Effect of irisin on metabolic and platelet functions in type 2 diabetic rats: role of soluble receptor of advanced glycation end products (sRAGE)

Irisin is an adipomyokine with a promising potential for the treatment of metabolic disturbances and endothelial dysfunction. This study aimed to explore the effect of irisin on metabolic and platelet functions, and to explore the possible involvement of soluble receptor of advanced glycation end product (sRAGE) in the type 2 diabetes mellitus (T2DM) rat model. Thirty-three adult male albino rats were divided into three groups: normal control, vehicle-treated T2DM group, and irisin-treated T2DM. At the end of the study period, metabolic parameters, platelet count, mean platelet volume, platelet distribution width, plateletcrit, and serum sRAGE were determined. Irisin significantly improved platelet function and metabolic derangements induced by T2DM and significantly increased sRAGE. sRAGE was significantly negatively associated with platelet function parameters and some glucometabolic parameters. Additionally, mean platelet volume showed a significant predictive value for the change in serum sRAGE. Irisin could have a protective role against diabetes-induced platelet dysfunction by increasing sRAGE levels, indicating the potential beneficial effects of sRAGE in the type 2 diabetic rat model.


Background
Type 2 diabetes mellitus (T2DM) is a serious, global metabolic health problem with a high prevalence and morbidity that is complicated by atherosclerosis and circulatory dysfunction [1]. The high incidence of thrombotic complications reported in T2DM patients is attributed to altered platelet morphology and function, since platelets have been found to be large and hyperactive with increased adhesion and aggregation [2].
Boström et al. [3] discovered irisin, which is a novel myokine that is secreted from exercised skeletal muscle cells by cleavage of the fibronectin type III domaincontaining protein 5 (FNDC5) via the activation of peroxisome proliferator-activated receptor γ (PPARγ) co-activator-1α (PGC-1α). Recent studies have shown that irisin is also secreted from white adipose tissue and may regulate several physiological and metabolic pathways that protect against obesity and complications from insulin resistance (IR) [4,5]. Recently, it has also been reported that irisin has anti-inflammatory, anti-oxidative, and anti-apoptotic properties, which play a significant role in the protection from many diseases, such as atherosclerosis and myocardial infarctions [5].
Hyperglycemia contributes to the formation of advanced glycation end products (AGEs), which are vital to the progression of diabetic micro-and macrovascular complications that occur when the receptors of AGE (RAGE) are bound [6]; however, soluble RAGE (sRAGE) inhibits AGES interaction with RAGE cell surfaces, thus attenuating the hazardous effects of AGES [7].
The aim of this study was to investigate the effect of irisin treatment on metabolic and platelet function, to explore the effect of irisin treatment on sRAGE, and to demonstrate the possible association between sRAGE and platelet function in experimentally induced T2DM in adult male albino rats.

Animals
This study was performed at the Physiology and Medical Biochemistry & Molecular Biology Departments, Faculty of Medicine, Zagazig University, Zagazig, Egypt.
A total of 33 adult male albino rats weighing 200 ± 15 g were obtained from the Animal House at the Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. The animals were kept in steel wire cages (5-6 animals/ cage) at the Animal House of Faculty of Medicine, Zagazig University, Zagazig, Egypt under hygienic conditions at room temperature with natural light/dark cycles and were fed standard chow with free access to food and water.

Grouping
After one week of acclimatization, animals were divided into two groups: Group I (control; n = 11) animals were fed the normal chow diet with 5% energy from fat, and total calorific values of 20 kJ/kg [8]; and Group II (T2DM group; n = 22). Group II rats were further divided into two subgroups: Group IIa (vehicle-treated diabetic group; n = 11) was treated with saline; Group IIb (irisin-treated diabetic group; n = 11) was treated with irisin, administered at 1 μg/kg daily for 14 days intra-peritoneally [9].

Induction of diabetes
The rats were injected subcutaneously with 230 mg/kg nicotinamide (NA). After 15 min, diabetes was induced by subcutaneous injection of streptozotocin (STZ; 35 mg/kg) in 10 mmol/L, freshly prepared, ice-cold citrate buffer (pH 4.5). A second dose of NA and STZ were administered 24 h later. After induction, the animals in this group were switched to a high fat diet (40% energy from fat) [10]. A week later, rats with a fasting blood glucose (FBG) between 180 and 220 mg/dL were considered diabetic and included in the study [11]. All drugs were obtained from Sigma (St. Louis, MO, USA). The diet was obtained from the Animal House of Faculty of Medicine, Zagazig University, Zagazig, Egypt.

