Christopher D. Church

Rapid depot-specific activation of adipocyte precursor cells at the onset of obesity

Excessive accumulation of white adipose tissue (WAT) is the defining characteristic of obesity. WAT mass is composed primarily of mature adipocytes, which are generated through the proliferation and differentiation of adipocyte precursors (APs). Although the production of new adipocytes contributes to WAT growth in obesity, little is known about the cellular and molecular mechanisms underlying adipogenesis in vivo. Here, we show that high-fat diet feeding in mice rapidly and transiently induces proliferation of APs within WAT to produce new adipocytes. Importantly, the activation of adipogenesis is specific to the perigonadal visceral depot in male mice, consistent with the patterns of obesogenic WAT growth observed in humans. Furthermore, we find that in multiple models of obesity, the activation of APs is dependent on the phosphoinositide 3-kinase (PI3K)-AKT2 pathway; however, the development of WAT does not require AKT2. These data indicate that developmental and obesogenic adipogenesis are regulated through distinct molecular mechanisms.

Characterization of Cre recombinase models for the study of adipose tissue

The study of adipose tissue in vivo has been significantly advanced through the use of genetic mouse models. While the aP2-CreBI and aP2-CreSalk lines have been widely used to target adipose tissue, the specificity of these lines for adipocytes has recently been questioned. Here we characterize Cre recombinase activity in multiple cell populations of the major adipose tissue depots of these and other Cre lines using the membrane-Tomato/membrane-GFP (mT/mG) dual fluorescent reporter. We find that the aP2-CreBI and aP2-CreSalk lines lack specificity for adipocytes within adipose tissue, and that the aP2-CreBI line does not efficiently target adipocytes in white adipose depots. Alternatively, the Adiponectin-CreERT line shows high efficiency and specificity for adipocytes, while the PdgfRα-CreERUCL and PdgfRα-CreERJHU lines do not efficiently target adipocyte precursor cells in the major adipose depots. Instead, we show that the PdgfRα-Cre line is preferable for studies targeting adipocyte precursor cells in vivo.

The Adipose Tissue Microenvironment Regulates Depot-Specific Adipogenesis in Obesity

The sexually dimorphic distribution of adipose tissue influences the development of obesity-associated pathologies. The accumulation of visceral white adipose tissue (VWAT) that occurs in males is detrimental to metabolic health, while accumulation of subcutaneous adipose tissue (SWAT) seen in females may be protective. Here, we show that adipocyte hyperplasia contributes directly to the differential fat distribution between the sexes. In male mice, high-fat diet (HFD) induces adipogenesis specifically in VWAT, while in females HFD induces adipogenesis in both VWAT and SWAT in a sex hormone-dependent manner. We also show that the activation of adipocyte precursors (APs), which drives adipocyte hyperplasia in obesity, is regulated by the adipose depot microenvironment and not by cell-intrinsic mechanisms. These findings indicate that APs are plastic cells, which respond to both local and systemic signals that influence their differentiation potential independent of depot origin. Therefore, depot-specific AP niches coordinate adipose tissue growth and distribution.

Use of osmium tetroxide staining with microcomputerized tomography to visualize and quantify bone marrow adipose tissue in vivo.

Adipocytes reside in discrete, well-defined depots throughout the body. In addition to mature adipocytes, white adipose tissue depots are composed of many cell types, including macrophages, endothelial cells, fibroblasts, and stromal cells, which together are referred to as the stromal vascular fraction (SVF). The SVF also contains adipocyte progenitors that give rise to mature adipocytes in those depots. Marrow adipose tissue (MAT) or marrow fat has long been known to be present in bone marrow (BM) but its origin, development, and function remain largely unknown. Clinically, increased MAT is associated with age, metabolic diseases, drug treatment, and marrow recovery in children receiving radiation and chemotherapy. In contrast to the other depots, MAT is unevenly distributed in the BM of long bones. Conventional quantitation relies on sectioning of the bone to overcome issues with distribution but is time-consuming, resource intensive, inconsistent between laboratories and may be unreliable as it may miss changes in MAT volume. Thus, the inability to quantitate MAT in a rapid, systematic, and reproducible manner has hampered a full understanding of its development and function. In this chapter, we describe a new technique that couples histochemical staining of lipid using osmium tetroxide with microcomputerized tomography to visualize and quantitate MAT within the medullary canal in three dimensions. Imaging of osmium staining provides a high-resolution map of existing and developing MAT in the BM. Because this method is simple, reproducible, and quantitative, we expect it will become a useful tool for the precise characterization of MAT.

Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content

Pigment epithelium‐derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF‐directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator‐activated receptor‐γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control‐treated cells (P<0.001). Differentiation to osteoblasts by PEDF resulted in a common pathway that involved PPARγ suppression (P<0.01). Canonical Wnt‐β‐catenin signaling results in a MSC differentiation pattern analogous to that seen with PEDF. Thus, adding PEDF enhanced Wnt‐β‐catenin signal transduction in human MSCs, demonstrating a novel Wnt agonist function. In PEDF knockout (KO) mice, total body adiposity was increased by >50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.—Gattu, A. K., Swenson, E. S., Iwakiri, Y., Samuel, V. T., Troiano, N., Berry, R., Church, C. D., Rodeheffer, M. S., Carpenter, T. O., Chung, C. Determination of mesenchymal stem cell fate by pigment epithelium‐derived factor (PEDF) results in increased adiposity and reduced bone mineral content. FASEB J. 27, 4384–4394 (2013). www.fasebj.org

