Interpretation
Our findings are consistent with some17-19 but not all20,21 previous studies in which C3 and C3a(desArg) levels were found to be increased in women with PCOS compared to controls. C3a(desArg), also known as acylation-stimulating protein (ASP), increases energy storage through a number of actions, including enhanced glucose uptake, reduced lipolysis and increased triglyceride clearance27. Consistent with these observations, C3a(desArg) and C3 levels are increased in subjects with obesity and type 2 diabetes28,29, whilst C3 knockout mice display reduced body weight and fat mass27. Longitudinal human data have additionally shown that changes in C3 levels are positively associated with increase in BMI30, incident obesity31 and metabolic syndrome32, implying a potential causal role for complement dysregulation in human obesity.
C3a(desArg) is synthesised in a two-step process involving C3, factor B and factor D; cleavage of the parent molecule C3 generates C3a, which subsequently undergoes desargination of the carboxyl terminus to generate C3a(desArg). In contrast to C3, fasting levels of factor B and factor D were only elevated in our PCOS subjects in unadjusted analyses, suggesting that obesity was the main factor accounting for these increases. This accords with similar findings from Daan et al 33, who found no difference in factor D levels in either hyperandrogenic or normoandrogenic PCOS subjects compared with unaffected controls, and with other studies showing elevated factor B and D concentrations in obesity28,34,35. In contrast, Gursoy Calan et al found that factor D levels were elevated in women with PCOS compared with age- and BMI-matched controls, and that circulating concentrations correlated with both BMI and HOMA-IR16.
Properdin and factor H are important regulators of alternative pathway activation: properdin acts as a stabilizer of C3 convertase leading to prolonged complement activation, whilst factor H functions as an inhibitor by dissociating C3 convertase; elevations in both reflect alternative pathway dysregulation. Whilst fasting properdin concentrations were not different in our unadjusted analyses, properdin levels were marginally lower after adjustment for BMI and age. In contrast, fasting factor H concentrations were higher in PCOS subjects only before adjustment for the group differences in BMI. This observation is consistent with previous cross-sectional and longitudinal studies which have shown positive associations between factor H concentrations and adiposity30,36. We also found elevated TCC concentrations in women with PCOS, indicating increased terminal pathway activation. Increased TCC levels are likely caused by alternative pathway dysregulation since inhibition of the alternative pathway reduces TCC concentrations by >80%37,38.
In view of the differences we observed in fasting complement protein levels of both activation and terminal pathways, we subsequently sought to compare the kinetics of complement protein generation in response to a fatty meal. We were keen to focus on the impact of lipaemia since postprandial triglycerides predict cardiovascular risk better than fasting levels39, not least because humans exist in a postprandial state for most of the day. Lipaemic challenge resulted in a substantial increase in triglyceride levels whilst C3, C4 and TCC increased to a similar extent in both groups. In contrast, whilst factor H levels fell in controls, an acute rise was observed in PCOS subjects, notably in the presence of co-existing obesity. Several complement components have been implicated in lipid metabolism. Chylomicrons, which transport dietary lipids, can spontaneously activate the alternative pathway and become coated in C3 opsonic fragments11. C3 fragments are also present in other lipoproteins, including Apolipoprotein-A1 (which associates with chylomicrons), and in HDL40. Factor H regulates chylomicron-induced C3 activation during lipid storage11 and alternative pathway complement activation on HDL41. Obesity appeared to be a significant driver for the elevated factor H levels in our PCOS subjects in both the fasting and post-prandial state. Insulin resistance also correlated significantly with postprandial factor H area under the curve. Since adiposity and insulin resistance are intimately linked, it is difficult to establish which of these parameters is the dominant driver. However, it is interesting to note that not only does insulin resistance correlate with omental fat factor H expression36 but also that addition of factor H to freshly isolated pancreatic islet cells reduces insulin secretion42. It is thus conceivable that a bidirectional effect may exist, whereby reduced insulin sensitivity is both a mediator and consequence of alternative pathway complement activation.
In contrast to C3, postprandial C3a(desArg) levels did not alter from baseline. At first glance this may seem surprising given the role of C3a(desArg) in increasing lipid uptake and storage. However, other studies have also shown unaltered circulating C3a(desArg) levels in response to a fat meal43,44. This may be attributable to local as opposed to systemic C3a(desArg) formation, and rapid adipocyte uptake upon subsequent C5L2 receptor binding. In line with this, veno-arterial gradient studies have confirmed that C3a(desArg) is generated in vivo by human adipocytes and that this production is exaggerated postprandially, especially in the presence of obesity45,46. Indeed, adipocytes are an important source of complement components, particularly those of the alternative pathway. Both visceral and subcutaneous adipose tissue are known to produce and secrete C3, C4, factor D, properdin and factor H, creating an environment for local generation of C3a(desArg)29,36,47.
In light of our findings of complement dysregulation in women with PCOS, therapeutic strategies to reduce alternative pathway activation might be important in alleviating or preventing complications associated with PCOS such as metabolic syndrome, type 2 diabetes and cardiovascular disease. In this context, it is interesting to note that C3a(desArg) levels are reduced by weight loss48 and physical activity49 in obesity, whilst C3 and C3a(desArg) levels fall in response to metformin therapy in women with PCOS18. Additional studies examining the effects of new compounds that selectively reduce alternative complement pathway activation50 would be of therapeutic interest, not only in PCOS but also in other cardiometabolic disorders associated with enhanced complement activation.