Associations of plasma phospholipid cis-vaccenic acid with insulin resistance markers in non-diabetic men with hyperlipidemia

The study addresses whether the proportion of cis-vaccenic acid (cVA, 18:1n-7) in plasma phospholipids relates to insulin resistance in non-diabetic men with hyperlipidemia. Fatty acids influence membrane properties, signaling, and serve as ligands and precursors for bioactive molecules. Dietary intake, endogenous synthesis, desaturase/elongase activities, and genetic factors shape FA profiles. Insulin resistance is implicated in metabolic syndrome, type 2 diabetes, and dyslipidemia. Different FA classes show distinct associations with insulin resistance; higher saturated fat and lower n-3 PUFA and MUFA often link to insulin resistance, whereas n-6 PUFA effects are mixed. Prior work has shown both beneficial and adverse associations for specific FA (e.g., palmitoleic acid). cVA, an elongation product of palmitoleic acid, has been inversely associated with heart failure risk and incident diabetes and with higher insulin sensitivity in some cohorts, though not consistently. Given these mixed findings and potential sex/ethnic differences in FA metabolism, the study aims to test associations between plasma PL cVA and markers of insulin resistance in men at high cardiovascular risk.

Prior evidence suggests complex, FA-specific relationships with insulin resistance and diabetes risk. Higher plasma linoleic acid associates with lower type 2 diabetes risk, while higher dihomo-γ-linolenic acid correlates with increased diabetes incidence. MUFA, especially oleic acid, have been linked to improved insulin sensitivity, though some studies report increased diabetes risk with higher oleic and palmitoleic acids. Palmitoleic acid shows both beneficial metabolic effects in experimental models and adverse associations (abdominal obesity, inflammation) in humans. cVA (18:1n-7), produced by elongation of palmitoleic acid (notably via ELOVL5), has been inversely associated with incident diabetes (Cardiovascular Health Study) and with higher insulin sensitivity and β-cell function over time in a Canadian cohort; higher cVA related to lower insulin resistance and lower diabetes risk across several ethnicities in MESA, though not among Caucasians. Conversely, EPIC-Potsdam found no association between erythrocyte membrane cVA and diabetes risk. These discrepancies may reflect differences in study design, compartments measured (plasma PL vs erythrocytes), populations, and metabolic regulation of desaturases/elongases.

Design: Cross-sectional analysis of 231 consecutive hyperlipidemic men (2020–2022) referred to a lipid clinic prior to hypolipidemic therapy consideration. A control group of 50 apparently healthy men (volunteers/university staff) was also examined. Ethics approval was obtained; written informed consent was collected. Inclusion/Exclusion: Patients had LDL-C >3.0 mmol/L and/or triacylglycerols >1.70 mmol/L; 24% had HDL-C <1.00 mmol/L. Exclusions included current lipid-lowering, antidiabetic, or antioxidant therapy; excessive alcohol (>30 g/day); hormone therapy; PUFA supplementation; manifest cardiovascular/cerebrovascular disease; diabetes; significant liver (except NAFLD) or kidney disease (creatinine >130 μmol/L); hypothyroidism; recent infection or malignancy. Prior to testing, participants were advised to follow AHA Step One diet and maintain stable dietary patterns for 6 weeks. Measurements: Standardized anthropometry (BMI, waist, WHCR), body fat by bioelectrical impedance (Tanita SC-240), and blood pressure (Omron M3) after 10 min rest. Laboratory assays included routine biochemistry, NEFA by enzymatic colorimetry (Randox), HOMA-IR calculation (insulin × glucose / 22.5), and eGFR by 2021 CKD-EPI creatinine equation. Fatty acid analysis: Plasma phospholipid FA methyl esters quantified by GC-FID (LN-FAME-HT 60 m column), enabling separation of MUFA isomers; retention times verified with commercial standards; detector linearity and response factors checked; analytical variability RSD 1.07% (16:0) to 8.60% (16:1n-9). Estimated enzyme activities computed as product/precursor FA ratios (e.g., D9D16=16:1n-7/16:0, D9D18=18:1n-9/18:0, D6D=18:3n-6/18:2n-6, D5D=20:4n-6/20:3n-6, ELOVL5=18:1n-7/16:1n-7, ELOVL2/5=22:5n-3/20:5n-3 and 22:4n-6/20:4n-6). Diet: 3-day dietary questionnaire analyzed with Nutrimaster SE v1.0 to estimate energy and macronutrient intakes, including fat and saturated fat. Statistics: Participants stratified into quartiles by plasma PL cVA; primary comparison between Q4 (highest) and Q1 (lowest) quartiles with investigators blinded to quartile assignment. Normality assessed by Shapiro–Wilk; group comparisons by t-test or Wilcoxon test as appropriate; categorical variables by frequency; Benjamini–Hochberg correction applied. Multivariate linear regression with backward stepwise selection assessed independent associations of MUFA species with insulin resistance markers. Post hoc power for HOMA-IR and insulin was 0.76 and 0.66, respectively.

