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Subcutaneous obesity is associated with better metabolic health than visceral obesity. Here, we leverage two mouse models with differing quantities of visceral and subcutaneous fat to assess the role of fat distribution in glucose homeostasis. Interestingly, we found genetic ablation of inguinal subcutaneous fat does not exacerbate obesity-associated impairments in glucose metabolism. Consistent with this observation, mutant mice that preferentially accrue subcutaneous fat display a similar metabolic profile to controls with equal fat mass. Importantly, the increased subcutaneous adiposity in these mice occurs downstream of androgen receptor deficiency and is not driven by elevated adiponectin activity. Rather, it is caused by diminished adipocyte precursor seeding in nascent visceral fat and proportionally greater growth of subcutaneous fat. Thus, the pattern of obesogenic fat mass expansion can be determined early in development without impacting glucose metabolism. This suggests that different mechanisms underlying biased fat accumulation exert different effects on glucometabolic health.

Over the past 2 decades, the incidence of childhood obesity has risen dramatically. This recent rise in childhood obesity is particularly concerning as adults who were obese during childhood develop type II diabetes that is intractable to current forms of treatment compared with individuals who develop obesity in adulthood. While the mechanisms responsible for the exacerbated diabetic phenotype associated with childhood obesity is not clear, it is well known that childhood is an important time period for the establishment of normal white adipose tissue in humans. This association suggests that exposure to obesogenic stimuli during adipose development may have detrimental effects on adipose function and metabolic homeostasis. In this study, we identify the period of development associated with puberty, postnatal days 18–34, as critical for the establishment of normal adipose mass in mice. Exposure of mice to high fat diet only during this time period results in metabolic dysfunction, increased leptin expression, and increased adipocyte size in adulthood in the absence of sustained increased fat mass or body weight. These findings indicate that exposure to obesogenic stimuli during critical developmental periods have prolonged effects on adipose tissue function that may contribute to the exacerbated metabolic dysfunctions associated with childhood obesity.

The production of new adipocytes requires the differentiation of adipocyte precursor (AP) cells residing within the adipose tissue stromal-vascular compartment. The objective was to obtain an immortalized primary adipogenic cell line derived from FACS isolated committed APs using the conditional expression of SV40 T antigen. Adipocyte precursors were isolated from white adipose tissue (WAT) using FACS to remove non-adipogenic cell populations from mice expressing a conditionally regulated SV40 T antigen. APs were maintained by continuous culture and induced to undergo adipogenic differentiation. Adipogenesis, determined by Oil Red O staining, was assessed with each passage and compared to wildtype controls. Adipogenic capability was rapidly lost with increased passage number in committed APs with concurrent reduction in cell proliferation and expression of essential late adipogenic genes, including Pparγ and C/ebpα. Thus, FACS purified committed APs have limited capability to undergo expansion and subsequent adipogenic differentiation in vitro even if they are immortalized with the SV40 T antigen.

The study of adipose tissue in vivo has been significantly advanced through the use of genetic mouse models. While the aP2-Cre(BI) and aP2-Cre(Salk) 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-Cre(BI) and aP2-Cre(Salk) lines lack specificity for adipocytes within adipose tissue, and that the aP2-Cre(BI) 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.

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 (P50% 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.

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 microenvironment niches coordinate adipose tissue growth and distribution.

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.

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.

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, Brandt's 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).

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.

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.

We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5-14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.