We investigated sphingolipid metabolism in human adipose tissue to identify pathways underlying increased ceramide concentrations in inflamed adipose tissue . Our data suggest that hydrolysis of sphingomyelin to ceramide by sphingomyelinases could explain, at least partly, this increase. Gene expression levels of SMPD3 correlate significantly with concentrations of various ceramides and sphingomyelins in subcutaneous adipose tissue, and are higher in the relatively more inflamed intra-abdominal compared to the subcutaneous depot in both obese and non-obese subjects. Sphingomyelinases are expressed by both adipocytes and macrophages in adipose tissue, but their expression is strongest in and around blood vessels. Our findings implicate a role for sphingomyelinase-mediated generation of ceramide in adipose tissue inflammation.
When comparing inflamed ceramide-rich and relatively less inflamed ceramide-poor subcutaneous adipose tissue of obese women, we report here for the first time that there were no differences in mRNA levels of genes involved in de novo ceramide synthesis. However, expression of sphingomyelinases SMPD1 and SMPD3 was significantly higher, while that of SMPD2 tended to be higher in the inflamed adipose tissue group (as reported ). Since sphingomyelinases catalyse the conversion of sphingomyelins to ceramide, this pathway rather than de novo ceramide synthesis, may underlie the increased ceramide content of the inflamed adipose tissue of these women. Sphingomyelinase activity is increased by oxidative stress both in vitro and in vivo and sphingomyelinase expression in adipose tissue increases in response to a high fat diet in mouse models [15, 27]. Since ceramides stimulate synthesis of pro-inflammatory cytokines by both adipocytes and macrophages [27, 28], increased sphingomyelinase activity in adipose tissue could exacerbate the inflammatory milieu and enhance recruitment of macrophages. Therefore investigation of sphingomyelinases in human adipose tissue in relation to inflammation and macrophage accumulation is motivated. To date the only report of sphingomyelinases in human adipose tissue found reduced acid sphingomyelinase, but unchanged neutral sphingomyelinase activity in obese compared to lean patients .
Although we observed increased sphingomyelinase mRNA expression in inflamed adipose tissue, sphingomyelinases did not localise only to inflammatory cells (determined by immunohistochemistry). Staining for SMPD1, -2 and −3 was seen in macrophages and also in adipocytes, but the strongest staining was seen in and around blood vessels, the latter being reminiscent of the secretory form of SMPD1 localising to the subendothelial matrix of atherosclerotic lesions . Additionally, the ceramide-metabolising enzyme ASAH1 was found in the vasculature, indicating that blood vessels are important sites for ceramide metabolism within adipose tissue. Indeed, immunohistochemical analysis revealed staining for apo B in areas containing inflammatory cells (positive for CD68) and within blood vessels, indicating access of adipose tissue sphingomyelinases to sphingomyelins within lipoproteins. Previously we found that not only ceramides, but also sphingomyelins were increased in adipose tissue of obese women with more inflamed adipose tissue. The increase in sphingomyelins did not appear to be accounted for by increased local synthesis (as discussed above), but might be explained by an increased delivery of sphingomyelin-rich lipoproteins produced by fatty liver , since these women also had increased hepatic fat content, but quantification of lipoprotein delivery to or retention within adipose tissue was not possible in this study. To pursue this idea we investigated the relationship between hepatic triacylglycerol accumulation and adipose tissue ceramide metabolism and inflammation in non-obese individuals. Inflammation in both subcutaneous and intra-abdominal adipose tissue (as assessed by RNA levels of the macrophage marker CD68) was positively related to the expression of genes in the liver reflecting triacylglycerol accumulation, but no such relationships were found for sphingomyelinase expression (SMPD1, -2 or −3) in either adipose tissue depot. This suggests that even in non-obese individuals, the number of macrophages within adipose tissue is linked to hepatic triacylglycerol metabolism. However, sphingomyelinases do not appear to be involved in this relationship in these subjects. Unfortunately no measurements of either adipose tissue ceramide concentrations or liver fat content were available, so no conclusions can be drawn as to the relationship between adipose tissue macrophage accumulation, ceramide concentration and hepatic triacylglycerol content, but our data suggest that sphingomyelinase-mediated generation of ceramide in adipose tissue does not play a major role in this context in non-obese subjects who are unlikely to have fatty livers.
Our data highlighted the potential importance of SMPD3 within adipose tissue in relation to ceramide generation and inflammation for two reasons. Firstly, mRNA levels of SMPD3 correlated significantly with ceramide and sphingomyelin concentrations within adipose tissue of obese women. Secondly, a relatively more inflamed adipose tissue depot, namely intra-abdominal fat, expressed SMPD3 mRNA at significantly greater levels than relatively less inflamed subcutaneous adipose tissue in both non-obese and obese subjects. It is possible that increased SMPD3 activity contributes to the greater ceramide concentrations in intra-abdominal compared to subcutaneous adipose tissue . Adipose tissue hypoxia is proposed to be a major underlying cause for insulin resistance and other disorders associated with obesity, promoting macrophage infiltration and angiogenesis . The location of sphingomyelinases to blood vessels within adipose tissue might indicate their involvement in angiogenesis. The increased expression of markers of hypoxia and endothelial activation, but no differences in markers of either angiogenesis or endothelial cell number in adipose tissue of women with inflamed compared to less inflamed adipose tissue, indicates the existence of hypoxia and an activated endothelium without an apparent decrease in vessel density or onset of angiogenesis. It is possible that the increased degree of inflammation is related to an inability of hypoxia and increased ceramide concentrations to induce angiogenesis within adipose tissue. Since particular importance was assigned to SMPD3 in hypoxic vasoconstriction in the lung , SMPD3 may play a role linking hypoxia, ceramide generation and inflammation within adipose tissue, however, this remains to be shown.
The observation that concentrations of some ceramide and sphingomyelin species correlated positively with gene expression levels of SPHK1 (sphingosine kinase) in adipose tissue in the cohort of obese women is also of interest. The product of SPHK1 action is sphingosine-1-phosphate (concentrations of which were not quantified in the present study), a sphingolipid that appears to have the opposite actions to that of ceramide, namely promoting cell survival and proliferation . Indeed, the balance between concentrations of ceramide and sphingosine-1-phosphate is proposed to be an important mechanism controlling cell fate . One interpretation of our data could be that the higher expression levels of SPHK1 in the more inflamed adipose tissue of the women with fatty livers, as compared to the less inflamed adipose tissue of women with normal liver fat content, and the correlations between SPHK1 expression and ceramide/sphingomyelin concentrations in the cohort as a whole reflect a protective mechanism to counteract the potentially detrimental consequences of ceramide accumulation, but this is speculative and future studies are needed to investigate this.
Strengths of our study include the investigation of human adipose tissue biopsies from three independent patient groups and two different depots, and the availability of measures of hepatic fat content in the obese women, and hepatic gene expression data in the non-obese subjects enabling us to investigate relationships between liver and adipose tissue. A limitation is that comparisons between the different patient groups cannot be made since the groups were not anlaysed at the same time, thus only within group comparisons can be made. Another limitation is the availability of only mRNA quantification of sphingomyelinase expression rather than protein concentration or enzyme activity. Nonetheless, protein expression of sphingomyelinase was confirmed and its location in adipose tissue established by immunohistochemistry. The small size of the biopsies obtained precluded more extensive analysis. Analysis of only adipocytes was not performed since we aimed to explore the expression of ceramide-metabolising enzymes in adipose tissue as a whole (including inflammatory cells, connective tissue, blood vessels etc.) rather than exclusively in adipocytes. However, immunohistochemical analysis provided information as to the cellular location of certain proteins.