Insulin is a hormone that plays a crucial role in various metabolic processes in peripheral tissues, including glucose uptake, glycogen synthesis, and lipid synthesis. It also prevents the breakdown of energy molecules like glycogen, triglycerides, and proteins, thus facilitating energy storage. Insulin resistance (IR) refers to a persistent loss of insulin sensitivity [1], and is closely associated with the development of obesity-related chronic non-communicable diseases such as type 2 diabetes, nonalcoholic fatty liver disease and cardiovascular disease. Several risk factors contribute to IR, including impaired glucose tolerance, hypercholesterolemia, hypertriglyceridemia, hyperuricemia and hypertension [2,3]. These risk factors often occur alongside obesity, which arises when the body is over-nourished and excess nutrients are stored as lipids in adipose tissue. Prolonged obesity can result in the accumulation of fat in internal organs, leading to the infiltration of immune cells around unhealthy adipose tissue [4]. The excessive production of reactive oxygen species and pro-inflammatory responses can result in chronic inflammation. This chronic inflammation, caused by obesity, can in turn lead to IR [5].

The pathogenesis of IR is not fully understood, but it is widely accepted that it involves the impairment of the insulin signaling pathway downstream of the insulin receptor. In normal circumstances, when insulin binds to the receptor, it activates a series of downstream phosphorylation reactions. This leads to the recruitment and phosphorylation of receptor substrates such as insulin receptor substrate (IRS) and src homology 2 domain containing (Shc) proteins. Shc then activates the Ras-MAPK pathway, which affects cell proliferation and transcription. On the other hand, the IRS protein connects insulin to its metabolism through the PI3-kinase (PI3K) and Akt pathways. IRS1 generates second messengers and recruits and activates phosphoinositide-dependent kinase (PDK), which in turn phosphorylates and activates protein kinase B (Akt) and protein kinase C (PKC). Akt is responsible for mediating most of the metabolic actions of insulin, including the regulation of glucose transport, lipid synthesis, gluconeogenesis, and glycogen synthesis. Among these actions, glucose transport serves as the primary checkpoint for insulin to function properly. Most cells can uptake glucose through the membrane translocation of the insulin-stimulated glucose transporter type 4 protein (GLUT4), which helps maintain blood sugar balance. However, the ectopic accumulation of lipids caused by obesity can disrupt the normal mechanism of insulin action, leading to IR [6,7].

The current pharmaceutical drugs for treating IR primarily focus on stimulating insulin secretion or improving insulin sensitivity. However, these drugs often have side effects. Alongside medication, the impact of a healthy diet on improving IR highlights the potential role of nutrients in this regard [8,9]. Eicosapentaenoic acid (EPA) (C20:5n-3) and docosahexaenoic acid (DHA) (C22: 6n-3) are long-chain polyunsaturated fatty acids (n-3 PUFAs) derived from fish oil. EPA and DHA are primarily synthesized by the liver in the human body. However, the synthesis of these omega-3 fatty acids is limited, particularly in unhealthy conditions like obesity [10,11], therefore, it is essential to investigate the potential preventive and therapeutic effects of actively consuming EPA and DHA in managing diseases. Recent studies have shown a negative correlation between their supplementation and various metabolic syndromes. Nevertheless, the effect of EPA and DHA on improving IR remains controversial, and further research is needed to understand the underlying mechanisms. While many studies found that EPA and DHA supplementation is beneficial for improving IR, some studies suggested that short-term interventions may be more effective [12,13]. These studies primarily utilized a combination of DHA and EPA for intervention, and the varying ratios used in each study may contribute to the differences in research findings. However, there is still limited research on the mechanism by which EPA and DHA enhance IR. It has been observed that G-protein-coupled receptor 120 (GPR120) functions as a cell membrane receptor for EPA and DHA, while peroxisome proliferator-activated receptor γ (PPARγ) acts as a nuclear receptor [14,15]. A recent study suggested that GPR120 and PPARγ have functional interactions, and the combined use of a GPR120 selective agonist and thiazolidinediones (TZDs), a therapeutic agent targeting PPARγ, could potentially mitigate the known adverse effects of TZDs [16]. Therefore, this study aims to investigate whether different concentrations of EPA and DHA yield varying effects on improving IR, and if these differential effects are mediated through the GPR120 and PPARγ pathways. Additionally, considering the close association between obesity and IR [17], we are also interested in exploring whether EPA and DHA can improve IR by positively impacting adipose tissue inflammation.

This study initially examined the effects of EPA and DHA on insulin resistance and adipose tissue inflammation in mice, followed by cell experiments using inhibitors, co-culture, and other techniques to confirm the signaling pathways through which EPA and DHA improve insulin resistance. If the mechanism is to be verified in mice experiments, it is necessary to use GPR120 or PPARy gene knockout mice and mice with both genes knocked out at the same time. The knockout of these signals may impact downstream pathways related to lipid metabolism and insulin resistance in mice, potentially increasing mortality rates and introducing uncontrollable variables in the experiment [18], [19], [20]. Therefore, we chosed to only demonstrate the signaling pathway that EPA and DHA improve IR in vitro experiments.