In this study, we have found significant differences in the parameters of glucose and lipid homeostasis between the patients belonging to the upper (group Q4) vs. lower (Q1) quartile of the cis-vaccenic acid content in plasma phospholipids (PL). Fatty acid profiles of the Q4 and Q1 groups significantly differed, as well. To our knowledge, it is the first study investigating associations of high vs. low proportions of cis-vaccenic acid in plasma phospholipids (PL) with markers of insulin resistance in men with hyperlipidemia.

The main finding of the study was that individuals in group Q4 differed from those in Q1 by the more favorable profile of insulin resistance markers. They had significantly lower plasma insulin, HOMA-IR values, and apolipoprotein B concentrations. Moreover, both quartiles differed significantly in the composition of FA in plasma PL. The individuals in the Q4 group had lower proportions of FA 14:0, 18:3n-6, 20:3n-6, and the sum of PUFA n-6. On the other hand, the Q4 group had, besides cVA, significantly elevated FA 16:1n-9, 16:1n-7, 18:1n-9, 20:1n-9, 18:3n-3, sum of MUFA and total plasma NEFA.

We did not prove different dietary habits between Q1 and Q4 subjects (data not shown). It is known that dietary assessment methods have many limitations. The accuracy for individual dietary components reaches a maximum of 70–80% [35].

While insulin and HOMA-IR values serve as surrogate markers of insulin resistance, elevated apolipoprotein B, concurrently with elevated serum triacylglycerols, increased small LDL particles, and reduced level of HDL characterize atherogenic dyslipidemia that is typical for insulin resistant states. Interestingly, the whole group of 231 men with hyperlipidemia had a significantly smaller proportion of cVA and oleic acid in their plasma PL, in comparison with a small control group of 50 normolipidemic men (see Supplementary Tables S2, S3).

Our findings of associations of plasma phospholipid cis-vaccenic acid (18:1n-7) with cardiometabolic risk factors are in line with the results of several studies. In participants of a prospective Cardiovascular Health Study, free of diabetes, concentrations of 18:1n-7 in plasma PL were inversely associated with incident T2DM [24]. From another perspective, in a 6-year study in the Canadian population at risk for diabetes, lower cis-vaccenic acid (cVA) predicted lower insulin sensitivity and β-cell function [23]. Ethnic factors may also play a role in the functioning of cVA. In Multi-Ethnic Study of Atherosclerosis (MESA) participants, higher levels of plasma cVA were inversely associated with insulin resistance scores across four races/ethnicities (Caucasian, Black, American Chinese, Hispanic), and related to 17%, 39%, and 32% lower risks of incident T2DM in Black, Chinese American, and Hispanic participants, respectively. In Caucasians, the significant association of cVA with incident T2DM was not found [36]. In other studies, different results were found, too. In the European Prospective Investigation into Cancer and Nutrition (EPIC)–Potsdam study, cVA by quintile of erythrocyte membrane fatty acid proportions did not correlate with the risk of T2DM [37].

The Q4 group differed from the Q1 group by a higher level of individual MUFAs (16:1n-9, 18:1n-9, 20:1n-9) and palmitoleic acid (16:1n-7, POA), which is a precursor of cVA. The levels of α-linolenic acid (18:3n-3, LnA) and total plasma NEFA were higher in the Q4 group, as well.

MUFA, mostly oleic acid, is recognized as beneficial in influencing insulin sensitivity and preventing DM2. However, not all FA in this class have the same effects. Oleic acid (OA) content has been linked with a number of mechanisms, such as reduced expression of pro-inflammatory genes, upregulation of enzymes responsible for fatty acid oxidation, beneficial effects on endothelial dysfunction, and others [38]. Negative correlations with T2DM were found for OA in both plasma CE [39] and plasma PL [13]. On the other hand, some studies also produced opposite results - in one prospective study, oleic and palmitoleic (POA) acids were independently associated with incident T2DM [40].

Q1 differed from Q4 by significantly higher concentrations of γ-linolenic acid (GLA) and dihomo-γ-linolenic acid (DGLA) while lower proportions of sum PUFA n-6 (at the expense of linoleic acid, LA) and α-linolenic acid (ALA, 18:3n-3). In our previous work, higher proportions of POA, GLA, and DGLA, while lower content of LA in plasma phosphatidylcholines were able to identify in the patients with metabolic syndrome those with higher IR or an oxidative stress marker [33]. Also, DGLA was associated with abdominal fat expressed as waist circumference or waist/hip ratio [41].

In this work, we observed higher levels of NEFA in the Q4 vs. Q1 group. It was generally accepted that elevated plasma NEFA is associated with insulin resistance and T2DM in humans [42]. NEFA was elevated in diabetic subjects with overweight/obesity in comparison with healthy subjects [43] and was prospectively associated with an increased risk of both impaired glucose tolerance and T2DM [44]. Nevertheless, individual NEFA may have different effects on the progress of insulin resistance and the development of type 2 diabetes mellitus [45]. Saturated NEFA causes insulin resistance, while monounsaturated FA increases insulin sensitivity in both diabetic [46] and healthy persons [45].

Still, some issues are to be resolved dealing with different NEFA contribution to insulin resistance and other pathologic states: FA profiles in plasma lipid compartments differ significantly between men and women and among different ethnic groups, as well [47], and reference range for main plasma FA in healthy subjects are missing [48].

