According to the results of the present research, there is a notable increase in BW following HFD utilization. Moreover, it was found that some strains (Wistar and Sprague-Dawley rats) are more vulnerable to obesity when receiving HFD, which may be due to the hyperphagic nature of these animals that are resistant to the insulin function and the reduced hypothalamic peptides. Furthermore, the alterations in BW gain mostly depend on a large extent fat composition of the diet (29–31), the deposition of the saturated fats resulted from the consumption of a diet rich in energy in different layers of body fat due to the reduced energy consumption compared to animals fed with a normal diet (32, 33). A non-significant BW loss was also shown in our results after the injection of STZ that can increase levels of leptin and adiponectin and a concurrency between adipogenesis and lipolysis in the diabetic rats (34, 35). The reduced BW in diabetic rats in the present study is consistent with the results of the Gundala’s study in 2019 (36). It was suggested that ω-3 PUFA enhances lipid oxidation through the activation of peroxisome proliferator-activated receptor α (PPAR α) and suppression of lipogenic enzymes, which make obesity resistance (37, 38). Additionally, Li et al. (2012) (39) in their study reported that iron can cause loss of BW in HFD/STZ-treated rats compared to the control group. However, its precise mechanism has not been determined yet (39).
Sahin et al. 2007 (40) suggested that the HFD/STZ model can be considered as a novel animal model for the induction of T2DM and the study of antidiabetic and anti-inflammatory compounds (40). Srinivasan et al. (2005) reported a significant increase in FBS in diabetic rats after the STZ injection compared to the normal control group (34). This is likely due to compensatory hyperinsulinemia to control glucose homeostasis (41). After the treatment period, FBS levels were significantly higher in diabetic groups compared to the normal control group. Besides, a non-significant decrease in FBS levels were observed in the D.ω group. The positive effect of ω-3 PUFA on increasing glycolysis and glycogenesis and its negative effect on lowering FBS levels via increasing insulin resistance and exacerbating glucose intolerance have also been reported (42) (43–46).
The mechanisms of STZ include elective devastation of pancreatic beta cells and a decreased activity of insulin secretion (47, 48). It was shown that chronic hyperglycemia leads to the formation of AGE through the glycation process of long half-life proteins and lipids. Accordingly, the accumulation of these compounds and their binding to RAGE cause cross-linking in collagen molecules, which consequently increase myocardial thickness, oxidative stress, and fibrosis, and eventually damage myocardial cells (7, 8). Furthermore, it has been reported that oxidative stress can lead to the development of the glycation process. In contrast, it was found that AGE increases its level by accelerating the oxidation process (4). Thus, the oxidative stress caused by the binding of AGE to its receptor, in turn, leads to numerous pathological changes in the gene expression and impairs the structure and function of the heart in diabetic patients (49, 50). According to the findings of the present study, the increased OSI was observed in the heart tissues of the groups D, D.F, and D.ω.F compared to the CN group. Moreover, there was a significant increase in the OSI level in the groups D, D.F, and D.ω.F compared to the D.ω group. Furthermore, the administration of ω-3 PUFA as a supplement, significantly reduced OSI compared to the group D.
In line with the findings pf a study by Flavia et al (2014), the effective role of iron supplements in the high production of ROS was one of the major mechanisms proposed to explain cardiac changes, myocardial cell death, and cardiac fibrosis in diabetic rats (51). Ginty and Conklin (2012) suggested that the ω-3 PUFA diet may improve the antioxidant capacity of diabetic rats (52). In the present study, it was expected that iron supplementation would have a negative outcome on the antioxidant effect of ω-3 PUFA supplementation. After comparing the group D.ω.F with the group D.F, it was found that the level of OSI has more significantly increased. Therefore, concurrent use of both supplements in diabetics can reduce the oxidative capacity, may be due to the negative effect of free radicals, resulted from the reactions of the divalent metal iron, on the antioxidant properties of ω-3 PUFA. In contrast, some studies have shown that diets containing fish oil, besides increasing cellular oxidative damage, produce antioxidant effects (30).
