Increased Gs Signaling in Osteoblasts Reduces Bone Marrow and Whole-Body Adiposity in Male Mice

Abstract

Bone is increasingly recognized as an endocrine organ that can regulate systemic hormones and metabolism through secreted factors. Although bone loss and increased adiposity appear to be linked clinically, whether conditions of increased bone formation can also change systemic metabolism remains unclear. In this study, we examined how increased osteogenesis affects metabolism by using an engineered G protein-coupled receptor, Rs1, to activate Gs signaling in osteoblastic cells in ColI(2.3)(+)/Rs1(+) transgenic mice. We previously showed that these mice have dramatically increased bone formation resembling fibrous dysplasia of the bone. We found that total body fat was significantly reduced starting at 3 weeks of age. Furthermore, ColI(2.3)(+)/Rs1(+) mice showed reduced O2 consumption and respiratory quotient measures without effects on food intake and energy expenditure. The mice had significantly decreased serum triacylglycerides, leptin, and adiponectin. Resting glucose and insulin levels were unchanged; however, glucose and insulin tolerance tests revealed increased sensitivity to insulin. The mice showed resistance to fat accumulation from a high-fat diet. Furthermore, ColI(2.3)(+)/Rs1(+) mouse bones had dramatically reduced mature adipocyte differentiation, increased Wingless/Int-1 (Wnt) signaling, and higher osteoblastic glucose utilization than controls. These findings suggest that osteoblasts can influence both local and peripheral adiposity in conditions of increased bone formation and suggest a role for osteoblasts in the regulation of whole-body adiposity and metabolic homeostasis.

Figure 1.

Osteoblastic G_s signaling reduces adipose tissue in ColI(2.3)⁺/Rs1⁺ mice. Body weight (A) and BMD (B) of 8- to 9-week-old control and ColI(2.3)⁺/Rs1⁺ male mice. Data are from n = 9 control and n = 6 (n = 5 for BMD) ColI(2.3)⁺/Rs1⁺ mice. Percent whole-body fat (C) and lean mass (D) of 3- and 8-week-old control and ColI(2.3)⁺/Rs1⁺ male mice. Data are from n = 7 control and n = 4 ColI(2.3)⁺/Rs1⁺ male mice at 3 weeks of age, and n = 9 control and n = 6 ColI(2.3)⁺/Rs1⁺ male mice at 8 weeks of age. Representative dorsal (E) and ventral (F) photos of control and ColI(2.3)⁺/Rs1⁺ male mice showing reductions of visceral adipose tissues with no significant effect on subscapular brown adipose tissue. Yellow outlines denote brown and gonadal adipose tissues. Scale bar, 5 mm. Tissue weights of (G) subscapular brown, (H) inguinal, and (I) gonadal fat pads from 8- to 9-week-old male mice. Data are from n = 5 control and n = 6 ColI(2.3)⁺/Rs1⁺ male mice for brown and gonadal adipose tissue, and n = 4 control and n = 5 ColI(2.3)⁺/Rs1⁺ male mice for inguinal adipose tissue. Statistical differences were determined by Student's t test; *, P < .05; **, P < .01; ***, P < .001; N.S., not significant.

Figure 2.

ColI(2.3)⁺/Rs1⁺ mice have increased fat utilization. Volume of oxygen (VO₂) (A), volume of carbon dioxide (VCO₂) (B), respiratory quotient (RQ) (C), and energy expenditure (D) of 8-week-old male control and an ColI(2.3)⁺/Rs1⁺ mice mouse subjected to a CLAMS on day 3, after 2 days of acclimation to the chamber. Total food intake (E), total water consumption (F), total ambulatory activity (G), and total energy expenditure (H) of 8-week-old control and ColI(2.3)⁺/Rs1⁺ mice after 5 days in the CLAMS. Data are from n = 3 control and n = 5 ColI(2.3)⁺/Rs1⁺ male mice. Statistical differences were determined by Student's t test; *, P < .05; N.S., not significant.

Figure 3.

