Hepatic modulation of apelin and galectin-3 by darbepoetin-alpha in Dexamethasone induced insulin-resistant rats

Abstract

Background

Insulin resistance (IR) is a central pathological mechanism underlying metabolic syndrome and associated disorders, including type 2 diabetes and non-alcoholic fatty liver disease. This study investigated the therapeutic effects of darbepoetin alfa (DA), an erythropoietin analogue, in a dexamethasone (Dexa)-induced IR model, focusing on its impact on metabolic regulation (apelin) and inflammatory pathways (galectin-3) in liver tissue.

Materials and methods

Thirty-two male Wistar rats were divided into four groups (n = 8/group): (saline 1 ml/kg/day intraperitoneal [ip] for 5 days), IR (Dexa 1 mg/kg/day ip for 5 days), DA (single 5 µg/kg ip dose on day 6), and IR + DA (Dexa followed by DA). Body weight, fasting glucose, insulin levels, and HOMA-IR were assessed. Liver function was evaluated via AST/ALT levels, while histopathological changes (hydropic degeneration, necrosis, fibrosis via Masson’s trichrome) and immunohistochemical expression of apelin and galectin-3 were analyzed.

Results

There were no significant differences in body weights between groups; only the IR group experienced weight loss. Fasting glucose and HOMA-IR levels were significantly higher in the IR group (p < 0.001, p = 0.008), while insulin levels showed no significant change. Darbepoetin treatment reduced glucose and HOMA-IR. Histologically, the IR group displayed marked hepatocyte degeneration, lipid accumulation, necrosis, and disorganized hepatic cords. Masson’s Trichrome staining showed increased collagen deposition around vessels in the IR group, which was reduced after darbepoetin. Immunohistochemically, apelin expression was significantly decreased and galectin-3 increased in the IR group compared to SHAM (p < 0.001). These changes were reversed in the IR + DA group. The SHAM and DA groups exhibited normal liver architecture without pathological alterations.

Conclusion

DA attenuated Dexa-induced IR by improving glycemic control, reducing hepatic injury, and modulating key metabolic (apelin↑) and inflammatory (galectin-3↓) markers. The restoration of liver histopathology and reduction in fibrosis suggest DA’s potential as a multifunctional agent against IR-associated complications. Further studies are needed to validate these findings in chronic models and clinical settings.

Introduction

Insulin resistance (IR) is a key mechanism underlying metabolic syndrome (MetS) a cluster of interrelated conditions including obesity, hyperglycemia, hyperlipidemia, and hypertension [1, 2]. MetS significantly increases the risk of cardiovascular diseases and contributes to higher morbidity and mortality rates. IR is a key factor in the development of type 2 diabetes, cardiovascular dysfunction, obesity, and non-alcoholic fatty liver disease (NAFLD), as it leads to excessive insulin secretion from pancreatic β-cells [3].

Dexamethasone (Dexa), a synthetic glucocorticoid analog, is frequently used in clinical and experimental models to induce insulin resistance. In rats treated with Dexa, insulin loses its ability to stimulate glycogen synthesis [4]. Specifically, suppression of the PI3K/Akt pathway impairs glycogen synthesis, disrupts glucose transport, and reduces glucose utilization in peripheral tissues [4–6]. IR drives metabolic dysfunction by promoting adipose tissue expansion and adipocyte growth. Insulin resistance promotes metabolic dysfunction by contributing to adipose tissue expansion and adipocyte hypertrophy. In obesity, the liver supports adipose tissue expansion and vascularization through the adipokin secretions such as apelin [7].

Adipokines, particularly molecules such as apelin and galectin-3, play important roles in the pathophysiology of insulin resistance (IR). Apelin binds to the G protein-coupled APJ receptor and is involved in several metabolic processes, including energy homeostasis, glucose uptake, and insulin sensitivity. In conditions such as obesity and IR, its levels increase and may act as a compensatory mechanism to counteract the effects of insulin resistance [7]. However, glucocorticoid exposure induced by Dexa can suppress apelin mRNA expression in adipocytes, thereby reducing apelin production and potentially exacerbating IR [8]. Apelin also stimulates lipolysis and enhances glucose uptake in muscle and adipose tissue, aiming to balance hyperglycemia. In chronic IR, the regulation of apelin becomes more complex and may be associated with metabolic disorders [9].

