use.
3.2. Body weight and GTT Body weight of mice in each group increased with time over the seven week duration (Fig. 1A). After seven weeks, the three HFD-fed mice groups weighed significantly more than mice fed NFD (P < 0.05). The weight gain was not suppressed in 0.1% CN-treated mice, but in 0.2% CN-treated mice (P < 0.05), when compared with CN-untreated HFD-fed mice (Fig. 1B). Moreover, the levels of fasting blood glucose were measured and significant differences were not observed in the four treatment groups (Fig. 1C). GTTs were also conducted (Fig. 1D) and there were no significant differences in the area under the curves (AUCs) among the four treatment groups (Fig. 1E). 3.3. Fat accumulation, weight, and pH of cecum The accumulation of epididymal, visceral, and subcutaneous fat were determined (Fig. 2A–C). The weights of epididymal, visceral, and sub cutaneous fat in CN-untreated and 0.1% CN-treated HFD-fed mice were significantly higher than those in NFD-fed mice (P < 0.01). In contrast, the fat accumulation in 0.2% CN treated HFD-fed mice was lower for epididymal and subcutaneous fat when compared with that in the CNuntreated HFD-fed mice (P < 0.05). In addition, no differences were
2.3. Bacterial DNA sequencing and taxonomic analysis Bacteria were recovered from fecal sample-lysates using the FastDNA Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA). PCR and sequencing of the V4 region (16S rDNA) was performed using the 2
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Fig. 1. Effects of cellulose nanofiber (CN) intake on body weight and glucose tolerant tests (GTTs) in high-fat diet (HFD)-fed mice. (A) Body weight change for seven weeks. (B) Body weight gain after seven weeks. (C) Fasting blood glucose levels. (D) GTTs at seventh week. (E) Area under the curves of blood glucose as shown in Fig. 1D. NFD, normal-fat diet-fed group; CN0%, CN-untreated HFD-fed group; CN0.1%, 0.1% CN-treated HFD-fed group; CN0.2%, 0.2% CN-treated HFD-fed group. The data are presented as the mean � standard error of the mean (SEM) in A and D; *p < 0.05; **p < 0.01.
Fig. 2. Effects of cellulose nanofiber intake on fat accumulation, cecum weight and pH in high-fat diet (HFD)-fed mice. (A) Epididymal fat mass. (B) Visceral fat mass. (C) Subcutaneous fat mass. (D) Cecum weight. (E) Cecum pH. NFD, normal-fat diet-fed group; CN0%, CN-untreated HFD-fed group; CN0.1%, 0.1% CN-treated HFDfed group; CN0.2%, 0.2% CN-treated HFD-fed group. *p < 0.05, **p < 0.01, ***p < 0.001.
3
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observed between 0.2% CN treated HFD-fed and NFD-fed mice groups for epididymal, visceral, and subcutaneous fat. Furthermore, the cecum weights (Fig. 2D) were significantly higher (P < 0.05), and pH levels of the cecum contents (Fig. 2E) were lower (P < 0.01) in NFD-fed mice than those in the three HFD-fed mice groups. In addition, the cecum weights were also higher, and pH in the cecum was significantly lower in 0.2% CN treated HFD-fed mice than those in 0.1% CN treated HFD-fed mice (P < 0.05).
The relative abundance of Streptococcaceae and Rikenellaceae increased with feeding HFD and decreased with higher CN intake in the HFD-fed mice (Fig. 5D and E). The relative abundance of Streptococca ceae was significantly lower in the NFD-fed and 0.2% CN-treated HFDfed mice than in the CN-untreated HFD-fed mice (P < 0.05; Fig. 5D). In contrast, the relative abundance of Rikenellaceae was significantly higher in the CN-untreated (P < 0.05) and 0.1% CN-treated (P < 0.01) HFD-fed mice than in the NFD-fed mice, and lower in the 0.2% CNtreated mice than in the 0.1% CN-treated mice (P < 0.05; Fig. 5E). Furthermore, the relative abundance of Lactobacillaceae increased with increasing CN consumption. The relative abundance of Lactobacillaceae was significantly higher in the 0.2% CN-treated HFD-fed mice compared with that in mice from the other three groups (P < 0.05, Fig. 5F).
3.4. Gut microbiota composition in mice Total sequence reads and number of OTUs derived by sequencing bacterial 16S rDNA are shown in the Supplementary table. The analysis of α- and β-diversities were performed to estimate bacterial richness and diversity (Fig. 3). Chao-1 index in the NFD-fed mice was significantly lower than that in the three HFD-fed mice groups (P < 0.01), and chao-1 index in 0.2% CN-treated HFD-fed mice was significantly higher than that in CN-untreated HFD-fed mice (P < 0.05; Fig. 3A). Furthermore, the PCA based on weighted UniFrac of β-diversity showed that the HFD-fed and NFD-fed mice separated on the PC1 axis (71.0% of explained vari ance). The oral administration of 0.2% CN, but not 0.1%CN, changed the composition of gut microbiota in the HFD-fed mice as evident on the PC2 axis (13.4% of explained variance) (Fig. 3B). The PCA (Fig. 4A) and gut microbiota composition (Fig. 4B) were determined at the phylum level. In the PCA, the microbiota composi tions of the NFD-fed and CN-untreated HFD-fed mice groups separated on the PC1 axis (39.1% of explained variance). In contrast, the micro biota compositions of the CN-untreated and 0.1% CN-treated HFD-fed mice groups did not separate on the PC1 axis. The microbiota compo sition of the 0.2% CN-treated HFD-fed mice shifted toward that of NFDfed mice and separated from that of CN-untreated HFD-fed mice on the PC1 axis. At the family level, the relative abundance of Erysipelotrichaceae (Fig. 5A) decreased with feeding HFD and was significantly lower in the three HFD-fed mice groups than in the NFD-fed mice (P < 0.01). In addition, the relative abundance of Erysipelotrichaceae was significantly higher in the 0.2% CN-treated mice than in the 0.1% CN-treated mice (P < 0.05). The relative abundance of Porphyromonadaceae also decreased with feeding HFD and was significantly lower in the 0.1% (P < 0.01) and 0.2% (P < 0.05) CN-treated HFD-fed mice than in the NFD-fed mice (Fig. 5B). In contrast, the relative abundance of Verrucomicrobiaceae increased with feeding HFD and was significantly higher in the three HFD-fed mice groups than in the NFD-fed mice (P < 0.01, Fig. 5C).
