Effect of Vaspin on insulin resistance in gestational diabetes and the involvement of the ROS/eNOS/NO pathway

Abstract

The global prevalence of gestational diabetes mellitus (GDM) is increasing, posing significant health risks to both mothers and infants. Visceral adipose tissue-derived serine protease inhibitor (Vaspin) has been identified as a potential insulin sensitizer that may mitigate insulin resistance (IR). However, the related mechanism by which Vaspin improves IR in patients with GDM remains unclear. This study aims to investigate whether Vaspin ameliorates IR in GDM via modulation of the ROS/eNOS/NO signaling pathway. Clinical samples from 58 pregnant women were collected and categorized into GDM and control (G) groups. Binary logistic regression analysis revealed significant associations between Vaspin, IR, and GDM. A GDM rat model was established using 50 female Sprague-Dawley (SD) rats, divided into GDM and G groups. Fasting blood glucose (FBG), fasting insulin (FINS), and Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) levels were measured using ELISA and steady-state model evaluation. Vaspin intervention significantly reduced FBG, FINS, and HOMA-IR levels in the GDM group, while L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, blocked these effects. INS-1 cells were used to establish a high-glucose model, and laser confocal microscopy, ELISA, and Griess reagent were employed to detect reactive oxygen species (ROS), eNOS, and nitric oxide (NO) levels. Exogenous Vaspin administration improved ROS, eNOS, and NO levels, but these effects were inhibited by L-NAME. Collectively, these results propose a model in which the protective effect of Vaspin against insulin resistance in GDM may be mediated, at least in part, through the ROS/eNOS/NO pathway.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12902-025-02157-y.

Introduction

Gestational diabetes mellitus (GDM) represents a common complication during pregnancy, significantly impacting both maternal and neonatal health [1, 2]. Current diagnostic guidelines estimate that approximately 14% of pregnant women are affected by GDM [3].

The pathogenesis of GDM involves a complex interplay of factors, with insulin resistance (IR) serving as a central pathophysiological element [4]. In a typical pregnancy, hormonal changes induce a physiological state of IR to redirect nutrients to the fetus; however, this process is markedly exacerbated in GDM [5].This heightened IR stems from alterations in adipocyte function, placental and hormonal changes, inflammation, and oxidative stress [6, 7], which collectively impair the insulin signaling cascade in skeletal muscle, liver, and other target tissues.

Obesity stands out as a major modifiable risk factor for GDM [3]. Adipose tissue not only serves as an energy reservoir but also functions as an endocrine organ, releasing bioactive molecules that regulate inflammation and maintain metabolic homeostasis [8]. Vaspin, a serine protease inhibitor primarily expressed in adipose tissue, has emerged as a significant adipokine implicated in glucose metabolism. Initially identified for its association with obesity and insulin sensitivity, subsequent studies have established a strong link between Vaspin and various forms of diabetes, including type 2 diabetes and GDM [9–11]. Its pleiotropic effects extend beyond enhancing insulin sensitivity; research in obese murine models has demonstrated that Vaspin administration not only improves glycemic control but also exhibits notable anti-inflammatory and anti-atherosclerotic properties [12–14]. Moreover, evidence from other metabolic disease models suggests that Vaspin can mitigate oxidative stress and improve endothelial function [15, 16]. Despite these compelling associations, the precise molecular mechanisms through which Vaspin regulates blood glucose, particularly in the context of GDM, remain inadequately defined.

Given Vaspin’s potential to improve metabolic parameters and mitigate oxidative stress, it is imperative to explore the relevant mechanistic pathways in GDM. A key mechanism implicated in GDM pathophysiology involves reactive oxygen species (ROS). ROS are known to play a critical role in blood glucose regulation [7, 17]. ROS can disrupt intracellular insulin signaling and impair the bioavailability of nitric oxide (NO), catalyzed by endothelial nitric oxide synthase (eNOS), a key mediator for insulin-mediated glucose uptake and vasodilation [18–20]. Evidence suggests that Vaspin can mitigate oxidative stress and improve endothelial function in other models of metabolic disease [15, 16], leading us to hypothesize that Vaspin ameliorates IR in GDM by modulating the ROS/eNOS/NO signaling pathway.