Galectin-3 is a lectin that modulates inflammation and immune responses. Its levels increase in conditions such as insulin resistance (IR), obesity, and inflammation. In obesity, elevated serum galectin-3 levels may reflect a compensatory response to chronic inflammation [10]. Galectin-3 has been shown to contribute to chronic inflammation by activating inflammatory signaling pathways, such as NF-κB, which can impair insulin signaling [11]. Moreover, galectin-3 may exacerbate cellular damage associated with oxidative stress and reduce insulin receptor sensitivity [12]. Higher galectin-3 levels have been observed in individuals with IR compared to those without IR. Studies have demonstrated a correlation between galectin-3 levels and IR, suggesting that galectin-3 may serve as a potential biomarker for insulin resistance [13].

Darbepoetin alfa (DA) is a synthetic erythropoietin (EPO) derivative that a threefold longer half-life than native EPO [14]. EPO produced by the kidneys, (with minor hepatic synthesis regulates erythropoiesis. Beyond its hematopoietic role, EPO exhibits regenerative properties, including anti-inflammatory [15], anti-apoptotic [16], and antioxidant [17] effects, making it beneficial in various disease models [18, 19]. Additionally, EPO has been used to regulate glucose metabolism through hematopoietic stimulation, modulate insulin signaling pathways, and enhance antioxidant activity by reducing oxidative stress [18–20].

This study investigates the effects of darbepoetin alfa on glucose homeostasis in liver tissue using a Dexa-induced IR model, focusing on the immune reactivities of apelin and galectin-3 (the latter of which increases under hypoxia). Histological and immunohistochemical analyses examined changes in inflammation- and IR-related proteins The goal was to elucidate how darbepoetin alfa influences apelin and galectin-3 expression in liver tissue during acute IR.

Materials and methods

Animals and experimental groups

This study was conducted at the Animal Research Center of Kırşehir Ahi Evran University. The experiments were carried out in accordance with international guidelines for the use of laboratory animals. A total of 32 male Wistar albino rats (130–135 gram) were used in the study. Before and during the experiment, all animals were housed under a 12-hour light and 12-hour dark cycle in rooms with a constant temperature of 22–24 °C. The animals were fed with pellet chow and provided with tap water, and there were no restrictions on their food or water intake.

They were randomly divided to four groups (n = 8 per group) as follows:

Sham group (SHAM): Saline was given intraperitoneal injection (ip) for five days (1 mL/kg/day).

Insulin Resistance group (IR): Insulin resistance was induced via ip injections of dexa at a dose of 1 mg/kg/day for 5 consecutive days (Dekort injectable solution, Deva Pharma Company, Turkey).

Darbepoetin alfa (DA): On fifth day, rats received a single i.p. dose of DA at 5 mcg/kg (Amgen Inc. ARANESP^® (darbopoetin alfa) injection 0,3mL. 100 mcg/mL).

Insulin Resistance + Darbepoetin alfa (IR + DA): Rats were administered dexa at 1 mg/kg/day i.p. for 5 days, followed by a single i.p. dose of DA (5 mcg/kg) on the sixth day (Fig. 1).

Fig. 1.

Schematic of the experimental design and results

Insulin resistance protocol

In the literature, there are various approaches to establishing an insulin resistance model using dexamethasone. In our study, the model was induced by intraperitoneal injection of dexamethasone at a dose of 1 mg/kg/day for 5 consecutive days, as applied by Inácio et al. [21]. Blood glucose levels were measured using a glucometer (Accu-check, USA) every day and at the end of the experiment. At the end of the experiment, insulin hormone levels in the blood were determined using the ELISA (Enzyme-Linked Immunosorbent Assay) method. Body weight variations of the animals were observed daily for six consecutive days.

HOMA-IR stands for “Homeostasis Model Assessment of Insulin Resistance.” It is a method used to evaluate insulin resistance. HOMA-IR is calculated in rats using a simple mathematical formula based on fasting blood glucose and insulin levels. HOMA-IR was calculated from the fasting glucose and insulin levels [22, 23].

