Sclerostin influences body composition by regulating catabolic and anabolic metabolism in adipocytes

Abstract

Sclerostin has traditionally been thought of as a local inhibitor of bone acquisition that antagonizes the profound osteoanabolic capacity of activated Wnt/β-catenin signaling, but serum sclerostin levels in humans exhibit a correlation with impairments in several metabolic parameters. These data, together with the increased production of sclerostin in mouse models of type 2 diabetes, suggest an endocrine function. To determine whether sclerostin contributes to the coordination of whole-body metabolism, we examined body composition, glucose homeostasis, and fatty acid metabolism in Sost^-/- mice as well as mice that overproduce sclerostin as a result of adeno-associated virus expression from the liver. Here, we show that in addition to dramatic increases in bone volume, Sost^-/- mice exhibit a reduction in adipose tissue accumulation in association with increased insulin sensitivity. Sclerostin overproduction results in the opposite metabolic phenotype due to adipocyte hypertrophy. Additionally, Sost^-/- mice and those administered a sclerostin-neutralizing antibody are resistant to obesogenic diet-induced disturbances in metabolism. This effect appears to be the result of sclerostin's effects on Wnt signaling and metabolism in white adipose tissue. Since adipocytes do not produce sclerostin, these findings suggest an unexplored endocrine function for sclerostin that facilitates communication between the skeleton and adipose tissue.

Keywords: Wnt; adipose; bone; metabolism; sclerostin.

Fig. 1.

Fat mass is reduced in Sost^−/− mice. (A) Representative computer renderings of bone structure in the distal femur of control (WT) and Sost^−/− mice. (B) Quantification of trabecular bone volume in the distal femur at 8 and 16 wk of age (n = 6–10 mice per group). (C) Body weight was assessed weekly (n = 6–10 mice per group). (D and E) qNMR analysis of fat mass and lean mass at 8 and 16 wk of age (n = 6–9 mice per group). (F) Adipose depot weights were assessed at 16 wk of age (n = 6–8 mice per group). (G) Wet tissue weights of major organs from WT and Sost^−/− mice (n = 6–8 mice per group). (H) Energy expenditure at 8 wk of age from indirect calorimetry. Data are normalized to lean body mass (n = 6 mice per group). (I) Representative histological sections of gonadal (gWAT) and inguinal (iWAT) fat pads and liver from 16-wk-old WT and Sost^−/− mice (10× magnification). (J) qPCR analysis of Wnt target gene expression in gWAT (n = 6 mice per group). (K) Immunoblot analysis of phosphorylated Smad1/5/9 levels in gWAT (n = 3 mice per group). (L) qPCR analysis of Axin2 expression in liver, muscle, and pancreas (n = 5 mice per group). All data are represented as mean ± SEM. *P < 0.05.

Fig. 2.

Sclerostin overproduction increases fat mass. Eight-week-old C57BL/6 mice were injected with AAV8 constructs encoding Sost or GFP. (A) Serum sclerostin levels 8 wk after injection (n = 11–12 mice). (B) Quantification of trabecular bone volume in the distal femur and L5 vertebrae 8 wk after injection (n = 11–12 mice). (C) Body weight was assessed weekly (n = 8 mice). (D and E) qNMR analysis of fat mass and lean mass (n = 11–12 mice). (F) Adipose depot weights of AAV-GFP and AAV-Sost mice (n = 6–7 mice). (G) Wet tissue weights of major organs from AAV-GFP and AAV-Sost mice (n = 6–7 mice). (H) Energy expenditure from indirect calorimetry. Data are normalized to lean body mass (n = 6 mice). (I) Representative histological sections of the gonadal fat pad (gWAT) (10× magnification). (J–L) qPCR analysis of Wnt target gene (J), adipocyte differentiation markers (K), and lipid storage genes (L) in gWAT (n = 6 mice). (M) Representative histological sections of the liver (10× magnification). All data are represented as mean ± SEM. *P < 0.05.

Fig. 3.

Sclerostin modulates glucose metabolism in mice. (A) Fasting and random fed glucose in 16-wk-old control (WT) and Sost^−/− mice (n = 8–14 mice). (B) Random fed insulin levels (n = 6–10 mice). (C) Glucose tolerance test (GTT) and (D) insulin tolerance test (ITT) at 16 wk of age (n = 6–8 mice). (E) Area under the curve (AUC) analysis for GTT and ITT. (F) Representative histological images of pancreatic islets after immunostaining for insulin at 16 wk of age (20× magnification). (G and H) Quantification of islet area per tissue area and mean islet area at 8 and 16 wk of age (n = 6 mice). (I) Steady-state glucose infusion rate, (J) basal hepatic glucose production, (K) clamp hepatic glucose production, (L) insulin-stimulated glucose uptake in skeletal muscle (gastrocnemius), and (M) white adipose tissue (gonadal) during hyperinsulinemic-euglycemic clamp performed on 16-wk-old control and Sost^−/− mice (n = 6–8 mice). (N) Immunoblotting of phosphorylated AKT and ERK in gWAT before and after insulin stimulation. (O) Quantification of insulin-stimulated phosphorylation (n = 8–9 mice). All data are represented as mean ± SEM. *P < 0.05.

