Gut microbiota induce IGF-1 and promote bone formation and growth

Abstract

Appreciation of the role of the gut microbiome in regulating vertebrate metabolism has exploded recently. However, the effects of gut microbiota on skeletal growth and homeostasis have only recently begun to be explored. Here, we report that colonization of sexually mature germ-free (GF) mice with conventional specific pathogen-free (SPF) gut microbiota increases both bone formation and resorption, with the net effect of colonization varying with the duration of colonization. Although colonization of adult mice acutely reduces bone mass, in long-term colonized mice, an increase in bone formation and growth plate activity predominates, resulting in equalization of bone mass and increased longitudinal and radial bone growth. Serum levels of insulin-like growth factor 1 (IGF-1), a hormone with known actions on skeletal growth, are substantially increased in response to microbial colonization, with significant increases in liver and adipose tissue IGF-1 production. Antibiotic treatment of conventional mice, in contrast, decreases serum IGF-1 and inhibits bone formation. Supplementation of antibiotic-treated mice with short-chain fatty acids (SCFAs), products of microbial metabolism, restores IGF-1 and bone mass to levels seen in nonantibiotic-treated mice. Thus, SCFA production may be one mechanism by which microbiota increase serum IGF-1. Our study demonstrates that gut microbiota provide a net anabolic stimulus to the skeleton, which is likely mediated by IGF-1. Manipulation of the microbiome or its metabolites may afford opportunities to optimize bone health and growth.

Keywords: IGF-1; SCFA; bone; microbiota.

Conflict of interest statement

W.S.G. is a Scientific Advisory Board member of Evelo Therapeutics and Synlogic and consults for Janssen Pharmaceuticals. J.F.C. receives author royalties from Up To Date, Inc.

Figures

Fig. 1.

Microbiota promote bone turnover and endochondral ossification after short-term colonization in adulthood. (A) Experimental procedure schematic. Two-month-old GF CB6F1 mice were colonized with unfractionated feces from SPF mice and evaluated 1 mo later. (B) Trabecular bone mass determined by micro-CT (n ≥ 15), with representative images of trabecular bone from the femurs of GF or colonized (Col) siblings. (C and D) Serum bone turnover markers CTX-I and P1NP in GF and Col mice (n ≥ 15). (E) Dynamic BFR/BS and MAR, determined by histomorphometry on double-labeled GF and Col mice (n ≥ 10). Representative double labeling in trabecular bone (Left) and secondary ossification center (Right) is shown. White arrows indicate bone formation in the interval between labels. (F) Growth plate thickness measured on 3D reconstructions of micro-CT images and on histologic specimens (Left). Representative H&E and toluidine blue staining is shown (Right). White dashed lines indicate the growth plate. Black arrows indicate hypertrophic chondrocytes. *P < 0.05; ***P < 0.001.

Fig. S1.

Effect of colonization on macroscopic data, vertebral and cortical bone parameters. (A) Body weight and length of GF and colonized (Col) littermates, 1 mo after colonization. (B–H) Comparison of GF and colonized littermates, 8 mo after colonization. Only female mice were examined, unless otherwise specified. (B) L5 vertebrae length. (C and D) Cortical bone thickness and porosity for female (C) and male (D) mice. (E) Body weight. (F) Vertebral L5 trabecular bone parameters. (G and H) Cortical dynamic bone formation indices in periosteal (Peri) and endosteal (Endo) areas. *P < 0.05; ns, not statistically significant.

Fig. 2.

Long-term colonization with gut microbiota promotes longitudinal and radial bone growth in adult mice. (A) Schematic diagram of the experimental procedure. Two-month-old GF CB6F1 female and male mice (n ≥ 6) were colonized with unfractionated feces from SPF mice and evaluated 8 mo later. (B–D) Bone parameters of GF and colonized (Col) mice determined by micro-CT. (B) Femur length (Left) with representative images (Right). (C) Periosteal (Left) and endosteal (Middle) area with representative 3D reconstructions of midshaft cortical bone (Right). (D) Trabecular bone mass. (E and F) Serum bone turnover markers CTX-I and P1NP in female GF and Col mice. *P < 0.05; **P < 0.01; ns, not statistically significant.

Fig. 3.

Microbiota increase systemic and local IGF-1. Serum IGF-1 and IGFBP3 levels in (A) long-term and (B) short-term colonized mice compared with GF siblings (n ≥ 6). (C) Relative expression of Runx2 in the epiphyseal bone tissue (n = 5). (D and E) Tissue IGF-1 production. (D) Liver and (E) abdominal fat pad total tissue IGF-1 (Left) was calculated by multiplying the IGF-1 production per gram tissue (Middle) by the weight of the tissue (Right). (F) Relative expression of Igf1 in the bone marrow (BM). *P < 0.05; **P < 0.01; ns, not statistically significant.

Fig. S2.