Erythropoietin administration

Following the induction of the insulin resistance model and the final corticosteroid administration, a single dose of 5 mcg/kg Darbepoetin alfa was administered intraperitoneally. This dose was selected based on previous reports demonstrating that erythropoietic agents at this level effectively cross biological barriers and reach target tissues, ensuring therapeutic efficacy [24].

On the sixth day, following the completion of all experimental protocols, euthanasia was performed under deep anaesthesia (ketamine 50 mg/kg, xylazine 10 mg/kg) to ensure the absence of pain and distress. Blood samples were collected via cardiac puncture for biochemical analysis, and serum was separated and stored at -80 °C. Livers were excised promptly and prepared for histopathological, immunohistochemical, and biochemical analyses.

Biochemical analysis

Blood samples were quickly centrifuged to separate the serum. Serum samples were then placed into Eppendorf tubes and stored at -80 °C for preservation. The ELISA technique was used to measure the insulin levels. The liver enzymes measurements were conducted according to the manufacturer’s instructions (Elabscience ELISA kits, Cat.No: E-EL-2466). Samples were analyzed by using an ELISA reader and autostrip washer. (BIO- TEK EL X 80, BIO TEK EL X 50). Levels of liver enzymes (AST, ALT) were analyzed with auto analyzer (MINDRAY BS-400, Cat.No: OttoBC127, OttoBC128).

Histopathological analysis

Liver tissues were fixed in 10% formalin for 24 h, dehydrated through a graded ethanol series (70%, 80%, 90%, 95%, 100%), embedded in paraffin, and sectioned at 5 μm (Leica Autocut 14051956472, Germany). Consecutive sections were stained with hematoxylin–eosin (Bio-Optica 05-06004/L Harris’ Hematoxylin; 05-10002/L Eosin 1%) to assess morphology and with Masson’s trichrome (GBL Masson Trichrome 5022, Turkey) to detect fibrosis. All slides were examined blindly under a standard light microscope (Nikon Eclipse Ni-U, 940728). Hydropic degeneration and coagulative necrosis of hepatocytes, sinusoidal dilation, vascular hyperemia (H&E), and perivascular collagen deposition (MT) were evaluated semi-quantitatively in 10 random fields per section. Each damage parameter was scored in 10 different fields per liver section, and the mean percentage values within each group were calculated. Histopathological changes were graded as follows: 0 (none) when observed in less than 5%, 1 (mild) when observed in less than 25% of liver cells and the peri-acinar region, 2 (moderate) when present in 25–50%, 3 (severe) when present in more than 50–75%, and 4 (very severe) when present in 75–100% (none = 0, mild = 1, moderate = 2, severe = 3, very severe = 4) [25].

Immunohistochemistry

The liver sections with a thickness of 5 μm, taken from paraffin blocks, were placed on polylysine-coated slides and then deparaffinized. Afterward, the sections were passed through graded alcohol series and boiled in a citrate buffer solution (pH: 6) in a microwave oven (750 W) for 12 min. Following the boiling process, the tissues were allowed to cool at room temperature and were then washed with phosphate buffered saline (PBS). To inhibit endogenous peroxidase activity, %3 hydrogen peroxide solution was applied for 15 min. After washing the tissues with PBS for 3 × 5 min, UV blocking solution was applied for 5 min. The tissues were then incubated at room temperature for 60 min in a humidified chamber with primary antibodies diluted at 1/200 (Galectin-3; Santa Cruz Biotechnology Cat# sc-20157, RRID: AB2136784, and Apelin; Abcam Cat# ab125213, RRID: AB10999708). After incubation, the tissues were washed with PBS for 3 × 5 min, followed by a 30-minute incubation with a secondary antibody compatible with the primary antibody. Finally, the tissues were washed again with PBS for 3 × 5 min and incubated with Streptavidin Peroxidase (TS–125-HR, Lab Vision Corporation, USA) for 30 min at room temperature in a humidified chamber, and then placed in PBS. After applying Diaminobenzidine (DAB) solution, the tissues were observed under a light microscope until the signal was detected, followed by simultaneous washing of all groups with PBS. The sections were counterstained with Mayer’s hematoxylin, passed through PBS and distilled water, and then mounted with an appropriate mounting solution (Large Volume Vision Mount, TA-125-UG, Lab Vision Corporation, USA). The prepared slides were examined and photographed using a Leica DM500 microscope (Leica DFC295). The histoscore was calculated based on the extent (0.1: <25%, 0.4: 26–50%, 0.6: 51–75%, 0.9: 76–100%) and intensity (0: none, + 0.5: very mild, + 1: mild, + 2: moderate, + 3: severe) of immunoreactivity, using the formula: Histoscore = extent × intensity (1x, 2x) [26].