Fig. 4.

Sost^−/− mice are resistant to high fat diet feeding. Control (WT) and Sost^−/− mice were fed a high fat diet (60% of calories from fat) from 6 wk of age. (A) Body weight was assessed weekly over the 10-wk study (n = 7–12 mice). (B) Adipose depot weights of high fat diet fed mice (n = 7–12 mice). (C) Random fed glucose levels (n = 7–12 mice). (D) Random fed insulin levels (n = 7–12 mice). (E) Serum-free fatty acids were assessed after 10 wk of high fat diet feeding (n = 7–12 mice). (F) Glucose tolerance testing and (G) insulin tolerance testing (n = 7–12 mice). (H) Representative histological images of pancreatic islets after immunostaining for insulin (20× magnification). (I) Representative histological sections of gonadal fat pad (gWAT) and liver after 10 wk of high fat diet feeding (10× magnification). (J) Frequency distribution of adipocyte size in the gonadal fat pad (n = 7–9 mice). (K) qPCR analysis of inflammatory and oxidative stress markers in the gonadal fat pad of high fat diet fed mice (n = 7–9 mice). (L) Assessment of liver triglycerides (n = 7–9 mice). (M and N) qPCR analsis of markers of steatosis and inflammation in the liver after high fat diet feeding (n = 7–9 mice). All data are represented as mean ± SEM. *P < 0.05.

Fig. 5.

Sclerostin regulates adipocyte metabolism by influencing Bmp4 expression. (A) De novo lipogeneis by gWAT, iWAT, and liver measured by the incorporation of ³H-acetate into tissue lipids (n = 6–7 mice). (B) Relative levels of oleate oxidation by gWAT, iWAT, BAT, and liver assessed by measuring the conversion of 1-¹⁴C-oleic acid to ¹⁴CO₂ (n = 4 mice). (C) qPCR analysis of Sost mRNA levels in femur, gWAT, iWAT, and primary mouse adipocytes, and CD45⁻, Sca1⁺, and Pdgfra⁺ adipoprogenitors (n = 5–6 mice). (D) qPCR analysis of Axin2 mRNA levels in primary mouse adipocytes isolated from WT and Sost^−/− mice 6 h after stimulation with Wnt10b or Wnt3a. (E) Oil Red O staining of primary adipocyte cultures treated with vehicle or recombinant mouse sclerostin (100 ng/mL). (F) qPCR analysis of adipocyte differentiation markers. (G) 3H-acetate incorporation in primary mouse adipocytes treated with vehicle or recombinant mouse sclerostin. (H) Oleate oxidation by primary mouse adipocytes treated with vehicle or recombinant mouse sclerostin. (I) qPCR of Wnt target genes and mediators of fatty acid metabolism in primary mouse adipocytes treated with vehicle or recombinant sclerostin. (J) qPCR analysis of Bmp expression in primary mouse adipocytes treated with vehicle, recombinant sclerostin, or Wnt3a. (K) Immunoblot analysis of phosphorylated Smad1/5/9 levels in primary mouse adipocytes treated with vehicle or recombinant mouse sclerostin. (L and M) qPCR analysis of Bmp4 and Bmpr1a in gonadal adipose tissue isolated from Sost^−/− mice (L) or AAV-Sost mice (M) and controls (n = 5–6 mice per group). (N–R) Primary mouse adipocytes were treated with vehicle, recominant mouse sclerostin (100 ng/mL), noggin (50 ng/mL), or a combination of the two. (N) qPCR analysis of Axin2 expression. (O) 3H-acetate incorporation. (P) Oleate oxidation. (Q) qPCR analysis of genes associated with adipocyte differentiation and fatty acid synthesis. (R) qPCR analysis of genes associated with fatty acid oxidation. In vitro studies were repeated in at least two independent experiments. All data are represented as mean ± SEM. *P < 0.05.

Fig. 6.

Sclerostin-neutralizing antibodies reduce fat mass in mice. (A) Serum sclerostin levels in mice fed a low fat (LFD) or high fat diet (HFD) and treated weekly with vehicle or sclerostin-neutralizing antibody (Scl Ab, 30 mg/kg) for 8 wk (n = 8–10 mice per group). (B and C) qPCR analysis of Axin2 mRNA levels in tissues of vehicle or Scl Ab-treated mice fed LFD (B) or HFD (C) (n = 6 mice per group). (D) Final body weight after 8 wk on LFD or HFD (n = 8–10 mice per group). (E and F) qNMR analysis of fat mass and lean mass (n = 8–10 mice per group). (G and H) Adipose depot weights after 8 wk of treatment (n = 8–10 mice per group). (I) Representative histological sections of gonadal (gWAT) and inguinal (iWAT) fat pads and liver LFD- and HFD-fed mice treated with vehicle or Scl Ab (10× magnification). (J) Relative levels of oleate oxidation by gWAT, iWAT, BAT, liver, and muscle assessed by measuring the conversion of 1-¹⁴C-oleic acid to ¹⁴CO₂ (n = 8–10 mice per group). (K and L) Random fed glucose and insulin levels for each treatment group (n = 8–10 mice per group). All data are represented as mean ± SEM. *P < 0.05.