While Vaspin is primarily expressed in adipose tissue and skin [21, 22], the specific mechanisms by which it mitigates IR in GDM remain poorly understood. Therefore, this study aims to investigate the effect of Vaspin on IR in GDM and its underlying mechanism, with a particular focus on validating its role in the ROS/eNOS/NO signaling pathway.

Materials and methods

Human subjects

Data were collected from pregnant women who visited the Department of Obstetrics and Gynecology at Tangshan Central Hospital between October 2022 and October 2023. Participants were categorized into two groups: a gestational diabetes mellitus (GDM) group (n = 16) and a control group (G) of healthy pregnant women (n = 42). GDM diagnosis was based on the International Diabetes and Pregnancy Study Group (IADPSG) criteria: fasting blood glucose (FPG) ≥ 5.1 mmol/L, 1-hour postprandial glucose (1 hPG) ≥ 10.0 mmol/L, or 2-hour postprandial glucose (2 hPG) ≥ 8.5 mmol/L. Meeting any of these criteria confirmed a GDM diagnosis.

Inclusion criteria for the GDM group included singleton pregnancy, first pregnancy, age between 20 and 45 years, absence of pre-pregnancy diabetes, willingness to participate, completion of oral glucose tolerance tests (OGTT), and no use of medications affecting glucose metabolism. Exclusion criteria were age below 20 or above 45 years, incomplete OGTT screening, pre-pregnancy diabetes or abnormal glucose tolerance, long-term use of medications affecting glucose metabolism, cognitive or communication impairments, organ dysfunction, loss to follow-up, or refusal to participate.

Clinical characteristics and blood samples were collected from all participants. Blood samples were centrifuged at 2000 × g for 20 min to isolate serum, which was stored at − 80 °C for further analysis.

All patient blood collection and testing methods were conducted in accordance with relevant guidelines and regulations. After signing the consent form, a blood sample was taken from the subject. This study was approved by the Ethics Committee of Tangshan Central Hospital. Clinical trial number: not applicable.

Animals

Female SD rats (150–200 g, specific pathogen-free [SPF]) were purchased from HFK Bioscience (Beijing, China) and housed in a controlled environment (20–24 °C, 50–60% humidity, 12-hour light/dark cycle) with free access to distilled water. All experiments adhered to the ARRIVE guidelines and complied with local and national regulations. The animal experiment program has been approved by the Experimental Animal Ethics and Welfare Committee of North China University of Science and Technology (application approval No. 2023-SY-44).

Culture of rat insulinoma cells

INS-1 cells were obtained from Sunncell (Wuhan, China) and cultured in RPMI-1640 medium supplemented with 20% fetal bovine serum, 1% penicillin-streptomycin, 4 mM L-glutamine, 110 mg/L sodium pyruvate, and 50 µM β-mercaptoethanol. Cells were maintained at 37 °C in a 5% CO₂ atmosphere. Cells in the logarithmic growth phase were subcultured and evenly distributed into culture dishes. They were then randomly assigned to four treatment groups: control (11.1 mM glucose), high glucose (33.3 mM glucose), high glucose + Vaspin (320 ng/mL Vaspin; Beyotime Biotechnology, Shanghai, China), and high glucose + Vaspin + L-NAME (50 mg/mL L-NAME; Beyotime Biotechnology, Shanghai, China). After 24 h of culture, treatments were applied, followed by another 24-hour incubation before analysis.

Binary logistic regression analysis

Participants were categorized into G and GDM groups based on diabetes status. Variables were assigned numerical values, and univariate logistic regression was used to assess the relationship between individual variables and GDM. After confirming no collinearity, forward stepwise regression was employed to screen independent variables. Multivariate logistic regression was then performed to evaluate associations between variables and GDM. Finally, a receiver operating characteristic (ROC) curve was generated to assess the model’s predictive accuracy.

