Discussion
In this pilot study, FN3K protein was detected both in the lungs and systemically in COPD. The levels of FN3K positive (FN3K+) epithelial cells in non-smoker, smoker and COPD airways separated into two distinct phenotypes: very low or high numbers of FN3K+ positive cells. However, a similar separation was not seen in the smaller number of COPD subjects in the metformin study. This separation of FN3K+ subjects into two distinct groups is similar to that reported in diabetic subjects.17 Moreover, these phenotypes correlated with cardiovascular risk, whereby low FN3K expression was associated with a larger G-gap and greater cardiovascular risk and vice versa.17
The accumulation and increased presence of AGEs or carbonyl stress along with the corresponding pathophysiological mechanisms are well documented31 and a known risk factor for developing CVD.23 In diabetes the formation of AGEs is linked to high glucose levels and the presence of oxidative stress through a process known as glycation. However, in COPD high levels of carbonyl stress are also present as a result of chronic inflammation, oxidative stress2 and elevated ribose levels resulting from metabolic reprogramming.10 Ribose is 20 times more reactive than glucose in glycating proteins and forming AGE/carbonyl adducts.32 Consequently, both COPD and diabetes are influenced by inflammation and oxidative stress resulting in increased carbonyl stress and AGEs although they are driven through different aetiological stimuli (figure 5). Increased carbonyl stress and AGEs have been linked to CVD23 as well as many other comorbidities associated with COPD.33 As FN3K is widely expressed throughout the body, it is plausible that FN3K may be functioning as a protective clearance mechanism to remove the damaging precursors to AGE formation and that low levels of FN3K expression will lead to increased CVD risk (figure 5). In support of our hypothesis is the observation that in control smokers with normal lung function, we observed an increase in the numbers of cells expressing FN3K protein within the lower airways, presumably to protect from the increase in carbonyl stress and AGE levels. However, this protective mechanism would appear to be impaired in COPD as the numbers of FN3K+ cells in the lung were reduced. Furthermore, this downregulation in the numbers of FN3K+ cells was modelled in a chronic smoking model of COPD. This might suggest that pathophysiological mechanisms prevalent in COPD to trigger downregulation in FN3K expression are being replicated in the chronic cigarette smoke exposure animal model used here. We have previously shown that both oxidative stress and carbonyl levels are elevated in the chronic smoke exposure model.34 Clearly further investigation is warranted into how chronic exposure to oxidative stress/cigarette smoke can lead to down regulation in FN3K expression. Consequently, as a result of our findings reported here, the chronic smoking model may provide a suitable platform for further investigations. Indeed, little is known about the mechanisms controlling FN3K expression but a complex interaction between SNPs within the FN3K locus, inducible protective mechanisms upregulating FN3K expression and activity in response to cigarette smoke and epigenetic changes may exist.35 It is also likely that oxidative stress may have a direct impact on FN3K activity. However, a limitation in the findings reported here was that there was insufficient sample to measure FN3K enzymic activity. To try and address a possible linkage between FN3K SNPs and CVD risk in COPD, we analysed the genotyping data from the SPIROMICS cohort consisting of at-risk smokers and COPD subjects. 6MWD is a measure of cardiorespiratory function determined by multiple factors that when evaluated in the context of the CVD risk factors determined by FN3K /FN3K-RP (body mass index, haemoglobin A1C, systolic blood pressure) it precludes these loci as genetic determinants for 6MWD.16 While 6MWD was the only data available to us at the time within this study cohort, a more reliable marker of CVD risk could be evaluated using more detailed phenotypes of CVD risk factors in COPD cohorts, such as MRI-calcium scores, lipid profiles, haemoglobin A1C or CT scan-based measures of adiposity in conjunction with multi-omic (transcriptomic/epigenomic) approaches. Consequently, our negative findings for FN3K and FN3K-RP variation in relation to 6MWD does not exclude an indirect contribution leading to increased CVD risk and comorbidity. Indeed, one of the SNPs identified in our study (rs1046875) is linked to variation associated with increased CVD risk traits.29 30
Figure 5Fructosamine-3-kinase (FN3K) as a potential gatekeeper to prevent the accumulation of damaging AGEs that may predispose subjects to increased risk of cardiovascular disease. Accumulation of AGEs and carbonyl stress may come directly through raised uncontrolled blood glucose levels in diabetes or indirectly through chronic oxidative stress from cigarette smoking in COPD resulting in mitochondrial dysfunction, an altered metabolic profile and elevated cellular ribose levels and increased carbonyl stress. AGE, advanced glycation endproduct; COPD, chronic obstructive pulmonary disease; CVD, cardiovasculardisease.
