Incidence and prevalence of type 2 diabetes mellitus (T2DM) are rising worldwide with an alarming speed (1), leading to increased cardiovascular and metabolic morbidity and mortality. T2DM is a chronic condition characterized by hyperglycemia due to inadequate insulin secretion and/or defective insulin action. In 2001, McGarry proposed a new concept of glycolipid metabolism in diabetes mellitus, suggesting that excessive deposition of circulating free fatty acids (FFAs) and triglycerides (TGs) are key factors for the regulation of insulin action (2, 3). In this context, TG levels are an important biomarker of dyslipidemia, with adverse outcomes in patients with increasing hypertriglyceridemia (HTG) levels above 2 mmol/l (4).

Elevated TGs are common in T2DM and contribute to a two-fold increase in atherosclerotic cardiovascular disease (ASCVD) in T2DM. However, recommendations for dyslipidemia control in T2DM focus mostly on lowering low-density lipoprotein cholesterol (LDL-C) (5). The role of TG might be underappreciated though, as a recent study demonstrated that even an increase in TGs within the normal range confers an increased risk of T2DM in healthy subjects without metabolic syndrome (6). Furthermore, recent data confirm that elevated fasting TG (above 2.25 mmol/l) were associated with an increased risk of cardiovascular disease (CVD) mortality in patients with T2DM (7). Patients with T2DM and HTG also seem to have a more atherogenic lipoprotein phenotype than patients with elevated TG without T2DM (8)

The involvement of omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in the cardiovascular system and diabetes has been a subject of considerable interest. (9, 10). It has been shown that n-3 PUFAs can mediate biological effects in human cardiovascular, neurological, inflammatory diseases and cancer through different mechanisms such as alteration of membrane composition and function, gene expression and signaling molecules (11, 12). Notably, n-3 PUFA is also well-known for lowering TG levels (13) and liver fat content (14). However, the results of several recent large studies have been inconsistent in terms of cardiovascular benefit. The ORIGIN and STRENGTH trials observed moderate TG reductions but no impact on cardiovascular outcomes by n-3 PUFA supplementation (15, 16). A cardiovascular benefit was described with albeit minimal changes of TG in the GISSI-Prevenzione and JELIS trials (17, 18), and a significant 18% TG-decrease in the REDUCE-IT trial (19).

N-3 and n-6 PUFA derived oxylipins also have potent biological activity, with their metabolism relying on three enzymatic pathways including cytochrome P450 (CYP), cyclooxygenase (COX) and lipoxygenase (LOX), as well as non-enzymatic autoxidation (20, 21). The role of these oxylipins is multifaceted. Epoxidation products from n-3 PUFA are involved in regulating cardiac function, as well as reduce TG levels by suppressing hepatic lipogenesis and increasing fatty acid oxidation. (22, 23). In addition, evidence suggests that specialized pro-resolving mediators (SPM), derived from n-3 PUFA via LOX action, could regulate inflammation resolution (4). This supports the concept of n-3 PUFA derived inflammation-dampening oxylipins (n-3 IDOs) (25–27). In contrast, n-6 PUFA metabolites may have opposite or neutral effects (28). In a study involving 123 Caucasian men, researchers found that plasma concentrations of 5-hydroxyeicosatetraeonic acid (5-HETE) and 11-HETE were significantly higher, in obese individuals (29). This finding was further supported by evidence from the obese zucker mouse model (30), which elevated levels of 5-HETE, 12-HETE, and leukotriene B4 (LTB4) were discovered in adipocytes. Moreover, several studies revealed that 12- and 20-HETE may promote vasoconstriction, endothelial dysfunction, and platelet aggregation effects, which contribute to a higher risk of CVD (31, 32).

A scientific statement from the American Heart Association emphasized the need to lower triglycerides in order to lower CVD risk, with an optimal goal of < 1.1 mmol/l for fasting TG and a screening threshold of < 2.25 mmol/l (33). The proposed interventions focus on lifestyle to optimize diet, reduce weight and increase aerobic exercise (33) plus aggressive LDL-lowering therapies and control of hypertension, optimal glycemic control in individuals with T2DM and antithrombotic therapies for secondary prevention of ASCVD. In addition, icosapent ethyl is promoted in eligible patients (34).