Establishment of the GDM rat model

Five-week-old female rats were acclimatized for one week and then randomly assigned to a gestational control (G) group (n = 25) or a GDM group (n = 25). The G group received a standard chow diet (3.28 kcal/g; 22% protein, 11% fat, 67% carbohydrate), while the GDM group was fed a high-fat/high-sucrose diet (3.94 kcal/g; 15.6% protein, 31% fat, 53.4% carbohydrate) for 8 weeks. All animals had ad libitum access to their respective diets and water throughout the study. At 14 weeks, female and male rats were paired at a 2:1 ratio. Pregnancy was confirmed by microscopic examination of vaginal smears for sperm presence, with gestational day 0 defined as the day of sperm detection. Non-pregnant rats were excluded. After 12 h of fasting, the GDM group received a single intraperitoneal injection of streptozotocin (STZ, 25 mg/kg). Blood glucose levels were measured for three consecutive days post-injection, and rats with levels > 16.7 mmol/L were considered successfully modeled. Rats failing to meet this criterion were excluded. Post-modeling, rats were divided into four groups: G, GDM, GDM + V (Vaspin 1 µg/kg/day, intraperitoneal injection), and GDM + V + L (L-NAME 25 mg/kg/day, oral gavage).

Glucose tolerance test (GTT) and Insulin tolerance test (ITT)

For GTT, rats were fasted for 16 h before receiving an intraperitoneal injection of glucose (2 g/kg). Blood glucose levels were measured at 0, 30, 60, 90, and 120 min post-injection via tail vein sampling. For ITT, rats were fasted for 4 h and injected intraperitoneally with insulin (1 U/kg). Blood glucose levels were measured at the same intervals.

Serum collection from rats

On the 14th day after pregnancy, rats were anesthetized with a 20% urethane solution (1 g/kg, intraperitoneal injection), and blood samples were collected. After allowing the blood to clot for 1 h, serum was isolated by centrifugation at 2000 × g for 15 min and stored at − 80 °C.

Enzyme-linked immunosorbent assay (ELISA)

Levels of human and rat Vaspin, insulin, C-peptide, and eNOS were measured using ELISA kits (Mlbio, Shanghai, China) according to the manufacturer’s instructions. Serum concentrations were quantified by diluting thawed samples to an appropriate ratio and then assaying them against a standard curve for quantification.

Measurement of SOD and NO levels

Superoxide dismutase (SOD) and NO levels in serum were quantified using commercial kits (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols. The analyte concentration was measured in both serum and cellular samples. For serum, samples were thawed and diluted to an appropriate ratio before the assay. For cellular analysis, cells were homogenized, and the supernatant was collected following centrifugation of the lysates at 12,000 g. All measurements were quantified against a standard curve.

Cell viability assay (CCK-8)

Cell viability was assessed using the CCK-8 kit (APExBIO, Houston, USA). Briefly, 10 µL of CCK-8 solution was added to each well of a 96-well plate, and cells were incubated for 1 h. Absorbance was measured at 450 nm. Results were compared with those of the control to assess cell viability.

Measurement of mitochondrial membrane potential, ROS, and NO levels

The mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS), and NO levels were assessed using specific fluorescent probes. Cells were seeded in confocal dishes and subjected to designated treatments. Following treatment, the cells were stained with JC-1, DCFH-DA, and DAF-FM DA probes (Beyotime Biotechnology, Shanghai, China) by incubating at 37 °C for 30 min. Fluorescent images were subsequently captured using an Olympus laser scanning confocal microscope (Japan). The fluorescence intensity was quantified from the acquired images for statistical analysis.

Statistical analysis

Data were analyzed using SPSS 23.0 (SPSS Inc., Chicago, IL, USA). Results are presented as mean ± standard deviation. Normality and homogeneity of variance were assessed, and differences between groups were analyzed using ANOVA or the Kruskal-Wallis test, as appropriate. Post-hoc pairwise comparisons were performed using the Games-Howell test. A p-value < 0.05 was considered statistically significant.

A post hoc power analysis was performed using SPSS software to assess the adequacy of the clinical sample size. Using the significant difference in serum Vaspin levels between the GDM and control groups as the primary indicator, the calculated Cohen’s d effect size was 2.39 (representing a very large effect). With an α level of 0.05 and sample sizes of 16 (GDM) and 42 (Control), the analysis demonstrated that the statistical power (1 – β) of this study exceeded 0.99, which is well above the conventional threshold of 0.80, indicating a very low risk of Type II error.