ColI(2.3)⁺/Rs1⁺ mice have increased insulin sensitivity. Fasting serum glucose (A) and insulin (B) of 8- to 9-week-old control and ColI(2.3)⁺/Rs1⁺ male mice. C, Homeostatic model assessment of insulin resistance (HOMA-IR) of 8- to 9-week-old male control and ColI(2.3)⁺/Rs1⁺ mice. Data are from n = 6–11 control and n = 4–8 ColI(2.3)⁺/Rs1⁺mice. D, Ratio of serum TAGs to serum protein of 8- to 9-week-old control and ColI(2.3)⁺/Rs1⁺ male mice. Data are from n = 4 control and n = 3 ColI(2.3)⁺/Rs1⁺ mice. Glucose (E) and insulin (F) tolerance test of mice injected with 2 g/kg of glucose (at 8 wk of age) or 0.75-U/kg body weight of insulin (at 10 wk of age), respectively. Data are from n = 6 control and n = 4 ColI(2.3)⁺/Rs1⁺ male mice. Control and ColI(2.3)⁺/Rs1⁺ mice in A–F are all free fed with regular chow. Whole-body fat (G) and lean body mass (H) of male 8- to 10-week-old control and ColI(2.3)⁺/Rs1⁺ mice subjected to paired feeding with regular chow starting at 3 weeks of age. Tissue weights of (I) subscapular brown, (J) inguinal, and (K) gonadal fat pads from 8- to 10-week-old male mice subjected to paired feeding starting at 3 weeks of age. Glucose (L) and insulin (M) tolerance tests of pair fed male mice. Data are from n = 3–4 control and n = 4–6 ColI(2.3)⁺/Rs1⁺ mice. Statistical differences were determined by Student's t test; *, P < .05; **, P < .01; ***, P < .001; N.S., not significant.

Figure 4.

ColI(2.3)⁺/Rs1⁺ mice on a HFD are resistant to increases in adiposity. A, Male control and ColI(2.3)⁺/Rs1⁺ mice were fed a HFD starting at 3 weeks of age and followed for 5.5 weeks. B, Representative ventral photos of adipose tissues from 1 control and 2 ColI(2.3)⁺/Rs1⁺ mice fed a HFD for 5.5 weeks. C, Whole-body weight normalized to food intake. D, Whole-body weight, percent body fat, and percent lean body mass of 8-week-old control and ColI(2.3)⁺/Rs1⁺ mice free fed a regular chow diet or a HFD for 5 weeks. E, BMD and BMC male 8-week-old control and ColI(2.3)⁺/Rs1⁺ mice free fed a regular chow diet or a HFD for 5 weeks. ColI(2.3)⁺/Rs1⁺ mice. F, Serum glucose from 8- to 9-week-old male mice free fed a regular chow diet or a HFD. G, Tissue weights of inguinal, gonadal, and subscapular brown fat pads from 8- to 9-week-old mice free fed a regular chow diet or a HFD. Data are from n = 10–14 control and n = 5–8 ColI(2.3)⁺/Rs1⁺ male mice free fed a regular chow diet and n = 9 control and n = 4 ColI(2.3)⁺/Rs1⁺ male mice fed a HFD. H, Change in somatic energy content, mean energy intake, and energy expenditure of control and ColI(2.3)⁺/Rs1⁺ mice fed a HFD between 3 and 8 weeks of age. Data are from n = 4 control and n = 4 ColI(2.3)⁺/Rs1⁺ male mice fed a HFD. Statistical differences were determined by Student's t test; *, P < .05; **, P < .01; ***, P < .001; N.S., not significant.

Figure 5.

ColI(2.3)⁺/Rs1⁺ mice have smaller adipocytes, reduced levels of serum adipogenic factors, and increased levels of serum osteocalcin. H&E-stained histological sections of (A) free-fed and (B) HFD-fed inguinal and gonadal adipose tissue in male 8-week-old control and ColI(2.3)⁺/Rs1⁺ mice. Scale bar, 10 μm. Histological counts of adipocytes from inguinal and gonadal adipose tissues (C) and histological counts normalized to % lipid content (D). Data in C and D are from 12 fields of 2 individual control and 2 individual ColI(2.3)⁺/Rs1⁺ male mice on a free fed or HFD. E, Serum resistin, adiponectin, and leptin. F, Serum ghrelin and PAI-1 from male 8- to 9-week-old control and ColI(2.3)⁺/Rs1⁺ mice. Data are from male n = 6 control and n = 5 ColI(2.3)⁺/Rs1⁺ male mice. G, Serum carboxylated osteocalcin, decarboxylated osteocalcin, and the ratio of decarboxylated to carboxylated osteocalcin from male 8- to 9-week-old control and ColI(2.3)⁺/Rs1⁺ mice. Data are from n = 5 control and n = 6 ColI(2.3)⁺/Rs1⁺ male mice. Statistical differences were determined by Student's t test; *, P < .05; **, P < .01; ***, P < .001; N.S., not significant.