On this background, we set out to analyze patients with T2DM to identify lipidomic changes that might modify liver and/or cardiovascular risk in patients with T2DM depending on their TG levels.

In this study, we found significant differences in BMI and CAP values for patients with T2DM and high TGs (TG ≥ 1.7mmol/l) as compared to those with TGs within the reference range (TG < 1.7mmol/l), but no increase in liver fibrosis parameters. Hypertriglyceridemia was strongly associated with an increased BMI, as well as increased CAP values in patients with T2DM. Furthermore, total amount of palmitic and oleic acid was significantly increased in blood plasma from diabetics with increased TGs. We also observed significant changes in plasma oxylipin profiles between these two groups. Metabolites tended to be higher in hypertriglyceridemic patients, possibly indicating a (subclinical) pro-inflammatory environment arising from the hypertriglyceridemic metabolic dysregulation.

Our findings regarding HTG and liver fat content are consistent with other findings. In a multivariate analysis of nearly 300 patients with and without NAFLD, TGs were strongly associated with liver fat content (36). In addition, this research also indicated that CAP values of patients with T2DM progressively increased with rising TG levels. A recent large trial by de Lédinghen et al. analyzed patients with suspected chronic liver disease (37) and demonstrated that CAP values significantly increased with an increasing pathology of metabolic syndrome (MetS) components.

Pathophysiological mechanisms of MetS are complex and manifold. FFAs and lipid toxicity lead to dysfunction of cell membrane structure, inhibition of glycogen synthesis, and an increase of IR (38). Moreover, fat accumulation promotes inflammation and a thrombotic state that exacerbates atherosclerosis (39). The relationship between elastography measurements and NAFLD has been extensively documented in clinical trials (40, 41). In summary, our data confirm that CAP levels are closely related to MetS components. In clinical practice, determination of MetS patients relies on clinical examination and blood tests. However, based on the non-invasive, rapid, simple, and reproducible characteristics of CAP, adding it to the clinical assessment of MetS development might improve detection and risk stratification. Even inclusion of NAFLD into the definition of the metabolic syndrome could be a step to better identify the population with metabolic risk (42).

As the n-3 PUFA have been proven to lower TGs and reduce cardiovascular risk (19), studies have shown that n-6 and n-3 PUFA serum levels are negatively correlated with TGs [e.g. (43)] and that n-3 PUFA administration lowers triglycerides also in healthy volunteers (20), as well as in patients (44). Both higher serum n-6 and n-3 PUFA were associated with a lower risk of NAFLD (45). However, our data do not show these inverse relationships between n-3 and n-6 PUFAs observed elsewhere. This might be due to the fact that we measured plasma and not serum samples. Indeed, except for significantly higher levels of palmitic acid, which is known to promote insulin resistance in T2DM patients (46), oleic acid, and lower levels of γ-LA, and 11-eicosenoic acid, we did not find significant differences, and not even a trend toward higher levels of n-3 and n-6 PUFAs in the high TG (and high CAP) group. Further investigation may be needed to consider not only different types of samples but also other potential factors contributing to the observed differences, such as variations in study populations, dietary habits, or methodology. This could help clarify the relationship between n-3 and n-6 PUFAs and TG levels.

As shown in the present study, monohydroxy metabolites of PUFAs were significantly higher in those patients with high TGs than in those with normal TGs, with a broad range of AA- as well as EPA- and DHA- metabolites displaying significant increases. In contrast, in the study by Schuchard et al. there were only slightly higher serum concentrations of 5-HEPE, a lower concentration of 12-HETE in hyperlipidemic patients compared to normolipidemic patients (47). In this population diabetes was not studied, though.

Increased TG levels can provide more FAs substrates for oxylipin production. Therefore, as TGs are broken down, they release FAs that can be converted into oxylipins through enzymatic oxidation (26). Recent research has further supported this observation, indicating a linear relationship between the generation of oxylipins and triglyceride oxidation (48). Meanwhile, gene expression also appears to influence the route of PUFA metabolism. Several studies have indicated that high TGs activate PPAR-alpha, leading to increased expression of genes involved in beta-oxidation and promoting the expression of inflammatory oxylipins (49). Moreover, excess TGs and their FFAs can cause accumulation of lipids, and then trigger the activation of stress-responsive signaling pathways. The activation of these pathways can lead to a comprehensive increase in the metabolites of PUFAs (50).

