Fructooligosaccharides act on the gut-bone axis to improve bone independent of Tregs and alter osteocytes in young adult C57BL/6 female mice

Abstract

Targeting the gut-bone axis with probiotics and prebiotics is considered as a promising strategy to reduce the risk of osteoporosis. Gut-derived short chain fatty acids (SCFA) mediate the effects of probiotics on bone via Tregs, but it is not known whether prebiotics act through a similar mechanism. We investigated how 2 different prebiotics, tart cherry (TC) and fructooligosaccharide (FOS), affect bone, and whether Tregs are required for this response. Eight-wk-old C57BL/6 female mice were fed with diets supplemented with 10% w/w TC, FOS, or a control diet (Con; AIN-93M) diet, and they received an isotype control or CD25 Ab to suppress Tregs. The FOS diet increased BMC, density, and trabecular bone volume in the vertebra (~40%) and proximal tibia (~30%) compared to the TC and control diets (Con), irrespective of CD25 treatment. Both prebiotics increased (P < .01) fecal SCFAs, but the response was greater with FOS. To determine how FOS affected bone cells, we examined genes involved in osteoblast and osteoclast differentiation and activity as well as genes expressed by osteocytes. The FOS increased the expression of regulators of osteoblast differentiation (bone morphogenetic protein 2 [Bmp2], Wnt family member 10b [Wnt10b] and Osterix [Osx]) and type 1 collagen). Osteoclasts regulators were unaltered. The FOS also increased the expression of genes associated with osteocytes, including (Phex), matrix extracellular phosphoglycoprotein (Mepe), and dentin matrix acidic phosphoprotein 1 (Dmp-1). However, Sost, the gene that encodes for sclerostin was also increased by FOS as the number and density of osteocytes increased. These findings demonstrate that FOS has a greater effect on the bone mass and structure in young adult female mice than TC and that its influence on osteoblasts and osteocytes is not dependent on Tregs.

Keywords: fructooligosaccharide; gut–bone axis; osteocytes; prebiotics; short chain fatty acids; tart cherry.

Graphical Abstract

Figure 1

Bone mass, structural alterations, and biomarkers in mice consuming control diet (Con), TC, or FOS with (+CD25; green) or without CD25 (−CD25; black) Ab: whole-body (A) BMC, (B) BMD, (C) lumbar vertebra trabecular bone volume relative total volume (BV/TV), (D) representative 3D images of the proximal tibia, (E) proximal tibia BV/TV, (F) tibia midshaft cortical thickness, (G) cortical area, (H) serum P1NP, and (I) CTX-1. Data presented as mean + SE. P-values < .05 are statistically different and P-values > .05 are not shown. ^*** indicates a main effect P < 0.0001, ^** indicates a main effect P < 0.01, and ^* indicates a main effect P < 0.05. Superscript letters show differences between groups with a significant CD25 ^* diet interaction.

Figure 2

The relative abundance of T lymphocytes using FACS in the lamina propria of the ileum after 8 wk of control (con), TC, or FOS diets with (+CD25; green) or without (−CD25; black) CD25 Ab. The percentage of (A) CD4⁺cells, (B) CD25⁺Foxp3⁺ Tregs, (C) Th-17⁺ cells, and the (D) ratio of Treg to Th-17 cells are shown. Data presented as mean + SE. P-values < .05 are statistically significant and P-values > .05 are not shown. ^*** indicates main effect P < 0.0001, ^** indicates main effect P < 0.01, and ^* indicates main effect P < 0.05. Superscript letters show differences between groups with a significant CD25 ^* diet interaction. Groups that do not have the same superscript letter are statistically different from each other (P < 0.05).

Figure 3

The FACS analysis of bone marrow T lymphocytes after 8 wk of control (Con), TC or FOS diets with (+CD25; green) or without (−CD25; black) CD 25 Ab. The percentage of (A) CD4⁺cells, (B) CD25⁺Foxp3⁺ Tregs, (C) Th-17⁺ cells, the (D) ratio of Treg to Th-17 cells, and (E) CD8⁺ cells are shown. Data presented as mean + SE. P-values < .05 are statistically significant and P-values > .05 are not shown. ^*** indicates a main effect P < 0.0001, ^** indicates a main effect P < 0.01, and ^* indicates a main effect P < 0.05. Superscript letters show differences between groups with a significant CD25 ^* diet interaction. Groups that do not have the same superscript letter are statistically different from each other (P < 0.05).

Figure 4

Supplementation with FOS compared to control (Con) diet with (CD + 25; green) or without (−CD25; black) CD25 Ab alters the abundance of mRNA of genes involved in osteoblast differentiation (A) Wnt10b, (B) Bmp2, (C) Osx, and (D) indicator of osteoblast activity Col1a1 over the course of the 8-wkstudy. Data presented as mean + SE. P-values < .05 are considered to be statistically significant. No interactions were observed and P-values for statistically significant main effects are shown.

Figure 5

Eight weeks of dietary supplementation with FOS compared to control (Con) diet with (CD +25; green) or without (−CD25; black) the CD25 Ab alters the abundance of genes expressed by osteocytes: (A) Phex, (B) Mepe, (C) Dmp1, (D) Cx43, (E) Sost, and (F) osteocyte number. Data presented as mean + SE. P-values < .05 are considered to be statistically significant. No interactions were observed and P-values for statistically significant main effects are shown.