Adiponectin regulates bone mass via opposite central and peripheral mechanisms through FoxO1

Abstract

The synthesis of adiponectin, an adipokine with ill-defined functions in animals fed a normal diet, is enhanced by the osteoblast-derived hormone osteocalcin. Here we show that adiponectin signals back in osteoblasts to hamper their proliferation and favor their apoptosis, altogether decreasing bone mass and circulating osteocalcin levels. Adiponectin fulfills these functions, independently of its known receptors and signaling pathways, by decreasing FoxO1 activity in a PI3-kinase-dependent manner. Over time, however, these local effects are masked because adiponectin signals in neurons of the locus coeruleus, also through FoxO1, to decrease the sympathetic tone, thereby increasing bone mass and decreasing energy expenditure. This study reveals that adiponectin has the unusual ability to regulate the same function in two opposite manners depending on where it acts and that it opposes, partially, leptin's influence on the sympathetic nervous system. It also proposes that adiponectin regulation of bone mass occurs through a PI3-kinase-FoxO1 pathway.

Figure 1

Adiponectin regulation of Rankl and bone mass. (A) Conservation of adiponectin, AdipoR1, AdipoR2 and T-cadherin sequences among vertebrate and invertebrate species. (B) Relative expression of Adiponectin in WT and Adiponectin−/− adipocytes, osteoblasts, and osteoclasts population. (C) Adiponectin regulation of gene expression analyzed by qPCR. Primary osteoblasts or osteoclasts cell populations treated with indicated concentration of adiponectin for 4 hours. (D) Bone histomorophometric analysis of 6 and 12 week-old WT (n>10) or Adiponectin−/− (n>10) mice. BV/TV: bone volume over tissue volume. Ob.N/T.Ar: number of osteoblasts per trabecular area. BFR: bone formation rate. OcS/BS: osteoclast surface per bone surface. ES/BS: Eroded surface per bone surface. (E) μCT analysis of 6 week-old WT (n=5) or Adiponectin−/− mice (n=5). Cort.Th: Cortical thickness. Conn-Den: connective density. Tb.N: trabecular number. (F–G) Three points bending test and Rankl expression in 6 week-old Adiponectin−/− bones. (H) Total and uncarboxylated osteocalcin serum levels in 6 week-old Adiponectin−/− mice. (I) Adiponectin serum levels in 12 week-old pLiv-Adiponectin (pLiv-Apn) mice. (J) Bone histomorphometric analysis of 12 week-old WT (n=10) and pLiv-Adiponectin mice (n=8). (K) Total and uncarboxylated osteocalcin serum levels in 12 week-old pLiv-Adiponectin mice. (L–M) Glucose tolerance test and glucose-stimulated insulin secretion test in 12 week-old Adiponectin−/− mice. For all panels, results are given as means ± standard error of mean (SEM). *, p < 0.05 by ANOVA and/or student t-test. NS: not significant. N.D: not determined. See also figure S1.

Figure 2

Adiponectin regulates osteoblast proliferation and apoptosis. (A, B) BrdU incorporation in 10 day-old Adiponectin−/− bones, or WT osteoblasts, myoblasts or hepatocytes treated with vehicle or adiponectin for 24 hours. (C–D) Cyclins accumulation in 6 week-old Adiponectin−/− bones, and WT osteoblasts treated with vehicle or adiponectin for 24 hours. (E–F) TUNEL assay in 10 day-old Adiponectin−/− bones, and WT osteoblasts, myoblasts or hepatocytes treated with vehicle or adiponectin for 24 hours. (G) Annexin-V positive 6 week-old Adiponectin−/− osteoblasts. (H) Cleaved-caspase 3 accumulation in WT osteoblasts treated with vehicle, adiponectin, or H₂O₂ (100μM) treated for 24 hours. (I, J) Quantification of malondialdehyde, 4-hydroxynonenal and reactive oxygen species (ROS) in 6 week-old Adiponectin−/− osteoblasts.