4. Discussion Our results demonstrated that 0.2% CN consumption via drinking water controlled obesity in mice fed HFD, as evidenced by reduced body weight gain and fat accumulation. It is likely that this beneficial effect of CN intake is due to the property of forming a solution of high viscosity. Clinical studies have indicated that highly viscous soluble DFs, such as guar gum and β-glucan, improve glycemic control, whereas insoluble DFs such as cellulose do not provide the beneficial effect (McRorie & McKeown, 2017). Sasaki and Kohyama (2012) reported a negative relationship between the α-amylase-mediated starch hydrolysis and apparent viscosity by adding non-starch polysaccharides. In addition, the postprandial increase in blood insulin was suppressed by CN in a concentration dependent manner (Shimotoyodome et al., 2011). In this study, mice were provided with 0.1% and 0.2% CN via drinking water, and HFD with no added/supplemented fibers. The HFDinduced body weight increased were suppressed by administration of 0.2% CN, but not 0.1% CN. The amount of CN intake was 4.63 � 0.57 mg/day and 9.34 � 1.07 mg/day for 0.1% and 0.2% CN-treated HFD-fed mice, respectively; these intakes correspond to approximately 9.35 � 0.42 g and 19.80 � 1.96 g for a 60 kg human per day. As per Japanese nutritional guidelines, 20 g dietary fiber intake per day is recommended for an adult man. It is likely that this guideline helps explain why 0.2% CN-treatment has a positive effect on obesity, whereas only a marginal effect is seen in 0.1% CN-treated mice. Obesity results from an imbalance in the body’s regulation of energy intake and expenditure (Dahiya et al., 2017). Recent evidence from studies in animal models and humans suggests that the gut microbiota has a significant role in energy acquisition and regulation. Its
Fig. 3. Effects of cellulose nanofiber (CN) intake on bacterial diversities in the gut microbiota of high-fat diet (HFD)-fed mice. (A) Chao-1 index (α-diversity), (B) Principal component analysis (PCA) based on weighted UniFrac (β-diversity). NFD, normal-fat diet-fed group; CN0%, CN-untreated HFD-fed group; CN0.1%, 0.1% CN-treated HFD-fed group; CN0.2%, 0.2% CN-treated HFD-fed group. *p < 0.05, **p < 0.01, ***p < 0.001. 4
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Fig. 4. Cellulose nanofiber (CN) intake affects the gut microbiota composition in high-fat diet (HFD)-fed mice at the phylum level. The gut microbiota composition was analyzed by sequencing the bacterial 16S ribosomal RNA genes, followed by principal component analysis (PCA) (A) and determination of the taxonomic distribution (B). NFD, normal-fat diet-fed group; CN0%, CN-untreated HFD-fed group; CN0.1%, 0.1% CN-treated HFD-fed group; CN0.2%, 0.2% CN-treated HFDfed group.
Fig. 5. Cellulose nanofiber (CN) supplementation influences the relative abundance of intestinal bacteria in high-fat diet (HFD)-fed mice at the family level. (A) Erysipelotrichaceae, (B) Porphyromonadaceae, (C) Verrucomicrobiaceae, (D) Streptococcaceae, (E) Rikenellaceae, (F) Lactobacillaceae. NFD, normal-fat diet-fed group; CN0%, CN-untreated HFD-fed group; CN0.1%, 0.1% CN-treated HFD-fed group; CN0.2%, 0.2% CN-treated HFD-fed group. The data are presented as the means � SEM; *p < 0.05; **p < 0.01.
