Fatty acid oxidation by the osteoblast is required for normal bone acquisition in a sex- and diet-dependent manner

Abstract

Postnatal bone formation is influenced by nutritional status and compromised by disturbances in metabolism. The oxidation of dietary lipids represents a critical source of ATP for many cells but has been poorly studied in the skeleton, where the prevailing view is that glucose is the primary energy source. Here, we examined fatty acid uptake by bone and probed the requirement for fatty acid catabolism during bone formation by specifically disrupting the expression of carnitine palmitoyltransferase 2 (Cpt2), an obligate enzyme in fatty acid oxidation, in osteoblasts and osteocytes. Radiotracer studies demonstrated that the skeleton accumulates a significant fraction of postprandial fatty acids, which was equal to or in excess of that acquired by skeletal muscle or adipose tissue. Female, but not male, Cpt2 mutant mice exhibited significant impairments in postnatal bone acquisition, potentially due to an inability of osteoblasts to modify fuel selection. Intriguingly, suppression of fatty acid utilization by osteoblasts and osteocytes also resulted in the development of dyslipidemia and diet-dependent modifications in body composition. Taken together, these studies demonstrate a requirement for fatty acid oxidation during bone accrual and suggest a role for the skeleton in lipid homeostasis.

Keywords: Bone Biology.

Figure 1. Bone takes up a substantial fraction of postprandial lipid.

(A and C) Biodistribution of [³H]-bromopalmitate (A) and [¹⁴C]-oleate (C) in 12-week-old C57Bl/6 mice (n = 5–6 mice). Uptake (cpm) is normalized for tissue weight. Skeleton represents the combined uptake by femur, tibia, and calvaria. (B and D) Levels of [³H]-bromopalmitate (B) and [¹⁴C]-oleate (D) uptake in the skeleton relative to the indicated tissue. (E) Autoradiographic analysis of ¹²⁵I-BMIPP uptake in the tibia, with whole mount tissue section on right (Representative of n = 4 mice). (F) Comparison of [³H]-acetate incorporation into tissue lipids in gonadal white adipose and the femur (n = 6-7 mice). All data are represented by mean ± SEM. *P < 0.05 by unpaired, two-tailed Student’s t test.

Figure 2. Fatty acid oxidation is required for the maintenance of normal bone structure in female mice.

(A) qPCR analysis of Cpt2 mRNA levels in the femur of 6-week-old control and ΔCpt2 mice (n = 5–7 mice). (B) Body weight was assessed weekly from 3–12 weeks of age (n = 7–11 mice). (C and D) Quantification of trabecular bone volume in the distal femur (C) and L5 vertebrae (D) of male control and ΔCpt2 mice (n = 6–12 mice). (E) Representative computer renderings of bone structure in the distal femur (top), L5 vertebrae (middle), and femoral mid-diaphysis (bottom) of 6-week-old female control and ΔCpt2 mice. (F–I) Quantification of trabecular bone volume per tissue volume (F and H, BV/TV), trabecular number (G and I, Tb.N) in the distal femur (F and G) and L5 vertebrae (H and I) of female control and ΔCpt2 mice (n = 7–11 mice). (J–L) Quantification of cortical tissue area (J, Tt.Ar), cortical thickness (K, Ct.Th), and cortical area per tissue area (L, Ct.Ar/Tt.Ar) at the mid-diaphysis of female control and ΔCpt2 mice (n = 7–10 mice). All data are represented by mean ± SEM. *P < 0.05 by unpaired, two-tailed Student’s t test.

Figure 3. In vitro differentiation of osteoblasts is impaired following the ablation of Cpt2 expression.

(A) qPCR analysis of Cpt2 mRNA levels in primary osteoblasts after infection with adenoviral constructs encoding GFP (control) or Cre-recombinase (ΔCpt2) (n = 6). (B) Relative levels of [¹⁴C]-oleate oxidation to ¹⁴CO₂ (n = 7). (C) Oxygen consumption rate (OCR) in control and ΔCpt2 osteoblasts in the presence of 200 μM oleate (n = 16). (D) qPCR analysis of genes associated with osteoblastic differentiation in control and ΔCpt2 osteoblasts cultured in the presence or absence of 10 nM 17β-estradiol (E2) (n = 5). (E) Staining for alkaline phosphatase activity (AP) on days 7 and 14 of in vitro differentiation and calcium deposition by alizarin red (ARS) on day 14 in cultures of control and ΔCpt2 osteoblasts. (F) Quantification of relative ARS levels (n = 6). (G) Relative levels of 2-deoxy-D-[³H]-glucose uptake in control and ΔCpt2 osteoblasts cultured in the presence or absence of E2 (n = 6). (H) Cellular lactate levels in control and ΔCpt2 osteoblasts (n = 5). (I) qPCR analysis of genes associated with glucose metabolism in control and ΔCpt2 osteoblasts cultured in the presence or absence of E2 (n = 5). (J and K) OCR (J) and extracellular acidification rate (ECAR, K) were assessed in osteoblasts cultured in the presence of 2.5 mM glucose (n = 15–16). (L) Relative levels of [¹⁴C]-glutamine uptake (n = 11–12). (M) Cellular ATP in control and ΔCpt2 osteoblasts (n = 10–12). (N) Immunoblots showing AMPK phosphorylation levels (representative of n = 4). All data are represented by mean ± SEM. *P < 0.05 by unpaired, two-tailed Student’s t test or ANOVA.