Statistical analysis

All statistical analyses were performed with GraphPad Prism v9 (GraphPad Software, San Diego, CA, USA). Data normality was assessed using the Shapiro–Wilk test. Variables exhibiting a normal (parametric) distribution are presented as mean ± standard deviation (SD) and were compared across groups using one-way analysis of variance (ANOVA), followed by Bonferroni’s post-hoc test for multiple comparisons. Variables that did not meet normality assumptions are expressed as median (min-max) and were analyzed with the non-parametric Kruskal–Wallis test, with Dunn’s post-hoc test applied when overall significance was detected. P-value < 0.05 was considered statistically significant for all analyses.

Results

Experimental groups’ body weight

There is no significant difference in the initial and final weights between the groups. Among the four groups, only IR group experienced weight loss, while the other three groups gained weight. At the end of the experiment, no statistically significant differences were observed among Control group (140.8), IR group (120.8), IR + DA group (136.8) and DA group (136.9) (SHAM vs. IR p = 0.378, SHAM vs. IR + DA p > 0.999, AM vs. DA p > 0.999, DA vs. IR p = 0.781, DA ve IR + DA p > 0.999) The weight loss in the IR group was 5.08%, while the weight gain in the SHAM, DA, and IR + DA groups were 8.6%, 2.16%, and 3.46%, respectively (Fig. 2).

Fig. 2.

Assessment of the body weight changes in the experimental groups throughout the course of the study. Data are presented as mean ± SD. *; p < 0.05, **; p < 0.01, ***; p < 0.001

Insulin resistance measurement

The results shows that rats treated with dexa had significantly higher glycemia (p < 0.001) compared to the SHAM group. Additionally, their insulinemia was not significantly high (p = 0.156) The HOMA-IR index, which indicates insulin resistance (with higher values indicating greater resistance), was also significantly higher in the IR group (p < 0.01). In the groups treated with darbepoetin, both glucose levels and the HOMA-IR index showed a decrease. Specifically, when fasting glucose was measured, the IR + DA group had significantly lower levels compared to the IR group (p < 0.05) (Fig. 3).

Fig. 3.

Assessment of insulin resistance indicators in the experimental groups. (a) Insulin levels (b) Fasting glucose levels (c) HOMA-IR index. *; p < 0.05, **; p < 0.01, ***;p < 0.001

Liver function analyses effects of darbepoietin on serum activities

Serum AST and ALT enzyme activities, which are indicators of liver function, are presented in Fig. 4. In the IR group, AST levels were significantly elevated compared to the SHAM group (P < 0.001). No significant differences were observed between the IR and IR + DA groups, nor between the SHAM and DA groups. However, AST levels in the IR + DA group were significantly higher than those in the DA group (P = 0.002). Significant differences were also observed in serum ALT levels across the groups. A statistically significant increase in ALT levels was observed between the SHAM group and the IR group (P = 0.014). Additionally, a significant reduction in ALT levels was noted between the IR and IR + DA groups (P < 0.001). No significant differences were observed between the remaining groups (Sham vs. IR + DA p = 0.686, SHAM vs. DA p = 0.601, IR vs. DA p = 0.199, IR + DA vs. DA p = 0.108) (Table 1; Fig. 4).

Fig. 4.

Assessment of liver function markers in the experimental groups following the termination of the experiments *; p < 0.05, **; p < 0.01, ***; p < 0.001

Table 1.