composition also differs between obese and lean animals and humans (Subramanian et al., 2015). In the present study, administration of 0.2% CN, but not 0.1%CN, increased α-diversity and induced changes in gut microbiota composition. In addition, the PCA plot at the phylum level showed a shift in the microbiota composition resulting from HFD feeding, and this difference in the PC1 axis disappeared between the 0.2% CN-treated HFD-fed and NFD-fed mice. These results indicate that administration of 0.2% CN, but not 0.1% CN, has increased effects on bacterial richness and changes in gut microbiota composition. The data provides a good correlation with the results that consumption of CN suppressed obesity in mice fed HFD. Moreover, the abundance of Ery sipelotrichaceae and Porphyromonadaceae decreased, whereas the relative abundance of Verrucomicrobiaceae, Streptococcaceae, and Rikenellaceae
increased in the CN-untreated HFD-fed mice when compared with the NFD-fed mice. In previous studies, the increase in Streptococcaceae and Rikenellaceae were correlated with HFD-induced obesity (Daniel et al., 2013; Jiang et al., 2015; Price et al., 2009; Zhang et al., 2013). Our study also showed that Streptococcaceae and Rikenellaceae decreased with increasing CN intake in HFD-fed mice. In previous studies, some strains of Streptococcaceae were also reported to induce mild inflammation in humans (Price et al., 2009). The high abundance of Streptococcaceae was also shown to be associated with diseases such as obesity, diabetes and colon cancer (Daniel et al., 2013). Furthermore, it was pointed out that decreasing Streptococcaceae was a potential strat egy for preventing metabolic diseases (Zhang et al., 2013). Moreover, the presence of Alistipes, a genus in Rikenellaceae, was also correlated to 5
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(to T.N.) and Interdepartmental Research Fund of Kawasaki University of Medical Welfare (to H.Y.). We thank TechnoSuruga laboratory (Shi zuoka, Japan) for taxonomic analysis of the 16S rDNA sequences. We would like to thank Editage (www.editage.jp) for English language editing.
type-2 diabetes in humans (Jiang et al., 2015). In this study, Lactobacillaceae increased with increasing CN con sumption. In former studies, effects of prebiotics supplementation were investigated in animal models and humans (Dahiya et al., 2017; Haar man & Knol, 2006; Park et al., 2013). Prebiotics fibers extensively studied are fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), and inulin (Dahiya et al., 2017). A mixture of FOS and GOS in infant milk formula stimulated the growth of Lactobacillus and Bifido bacterium and the resulting microbiota was comparable to the micro biota of breast-fed infants. These prebiotic fibers also modulated the gut microbiota and increased populations of Lactobacillus and Bifidobacte rium in the gut (Haarman & Knol, 2006). In other studies, Lactobacillus strains were studied as probiotics used to modulate gut microbiota composition in the context of obesity (Park et al., 2013; Wang et al., 2014). Oral feeding of L. curvatus HY7601 and L. plantarum KY1032 to HFD mice shifted the gut microbiota composition and reduced obesity (Park et al., 2013). Three probiotic strains, L. paracasei, L. rhamnosus, and B. animalis, were administrated to HFD fed-mice. Dietary supple mentation with these strains attenuated HFD-induce weight increase and modulated the gut microbiota composition (Wang et al., 2014). Although many studies documented anti-obesity effect of Lactobacillus as discussed above, several investigations found increase in the relative abundance of Lactobacillus in obese subjects and this increase correlated with obesity (Dahiya et al., 2017). Limitations of this study involved issues concerned with study sub jects and microbial functionality. As the animal experiments were con ducted in a very controlled condition, human trials are necessary to extrapolate the beneficial effects of CN consumption on obesity. Compositional changes in the gut microbiota showed a decrease in Streptococcaceae and Rikenellaceae, which are obesity-related bacteria, whereas an increase in probiotic microorganisms such as Lactobacilla ceae, with higher CN intake in HFD-fed mice. However, we do not assess changes in microbial functionality. In another study by our group, we investigated effects of dietary CN and exercise on obesity and gut microbiota in HFD-fed mice. Exercise, but not CN intake, increased the amount of acetate in the cecum (Nagano & Yano, 2020). The major products from gut microbiota are short-chain fatty acids (acetate, pro pionate, and butyrate) that can activate G protein-coupled receptors (GPR)43 and GPR41, and thereby exert anti-obesity effects (Koh, De €ckhed, 2016). Further studies are Vadder, Kovatcheva-Datchary, & Ba necessary to elucidate the relationship between the anti-obesity effects of CN intake and its effects on the gut microbiota composition. In conclusion, our data provide evidence that CN consumption control HFD-induced obesity in mice. In addition, our analysis of the gut microbiota showed that the increase in CN intake increased bacterial diversity and reduced the changes in gut microbiota composition caused by feeding HFD. The PCA plot of gut microbiota structure at the phylum level showed a shift in the microbiota composition resulting from feeding HFD. This shift in the PC1 axis was reversed with 0.2% CN intake. These results suggest that the consumption of CN has beneficial effects on obesity by controlling the balance of intestinal microbiota.
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Declaration of competing interest The authors declare no conflict of interest. CRediT authorship contribution statement Takao Nagano: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing, Funding acquisition. Hiromi Yano: Investigation, Methodology, Writing - review & editing, Funding acquisition. Acknowledgements This work was supported by the Fuji Foundation for Protein Research 6
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Subramanian, S., Blanton, L. V., Frese, S. A., Charbonneau, M., Mills, D. A., & Gordon, J. I. (2015). Cultivating healthy growth and nutrition through the gut microbiota. Cell, 161(1), 36–48. Takahashi, S., Tomita, J., Nishioka, K., Hisada, T., & Nishijima, M. (2014). Development of a prokaryotic universal primer for simultaneous analysis of bacteria and archaea using next-generation sequencing. PloS One, 9(8), e105592. Wang, J., Tang, H., Zhang, C., Zhao, Y., Derrien, M., Rocher, E., et al. (2014). Modulation of gut microbiota during probiotic-mediated attenuation of metabolic syndrome in high fat diet-fed mice. The ISME Journal, 9, 1–15. Weitkunat, K., Stuhlmann, C., Postel, A., Rumberger, S., Fankh€ anel, M., Woting, A. P., et al. (2017). Short-chain fatty acids and inulin, but not guar gum, prevent diet-
induced obesity and insulin resistance through differential mechanisms in mice. Scientific Reports, 7(1), 6109. Wu, J., Shi, S., Wang, H., & Wang, S. (2016). Mechanisms underlying the effect of polysaccharides in the treatment of type 2 diabetes: A review. Carbohydrate Polymers, 144, 474–494. Zhai, X., Lin, D., Zhao, Y., Li, W., & Yang, X. (2018). Effects of dietary fiber supplementation on fatty acid metabolism and intestinal microbiota diversity in C57BL/6J mice fed with a high-fat diet. Journal of Agricultural and Food Chemistry, 66 (48), 12706–12718. Zhang, C., Li, S., Yang, L., Huang, P., Li, W., Wang, S., et al. (2013). Structural modulation of gut microbiota in life-long calorie-restricted mice. Nature Communications, 4, 2163.