Figure 3

Analysis of older Adiponectin−/− mice. (A) Bone histomorophometric analysis of 24 and 36 week-old WT (n>10) and Adiponectin−/− (n>10) mice. (B–C) Cyclin expression, Cyclin D1 accumulation in 36 week-old Adiponectin−/− bones. (D–F) Serum CTx levels, Rankl expression, Energy expenditure in 36 week-old Adiponectin−/− mice. (G–J) Fat pad weight, body weight, blood pressure and heart rate of 12 and/or 36 week-old Adiponectin−/− mice. (K) Adiponectin accumulation in brain. 6 week-old Adiponectin−/− mice received 5 μg/day of adiponectin peripherally for 7 days, adiponectin levels in hypothalamus, brainstem, cortex, and cerebellum were then measured. ND: not detectable. (L) Adiponectin binding to locus coeruleus (LC), visualized with anti-biotin antibody (red) together with locus coeruleus-specific marker DBH using anti-DBH antibody (green). (M) Norepinephrine content in the brainstem of 36 week-old Adiponectin−/− mice. (N) Ucp1 expression in brown fat of 6, 12 and 36 week-old Adiponectin−/− and 12 week-old pLib-Adiponectin mice. (O) Urinary epinephrine elimination of 6, 12 and 36 week-old Adiponectin−/− mice. (P) Ucp1 expression in 36 week-old Adiponectin−/− subcutaneous fat (sub. fat). See also figure S2.

Figure 4

Analysis of Adiponectin−/−;Dbh+/−, Adiponectin−/−;Adrb2_osb+/− and Adipoenctin−/−;ob/ob mice. (A) Bone histomorophometric analysis of 36 week-old Adiponectin−/−;Dbh+/− mice. WT (n>10), Dbh+/− (n>10), Adiponectin−/− (n>10), or Adiponectin−/−;Dbh+/− (n>10). *, P<0.05 between WT and Adiponectin−/−. #, P<0.05 between Adiponectin−/− and Adiponectin−/−;Dbh+/−. **, P<0.05 between WT and Adiponectin−/−;Dbh+/−. (B–F) Serum CTx levels, Rankl expression, Cyclin D1 accumulation, energy expenditure, and fat pad weight in 36 week-old Adiponectin−/−;Dbh+/− mice. *, P<0.05 between WT and Adiponectin−/−. #, P<0.05 between Adiponectin−/− and Adiponectin−/−;Dbh+/−. (G) Bone mass of 12 week-old Adiponectin−/−;Adrb2_osb+/− mice. (H) Ucp1 expression in 6 week-old Adiponectin−/−;ob/ob brown fat. (I–L) Energy expenditure, fat pad weight, blood pressure, heart rate in 6 week-old Adiponectin−/−;ob/ob mice. (M–N) Bone histomorphometric analysis of 10 week-old Adiponectin−/−;ob/ob vertebrae and femora. WT (n=9), ob/ob (n=8), Adiponectin−/− (n=7), Adiponectin−/−;ob/ob (n=7) *, P<0.05 between WT and Adiponectin−/−, **, P<0.05 between WT and ob/ob, ***, P<0.05 between WT and Adiponectin−/−;ob/ob. # P<0.05 between Adiponectin−/− and ob/ob. ##, P<0.05 between Adiponectin−/− and Adiponectin−/−;ob/ob. $, P<0.05 between Adiponectin−/−;ob/ob and ob/ob. See also figure S3.

Figure 5

Known adiponectin receptors and signaling pathways do not mediate adiponectin function in osteoblasts. (A–C) AdipoR1, AdipoR2, and T-cadherin expression in osteoblasts, muscle or liver. (D–G) Bone histomorophometric analyses of 12 week-old AdipoR1_osb−/−, AdipoR2 _osb−/−, AdipoR1_osb−/−; AdipoR2_osb−/−, or T-cadherin _osb−/− mice. (H) Rankl expression in 12 week-old AdipoR1_osb−/−, AdipoR2 _osb−/−, T-cadherin _osb−/− and AdipoR1_osb−/−; AdipoR2_osb−/− or fl/fl osteoblasts treated with vehicle or adiponectin. See also figure S4.