Isolation and Study of Adipocyte Precursors

White adipose tissue (WAT) is a heterogeneous tissue composed of lipid-filled adipocytes and several nonadipocyte cell populations, including endothelial, blood, uncharacterized stromal, and adipocyte precursor cells. Although lipid-filled adipocytes account for the majority of WAT volume and mass, nonadipocyte cell populations have critical roles in WAT maintenance, growth, and function. As mature adipocytes are terminally differentiated postmitotic cells, differentiation of adipocyte precursors is required for hyperplastic WAT growth during development and in obesity. In this chapter, we present methods to separate adipocyte precursor cells from other nonadipocyte cell populations within WAT for analysis by flow cytometry or purification by fluorescence-activated cell sorting. Additionally, we provide methods to study the adipogenic capacity of purified adipocyte precursor cells ex vivo.

Imaging of adipose tissue.

Adipose tissue is an endocrine organ that specializes in lipid metabolism and is distributed throughout the body in distinct white adipose tissue (WAT) and brown adipose tissue (BAT) depots. These tissues have opposing roles in lipid metabolism with WAT storing excessive caloric intake in the form of lipid, and BAT burning lipid through nonshivering thermogenesis. As accumulation of lipid in mature adipocytes of WAT leads to obesity and increased risk of comorbidity (Pi-Sunyer et al., 1998), detailed understanding of the mechanisms of BAT activation and WAT accumulation could produce therapeutic strategies for combatting metabolic pathologies. As morphological changes accompany alterations in adipose function, imaging of adipose tissue is one of the most important tools for understanding how adipose tissue mass fluctuates in response to various physiological contexts. Therefore, this chapter details several methods of processing and imaging adipose tissue, including bright-field colorimetric imaging of paraffin-sectioned adipose tissue with a detailed protocol for automated adipocyte size analysis; fluorescent imaging of paraffin and frozen-sectioned adipose tissue; and confocal fluorescent microscopy of whole mounted adipose tissue. We have also provided many example images showing results produced using each protocol, as well as commentary on the strengths and limitations of each approach.

The first international mini-symposium on methionine restriction and lifespan.

It has been 20 years since the Orentreich Foundation for the Advancement of Science, under the leadership Dr. Norman Orentreich, first reported that low methionine (Met) ingestion by rats extends lifespan (Orentreich et al., 1993). Since then, several studies have replicated the effects of dietary methionine restricted (MR) in delaying age-related diseases (Richie et al., 1994; Miller et al., 2005; Ables et al., 2012; Sanchez-Roman and Barja, 2013). We report the abstracts from the First International Mini-Symposium on Methionine Restriction and Lifespan held in Tarrytown, NY, September 2013. The goals were (1) to gather researchers with an interest in MR and lifespan, (2) to exchange knowledge, (3) to generate ideas for future investigations, and (4) to strengthen relationships within this community. The presentations highlighted the importance of research on cysteine, growth hormone (GH), and ATF4 in the paradigm of aging. In addition, the effects of dietary restriction or MR in the kidneys, liver, bones, and the adipose tissue were discussed. The symposium also emphasized the value of other species, e.g., the naked mole rat, Brandts bat, and Drosophila, in aging research. Overall, the symposium consolidated scientists with similar research interests and provided opportunities to conduct future collaborative studies (Figure 3).

WAT is a functional adipocyte

In vertebrates, adipose tissue is the main storage site for lipids within specialized lipid-laden mature adipocytes. While many species have evolved cells capable of lipid storage, the adipocyte represents a unique specialized cell involved in fuel storage, endocrine, nervous and immune function. However, the adipocytes are not the only cell type in mammals that can accumulate lipid droplets. The ectopic accumulation of lipid in non-adipose tissues including the liver, skeletal muscle, bone, pancreas, and heart in combination with its excessive accumulation in adipose tissue contributes to metabolic disease. Determining the lipid processing components that are necessary and sufficiently for lipid accumulation in adipose and non-adipose tissues, in addition to endocrine function, will lead to a clearer definition of an adipocyte.

Methods in Enzymology (MIE): Methods of Adipose Tissue Biology-: Chapter 7: Imaging of Adipose Tissue

Adipose tissue is an endocrine organ that specializes in lipid metabolism and is distributed throughout the body in distinct white adipose tissue (WAT) and brown adipose tissue (BAT) depots. These tissues have opposing roles in lipid metabolism with WAT storing excessive caloric intake in the form of lipid, and BAT burning lipid through nonshivering thermogenesis. As accumulation of lipid in mature adipocytes of WAT leads to obesity and increased risk of comorbidity (Pi-Sunyer et al., 1998), detailed understanding of the mechanisms of BAT activation and WAT accumulation could produce therapeutic strategies for combatting metabolic pathologies. As morphological changes accompany alterations in adipose function, imaging of adipose tissue is one of the most important tools for understanding how adipose tissue mass fluctuates in response to various physiological contexts. Therefore, this chapter details several methods of processing and imaging adipose tissue, including bright-field colorimetric imaging of paraffin-sectioned adipose tissue with a detailed protocol for automated adipocyte size analysis; fluorescent imaging of paraffin and frozen-sectioned adipose tissue; and confocal fluorescent microscopy of whole mounted adipose tissue. We have also provided many example images showing results produced using each protocol, as well as commentary on the strengths and limitations of each approach.