Effects of colonization on serum IGF-1, GH, and tissue Igf1 expression. GF and colonized (Col) littermates were compared at the points indicated in the legend. (A) Serum IGF-1 concentration, 1 wk after colonization. (B) Serum GH concentration, 1 mo after colonization. (C and D) Relative expression of Igf1 and Igfbp3 in liver and adipose tissue, 1 mo after colonization. (E) Relative expression of Igf1 in muscle tissue, 1 mo after colonization. *P < 0.05; ns, not statistically significant.

Fig. S3.

Igf1 and Igf1r expression in bone. Relative expression of (A) Igf1 and (B) Igf1r in epiphyseal and cortical bone tissue. ns, not statistically significant.

Fig. S4.

Effects of colonization on pro-osteoclastogenic cytokine expression in the bone marrow and colon. Relative expression of Rankl, Tnfa, Il1b, and Il6 in the (A) bone marrow and (B) colon from GF and colonized mice. *P < 0.05; **P < 0.01, ns, not statistically significant.

Fig. 4.

Antibiotic treatment decreases IGF-1 and increases bone mass. (A) Experimental procedure schematic. Two-month-old BALB/c mice raised under SPF conditions were treated with either broad-spectrum antibiotics (B–E) or vancomycin (H–K) for 1 mo. Serum levels of IGF-1 after (B) antibiotic mixture (Abx) or (H) vancomycin treatment. Trabecular bone mass after treatment with (C) Abx or (I) vancomycin was determined by micro-CT. Serum P1NP concentrations after treatment with (D) Abx or (J) vancomycin. Serum CTX-I concentrations after treatment with (E) Abx or (K) vancomycin. (F) Serum was collected from SPF mice before and 1 wk after antibiotic treatment and (G) CTX-I concentration. *P < 0.05; **P < 0.01; ns, not statistically significant.

Fig. S5.

Validation of bacterial depletion efficiency after antibiotic treatment. Genomic DNA was isolated from equal amounts of fecal material of SPF and antibiotic-treated mice. Relative quantification of 16S rRNA gene copies of total bacteria were determined by qPCR, using universal 16S rRNA primers. *P < 0.05.

Fig. S6.

Effects of antibiotic treatment on pro-osteoclastogenic cytokine expression in the bone marrow and colon. Relative expression of Rankl, Tnfa, Il1b, and Il6 in the bone marrow (A and B) and colon (C and D) of SPF mice treated with either of two antibiotic mixtures (Abx) or control treated (Ctrl). *P < 0.05; ns, not statistically significant.

Fig. S7.

Cecal content SCFA concentration is increased by colonization and decreased by antibiotic treatment. Acetate, propionate, and butyrate concentrations in cecal contents were determined after colonization of GF mice (A and B) and after antibiotic treatment (C). (A and B) Cecal SCFA concentrations in GF mice compared with littermates colonized for 1 mo (A) or 8 mo (B). (C) Cecal SCFA concentrations in SPF mice compared with SPF mice treated with either of two broad-spectrum antibiotic mixtures (Abx 1, Abx 2) or vancomycin. *P < 0.05; ***P < 0.001; ns, not statistically significant.

Fig. 5.

SCFA supplementation increases IGF-1 and decreases bone mass in antibiotic-treated mice. (A) Experimental procedure schematic. Mice were treated with broad-spectrum antibiotics for 2 wk to perturb microbiota, followed by supplementation of antibiotic water with either SCFA (Abx+SCFA) or sodium chloride control (Abx) for another 4 wk. Control (Ctrl) group received water with NaCl for 6 wk. (B) Serum IGF-1 and IGFBP3 levels. (C) Trabecular bone mass was determined by micro-CT. (D and E) Tissue IGF-1 production. (D) Abdominal fat pad and (E) liver total tissue IGF-1 (Left) was calculated by multiplying the IGF-1 production per gram tissue (Middle) by the weight of the tissue (Right). *P < 0.05; **P < 0.01; ns, not statistically significant.

Fig. S8.

SCFA supplementation of antibiotic-treated mice does not affect bone turnover markers. Bone turnover markers were measured in serum collected from the mice described in Fig. 5. (A) P1NP and (B) CTX-I levels at termination of the experiment. **P < 0.01; ns, not statistically significant.

Fig. S9.

Effects of antibiotics and SCFA on IGF-1 levels are consistent across antibiotic mixtures. SPF mice were treated as outlined in Fig. 5A, but antibiotic treatment was with antibiotic mixture 2, described in the Materials and Methods. (A) Serum IGF-1 and IGFBP3 levels. (B) Trabecular bone mass determined by micro-CT. (C and D) Tissue IGF-1 production (Left) was calculated by multiplying IGF-1 production per gram tissue (Middle) by the weight of the tissue (Right) for both liver (C) and fat pad (D). *P < 0.05; **P < 0.01; ns, not statistically significant.

Fig. S10.

Antibiotic treatment does not alter muscle Igf1 expression. Relative expression of Igf1 in the muscle of SPF mice treated with either of two antibiotic mixtures (A and B) or vancomycin (C) compared with control treated mice. ns, not statistically significant.