Figure 6.

ColI(2.3)⁺/Rs1⁺ whole bones show reduced adiposity with high levels of Wnt related gene expression. H&E-stained decalcified 8-week-old tibia (A) and tibia TAG to protein ratio from control and ColI(2.3)⁺/Rs1⁺ male mice (B). H&E-stained decalcified 8-week-old femur (C) and femur TAG to protein ratio from control and ColI(2.3)⁺/Rs1⁺ male mice (D). Yellow arrows indicate adipocytes. Scale bar, 10 μm. Serum TAGs are from n = 6 control and n = 4 ColI(2.3)⁺/Rs1⁺ male mice. E, qPCR of Pparγ, Cepbα, Cepbβ, and Cepbδ. Data are from n = 3–5 control n = 3–5 and ColI(2.3)⁺/Rs1⁺ male mice. F, qPCR of adipogenic gene expression of Adipoq, Pref1, Lep, and Fabp4. Data are from n = 5–6 control and n = 3–5 ColI(2.3)⁺/Rs1⁺ male mice. G, qPCR of mature adipogenic gene expression of Lepr and Pdgfrβ. Data are from n = 5–6 control and n = 5 ColI(2.3)⁺/Rs1⁺ male mice. H, qPCR of brown adipose tissue genes Ucp1 and Prdm16. Data are from n = 6 control and n = 5 ColI(2.3)⁺/Rs1⁺ male mice. I, qPCR of Bglap and Col1a1. Data are from n = 5 control and n = 5 ColI(2.3)⁺/Rs1⁺ male mice. J, qPCR of Wnt6, Wnt10a, and Wnt10b. Data are from n = 6 control and n = 5 ColI(2.3)⁺/Rs1⁺ male mice. K, qPCR of Ccnd1, Tcf7, and Axin. Data are from n = 3–4 control and n = 3 ColI(2.3)⁺/Rs1⁺ male mice. L, qPCR of Dkk1 and Sost. Data are from n = 3 control and n = 5 ColI(2.3)⁺/Rs1⁺ male mice. M, Western blottings of active β-CATENIN and total β-CATENIN from crushed femurs with GAPDH as a loading control. Data are from n = 4 control and n = 4 ColI(2.3)⁺/Rs1⁺ male mice. Active β-CATENIN and GAPDH control images were taken at 45-second exposure. Total β-CATENIN and GAPDH control images were taken at 60- and 15-second exposures, respectively. All data are from crushed femurs of control and ColI(2.3)⁺/Rs1⁺ mice. Statistical differences were determined by Student's t test; *, P < .05; **, P < .01; ***, P < .001; N.S., not significant.

Figure 7.

ColI(2.3)⁺/Rs1⁺ osteoblastic cells have increased glycolytic demand without alterations to oxidative metabolism. Seahorse (A) mitochondrial and (B) glycolytic stress tests of isolated bone cells cultured for 7 days. Data are from 5 wells from 2 separate male control mice and 6 wells from 3 separate male ColI(2.3)⁺/Rs1⁺ mice. C, Glut1, Glut3, and Glut4 receptor expression from crushed tibias of control and ColI(2.3)⁺/Rs1⁺ mice. D, Cpt1b and Cyc1 expression from crushed tibias of control and ColI(2.3)⁺/Rs1⁺ mice. Data are from n = 5 control and n = 5 ColI(2.3)⁺/Rs1⁺ male mice. Statistical differences were determined by Student's t test; *, P < .05; **, P < .01; ***, P < .001; N.S., not significant.

Figure 8.

Model for the reduction of bone marrow and whole-body adiposity in ColI(2.3)⁺/Rs1⁺ mice. Osteoblastic (OBC) G_s-GPCR signaling in ColI(2.3)⁺/Rs1⁺ mice results in increased osteocalcin secretion that likely contributes to increased insulin sensitivity and influences fat utilization. This also reduces peripheral adiposity, serum TAGs, and secretion of adipokines by peripheral adipose tissues, which in turn affects sympathetic nervous system signaling to the bone and other tissues. In the ColI(2.3)⁺/Rs1⁺ mice bones, OBC G_s signaling results in reduced adipocytes development (ADCs) and increased OBC energy consumption, possibly from Wnt-mediated bone formation at the expense of adipogenesis.