Adipose tissue has been well documented as an endocrine organ in the last two decades [42
]. The recognition that ASP (aka C3adesArg) is produced by adipose tissue, and that the three precursor proteins C3, factor B and adipsin, all increase with differentiation of adipocytes [43
] was one of the first demonstrations that hormones with both paracrine and endocrine functions were produced within the adipose milieu. The primary function of ASP in adipocytes appears to be promotion of lipid storage, and this is achieved through increases in fatty acid esterification and glucose uptake [44
A number of human studies have demonstrated positive associations between ASP and various metabolic disturbances (diabetes, metabolic syndrome, cardiovascular disease, nephrotic syndrome, polycystic ovary syndrome) with or without the presence of obesity. Previous studies have demonstrated that circulating plasma ASP is associated with TG and fatty acid levels [15
], with a strong correlation between fasting ASP and postprandial TG clearance even in non-obese individuals [22
]. The demonstration of direct adipose tissue production of ASP postprandially in human studies [19
], coupled to evidence that dietary TG in the form of chylomicrons directly stimulate C3 and ASP production in cultured human adipocytes [45
] provides a strong link between plasma ASP and TG. Consequently, the aim of the present study was to evaluate microarray expression in subcutaneous vs. omental adipose tissue in relation to plasma ASP and TG (lipemic index), and genes directly related to ASP production, storage lipid metabolism and inflammation.
However, there are several limitations to this study. The samples were derived from morbidly obese subjects, limiting interpretations to that population. Further, although there were often large relative differences between the two groups (LAT vs HAT), the limited number of samples restricted the statistical power. There was also no functional analysis of the adipose tissue. On the other hand, the strengths of the study are that no samples were pooled and microarray analysis was conducted individually on all 21 samples. It should be noted that intact adipose tissue was specifically chosen for the analysis. This tissue represents a pool of adipocytes as well as preadipocytes, macrophages, and endothelial cells. Direct analysis of the tissue prevented the introduction of artefactual changes resulting from processing and separating the various cell populations. Hence, this can be viewed as both a strength and a weakness.
Interestingly, while the design of the study allowed for a comprehensive comparison of gene expression between SC and OM adipose tissue, we discovered that the significant differences were found primarily in the SC tissue. SAM analysis determined that OM tissue did not have significant differences between the LAT and HAT groups. Targeted analysis of specific biological pathways allowed us to compare genes from many different gene families simultaneously. Differential results between LAT and HAT in the SC tissue included genes involved in the alternative complement system, lipid synthesis, lipolysis, oxidation, fatty acid binding proteins, markers of differentiation, pro-inflammatory, anti-inflammatory, macrophage recruitment, and matrix remodelling genes. This contrasts markedly with a previous study comparing insulin resistant obese with insulin sensitive obese where differences were primarily noted in OM tissue, with no differences in SC tissue [12
]. This is a particularly interesting point. While differences in omental adipose tissue in relationship to insulin resistance have been noted (in microarray studies and others), our present study specifically identified differences in subcutaneous adipose tissue with respect to ASP related metabolism. While differences in subcutaneous adipose tissue have been identified using other biochemical parameters (such as differences in triglyceride synthesis in subcutaneous adipose tissue in non-obese vs obese subjects) this is the first to identify this based on ASP metabolism. We speculate (as a hypothesis to be tested) that this may indicate a compensatory mechanism, whereby genes related to ASP are concomitantly increased with genes related to lipid storage, however this remains to be tested in future studies.
In contrast to the demonstrated changes in the gene families described above, there were no significant differences in housekeeping genes, insulin signalling genes and non-related complement genes between these two groups (LAT vs HAT). We demonstrated that four genes commonly reported as housekeeping genes [30
], B2M, GUSB, PPIA, and TFRC, were not significantly different between the LAT and HAT groups nor did they correlate with age or BMI. Further, not only were plasma glucose and insulin comparable between the 2 groups, the expression of genes related to insulin resistance and insulin signalling such as insulin receptor InsR, IRS-1, IRS-4, IGF1, and IGF2 were comparably expressed between LAT and HAT groups, and did not correlate with age or BMI. Further, the expression of other complement genes (C5) and receptors (C3aR, C5aR) that are not involved in ASP production or signalling were not different between groups (LAT and HAT). This suggests that there is selective regulation of metabolic genes directly related to the role of ASP, independent of insulin or other immune related genes.