- Cohort characteristics (n=231 hyperlipidemic men): median age 50 years; BMI 28.5 kg/m² [26.01–30.89]; high prevalence of hypertension (61.5%), TAG ≥1.7 mmol/L (68.5%), LDL-C ≥3.0 mmol/L (73.1%). - Diet: No significant differences between Q4 and Q1 in energy or macronutrient intake, including total fat, SFA, MUFA, and PUFA (sum n-3 and n-6). - Insulin resistance markers (Q4 vs Q1; medians [IQR]): insulin 8.79 [5.83–11.78] vs 10.90 [7.70–17.19] mU/L (p<0.05); HOMA-IR 2.09 [1.42–2.76] vs 2.82 [1.84–4.36] (p<0.05); apolipoprotein B 1.12 [0.90–1.42] vs 1.29 [1.08–1.55] g/L (p<0.01). Fasting glucose similar. - NEFA and oxidative stress: NEFA higher in Q4 0.72 [0.57–1.26] vs 0.42 [0.35–0.56] mmol/L (p<0.01). CD-LDL lower in Q4 57.4 [43.8–67.5] vs 62.1 [51.0–72.7] μmol/L (p<0.05). hs-CRP similar. - Lipids and anthropometrics: No significant differences between Q4 and Q1 in total cholesterol, TAG, HDL-C, LDL-C, non-HDL-C, apoA-I, age, BMI, waist, WHCR, fat mass, or blood pressure; similar proportions of smokers and hypertensives. - Plasma PL fatty acid profile (mol%): Q4 vs Q1 showed higher MUFA sum 14.34 vs 11.19 (p<0.001); higher 18:1n-9 (oleic) 11.53 vs 9.22 (p<0.001); higher 18:1n-7 (cVA) 1.97 vs 1.23 (p<0.001); higher 16:1n-7 0.62 vs 0.56 (p<0.05); higher 16:1n-9 0.13 vs 0.09 (p<0.001); higher 20:1n-9 0.17 vs 0.11 (p<0.001). Lower n-6 PUFA sum 35.23 vs 38.52 (p<0.001) due to lower 18:3n-6 (γ-linolenic) 0.07 vs 0.11 (p<0.001) and 20:3n-6 (dihomo-γ-linolenic) 3.04 vs 3.33 (p<0.05); no significant differences in linoleic or arachidonic acids. ALA (18:3n-3) higher 0.23 vs 0.16 (p<0.001). - Estimated enzyme indices: Higher D9D16 (2.06 vs 1.82, p<0.05) and D9D18 (0.76 vs 0.66, p<0.001) in Q4; higher ELOVL5 (18:1n-7/16:1n-7) 3.13 vs 2.15 (p<0.001); higher ELOVL2/5 ratios 22:5n-3/20:5n-3 1.04 vs 0.88 (p<0.01) and 22:4n-6/20:4n-6 2.97 vs 2.76 (p<0.05); lower D6D 18:3n-6/18:2n-6 0.33 vs 0.50 (p<0.01). - Correlations (n=231): cVA inversely correlated with insulin (r=-0.183, p<0.01), HOMA-IR (r=-0.196, p<0.01), and apoB (r=-0.203, p<0.01). Backward stepwise regression across MUFA species identified only 18:1n-7c (cVA) as independently associated with insulin (p=0.015) and HOMA-IR (p=0.006). - Case-control comparison: Controls (n=50) had higher cVA and MUFA sums in plasma PL than hyperlipidemic patients (supplementary data).

Individuals with higher plasma phospholipid cVA (Q4) exhibited a more favorable insulin resistance profile (lower fasting insulin and HOMA-IR) and lower apoB, despite similar adiposity and lipid levels, suggesting cVA may be linked to improved insulin sensitivity in hyperlipidemic men. The FA profile differences—higher MUFA (oleic, palmitoleic, cVA) and lower n-6 PUFA (notably GLA and DGLA)—align with literature associating MUFA and lower DGLA with better metabolic health. Estimated enzyme activity patterns (higher D9D and ELOVL5/ELOVL2/5, lower D6D) support an endogenous lipid metabolic state favoring MUFA elongation/desaturation and reduced n-6 PUFA conversion, potentially contributing to improved insulin signaling. The inverse associations of cVA with insulin, HOMA-IR, and apoB, and the independent association of cVA (but not other MUFA) with insulin resistance markers after accounting for intercorrelations, point to a specific role of cVA rather than generic MUFA effects. Although NEFA was higher in Q4, which is often linked to insulin resistance, the literature indicates NEFA species differ in metabolic effects; higher MUFA NEFA could be less deleterious or even beneficial, and elevated elongase activity (ELOVL5) may relate to lower ectopic fat and improved hepatic metabolism. Lower oxidative stress (reduced CD-LDL) in Q4 is consistent with better insulin sensitivity. Findings corroborate cohort studies linking higher cVA with lower incident T2DM and better insulin sensitivity, while acknowledging heterogeneity across populations and compartments. Overall, the data suggest cVA may be a biomarker (and possibly mediator) of improved insulin sensitivity in hyperlipidemic men.

In non-diabetic men with hyperlipidemia, a higher proportion of cis-vaccenic acid in plasma phospholipids is associated with lower fasting insulin, lower HOMA-IR, lower apoB, distinct FA profiles (higher MUFA, lower n-6 PUFA), altered desaturase/elongase indices, and lower LDL lipid peroxidation. These results support cVA as a potential marker of insulin sensitivity and highlight that individual fatty acids within the same class can have divergent metabolic associations. Future work should include longitudinal and interventional studies to assess causality, detailed profiling of NEFA species, assessment of dietary FA subtypes and physical activity, and extension to other populations (women, healthy individuals, and patients with type 2 diabetes).

Cross-sectional design limits causal inference. Plasma NEFA species composition was not measured. Dietary questionnaires did not capture individual MUFA and PUFA species. Physical activity data were unavailable. The control group was relatively small. Post hoc power was moderate for insulin-related endpoints.