Expression and correlation analysis of silent information regulator 1 (SIRT1), sterol regulatory element-binding protein-1 (SREBP1), and pyroptosis factor in gestational diabetes mellitus

Abstract

Background and aim

Globally, the prevalence of gestational diabetes mellitus (GDM) is rising each year, yet its pathophysiology is still unclear. To shed new light on the pathogenesis of gestational diabetes mellitus and perhaps uncover new therapeutic targets, this study looked at the expression levels and correlations of SIRT1, SREBP1, and pyroptosis factors like NLRP3, Caspase-1, IL-1, and IL-18 in patients with GDM.

Methods

This study involved a comparative analysis between two groups. The GDM group consisted of 50 GDM patients and the control group included 50 pregnant women with normal pregnancies. Detailed case data were collected for all participants. We utilized real-time quantitative PCR and Western Blot techniques to assess the expression levels of SIRT1 and SREBP1 in placental tissues from both groups. Additionally, we employed an enzyme-linked immunosorbent assay to measure the serum levels of SIRT1, SREBP1, and pyroptosis factors, namely NLRP3, Caspase-1, IL-1β, and IL-18, in the patients of both groups. Subsequently, we analyzed the correlations between these factors and clinical.

Results

The results showed that there were significantly lower expression levels of SIRT1 in both GDM group placental tissue and serum compared to the control group (p < 0.01). In contrast, the expression of SREBP1 was significantly higher in the GDM group than in the control group (p < 0.05). Additionally, the serum levels of NLRP3, Caspase-1, IL-1β, and IL-18 were significantly elevated in the GDM group compared to the control group (p < 0.01). The expression of SIRT1 exhibited negative correlations with the expression of FPG, OGTT-1h, FINS, HOMA-IR, SREBP1, IL-1β, and IL-18. However, there was no significant correlation between SIRT1 expression and OGTT-2h, NLRP3, or Caspase-1. On the other hand, the expression of SREBP1 was positively correlated with the expression of IL-1β, Caspase-1, and IL-18, but has no apparent correlation with NLRP3.

Conclusions

Low SIRT1 levels and high SREBP1 levels in placental tissue and serum, coupled with elevated levels of pyroptosis factors NLRP3, Caspase-1, IL-1β, and IL-18 in serum, may be linked to the development of gestational diabetes mellitus. Furthermore, these three factors appear to correlate with each other in the pathogenesis of GDM, offering potential directions for future research and therapeutic strategies.

Keywords:

• Gestational diabetes mellitus
• silent information regulator 1
• sterol regulatory element binding protein 1
• pyroptosis
• insulin resistance

Introduction

Gestational Diabetes Mellitus (GDM) is a condition characterized by varying degrees of hyperglycemia resulting from impaired carbohydrate tolerance that either develops or is first detected during pregnancy [1]. In recent years, there has been a steady increase in the global prevalence of GDM, likely attributed to shifts in lifestyle and the implementation of the two-child policy. However, despite its growing incidence, the underlying pathogenesis of GDM remains incompletely understood, posing a significant challenge for healthcare professionals. Research has highlighted the close associations between GDM and various factors, such as genetics, obesity, insulin resistance, oxidative stress, and inflammation [2–4]. Silent information regulator 1 (SIRT1) is an evolutionarily conserved enzyme that relies on NAD + and serves as a histone and non-histone deacetylase [5]. It is widely expressed in metabolic organs like the liver, pancreatic islets, adipose tissue, and skeletal muscle, playing a critical role in diverse biological processes, including responses to oxidative stress and the development of insulin resistance [6,7]. Sterol regulatory element-binding protein-1 (SREBP1) is a transcription factor primarily responsible for regulating the synthesis of lipids, including fatty acids and total cholesterol. It also plays a role in insulin signaling pathways [8]. Pyroptosis is a Caspase-1-dependent programmed cell death directly related to inflammation [9]. The inflammatory response in the placenta is believed to be a significant factor in insulin resistance in GDM [10]. However, there is a relative scarcity of research exploring potential correlations between SIRT1 and SREBP1 and pyroptosis factors in the context of GDM. This study seeks to investigate the expression of SIRT1 and SREBP1 in placental tissue, along with the serum levels of SIRT1, SREBP1, and pyroptosis factors, including NLRP3, Caspase-1, IL-1β, and IL-18 in GDM patients. Additionally, it aims to analyze the potential correlations between these factors. The ultimate goal is to gain a deeper understanding of the pathogenesis of gestational diabetes mellitus and provide novel insights into its prevention and treatment strategies.

