To our knowledge, this study is the first to show that in patients with T2D and angiographically documented ASCVD, a higher LI of serum phospholipids, reflecting increased overall FA lipophilicity and lower fluidity of the human biological membranes, characterizes individuals with worse glucometabolic control, increased vascular inflammation and higher platelet reactivity to arachidonic acid during aspirin treatment at COX-1–selective doses.

A unique property of FAs is their melting point, which depends on the length and degree of unsaturation of the FA chains, and reflects the lipophilicity of FAs [19]. There is increasing evidence that the lipophilicity of FAs determines the lipophilicity and fluidity of lipoproteins and biological membranes [28]. In turn, the fluidity of cell membranes and plasma lipoproteins may play an important role in the etiology of cardiovascular diseases and T2D through the mechanisms associated with lipid metabolism, blood pressure, endothelial function or insulin action in target cells [28]. The LI, which is the mean of the melting points of individual FAs, can be assessed in various biological samples, including serum or plasma phospholipids, TG, cholesterol esters, as well as in the phospholipids of erythrocyte cell membranes or, less frequently, other cells [14]. Phospholipids, assessed in both serum and plasma, have the advantage of being stable during long-term storage and are considered a good marker of the fatty acid composition of cell membranes and plasma lipoproteins, as well as medium-term (weeks to months) dietary fat intake [29,30,31,32]. In our study, the assessment of FA composition of serum phospholipids was performed by gas chromatography, which is a high-sensitivity and high-resolution analytical separation technique, widely used to analyze a variety of samples in clinical, pharmaceutical, biochemical, forensic and food science laboratories. Despite its many advantages, this technique also has some limitations, including the inability to analyze thermolabile compounds and its relative time consumption [33]. On the other hand, the advances in the implementation of novel techniques (e.g. low-pressure gas chromatography, low-pressure gas chromatography - mass spectrometry, low-pressure gas chromatography - mass spectrometry with low thermal mass) make it possible to significantly improve the process of sample analysis (even < 1 min) [34]. In our opinion, this method, after its standardization and validation, could be more widely used for clinical purposes in the future.

In this study, the n-6/n-3 PUFA ratio in serum phospholipids, reflecting their medium-term dietary intake, and both desaturation indices, showing endogenous biosynthesis of MUFAs from SFAs, were predictors of LI above the median. Previous studies have demonstrated that increased activity and/or overexpression of SCD1 (EC 1.14.19.1), a highly conserved member of the ferrous Δ-9 desaturases family, are relevant contributing factors for T2D [35]. Moreover, higher desaturation indices were associated with IR, T2D, obesity and metabolic syndrome [36]. Our findings highlight the importance of both diet and endogenous FA biosynthesis in modulating the fluidity of human biomembranes in diabetic patients, which is consistent with the results of previous studies [7,8,9,10,11].

We showed that in T2D patients with ASCVD, a higher serum phospholipid LI is associated with increased serum concentrations of TCh and LDL-Ch. Previous population-based studies have shown a link between LI and abnormal lipid profile, including higher levels of LDL-Ch and TG [15, 37, 38]. However, the relationship between serum phospholipid LI and lipid parameters has not been evaluated in patients with T2D, who usually have complex lipoprotein abnormalities known as atherogenic dyslipidemia and characterized by elevated fasting and postprandial TG levels, low HDL-Ch, and increased small dense LDL particles. The effect of ASCVD on the link between LI and abnormal lipid profile was also unknown. Thus, our study deepens the understanding of the interrelationship between FA and lipoprotein metabolism and the fluidity of biological membranes and, thereby, probably insulin sensitivity and glycemic control in patients with T2D and ASCVD. This supports another finding from our study that higher LI values are associated with worse glycemic control and higher HbA1c levels. Growing evidence suggests that reduced fluidity of biological membranes has an adverse effect on the insulin signal transduction pathways, decreases glucose transporter type 4 (GLUT-4) transport to the membrane and glucose uptake [9, 39]. It is tempting to hypothesize that improved glycemic control may have the effect of reducing LI and increasing membrane fluidity in patients with T2D and ASCVD, which in turn may translate into improved insulin sensitivity, better glucose utilization efficiency, and beneficial clinical outcomes. However, it is worth highlighting that the associations observed in our study between LI and selected parameters of metabolic control in T2D may also be due to ASCVD, which requires further targeted studies.

This study also showed an association between serum phospholipid LI and vascular inflammation defined as elevated serum levels of Lp-PLA2 (a marker of vascular inflammation) and interleukin-6, which is a novel finding. T2D is closely related to chronic low-grade inflammation [1,2,3,4]. In turn, vascular inflammation has a crucial role in all phases of atherogenesis, from the formation of fatty streaks to the rupture/erosion of the vulnerable plaque, which can result in acute coronary syndrome [39,40,41,42]. Growing evidence suggests that inflammation in T2D is associated with abnormalities in membrane phospholipid and PUFA composition [39, 40]. Our findings seem to confirm that high serum phospholipid LI may be a biomarker of active vascular inflammation and ASCVD in diabetic patients and identify subjects at particularly high cardiovascular risk. This is especially important because most patients at the time of diagnosis of T2D have macroangiopathy, which is responsible for the majority of deaths in this population [1,2,3,4,5,6].