Figure 6

FoxO1 mediates adiponectin functions in osteoblasts and neurons. (A) Western blot analysis of phospho-AMPK in WT osteoblass treated with vehicle, 15 μg/ml adiponectin or AICAR (0.5 mM) for 5 or 30 min. (B) Ceramide contents in 6 week-old Adiponectin−/− bones. (C–D) Western blot analysis of phospho-FoxO1 in WT and 6 week-old Adiponectin−/− bones, and osteoblasts treated with vehicle, adiponectin or insulin for 15 min. (E) FoxO1 luciferase assay in ROS 17/6.2 cells treated with adiponectin for 24 hours. (F) Expression of FoxO1 target genes in 12 week-old Adiponectin−/− or pLiv-Adiponectin bones. (G) Bone histomorphometric analysis of 12 week-old Adiponectin−/−;FoxO1_osb+/− bones. Controls (n=8), Adiponectin−/− (n=8), Adiponectin−/−;FoxO1_osb+/− (n=6). *, P<0.05 between Controls and Adiponectin−/−. **, P<0.05 between Adiponectin−/− and Adiponectin−/−;FoxO1_osb+/−. (H) BrdU incorporation in 10 day-old Adiponectin−/−;FoxO1_osb+/− bones. (I) CyclinD1 accumulation in 12 week-old Adiponectin−/−;FoxO1_osb+/− bones. (J) Energy expenditure of 36 week-old Adiponectin−/−;FoxO1_LC+/− mice. (K) Ucp1 expression in 36 week-old Adiponectin−/−;FoxO1_LC+/− brown fat. (L) Fat pad weight of 36 week-old Adiponectin−/−;FoxO1_LC+/− mice. (M) Bone histomorphometric analysis of 36 week-old Adiponectin−/−;FoxO1_LC+/− mice. Controls (n=5), Adiponectin−/− (n=6), Adiponectin−/−;FoxO1_LC+/− (n=5). *, P<0.05 between Controls and Adiponectin−/−. **, P<0.05 between Adiponectin−/− and Adiponectin−/−;FoxO1_LC+/−. (N) Rankl expression in 36 week-old Adiponectin−/−;FoxO1_LC+/− bones. (O) CTx levels of 36 week-old Adiponectin−/−;FoxO1_LC+/− mice. See also figure S5.

Figure 7

Adiponectin signaling in osteoblasts. (A) Accumulation of phosphatidylinositol 3-phosphate in osteoblasts treated with vehicle, adiponectin or insulin (10 nM) in the presence PI3 kinase inhibitor of LY294002 (10 μM) for 5 min. (B) Western blot analysis of phospho-AKT in osteoblasts treated with vehicle, adiponectin, insulin (10 nM) or PTH (10 nM) for 5 min. (C) Western blot analysis of phospho-AKT and phospho-FoxO1 in the presence of LY294002 (10 μM) for 5 min (P-AKT) and 15 min (P-FoxO1). (D–E) Rankl induction and phosphorylation of AKT by adiponectin in the presence of G_s inhibitor (NF449), G_i inhibitor (NF023), G_ii inhibitor (Gallein), or PLC inhibitor (ET-18-OCH₃). (F) Western blot analysis of phospho-tyrosine in osteoblasts treated with vehicle, adiponectin or PTH (10 nM) for 2 min. Arrowhead, the phosphorylated band around 170 kDa. (G) Evolution of the bone phenotype of Adiponectin−/− mice overtime. (H) Schematic representation of the diverse functions exerted by adiponectin in mice fed a normal diet and of its two sites of action. See also figure S6.