Figure 4. Cpt2 loss of function in osteoblasts alters glucose distribution, lipid homeostasis, and body composition.

(A and B) [¹⁸F]-fluorodeoxyglucose (FDG) biodistribution in 12-week-old male (A) and female (B) ΔCpt2 mice and their control littermates (n = 5–8 mice). (C) qPCR analysis of genes associated with glucose metabolism in the femurs of control and ΔCpt2 mice (n = 5–6 mice). (D and E) qNMR analysis of fat (D) and lean (E) body mass (n = 9–15 mice). (F and G) Wet weights of adipose depots in 12-week-old male (F) and female (G) mice (n = 7–11 mice) . (H) Representative histological images of the gonadal fat pad. Original magnification, 10× (representative of n = 6–11 mice). (I and J) Frequency distribution of adipocyte size in the gonadal fat pad of male (I) and female (J) mice (n = 6–11 mice). (K) Representative histological image of liver in 12 week old male control and ΔCpt2 mice. Original magnification, 10× (representative of n = 6-11 mice). (L) Random-fed blood glucose (n = 11–17 mice). (M–Q) Random-fed serum lipid analyses (n = 7–11 mice). All data are represented by mean ± SEM. *P < 0.05 by unpaired, two-tailed Student’s t test.

Figure 5. High-fat diet feeding reduces bone volume in male ΔCpt2 mice.

(A) Serum estradiol levels in male mice fed a low- or high-fat diet (n = 11–12 mice). (B–I) Control and ΔCpt2 mice were fed a high-fat diet (60% of calories from fat) from 4–12 weeks of age. (B) Body weight was assessed weekly (n = 11–13 mice). (C) Representative computer renderings of bone trabecular structure in the distal femur of control and ΔCpt2 mice. (D–F) Quantitation of trabecular bone volume per tissue volume (D, BV/TV), trabecular number (E, Tb.N), and trabecular thickness (F, Tb.Th) in the distal femur and L5 vertebrae of high-fat diet–fed mice (n = 7–10 mice). (G) Change in trabecular bone volume of high-fat diet–fed control and ΔCpt2 mice relative to mice fed a chow diet (n = 7–10 mice). (H and I) Serum analysis of the bone turnover markers procollagen type 1 N-terminal propeptide (H, P1NP) and collagen Type I C-terminal telopeptide (I, CTx) (n = 11–12 mice). All data are represented by mean ± SEM. *P < 0.05 by unpaired, two-tailed Student’s t test.

Figure 6. ΔCpt2 mice are sensitized to high-fat diet feeding–induced disturbances in metabolism.

Control and ΔCpt2 mice were fed a high-fat diet (60% of calories from fat) from 4–12 weeks of age. (A) Adipose depot weights (n = 8–10 mice). (B) Wet weights of major organs (n = 8–10 mice). (C) Representative histological images of liver, gonadal adipose, and inguinal adipose. Original magnification, 10× (representative of n = 6–7 mice). (D and E) Frequency distribution of adipocyte size in the gonadal (D) and inguinal (E) fat pads (n = 6–7 mice). (F) Liver triglycerides (n = 7 mice). (G–K) Random-fed serum lipid analyses (n = 7–10 mice). (L) Random-fed blood glucose (n = 11–13 mice). (M) Random-fed serum insulin (n = 7–10 mice). (N) Glucose tolerance testing and (O) insulin tolerance testing (n = 7–10 mice). (P) Representative histological images of pancreatic islets after immunostaining for insulin. (Q) Quantification of mean islet area (n = 7 mice). (R and S) Analysis of Akt phosphorylation in gonadal adipose, liver, and bone after a bolus of insulin (n = 6 mice). (T) Serum Glu-osteocalcin (n = 5–11 mice). All data are represented by mean ± SEM. *P < 0.05 by unpaired, two-tailed Student’s t test.