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Bioactive Carbohydrates and Dietary Fibre journal homepage: http://www.elsevier.com/locate/bcdf
Dietary cellulose nanofiber modulates obesity and gut microbiota in high-fat-fed mice Takao Nagano a, *, Hiromi Yano b a
Department of Food Science, Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa, 921-8836, Japan b Department of Health & Sports Science, Faculty of Health Science and Technology, Kawasaki University of Medical Welfare, 288 Matsushima, Kurashiki, Okayama, 719-1197, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Cellulose nanofiber Gut microbiota High-fat diet Lactobacillaceae Obesity 16S ribosomal RNA genes
Cellulose nanofiber (CN) has a unique feature of dispersing in water and producing a dispersion of high viscosity, similar to highly viscous soluble dietary fibers (DFs). The beneficial effects of soluble DFs on glycemic control are associated with viscosity, thus making CN a potential option to attenuate obesity and related diseases. Here, we studied the effects of CN intake on obesity and gut microbiota in high-fat diet (HFD)-fed mice. The CN-treated mice were provided 0.1% or 0.2% CN dispersions via drinking water for seven weeks. HFD-fed mice weighed more than mice fed a normal-fat diet (NFD). The weight gain in HFD-fed mice was suppressed in 0.2% CN-treated group, but not in 0.1% CN-treated group, when compared with CN-untreated group. Also, the accumulation of epididymal and subcutaneous fat in HFD-fed mice was lower in 0.2% CN treated group than in the CN-untreated group. Moreover, the fecal gut microbiota was analyzed by sequencing bacterial 16S ribosomal RNA genes. Oral administration of 0.2% CN, but not 0.1% CN, increased bacterial diversity and induced changes in the gut microbiota composition of HFD-fed mice. The principal component analysis (PCA) at the phylum level showed a shift in the microbiota composition resulting from feeding HFD. This shift in the PC1 axis was reversed by 0.2% CN intake. In addition, the relative abundance of Streptococcaceae and Rikenellaceae decreased while that of Lactobacillaceae increased, upon higher intake of CN. These results suggest that CN consumption has inhibitory effects on obesity mediated via control of gut microbiota balance.
1. Introduction In recent decades, obesity is a growing epidemic in developed countries and induces related metabolic diseases, such as type 2 diabetes and cardiovascular disease. Diets comprising high fat and low dietary fiber are the most common causes of obesity. In addition, the develop ment of obesity has been correlated with gut microbiota structures that may play a significant role in energy homeostasis (Cani & Everard, 2016; Dahiya et al., 2017; Lynch & Pedersen, 2016; Subramanian et al., 2015). For these reasons, many researchers have been investigating anti-obesity food components using high-fat diets (HFD)-induced obesity animal models to find solutions to obesity and other metabolic diseases and thereby provide potential health benefits for humans (Genda et al., 2018; Miyamoto et al., 2018; Weitkunat et al., 2017; Zhai, Lin, Zhao, Li, & Yang, 2018).
Dietary fibers are one of the most important food components and their beneficial effects on our health dependent on their physicochem ical properties, such as solubility, viscosity, and fermentability by the gut microbiota (Gidley & Yakubov, 2019; Kaczmarczyk, Miller, & Freund, 2012; Wu, Shi, Wang, & Wang, 2016). Cellulose is the main component of plants and the most consumed dietary fiber. Dhital, Gid ley, and Warren (2015) investigated the inhibition of α-amylase activity by cellulose. They found that cellulose reduced α-amylase-mediated starch hydrolysis in a concentration dependent manner, and its inhibi tory activity was exerted by non-specific adsorption of α-amylase to cellulose. However, the inhibition of enzyme activity by cellulose is much lower than that by a polysaccharide thickener, guar gal actomannan, as cellulose is not soluble in water and shows low viscosity at 0.1–3% concentration (Slaughter, Ellis, Jackson, & Butterworth, 2002). Sasaki and Kohyama (2012) studied starch digestibility by
* Corresponding author. Department of Food Science, Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, 1-308, Suematsu, Nonoich, Ishikawa, 921-8836, Japan. E-mail address: [email protected] (T. Nagano). https://doi.org/10.1016/j.bcdf.2020.100214 Received 16 June 2019; Received in revised form 29 January 2020; Accepted 17 February 2020 Available online 20 February 2020 2212-6198/© 2020 Published by Elsevier Ltd.
