Introduction
Polycystic ovary syndrome (PCOS) is the most common complicated endocrine disease in women of childbearing age. It is characterized by hyperandrogenism, usually associated with hyperinsulinemia and insulin resistance (IR), and often accompanied by morphological manifestations of polycystic ovaries [
1]. Women with PCOS often have irregular menstrual cycles, infertility, obesity, hirsutism or acne. The incidence rate of PCOS in women of childbearing age is approximately 6–10% [
2]. The rising prevalence rate is the main cause of infertility in women of childbearing age. In addition, women who have polycystic ovary syndrome are recognized to have an elevated risk of experiencing various metabolic, obstetric, psychological and even tumor-related complications in their lifetime, posing a great threat to women’s health [
3].
However, the pathophysiological mechanism underlying PCOS remain unclear. Interestingly, chronic low-grade inflammation is recognized as a significant contributor to the development and occurrence of PCOS. PCOS is also a chronic inflammatory mediator disorder [
4]. It is well known that C-reactive protein (CRP) is a commonly used and dependable acute-phase reactant inflammation biomarker in clinical. Reports have shown that patients with polycystic ovary syndrome exhibit elevated levels of CRP, and this indicator is associated with infertility as well as diabetes [
5] and cardiovascular disease [
6]. These findings imply that CRP may play a multiple action in PCOS patients [
7]. Although these strong correlations have been found in clinical studies, the current research is limited to the observation of the PCOS population. Up to now, there are no reports on the pathophysiological mechanism of CRP in the progression of PCOS and whether CRP affects insulin resistance, glucose tolerance, and even hepatic glucose flux in PCOS.
Leptin is a peptide composed of 167 amino acids that is secreted by adipose tissue. As an adipokine, leptin has important effects on food intake, energy storage and body weight. The development of obesity, insulin resistance, and type 2 diabetes is closely associated with the significant feature of leptin resistance [
8]. Previous studies have shown that patients with PCOS have hyperleptinemia, which is positively correlated with obesity, insulin and body mass index [
9]. Because hyperleptinemia often reflects the existence of leptin resistance, it can be inferred that patients with PCOS also have leptin resistance. Studies have found that in insulin-resistant people, binding of human C-reactive protein (CRP) to leptin inhibits leptin signaling and impairs its function, thus exacerbating leptin resistance [
10]. Our previous studies have shown that CRP can directly regulate energy balance, body weight, insulin sensitivity, and glucose homeostasis by affecting leptin’s central effects in the hypothalamus [
11].
PCOS is a metabolism-related disease that integrates chronic low-grade inflammation, insulin resistance and leptin resistance [
9]. CRP concentrations are elevated in women with PCOS [
12] and has been closely linked to the insulin resistance. Collectively, these evidences suggest that CRP may exert some of its metabolic actions in part via interaction with leptin in PCOS. Accordingly, we used CRP knockout (KO) rats to construct a PCOS model to explore whether CRP gene deletion affects insulin resistance, hepatic glucose homeostasis and leptin bioactivity in PCOS rats. We hypothesize that the amplification of leptin action to lower hepatic glucose production (HGP) and promote insulin sensitivity in the absence of CRP in individuals with the hyperandrogen environment.
Materials and methods
Animal experiments
Animal experiments were performed according to protocols approved by the Animal Care Committee of Chongqing Medical University. CRP gene knockout rats were donated by Professor Zhao Zijian of Nanjing Medical University and reproduced in the Animal Experiment Center of Chongqing Medical University. Detailed production method as described previously [
11]. The animals were housed in a facility equipped with temperature and humidity control, following a 12-hour light/dark cycle. They were provided with ad libitum access to food and water. The animals were randomly allocated to different treatment groups.
