Increased Bone Marrow Adiposity in a Context of Energy Deficit: The Tip of the Iceberg?

Elevated bone marrow adiposity (BMA) is defined as an increase in the proportion of the bone marrow (BM) cavity volume occupied by adipocytes. This can be caused by an increase in the size and/or number of adipocytes. BMA increases with age in a bone-site-specific manner. This increase may be linked to certain pathophysiological situations. Osteoporosis or compromised bone quality is frequently associated with high BMA. The involvement of BM adipocytes in bone loss may be due to commitment of mesenchymal stem cells to the adipogenic pathway rather than the osteogenic pathway. However, adipocytes may also act on their microenvironment by secreting factors with harmful effects for the bone health. Here, we review evidence that in a context of energy deficit (such as anorexia nervosa (AN) and restriction rodent models) bone alterations can occur in the absence of an increase in BMA. In severe cases, bone alterations are even associated with gelatinous BM transformation. The relationship between BMA and energy deficit and the potential regulators of this adiposity in this context are also discussed. On the basis of clinical studies and preliminary results on animal model, we propose that competition between differentiation into osteoblasts and differentiation into adipocytes might trigger bone loss at least in moderate-to-severe AN and in some calorie restriction models. Finally, some of the main questions resulting from this hypothesis are discussed.

Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation.

BACKGROUND: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the adipogenic pathway. Supporting this hypothesis the competition between adipogenic and osteogenic lineages was widely demonstrated on partially homogeneous cell populations. However, some data from mouse models showed the existence of an independent relationship between bone mineral content and bone marrow adiposity. Therefore, the combination of adipogenesis and osteogenesis in primary culture would be helpful to determine if this competition would be observed on a whole bone marrow stromal cell population in a culture medium allowing both lineages. In this aim, mouse bone marrow stromal cells were cultured in a standard osteogenic medium added with different concentrations of Dexamethasone, known to be an important regulator of mesenchymal progenitor cell differentiation. RESULTS: Gene expression of osteoblast and adipocyte markers, biochemical and physical analyses demonstrated the presence of both cell types when Dexamethasone was used at 100 nM. Overall, our data showed that in this co-differentiation medium both differentiation lineages were enhanced compared to classical adipogenic or osteogenic culture medium. This suggests that in this model, adipocyte phenotype does not seem to increase at the expense of the osteoblast lineage. CONCLUSION: This model appears to be a promising tool to study osteoblast and adipocyte differentiation capabilities and the interactions between these two processes.

Adipogenic RNAs are transferred in osteoblasts via bone marrow adipocytes-derived extracellular vesicles (EVs).

BACKGROUND: In osteoporosis, bone loss is accompanied by increased marrow adiposity. Given their proximity in the bone marrow and their shared origin, a dialogue between adipocytes and osteoblasts could be a factor in the competition between human Mesenchymal Stem Cells (hMSC) differentiation routes, leading to adipocyte differentiation at the expense of osteoblast differentiation. The adipocyte/osteoblast balance is highly regulated at the level of gene transcription. In our work, we focused on PPARgamma, CEBPalpha and CEBPdelta, as these transcription factors are seen as master regulators of adipogenesis and expressed precociously, and on leptin and adiponectin, considered as adipocyte marker genes. In 2010, our group has demonstrated, thanks to a coculture model, that in the presence of hMSC-derived adipocytes (hMSC-Adi), hMSC-derived osteoblasts (hMSC-Ost) express lesser amounts of osteogenic markers but exhibit the expression of typical adipogenic genes. Nevertheless, the mechanisms underlying this modulation of gene expression are not clarified. Recently, adipocytes were described as releasing extracellular vesicles (EVs), containing and transferring adipocyte specific transcripts, like PPARgamma, leptin and adiponectin. Here, we investigated whether EVs could be the way in which adipocytes transfer adipogenic RNAs in our coculture model. RESULTS: We observed in hMSC-Ost incubated in hAdi-CM an increase in the adipogenic PPARγ, leptin, CEBPα and CEBPδ transcripts as well as the anti-osteoblastic miR-138, miR30c, miR125a, miR-125b, miR-31 miRNAs, probably implicated in the observed osteocalcin (OC) and osteopontin (OP) expression decrease. Moreover, EVs were isolated from conditioned media collected from cultures of hMSC at different stages of adipocyte differentiation and these specific adipogenic transcripts were detected inside. Finally, thanks to interspecies conditioned media exposition, we could highlight for the first time a horizontal transfer of adipogenic transcripts from medullary adipocytes to osteoblasts. CONCLUSIONS: Here, we have shown, for the first time, RNA transfer between hMSC-derived adipocytes and osteoblasts through EVs. Additional studies are needed to clarify if this mechanism has a role in the adipocytic switch driven on osteoblasts by adipocytes inside bone marrow and if EVs could be a target component to regulate the competition between osteoblasts and adipocytes in the prevention or in the therapy of osteoporosis and other osteopenia.

