*3.5. Publication Bias*

Funnel plots of insulin levels and HOMA-IR were drawn with Review Manager version 5.3, and Egger's test was conducted to quantify the publication bias with Stata version 17.0. The two funnel plots were substantially symmetrical (Figure 14). The results of Egger's test for insulin levels and HOMA-IR were *p* = 0.678 and *p* = 0.908, respectively, indicating that there was no publication bias.

**Figure 14.** Funnel plot of insulin levels and HOMA-IR. (**A**) Insulin levels, (**B**) HOMA-IR.

#### **4. Discussion**

Over the last decade, increasing attention has been paid to the selenium status in patients with various cardiometabolic diseases and the association between selenium status and mortality risk of various cardiometabolic diseases. Several meta-analyses have indicated that individuals with cardiometabolic diseases tend to have lower selenium levels than healthy individuals [50–52], and that selenium levels are negatively associated with mortality among patients with MS, T2DM, and CVD [53–55]. Alterations in the metabolism of glucose and lipids characterize metabolic disorders [56]. Insulin has regulatory effects on glucose, which are mainly classified into two aspects: promoting glucose absorption into liver cells, muscle cells, and adipose tissue, and inhibiting glycogenolysis and gluconeogenesis in the liver [57]. Insulin resistance is a common pathophysiological status in which individuals have decreased insulin sensitivity and impaired glucose regulation by insulin, resulting in glucose intolerance [58]. It is well-established that insulin resistance underpins many chronic metabolic diseases [59], and cardiometabolic risks such as obesity and dyslipidemia can exacerbate insulin resistance and impel the progression of CMDs [1]. Studies have shown that the micronutrient selenium can regulate the human body's insulin sensitivity, and selenium in the form of selenate is known to act as an insulin mimetic with a role in maintaining blood sugar homeostasis [60,61]. Expression of selenoprotein *P* plays a crucial role in pancreatic β-cell function by preventing ferroptosis and thus maintaining glutathione peroxidase 4 (Gpx4) and cell viability, and also by inhibiting stress-induced degradation of nascent granules, a novel regulatory pathway for insulin [62], thus maintaining cellular proinsulin levels [63]. Furthermore, a meta-analysis performed by Tabrizi et al. [64] supported the positive effect of selenium supplements on lipid profiles. Of note, that selenium supplementation makes the most sense when there is deficit of selenium [14,65].

In this meta-analysis, we examined the impact of selenium supplementation on the markers of insulin resistance, glucose, as well as blood lipid profiles in patients with CMDs. The level of glucose in the blood is one of the most significant homeostatic indicators and is strictly regulated [66]. The pathways and regulation of glucose metabolism include glycolysis/glycogenolysis, gluconeogenesis, pentose phosphate pathway (PPP), insulin signaling pathway, and many others [67], some of which can be regulated by insulin [57]. HOMA-IR is often used in investigating and quantifying insulin resistance because of the simplicity of the underlying mathematical model (HOMA-IR = fasting glucose [mg] × fasting insulin [mu/L]/22.5) [68]. Furthermore, HbA1c is an essential marker of longterm glycemic control that reflects a cumulative glycemic level over the past two to three months [69]. Therefore, insulin levels, HOMA-IR, FPG and HbA1c were used to evaluate the selenium supplementation on glycemic control. In this study, comprehensive pooled results from 10 RCTs involving 526 patients supported the favorable effects of selenium supplementation in decreasing serum insulin levels and HOMA-IR. Moreover, selenium supplementation may increase HDL-C levels, but the effectiveness of selenium supplementation on FPG, TC, TG, LDL-C, and VLDL-C levels was unclear. The current results suggest that selenium supplementation may be an effective treatment for reducing insulin resistance.