Results

Association of Vaspin and IR with GDM

The clinical characteristics of the 140 participants were analyzed and are detailed in Table 1. Significant differences were observed between the GDM and G groups in age, glycosylated hemoglobin (HbA1c), fasting blood glucose (FBG), fasting insulin (FINS), and Vaspin levels (P < 0.05). Notably, serum Vaspin levels were approximately 1.5 times higher in the GDM group compared to the control group (5.05 ± 1.05 ng/ml vs. 3.39 ± 0.43 ng/ml, P = 0.001). These variables were quantified (Table 2), and univariate logistic regression was conducted to assess the relationship between individual indicators and gestational diabetes. The results, presented in Table 3, indicated that all factors except body mass index (BMI) were associated with GDM. The independence of all indicators was confirmed by the calculation of tolerance (TOL) and variance inflation factor (VIF) to assess multicollinearity (Table 4). After excluding collinearity, multifactor logistic regression was performed. Using forward stepwise regression, age, Vaspin, and HOMA-IR were identified as independent risk factors for GDM (Table 5). The correlation coefficients for age, FBG, FINS, Vaspin, and HOMA-IR were positive and statistically significant (P < 0.05), suggesting their association with GDM. Survival probabilities derived from these results were used to construct the ROC curve (Fig. 1), which yielded an area under the curve (AUC) of 0.892 (95% CI: 0.784–0.994) (Table 6). The model incorporating age, Vaspin, and HOMA-IR exhibits excellent power for differentiating GDM patients from healthy controls, supported by an AUC of 0.892. These findings demonstrate that Vaspin and IR are associated with GDM.

Table 1.

Clinical and laboratory indicators of the population samples

Variables	G group (n = 42)	GDM group (n = 16)	P
Age (years)	27.86 ± 4.39	31.14 ± 4.87	0.021
BMI	21.95 ± 3.30	23.21 ± 3.76	0.235
HbA1c (%)	4.95 ± 0.27	5.16 ± 0.44	0.033
FBG (mmol/L)	4.28 ± 0.25	5.25 ± 0.62	0.001
FINS (mIU/L)	9.85 ± 1.30	12.35 ± 2.25	0.001
Vaspin (ng/mL)	3.39 ± 0.43	5.05 ± 1.05	0.001
HOMA-IR	1.90 ± 0.30	2.79 ± 0.39	0.001

Data are expressed as mean ± standard deviation

Table 2.

Assignment of variables that May be associated with gestational diabetes mellitus

Variables	assignment
Age (years)	< 30 = 0, ≥ 30 = 1
BMI	18.5–22.9 = 0, 23-24.9 = 1, 25-29.9 = 2, ≥ 30 = 3
HbA1C (%)	4.0–6.0 = 0, > 6.0 = 1
FBG (mmol/L)	< 5.1 = 0, ≥ 5.1 = 1
FINS (mIU/L)	< 9.17 = 0, 9.18–10.36 = 1, 10.37–11.46 = 2, > 11.46 = 3
Vaspin (ng/mL)	< 3.01 = 0, 3.02–3.76 = 1, 3.77–4.24 = 2, > 4.24 = 3
HOMA-IR	< 1.76 = 0, 1.76–2.07 = 1, 2.08–2.31 = 2, > 2.31 = 3
GDM	no = 0, yes = 1

Table 3.

Univariate logistic regression analysis of the risk factors for GDM

Variables	β	SE	Wals	OR	P	OR 95% CI
						Lower-bound	Upper-bound
Age	2.715	0.746	12.233	15.111	0.001	3.499	65.264
BMI	-0.057	0.343	0.028	0.944	0.867	0.482	1.848
HbA1c	0.477	0.316	2.279	0.631	0.131	0.867	2.994
FBG	3.664	0.832	19.387	39.000	0.001	7.635	199.208
FINS	1.134	0.371	9.370	3.110	0.002	1.504	6.429
Vaspin	3.048	0.821	13.773	21.070	0.001	4.213	105.367
HOMA-IR	3.228	0.905	12.725	25.238	0.001	4.283	148.729

Table 4.