The reasons for the higher NEFA concentration in the Q4 group are not clear. Some findings suggest that increased content of cis vaccenic acid, a product of ELOVL5 activity, could be related to a decrease in ectopic fat deposition. Supplementation of obese rats with trans-vaccenic acid (replacing oleic acid) led to increased insulin sensitivity, reduced hepatic steatosis, and decreased nonalcoholic fatty liver disease activity scores. The action of cVA on PPARγ (peroxisome proliferator activator receptor γ) in adipose tissue may counteract excessive ectopic lipid accumulation [49]. In our study, in set of men with hyperlipidemia group of patients belonging to the fourth quartile of serum PL cVA proportions had higher values of estimated ELOVL2/5 activity, calculated both as a 22:5n-3/20:5 n-3 and 22:4n-6/20:4n-6 ratios and activity of ELOVL5 calculated as a ratio 18:1n-7/16:1n-7. In experimental work, increasing hepatic ELOVL5 activity improved glycemia, insulinemia, HOMA-IR, and glucose tolerance to normal values in obese mice [50]. In mice with a knockout of the Elovl5 gene, the lack of endogenously formed long-chain PUFA leads to the derepression of SREBP-1c, the activation of lipogenesis, and hepatic steatosis [51]. ELOVL5 activity was also associated with increased catabolism of triacylglycerols, suppressed expression of enzymes involved in gluconeogenesis [52], and induced the expression of fibroblast growth factor 21 (FGF21), which increases hepatic insulin sensitivity, decreases lipogenesis, potentiates fatty acid β-oxidation, reduces hepatic ER (endoplasmic reticulum) stress, and diminishes VLDL (very low-density lipoproteins) delivery to the liver [53] and can improve ectopic lipid deposition in liver and muscle [54].

The patients in the Q4 group had significantly higher estimated activities of delta-9-desaturase (D9D) both for 16:0 (D9D16) and 18:0 (D9D18), whereas lower estimated activity of D6D (calculated as 18:3n-6/18:2n-6 ratio). Elevated activity of D9D is a marker of de novo lipogenesis, and in this study, both D9D16 and D9D18 correlated significantly with the total plasma NEFA levels and with all principal n-9 FA (16:1n-7, 18:1n-9, 20:1n-9, and cVA.

Interestingly, the estimated activity (D9D18) index correlated negatively with markers of insulin resistance, whereas the D9D16 index correlated positively (see Supplementary Table 1). This probably could be related to the possible positive association of POA with insulin resistance in this set of patients.

Proportion of cVA correlated negatively with insulin, HOMA-IR, and apolipoprotein B, showing that in our set of hyperlipidemic patients with obesity/overweight, cVA in plasma PL may function to improve insulin sensitivity. In contrast, POA correlated rather differently both with anthropometric parameters and markers of glucose and lipid homeostasis. Its proportion in plasma PL correlated positively with BMI, WHCR, fat mass, and triacylglycerols. This finding corresponds with some results of previous human studies, indicating unfavorable effects of POA on glucose homeostasis [19], though not all studies did prove such results [24].

Patients of Q4, in comparison with the Q1 group, had significantly lower values of an estimated D6D (18:3n6/18:2n6) (p < 0.001). This finding is in line with the results of other studies, where elevated estimated activity of D6D was described in patients with metabolic syndrome and T2DM [31], impaired fasting glycemia [30], and other pathologic conditions such as cardiovascular disease [55], tumors [9], or depression [10] Inverse correlations have been found between POA and cVA with estimated D6D activity (while POA correlated positively, cVA correlated negatively, both p < 0.001).

We have analyzed in our group of hyperlipidemic men markers of oxidative stress and inflammation (conjugated dienes in LDL and hs-CRP), as these conditions are associated with insulin resistance. Probands in the Q4 group had significantly lower concentrations of CD-LDL in comparison with the Q1 group. Concentrations of CD-LDL are considered a marker of systemic oxidative stress, partly reflecting the levels of minimally modified LDL, in which only the lipid component is oxidatively modified [56]. This finding is in line with the results of our earlier studies dealing with hypertriglyceridemia, severity of metabolic syndrome, or pancreatic cancer, as well [9, 26, 57]. The significance of lipid peroxidation in the pathogenesis of insulin resistance has been proven in experimental and clinical studies [58].

On the other hand, we were not able to find differences in hs-CRP concentrations. To our knowledge, this is the first work describing significant associations of serum PL cVA with parameters of insulin sensitivity in male patients with hyperlipidemia.

The study was conducted in a relatively large set of men with overweight or obesity, who were not treated with lipid-lowering drugs, supplements of n-3 or n-6 PUFA, or antioxidants. Limitations include a cross-sectional type of study, so causality could not be proved. Moreover, the profile of individual plasma NEFA was not investigated, and the content of individual MUFA and PUFA was not assessed in dietary questionnaires, as well. Also, the data about the physical activity were not available.

Our results support the hypothesis that plasma PL cis-vaccenic acid could be associated with insulin sensitivity in men with hyperlipidemia and high cardiovascular risk. Moreover, the results indicate that individual FA in the same class can have different pathophysiological effects. The results should be further studied and applied to other populations, e.g. healthy subjects, women, or type 2 diabetics.