Emily C. Graff, DVM, Dipl. ACVP - The ACVP/STP Coalition for

Identification and Characterization of Adipose
Tissue Macrophage Populations in Cats
During Development of Obesity
Emily C. Graff, DVM, Dipl. ACVP (clinical pathology)
U.S. Obesity Trends
Obesity = Adipose Tissue Dysregulation
Adipose tissue- cytology
Radin et al. Vet Clin Pathol 2009
Adipose tissue remodeling =
altered adipokine regulation and chronic low grade inflammation
Macrophages (MΦ) & Crown-Like Structures
Weisberg et al. J Clin Invest. 2003
Cinti et al Journal of Lipid Research, 2005
“Adipose tissue macrophage numbers
increase in obesity and participate in
inflammatory pathways that are activated
in adipose tissues of obese individuals.”
Weisberg et al. JCI 112 (2003)
Macrophage Polarity
Obesity-Associated Adipose Tissue Inflammation
Ouchi et al Nature Reviews immunology. Feb. 2011
M2 > M1
M1 > M2
Companion Animal Obesity
The Cat as a Model for Human Obesity,
Nutrition and Diabetes
• Similarities:
Nutritional disorder
Risk factor for T2DM
• Amyloid deposition
• β-cell loss
– GLUT-4 expression
O’brien Molecular and Cellular Endocrinology 2002
• Differences:
– Obligate carnivores
– Do not develop atherosclerosis or hypertension
Do Obese Cats Develop a Systemic
Inflammatory Response? – Possibly No!
• Tvarijonaviciute et al. Domestic Animal Endocrinology 2012
– Study: Weight-loss in 37 client owned cats
– Cats: No change in serum amyloid A or serum haptaglobin
– People: Weight gain is associated with increased C-reactive
• Hoenig et al. Obesity 2013
– Study: Longitudinal feline obesity model with 100% weight gain
over 1 year
– Cats: Significant alterations in serum adipokines, but no significant
increase in serum concentrations of IL-1, IL-6 or TNF-α
– People: Weight gain is associated with marked adipokine
dysregulation and significant increases in inflammatory cytokines
Do Obese Cats Develop a Systemic
Inflammatory Response? – Possibly Yes!
• Miller et al. Journal of Nutrition 1998
– Tumor Necrosis Factor-α Levels in Adipose Tissue of Lean
and Obese Cats
• Van de Velde et al. British Journal of Nutrition 2013
– The cat as a model for human obesity: insights into depotspecific inflammation associated with feline obesity
– Increased adipocyte cell size, altered adipokine expression
and increased pro-inflammtory cytokines in tissue
Identification of Depot Specific Inflammation
Associated with Feline Obesity
Gap in Knowledge
• What is actually contributing to the adipose
tissue inflammation?
– Adipocytes
– Adipose tissue macrophages
• What, if any, are the systemic inflammatory
patterns associated with obesity in cats?
– Biomarkers of inflammation (cytokines)
– Cellular markers of inflammation
Initial Objective – Preliminary Data
Identify adipose tissue macrophages (ATM) and
crown-like structures (CLS) in feline adipose
Two methods:
1. Immunohistochemistry of formalin fixed
adipose tissue
2. Isolation of the stromal vascular fraction
(SVF) followed by flow cytometric analysis
Subcutaneous Adipose Tissue
H&E 200X
CD18 200X
Lysozyme 200X
Alpha-1 Antitrypsin
Flow Cytometric Evaluation of SVF
• 4 g adipose tissue digested in collagenase
• Isolation of SVF via filtration and differential
• Accuri C6 flow cytometer and Beckman Coulter
MoFlo XDP for FACS to identify and sort cell
• PI stain to determine cell viability
• Prepared cytospin preparation of the cells for
cytologic identification
Flow Cytometry & Cytospin Results
Preliminary Data Results
• Immunohistochemistry:
– Lean cats do contain rare adipose tissue
• Isolation of SVF and flow cytometry:
– Concentration of adipose tissue macrophages
– Viable macrophages can be isolated from feline
adipose tissue
Identify and characterize feline macrophage
populations in adipose tissue during the
development of obesity
Central hypothesis: During the development of obesity
cats will demonstrate…
increased numbers of macrophages
increased proportion of M1 type macrophages
increased adipose tissue inflammation
adipokine dysregulation
systemic inflammation
Specific Aims
• Specific Aim 1:
– Identify and enumerate macrophages in lean and
obese feline adipose tissue
• Specific Aim 2:
– Confirm phenotype of the populations based on
surface receptor expression and cytokine mRNA
• Specific Aim 3:
– Determine circulating feline adipokine profiles,
specifically adiponectin, leptin, serum amyloid-A, TNFα and IL-6 in cats as changes in adipose inflammation
Feline Obesity Model
End of Study
• Each cat will serve as their own control
• Cats will be fed same diet throughout the study
• CT scan to evaluate adipose tissue deposition (baseline and end of
• Samples collected
– Adipose tissue – Visceral and subcutaneous (baseline and end of study)
– Serum ( at each 10% increase in weight gain)
cat images from: http://www.healthyweightpet.com/uk/cat
Sample Evaluation
• Perform IHC evaluation
– Adipose tissue macrophages
– Crown-like-structures
• Isolate SVF & sort cell populations
– Identify and quantify cell populations
• Evaluate systemic markers of Inflammation
– Adipokines
– Inflammatory cytokines
– Acute phase proteins
• Evaluate insulin sensitivity
Expand Evaluation of SVF
• Evaluate M1 and M2 macrophage populations
– Apply specific macrophage surface markers
– RT-PCR for M1 and M2 cytokine profiles
Classical - M1
Alternative - M2
Surface marker
TNFα, IL-6, IL-12
IL-10, IL-1
Induced by
LPS (th1 cytokines) IL-10 (th2 cytokines)
Cat Colony - Growth and Weight
Neutering and recovery
Expected Outcomes
As obesity develops we expect to find:
– Increased numbers of adipose macrophages
– Increased percentage of M1 type macrophages
– Increased adipose tissue inflammation
– Altered systemic and tissue adipokine regulation
– Increased circulating inflammatory markers
• Obesity and obesity-induced peripheral
insulin resistance are epidemic in both people
and companion animals
• Adipokine dysregulation and chronic
inflammation are features of obesity-induced
peripheral insulin resistance in humans
• Cats are naturally occurring model of human
obesity and T2DM
• Further work is needed to evaluate feline
obesity- associated inflammation
Thank you
Dr. Robert L. Judd
Dr. Desiree Wanders
Olga Norris
Ally Emmert
Nathan Gray
Dr. Elizabeth G. Welles
Dr. Beth Spangler
Dr. Robert Kemppainen
Dr. Ellen Behrend
Dr. Ray Dillon
Dr. D.M. Tillson
Sharron Barney
Dr. R.C. Bird
Ali Bird
Dr. Pat Rynders
Dr. Bettina Schemera
Dr. Bob Cole
Dr. Emily Graff was partially supported by the Charles and Sharron Capen
Fellowship in Veterinary Pathology organized by the American College of
Veterinary Pathologists and Society of Toxicologic Pathology Coalition for
Veterinary Pathology Fellows.

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