Serum AST and ALT enzyme activities

Group (n = 8)	AST (U/L)	ALT (U/L)
SHAM	165.5 ± 36.82a	79.81 ± 10.93a
IR	229.6 ± 15.49b	120.6 ± 11.38b
IR + DA	217.5 ± 29.67ab	66.03 ± 34.60c
DA	163.0 ± 19.98a	95.33 ± 31.87ac

Values are presented as meean, ±SD. SHAM: Sham group; IR: Insulin resistance group; DA: Darbepoetin Alfa. Different superscript letters (a, b, c) within the same column indicate statistically significant differences between groups (p < 0.05, one-way ANOVA followed by Bonferroni’s post hoc test)

Histopathological findings of the liver

When the livers of rats in SHAM and DA groups were examined, it was found that they were in normal histological structure. In the liver sections, hepatocytes with prominent nuclei are arranged in an orderly manner around the central vein, forming well-organized cords. The sinusoids between the cell cords exhibit a normal appearance. In the IR group rats, degeneration was observed in the hepatocytes around the central veins of the liver, along with lipid vacuoles in some hepatocytes, necrosis, irregularities in the hepatic cords, and vacuolization. In liver sections stained with Masson’s Trichrome (MT), the blue-stained areas indicate regions with high collagen fiber density. In the IR group, an increase in fiber density around the vessels was observed compared to the SHAM and DA groups. In the IR group treated with DA, a reduction in collagen fiber content was noted (Fig. 5a).

Fig. 5.

Histology of liver sections hematoxylin–eosin (H&E) (a), Masson’s trichrome (MT) (b). Bar: 100 μm. a. SHAM group, liver tissue, Normal histological appearance. IR group, liver tissue, irregularities and vacuolization in cords, fat vacuoles and hidropic degenaration in hepatocytes of central region. IR + DA group, liver tissue, mild hydropic degeneration in hepatocytes. DA group, liver tissue, normal histological appearance. Black Arrow: hydropic degeneration. Red Arrow: Necrosis. b. MT staining shows collagen deposition in the periportal and perisinusoidal regions. In the IR group, a marked increase in collagen fibers is observed, indicating fibrosis, whereas the IR + DA group shows a notable reduction in collagen deposition compared to IR. The SHAM and DA groups display minimal collagen fibers, reflecting normal liver architecture

When evaluating the histopathological scores of liver tissues, statistically significant differences were observed between the experimental groups in all evaluated parameters: hydropic degeneration, coagulation necrosis, sinusoidal dilatation, lipid vacuolization, and collagen increase (fibrosis) (p < 0.001 for all). When comparing the IR and IR + DA groups, darbepoetin alfa administration resulted in a significant reduction in histopathological parameters, including coagulation necrosis (p = 0.024), sinusoidal dilatation (p = 0.010), lipid vacuolization (p = 0.031), and fibrosis (p = 0.031) (Table 2; Fig. 5b).

Table 2.

Histopathological score in liver tissue

Group (n = 8)	Hydropic degeneration	Coagulation necrosis	Sinusoidal dilatation	Lipid vacuolization	Collagen increase (Fibrosis)
SHAM	0.0 [0.0–1.0]a	0.0 [0.0–1.0]a	0.0 [0.0–1.0]a	0.0 [0.0–0.0]a	0.0 [0.0–2.0]a
IR	4.0 [3.0–4.0]b	3.5 [2.0–4.0]b	3.0 [2.0–4.0]b	3.0 [2.0–4.0]b	4.0 [3.0–4.0]b
IR + DA	1.0 [0.0–1.0]c	1.0 [0.0–1.0]c	0.5 [0.0–2.0]c	1.0 [0.0–1.0]c	1.0 [0.0–2.0]c
DA	0.0 [0.0–3.0]ac	0.0 [0.0–1.0] ac	0.0 [0.0–1.0] ac	0.0 [0.0–1.0] ac	1.0 [1.0–1.0] ac

Values are presented as median [min–max]. SHAM: Sham group; IR: Insulin resistance group; DA: Darbepoetin Alfa; Different superscript letters (a, b, c) within the same column indicate statistically significant differences between groups (p < 0.05, Kruskal–Wallis test followed by Dunn’s post hoc test)

Apelin and galectin-3 immunoreactivity

Immunohistochemical staining for apelin expression in liver tissue revealed detectable apelin immunoreactivity. Apelin immunoreactivity was similar between the SHAM and DA groups (P = 0.252). However, compared to the SHAM group, apelin immunoreactivity was significantly reduced in the IR group (P < 0.001). In contrast, apelin immunoreactivity was significantly increased in the IR + DA group compared to the IR group (P < 0.001) (Fig. 6) (Table 3).