Materials and methods

Participants and sample collection

A total of 100 pregnant women who received routine prenatal care at the obstetrics outpatient clinic of the Third Affiliated Hospital of Zhengzhou University between September 2021 and May 2022 were included in this study. These participants were categorized into two groups: 50 pregnant women with normal pregnancies and 50 pregnant women diagnosed with gestational diabetes mellitus (GDM) based on the results of the mid-pregnancy (24–28 weeks) 75 g oral glucose tolerance test (OGTT) [11]. Inclusion criteria for the study were as follows: Singleton pregnancy with a live fetus. No history of acute or chronic diseases before pregnancy. Receiving regular obstetric care at our hospital throughout the perinatal period and planning to deliver at our hospital. Subjects provided informed consent. Exclusion criteria included: Twin or multiple pregnancies. Requirement for insulin treatment during pregnancy. A preexisting diagnosis of diabetes mellitus (including type 1 and type 2) before pregnancy. Co-occurrence of serious systemic diseases, such as severe liver or renal impairment, cardio-cerebral and cerebral vascular diseases, etc. Presence of endocrine disorders, such as abnormalities in thyroid function. Combined pregnancy complications, such as hypertensive disorders of pregnancy, pre-eclampsia, intrahepatic cholestasis in pregnancy, etc. This clinical trial received approval from the Medical Ethics Committee of our hospital, and informed consent was obtained from the participating patients. Ethical Approval No. 2021-056-01.

For the serum sample collection, ∼5 ml of blood was drawn from the median elbow vein of the pregnant women while fasting, before their delivery. The collected blood was then centrifuged at 4 °C for 10 min at 3000 revolutions per minute (r/min) to obtain the supernatant. The supernatant was carefully preserved by storing it in a refrigerator at −80 °C.

As for placenta sample collection, within 30 min of full-term delivery, a portion of the placental tissue from the maternal side, approximately 3 *3*1 mm in size, was carefully obtained to avoid any bleeding, necrosis, or calcification. The surface of the tissue was cleaned to remove any blood stains using physiological saline. The tissue block obtained was rapidly frozen in liquid nitrogen and then stored in a −80 °C refrigerator for subsequent measurements.

ELISA

The serum levels of SIRT1, SREBP1, NLRP3, Caspase-1, IL-1β, and IL-18 in each group of pregnant women were determined using Enzyme-Linked Immunosorbent Assay (ELISA). The assay kits used for SIRT1 and SREBP1 were obtained from Wuhan Huamei Bioengineering Co., while the kit for pyroptosis factors was purchased from Jiangsu Enzyme Immunity Industry Co. All procedures were carried out meticulously following the kit instructions.

Furthermore, a fully automatic electrochemiluminescence immunoassay analyzer was employed to measure the fasting glucose (FPG), HbA1c (%), and fasting insulin levels (FINS) of pregnant women. The Homeostasis Model Assessment-Insulin Resistance (HOMA-IR) was calculated using the formula: HOMA-IR = (FPG * FINS)/22.5.

Real-time quantitative PCR (qRT-PCR)

qRT-PCR was employed to assess the mRNA expression levels of SIRT1 and SREBP1 in placental tissues. The following steps were carried out: Trizol reagent (Beijing Seven Biotech Co., Ltd) was used to extract total RNA from placental tissues. The extracted total RNA was reverse-transcribed into cDNA. The total reaction system (20 μL) of the qRT-PCR kit (Beijing TransGen Biotech Co., Ltd.) consisted of 5 μL of cDNA, 0.4 μL of each of the upstream and downstream primers (Beijing Tsingke Biotech Co., Ltd.), 10 μL of 2× PerfectStart Green qPCR SuperMix, and 4.2 μL of enzyme-free water. The 2^−ΔΔCt method was used to calculate the relative expression levels of SIRT1 and SREBP1 mRNAs. Table 1 contains a list of the primer sequences used in qRT-PCR.

