Volume 24, Issue 12, Pages (September 2018)

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Volume 24, Issue 12, Pages 3087-3098 (September 2018) Mannose Alters Gut Microbiome, Prevents Diet-Induced Obesity, and Improves Host Metabolism  Vandana Sharma, Jamie Smolin, Jonamani Nayak, Julio E. Ayala, David A. Scott, Scott N. Peterson, Hudson H. Freeze  Cell Reports  Volume 24, Issue 12, Pages 3087-3098 (September 2018) DOI: 10.1016/j.celrep.2018.08.064 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 24, 3087-3098DOI: (10.1016/j.celrep.2018.08.064) Copyright © 2018 The Authors Terms and Conditions

Figure 1 Mannose Induces a Lean Phenotype, Reduces Liver Steatosis, and Improves Glucose Tolerance (A–E) Body weights were measured weekly for C57BL/6J mice weaned at the age of 3 weeks on either ND or 45% HFD without or with (A) 2% mannose at weaning, (B) 2% galactose at weaning, (C) 2% mannose started 3 weeks PW, (D) 2% mannose started at weaning and continued for 16 weeks followed by its removal, and (E) 2% mannose at weaning. (F–H) Fat mass measurements. Total fat mass is the sum of all fat pads collected at age 16 weeks (F). Mice weaned on 45% HFD, and mannose started at 3 weeks PW (G). Fat mass of mice weaned on 60% HFD and 2% mannose (H). (I) Histology images are representative of analysis from 4 mice in each diet group. Top: H&E staining of epididymal adipocytes is shown. Middle and bottom: oil red O staining of frozen liver sections from mice is shown. (J–M) Three-week-old C57BL6/J mice were weaned on ND or HFD or HFD+2% mannose. (J and K) Glucose tolerance test: fasted mice were injected with glucose (1.5 g/kg), and blood glucose was measured at 0, 15, 30, 60, and 90 min. Glucose clearance (J). Area under the curve indicates glucose levels (K). (L and M) Insulin tolerance test: mice were injected with insulin (0.5 U/kg), and blood glucose levels were measured at 0, 15, 30, 60, and 90 min. Glucose clearance (L). The fall in the slope of the curve is the measure of insulin sensitivity (M). The data presented in the graphs are the mean ± SEM of indicated number of mice (N). Significance was calculated for HFD+M versus HFD using Student’s t test. ∗∗∗∗p < 0.00005; ∗∗∗p < 0.0005; ∗∗p < 0.005; ∗p < 0.05. See also Figures S1 and S2 and Table S1. Cell Reports 2018 24, 3087-3098DOI: (10.1016/j.celrep.2018.08.064) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Mannose Supplementation Changes Gut Microbial Composition (A) Schematic shows the experimental design. 16S rDNA sequencing was performed on 6–8 mice in each diet group: ND, HFD, or HFD+2% mannose where mannose was supplemented in water at 0 weeks (N0, H0, and HM0), 3 weeks (N3, H3, and HM3), or 8 weeks (N8, H8, and HM8) PW. (B) Changes in phyla with different diets. Significance was calculated for HFD+M versus HFD using Student’s t test. ∗∗∗∗∗∗p = 8.26E-10; ∗∗∗∗∗p < 0.000005; ∗∗∗∗p < 0.00005; ∗∗∗p < 0.0005; ∗∗p < 0.005; ∗p < 0.05. (C and D) The heatmaps generated using R Studio show the relative abundance of relevant genus (C) and species (D) in HFD and HFD+M groups. Percentage is the % identity to taxonomy (best hit) in NCBI 16S database. The accompanying box shows the color scale for relative abundance. Black color shows undetectable genera or species. Also see Figure S3. Cell Reports 2018 24, 3087-3098DOI: (10.1016/j.celrep.2018.08.064) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Mannose Modulates Transcriptome of Gut Microbiota RNAs from cecal contents were pooled from 8 mice in each group weaned on HFD with mannose (HM) or without 2% mannose (H) in drinking water started at 3 and 8 weeks PW. Differential abundances of the contigs between HFD and HFD+M were calculated for both 3 weeks and 8 weeks PW. The comparisons were drawn between 3 weeks PW (mannose-responsive) mice and 8 weeks PW (non-responsive) mice. (A and B) Taxonomic analysis: comparison of the number of differentially abundant contigs mapped to various phyla (A) and genus (B). (C–E) Functional analysis: heatmaps of relative transcript abundance based on RPKM values using R Studio. The accompanying box shows the color scale for relative abundance with red being the most abundant (set to a value of 1) and dark green being the least abundant. Black color in the heatmap depicts no differential abundance. (C) Relative transcript distribution in various metabolic pathways. (D and E) Relative transcript abundance of most relevant and abundant genes within the subsets of glycosyl hydrolases (D) and carbohydrate metabolism (E). Asterisk indicates the data for transcripts that did not meet the initial cutoff of ≥3 fold change; Kal’s p < 10−5 for individual contigs, however, are shown for comparison. Also see associated Tables S2, S3, S4, S5, S6, S7, and S8. (F) Summary of mannose effects and the potential mechanism. Cell Reports 2018 24, 3087-3098DOI: (10.1016/j.celrep.2018.08.064) Copyright © 2018 The Authors Terms and Conditions