Glucose Uptake and Runx2 Synergize to Orchestrate Osteoblast Differentiation and Bone Formation

Abstract

The synthesis of type I collagen, the main component of bone matrix, precedes the expression of Runx2, the earliest determinant of osteoblast differentiation. We hypothesized that the energetic needs of osteoblasts might explain this apparent paradox. We show here that glucose, the main nutrient of osteoblasts, is transported in these cells through Glut1, whose expression precedes that of Runx2. Glucose uptake favors osteoblast differentiation by suppressing the AMPK-dependent proteasomal degradation of Runx2 and promotes bone formation by inhibiting another function of AMPK. While RUNX2 cannot induce osteoblast differentiation when glucose uptake is compromised, raising blood glucose levels restores collagen synthesis in Runx2-null osteoblasts and initiates bone formation in Runx2-deficient embryos. Moreover, RUNX2 favors Glut1 expression, and this feedforward regulation between RUNX2 and Glut1 determines the onset of osteoblast differentiation during development and the extent of bone formation throughout life. These results reveal an unexpected intricacy between bone and glucose metabolism.

Figure 1. Insulin-independent glucose uptake in osteoblasts

A. Oxygen consumption rate (OCR) of osteoblasts, C2C12 myoblasts or hippocampal neurons incubated with vehicle, 10mM glucose, 2mM glutamine or 300μM palmitate in 1X KHB buffer for 2hrs (n=8). B. Glucose uptake measured by euglycemic hyperinsulinemic clamps in femurs, white adipose tissue and gastrocnemius muscle of WT mice before or after insulin infusion (2.5 mU/kg/min) (n=4); C. Uptake rate of 2-DG in osteoblasts (Osb), osteoclasts (Ocl) and myoblasts (n=3). D. Expression of class I Gluts in osteoblasts and osteoclasts assayed by qPCR. E. Uptake rate of 2-DG in Glut1fl/fl, Glut1−/−, WT and α1(I)Col-Glut1 osteoblasts (n=6-8). F. In situ hybridization analysis of Glut1 (a-e), α1(I) Collagen (f-j), Runx2 (k-o) Runx1(p-t), Runx3 (u-v), α1(II) Collagen (z-b1) and α1(X) Collagen (c1-e1) in hind-limbs during embryonic development.

Figure 2. Glucose uptake is necessary for osteoblast differentiation during development

A. Alcian blue/alizarin red staining of skeletal preparations of E14.0 and 14.5 Glut1_dermo1−/− and Glut1fl/fl embryos. B. Von Kossa staining of skull sections of E14.5 Glut1_dermo1−/− and Glut1fl/fl embryos. C and F. Alcian blue/alizarin red staining of skeletal preparations of E15.5 (C) and 18.5 (F) Glut1_osx−/−, Glut1fl/fl and Osx-cre embryos. D and G. Von Kossa or alcian blue staining of femur from E15.5 (D) and 18.5 (G) Glut1_osx−/−, Glut1fl/fl and Osx-cre embryos. E and H. In situ hybridization analysis of Osteocalcin, α1(I), α1(X) Collagen and Runx2 expression in E15.5 (E) and 18.5 (H) Glut1_osx−/− and Glut1fl/fl femurs. I. Expression of osteoblast markers in E18.5 Glut1_osx−/− and Glut1fl/fl femurs (n=9). J. Expression of osteoblast marker genes in Glut1fl/fl and Glut1−/− osteoblasts. (n=6) K. α1(I) Collagen and Runx2 accumulations in E18.5 Glut1_osx−/− and Glut1fl/fl femurs and Glut1fl/fl and Glut1−/− osteoblasts. L. ³H-proline incorporation in collagen in Glut1fl/fl and Glut1−/− osteoblasts (n=6).

Figure 3. Glucose uptake in osteoblasts is necessary for bone formation and glucose homeostasis post-natally

A and B. Histomorphometric analysis of L4 vertebrae of 3 month-old lut1fl/fl, Glut1_ocn−/−, WT and α1(I)Col-Glut1 male mice (n=9-11). C. μCT analysis of proximal femurs of Glut1fl/fl and Glut1_ocn−/− male mice (n=7). D. Expression of Ccnd2, Ccne1 and Cdk4 in femurs of 3 month-old Glut1fl/fl, Glut1_ocn−/−, WT and α1(I)Col-Glut1 mice (n=6). E. BrdU incorporation in calvaria of P14 Glut1fl/fl, Glut1_ocn−/−, WT and α1(I)Col-Glut1 mice (n=5-8). F. Expression of osteoblast marker genes in femurs of 3 month-old Glut1fl/fl, Glut1_ocn−/−, WT and α1(I)Col-Glut1 mice (n=8). G. α1(I) Collagen and Runx2 accumulations in femurs of 3 month-old Glut1fl/fl, Glut1_ocn−/−, WT and α1(I)Col-Glut1 mice. H-I. GSIS in 3 month-old Glut1fl/fl, Glut1_ocn−/−, WT and α1(I)Col-Glut1 mice (n=10-11). J-K. GTT in 3 month-old Glut1fl/fl, Glut1_ocn−/−, WT and α1(I)Col-Glut1 mice (n=5-10). L-N. ITT in 3 month-old Glut1fl/fl, Glut1_ocn−/−, WT and α1(I)Col-Glut1 mice (n=8-10). M. Glucose infusion rate in 3 month-old Glut1fl/fl and Glut1_ocn−/− mice (n=6).