Table 1. Primer sequences.

	Primer sets
SIRT1	Forward 5′-CAAAGGAGCAGATTAGTAGGCG-3′
	Reverse 5′-CTCTGGCATGTCCCACTATCAC-3′
SREBP1	Forward 5′-GCCTATTTAACCCACCCTATG-3′
	Reverse 5′-TGGCACTGACTCTTCCTTGAT-3′
GAPDH	Forward 5′-GGAGTCCACTGGCGTCTTCA-3′
	Reverse 5′-GTCATGAGTCCTTCCACGATACC-3′

Western blot

Placental tissue proteins were extracted using radioimmunoprecipitation (RIPA) lysate. The protein concentration in the extracted samples was determined using a BCA assay kit (Beijing Seven Biotech Co., Ltd.). Samples containing 25 μg of protein per well were loaded onto a polyacrylamide gel (SDS-PAGE) (Epizyme Biotech) for electrophoresis. The proteins were separated by electrophoresis on the SDS-PAGE gel. After electrophoresis, the proteins on the gel were transferred onto a PVDF membrane at a constant flow of 400 mA. The PVDF membrane was blocked with 5% skimmed milk powder at room temperature for 1 h. The membrane was then incubated with diluted anti-SIRT1 (1:4500 dilution, #13161-1-AP Proteintech), anti-SREBP1 (1:3000 dilution, #14088-1-AP Proteintech), and anti-β-actin (1:8000 dilution, BM0627, Boster) antibodies at 4 °C on a shaking bed overnight. After incubation, the membrane was washed with TBST. Goat Anti-Rabbit IgG-HRP antibody (1:8000 dilution, BA1054, Boster) was added to SIRT1 and SREBP1 membranes, Goat Anti-Mouse IgG-HRP antibody (1:15,000 dilution, 511103, Zenbio) was added to β-actin membrane, then incubated at room temperature for 2 h. ECL chemiluminescent solution was added to develop the image. The gray value of the bands on the membrane was analyzed using Image J software.

Statistical analysis

The Statistical Package for the Prism 9.0 software was used for the data analysis. Date that conformed to normal distribution were expressed as means ± SDs, and comparisons between the two groups were made using the independent samples t-test, and correlation analyses were performed using Pearson’s correlation test, while those that did not conform to normal distribution were expressed as [M(P25,P75)], and comparisons between the two groups were made using the Mann–Whitney U test, and correlation analyses were performed using Spearman’s correlation test. p-Values <0.05 were considered to be significant.

Results

Pre-pregnancy BMI, FPG, OGTT-1h, OGTT-2h, FINS, HOMA-IR, HbA1c, neonatal weight, and gestational week of delivery differed significantly between the control and GDM groups, but differences in age, gestational weight gain, and placental mass were not statistically significant. Table 2 summarizes the clinical materials and laboratory characteristics of the participants.

Table 2. Clinical materials and laboratory characteristics of the participant.

Characteristics	Control (n = 50)	GDM (n = 50)	p-Value
Age (years)	30.82 ± 3.53	31.9 ± 3.16	0.110
Pre-pregnancy BMI (kg/m^2)	21.92 ± 2.30	23.18 ± 3.50	0.037
Gestational weight gain (kg)	11.74 ± 4.5	11.84 ± 4.67	0.914
Gestational week of labor	39.57 ± 0.87	38.75 ± 1.11	<0.0001
FPG (mmol/L)	4.54 ± 0.39	4.96 ± 0.41	<0.0001
OGTT-1h (mmol/L)	7.16 ± 1.32	9.62 ± 1.32	<0.0001
OGTT-2h (mmol/L)	6.77 ± 1.04	8.54 ± 0.98	<0.0001
FINS	10.34 ± 1.78	11.80 ± 1.83	0.0001
HOMA-IR	2.08 ± 0.38	2.61 ± 0.49	<0.0001
HbA1c (%)	5.10 ± 0.25	5.44 ± 0.32	<0.0001
Neonatal weight (kg)	3.24 ± 0.23	3.37 ± 0.33	0.028
Placenta mass (g)	610.3 ± 52.50	630.6 ± 72.91	0.114