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adding non-starch polysaccharides, such as pectin, konjac glucomannan, guar gum, and xanthan gum. They found that increased viscosity due to addition of the non-starch polysaccharides reduced starch digestibility in an in vitro assay. These results indicate that viscosity is a key factor in inhibition of α-amylase activity. The Isogai group developed a method to prepare cellulose nanofiber (CN) from plant cellulose by catalytic oxidation with 2,2,6,6-tetramethl piperidine-1-oxyl radical (TEMPO). This CN has unique features of dispersing in water and high viscosity (Saito, Kimura, Nishiyama, & Isogai, 2007). Since CN dispersions showed high viscosity, they also investigated inhibitory effects of CN on postprandial increases in blood glucose and insulin in mice. The postprandial increase in blood insulin was suppressed by CN in a concentration dependent manner. These re sults suggest that CN is a potential option to attenuate obesity and related disorders (Shimotoyodome, Suzuki, Kumamoto, Hase, & Isogai, 2011). However, the beneficial effects of CN intake on obesity and gut microbiota remain unclear. In the present study, we investigated the effect of CN supplementa tion on obesity in a HFD-fed mouse model of obesity. In addition, the gut microbiota composition was analyzed by sequencing bacterial 16S ri bosomal RNA genes (16S rDNA) to elucidate the effect of CN intake on the intestinal microbiota in HFD-fed mice.
Illumina Miseq platform (Illumina, San Diego, CA, USA), as described previously (Nagano, Katase, & Tsumura, 2019). All sequence reads were analyzed by the TechnoSuruga laboratory (Shizuoka, Japan). The paired reads were joined together with the fastq-join program (http://code.goo gle.com/p/ea-utils/). Only reads that had quality value (QV) scores of �20 for more than 99% of the sequence were extracted. Chimeras were detected and omitted by USEARCH 6.1.544_i86 (Edgar, Haas, Clemente, Quince, & Knight, 2011). Operational taxonomic units (OTUs) were determined by clustering sequences with a similarity threshold of 97%, and α- and β-diversities of the gut microbiota were analyzed using Quantitative Insight into Microbial Ecology (QIIME ver1.8.0) (Caporaso, Kuczynski, Stombaugh et al., 2010). Taxonomic analysis of the 16S rDNA sequences was also conducted based on the Ribosomal Database Project, using the Metagenome@KIN analysis software (World Fusion, Tokyo, Japan) (Takahashi, Tomita, Nishioka, Hisada, & Nishijima, 2014). 2.4. Statistical analysis
2. Methods
The statistical significance of differences was analyzed using nonparametric Kruskal-Wallis test followed by Mann-Whitney’s post-hoc tests, and the principal component analysis (PCA) was conducted using Origin 2019 software (OriginLab, Northampton, MA, USA). The data were considered significant at P < 0.05.
2.1. Materials
3. Results
The normal fat diet (NFD, 10% kcal fat; D12450J) and HFD (60% kcal fat; D12492G) with no added cellulose were purchased from Research Diets, New Brunswick, NJ, US. The CN (BiNFi-s™, WFo10002) was obtained from Sugino Machine (Uozu, Japan).
3.1. Feed, water, and CN intake Feed intake was 11.7 � 0.4 kcal/day, 11.4 � 0.6 kcal/day, 11.4 � 0.7 kcal/day, and 11.3 � 6.0 kcal/day for NFD-fed and CN-untreated, 0.1% and 0.2% CN-treated HFD-fed mice groups, respectively. Feed intake was significantly higher in the NFD group compared with that in HFD groups based on weight (P < 0.01), but not calories. Water intake was 3.65 � 0.75 mL/day, 3.66 � 0.64 mL/day, 4.63 � 0.57 mL/day, and 4.69 � 0.85 mL/day for NFD-fed and CN-untreated, 0.1% and 0.2% CNtreated HFD-fed mice groups, respectively. There was no difference in water intake between NFD-fed and CN-untreated HFD-fed mice groups. However, mice from these groups consumed lower amounts of water than the mice in the 0.1% and 0.2% CN-treated HFD-fed groups (P < 0.01). As mice were provided CN via drinking water, CN intake was 4.63 � 0.57 mg/day and 9.34 � 1.07 mg/day for 0.1% and 0.2% CN-treated HFD-fed mice, respectively.
2.2. Animal experiments Eight-week-old male C57BL/6N mice were obtained from Clea Japan (Tokyo, Japan). Four mice were housed per cage in a temperaturecontrolled room (22 � 1� C, 12-h light/dark cycle) and conventional conditions. The experiment involving the mice including the procedures performed, were approved by the Institutional Animal Care and Use Committee of Kawasaki University of Medical Welfare (#18–015). After three-day acclimatization, the mice were randomly divided into NFDfed (NFD, n ¼ 8), CN-untreated HFD-fed (CN0%, n ¼ 7), 0.1% CNtreated HFD-fed (CN0.1%, n ¼ 8), and 0.2% CN-treated HFD-fed (CN0.2%, n ¼ 8) groups. The NFD group was fed with NFD only, while CN0%, CN0.1%, and CN0.2% groups were fed with HFD. CN0.1% and CN0.2% groups were orally administered 0.1 wt% and 0.2 wt% CN dispersions, respectively, via drinking water provided through a water bottle, throughout the experiment (seven weeks). For glucose tolerance test (GTT) conducted at seventh week, each mouse was lightly anes thetized by the inhalation of Isoflurane prior to glucose administration (2 g/kg, i.p.). Blood samples were collected from the tail vein, and blood glucose levels were measured using the glucose monitoring device AccuChek, (Roche, Basel, Schweiz) immediately prior to, and at 15, 30, 60 and 120 min after glucose administration (Bradley, Jeon, Liu, & Maratos-Flier, 2008). On the final day of the experiment, mice were rendered unconscious by Isoflurane exposure and sacrificed by cervical dislocation for euthanasia. Epididymal, visceral, and subcutaneous fat were taken and weighed. The cecum was collected and pH levels of the cecum contents were measured. The fecal contents were collected from the proximal colon. Collected samples were stored at 80� C until use.