Figure 4. Glucose uptake favors osteoblast differentiation and bone formation by inhibiting AMPK

A. ADP content and ADP/ATP ratio in WT and Glut1−/− osteoblasts (n=6). B-C. AMPK, ACC1, Raptor (B), p70S6K, 4E-BP1 and eIF4G phosphorylation in WT and Glut1−/− osteoblasts. D. Kinase assay of immune-precipitated mTORC1 complex in WT and Glut1−/− osteoblasts (n=3). E. Raptor, p70S6K phosphorylation and α1(I)Collagen accumulations in WT and Raptor−/− osteoblasts. F. TSC1, TSC2, and p70S6K phosphorylation, α1(I)Collagen and Runx2 accumulations in WT and Glut1−/− osteoblasts transfected with SiRNAs targeting Tsc1 and Tsc2 or scrambled SiRNA. G. Expression of various AMPK subunits in osteoblasts. H. AMPK and p70S6K phosphorylation, Runx2 and α1(I)Collagen accumulations in Glut1fl/fl, Glut1_osx−/− and Glut1_osx−/−;Ampka1_osx+/− osteoblasts. I-J. Alcian blue/alizarin red staining of skeletal preparations of E15.5 (I) and 18.5 (J) Glut1fl/fl, Glut1_osx−/−, Ampka1_osx+/− and Glut1_osx−/−;Ampka1_osx+/− embryos. K. Alcian blue staining of histological sections of femurs of E18.5 Glut1fl/fl, Glut1_osx−/−, Ampka1_osx+/− and Glut1_osx−/−;Ampka1_osx+/− embryos. L. In situ hybridization analysis of Osteocalcin, α1(I), α1(II) and α1(X) Collagen expression in femurs of E18.5 Glut1fl/fl, Glut1_osx−/−, Ampka1_osx+/− and Glut1_osx−/−;Ampka1 _osx+/− embryos. M. Histomorphometric analysis of L4 vertebrae of 3 month-old of Glut1fl/fl, Glut1_ocn−/−, Ampka1_ocn+/− and Glut1_ocn−/−;Ampka1_ocn+/− male mice (n=7-10). N. AMPK phosphorylation, Runx2 and α1(I)Collagen accumulations in osteoblasts treated with vehicle or AICAR (0.1mM) for 16 hrs. O. Histomorphometric analysis of L4 vertebrae of 3 month-old of WT mice treated with vehicle or AICAR (250mg/kg/day) for 8 weeks (n=6).

Figure 5. Runx2 cannot induce proper osteoblast differentiation if glucose uptake is hampered

A. Ubiquitination of immune-precipitated Runx2 in WT and Glut1−/− osteoblasts treated with 25nM Bortezomib for 16hrs. B. Ubiquitination of immune-precipitated Runx2 in WT, Glut1−/−, Glut1−/−Ampka1+/− and Glut1−/− Ampka1−/− osteoblasts treated with 25nM Bortezomib for 16hrs. C. In vitro AMPK phosphorylation assay of Smurf1 and Smurf1-S148/A. D. In vitro ubiquitination assay of Smurf1 and Smurf1-S148/A phosphorylated by AMPK. E-F. Runx2 and α1(I)Collagen accumulations, AMPK and Smurf1 phosphorylation in WT, Smurf1−/− (E) and Ampka1−/− (F) osteoblasts cultured with or without glucose for 16 hrs. G. Co-immunoprecipitation of AMPKα1 and Smurf1 in WT and Glut1−/− osteoblasts. H. Runx2 and α1(I)Collagen accumulations, AMPKα1, p70S6K and Smurf1 phosphorylation in WT, Glut1−/−, Glut1−/− Ampka1+/− and Glut1−/− Ampka1−/− osteoblasts. I. Runx2 and α1(I)Collagen accumulations and p70S6K phosphorylation in Glut1fl/fl, Glut1_osx−/− and Glut1_osx−/−;Smurf1+/− osteoblasts. J. ³H-proline incorporation in collagen molecules in Glut1fl/fl, Glut1−/− and Glut1−/−;Smurf1+/− osteoblasts (n=6). K-L. Alcian blue/alizarin red staining of skeletal preparations of E15.5 (G) and 18.5 (I) Glut1fl/fl, Smurf1+/−, Glut1_osx−/− and Glut1_osx−/−;Smurf1+/− embryos. M. Alcian blue staining of sections of femurs of E18.5 Glut1fl/fl, Glut1_osx−/− and Glut1_osx −/−;Smurf1+/− embryos. N. In situ hybridization analysis of Osteocalcin, α1(I) and α1(X) Collagens expression, in femurs of E18.5 Glut1fl/fl, Glut1_osx−/− and Glut1_osx−/−;Smurf1+/− embryos. O. Schematic representation of the pathways triggered by glucose uptake in osteoblasts.