Serum glucometabolic and platelet parameters
Twenty-four hours after the last irisin injection, overnight fasted rats were weighed, then killed under ether anesthesia, and 1 ml blood samples were collected in EDTA containing tubes. These samples were used immediately to determine platelet count, mean platelet volume (MPV), platelet distribution width (PDW), and plateletcrit using an automated cell counter (Benesphera, the Netherlands). Additional 3 ml blood samples were collected in clean, plastic centrifuge tubes, and allowed to coagulate. Afterward, the serum was separated by centrifugation at 3000 rpm for 15 min. The separated serum was stored at − 20 °C until assayed for glucose using the enzymatic colorimetric method (Spinreact, Spain) [12]. Insulin levels were measured using rat insulin enzymelinked immunosorbent assay (ELISA) kits (BioSource, Europe S.A.-Rue de l'Industrie, 4-A-1300 Nivelles-Belgium) [13].

Statistical analysis
The results of this study were expressed as mean ± SD. Data were analyzed using one-way ANOVA, followed by the Post hoc test (LSD) to test for differences between groups. Pearson correlations were also performed. Multiple linear regression analysis was performed with sRAGE as the dependent variable. p < 0.05 was considered significant. The statistical analysis was performed using the SPSS program (version 18 for windows; SPSS Inc. Chicago, IL, USA).

Effect of irisin treatment on metabolic parameters
There was a significant increase in the final body weights (BW) of both diabetic groups (IIa and IIb) compared to the control group (p < 0.05) but there was no significant difference in the BWs of the two groups (p > 0.05; Table 1).
There was a significant increase in serum glucose, insulin and HOMA-IR in group (IIa) compared to the control group (p < 0.05). The irisin-treated diabetic rats (IIb) showed a significant reduction in the above-mentioned parameters compared to the diabetic group (IIa; p < 0.05, p < 0.05, p < 0.001, respectively). Serum glucose, insulin, and HOMA-IR were significantly higher compared to the control (p < 0.05, p < 0.05, p < 0.001, respectively; Table 1).
The lipid profiles showed a significant increase in TC, TG, LDL, and VLDL in the diabetic groups (IIa and IIb) compared to the control group (p < 0.001, p < 0.001, p < 0.05, p < 0.001, respectively); however, these parameters were significantly decreased in group IIb after irisin treatment compared to group IIa (p < 0.05; Table 1). In addition, serum HDL showed a significant decrease in group IIa compared to the control (p < 0.05). HDL was significantly increased in group IIb compared to group IIa (p < 0.05), with no significant change compared to the control group (p > 0.05; Table 1).

Effect of irisin treatment on sRAGE
Serum sRAGE was significantly decreased in diabetic group IIa compared to the control group (p < 0.05). Group IIb displayed a significant increase in serum sRAGE (p < 0.05) compared to the diabetic group (IIa; p < 0.05), but the difference was not significant compared to the control group (p > 0.05; Fig. 1).

Effect of irisin treatment on platelet function
T2DM (group IIa) induced a significant increase in platelet count, MPV, PDW, and plateletcrit compared to the control group (p < 0.001, p < 0.001, p < 0.05, and p < 0.001, respectively). Intraperitoneal administration of irisin induced a significant reduction of all of these parameters compared to the untreated diabetic group (IIa; p < 0.05). In contrast, platelet count, PDW, and plateletcrit were significantly higher after irisin treatment in group IIb in comparison to the control group (p < 0.01, p < 0.05, p < 0.05, respectively). No significant change in MPV was found between group IIb and the control (p > 0.05; Table 2).
The results of the linear regression analysis showed a significant linear regression with 85% predictive value of MPV to the change in the serum level of sRAGE (β coefficient = 0.851, p < 0.05); however, none of the other independent variables showed significant predictive value for sRAGE (Table 4).