3.2. Body weight and GTT Body weight of mice in each group increased with time over the seven week duration (Fig. 1A). After seven weeks, the three HFD-fed mice groups weighed significantly more than mice fed NFD (P < 0.05). The weight gain was not suppressed in 0.1% CN-treated mice, but in 0.2% CN-treated mice (P < 0.05), when compared with CN-untreated HFD-fed mice (Fig. 1B). Moreover, the levels of fasting blood glucose were measured and significant differences were not observed in the four treatment groups (Fig. 1C). GTTs were also conducted (Fig. 1D) and there were no significant differences in the area under the curves (AUCs) among the four treatment groups (Fig. 1E). 3.3. Fat accumulation, weight, and pH of cecum The accumulation of epididymal, visceral, and subcutaneous fat were determined (Fig. 2A–C). The weights of epididymal, visceral, and sub cutaneous fat in CN-untreated and 0.1% CN-treated HFD-fed mice were significantly higher than those in NFD-fed mice (P < 0.01). In contrast, the fat accumulation in 0.2% CN treated HFD-fed mice was lower for epididymal and subcutaneous fat when compared with that in the CNuntreated HFD-fed mice (P < 0.05). In addition, no differences were
2.3. Bacterial DNA sequencing and taxonomic analysis Bacteria were recovered from fecal sample-lysates using the FastDNA Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA). PCR and sequencing of the V4 region (16S rDNA) was performed using the 2
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Fig. 1. Effects of cellulose nanofiber (CN) intake on body weight and glucose tolerant tests (GTTs) in high-fat diet (HFD)-fed mice. (A) Body weight change for seven weeks. (B) Body weight gain after seven weeks. (C) Fasting blood glucose levels. (D) GTTs at seventh week. (E) Area under the curves of blood glucose as shown in Fig. 1D. NFD, normal-fat diet-fed group; CN0%, CN-untreated HFD-fed group; CN0.1%, 0.1% CN-treated HFD-fed group; CN0.2%, 0.2% CN-treated HFD-fed group. The data are presented as the mean � standard error of the mean (SEM) in A and D; *p < 0.05; **p < 0.01.
Fig. 2. Effects of cellulose nanofiber intake on fat accumulation, cecum weight and pH in high-fat diet (HFD)-fed mice. (A) Epididymal fat mass. (B) Visceral fat mass. (C) Subcutaneous fat mass. (D) Cecum weight. (E) Cecum pH. NFD, normal-fat diet-fed group; CN0%, CN-untreated HFD-fed group; CN0.1%, 0.1% CN-treated HFDfed group; CN0.2%, 0.2% CN-treated HFD-fed group. *p < 0.05, **p < 0.01, ***p < 0.001.
3
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observed between 0.2% CN treated HFD-fed and NFD-fed mice groups for epididymal, visceral, and subcutaneous fat. Furthermore, the cecum weights (Fig. 2D) were significantly higher (P < 0.05), and pH levels of the cecum contents (Fig. 2E) were lower (P < 0.01) in NFD-fed mice than those in the three HFD-fed mice groups. In addition, the cecum weights were also higher, and pH in the cecum was significantly lower in 0.2% CN treated HFD-fed mice than those in 0.1% CN treated HFD-fed mice (P < 0.05).
The relative abundance of Streptococcaceae and Rikenellaceae increased with feeding HFD and decreased with higher CN intake in the HFD-fed mice (Fig. 5D and E). The relative abundance of Streptococca ceae was significantly lower in the NFD-fed and 0.2% CN-treated HFDfed mice than in the CN-untreated HFD-fed mice (P < 0.05; Fig. 5D). In contrast, the relative abundance of Rikenellaceae was significantly higher in the CN-untreated (P < 0.05) and 0.1% CN-treated (P < 0.01) HFD-fed mice than in the NFD-fed mice, and lower in the 0.2% CNtreated mice than in the 0.1% CN-treated mice (P < 0.05; Fig. 5E). Furthermore, the relative abundance of Lactobacillaceae increased with increasing CN consumption. The relative abundance of Lactobacillaceae was significantly higher in the 0.2% CN-treated HFD-fed mice compared with that in mice from the other three groups (P < 0.05, Fig. 5F).