FPG: fasting plasma glucose; OGTT-1h: 1-h oral glucose tolerance test; OGTT-2h: 2-h oral glucose tolerance test; FINS: fasting insulin; HOMA-IR: homeostatic model assessment of insulin resistance; HbA1c: insulin, glycated hemoglobin A1c.

Data are presented as mean ± standard deviation.

The serum level of SIRT1 was significantly lower in the GDM group compared with the control group, whereas SREBP1, NLRP3, Caspase-1, IL-1β, and IL-18 were significantly higher (p < 0.01). The results are summarized in Table 3.

Table 3. The results of ELISA for SIRT1, SREBP1, and pyroptosis factor in two groups.

	Control (n = 50)	GDM (n = 50)	p-Value
SIRT1 (ng/L)	344.8 ± 175.3	238.8 ± 125.2	0.0008
SREBP1 (ng/L)	1905 ± 1121	2546 ± 1174	0.006
NLRP3 (ng/L)	492.7 ± 114.6	633.2 ± 106.7	<0.0001
Caspase-1 (ng/L)	27.36 ± 11.88	45.71 ± 12.46	<0.0001
IL-1β (ng/L)	34.55 ± 9.31	45.36 ± 8.42	<0.0001
IL-18 (ng/L)	140.5 ± 43.15	209.7 ± 35.71	<0.0001

Data are presented as mean ± standard deviation.

SIRT1 mRNA expression was considerably lower in the GDM group compared to the control group, although SREBP1 mRNA expression was significantly higher (p < 0.01), as shown in Figure 1. SIRT1 protein expression was significantly reduced in the GDM group (p < 0.001) compared to the control group, but SREBP1 protein expression was significantly increased (p < 0.05), as shown in Figure 2.

Figure 1. Relative mRNA expression of SIRT1 and SREBP1 in control and GDM groups.

Figure 2. Relative protein expression of SIRT1 and SREBP1 in control and GDM groups. (A) Western blot detection of SIRT1 protein expression levels in placental tissues. (B) Western blot detection of SREBP1 protein expression levels in placental tissues. (C) SIRT1 and SREBP1 were significantly differentially expressed in the placental tissues of the two groups.

The serum levels of SIRT1 were found to have negative correlations with FPG, OGTT-1h, FINS, HOMA-IR, SREBP1, IL-1β, and IL-18. However, there was no significant correlation observed with OGTT-2h, NLRP3, and Caspase-1. Additionally, the serum level of SREBP1 was positively correlated with IL-1β, Caspase-1, and IL-18, but not with NLRP3, as shown in Figures 3 and 4.

Figure 3. Correlations of SIRT1 with biochemical parameters and pyroptosis factors.

Figure 4. Correlations of SREBP1 with pyroptosis factors.

Discussion

Gestational diabetes mellitus (GDM) is a prevalent metabolic disorder during pregnancy that is increasingly affecting women worldwide. This condition has significant implications for the health of pregnant women, fetuses, and newborns, including an elevated risk of complications, such as gestational hypertension, preterm labor, macrosomia, neonatal hypoglycemia, and an increased likelihood of developing type 2 diabetes mellitus in both the affected individuals and their offspring [12]. Despite its prevalence, the exact mechanisms underlying the development of GDM remain unclear. However, existing research suggests that insulin resistance, inflammation, and abnormal lipid metabolism play crucial roles in its pathogenesis [3]. Currently, there is limited scientific literature exploring the involvement of factors, such as SIRT1, SREBP1, and pyroptosis factors in GDM. Therefore, this study aims to investigate the expression of these factors in GDM patients and propose a potential mechanism by which SIRT1, SREBP1, and pyroptosis factors contribute to the development of GDM.