Collinearity in the diagnosis of each contributing factor

Model	TOL	VIF
Age	0.816	1.255
BMI	0.830	1.205
HbA1c	0.854	1.170
FBG	0.538	1.858
Vaspin	0.295	3.393
FINS	0.411	2.432
HOMA-IR	0.183	5.459

Table 5.

Binary logistic regression analysis

Variables	β	SE	Wals	OR	P	OR 95% CI
						Lower-bound	Upper-bound
Age	4.107	1.776	5.409	16.683	0.020	2.850	42.451
Vaspin	2.986	1.432	4.348	19.815	0.037	1.196	328.218
HOMA-IR	2.156	1.047	4.242	8.636	0.039	1.110	67.179

Fig. 1.

ROC curve

Table 6.

Area under curve

AUC	SE	P	95% CI
			Lower-bound	Upper-bound
0.892	0.041	0.001	0.784	0.994

General characteristics of the GDM rats

Key metabolic parameters were evaluated to characterize the successfully established GDM rat model (Fig. 2). Compared with the control group, the GDM group showed significantly lower body weight and higher blood glucose levels (Figs. 2A, B). Furthermore, both glucose tolerance and insulin tolerance were markedly impaired in the GDM group, as evidenced by significantly larger AUC values for GTT and ITT (Figs. 2C–F). These results collectively confirm impaired glucose clearance and pronounced insulin resistance in the GDM model, recapitulating the core clinical features of the human condition.

Fig. 2.

General characteristics of the GDM rat. (A) Body weight comparison between GDM and control (G) groups; (B) Blood glucose levels (n = 18); (C) Intraperitoneal glucose tolerance test (GTT); (D) Area under the GTT curve (n = 6); (E) Intraperitoneal insulin tolerance test (ITT); (F) Area under the ITT curve (n = 6). Data are expressed as mean ± standard deviation. Statistical significance was determined using Student’s t-test (P < 0.05 vs. Group G)

Vaspin enhances pancreatic islet function in GDM rats

Vaspin has been shown to enhance pancreatic islet function in rats with gestational diabetes mellitus (GDM). On the 14th day post-pregnancy, several indicators were assessed. Serum Vaspin levels were measured in both the GDM and control (G) groups. Results indicated a significant increase in serum Vaspin levels in the GDM group compared to the control group (P < 0.05), aligning with clinical sample findings (Fig. 3A). Figures 3B–C illustrate that fasting blood glucose (FBG) levels at 12 h were significantly higher in the GDM group than in the control group (P < 0.05), with no significant difference observed between the GDM + V (GDM rats treated with Vaspin) and GDM groups (P > 0.05). Insulin levels were also significantly elevated in the GDM group compared to the control group, likely due to a compensatory increase in insulin. This elevation also confirms pancreatic function in rats, as insulin’s metabolic cycle is only a few hours, and this test was conducted 13 days after STZ injection. Vaspin intervention significantly reduced insulin levels in the GDM + V group compared to the GDM group (P < 0.05). This reduction in FINS following Vaspin treatment suggests an improvement in systemic insulin sensitivity. Furthermore, the GDM group exhibited more severe IR (P < 0.05), although C-peptide levels remained unchanged (P > 0.05), suggesting no significant alteration in insulin production. However, intraperitoneal administration of Vaspin significantly alleviated IR and reduced C-peptide production (P < 0.05). The decrease in C-peptide levels upon Vaspin treatment, coupled with improved HOMA-IR, indicates that Vaspin’s beneficial effect stems from enhancing insulin sensitivity in peripheral tissues rather than directly stimulating excessive insulin secretion from pancreatic β-cells. These findings suggest that Vaspin may confer a protective effect by mitigating IR in GDM.

Fig. 3.