3.4. Gut microbiota composition in mice Total sequence reads and number of OTUs derived by sequencing bacterial 16S rDNA are shown in the Supplementary table. The analysis of α- and β-diversities were performed to estimate bacterial richness and diversity (Fig. 3). Chao-1 index in the NFD-fed mice was significantly lower than that in the three HFD-fed mice groups (P < 0.01), and chao-1 index in 0.2% CN-treated HFD-fed mice was significantly higher than that in CN-untreated HFD-fed mice (P < 0.05; Fig. 3A). Furthermore, the PCA based on weighted UniFrac of β-diversity showed that the HFD-fed and NFD-fed mice separated on the PC1 axis (71.0% of explained vari ance). The oral administration of 0.2% CN, but not 0.1%CN, changed the composition of gut microbiota in the HFD-fed mice as evident on the PC2 axis (13.4% of explained variance) (Fig. 3B). The PCA (Fig. 4A) and gut microbiota composition (Fig. 4B) were determined at the phylum level. In the PCA, the microbiota composi tions of the NFD-fed and CN-untreated HFD-fed mice groups separated on the PC1 axis (39.1% of explained variance). In contrast, the micro biota compositions of the CN-untreated and 0.1% CN-treated HFD-fed mice groups did not separate on the PC1 axis. The microbiota compo sition of the 0.2% CN-treated HFD-fed mice shifted toward that of NFDfed mice and separated from that of CN-untreated HFD-fed mice on the PC1 axis. At the family level, the relative abundance of Erysipelotrichaceae (Fig. 5A) decreased with feeding HFD and was significantly lower in the three HFD-fed mice groups than in the NFD-fed mice (P < 0.01). In addition, the relative abundance of Erysipelotrichaceae was significantly higher in the 0.2% CN-treated mice than in the 0.1% CN-treated mice (P < 0.05). The relative abundance of Porphyromonadaceae also decreased with feeding HFD and was significantly lower in the 0.1% (P < 0.01) and 0.2% (P < 0.05) CN-treated HFD-fed mice than in the NFD-fed mice (Fig. 5B). In contrast, the relative abundance of Verrucomicrobiaceae increased with feeding HFD and was significantly higher in the three HFD-fed mice groups than in the NFD-fed mice (P < 0.01, Fig. 5C).
4. Discussion Our results demonstrated that 0.2% CN consumption via drinking water controlled obesity in mice fed HFD, as evidenced by reduced body weight gain and fat accumulation. It is likely that this beneficial effect of CN intake is due to the property of forming a solution of high viscosity. Clinical studies have indicated that highly viscous soluble DFs, such as guar gum and β-glucan, improve glycemic control, whereas insoluble DFs such as cellulose do not provide the beneficial effect (McRorie & McKeown, 2017). Sasaki and Kohyama (2012) reported a negative relationship between the α-amylase-mediated starch hydrolysis and apparent viscosity by adding non-starch polysaccharides. In addition, the postprandial increase in blood insulin was suppressed by CN in a concentration dependent manner (Shimotoyodome et al., 2011). In this study, mice were provided with 0.1% and 0.2% CN via drinking water, and HFD with no added/supplemented fibers. The HFDinduced body weight increased were suppressed by administration of 0.2% CN, but not 0.1% CN. The amount of CN intake was 4.63 � 0.57 mg/day and 9.34 � 1.07 mg/day for 0.1% and 0.2% CN-treated HFD-fed mice, respectively; these intakes correspond to approximately 9.35 � 0.42 g and 19.80 � 1.96 g for a 60 kg human per day. As per Japanese nutritional guidelines, 20 g dietary fiber intake per day is recommended for an adult man. It is likely that this guideline helps explain why 0.2% CN-treatment has a positive effect on obesity, whereas only a marginal effect is seen in 0.1% CN-treated mice. Obesity results from an imbalance in the body’s regulation of energy intake and expenditure (Dahiya et al., 2017). Recent evidence from studies in animal models and humans suggests that the gut microbiota has a significant role in energy acquisition and regulation. Its
Fig. 3. Effects of cellulose nanofiber (CN) intake on bacterial diversities in the gut microbiota of high-fat diet (HFD)-fed mice. (A) Chao-1 index (α-diversity), (B) Principal component analysis (PCA) based on weighted UniFrac (β-diversity). NFD, normal-fat diet-fed group; CN0%, CN-untreated HFD-fed group; CN0.1%, 0.1% CN-treated HFD-fed group; CN0.2%, 0.2% CN-treated HFD-fed group. *p < 0.05, **p < 0.01, ***p < 0.001. 4
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Fig. 4. Cellulose nanofiber (CN) intake affects the gut microbiota composition in high-fat diet (HFD)-fed mice at the phylum level. The gut microbiota composition was analyzed by sequencing the bacterial 16S ribosomal RNA genes, followed by principal component analysis (PCA) (A) and determination of the taxonomic distribution (B). NFD, normal-fat diet-fed group; CN0%, CN-untreated HFD-fed group; CN0.1%, 0.1% CN-treated HFD-fed group; CN0.2%, 0.2% CN-treated HFDfed group.