The findings reveal that selenium supplementation could reduce insulin levels and HOMA-IR in patients with CMDs, but the effect on FPG was ambiguous. This result is similar to that of the meta-analysis conducted by Strozyk et al. in 2017, which included four RCTs [70]. Their study focused on assessing the efficacy of selenium supplementation in T2DM, and the results supported that selenium supplementation significantly improved insulin resistance. However, our study also focused on other cardiometabolic diseases, such as cardiovascular disease, and collected RCTs that have been updated recently. Therefore, 10 RCTs were included in this meta-analysis. The pooled results derived from the included studies are in line with those of previous animal studies [71–73], in that supplemental selenium therapy had a significantly protective anti-diabetic effect. Selenium nanoparticles (selenium-NPs) are a new supplemental form of selenium that is readily absorbed and

has low toxicity. According to Abdulmalek et al. [74], treating diabetic rats with selenium-NPs (0.1 and 0.4 mg/kg) and/or metformin (100 mg/kg) separately or together, led to a remarkable decrease in FBG and insulin levels, suggesting that selenium and its derivatives play a significant role in the maintenance of glucose homeostasis. There is a lack of positive evidence regarding the effect of selenium supplementation on HbA1c. A crosssectional study reported that obese participants with lower selenium intakes exhibited higher concentrations of HbA1c [75]. In this regard, the effect of selenium supplementation on long-term glycemic control deserves further attention.

The pooled results of this study also demonstrated that selenium supplementation increased HDL-C levels, but had little effect on other blood lipids. In addition, in the subgroup of disease types, we found that selenium supplementation increased HDL-C levels more significantly in participants with cardiovascular disease, followed by those with diabetes mellitus. HDL-C is essential for reverse cholesterol transport and has antiinflammatory, anti-atherogenic, and anti-thrombotic effects [76]. The interaction of these properties contributes to the cardioprotective properties of HDL-C. Thus, appropriate selenium supplementation may contribute to the improvement of CMDs, particularly cardiovascular disease.

Some statistical heterogeneity was discovered in the pooled analysis of insulin levels, HOMA-IR, FPG, and HDL-C. The results were similar before and after sensitivity analysis, suggesting that the results were stable and reliable. In terms of insulin levels and HOMA-IR outcomes, we considered that Faghihi's study [48] administered inconsistent types or combinations of antidiabetic drugs at baseline, which might be an essential source of heterogeneity in the pooled analysis. In Faghihi's study [48], approximately 85.2% of participants in the control group received combined antidiabetic medication, compared to only 66.6% of participants in the selenium group. Additionally, subgroup analysis suggested that the inconsistency of participants' underlying diseases is a major source of heterogeneity in terms of FPG and HDL-C. In the future, more evaluable studies should be included in the analysis to better systematically evaluate the effectiveness of selenium supplementation in different types of CMDs.

This meta-analysis has several strengths. First, two reviewers independently used a comprehensive search strategy to identify all trials evaluating the effect of selenium supplementation in patients with CMDs and used standardized templates to extract data from included trials to guarantee data accuracy and reduce the impact of study design faults. Second, most included RCTs were high-quality with appropriate randomization, allocation concealment, and double-blinding methods. Third, no publication bias was found in this meta-analysis. Furthermore, we performed a thorough sensitivity analysis to examine the stability of our results. However, there are several limitations that should be considered. First, as the control group was placebo instead of different doses of selenium, the optimal dose of selenium supplementation cannot be accurately determined in this study. Second, the number of participants in the included RCTs was relatively small, with none having more than 100. Third, as only two studies evaluated HbA1c and there was high heterogeneity in the pooled analysis results, we could only perform a systematic review, which may affect the reliability and comprehensiveness of the evaluation of the efficacy of selenium supplementation on the HbA1c control. Fourth, although trials of the effects of selenium supplementation on CMDs have been conducted in countries other than Iran, trials in which other nutritional supplements were supplemented in addition to selenium in the intervention [77], and trials in which markers related to glucolipid metabolism were not reported [78] were excluded from this meta-analysis, and the final relevant included studies were all conducted in Iran, which may limit the generalizability. According to this, the recommendations of potential effects of selenium supplementation conclusions should be drawn with caution. Finally, we suggest that future clinical trials should pay more attention to the different doses of selenium supplements and the baseline level of serum selenium of CMDs patients, in order to further illuminate the therapeutic effects of selenium supplementation on CMDs.
