Dataset Information

Comparison of Methods for Analyzing Human Adipose Tissue Macrophage Content.

ABSTRACT:

Objective

The relationship between inflammation, obesity, and adverse metabolic conditions is associated with adipose tissue macrophages (ATM). This study compared the measurements of human ATM using flow cytometry, immunohistochemistry (IHC), and real-time polymerase chain reaction (RT-PCR) of ATM markers.

Methods

A new software program (AMCounter) was evaluated to help measure ATM using IHC, and this was compared to flow cytometry and RT-PCR.

Results

IHC had good intraindividual reproducibility for total (CD68), proinflammatory (CD14), and anti-inflammatory (CD206) ATM. The AMCounter improved interreader agreement and was more time efficient. Flow cytometry had acceptable intraindividual reproducibility for the percentage of CD68⁺ cells that were CD14⁺ or CD206⁺ , but not for ATMs per gram of tissue. ATMs per gram of tissue was much greater using IHC than flow cytometry. The flow cytometry and IHC measures of ATM from the same biopsies were not correlated. There were statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14, and CD206 ATMs per 100 adipocytes. Also of interest were statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14, and adipose flow cytometry measures of CD68⁺ , CD68⁺ /CD14⁺ , and CD68⁺ /CD206⁺ ATMs per gram of tissue.

Conclusions

The AMCounter software helps provide reproducible and efficient measures of IHC ATMs. Flow cytometry, IHC, and RT-PCR measures of adipose inflammation provide somewhat different information.

SUBMITTER: Morgan-Bathke M

PROVIDER: S-EPMC5705319 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Comparison of Methods for Analyzing Human Adipose Tissue Macrophage Content.

Morgan-Bathke Maria M Harteneck Debra D Jaeger Philippa P Sondergaard Esben E Karwoski Ron R Espinosa De Ycaza Ana A Carranza-Leon B Gisella BG Faubion William A WA Oliveira Andre M AM Jensen Michael D MD

Obesity (Silver Spring, Md.) 20171006 12

<h4>Objective</h4>The relationship between inflammation, obesity, and adverse metabolic conditions is associated with adipose tissue macrophages (ATM). This study compared the measurements of human ATM using flow cytometry, immunohistochemistry (IHC), and real-time polymerase chain reaction (RT-PCR) of ATM markers.<h4>Methods</h4>A new software program (AMCounter) was evaluated to help measure ATM using IHC, and this was compared to flow cytometry and RT-PCR.<h4>Results</h4>IHC had good intraind ...[more]

PMID: 28985040

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Project description:Insulin has anti-inflammatory effects in short-term experiments. However, the effects of chronic insulin administration on inflammation are unknown. We hypothesised that chronic insulin administration would beneficially alter adipose tissue inflammation and several circulating inflammatory markers.We administered two forms of long-acting insulin, insulin glargine (A21Gly,B31Arg,B32Arg human insulin) and insulin detemir (B29Lys[?-tetradecanoyl],desB30 human insulin), to LDL-receptor-deficient mice. After 8 weeks on a diet that causes obesity, hyperglycaemia, adipose tissue macrophage accumulation and atherosclerosis, the mice received subcutaneous glargine, detemir or NaCl (control) for 12 weeks. Serum amyloid A (SAA) and serum amyloid P (SAP), metabolic variables, adipose tissue macrophages and aortic atherosclerosis were evaluated.Weight gain was equivalent in all groups. The glycated haemoglobin level fell equivalently in both insulin-treated groups. Plasma cholesterol and triacylglycerol levels, and hepatic triacylglycerol level significantly improved in the glargine compared with the detemir or control groups. Levels of mRNA expression for monocyte chemotactic protein-1 and F4/80, a macrophage marker, in adipose tissue were decreased only in the glargine group (p?<?0.05). Visceral adipose tissue macrophage content decreased in both insulin groups (p?<?0.05), whereas atherosclerosis decreased only in the glargine group. Circulating SAA and SAP did not decrease in either insulin-treated group, but IL-6 levels fell in the glargine-treated mice.While chronic insulin administration did not decrease SAA and SAP, administration of glargine but not detemir insulin improved dyslipidaemia, IL-6 levels and atherosclerosis, and both insulins reduced macrophage accumulation in visceral adipose tissue. Thus, chronic insulin therapy has beneficial tissue effects independent of circulating inflammatory markers in this murine model of diet-induced obesity and diabetes.

Dataset Information

Comparison of Methods for Analyzing Human Adipose Tissue Macrophage Content.

Objective

Methods

Results

Conclusions

Publications

Comparison of Methods for Analyzing Human Adipose Tissue Macrophage Content.

Similar Datasets

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