Fig. 5. Cellulose nanofiber (CN) supplementation influences the relative abundance of intestinal bacteria in high-fat diet (HFD)-fed mice at the family level. (A) Erysipelotrichaceae, (B) Porphyromonadaceae, (C) Verrucomicrobiaceae, (D) Streptococcaceae, (E) Rikenellaceae, (F) Lactobacillaceae. NFD, normal-fat diet-fed group; CN0%, CN-untreated HFD-fed group; CN0.1%, 0.1% CN-treated HFD-fed group; CN0.2%, 0.2% CN-treated HFD-fed group. The data are presented as the means � SEM; *p < 0.05; **p < 0.01.
composition also differs between obese and lean animals and humans (Subramanian et al., 2015). In the present study, administration of 0.2% CN, but not 0.1%CN, increased α-diversity and induced changes in gut microbiota composition. In addition, the PCA plot at the phylum level showed a shift in the microbiota composition resulting from HFD feeding, and this difference in the PC1 axis disappeared between the 0.2% CN-treated HFD-fed and NFD-fed mice. These results indicate that administration of 0.2% CN, but not 0.1% CN, has increased effects on bacterial richness and changes in gut microbiota composition. The data provides a good correlation with the results that consumption of CN suppressed obesity in mice fed HFD. Moreover, the abundance of Ery sipelotrichaceae and Porphyromonadaceae decreased, whereas the relative abundance of Verrucomicrobiaceae, Streptococcaceae, and Rikenellaceae
increased in the CN-untreated HFD-fed mice when compared with the NFD-fed mice. In previous studies, the increase in Streptococcaceae and Rikenellaceae were correlated with HFD-induced obesity (Daniel et al., 2013; Jiang et al., 2015; Price et al., 2009; Zhang et al., 2013). Our study also showed that Streptococcaceae and Rikenellaceae decreased with increasing CN intake in HFD-fed mice. In previous studies, some strains of Streptococcaceae were also reported to induce mild inflammation in humans (Price et al., 2009). The high abundance of Streptococcaceae was also shown to be associated with diseases such as obesity, diabetes and colon cancer (Daniel et al., 2013). Furthermore, it was pointed out that decreasing Streptococcaceae was a potential strat egy for preventing metabolic diseases (Zhang et al., 2013). Moreover, the presence of Alistipes, a genus in Rikenellaceae, was also correlated to 5
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(to T.N.) and Interdepartmental Research Fund of Kawasaki University of Medical Welfare (to H.Y.). We thank TechnoSuruga laboratory (Shi zuoka, Japan) for taxonomic analysis of the 16S rDNA sequences. We would like to thank Editage (www.editage.jp) for English language editing.
type-2 diabetes in humans (Jiang et al., 2015). In this study, Lactobacillaceae increased with increasing CN con sumption. In former studies, effects of prebiotics supplementation were investigated in animal models and humans (Dahiya et al., 2017; Haar man & Knol, 2006; Park et al., 2013). Prebiotics fibers extensively studied are fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), and inulin (Dahiya et al., 2017). A mixture of FOS and GOS in infant milk formula stimulated the growth of Lactobacillus and Bifido bacterium and the resulting microbiota was comparable to the micro biota of breast-fed infants. These prebiotic fibers also modulated the gut microbiota and increased populations of Lactobacillus and Bifidobacte rium in the gut (Haarman & Knol, 2006). In other studies, Lactobacillus strains were studied as probiotics used to modulate gut microbiota composition in the context of obesity (Park et al., 2013; Wang et al., 2014). Oral feeding of L. curvatus HY7601 and L. plantarum KY1032 to HFD mice shifted the gut microbiota composition and reduced obesity (Park et al., 2013). Three probiotic strains, L. paracasei, L. rhamnosus, and B. animalis, were administrated to HFD fed-mice. Dietary supple mentation with these strains attenuated HFD-induce weight increase and modulated the gut microbiota composition (Wang et al., 2014). Although many studies documented anti-obesity effect of Lactobacillus as discussed above, several investigations found increase in the relative abundance of Lactobacillus in obese subjects and this increase correlated with obesity (Dahiya et al., 2017). Limitations of this study involved issues concerned with study sub jects and microbial functionality. As the animal experiments were con ducted in a very controlled condition, human trials are necessary to extrapolate the beneficial effects of CN consumption on obesity. Compositional changes in the gut microbiota showed a decrease in Streptococcaceae and Rikenellaceae, which are obesity-related bacteria, whereas an increase in probiotic microorganisms such as Lactobacilla ceae, with higher CN intake in HFD-fed mice. However, we do not assess changes in microbial functionality. In another study by our group, we investigated effects of dietary CN and exercise on obesity and gut microbiota in HFD-fed mice. Exercise, but not CN intake, increased the amount of acetate in the cecum (Nagano & Yano, 2020). The major products from gut microbiota are short-chain fatty acids (acetate, pro pionate, and butyrate) that can activate G protein-coupled receptors (GPR)43 and GPR41, and thereby exert anti-obesity effects (Koh, De €ckhed, 2016). Further studies are Vadder, Kovatcheva-Datchary, & Ba necessary to elucidate the relationship between the anti-obesity effects of CN intake and its effects on the gut microbiota composition. In conclusion, our data provide evidence that CN consumption control HFD-induced obesity in mice. In addition, our analysis of the gut microbiota showed that the increase in CN intake increased bacterial diversity and reduced the changes in gut microbiota composition caused by feeding HFD. The PCA plot of gut microbiota structure at the phylum level showed a shift in the microbiota composition resulting from feeding HFD. This shift in the PC1 axis was reversed with 0.2% CN intake. These results suggest that the consumption of CN has beneficial effects on obesity by controlling the balance of intestinal microbiota.
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Declaration of competing interest The authors declare no conflict of interest. CRediT authorship contribution statement Takao Nagano: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing, Funding acquisition. Hiromi Yano: Investigation, Methodology, Writing - review & editing, Funding acquisition. Acknowledgements This work was supported by the Fuji Foundation for Protein Research 6
T. Nagano and H. Yano
Bioactive Carbohydrates and Dietary Fibre 22 (2020) 100214
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