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.