Oxylipins are bioactive lipids, and changes in their levels reflect underlying inflammation or metabolic disturbances. There have been recent attempts to use oxylipins for stratification of MetS patients, showing that levels of a number of mono- and dihydroxy as well as epoxy metabolites were higher in MetS (51). In another study, it was shown that 5-/8-/and 12-HETE were increased in obese subjects with low-grade inflammation. However, weight reduction over an 8-week intervention led to a significant reduction of these oxylipins (52). There is also work ongoing to use oxylipin for liver disease risk evaluation [reviewed in (53)]. A comprehensive study was conducted to assess oxylipins as progression indicators from NAFLD to NASH and observed increases in various oxylipin classes with advancing disease stage (54). Another study has previously demonstrated in a lipidomic analysis of NAFLD/NASH that significantly increased levels of AA-products such as 5-HETE, 8-HETE and 15-HETE and 11-HETE indicate progression towards NASH (55). In a study of children with NAFLD, hepatic epoxyeicosanoids significantly increased with higher grades of steatosis (56). Therefore, our lipidomic analysis may also provide valuable insights into the metabolic risk stratification of T2DM.

Additionally, it is well-established that elevated levels of CYP- and LOX-metabolites, such as HETEs and LTs leads to inflammation, oxidative stress, endothelial dysfunction, and peripheral vascular resistance, and are generally associated with increased CVD risk. (57). 20-HETE has been reported to be associated with vascular inflammation and injury, angiogenesis, which are crucial factors in the development and progression of CVD (58). 9-HETE as a marker of oxidative stress was elevated in patients with coronary artery disease (57). In contrast, EETs help dilate arteries and reduce inflammation, but their sEH products may negate the protective effects (59). Given the experimental data implicating n-3 PUFA derived inflammation-dampening oxylipins in the alleviation of metabolic liver disease (25–27, 60), our data presented here support a concept of optimizing n-3 PUFA levels by identifying individuals with fatty acid imbalance and evidence of excess inflammation, rebalancing oxylipin profiles could significantly impact outcomes. This could alleviate metabolic disease-associated inflammation due to increased n-3 inflammation dampening oxylipin (n-3 IDO) formation with increased TGs, and liver fat, in patients with T2DM. Our study has several limitations. The majority of patients were enrolled during presentation to our gastroenterology and endocrinology unit, ranging from routine to presentations due to comorbidities (see Table 1). Although in multivariate linear regression, the positive effect indicators for two groups of HTG and non-HTG patients with TG levels showed no significant between-group associations (Supplementary Table 2), there may still be some confounding factors in our results, which should be interpreted with caution. Specifically, first, our patients with a higher BMI and higher triglycerides showed slightly better blood glucose control, which may be contrary to the experience in clinical practice. Second, there was probably some undertreatment with regard to guideline-based therapies, as evidenced by low prescription rates of statins. Last, our focus on diabetic patients in this study was based on the well-established association between diabetes and HTG, as well as a higher risk of cardiovascular disease. Future studies with more resources could expand the scope to investigate non-diabetic individuals as well.

The findings presented here indicate a clear association between TGs, BMI, and hepatic steatosis as measured by CAP in patients with T2DM. Compared to normal TG subjects, the unique feature of high TG subjects was the upregulation of CYP450 and LOX activity, accompanied by elevated oxylipin levels, potentially contributing to an inflammatory metabolic state.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

The study involving human participants was reviewed and approved by Ethics Committee of Brandenburg Medical School. The patients/participants provided their written informed consent to participate in this study.

Conceptualization: UE, YX, and KW. Methodology: AJ, MR, AP, NR, and KW. Analysis: YX, UE, and KW. Investigation: YX, UE, AJ, AP, and KW. Data curation: AJ, AP, and YX. Writing—original draft preparation: YX and KW. Writing—review and editing: YX, UE, and KW. Visualization: YX, and KW. Supervision: UE and KW. Project administration: UE and KW. All authors have read and agreed to the published version of the manuscript. All authors contributed to the article.