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[Preprint]. 2023 Jan 18:2023.01.16.524248.
doi: 10.1101/2023.01.16.524248.

Osteoblast-intrinsic defect in glucose metabolism impairs bone formation in type II diabetic mice

Fangfang Song  1   2 , Won Dong Lee  3 , Tyler Marmo  1 , Xing Ji  1 , Chao Song  1 , Xueyang Liao  1 , Rebbeca Seeley  1 , Lutian Yao  1 , Haoran Liu  4 , Fanxin Long  1   5
Affiliations

Osteoblast-intrinsic defect in glucose metabolism impairs bone formation in type II diabetic mice

Fangfang Song et al. bioRxiv. .

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Abstract

Skeletal fragility is associated with type 2 diabetes mellitus (T2D), but the underlying mechanism is not well understood. Here, in a mouse model for youth-onset T2D, we show that both trabecular and cortical bone mass are reduced due to diminished osteoblast activity. Stable isotope tracing in vivo with 13 C-glucose demonstrates that both glycolysis and glucose fueling of the TCA cycle are impaired in diabetic bones. Similarly, Seahorse assays show suppression of both glycolysis and oxidative phosphorylation by diabetes in bone marrow mesenchymal cells as a whole, whereas single-cell RNA sequencing reveals distinct modes of metabolic dysregulation among the subpopulations. Metformin not only promotes glycolysis and osteoblast differentiation in vitro, but also improves bone mass in diabetic mice. Finally, targeted overexpression of Hif1a or Pfkfb3 in osteoblasts of T2D mice averts bone loss. The study identifies osteoblast-intrinsic defects in glucose metabolism as an underlying cause of diabetic osteopenia, which may be targeted therapeutically.