Fig. 6.

Immunohistochemical analysis of liver sections. Bar: 50 μm. Apelin and galectin-3 immunoreactivity. Apelin immunoreactivity was reduced in the IR group and restored in the IR + DA group, while galectin-3 immunoreactivity was markedly increased in the IR group and decreased following darbepoetin alfa treatment. SHAM and DA groups showed near-normal staining patterns

Table 3.

Apelin and galectin-3 histoscore

Group (n = 8)	Apelin histoscore (Median [Min–Max])	Galectin-3 histoscore (Median [Min–Max])
SHAM	0.80 [0.60–1.20]	0.80 [0.60–0.90]
IR	0.15 [0.10–0.30]	1.20 [0.80–1.20]
IR + DA	0.80 [0.40–1.20] a	0.30 [0.10–0.60] a
DA	0.90 [0.80–1.20] b	0.70 [0.60–1.20] b

Values are presented as median (min–max). SHAM: Sham group; IR: Insulin resistance group; DA: Darbepoetin Alfa. a: p < 0.001 vs. IR group, b: p < 0.001 vs. SHAM group Statistical test: Kruskal–Wallis with Dunn’s post hoc test

Immunohistochemical staining for galectin-3 expression in liver tissue revealed detectable galectin-3 immunoreactivity. Galectin-3 immunoreactivity was similar between the control and DA groups (P = 0.913). However, compared to the control group, galectin-3 immunoreactivity was significantly increased in the IR group (P < 0.001). In contrast, galectin-3 immunoreactivity was significantly reduced in the IR + DA group compared to the IR group (P < 0.001) (Fig. 6), (Table 3).

Discussion

IR is a central pathological mechanism underlying metabolic syndrome, contributing to the development of various disorders such as type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease [1–3]. This study used dexa-induced IR to investigate the therapeutic effects of darbepoetin alfa on liver tissue, particularly focusing on apelin and galectin-3, two key markers involved in metabolic regulation and inflammation.

Dexa is a widely utilized agent for inducing insulin resistance (IR) in animal models, offering a faster and more reproducible alternative to high-fat diet (HFD)-induced models for establishing systemic IR within a relatively short experimental period [4–6, 27]. In this study, dexa administration significantly increased fasting blood glucose and HOMA-IR levels, accompanied by weight loss, confirming the establishment of insulin resistance in rats. In the IR + DA group, darbepoetin effectively regulated and significantly reduced both fasting glucose and HOMA-IR levels. While dexa or darbepoetin administration caused changes in fasting insulin levels, these changes were not statistically significant. Previous studies have demonstrated that varying doses and durations of dexa administration consistently lead to increases in insulin, fasting blood glucose, and HOMA-IR levels, thereby inducing IR [4, 21, 28]. Furthermore, DA treatment has shown positive effects on IR in rat models of polycystic ovary syndrome (PCOS), resulting in decreased insulin levels [29]. In patients with chronic kidney disease undergoing hemodialysis, DA therapy has been shown to improve IR, with significant reductions in HOMA-IR, HbA1C, and fasting glucose levels [30].