Given that increased ribose levels can accrue in COPD subjects as a result of an increase in the pentose phosphate pathway10 and poorly controlled diabetics also exhibit increased ribose levels, both of which can lead to increased AGE and corresponding increased CVD risk.6 It is plausible that FN3K-RP genetic variation results in alterations in ribose derived AGE products subsequently linked to CVD risk. While the case is not fully proven here in COPD subjects, it does offer the possibility that there may still be a linkage, although indirectly, between FN3K polymorphisms and CVD risk in COPD subjects. Indeed, SNPs in FN3K have been strongly associated with changes in sRAGE,36 which in turn has been linked to CVD risk.37 However, a recent study by Sartore et al18 has reported that a cluster of FN3K SNPs when expressed together were associated with increased risk of microvascular and macrovascular complications. It is possible therefore that no single direct linkage between FN3K variation and CVD may exist but instead requires interactions between different FN3K polymorphisms, and possibly RAGE expression as well, to confer increased risk to CVD within COPD subjects.
Metformin administration to COPD patients with AECOPD significantly increased serum FN3K levels over the course of an exacerbation following admission into hospital irrespective of the basal level. In contrast, serum FN3K levels did not change with placebo. This increase was mirrored by a corresponding fall in carbonyl (AGE) levels from entry into the study to follow-up in both the placebo and metformin groups. Although there was a 10% difference in carbonyl levels between the two groups, this just failed to reach significance (p=0.08). This was in contrast to that seen for fructosamine levels in the same study where metformin, but not placebo, caused a significant fall in serum fructosamine levels.24 It is possible that metformin could not have a significant effect on carbonyl levels in our study, as the exacerbation and subsequent production of carbonyls had already begun before metformin was administered. Indeed, in a study by Dallak et al38 when metformin was administered prior to the induction of a diabetic state subsequent increases in carbonyl (AGE) levels were prevented. This failure to reach significance may reflect the importance of other unknown mechanisms that aid the resolution of the exacerbation episode or that this study was not sufficiently powered to detect these changes. Indeed, a potential major limitation for the two pilot studies involving stable COPD and AECOPD reported here were the low number of subjects involved. However, post-hoc analysis of statistical power revealed that both studies achieved 100% power to discriminate significant differences (p<0.001) in FN3K expression between the different cohorts under investigation. Metformin has been shown to decrease CVD risk and resultant mortality in both diabetics and non-diabetics.14 39 40 It is unclear how metformin’s impact on FN3K expression may affect pathophysiological mechanisms and subsequent comorbidity risk of CVD in COPD patients, although Dallak et al have shown that metformin can reduce levels of AGEs.38 Several pathophysiological mechanisms such as endothelial dysfunction and increased coagulopathy within the vasculature are associated with increased risk of CVD in COPD.41 Moreover, AGEs have been implicated in vascular stiffness, atherosclerotic plaque formation, thrombogenesis, increased coagulopathy, vascular calcification and endothelial dysfunction with consequential impacts on the risk of mortality.42 Therefore, the ability of FN3K to remove fructosamine and ribosamine residues on modified proteins and thereby reduce the carbonyl load and AGE formation may have a clinical impact on these pathologies and survivability. Metformin can also directly lower AGE levels by reacting with AGE adducts containing dicarbonyls,43 in particular methyl glyoxal,44 a highly reactive dicarbonyl and a by-product of increased glycolysis,45 a pathway that has been shown to be elevated in COPD.10 Given that the manipulation of upstream metabolic events involving fructosamine will only impact on subsequent AGE formation, and the removal of existing ‘long-lived’ AGE/ carbonyls may take considerably more time, a longer time course may therefore be needed to observe whether changes in FN3K expression would result in notable changes in basal AGE/carbonyl levels and in turn clinical outcome. The limitations of the present assay in measuring all carbonyls may also be inappropriate, and more specific assays detecting unique AGE/carbonyl adducts known to be associated with increased CVD risk may be more appropriate. However, a recent study by Ho et al46 indicated that metformin had a beneficial impact on survivability in COPD patients with diabetes which may be attributed to the metformin-induced increase in FN3K expression reported here, thereby reducing AGEs and subsequent mortality.
In conclusion, this pilot study shows that the distribution and expression of FN3K protein in COPD and age-matched control subjects (both non-smokers and smokers with normal lung function) appears to fall into one of two groups, those with low levels of FN3K protein or those with high levels of FN3K protein expression. Moreover, smoking appears to trigger a protective mechanism whereby FN3K protein levels are increased, but this is apparently lost in most but not all COPD subjects. Furthermore, metformin elevates FN3K expression which can reduce carbonyl stress (AGEs) thereby potentially reducing the risk of developing CVD. Clearly further work needs to be done to demonstrate that a clear mechanistic link is present between carbonyl stress, FN3K expression and CVD risk in COPD, with or without diabetes, and potentially in ageing where systemic AGE levels are also known to rise. Finally, this study highlights a potential mechanistic explanation for the benefits of providing metformin to lower CVD and other disease risks associated with increasing AGE levels in COPD.