Pyroptosis

Pyroptosis is a form of programmed cell death that is associated with inflammation and is implicated in the pathogenesis of inflammatory and metabolic diseases. It has been observed that a moderate level of pyroptosis can contribute to the maintenance of homeostasis in the body’s internal environment. Excessive stimulation of the body, such as an overabundance of nutrients like glucose and lipids, can result in the activation of pyroptosis, exacerbating the inflammatory response and causing damage to tissues and organs to some extent [13]. This process can occur through various pathways, including the classical pathway, the non-classical pathway, the Caspase-3/8-mediated pathway, and the Granzyme-mediated pathway [14]. Among these pathways, the classical pathway is initiated by the activation of the inflammasome, in response to external stimuli or internal stress. This activation leads to the cleavage of Gasdermin D (GSDMD) by caspase-1 at the Asp275 site, resulting in the formation of a 22 kDa C-terminal fragment (C-GSDMD) and a 31 kDa N-terminal fragment (N-GSDMD) [15]. The N-GSDMD fragments subsequently generate nonselective pores in the cell membrane, with an inner diameter of ∼10–14 nm. This process results in cell swelling and lysis [16], as well as the release of pro-inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-18 (IL-18). Ultimately, these events lead to pyroptosis and elicit inflammatory and immune responses [17]. It has been demonstrated in previous studies that type 2 diabetes is characterized by chronic low-grade inflammation. Additionally, it has been observed that NLRP3 inflammasome can lead to insulin resistance through downstream signaling of IL-1β [18].

According to this study, pregnant women with GDM had higher serum levels of NLRP3, Caspase-1, IL-1, and IL-18 than pregnant women with normal pregnancies. This finding suggests that pyroptosis factors may play a role in the pathogenesis of GDM by promoting chronic inflammation. Hu et al. [19] discovered that the expression levels of NLRP3 and Caspase-1 were markedly increased in the placental tissues of pregnant women with GDM. Additionally, their experiment using high glucose-treated HTR-8/SVeno cells demonstrated that inhibiting NLRP3 inflammatory vesicles led to a significant reduction in the expression of Caspase-1, IL-1β, and IL-18. This finding supports the notion that high glucose may contribute to the development of GDM by activating NLRP3 inflammasome and promoting the release of IL-1β and IL-18. Schulze et al. [20] also observed a significant improvement in glucose tolerance in GDM mice following the inhibition of IL-1β expression in vivo. Therefore, there exists a close relationship between pyroptosis and GDM, suggesting that it may serve as one of the pathogenic mechanisms underlying GDM.

Sterol regulatory element-binding protein 1 (SREBP1)

Sterol Regulatory Element-Binding Protein 1 (SREBP1) is a transcription factor classified within the basic Helix-Loop-Helix Leucine Zipper family (bHLH-Zip) [21]. SREBP1 plays a crucial role in the regulation of lipid and cholesterol metabolism. The expression of SREBP1 is elevated in various metabolic disorders, including obesity, diabetes mellitus, and nonalcoholic fatty liver disease [22]. Within the cell, SREBP1 exists in an inactive form and is protected by the SREBP cleavage-activating protein (SCAP). When the body experiences a decrease in cholesterol levels or an increased demand for lipids, the SREBP-SCAP complex dissociates from INSIG (insulin-inducible gene) and moves from the endoplasmic reticulum (ER) to the Golgi apparatus. This translocation process enhances the expression of enzymes involved in cholesterol and lipid synthesis [23]. Petersen et al. [24] discovered that the activation of SREBP1 leads to the accumulation of fatty acid derivatives in cellular membranes. This accumulation causes an increase in the production of reactive oxygen species (ROS), activates atypical PKC, and phosphorylates serine residues in the insulin receptor (IRS), ultimately leading to insulin resistance. Thus, the transcription factor SREBP1 exerts regulatory control over factors involved in insulin secretion and plays a crucial role in the functioning of pancreatic islets.