Dexa-induced weight loss is a well-documented phenomenon in rodent models and is primarily attributed to its catabolic effects on muscle and adipose tissue. Glucocorticoids such as dexa promote protein catabolism and lipolysis, leading to a reduction in lean body mass and fat stores [4–6, 28]. This effect is exacerbated by impaired insulin signaling, which diminishes glucose uptake and utilization in peripheral tissues, further contributing to the observed weight loss [4, 5]. The significant weight loss observed in the IR group of this study aligns with previous reports demonstrating that chronic glucocorticoid exposure induces metabolic alterations that mimic cachexia-like states [27, 28]. Therefore, the decrease in body weight should not only be considered a marker of systemic catabolism but also as an indicator of successful IR induction. Additionally, the partial prevention of weight loss in the IR + DA group may reflect the metabolic protective effects of darbepoetin alfa, which could be linked to improved insulin sensitivity and enhanced energy metabolism [20, 29, 30]. These observations suggest that DA may exert anti-catabolic effects in glucocorticoid-induced IR models, which warrants further investigation.

In rats where insulin resistance (IR) was induced by dexa administration, serum levels of liver enzymes AST and ALT were elevated. IR disrupts glucose metabolism in the liver, leading to impaired energy metabolism in hepatocytes and consequently causing cellular damage. The increase in metabolic and oxidative stress contributes to the elevation of AST and ALT levels in the blood [31]. The AST and ALT enzymes are primarily released from hepatocytes during cellular damage. While both enzymes are indicators of liver injury, AST is also present in other tissues such as the heart and muscles, which could explain why AST levels did not show a significant reduction in our study. In contrast, ALT is more liver-specific, and its reduction in the DA-treated group suggests a more targeted improvement in hepatic injury. Similar findings have been supported by other studies [31–33]. In this study, DA administration on day 6 in the IR + DA group did not result in a significant reduction in AST levels, while a significant decrease in ALT levels was observed. This selective reduction in ALT suggests that darbepoetin alfa might specifically mitigate hepatocellular damage, which is further supported by studies that demonstrate DA’s role in reducing oxidative stress and enhancing cellular repair mechanisms in liver tissue [34]. Another study investigating treatments in IR-induced rats examined the effects of vitamin D, DA, and their combined therapies, reporting a decrease in enzyme levels [35].

The induction of insulin resistance (IR) through dexa exposure in rats resulted in significant alterations in liver histoarchitecture. Hydropic degeneration in hepatocytes was observed as the most prominent change. Additionally, necrotic cells, vacuolization, and pronounced sinusoidal dilatation were noted. These pathological changes are likely due to the combined effects of glucocorticoid-induced metabolic dysregulation and increased oxidative stress, which compromise hepatocyte function and structure. The degeneration was more concentrated around the central vein (vena centralis). The increase in collagen fibers further supported the degenerative changes. The observed fibrosis is indicative of ongoing tissue damage and a fibrotic response, which may have been mediated by the upregulation of pro-fibrotic markers like galectin-3 in the IR group. In the IR model treated with DA, hydropic degeneration, necrotic structures, and lipid vacuolization in hepatocytes were reduced, and the sinusoids appeared more normalized. This improvement is likely attributed to DA’s anti-inflammatory and anti-fibrotic effects, which may reduce macrophage activation and prevent further hepatic fibrosis. Studies involving IR induction through dexa have reported degenerative hepatocytes, dilated blood vessels with vascular damage, balloon-like structures, and blurred liver lobules [27, 33]. Similarly, in studies where IR was induced through a high-fat diet for 14 weeks, microvesicular and macrovesicular steatosis, inflammation, and severe hepatocyte necrosis were observed [34, 36]. In one of these studies, DA treatment administered during the last two weeks demonstrated improvements, including minimal activation of Kupffer cells and very mild hydropic degeneration in hepatocytes [34]. The reduction in liver damage in the DA-treated groups may be due to its ability to regulate inflammatory cytokines and modulate oxidative stress pathways, thus limiting hepatocyte injury and promoting tissue repair.