Based on the evidence, ferrous sulfate causes damage to cell membranes and their lipoproteins. This damage occurs through high production of toxic hydroxyl radicals resulted from the Fenton/Haber-Weiss reaction, followed by the free radical chain reaction, called lipid peroxidation, and the increased production of malondialdehyde (53). It is noteworthy that some factors like AGEs cause a substantial rise in PUFA peroxidation and MDA production in the presence of Fe+ 2 and Fe+ 3 (54). In the present study, it was found that the groups D, D.F, and D.ω.F had a significant increase in MDA level, as a lipid peroxidation index, in comparison with the group CN, probably due to the effect of divalent metals on raising the amount of lipid peroxidation. However, there was a non-significant decrease in lipid peroxidation in the group D.ω compared to the group D. Additionally, a study by Wei Bao et al. (2012) showed that there is no association between dietary or supplementation of iron and the risk of T2DM (55).
According to the results, gene expression of RAGE significantly increased in the heart tissues of the group D compared to the group CN. In addition, compared to CN group, the induction of T2DM in group D more increased gene expression and tissue level of TNF-α, as an inflammatory cytokine, as well as gene expressions of ICAM-1 and VCAM-1, as cardiac inflammation markers. These results are consistent with the findings of the studies by Evangelista et al. (2019) and Cheng et al. (2017) (9, 56).
According to the previous reports, it can be concluded that by increasing tissue level of AGE, tissue level of ROS; the gene expressions of RAGE, ICAM-1, VCAM-1; and inflammatory cytokines such as TNF-α increase. However, the increased levels of these factors eventually lead to some cardiac complications and diabetic cardiomyopathy (9, 57).
Tschöpe et al. in 2005 suggested a possible mechanism regarding the correlation among the genes expression of CAMs, cardiac inflammation, and cardiomyopathy in diabetic situation. According to their report, several pathogenic mechanisms such as the expression of pro-inflammatory cytokines, oxidative stress, and hyperglycemia, are responsible for the induction of endothelial CAMs. So, there is a significant relationship between cardiac parameters and the inflammatory endothelial activation through the elevated levels of CAMs (ICAM-1 and VCAM-1) and proinflammatory cytokines (TNF-α and IL-1β) induced by CAMs (6). Under the hyperglycemic conditions, the role of transitional metals such as copper and iron in protein glycation process and ultimately in the pathogenesis of cardiac diabetic complications have been identified. The high affinity of the glycosylated proteins to transitional metals leads to the maintenance of the reduced and active state of the metal and ultimately catalytic oxidation (58). Nevertheless, prescribing iron and vitamin supplements as well as the supplements containing DHA and EPA, as anti-inflammatory agents, to T2DM patients are recommended to control and improve metabolic disorders effectively (11, 12). Omega-3 lowers obstruction, inflammation, fibrosis, and hypertrophy caused by heart disease through disrupting JNK signaling, reducing TNF-α expression, and producing molecules involved in cardiac inflammation such as ICAM-1 and VCAM-1 (59–61). Besides, the expression of genes and tissue level of the TNF-α have significantly induced in the D.ω.F group compared to the D.ω group. In the current study, it was found that only the D.ω group significantly decreased gene expressions of RAGE and TNF-α and tissue level of TNF-α protein. It was also found that the gene expressions of RAGE and TNF-α, and tissue level of TNF-α protein significantly increased in the groups D.F, D.ω.F, CN, and D.F compared to the group D.ω. Although the administration of omega-3 alone decreased genes expression of ICAM-1 and VCAM-1 compared to the group D, co-treatment with Fe and omega-3 in diabetic rats led to a significant increase and a non-significant increase in the levels of genes ICAM-1 and VCAM-1, respectively. It was also found that in hyperglycemia, iron supplementation has a negative effect on the anti-inflammatory properties of ω-3 PUFAs by catalyzing their oxidation (62). In contrast, a study by Kosacka (2016) reported that a lack of iron in the diet caused a mild inflammation in diabetic rats (63).
