With lactation, FNDC5 global KO mice lose less bone and are mechanically stronger compared to WT

A: Respective µCT images of femoral midshafts from WT virgin (WT), KO virgin (KO), WT lactation (WT L), and KO lactation (KO L) mice.

B: µCT analysis of femoral cortical bone parameters of virgin and lactating WT and KO female mice reported as cortical bone area (Ct. B.Ar), cortical bone area fraction (Ct. B.Ar/ T.Ar %), and cortical thickness (Ct. Th).

C: Ex vivo DXA analysis for BMD and BMC of femurs from virgin and lactating WT and KO female mice.

D: 3-point bending analysis of WT and KO virgin and lactating mice reported as ultimate force and stiffness.

E: Representative TRAP-stained images of cortical bone from WT virgin (WT), WT lactation (WT L), KO virgin (KO), and KO lactation (KO L) mice.

F: Representative backscatter scanning electron microscope (BSEM) images of WT virgin (WT), KO virgin (KO), WT lactation (WT L), and KO lactation (KO L) mice femur at 400X magnification.

G: Percent TRAP-positive osteocytes (TRAP +ve) in tibia from virgin and lactating WT and KO mice.

H: Average osteocyte lacunar area in femurs from virgin and lactating WT and mice.

I: Osteoclast number per bone perimeter in tibia from virgin and lactating WT and KO mice.

J: Serum RANKL levels in virgin and lactating WT and KO mice.

4-5-month-old WT and KO virgin and lactating mice, n= 5-8/group. a= Significantly different from WT, b= Significantly different from KO, *= p< 0.05, **= p< 0.01, ***= p< 0.001. 2-way ANOVA with Tukey’s post hoc test was performed for statistical analysis.

FNDC5 KO female and male mice have opposite responses to a low-calcium diet compared to WT female and male mice where female KO mice are protected but male KO mice have greater bone loss than WT.

FNDC5 KO female and male mice have opposite responses to a low-calcium diet with regard to bone composition, structure, and mechanics, and irisin injection rescues FNDC5 KO male mice phenotype under a low-calcium diet

A: Representative µCT images of femoral midshaft cortical bones from WT low-calcium diet female mouse (WT lc) and KO low-calcium diet female mouse (KO lc).

B: Female femoral midshaft cortical bone parameters of WT control (WT), WT low-calcium diet (WT lc), KO control (KO), and KO low-calcium diet (KO lc) mice reported as cortical bone area fraction (Ct. B.Ar/T.Ar%) and cortical thickness (Ct.Th).

C: Mechanical properties of femurs from female WT and KO control and low-calcium diet reported as ultimate force and stiffness.

D: Representative µCT images of femoral midshaft cortical bones from WT low-calcium diet male mice (WT lc) and KO low-calcium diet male mice (KO lc).

E: Male femoral midshaft cortical bone parameters of WT control (WT), WT low-calcium diet (WT lc), KO control (KO), and KO low-calcium diet (KO lc) mice reported as cortical bone area fraction (Ct. B.Ar/T.Ar%) and cortical thickness (Ct. Th).

F: Mechanical properties of femurs from male WT and KO control and low-calcium diet reported as ultimate force and stiffness.

n= 4-5/group. a= Significantly different from WT, b= Significantly different from KO, *= p< 0.05, **= p< 0.01. 2-way ANOVA with Tukey’s post hoc test. As depicted here, red is female, and blue is male.

G: µCT measurement of femoral cortical bone of AAV8-GFP or AAV8-irisin injected male KO mice after a 2-week low calcium diet, reported as cortical bone area fraction (Ct. B.Ar/T.Ar%), cortical thickness (Ct. Th), periosteal parameter (Ps.Pm), and endosteal parameter (Es.Pm).

H. Mechanical properties of femurs from male KO low-calcium diet mice injected with AAV8-GFP or AAV8-irisin reported as ultimate force and stiffness.

n= 5-7/group, *= p< 0.05. Student’s t-test was performed for statistical analysis between male KO GFP vs irisin-injected mice. As depicted here, green shaded bars represent GFP-injected mice.

Osteocytes from female and male KO mice respond differently to a low-calcium diet

A: Percentage of TRAP-positive (+-ve) osteocytes in female and male WT and KO mice given a normal or a low-calcium diet.