Discussion
RAGE and sRAGE are involved in DM-related disorders. sRAGE is composed of two components: the endogenously secretory form which is the real receptor and the membrane-cleaved form which is an inflammatory marker [17].
In this study, STZ displays cytotoxic action caused by increases in the production of reactive oxygen species (ROS) by pancreatic β-cells, which induces DM. Additionally, the combination of high fat diet (HFD) with STZ has been reported to induce IR and hyperglycemia [18]. Nicotinamide has been shown to mediate a partial protection against cellular damage induced by STZ [19]; thus, this animal model mimics human T2DM with IR [10,20].
We found that the body weights in the diabetes group were significantly increased compared to the control group. This is in line with the findings of Veerapur et al. [21]. Additionally, the intraperitoneal administration of STZ significantly elevated FBG levels, with a significant increase in serum insulin levels, and HOMA-IR, which has also been reported previously [22,23].
Our results also showed that irisin administration caused a significant reduction in serum glucose and insulin levels in STZ diabetic rats, which agrees with Liu et al. [24], who explained that by the promotion of β cell proliferation. Irisin enhances glucose uptake and inhibits gluconeogenesis, thus leading to improved glucose utilization in T2DM rats [25]. These effects occur via activation of the 5′ adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, and acetyl-CoA-carboxylase in hepatic cells, an effect that plays a significant role in cellular energy homeostasis [26].
We also found a significant increase in TC, TG, and LDL-C in the untreated, diabetic group compared to the control. These results are in line with those of other investigators [22,27]. Irisin administration resulted in a significant decrease of TC and TG in the STZ diabetic   rats. Similar results also reported that irisin administration decreased plasma TC and TG levels, suggesting that irisin improves energy metabolism in adipose tissue [2]. The platelet parameters showed a significant increase in platelet count, MPV, PDW, and PCT in the diabetic group compared to the control group, which has also been previously noted [27]. Irisin treatment significantly reduced these parameters compared to the untreated diabetic group. Increased MPV is an indicator of platelet size in T2DM and is attributed to the osmotic swelling of platelets caused by high levels of glucose metabolites [2]. This finding is consistent with other studies that have also reported increased MPV in diabetic patients compared to non-diabetic controls [2,28]. In diabetic patients with atherothrombotic vascular complications, MPV could also be elevated as result of the compensatory production of reticulated platelets due to the increased consumption of small platelets [2]. The increased PDW was attributed to enhanced platelet production that caused qualitative changes, such as the production of different sized platelets and activated platelets that change from a discoid to a spherical shape with pseudopodia formation, leading to a change in PDW [29]. These findings correspond to those of Alhadas et al. [27] and Dalamaga et al. [30], who noticed that PDW was higher in diabetic patients compared to non-diabetics. Increased PCT could be caused by the increased platelet size and reactivity in type 2 diabetic patients, as these can lead to increases in platelet mass, thus resulting in increased PCT [27].
In DM, the high levels of AGE and RAGE ligands can induce pro-inflammatory and pro-thrombotic states, endothelial cells dysfunction, and vascular leakage [31]. In addition, many factors interact to cause platelet hyperreactivity, such as hyperglycemia and hyperlipidemia, IR, overexpression of inflammatory and oxidant stress mediators, glycoprotein receptors, and growth factors [2]. Hyperglycemia decreases platelet membrane fluidity, thus increasing platelet activation [32]. Dyslipidemia induces a pro-thrombotic state, which is associated with platelet hyper-reactivity [33]. A decreased platelet count in diabetic type 2 patients compared to non-diabetic patients, and the presence of other factors affecting platelet counts, such as the mean platelet survival, platelet production, and their turnover rate has been previously suggested [34], which disagrees with our results. This difference could be related to differences in the genetics, species, or disease duration in the respective studies.
The primary finding of this study was the significant decrease of platelet parameters in the irisin-treated group compared to the untreated diabetic group. This finding is attributed to irisin's effect on blood glucose, as evidenced by the significant reductions in serum glucose, insulin, and HOMA-IR, accompanied by significant improvements in lipid metabolism, which potentially plays a role in the reduction of platelet hyperactivity.
Also, we found a significant decrease in sRAGE levels in the diabetic group compared to the control group; however, irisin treatment in the treated diabetic group significantly increased sRAGE levels compared to the diabetic group.
Irisin is a promising key player in metabolic diseases and other diseases which improve with exercise [35]. Irisin has anti-inflammatory properties that inhibit the expression and release of inflammatory mediators [6]. Li et al. [6] found that patients with endothelial dysfunction had low irisin levels, which were negatively correlated with AGE [6]. Moreover, irisin decreased the atherosclerotic plaque area and inflammatory response in STZinduced diabetic apolipoprotein E-knockout mice [36]. Irisin reduced oxidative stress, inhibited glucose-induced apoptosis, and upregulated the proliferation and angiogenesis of human endothelial cell lines [37,38].
We found a significant negative correlation between sRAGE and platelet count, MPV, PDWs, and PCT. In addition, linear regression analysis demonstrated that MPV had a significant predictive value for the change in serum sRAGE. Our results agree with those of Fujisawa et al. [42], who reported that sRAGE was negatively associated with metabolic syndrome, and that its low level was associated with increased cardiovascular risk factors in this condition. Interestingly, the significant negative correlation between sRAGE and serum FBG, HOMA-IR, TC, TG, LDL, and VLDL levels in this study supports these findings [39]. sRAGE has anti-inflammatory effects by blocking the interactions between AGEs and membrane-bound RAGE, and by preventing the effects of AGEs [40,41]. Thus, increased sRAGE prevents T2DMinduced endothelial cell injury and inhibits the hyperactivation of platelets [42].

Conclusions
It could be deduced from our results that circulating serum sRAGE levels could represent a novel biomarker of vascular complications. The treatment of STZ-induced type 2 diabetic rats with I.P. irisin for 14 days improved platelet profile and metabolic status. Moreover, mean platelet volume is an independent predictive factor for serum sRAGE level in diabetic rats. These results suggest that the treatment with irisin might be a novel mechanism for the beneficial effects in patients with type 2 diabetes.

Limitations and recommendations
The data from this study on the effects of irisin on the AGE-RAGE axis are limited. Further studies are needed to understand the clinical significance of irisin supplementation in the context of RAGE and sRAGE regulation. Therapeutic antagonism of RAGE-dependent signaling could provide a new target for prevention of diabetes mellitus complications, particularly those associated with the increased risk of thrombosis.