Vaspin enhances pancreatic islet function in GDM rats (n = 6). (A) Serum Vaspin concentration in GDM and G rats; (B) FBG levels in SD rats; (C) FINS) levels in GDM and control rats; (D) Serum C-peptide levels in SD rats; (E) HOMA-IR values, calculated as FBG × FINS / 22.5. Data are expressed as mean ± standard deviation. *P < 0.05 vs. Group G; #P < 0.05 vs. GDM group

Vaspin attenuates IR in GDM rats via modulation of the ROS/eNOS/NO pathway

A high-glucose environment induces oxidative stress, resulting in eNOS decoupling and reduced NO production [23]. To investigate this mechanism, oxidative stress levels and the ROS/eNOS/NO pathway were assessed in the experimental rats. SOD, a critical antioxidant metalloenzyme, plays a crucial role in maintaining the oxidative-antioxidative balance. In this study, L-NAME, an eNOS inhibitor, was introduced to further explore the pathway. SOD, eNOS, and NO levels were measured, and HOMA-IR values were calculated.

As shown in Fig. 4A, SOD levels in the GDM group were significantly lower than those in the control (G) group (P < 0.05), indicating reduced antioxidant capacity and confirming that high glucose levels in GDM induce oxidative stress. Conversely, SOD levels in the GDM + V group (GDM rats treated with Vaspin) were significantly higher than those in the GDM group (P < 0.05), demonstrating that Vaspin enhances antioxidant capacity. However, SOD levels in the GDM + V + L group (GDM + V rats treated with L-NAME) were significantly lower than those in the GDM + V group (P < 0.05), suggesting that Vaspin exerts its antioxidant effects through eNOS.

Fig. 4.

Vaspin attenuates IR in GDM rats via modulation of the ROS/eNOS/NO pathway (n = 6). (A) Serum SOD levels in rats; (B) Serum eNOS levels in rats; (C) Serum (NO levels in rats; (D) HOMA-IR values in rats. Data are expressed as mean ± standard deviation, * P < 0.05 vs. G group; # P < 0.05 vs. GDM group; & P < 0.05 vs. GDM + V group

Further analysis of the ROS/eNOS/NO pathway revealed that Vaspin increased antioxidant levels, ultimately reducing NO production in a high-glucose environment. This conclusion was supported by the use of L-NAME, an eNOS inhibitor (Fig. 4B-C). Specifically, Vaspin treatment significantly increased serum eNOS and NO levels compared to the GDM group (P < 0.05), and this effect was abolished by L-NAME co-administration. NO contributes to IR by nitrating tyrosine residues on insulin receptor substrate 1 (IRS-1) and directly affecting downstream signaling molecules, such as PKB/Akt, in the insulin signaling pathway [24].

HOMA-IR values were evaluated to assess IR. The results (Fig. 4D) indicated that the GDM group exhibited significantly higher HOMA-IR values compared to the control group (P < 0.05). However, HOMA-IR values in the GDM + V group were significantly lower than those in the GDM group (P < 0.05). Upon the administration of L-NAME, the IR index in the GDM + V group increased significantly (P < 0.05). Notably, the beneficial effects of Vaspin on FBG, FINS, SOD, eNOS, NO, and HOMA-IR were significantly attenuated or reversed by L-NAME. This provides compelling pharmacological evidence that the ROS/eNOS/NO pathway is central to Vaspin’s mechanism of action. These findings collectively suggest that Vaspin ameliorates IR by modulating the ROS/eNOS/NO signaling pathway.

Vaspin attenuates mitochondrial oxidative stress via the ROS/eNOS/NO pathway

The expression of the conventional internal reference protein displayed significant tissue specificity, making it unreliable for cross-tissue comparisons. Consequently, we evaluated two additional reference proteins, but neither showed consistent expression across all tissues. Therefore, we employed absolute quantification for our analysis. The results indicated that the Vaspin content per 30 µg of total protein was highest in myocardial and pancreatic tissues (Fig. 5A). Given the correlation between GDM and insulin-secreting cells, we selected islet cells (INS-1) for further investigation. INS-1 cells were divided into four groups: control (C), high glucose (H), high glucose + Vaspin (H + V), and high glucose + Vaspin + L-NAME (H + V + L). The CCK-8 assay revealed that INS-1 cells cultured in 33.3 mmol/L glucose exhibited lower viability than those cultured in 11.1 mmol/L glucose. Treatment with 320 ng/mL Vaspin mitigated the adverse effects of high glucose on cell viability, increasing it by approximately 7% compared to the H group. However, the protective effect of Vaspin was negated by co-treatment with 50 mg/mL L-NAME, an eNOS inhibitor (Fig. 5B).