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Figures

Figure 1.
Figure 1.. Combination of HFD and low-dose STZ induces T2D in mice.
(A) A schematic for experimental design. Low-dose STZ: 30 mg/kg body weight. (B, C) Body weight (B, CTRL, n=8 T2D, n=10) and body composition by DXA (C, CTRL, n=6; T2D, n=9) at harvest. (D) Glucose levels after 6 hr fasting immediately before harvest. n=8. (E, F) Glucose tolerance test (GTT) curve (E) and area under curve (AUC) (F). CTRL, n=6; T2D, n=9. (G, H) Insulin tolerance test (ITT) glucose curve (G), AUC (H) and percentage of baseline (I). CTRL, n=6; T2D, n=7. (J, K) Serum insulin (J, CTRL, n=6; T2D, n=8) and Igf1 (K, CTRL, n=9; T2D, n=13) levels. (L) HOMA-IR graphs. CTRL, n=6; T2D, n=8. All bar graphs are presented as mean ± SD. unpaired student’s t test (B–D, F, H, J-L) or two-way ANOVA with Sidak’s multiple comparisons test (E, G, I). *: P < 0.05.
Figure 2.
Figure 2.. T2D causes low turnover osteopenia.
(A) BMD for whole body (minus head) and one leg (femur and tibia). CTRL, n=6; T2D, n=9. (B) Representative μCT images. (C) Quantitative analysis of trabecular bone of distal femur. n=6. (D) Quantitative analysis of cortical bone in femur. CTRL, n=6; T2D, n=7. (E) Serum bone formation (P1NP) and resorption (CTX-1) markers. CTRL, n=6; T2D, n=9. (F, G) Representative images (F) and quantification (G) for double labeling of trabecular bone in distal femur. n=5. Data are mean ± SD. Unpaired Student’s t tests. *: P < 0.05.
Figure 3.
Figure 3.. T2D suppresses glucose metabolism in bone.
(A) A diagram for carbon tracing with 13C6-Glc. (B) Relative pool size of glycolytic and TCA metabolites. CTRL, n=8; T2D, n=6. (C) Enrichment of specific isotopologues of metabolites relative to own pool. CTRL, n=8; T2D, n=6. (D) Relative enrichment of specific isotopologues normalized to Glc(m+6). CTRL, n=8; T2D, n=6. (E) Carbon enrichment of metabolites relative to own pool (left) and normalized to Glc(m+6) (right). CTRL, n=8; T2D, n=6. (F, G) Western blot images (F) and quantifications (G). Dashed line denotes two separate gels in F. n=3. Data presented as mean ± SD. *: P < 0.05, Two-tailed unpaired t test.
Figure 4.
Figure 4.. scRNA-seq detects metabolic dysregulation in bone marrow mesenchymal cells of T2D mice.
(A) UMAP clusters with annotations to right. Percentages denote relative abundance of osteoblast (OB), CAR and PaS cells among mesenchymal cells. (B) Violin plots of example feature genes for each cluster. (D) GSEA of T2D over CTRL for OXPHOS pathway in clusters as indicated.
Figure 5.
Figure 5.. T2D impairs glucose metabolism and osteogenic differentiation in BMSC.
(A) Glucose uptake and lactate production rates. n=3. (B, C) Seahorse measurements of OCR (B) and ECAR (C). n=8. (D) Projected ATP production glycolysis (glycoATP) and mitochondria (mitoATP) based on Seahorse. n=8. (E, F) qPCR for osteoblast markers at day 4 (E) and day 7 (F) of differentiation. n=3. (G) Alizarin red staining. Data presented as mean ± SD. *P < 0.05, Unpaired student’s t test.
Figure 6.
Figure 6.. Metformin improves bone mass and glucose metabolism impaired by T2D.
(A) A schematic for metformin regimen. T2D animals were randomly divided into Control (Ctrl) and metformin (Met) groups. (B, C) GTT curve (B) and area under curve (C). Ctrl, n=7; Met, n=8. (D, E) ITT curve (D) and area under curve (E). n=8. (F) Representative uCT images and quantification of trabecular bone in distal femur. Ctrl, n=9; Met, n=8. (G, H) Representative images (G) and quantification (H) of double labeling in trabecular bone of distal femur. Ctrl, n=5 Met, n=4. (I) Carbon enrichment of TCA metabolites normalized to Glc(m+6). Ctrl, n=6; Met, n=5. (J, K) Seahorse measurements of OCR (J) and ECAR (K) with or without 1 mM metformin treatment for 24 hrs. n=8. (L, M) qPCR analyses of osteoblast markers (L) and glycolysis-related genes (M) in BMSC at day 7 of differentiation with or without 1 mM metformin treatment. n=3. (N) Alizarin red staining at day 7 of osteoblast differentiation. Data are presented as mean ± SD. *: P < 0.05, two-way ANOVA followed by Sidak’s multiple comparisons (B, D) or Student’s t test (all others).
Figure 7.
Figure 7.. Targeted overexpression of Hif1a in osteoblasts rescues low bone mass in T2D mice.
(A) A schematic for Hif1a overexpression (Hif1OE). (B) qPCR analyses of glycolysis related genes in bone. n=4. (C, D) Representative uCT images (C) and quantification of distal trabecular bone and cortical bone (D) in femur. n=10. (E) Representative images and quantification of double labeling in trabecular bone of proximal tibia. Ctrl, n=5, Hif1OE, n=4. Data presented as mean ± SD. *: P < 0.05 Cinpaired Student’s t test.
Figure 8.
Figure 8.. Osteoblast-directed Pfkfb3 overexpression corrects bone loss in T2D.
(A) A schematic for experimental design. Representative uCT images (B) and quantification (C) of trabecular bone in distal femur. (D) Representative uCT images and quantification of cortical bone in femur. Ctrl NORMAL, n = 8; Pfkfb3OE NORMAL, n = 8; Ctrl T2D, n = 11; Pfkfb3OE T2D, n = 9. (E) Representative images and quantification of double labeling in trabecular bone of proximal tibia. Ctrl T2D, n = 4; Pfkfb3OE T2D, n = 5. (F) Serum markers for bone formation (P1NP) and resorption (CTX-1). Ctrl T2D, n = 12; Pfkfb3OE T2D, n = 9. (G) qPCR analyses of glycolysis related genes in bone. Ctrl T2D, n = 7; Pfkfb3OE T2D n = 4. Data presented as mean ± SD. *: P < 0.05, Two-way ANOVA followed by Fisher’s LSD test. (C, D) or Student’s t test (all others).
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