B. Osteoclast number (N.Oc/B.Pm) in WT and KO female and male mice given a normal or a low-calcium diet.

C: Representative BSEM images depicting osteocyte lacunar area in femurs from WT female (WT F) and WT male (WT M) given a normal diet at 450X magnification.

D. Osteocyte lacunar area in WT and KO female and male mice given a normal diet

E: Lacunar area in female and male WT and KO mice given a normal or a low-calcium diet.

F: Serum RANKL levels in female and male WT and KO mice given either a normal diet or a low-calcium diet.

G: Serum PTH levels in female and male WT and KO mice given either a normal diet or a low-calcium diet.

H: Serum calcium levels in female and male WT and KO mice given either a normal diet or a low-calcium diet.

n= 4-5/group. a= Significantly different from WT, b= Significantly different from KO, *= p< 0.05, **= p< 0.01. 2-way ANOVA with Tukey’s post hoc test. As depicted here, red is female, and blue is male.

Female and male wildtype osteocyte transcriptomes are distinctly different; however, female and male KO osteocyte transcriptomes have fewer differences compared to WT female and male transcriptomes.

A: Volcano plot showing the significantly regulated genes between WT female control (WT F) and WT male control (WT M) osteocyte transcriptome

B: Volcano plot showing the significantly regulated genes between KO female control (KO F) and KO male control (KO M) osteocyte transcriptome

C: Volcano plot showing the significantly regulated genes between WT male control (WT M) and KO male control (KO M) osteocyte transcriptome

D: Volcano plot showing the significantly regulated genes between WT female control (WT F) and KO female control (KO F) osteocyte transcriptome

E: Heat map showing the differentially expressed genes among WT female control (WT F), WT male control (WT M), KO female control (KO F), and KO male control (KO M) osteocyte transcriptome

F: Gene set enrichment analysis of Gene Ontology (GO) analysis of the significantly regulated genes between WT female control (WT F) and WT male control (WT M) osteocyte transcriptome, between KO female control (KO F) and KO male control (KO M) osteocyte transcriptome, WT male control (WT M) and KO male control (KO M) osteocyte transcriptome, and WT female control (WT F) and KO female control (KO F) osteocyte transcriptome. The figure shows the union of the top 10 GO terms of each analysis. If a term in the union, besides the top 10, is also significant (adjusted p-value less than 0.05) in an analysis, it is also included in the figure. n=3/group

The Osteocyte transcriptomes from female WT and KO mice are distinct when challenged with a low-calcium diet.

A: Volcano plot showing the significantly regulated genes between WT female control (WT C) and WT female low-calcium diet-fed mice (WT lc) osteocyte transcriptome

B: Volcano plot showing the significantly regulated genes between KO female control (KO C) and KO female low-calcium diet-fed mice (KO lc) osteocyte transcriptome

C: Volcano plot showing the significantly regulated genes between WT female low-calcium diet-fed mice (WT lc) and KO female low-calcium diet-fed mice (KO lc) osteocyte transcriptome

D: Heat map showing the differentially expressed genes among WT female control (WT C), WT female low-calcium diet-fed mice (WT lc), KO female control (KO C), and KO female low-calcium diet-fed mice (KO lc) osteocyte transcriptome

E: Gene set enrichment analysis of Gene Ontology (GO) analysis of the significantly regulated genes between WT female control (WT C) and WT female low-calcium diet-fed mice (WT lc) osteocyte transcriptome, between KO female control (KO C) and KO female low-calcium diet-fed mice (KO lc) osteocyte transcriptome, and WT female low-calcium diet-fed mice (WT lc) and KO female low-calcium diet-fed mice (KO lc) osteocyte transcriptome. The figure shows the union of the top 10 GO terms of each analysis. If a term in the union, besides the top 10, is also significant (adjusted p-value less than 0.05) in an analysis, it is also included in the figure. n=2-3/group

The Osteocyte transcriptomes from male WT and KO mice are dis-tinct when challenged with a low-calcium diet.

