Fat occupies a significant portion of bone cavity however its function is largely unknown. Marrow fat expands during aging and in conditions which affect energy metabolism, indicating that fat in bone is under similar regulatory mechanisms as other fat depots. On the other hand, its location may determine specific functions in the maintenance of the environment for bone remodeling and hematopoiesis. We have demonstrated that marrow fat has a distinctive phenotype, which resembles both, white and brown adipose tissue (WAT and BAT, respectively). Marrow adipocytes express gene markers of brown adipocytes at levels characteristic for the BAT, including transcription factor Prdm16, and regulators of thermogenesis such as deiodinase 2 (Dio2) and PGC1α. The levels of expression of BAT-specific gene markers are decreased in bone of 24 mo old C57BL/6 and in diabetic yellow agouti Avy/a mice implicating functional changes of marrow fat occurring with aging and diabetes. Administration of antidiabetic TZD rosiglitazone, which sensitizes cells to insulin and increases adipocyte metabolic functions, significantly increased both, BAT (UCP1, PGC1α, Dio2, β3AR, Prdm16, and FoxC2) and WAT (adiponectin and leptin) gene expression in marrow of normoglycemic C57BL/6 mice, but failed to increase the expression of BAT, but not WAT, gene markers in diabetic mice. In conclusion, the metabolic phenotype of marrow fat combines both BAT and WAT characteristics. Decrease in BAT-like characteristics with aging and diabetes may contribute to the negative changes in the marrow environment supporting bone remodeling and hematopoiesis.

It is known that insulin resistance and type 2 diabetes mellitus are associated with increased fractures and that brown adipose tissue (BAT) counteracts many if not all of the symptoms associated with type 2 diabetes. By the use of FoxC2(AD)(+/Tg) mice, a well-established model for induction of BAT, or beige fat, we present data extending the beneficial action of beige fat to also include a positive effect on bone. FoxC2(AD)(+/Tg) mice are lean and insulin-sensitive and have high bone mass due to increased bone formation associated with high bone turnover. Inducible BAT is linked to activation of endosteal osteoblasts whereas osteocytes have decreased expression of the Sost transcript encoding sclerostin and elevated expression of Rankl. Conditioned media (CM) collected from forkhead box c2 (FOXC2)-induced beige adipocytes activated the osteoblast phenotype and increased levels of phospho-AKT and β-catenin in recipient cells. In osteocytes, the same media decreased Sost expression. Immunodepletion of CM with antibodies against wingless related MMTV integration site 10b (WNT10b) and insulin-like growth factor binding protein 2 (IGFBP2) resulted in the loss of pro-osteoblastic activity, and the loss of increase in the levels of phospho-AKT and β-catenin. Conversely, CM derived from cells overexpressing IGFBP2 or WNT10b restored osteoblastic activity in recipient cells. In conclusion, beige fat secretes endocrine/paracrine activity that is beneficial for the skeleton.

Peroxisome proliferator activated receptor gamma (PPARγ) controls both glucose metabolism and an allocation of marrow mesenchymal stem cells (MSCs) toward osteoblast and adipocyte lineages. Its activity is determined by interaction with a ligand which directs posttranscriptional modifications of PPARγ protein including dephosphorylation of Ser112 and Ser273, which results in acquiring of pro-adipocytic and insulin-sensitizing activities, respectively. PPARγ full agonist TZD rosiglitazone (ROSI) decreases phosphorylation of both Ser112 and Ser273 and its prolonged use causes bone loss in part due to diversion of MSCs differentiation from osteoblastic toward adipocytic lineage. Telmisartan (TEL), an anti-hypertensive drug from the class of angiotensin receptor blockers, also acts as a partial PPARγ agonist with insulin-sensitizing and a weak pro-adipocytic activity. TEL decreased S273pPPARγ and did not affect S112pPPARγ levels in a model of marrow MSC differentiation, U-33/γ2 cells. In contrast to ROSI, TEL did not affect osteoblast phenotype and actively blocked ROSI-induced anti-osteoblastic activity and dephosphorylation of S112pPPARγ. The effect of TEL on bone was tested side-by-side with ROSI. In contrast to ROSI, TEL administration did not affect bone mass and bone biomechanical properties measured by micro-indentation method and did not induce fat accumulation in bone, and it partially protected from ROSI-induced bone loss. In addition, TEL induced “browning” of epididymal white adipose tissue marked by increased expression of UCP1, FoxC2, Wnt10b and IGFBP2 and increased overall energy expenditure. These studies point to the complexity of mechanisms by which PPARγ acquires anti-osteoblastic and pro-adipocytic activities and suggest an importance of Ser112 phosphorylation status as being a part of the mechanism regulating this process. These studies showed that TEL acts as a full PPARγ agonist for insulin-sensitizing activity and as a partial agonist/partial antagonist for pro-adipocytic and anti-osteoblastic activities. They also suggest a relationship between PPARγ fat “browning” activity and a lack of anti-osteoblastic activity.

