Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.
Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.
Adipose made a notable appearance in the media spotlight, due to obesity’s dramatic increase during the last few decades of the 20th century. Obesity currently affects more than one-third of adults and 17% of children and adolescents in the United States (US)1. Spanning all ethnic groups, statistical research surrounding the obesity epidemic has shown that non-Hispanic blacks have the highest age-adjusted rate of obesity (49.5%) compared with Mexican Americans (40.4%), all Hispanics (39.1%), and non-Hispanic whites (34.3%)2. The economic effect of obesity is also a growing concern for the healthcare system. In 2012, it was estimated that the annual medical cost of care for obesity in the US in 2005 was $190.2 billion, nearly 21% of the overall medical spending budget. Sadly, childhood obesity was estimated to be responsible for $14 billion in direct medical costs alone. Statistically, it was determined that the average medical cost of individuals with obesity was $2,741 higher a year than those without this morbidity3-5.
Obesity is a major risk factor for a variety of conditions such as: type 2 diabetes, dyslipidemia, cardiovascular disease, cancer, muscular skeletal disorders and chronic inflammation. Obesity is deeply tied to the pathogenesis of metabolic syndrome and other chronic diseases6-8. With such drastic and negative connections between obesity and other comorbidities, scientific research has focused attention to better understand the current epidemic and the diverse and pivotal roles played by adipose.
Historically, adipose tissue was considered inconsequential and was viewed merely as a simple filling tissue. Currently, adipose has been shown to play many essential roles in the body’s function in: metabolism, hormone regulation, inflammation, protection and insulation9. Adipose tissue is composed primarily of adipocytes but also contains pericytes, endothelial cells, monocytes, macrophages and pluripotent stem cells8. Adipose tissue is distributed throughout the body in distinct depots. The principal depots can be found subdermally, subcutaneously, intramuscularly, and viscerally10. Adipose depots have been shown to have depot specific metabolic profiles, which have shown a depot specific susceptibility to obesity and related disorders8.
Traditionally, adipose tissue has been classified into two major types: white adipose tissue (WAT) and brown adipose tissue (BAT); although recent literature indicates the presences of a third group christened brite or beige adipose11. Adipose tissue has been shown to have different colors, morphologies, metabolic functions, biochemical features and genetic patterns of expression10. Adipocytes in WAT have a single, large lipid droplet and variable amounts of mitochondria. WAT is dominantly found in subcutaneous and visceral localities of the body. WAT functions primarily as a site of energy storage and organ protection. Adipocytes in BAT have a multilocular morphology and abundant mitochondria. BAT is located primarily in the neck and large blood vessels of the thorax, as well as the scapulae12. BAT primarily functions in energy-expending behaviors that regulate thermogenesis7. Brite or beige adipose has been shown to share an analogous morphology and expression to BAT but has been found to be originated from white adipocytes11.
The described surgical method in this manuscript provides researchers with the capacity to analyze different effects that factors such as: environment, pharmaceuticals, and genetics, have on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Also, providing a way to identify and isolate different types of adipose tissue to allow for better understand of the biochemical relationships and differences between depots. This can aid in determining the relationship between location, function, and types of fat within the body. This described method accomplishes this by providing the means for gross visualization, gene expression analysis, protein expression analysis, histological examination, and isolation of primary cell lines for in vitro studies. Currently there are many articles that provide insight into the metabolic behavior of different adipose depots, as well as their anatomic locations; but do not provide an in-depth method on how to specifically localize, identify, and isolate these depots. This surgical method provides a precise technique that allows for isolation of multiple depots with a minimal quantity of dissection and contamination compared to others methods designed for the isolate of one or two depots13-14.
The goal of this protocol is to provide a precise method for the identification and isolation of different types of fat depots from multiple anatomic locations.
NOTE: All animal procedures were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati and in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH Publication No. 85-23, Revised 1996).
1. Euthanize and Sterilize the Mouse
2. Identification and Isolation of Three Different Adipose Depots
3. Isolation of Perivascular Adipose Tissue (PVAT)
Identification, and localization of inguinal subcutaneous adipose, interscapular brown adipose, visceral epididymal adipose (Figure 1), as well as the aortic arch perivascular adipose, thoracic aortic adipose, suprarenal aortic adipose and the infrarenal aortic adipose (Figure 2) was achieved successfully using the described surgical method. Histological examination and differentiation between BAT and WAT samples were positively evaluated using Hematoxylin and Eosin (H&E) staining (Figure 3). Analysis of RNA levels of adiponectin (AdipoQ), peroxisome proliferator-activated receptor gamma (PPAR-γ), cell death-inducing DFFA-like effector a (CIDEA), and other fat specific markers were measured for all the above isolated and excised depots (data not shown).