On the other hand, there was a strong association between increased plasma ASP and TG (lipemic index) and expression of the genes related directly to ASP production and function. Thus complement C3, factor B and adipsin were generally increased in the microarray in HAT vs LAT groups. Further, C5L2, the receptor for ASP, was assessed by real-time PCR, and expression was also increased in HAT vs LAT. C5L2, a G protein coupled receptor, has been shown to be present on human adipocytes and preadipocytes [17
], but this is the first quantitative evaluation of C5L2 reported in human adipose tissue. While C5L2 is increased in the HAT group, the expression of C5L2 in the LAT group is very similar to the non-obese control group in both SC and OM tissue. Further, in the present study there was a significant correlation between C5L2 expression in SC and OM adipose tissue. These results are consistent with increased binding of ASP to adipose plasma membranes in obese subjects vs non-obese subjects, although the C5L2 receptor was as yet unrecognized at that time, and no plasma data was available [47
]. We have also previously demonstrated an association between SC and OM adipose tissue for radiolabelled ASP binding to plasma membranes, across a range of BMI from obese and non-obese subjects. On the other hand, in our previous study, ASP binding and affinity was greater in SC vs OM adipose tissue, while in the present study, C5L2 mRNA expression was not different in SC vs OM, which could reflect a further level of regulation at the plasma membrane.
This is the first study evaluating expression of ASP related genes, including the receptor C5L2, in the context of plasma ASP. The few studies that have examined complement C3 gene expression in adipose tissue have demonstrated associations with BMI, adipose tissue site, insulin sensitivity, age and postprandial lipemia [48
]. Only one study examined the levels of plasma ASP in relationship to adipose tissue gene expression and demonstrated significant associations between C3 mRNA, plasma ASP and both insulin sensitivity and postprandial TG [51
This raises the question whether chronically increased plasma ASP represents an ASP resistant state (analogous to increased plasma insulin reflecting insulin resistance), or whether the increased ASP is compensatory. Hypothetically, the presence of increased plasma ASP, increased expression of C3, factor B and adipsin in the microarray and increased C5L2 based on real-time PCR would suggest a compensatory mechanism, indicative of a more effective response, and interpretation of the results are evaluated in that context below.
There was a striking correlation between the three genes most relevant to ASP production: C3, Factor B, and adipsin (aka Factor D), constituting an ASP triad. Other complement genes (C3aR and C5aR) that are neither related to ASP production nor signalling do not correlate with the "ASP triad." This selectivity within the alternative complement pathway and the correlation with many metabolic genes points to the physiological relevance. A general trend observed was that the HAT group has up-regulated genes for lipid TG storage. Along with the high plasma ASP and high C5L2 receptor, there are multiple genes including intracellular lipid storage, lipoprotein lipase, and fatty acid trafficking genes that are increased in the HAT group. The simultaneous increase in both lipid storage and lipolytic genes suggests an overall increase in substrate cycling and fatty acid flux [2
]. Concurrently, de novo TG synthesis and oxidative genes were down-regulated. Taken together, the cells appear to be responding to an increased plasma TG level in the HAT group and are increasing their uptake and storage of TG. Many of the increased storage genes correlated with the "ASP triad" of genes including DB1 and LPL. Among the oxidative genes, ACC correlates negatively with the ASP triad.
Changes in adipose tissue function implicate alterations in differentiation genes, remodelling genes, and inflammatory complications [40
]. The HAT group is characterized by increased in differentiation factors CEBPα, β, and CEBPδ as well as PPAR γ, consistent with the increased TG storage genes. Interestingly, we have previously demonstrated that ASP stimulates adipogenesis in cultured 3T3-L1 and 3T3-F442A cells, reflected by early increases in CEBP, followed later by increases in PPARγ, DGAT-1, adipsin and TG accumulation [57
The HAT group is also characterized by increased anti-inflammatory gene TGFβ1 and inhibitors of metalloproteinases TIMP1, TIMP3 and TIMP4 and decreased metalloproteinase MMP20 and MMP10. On the other hand, there were few increases in pro-inflammatory genes, although pro-inflammatory and cell remodelling factors such as PIG7, CCR2 and MMP2 were increased. An interesting link between the role of the alternative complement pathway factors ASP and C5L2 and immune and adipose have been supported by active demonstration of adipose tissue macrophage infiltration, apoptosis and tissue remodelling in obesity, and the differences demonstrated between HAT and LAT.