Figure 6. Glucose can initiate bone formation in Runx2-deficient embryos

A. Glucose consumption rate in WT and Runx2−/− osteoblasts cultured with 5 or 10mM glucose for 16hrs. B. Runx2, α1(I)Collagen and Glut1 accumulations, AMPKα1 and p70S6K phosphorylation in WT and Runx2−/− osteoblasts cultured with 5 or 10mM glucose for 14 days. C. ³H-proline incorporation in collagen molecules in WT and Runx2−/− osteoblasts cultured with 5 or 10mM glucose for 14 days (n=5). D. qPCR analysis of osteoblast marker genes in WT and Runx2−/− osteoblasts cultured with 5 or 10mM glucose for 14 days (n=6). E. Blood glucose and insulin levels in STZ (50, 100, 150mg/kg) and vehicle-treated Runx2+/- mothers and progenies at E18.5 (n=5-12). F-G. Alcian blue/alizarin red staining of the skull (F), clavicles and interparietal bones (G) of E18.5 Runx2+/- embryos carried by STZ (50, 100, or 150mg/kg) and vehicle-treated mothers. H-I. Von Kossa/van Gieson staining (H) and H&E staining (I) of clavicles of E18.5 Runx2+/- embryos carried by STZ- (150mg/kg) or vehicle-treated mothers. J. Immunohistochemical detection of Type I and type X collagens in clavicles of E18.5 Runx2+/- embryos carried by STZ- (150mg/kg) or vehicle-treated mothers. K. Runx2 and α1(I)Collagen accumulations in femurs of E18.5 WT and Runx2+/- embryos carried by STZ- (150mg/kg) or vehicle-treated mothers. L. α1(I) and α2(I) Collagen expression in femurs of E18.5 WT and Runx2+/− embryos carried by STZ- (150mg/kg) or vehicle-treated mothers (n=8). M. Runx2 and α1(I)Collagen accumulations in femurs of E18.5 WT and Runx2−/− embryos carried by STZ- (150mg/kg) or vehicle-treated mothers. N. Akp2 expression in femurs of E18.5 of WT and Runx2−/− embryos carried by STZ- (150mg/kg) or vehicle-treated mothers (n=6).

Figure 7. The reciprocal regulation between Runx2 and Glut1 determines osteoblast differentiation and bone formation

A. Glut1 expression in femurs of 3 month-old Runx2+/− mice (n=8). B. Uptake rate of 2-DG in the calvaria of 6 week-old WT and Runx2+/− (n=4-5)mice. C. Runx binding sites in the Glut1 promoter of several species. D. ChIP assay of Runx2 binding to the promoter of mouse Glut1. E. Luciferase assay of pGlut1-WT-Luc or pGlut1-mut-Luc in COS cells cotransfected with Runx2 or Runx1 expression vector (n=6). F. Expression of osteoblast marker genes in WT, Runx2−/− and Shn3−/−osteoblasts (n=6). G. Glucose uptake in WT, Runx2−/− and Shn3−/− osteoblasts measured by the uptake rate of 2-DG (n=6). H-I. Alcian blue/alizarin red staining of skeletal preparations of E16.5 (H) and 18.5 (I) Glut1_osx+/−, Runx2+/−, Glut1_osx+/−;Runx2+/- and Runx2−/− embryos. J. Alcian blue staining of histological sections of femurs of E18.5 Glut1_osx+/−, Runx2+/−, Glut1_osx+/−;Runx2+/− and Runx2−/− embryos. K. In situ hybridization analysis of Osteocalcin, Bsp and α1(X) Collagen expression, in E18.5 Glut1_osx+/−, Runx2+/− and Glut1_osx+/−;Runx2+/− femurs. L. Bone histomorphometric analysis of L4 vertebrae of 3 month-old of Glut1fl/+, Glut1_ocn+/−, Runx2+/− and Glut1_ocn+/−;Runx2+/− female mice (n=9-12). M. Runx2 and α1(I)Collagen accumulations and AMPKα1 and Smurf1 phosphorylation in femurs of E18.5 Glut1fl/+, Glut1_osx+/−, Runx2+/−, and Glut1_osx+/−; Runx2+/− embryos. N. Alcian blue/alizarin red staining of skeletal preparations of E18.5 Glut1_osx+/− ;Runx2+/− embryos carried by mothers treated with STZ (150mg/kg) or vehicle.