Our study showed no effect of FA class on the serum phospholipid LI. Individual FAs, particularly n-3 and n-6 PUFAs, had a differential effect on LI values which is consistent with the results of previous studies [14, 19, 28]. As a higher content of n-3 PUFAs - eicosapentaenoic (EPA) and docosahexaenoic (DHA) - has a beneficial effect on the fluidity of biological membranes and on reducing the value of LI, and consumption of marine fish rich in these FAs significantly reduces LI of serum phospholipids and erythrocyte membranes [37], it can be supposed that the association observed in our study between serum phospholipid LI and vascular inflammation is related to the anti-inflammatory properties of these n-3 PUFAs. Moreover, arachidonic acid, a member of the n-6 PUFA family whose anti-inflammatory properties are well known [43, 44], has a similar effect on LI. On the other hand, however, our previous observations did not show that 3-month supplementation of 1 g/d of EPA and 1 g/d of DHA had any effect on systemic inflammation and Lp-PLA2 levels in patients with T2D and ASCVD [20]. This may suggest the existence of other mechanisms responsible for the association of high LI, reflecting reduced biomembrane fluidity, with enhanced vascular inflammation.

Our study showed that platelets of patients with LI above the median were characterized by increased reactivity to AA despite taking aspirin at COX-1-selective doses. One of the major causes of vascular complications in T2D is increased platelet aggregation and their hyperreactivity [45, 46]. The fluidity of membrane phospholipids is thought to play a key role in signal transduction and affect platelet activation [47]. One of the main mechanisms responsible for platelet hyperreactivity in T2D patients is a decrease in platelet membrane fluidity due to non-enzymatic glycosylation of membrane proteins and altered architecture of the phospholipid bilayer [47]. These observations are consistent with our findings that higher LI is associated with increased AA-induced maximal platelet aggregation. Importantly, platelets can actively take up phospholipids and FAs from the serum pool, incorporating them into their membranes [48]. Recent findings suggest an even greater significance of the FA uptake by platelets and their precursors, megakaryocytes, via the CD36 receptor, which is likely to be crucial in megakaryopoiesis and platelet biology [49]. AA stimulates platelet aggregation after conversion by COX-1 to prostaglandin G2 and H2 and then to thromboxane A2 (TXA2), a potent platelet agonist and vasoconstrictor acting through its selective receptor belonging to the G-protein coupled receptor family [42, 50]. In turn, this effect is prevented by COX-1 inhibitors, including aspirin at COX-1-selective doses (75–150 mg/d), which irreversibly acetylates a serine residue at position 529 of human platelet COX-1 [50]. Increased platelet reactivity to AA in patients with higher LI, despite aspirin treatment (75 mg/d) in all participants of our study, may indicate novel mechanisms of aspirin hyporesponsiveness in T2D with concomitant ASCVD.

It is worth noting, however, that the relationship between higher serum phospholipid LI and increased platelet activity observed in our study may not have been due to changes in the fatty acid composition and the fluidity of platelet biological membranes (these variables were not evaluated) but could have been a coincidental finding. Subjects with T2D present the plethora of the abnormalities in the hemostasis, including increased platelet activity, endothelial dysfunction, hyperfibrinogenemia, and hypofibrinolysis, which aggravate with the worse glucometabolic control and inflammation [51,52,53]. Therefore, T2D patients with higher serum phospholipid LI index, who have worse glucometabolic control and increased systemic inflammation, will probably show the enhanced platelet activity. However, this requires further studies.

The cross-sectional nature of this study does not allow for inferences to be drawn as to the causal relationship between serum phospholipid LI and metabolic control, vascular inflammation and platelet activation. It is tempting to speculate that by affecting serum phospholipid LI and biomembrane fluidity, it would be possible to improve insulin sensitivity, metabolic control, reduce vascular inflammation and platelet reactivity in patients with T2D and ASCVD.

This study had several limitations. First, the sample size was limited though well characterized and representative for T2D with macroangiopathy. Second, the study did not include the control group(s). Third, the LI was assessed only in the serum phospholipid fraction and not in other blood lipid pools or in erythrocyte membranes or other tissues (e.g. adipose tissue). The dietary fat intake was not assessed precisely using a food frequency questionnaire (FFQ), however, study participants were provided with dietary recommendations regarding low-fat and low-carbohydrate foods and caloric values. Furthermore, the study was conducted at a time when SGLT-2 inhibitors were not widely used in the pharmacotherapy of diabetes, hence none of the patients received such treatment. Finally, the associations presented here do not necessarily mean the cause-effect relationship, however they should be perceived a hypothesis generating observation, which deserves further studies.