Previous research [25–27] has demonstrated that the activation of SREBP1 triggers endoplasmic reticulum stress, subsequently resulting in cell death through the activation of NLRP3 inflammasome and the subsequent release of cellular IL-1β. In contrast, in our study, serum SREBP1 expression was positively correlated with Caspase-1, IL-18, and IL-1β, but not with NLRP3. This may be related to our small sample size. The mRNA and protein expression levels of SREBP1 were significantly higher in both placental tissue and serum of patients with GDM compared to controls in our study. This finding is in line with the research conducted by Hua et al. [28] which demonstrated a significant upregulation of SREBP1 mRNA and protein expression in pregnant db/+ mice.

Silent information regulator 1 (SIRT1)

Silent Information Regulator 1 (SIRT1) is a highly conserved NAD+-dependent deacetylase that plays a pivotal role in numerous biological processes. These processes encompass the regulation of energy metabolism, anti-inflammatory responses, insulin resistance, and glucolipid metabolism, among numerous other diverse biological processes [7]. SIRT1 accomplishes these functions through the deacetylation of a broad spectrum of substrates, which include histones, non-histone proteins, transcription factors, and various signaling molecules [29].

Wang et al. [30] discovered that the expression of SIRT1 was reduced in mice treated with the NAMPT inhibitor FK866, which is a key enzyme in systemic NAD + biosynthesis. This reduction in SIRT1 expression led to a significant increase in the expression of SREBP1 and its downstream factors, acetyl coenzyme A carboxylase (ACC) and fatty acid synthase (FASN). Conversely, treatment of cells with resveratrol, an activator of SIRT1, resulted in a significant decrease in the expression and phosphorylation of ACC. Resveratrol exhibited a similar inhibitory effect on the expression of factors involved in the cellular pyroptosis signaling pathway, including NLRP3 inflammasome, in the hippocampal tissues of obese mice [31]. The expression of SREBP1 in the livers of mice overexpressing SIRT1 was found to be significantly reduced, leading to an improvement in lipid metabolism disorder [32].

Relevant studies have demonstrated that SIRT1 exerts a significant inhibitory effect on the expression of NLRP3 in human umbilical vein endothelial cells. This inhibition subsequently leads to the suppression of Caspase-1 cleavage and IL-1β secretion, thereby effectively attenuating the inflammatory response [33]. In our study, we found a negative correlation between SIRT1 was negatively correlated with the expression of SREBP1, IL-1β, and IL-18. However, there was no correlation between NLRP3 and Caspase-1. Additionally, the expression of SIRT1 was significantly lower in placental tissues and sera of patients with GDM compared to controls. Ulubasoglu et al. [34] similarly found that the levels of SIRT1 are lower in the serum of pregnant women with GDM compared to normal pregnant women. In addition, low serum SIRT1 levels have been found to be highly significant in predicting GDM. Sin et al. [35] discovered that in skeletal muscle, resveratrol reduces the activity of Foxo1 by deacetylating its modification through the activation of SIRT1. This inhibits the inhibitory effect of Foxo1 on insulin target genes and enhances insulin signaling.

Conclusions

In summary, the mRNA and protein levels of SIRT1 in placental tissues and serum of GDM patients were significantly decreased. Meanwhile, SREBP1 levels were significantly increased, and the expression of pyroptosis NLRP3, Caspase-1, IL-1β, and IL-18 was significantly increased in the serum of GDM patients. Additionally, SIRT1 showed a negative correlation with SREBP1, IL-1β, and IL-18. SREBP1 showed a positive correlation with caspase-1, IL-18, and IL-1β. Thus, SIRT1, SREBP1, and pyroptosis factors interact with each other in GDM to form a complex regulatory network involved in the genesis and development of the condition.

However, the scope of this study was limited to the analysis of expression levels of SIRT1, SREBP1, and pyroptosis factors in human subjects. The study did not extensively investigate the functions and regulatory mechanisms of these factors. To comprehend the interactions of these factors in GDM. we will validate their roles through additional cellular experiments and animal models in the future.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author, Ning Han, upon reasonable request.

Additional information

Funding

This work was supported by the Science and Technology Department of Henan Province under Grant number 222102310129.