A diabetic animal model with STZ-HFD induced mild hyperglycemia, hypertriglyceridemia, hypercholesterolemia, and conditions similar to insulin resistance in pre-diabetic humans. In this regard, it seems that the induction of diabetes mainly occurs through the glucose-fatty acid cycle or Randle cycle (64). Following diabetes disease and its significant effect on changing plasma and tissue lipid profiles, the increased tissue deposition of cholesterol and triglycerides and the increased risk of cardiomyopathy occur in patients with T2DM. Naidu et al. in their study in 2015 reported that the increased levels of blood triglyceride and cholesterol followed by a significant increase in their tissue levels have occurred in the hearts of HFD-STZ-induced diabetic rats compared with the normal group. By inducing T2DM in an animal model, following the consumption of HFD, uptake, and formation of triglycerides in the form of chylomicrons, the production of endogenous very-low-density lipoprotein rich of TG (VLDL-1, VLDL-2) has increased, and triglyceride uptake by peripheral tissues through a low lipoprotein lipase (LPL) activity has decreased. Therefore, it can be stated that hypertriglyceridemia and its cardiovascular complications are caused by diabetes (10). The findings of the present study showed that the levels of cholesterol and triglycerides were significantly higher in the heart muscle tissues of the group D compared to the group CN. On the other hand, several studies have shown the relationship of the level of triglyceride accumulation in heart tissue with heart dysfunction in patients with T2DM (65, 66). However, this relationship has been rejected in a study by by McGavock et al. in 2012 (67). A prominent feature in several models of cardiomyopathy is the intracardiac accumulation of TG due to the impaired cardiac free fatty acids (FFAs) metabolism and FFAs leakage into the heart. Additionally, adipose tissue, as the source of most of the circulatory FFAs due to lipolysis, can indirectly affect cardiac metabolism by affecting the serum concentrations of the FFAs. In addition, it has been shown that total body fat and circulating FFAs levels can lead to a sudden and significant increase in insulin resistance through the accumulation of high rate of TG in the heart (68). The reduced insulin's role, as a regulator of lipid homeostasis in insulin resistance, is accompanied with the increased lipogenesis in hepatocyte and lipolysis in adipocytes leading to the increased blood FFA and TG as well as the impaired handling lipids levels in adipose tissue, liver, and muscle. Thus, insulin resistance leads to some metabolic and functional disorders of the heart and the increased insulin levels stimulate the transfer of FFAs to cardiomyocytes (69, 70). Subsequently, hyperlipidemia and compensatory hyperinsulinemia enhance the transfer and utilization of FFAs in myocytes. Accordingly, if myocytes receive FFAs neyond their oxidative capacity, they would be accumulated leading to lipotoxicity. This phenomenon increases cardiac dysfunction through various mechanisms such as ROS production and the increased insulin signaling disorder (68). Accordingly, the findings of the lipid profile in the heart tissues of the studied rats showed that the tissue levels of total cholesterol and triglyceride had a significant increase in the groups D and D.F compared to the group CN. Due to the wide application of iron supplements and multivitamins, and on the other hand, the extensive tissue damage in diabetes due to iron storage through high production of ROS, low iron intake in the diet is recommended to have a beneficial effect on lowering serum and tissue cholesterol and triglycerides levels as well as heart pathology.
Additionally, it was indicated that the increased liver iron levels increase cholesterol by producing glutathione, which increases the activity of HMG-CoA reductase, a rate-limiting enzyme in cholesterol biosynthesis. Therefore, the increased liver iron resulted from supplements is likely to cause lipemia, cardiac TG accumulation, and severe pathological changes (51, 71, 72). The findings of the present study confirmed the possible effect of ω-3 PUFAs supplementation on lowering triglycerides and total cholesterol levels in type 2 diabetics, which is line with the findings reported by Friday et al. (1989) and Rudkowska (2010) (44, 73). Considering the relationship between blood FFAs levels and cardiac TGs accumulation (74), it is expected that ω-3 PUFAs are effective on reducing cardiac TGs through reducing plasma TG levels, improving lipemia by stimulating the intestine to reduce chylomicron secretion, increasing LPL activity and tissue blood flow, and improving the stimulatory effect of insulin and PPARs receptors on increasing chylomicron clearance (75, 76). The present study revealed the reduced tissue levels of total cholesterol and TG in the group D.ω compared to the other diabetic groups; however, this reduction was only significant in comparison with the group D.F.
Histological findings revealed some pathological changes caused by diabetes in the heart of animals. Following the defects in the proteins that make up the hallmark of the heart muscle, the intercalated discs (IDs) as an intercellular communication surrounding, has emerged cardiomyopathy or other heart disorders (77, 78). Besides, changes in the organization of the layered structure of heart tissue along with changes in the deformation and location of the myocytes nucleus, which is usually spindle-shaped and located along with the longitudinal axis or in the center of the cell, may indicate a change in the morphology of the heart muscle cell in diabetes (79, 80). In conclusion, the findings of this study confirmed the devastating effects of iron on the anti-inflammatory effect of omega-3 polyunsaturated fatty acids on the heart of diabetic rats