Primary cell lines of subcutaneous adipocytes and perivascular adipocytes were cultured and differentiated from preadipocytes to adipocytes successfully for microarray analysis. The cultured preadipocytes converted to adipocytes were confirmed with Oil Red O staining (Figure 4). Successful isolation, culture and differentiation of adipocytes was achieved for use in in vitro studies, and protein activity was successfully measured. Enzymatic activity of matrix metaloprotease-2 (MMP2) was measured in a treatment group compared to the control. The activity of MMP2 was measured in situ in a primary perivascular adipocyte line via zymography (Figure 5).
Figure 1. Anatomical locations of C57BL/6 male mouse adipose depots. (A) Interscapular brown adipose fat depot. (B) Inguinal subcutaneous adipose fat depot. (C) Visceral epididymal adipose fat depot. Please click here to view a larger version of this figure.
Figure 2. Anatomical locations of PVAT depots in a C57BL/6 male mouse. (A) Aortic arch perivascular adipose depot. (B) Thoracic aortic perivascular adipose depot. (C) Suprarenal aortic perivascular adipose depot. (D) Infrarenal aortic perivascular adipose depot. Please click here to view a larger version of this figure.
Figure 3. H&E staining of BAT and WAT adipose. (A) H&E staining of paraformaldehyde fixed, paraffin embedded C57BL/6 male mouse sample of WAT adipose at a 40X magnification. (B) H&E staining of paraformaldehyde fixed, paraffin embedded C57BL/6 male mouse sample of BAT at a 40X magnification. Please click here to view a larger version of this figure.
Figure 4. Oil Red O staining of cultured PVAT preadipocytes and adipocytes. (A) Oil Red O staining of cultured aortic perivascular preadipocytes at baseline of differentiation at 20X magnification with phase contrast. (B) Oil Red O staining of cultured aortic perivascular adipocytes after 5 days of differentiation at 20X magnification with phase contrast. Please click here to view a larger version of this figure.
Figure 5. Zymography of differentiated adipocytes, isolated from perivascular adipose tissue, demonstrated a decreased activity of MMP2 released after treatment compared to untreated (control) cells, *P < 0.01 compared to control.
Obesity can lead to a large host of morbidities and the full understanding of the role that adipose plays is not fully understood; therefore continued research in the field of adipose is necessary. Animal models, specifically murine models are ideal for initial research in the progression of diseases and testing of potential pharmaceutical treatments. In using these models, precise isolation and excision of adipose depots is an extremely important and necessary tool in the study of pathology of adipose affected diseases.
In current literature regarding adipose depots, there is a substantial quantity of literature regarding metabolic behavior and variation between adipose depots, as well as their anatomic locations. However, there are few that provide an in-depth method on how to specifically localize, identify, and isolate these depots. Based on the review of current isolation methods of adipose, there is a small subset of protocols that provide methodology on how to isolate one or two depots at a time. However, a precise technique that allows for isolation of multiple depots with a minimal quantity of dissection and contamination, as well as addresses various methods of studying the samples collected is distinctive to this protocol13-14.
Within this methodology, there are several steps that are vitally important to the isolation and purity of the sample. Cleaning tools, gloves and surfaces often to remove hair and contaminants is an imperative step to avoiding depot contamination. When cutting the skin transversely around the circumference of the mouse to expose the peritoneum for degloving, it is vital to avoid cutting too deeply. Cutting the peritoneum will make degloving very difficult and will raise the potential for contamination of the sample. When excising the SQ adipose depots it is vital to identify the triangular boundaries of the depot before excising any of the adipose. Also, careful cuts should be made to avoid muscle, and adjacent vessels, glands and adipose. This will prevent contamination of the sample from alternate adipose, glandular tissue, muscle or blood.
The major limitation to isolation and excision of adipose depots, in this method and other comparable methods can be found in the defining of the boundaries of certain depots. Due to the poorly defined borders in depots, such as the subcutaneous depots, isolation lacking a small amount of contamination from neighboring adipose can be challenging. Another limitation can be found in ensuring enough tissue is collected for supplemental experimentation in vascular associated depots. This limitation sometimes requires pooling of samples, although this is dependent on the site of isolation and the diet associated with the animal.
After the adipose depots are isolated, they can be utilized for a variety of assays. The adipose can be used for molecular studies such as protein expression, enzyme activity and gene expression analysis. Additionally, one can isolate adipocytes for primary cell line in vitro studies. Immortalized cells lines can also be used for in vitro studies, however immortal cells are not as credible as primary isolated cell lines. Finally, the adipose can be fixed or frozen in OCT for histological examination to identify leukocyte infiltration, protein localization, as well as characterization of adipocyte morphology.
The authors have nothing to disclose.
The authors have no acknowledgements.
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
isoflurane | Med-Vet International | #RXISO-250 | |
70% Ethanol | Fisher | 07-678-001 | |
DMEF-12 | Sigma Aldrich | D-6421 | Warm in waterbath before putting on tissue |
2 mL microcentrifuge tubes | Midsci | MCT-200-C-S | |
Phosphate buffered saline | Sigma Aldrich | P5368-10PAK |