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P&F Grant Awards

Year 9


Grant # 18

Extracellular Determinants of Marrow Adipocyte Function in the Skeletal Niche.

PI: Clarissa Craft, PhD

 

Specific Aims

Marrow adipocytes are increasingly becoming recognized as playing an active role in metabolic and skeletal homeostasis – with potential to function as an energy reservoir, endocrine regulator, and thermal insulator (Fig.1). Recent work has revealed that the relationship between marrow adipose tissue (MAT) and bone is far more nuanced than previously appreciated, being subject to regulation not only between disease models but also by skeletal site. Its inverse relationship with bone mass, in particular, has come under increasing scrutiny in recent years. For example, distal, fatty marrow regions are filled with MAT adipocytes, yet they actually have comparable or even higher basal cancellous bone volume and increased trabecular thickness relative to proximal red marrow sites (1–3). Consideration of mouse strains is also informative. C3H/HeJ mice have significantly more MAT and more bone than C57BL/6J, counterintuitive to the assumption that MAT causes bone loss in all contexts (4). We hypothesize that this discrepancy can be explained by sitespecific differences in the biochemical nature of the marrow adipocyte and its extracellular niche. Indeed, previous work has shown that the phenotype of marrow fat varies depending on its location in the skeleton (4) (Fig.2A). Specifically, regulated MAT (rMAT) adipocytes increase gradually throughout life in the proximal and central regions of the skeleton and readily undergo changes in cell number and/or size when acted on by external forces such as prolonged cold exposure (4), induction of sympathetic tone (5,6), and stimulated hemolysis (7). By contrast, constitutive MAT (cMAT) adipocytes in the yellow marrow are refractory to change (4). Reports that correlate MAT accumulation with decreases in bone mineral density or formation and increased bone loss are generally based on rMAT-enriched sites such as the proximal femur, hip and lumbar spine (reviewed in (8)). Conversely, studies demonstrating that MAT protects against bone loss have all selected cMAT-enriched sites as their area of interest (e.g. distal tibia and tail vertebrae) (1–3).

Though there are many potential mechanisms underlying these findings, we have pinpointed two wellsupported, yet understudied, components of MAT function that we will investigate using this pilot funding, thus providing preliminary data for future NIH grant applications. First, we will definitively answer the hotly contested question of whether MAT has the ability to undergo a beige-like transition during adrenergic stimulation. This is important because recent evidence suggests that brown adipocytes may have the capacity to support bone formation (9), thus, this may be one mechanism by which certain populations of MAT adipocytes promote skeletal maintenance while others drive bone loss. Second, we will characterize extracellular matrix components of the MAT adipocyte niche in health and obesity. Our central hypothesis is that degeneration of the MAT adipocyte niche in the obese state predisposes to pathologic marrow fat hypertrophy and phenotypic change, negatively impacting skeletal health.

Aim 1. Determine whether marrow adipocytes undergo stimulated ‘beiging’ or regulated lipolysis in response to adrenergic stimulation.
There are currently two highly-contested hypotheses that seek to explain the phenotype and subsequent function of marrow adipocytes within the skeletal niche (reviewed in (11)). In the first, MAT adipocytes are regarded as white adipose tissue-like cells with the capacity for energy storage and release by lipogenesis and lipolysis respectively. In the second, MAT adipocytes are regarded as “beige” with the capacity for stimulated browning, leading to uncoupling of mitochondrial respiration and utilization of bioenergetic substrates for local heat production. Both hypotheses have significant, but unique, implications for bone. In the first situation, the benefit of MAT for bone would stem from its ability to provide energy in the form of lipid to surrounding hematopoietic and skeletal cells. By contrast, a ‘beige’ MAT adipocyte would instead remove energy substrates (i.e. glucose and fatty acids) from the bone marrow and convert them into heat. Both cells also have a unique capacity for cytokine secretion, further diversifying their impact on local and distant cells. Thus, assigning a phenotype to the marrow adipocyte (white vs beige) is crucial to inform future studies pertaining to the role of the MAT adipocyte in skeletal and metabolic disorders. The objective of this aim is to leverage immunohistochemical and genetic techniques, including a novel UCP1-DTA mouse model, to define the ability of MAT adipocytes to undergo stimulated beiging. We hypothesize that MAT in the proximal tibia (rMAT), but not in the distal tibia or tail (cMAT), will undergo stimulated beiging, as evidenced by UCP1 expression, with β- adrenergic stimulation. The rationale for this work is that ability of the MAT adipocyte to either supply or remove energy from the niche has important implications for the function of skeletal and hematopoietic cells.

Aim 2. Identify regulators of marrow adipocyte phenotype and function by the characterizing the matricellular components of the skeletal niche in health and obesity.
All adipocytes are not the same. White adipocytes primarily function to store excess energy as lipids. Brown adipocytes store lipids, but are also highly efficient at energy dissipation. A third adipocyte phenotype is termed “beige”. Beige adipocytes store lipids, however, under the right stimulus take on brown-like properties. Expression of a protein called UCP1 is a hallmark of brown and beige adipocytes, and is the primary protein that allows these cells to dissipate energy through heat production. The discovery that brown and beige adipocytes exist in adult humans, and that “browning” combats both obesity and insulin resistance, has led to significant interest in identifying pharmacological mechanisms to support adipocyte “browning” (12,13). It is well established that adipocytes communicate with the skeletal cells. Specifically, obesity and obesity-associated diabetes correlates with skeletal fragility and predisposition to fracture (10,14,15). By contrast, brown and beige adipocytes have been recently proposed to support bone quality (9,16). In this aim, we will use both UCP-1 knock-out and UCP-1-DTA mice to define the ability of the MAT adipocyte to undergo stimulated beiging. Preliminary Data: Expression of Ucp1 and multi-locularity of the lipid droplet are the standard qualifications for identifying brown and beige adipocytes, we treated mice with CL316,243 – a β3 adrenergic receptor (β3AR) agonist – which is known to induce beiging of inguinal white adipose tissue (WAT) depots. Unexpectedly, we observed that a subset of MAT adipocytes took on a ‘multilocular’ appearance after 72-hour β3AR stimulation (Fig.2B). This occurred selectively in regions of rMAT adipocytes in the femur and not in cMAT regions such as the tail vertebrae, suggesting that MAT adipocytes may undergo selective ‘beiging’ at proximal sites. My previous work also provides correlative evidence suggesting that UCP1 may be functionally important in the MAT adipocyte: male UCP1 knock-out mice have increased MAT and both mice at thermoneutrality and MAGP1 knock-out animals have increases in MAT/decreased UCP1 expression.