Fig. 5.

Vaspin attenuates mitochondrial oxidative stress via the ROS/eNOS/NO pathway. (A) The expression of Vaspin and insulin in tissues closely related to glucose metabolism; (B) INS-1 cell viability assessed by CCK-8 assay (n = 6); (C) Representative fluorescence images of MMP; (D) Quantification of MMP red-to-green fluorescence ratio (n = 3); (E) Intracellular ROS levels assessed using DCFH-DA fluorescent probe; (F) Quantification of ROS fluorescence intensity (n = 3); (G) eNOS protein levels in INS-1 cells (n = 6); (H) NO levels measured using DAF-FM fluorescent probe; (I) Quantification of NO fluorescence intensity (n = 3). Data are expressed as mean ± standard deviation, * P < 0.05 vs. C group; # P < 0.05 vs. H group; & P < 0.05 vs. H + V group

Mitochondria are the primary sites of ROS production, and mitochondrial membrane potential (MMP) is a critical indicator of mitochondrial function [23]. To assess mitochondrial function, JC-1 fluorescent probes were used to measure MMP. A higher red-to-green fluorescence ratio indicates better mitochondrial integrity and reduced ROS production (Fig. 5C-D). The results demonstrated that Vaspin mitigated high glucose-induced MMP damage, as evidenced by a significantly higher red/green fluorescence ratio in the H + V group compared to the H group (P < 0.05), whereas this protective effect was abolished by L-NAME treatment (P < 0.05).

To further confirm ROS production, the DCFH-DA fluorescent probe was employed. The results were consistent with MMP predictions, showing increased ROS levels following MMP disruption (Fig. 5E-F). Quantification of fluorescence intensity revealed that Vaspin treatment significantly reduced high glucose-induced ROS overproduction, and this reduction was reversed by L-NAME. Additionally, eNOS and NO levels were measured (Fig. 5G-I). High glucose reduced both eNOS expression and NO production in INS-1 cells, while Vaspin treatment attenuated these effects. However, the beneficial effects of Vaspin were blocked by L-NAME (P < 0.05). This series of experiments demonstrates a clear causal chain: Vaspin preserves mitochondrial function (MMP), thereby reducing ROS production, which in turn prevents the downregulation of eNOS and the subsequent loss of NO bioavailability under high glucose conditions.

Discussion

Gestational diabetes mellitus (GDM) is a prevalent metabolic disorder characterized by insulin resistance (IR), which poses significant risks to both maternal and neonatal health [4, 25]. The search for adipokines involved in metabolic homeostasis has identified Vaspin as a potential key player. Initially discovered in a state of insulin resistance, Vaspin secretion appears to peak during compensatory phases, suggesting its elevation might be a protective response to metabolic stress [26]. Our study aimed to dissect the association between Vaspin and GDM and to explore its potential relationship with the ROS/eNOS/NO pathway.

Our clinical findings solidify the connection between Vaspin, IR, and GDM. The significantly elevated levels of Vaspin in the GDM group, alongside traditional markers like FBG, FINS, and HOMA-IR, are consistent with previous reports [27–29]. Furthermore, multivariate logistic regression identified Vaspin as an independent risk factor, reinforcing its potential role in GDM pathophysiology beyond a simple association with hyperglycemia. To mechanistically investigate this relationship, we employed a well-established GDM rat model induced by a high-fat/high-sucrose diet combined with a low-dose STZ injection [30]. This model successfully recapitulated key clinical features of GDM, including hyperglycemia, impaired glucose tolerance, and insulin resistance. A pertinent consideration is the use of STZ, which can impair pancreatic β-cell function. However, the low dose (25 mg/kg) used in our protocol, coupled with the short experimental timeline (assessment on gestational day 14), likely induced a partial and compensated β-cell dysfunction rather than complete ablation. This is supported by the detectable and dynamic levels of fasting insulin and C-peptide in our GDM rats, indicating preserved, albeit dysregulated, insulin secretion capacity. Therefore, this model remains a valuable tool for studying the insulin-sensitizing effects of interventions during the acute phase of GDM [31].