Apelin is a cardioprotective peptide that binds to the APJ receptor, regulating glucose metabolism, supporting insulin sensitivity [37], and preventing age-related muscle loss [38]. It also exhibits anti-obesity and anti-diabetic properties [39]. The relationship between apelin levels and dysmetabolic conditions such as type 2 diabetes remains controversial [40]. In this study, apelin immunoreactivity in liver tissue was reduced in the IR group compared to the SHAM and DA groups. This reduction has been associated with the downregulation of apelin mRNA levels, as observed in another study using dexa, a glucocorticoid antagonist [8]. In a separate study, a high-fat diet (HFD) administered during pregnancy and lactation increased maternal serum apelin levels while reducing serum apelin levels in adult male offspring [41]. Similarly, another study reported that rapid muscle wasting associated with IR and imbalanced protein homeostasis in cancer cachexia resulted in decreased apelin levels [42]. In the IR group treated with DA, apelin levels were elevated. However, no direct studies in the literature have examined the effects of DA on apelin levels in the context of improved IR. In a study investigating the hypermetabolic state caused by burns, DA treatment reduced muscle loss and regulated apoptotic and profibrotic pathways [43]. DA’s role in preserving muscle mass might indirectly contribute to maintaining apelin levels [44].

In the IR model induced by dexa, a significant increase in galectin-3 expression was observed, indicating the activation of inflammatory pathways. Galectin-3 is a pro-inflammatory lectin that is upregulated in response to tissue injury and metabolic stress, particularly under conditions of IR. Inflammatory cytokines such as TNF-α and IL-6, which are elevated in IR states, stimulate galectin-3 secretion from immune cells, especially macrophages [45]. Galectin-3 not only mediates macrophage recruitment but also amplifies inflammation by activating key signaling cascades such as NF-κB, thereby promoting the release of additional proinflammatory mediators [11]. This cascade contributes to tissue remodeling and fibrosis, as evidenced by the increased collagen deposition in liver tissue [46]. The elevated galectin-3 expression observed in the IR group (Fig. 5b) may therefore serve as a critical link between chronic low-grade inflammation and the progression of hepatic insulin resistance and fibrotic changes, a notion further supported by previous findings indicating that increased galectin-3 levels impair insulin receptor signaling and exacerbate systemic metabolic dysfunction [13, 47] A review addressing personalized antifibrotic therapies in the progression of chronic kidney disease demonstrated that the inhibition of galectin-3 genes targeted during treatment reduced fibrosis, inflammation, and kidney damage [48]. DA treatment in the IR-induced group led to a significant reduction in galectin-3 levels. This inhibition of galectin-3 also reduced fibrosis, as evidenced by the decreased collagen fibrils in the DA-treated group. The findings of this study indicate that DA may exert its anti-inflammatory effects, at least in part, through the downregulation of galectin-3 expression and its associated fibrotic signaling pathways. Considering the established role of galectin-3 in mediating inflammation via NF-κB signaling, these results underscore its critical involvement in the development of IR and hepatic injury in the dexa-induced model.

Conclusion

This study demonstrates that darbepoetin alfa administration may have beneficial effects on metabolic and inflammatory markers in a dexa-induced insulin IR model. The findings suggest that darbepoetin alfa treatment can enhance insulin sensitivity by increasing apelin levels and contribute to the suppression of inflammatory and fibrotic processes by reducing galectin-3 levels. Improvements in liver histology and the reduction of collagen fibrils further indicate the potential of this therapeutic approach as an antifibrotic agent.

However, the model used in this study was limited to dexa-induced IR, and it remains to be explored whether similar effects would be observed in other causes of insulin resistance. Moreover, more comprehensive studies are needed to evaluate the long-term safety and efficacy profile of darbepoetin alfa treatment. Future research should focus on testing therapeutic agents like darbepoetin alfa in diverse metabolic models and human studies to better understand their clinical applicability in IR and related complications. In this context, personalized treatment approaches could open new horizons, particularly in suppressing fibrosis and inflammation, thereby contributing to the development of more effective strategies.

Author contributions

H.T.Y.: İnvestigation, conceptualization, data curation, resources, writing-original draft, and writing-review and editing. A.T.: methodology, data curation, validation, formal analysis. E.K.: methodology, data curation, supervision, visualization, K.T.K.: project administration, methodology, data curation, software.

Funding

No funding has been received.

Data availability

Data supporting the findings of this study areavailable from the corresponding author upon reasonablerequest.

Declarations

Ethical approval

The experiments were conducted in accordance with The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All experimental protocols were approved by the Local Ethic Committee of Animal Experiments of Kırşehir Ahi Evran University (approval no. 2023-23-06) in Turkiye.

Competing interests

The authors declare no competing interests.