Lineage allocation of the marrow mesenchymal stem cells (MSCs) to osteoblasts and adipocytes is dependent on both Wnt signaling and PPARγ2 activity. Activation of PPARγ2, an essential regulator of energy metabolism and insulin sensitivity, stimulates adipocyte and suppresses osteoblast differentiation and bone formation, and correlates with decreased bone mass and increased fracture rate. In contrast, activation of Wnt signaling promotes osteoblast differentiation, augments bone accrual and reduces total body fat. This study examined the cross-talk between PPARγ2 and β-catenin, a key mediator of canonical Wnt signaling, on MSC lineage determination. Rosiglitazone-activated PPARγ2 induced rapid proteolytic degradation of β-catenin, which was prevented by either inhibiting glycogen synthase kinase 3 beta (GSK3β) activity, or blocking pro-adipocytic activity of PPARγ2 using selective antagonist GW9662 or mutation within PPARγ2 protein. Stabilization of β-catenin suppressed PPARγ2 pro-adipocytic but not anti-osteoblastic activity. Moreover, β-catenin stabilization decreased PPARγ2-mediated insulin signaling as measured by insulin receptor and FoxO1 gene expression, and protein levels of phosphorylated Akt (pAkt). Cellular knockdown of β-catenin with siRNA increased expression of adipocyte but did not affect osteoblast gene markers. Interestingly, the expression of Wnt10b was suppressed by anti-osteoblastic, but not by pro-adipocytic activity of PPARγ2. Moreover, β-catenin stabilization in the presence of activated PPARγ2 did not restore Wnt10b expression indicating a dominant role of PPARγ2 in negative regulation of pro-osteoblastic activity of Wnt signaling. In conclusion, β-catenin and PPARγ2 are in cross-talk which results in sequestration of pro-adipocytic and insulin sensitizing activity. The anti-osteoblastic activity of PPARγ2 is independent of this interaction.

Thiazolidinediones (TZDs), peroxisome proliferator-activated receptor gamma activators, and insulin sensitizers represent drugs used to treat hyperglycemia in diabetic patients. Type 2 diabetes mellitus (T2DM) is associated with a twofold increase in fracture risk, and TZDs use increases this risk by an additional twofold. In this study, we analyzed the effect of systemic administration of the TZD rosiglitazone on new bone formation in two in vivo models of bone repair, a model of drilled bone defect regeneration (BDR) and distraction osteogenesis (DO) and a model of extended bone formation. Rosiglitazone significantly inhibited new endosteal bone formation in both models. This effect was correlated with a significant accumulation of fat cells, specifically at sites of bone regeneration. The diminished bone regeneration in the DO model in rosiglitazone-treated animals was associated with a significant decrease in cell proliferation measured by the number of cells expressing proliferating cell nuclear antigen and neovascularization measured by both the number of vascular sinusoids and the number of cells producing proangiogenic vascular endothelial growth factor at the DO site. In summary, rosiglitazone decreased new bone formation in both BDR and DO models of bone repair by mechanisms which include both intrinsic changes in mesenchymal stem cell proliferation and differentiation and changes in the local environment supporting angiogenesis and new bone formation. These studies suggest that bone regeneration may be significantly compromised in T2DM patients on TZD therapy.