Our central finding is that Vaspin administration ameliorated IR in GDM rats, as evidenced by improved FBG, FINS, and HOMA-IR. We further investigated the potential involvement of the ROS/eNOS/NO axis in this process. A high-glucose environment can induce oxidative stress, which may uncouple eNOS, reducing the production of NO and contributing to IR [32, 33]. Our data demonstrate that Vaspin treatment was associated with enhanced antioxidant capacity (increased SOD), reduced oxidative stress, and concurrently improved eNOS and NO levels. The observation that L-NAME, a specific eNOS inhibitor, attenuated the beneficial effects of Vaspin on these parameters suggests that eNOS activity may be an important component in the mechanism of Vaspin’s action.

This insight was further explored in INS-1 cells under high-glucose conditions. Vaspin appeared to protect against high glucose-induced cytotoxicity, mitochondrial dysfunction, and oxidative stress. It also was associated with the prevention of eNOS downregulation and loss of NO. The co-administration of L-NAME reduced these protective effects, which is consistent with a potential role for eNOS in the cellular actions of Vaspin. The concordance between the in vivo and in vitro findings suggests that Vaspin may mitigate high-glucose-induced metabolic disturbances, potentially in part through mechanisms involving eNOS and NO signaling.

While our study provides insights, several limitations must be acknowledged. First, the primary evidence for the mechanism’s dependence on eNOS relies on pharmacological inhibition. To further strengthen this finding, future studies could employ genetic approaches, such as siRNA-mediated eNOS knockdown, and directly assess eNOS phosphorylation status to better understand Vaspin’s influence on eNOS activation [20]. Second, the STZ-induced GDM model, while useful, does not fully mirror the natural pathogenesis of human GDM. The irreversible component of β-cell damage by STZ is a constraint; thus, investigating Vaspin in other models would be informative. Finally, the precise upstream signaling events by which Vaspin influences mitochondrial ROS and eNOS function remain to be elucidated.

In conclusion, our data demonstrate that Vaspin is associated with GDM and that its insulin-sensitizing effects, both in vivo and in vitro, appear to be partially mediated through eNOS. The protective effects of Vaspin may involve the mitigation of mitochondrial oxidative stress and the subsequent preservation of eNOS/NO signaling. We propose that the elevated Vaspin levels observed in GDM could represent a compensatory mechanism to counteract insulin resistance. These findings position Vaspin as a candidate for further investigation in GDM, and targeting the ROS/eNOS/NO axis may represent a potential therapeutic strategy, though further research is necessary to fully establish the causal relationships and detailed mechanisms.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

W.T. and J.X., Conceptualization; design; writing-review and editing. X.Z., L.W. and X.H., Writing-original draft; investigation; validation and formal analysis. J.Y., Q.H., Investigation; Methodology. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grant QNRC202313 from Youth Talent Promotion Program of School of Public Health, North China University of Science and Technology and grant 226Z7711G from the Hebei Provincial Fund for Central Guiding Local Science and Technology Development Plan. North China University of Science and Technology and grant 246Z7715G from the Hebei Provincial Fund for Central Guiding Local Science and Technology Development Plan.

Data availability

The data that support this study are available within the article and supplementary material.

Declarations

Ethics approval and consent to participate

This study was conducted in accordance with the principles of the Declaration of Helsinki. The study involving human participants was reviewed and approved by the Ethics Committee of Tangshan Central Hospital. All participants provided written informed consent prior to blood collection and participation in the study. The animal experiment was approved by the Experimental Animal Ethics and Welfare Committee of North China University of Science and Technology (Approval No. 2023-SY-44). All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

The authors affirm that human research participants provided informed consent for publication. Consent to Publish declaration: not applicable.

Informed consent

Not applicable.

Competing interests

The authors declare no competing interests.