Long-term physiological alterations and recovery in a mouse model of separation associated with time-restricted feeding: a tool to study anorexia nervosa related consequences.

BACKGROUND: Anorexia nervosa is a primary psychiatric disorder, with non-negligible rates of mortality and morbidity. Some of the related alterations could participate in a vicious cycle limiting the recovery. Animal models mimicking various physiological alterations related to anorexia nervosa are necessary to provide better strategies of treatment. AIM: To explore physiological alterations and recovery in a long-term mouse model mimicking numerous consequences of severe anorexia nervosa. METHODS: C57Bl/6 female mice were submitted to a separation-based anorexia protocol combining separation and time-restricted feeding for 10 weeks. Thereafter, mice were housed in standard conditions for 10 weeks. Body weight, food intake, body composition, plasma levels of leptin, adiponectin, IGF-1, blood levels of GH, reproductive function and glucose tolerance were followed. Gene expression of several markers of lipid and energy metabolism was assayed in adipose tissues. RESULTS: Mimicking what is observed in anorexia nervosa patients, and despite a food intake close to that of control mice, separation-based anorexia mice displayed marked alterations in body weight, fat mass, lean mass, bone mass acquisition, reproductive function, GH/IGF-1 axis, and leptinemia. mRNA levels of markers of lipogenesis, lipolysis, and the brown-like adipocyte lineage in subcutaneous adipose tissue were also changed. All these alterations were corrected during the recovery phase, except for the hypoleptinemia that persisted despite the full recovery of fat mass. CONCLUSION: This study strongly supports the separation-based anorexia protocol as a valuable model of long-term negative energy balance state that closely mimics various symptoms observed in anorexia nervosa, including metabolic adaptations. Interestingly, during a recovery phase, mice showed a high capacity to normalize these parameters with the exception of plasma leptin levels. It will be interesting therefore to explore further the central and peripheral effects of the uncorrected hypoleptinemia during recovery from separation-based anorexia.

TNF-alpha's effects on proliferation and apoptosis in human mesenchymal stem cells depend on RUNX2 expression.

RUNX2 is a bone-specific transcription factor that plays a critical role in prenatal bone formation and postnatal bone development. It regulates the expression of genes that are important in committing cells into the osteoblast lineage. There is increasing evidence that RUNX2 is involved in osteoblast proliferation. RUNX2 expression increases during osteoblast differentiation, and recent data even suggest that it acts as a proapoptotic factor. The cytokine tumor necrosis factor alpha (TNF-alpha) is known to modulate osteoblast functions in a manner that depends on the differentiation stage. TNF-alpha affects the rate at which mesenchymal precursor cells differentiate into osteoblasts and induces apoptosis in mature osteoblasts. Thus we sought to establish whether or not the effects of TNF-alpha and fetal calf serum on proliferation and apoptosis in human mesenchymal stem cells (hMSCs) were dependent on RUNX2 level and activity. We transfected hMSCs with small interfering RNAs (siRNAs) directed against RUNX2 and found that they proliferated more quickly than control hMSCs transfected with a nonspecific siRNA. This increase in proliferation was accompanied by a rise in cyclin A1, B1, and E1 expression and a decrease in levels of the cyclin inhibitor p21. Moreover, we observed that RUNX2 silencing protected hMSCs from TNF-alpha's antiproliferative and apoptotic effects. This protection was accompanied by the inhibition of caspase-3 activity and Bax expression. Our results confirmed that RUNX2 is a critical link between cell fate, proliferation, and growth control. This study also suggested that, depending on the osteoblasts' differentiation stage, RUNX2 may control cell growth by regulating the expression of elements involved in hormone and cytokine sensitivity.