A: Volcano plot showing the significantly regulated genes between WT male control (WT C) and WT male low-calcium diet-fed mice (WT lc) osteocyte transcriptome

B: Volcano plot showing the significantly regulated genes between KO male control (KO C) and KO male low-calcium diet-fed mice (KO lc) osteocyte transcriptome

C: Volcano plot showing the significantly regulated genes between WT male low-calcium diet-fed mice (WT lc) and KO male low-calcium diet-fed mice (KO lc) osteocyte transcriptome

D: Heat map showing the differentially expressed genes among WT male control (WT C), WT male low-calcium diet-fed mice (WT lc), KO female control (KO C), and KO male low-calcium diet-fed mice (KO lc) osteocyte transcriptome

E: Gene set enrichment analysis of Gene Ontology (GO) analysis of the significantly regulated genes between WT male control (WT C) and WT male low-calcium diet-fed mice (WT lc) osteocyte transcriptome, between KO male control (KO C) and KO male low-calcium diet-fed mice (KO lc) osteocyte transcriptome, and WT male low-calcium diet-fed mice (WT lc) and KO male low-calcium diet-fed mice (KO lc) osteocyte transcriptome. The figure shows the union of the top 10 GO terms of each analysis. If a term in the union, besides the top 10, is also significant (adjusted p-value less than 0.05) in an analysis, it is also included in the figure.

n=3/group

Graphical abstract

(image was created using BioRender.com)

  • No differences are observed in bone from Fndc5 /irisin null female, whereas null male skeletons are larger but weaker compared to wildtype controls.

  • With calcium deficiency, lactating female null mice are protected from bone loss due to osteocytic osteolysis, whereas male null mice on a low calcium diet lose greater amounts of bone compared to their wildtype controls.

  • The osteocyte transcriptomes show wildtype males have higher expression of the steroid, lipid and fatty acid pathways which are lower in the null males, whereas the wildtype females have higher expression of genes regulating osteocytic osteolysis than null females.

  • With calcium deficiency, female null osteocytes have lower while male null osteocytes have higher expression of osteocytic osteolysis genes compared to wildtype controls.

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Neither genotype nor dietary calcium alters muscle functions in vivo or ex vivo

Panels A and C show in vivo muscle plantarflexion force (reported as plantarflexion torque and plantarflexion fatigue) in WT and KO female (A) and male (C) mice on a control or a low calcium diet, panels B and D show muscle electrophysiology parameters of CMAP, SMUP, and MUNE in WT and KO female (B) and male (D) mice, and panels E and F show ex vivo EDL functional measurement (reported as specific force frequency, maximum rate of contraction, maximum rate of relaxation, half-relaxation time, and % fatigue) in WT and KO female (E) and male (F) mice 2-way ANOVA with Tukey’s post hoc test was done. n= 4-5/group. As depicted here, red is female, and blue is male.

Quality control and validation of RNA sequencing

Sanity check of data on the sample’s sex. A: Boxplot of proportional of reads on chromosome Y. Male should have a higher value than female. B: Boxplot of RPKM of Xist. Males should have very low expression of Xist. C: Scatter plot of PC1 and PC2 from Principal Component Analysis (PCA) of gene expression data. D: qPCR analysis of Tnsfs11, Acp5, Sost, and Ctsk genes from osteocyte-enriched bone chips from female samples. n= 3-4/sample. Two-way ANOVA with Tukey’s post-hoc test was performed for statistical analysis. Gene fold-change was normalized using β-2-microglobulin as the housekeeping gene. a= Significantly different from WT, b= Significantly different from KO, *= p< 0.05. E: qPCR analysis of Tnsfs11, Acp5, Sost, and Ctsk genes from osteocyte-enriched bone chips from male samples. n= 3-4/sample. Two-way ANOVA with Tukey’s post-hoc test was performed for statistical analysis. Gene fold-change was normalized using β-2-microglobulin as the housekeeping gene. a= Significantly different from WT, b= Significantly different from KO, *= p< 0.05.

FNDC5 KO mice femurs are partially resistant to lactation-induced bone loss.

Femoral cortical and trabecular bone parameters of WT and FNDC5 KO female virgin and lactation mice. n = 5-8/group. a= significant compared to WT control, b= significant compared to KO control, c= significant compared to WT low Ca diet, 2-way ANOVA with Tukey’s post hoc test, significance <0.05, n= 8/group. Percentage change in different bone and serum parameters in WT and FNDC5 KO female mice with lactation. *= p<0.05 compared to WT.

WT and FNDC5 KO female and male mice bone responds differently to a low-calcium diet

Femoral BMD, BMC, cortical and trabecular bone parameters, and mechanical properties of 4-5-month-old WT and KO female and male mice under a normal diet or a 2-week low calcium diet. n = 5/group. a= significant compared to WT control, b= significant compared to KO control, c= significant compared to WT low Ca diet, 2-way ANOVA with Tukey’s posthoc test, significance <0.05, n= 4-5/group.