Expression of ceramide-metabolising enzymes in subcutaneous and intra-abdominal human adipose tissue

Abstract

Background: Inflammation and increased ceramide concentrations characterise adipose tissue of obese women with high liver fat content compared to equally obese women with normal liver fat content. The present study characterises enzymes involved in ceramide metabolism in subcutaneous and intra-abdominal adipose tissue.

Methods: Pathways leading to increased ceramide concentrations in inflamed versus non-inflamed adipose tissue were investigated by quantifying expression levels of key enzymes involved in ceramide metabolism. Sphingomyelinases (sphingomyelin phosphodiesterases SMPD1-3) were investigated further using immunohistochemistry to establish their location within adipose tissue, and their mRNA expression levels were determined in subcutaneous and intra-abdominal adipose tissue from both non-obese and obese subject.

Results: Gene expression levels of sphingomyelinases, enzymes that hydrolyse sphingomyelin to ceramide, rather than enzymes involved in de novo ceramide synthesis, were higher in inflamed compared to non-inflamed adipose tissue of obese women (with high and normal liver fat contents respectively). Sphingomyelinases were localised to both macrophages and adipocytes, but also to blood vessels and to extracellular regions surrounding vessels within adipose tissue. Expression levels of SMPD3 mRNA correlated significantly with concentrations of different ceramides and sphingomyelins. In both non-obese and obese subjects SMPD3 mRNA levels were higher in the more inflamed intra-abdominal compared to the subcutaneous adipose tissue depot.

Conclusions: Generation of ceramides within adipose tissue as a result of sphingomyelinase action may contribute to inflammation in human adipose tissue.

Figure 1

Schematic diagram depicting enzymatic reactions involved in the synthesis and degradation of ceramide and sphingomyelin.

Figure 2

Localisation of sphingomyelinases in subcutaneous adipose tissue. Representative pictures of immunohistochemical staining of macrophages and multinuclear giant cells (aggregated macrophages) (A-D) and a small blood vessel (E-J) within subcutaneous adipose tissue from an obese woman. Brown coloration indicates macrophage-specific CD68 (A and E), SMPD1 (B and F), SMPD2 (C and G), SMPD3 (D and H) and PECAM-1 (I). All sections were counterstained with haematoxylin (coloured blue). Collagen staining (J) was visualized with polarized light to reveal mature, extracellular collagen (coloured red).

Figure 3

Gene expression in subcutaneous and intra-abdominal adipose tissue from non-obese and obese individuals. (A) Relative gene expression levels of SMPD1-3, CD68, CCL2, CCL3, TNFα, IL6 and adiponectin in subcutaneous (black bars) and intra-abdominal (white bars) adipose tissue from 23 non-obese individuals. (B) Relative gene expression levels of SMPD1-3 in subcutaneous (black bars) and intra-abdominal (white bars) adipose tissue from 8 obese patients. Expression is in arbitrary units normalized to housekeeping genes RPLP0 and TBP, and set to 1 for the subcutaneous depot. * P < 0.05, ** P <0.01, *** P < 0.001 compared to subcutaneous adipose tissue.

Figure 4

Localisation of sphingomyelinase SMPD3 and ceramidase ASAH1 in subcutaneous and intra-abdominal adipose tissue. Representative pictures of immunohistochemical staining of a vessel within subcutaneous (A, B and E) and intra-abdominal (C and D) adipose tissue from a non-obese individual. Positive staining for SMPD3 (A and C) and ASAH1 (E) is shown as brown coloration. Negative staining is shown in panels B and D. All sections were counterstained with haematoxylin (coloured blue).

Figure 5

Localisation of apo B in subcutaneous and intra-abdominal adipose tissue. Representative pictures of immunohistochemical staining of apo B (A, C) and CD68 (B) in human adipose tissue. (A) and (B): Serial sections of subcutaneous adipose tissue from an obese woman stained for apo B and CD68 respectively. (C): Intra abdominal adipose tissue from a non-obese individual stained for apo B. Positive staining for apo B (A and C) and CD68 (B) is shown as brown coloration. All sections were counterstained with haematoxylin (coloured blue).