Wnt-Lrp5 signaling regulates fatty acid metabolism in the osteoblast

Abstract

The Wnt coreceptors Lrp5 and Lrp6 are essential for normal postnatal bone accrual and osteoblast function. In this study, we identify a previously unrecognized skeletal function unique to Lrp5 that enables osteoblasts to oxidize fatty acids. Mice lacking the Lrp5 coreceptor specifically in osteoblasts and osteocytes exhibit the expected reductions in postnatal bone mass but also exhibit an increase in body fat with corresponding reductions in energy expenditure. Conversely, mice expressing a high bone mass mutant Lrp5 allele are leaner with reduced plasma triglyceride and free fatty acid levels. In this context, Wnt-initiated signals downstream of Lrp5, but not the closely related Lrp6 coreceptor, regulate the activation of β-catenin and thereby induce the expression of key enzymes required for fatty acid β-oxidation. These results suggest that Wnt-Lrp5 signaling regulates basic cellular activities beyond those associated with fate specification and differentiation in bone and that the skeleton influences global energy homeostasis via mechanisms independent of osteocalcin and glucose metabolism.

FIG 1

ΔLrp5 mice, but not ΔLrp6 mice, have increased body fat. (A) Whole-body bone mineral density assessed by DXA at 12 weeks of age (n = 11 to 21 mice). There were no differences in the bone mineral densities of Lrp5^flox and Lrp6^flox mice, so data were combined as a control. (B) Representative micro-CT images of the L5 vertebrae at 24 weeks of age. (C) Quantitation of trabecular bone volume per tissue volume (BV/TV, %) in the L5 vertebrae at 24 weeks of age (n = 4 to 9 mice). (D) Representative images of control and ΔLrp5 mice at 8 weeks of age showing normal longitudinal growth. (E) Body weights (n = 7 to 20 mice). (F) Fat mass assessed by qNMR at 8 and 24 weeks of age (n = 6 to 11 mice). (G) Lean mass assessed by qNMR. (H) Representative images of the gonadal fat pad isolated from control and ΔLrp5 mice at 24 weeks of age. (I) Gonadal fat pad mass assessed at 8 and 24 weeks of age (n = 6 to 11 mice). (J and K) Wet tissue weights of major organs (J) and muscle groups (K) of 8-week-old control and ΔLrp5 mice (n = 5 to 6 mice). (L) Representative histological sections showing the distal femur of 24-week-old control and ΔLrp5 mice. No difference in marrow adiposity is apparent. (M) Daily food intake at 4 and 8 weeks of age (n = 4 to 6 mice). (N) Ambulatory activity measured via beam breaks in Oxymax system in 24 h (n = 4 to 6 mice). (O to Q) Indirect calorimetry at 8 weeks of age (n = 4 to 6 mice). (O) VO₂ (ml/kg/h). (P) Respiratory exchange ratio (RER). (Q) Energy expenditure (kcal/kg/h). The data are represented as means ± the SEM. *, P < 0.05.

FIG 2

Plasma lipids are increased in ΔLrp5 mice. (A) Blood glucose levels in random fed control and ΔLrp5 mice and after an overnight fast at 8 weeks of age (n = 5 to 12 mice). (B) Insulin levels in mice after an overnight fast at 8 weeks of age (n = 8 mice). (C) Glucose tolerance testing at 8 weeks of age (n = 7 to 12 mice). (D) Insulin tolerance testing at 8 weeks of age (n = 7 to 8 mice). (E) Area under the curve (AUC) analysis for GTT (C) and ITT (D). (F) Undercarboxylated osteocalcin (Glu) levels in 8-week-old mice (n = 10 mice). (G to J) Plasma analysis of random fed or fasted 8-week-old control and ΔLrp5 mice (n = 8 to 10 mice). (G) Plasma triglyceride levels (mg/dl). (H) Free fatty acid levels (mmol/liter). (I) Glycerol levels (mg/dl). (J) Cholesterol levels (mg/dl). (K and L) Plasma triglyceride levels (K) and free fatty acid levels (L) in random fed 24-week-old control and ΔLrp6 mice (n = 5 to 7 mice). (M) β-Hydroxybutyrate levels (mmol/liter) in randomly fed or fasted 8-week-old control and ΔLrp5 mice (n = 8 to 10 mice). (N) Representative images of the liver isolated at 8 weeks of age. (O to S) qPCR analysis of tissues isolated from 8-week-old control and ΔLrp5 mice (n = 6 mice). (O) Lrp5, Ldlr, and Vldlr expression in liver. (P) Expression of genes involved in lipid synthesis in the liver. (Q) Expression of genes involved in lipid oxidation in the liver. (R) Lrp5, Ldlr, and Vldlr expression in the gonadal fat pad. (S) Expression of genes involved in lipid storage in the gonadal fat pad. (T) Representative histological images of adipocyte morphology in the gonadal fat pad. The data are represented as means ± the SEM. *, P < 0.05.

FIG 3

Lrp5 regulates the expression of genes involved in lipid oxidation in osteoblasts. (A to C) qPCR analysis of genes associated with lipid storage in primary preadipocytes after overnight treatment with vehicle or 100 μM stearate (A) or medium conditioned by control and ΔLrp5 osteoblasts (B) or control and Lrp5^G171V osteoblasts (C). (D and E) qPCR analysis of genes involved in lipid metabolism in control and ΔLrp5 osteoblasts after 7 days of differentiation (D) and in mRNA samples isolated from the femur of 8-week-old control and ΔLrp5 mice (E, n = 5 mice). (F) Oxidation of [¹⁴C]oleate to ¹⁴CO₂ by differentiating osteoblasts. (G) qPCR analysis of genes involved in lipid metabolism in differentiating osteoblasts. (H to J) Impact of etomoxir (ETO; 100 μM) on osteoblast function after 7 days of differentiation. (H) Oxidation of [¹⁴C]oleate to ¹⁴CO₂. (I) qPCR analysis of genes involved in osteoblast differentiation. (J) Alizarin red staining for calcium deposition. (K to N) Oxidation of ¹⁴C-labeled substrates by control and ΔLrp5 osteoblasts after 7 days of differentiation. The results are normalized to protein concentration and presented as relative to control. (K) [¹⁴C]oleate to ¹⁴CO₂. (L) [¹⁴C]oleate to ¹⁴C-labeled acid-soluble metabolites (ASM). (M) [¹⁴C]pyruvate to ¹⁴CO₂. (N) [¹⁴C]glucose to ¹⁴CO₂. (O) Visualization of DiI-labeled LDL uptake by control and ΔLrp5 osteoblasts after 7 days of differentiation. (P and Q) Cellular lactate levels in cultures of control and ΔLrp5 osteoblasts after 7 days of differentiation (P) and in undifferentiated calvarial cells (Q). (R) qPCR analysis of genes involved in glucose metabolism in cultures of undifferentiated control and ΔLrp5 osteoblasts. (S) qPCR analysis of genes involved in lipid metabolism in control and ΔLrp6 osteoblasts after 7 days of differentiation. The data are represented as means ± the SEM. *, P < 0.05.

FIG 4

Expression of Lrp5^G171V increases lipid oxidation in vitro and reduces adiposity in vivo. (A and B) Detection of Lrp5^G171V-FLAG expression in calvarial osteoblasts by qPCR (A) and immunoblotting (B). (C) qPCR analysis of osteoblastic markers after 7 days of differentiation. (D) qPCR analysis of axin2 and Nkd2 mRNA levels in osteoblasts differentiated for 7 days. (E) Oxidation of [¹⁴C]oleate to ¹⁴CO₂. (F) Oxidation of [¹⁴C]oleate to ¹⁴C-labeled acid-soluble metabolites (ASM). (G) qPCR analysis of genes involved in lipid metabolism in control and Lrp5^G171V-expressing osteoblasts after 7 days of differentiation. (H) Oxidation of [¹⁴C]glucose to ¹⁴CO₂. (I) Cellular lactate levels. (J) Representative micro-CT images of the distal femur of 8-week-old controls and mice expressing LRP5^G171V in osteoblasts. (K) Representative micro-CT images of the L5 vertebrae from 8-week-old control and Lrp5^G171V mice. (L) Quantitation of trabecular bone volume in the distal femur and L5 vertebrae (n = 6 to 9 mice). (M) Body weights of 8-week-old control and Lrp5^G171V mice (n = 6 to 9 mice). (N and O) qNMR analysis of fat mass (N) and lean mass (O) in 8-week-old control and Lrp5^G171V mice (n = 6 to 9 mice). (P) Gonadal fat pad mass assessed at 8 weeks of age (n = 12 to 14 mice). (Q to W) Blood and plasma analysis of random fed mice at 8 weeks of age (n = 7 to 12 mice). (Q) Plasma triglyceride levels. (R) Free fatty acid levels. (S) Glycerol levels. (T) Cholesterol levels. (U) β-Hydroxybutyrate levels. (V) Blood glucose. (W) Insulin levels. (X) Undercarboxylated osteocalcin (Glu) levels. (Y) qPCR analysis of genes involved in lipid metabolism in mRNA samples isolated from the femurs of 8-week-old controls and mice expressing LRP5^G171V in osteoblasts (n = 5 mice). The data are represented as means ± the SEM. *, P < 0.05.

FIG 5

Acute Wnt stimulation increases lipid oxidation. (A to D) Primary osteoblasts were differentiated for 7 days and then treated for 18 h with 20 mM LiCl to stimulate Wnt/β-catenin signaling or NaCl (20 mM) as a control. (A) qPCR analysis of axin2 and Nkd2 mRNA levels. (B) Oxidation of [¹⁴C]oleate to ¹⁴CO₂. (C) Oxidation of [¹⁴C]oleate to ¹⁴C-labeled acid-soluble metabolites (ASM). (D) qPCR analysis of genes involved in lipid metabolism. (E and F) Primary osteoblasts expressing or deficient for Lrp5 were differentiated for 7 days and then treated with 100 ng of Wnt10b/ml for 18 h. (E) Oxidation of [¹⁴C]oleate to ¹⁴CO₂. (F) qPCR analysis of Acaa1a, Acadvl, and Cpt1b mRNA levels. (G to J) Primary osteoblasts differentiated for 4 days after infection with adenovirus expressing green fluorescent protein (ad-GFP) or β-catenin (ad-Ctnnb1). (G) Immunoblot analysis of β-catenin protein levels. (H) qPCR analysis of Ctnnb1, axin2, and Nkd2 mRNA levels. (I) Oxidation of [¹⁴C]oleate to ¹⁴CO₂. (J) qPCR analysis of genes involved in lipid metabolism. The data are represented as means ± the SEM. *, P < 0.05.