Rat models
A total of 150 4-week-old female rats, either CRP knockout or littermate wild-type, were randomly assigned to either a normal chow diet (ND) or a high-fat diet (HFD) feeding group. Additionally, the rats were randomly allocated to different treatment groups. Female rats were injected daily subcutaneous with dehydroepiandrosterone (DHEA) (Sigma Aldrich) at a dose of 60 mg/kg/day [
13] dissolved in 0.2 ml sesame oil for up to 90 days, while the control group were injected with 0.2 ml sesame oil for an equivalent length of time. Vaginal exfoliative cytology was performed for 10 consecutive days on the 20th day after the start of subcutaneous administration, and the rats that exhibited successful development of the PCOS pathology were chosen for follow-up experiments.
Light microscope analysis
The ovarian tissues of rats were fixed in 4% paraformaldehyde for 24 h, thereafter, they were embedded in paraffin wax. The paraffin-embedded ovary Sect. (4 𝜇m) were stained using hematoxylin and eosin and calculated the number of follicles using a light microscope as described previously [
14].
Third ventricle and jugular arteriovenous cannulations
The stereotaxic brain surgeries were performed 7 days before the jugular arteriovenous cannulations. Indwelling catheters were stereotactically inserted into the third ventricle of the brain. Correct intubation position was confirmed as described previously [
15]. For the euglycemic hyperinsulinemic clamp (EHC) study, indwelling catheters were inserted into the internal jugular vein and carotid artery for infusion and blood sampling seven days after stereotactic surgery. Recovery from the surgery was monitored for a four-day period by measuring food intake and weight.
Daily body weight measurements were taken from rats aged 4–16 weeks. Food intake and rectal temperature were recorded at specific time points and durations. The rectal temperature was measured using an electronic thermometer. Energy expenditure was determined by measuring the 24-hour rates of VO2 and VCO2 production, as described previously [
11].
Intraperitoneal glucose and insulin tolerance experiments
Following a 6-hour fast, rats received an intraperitoneal injection of 25% glucose (2.0 g/kg) for the glucose tolerance test (GTT) or insulin (1.0 U/kg, Novolin R, Nordisk, Bagsvaerd, Denmark) for the insulin tolerance test (ITT). Venous blood was collected at the designated time before and after injection in order to quantitatively assess levels of glucose and insulin.
EHC and intracerebroventricular (ICV) infusion
Following a 12-hour fast, EHC was performed while the subjects were conscious. In brief, a continuous infusion of [3-H3] glucose that had undergone high-performance liquid chromatography (HPLC) purification (PerkinElmer, Waltham, MA) was started at t = 0 min with a 6 µCi bolus followed by a 0.2 µCi/min rate of infusion, which continued for a total of 240 min. The EHC was started at 120 min after the tracer infusion. Somatostatin (3 µg/kg/min) was infused intravenously together with insulin (6.0 mU/kg/min) to inhibit endogenous insulin secretion. A variable infusion of 25% glucose was started and adjusted every 10 min to maintain blood glucose at the baseline level. Subgroups of rats undergoing intravenous or ICV administration of leptin procedures, recombinant rat leptin (0.5ug/kg/min for peripheral, 0.83 µg/h for central, R&D Systems) was initiated at t = − 120 and continued until the end of the clamp at t = 240 min. To determine insulin, free fatty acids (FFA), and glucose-specific activity, blood samples were collected from the jugular vein catheter at 0, 120, 200, 220, 230, and 240 min. Subsequently, the rats were anesthetized, and tissue samples were freeze-clamped in situ using aluminum tongs pre-cooled with liquid nitrogen and stored at -80ºC for further analysis.
Biochemical analysis
Plasma glucose was measured using the glucose oxidase method. Plasma insulin was measured using a commercial RIA kit (HTA CO.LTD. Beijing, China). Plasma levels of leptin, CRP, estradiol (E2) ,testosterone (T), progesterone (P), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were detected by commercial enzyme-linked immunosorbent assay kits (Jingmei Biotechnology, Yancheng, China). [3-3 H] Glucose radioactivities was detected by scintillation counter.
mRNA analysis
To measure the expression levels of mRNA, we used Quantitative Real-Time-PCR (qRT-PCR). We analyzed mRNA expressions using the comparative threshold cycle method and normalized with β-actin. The following primers were used: 5’-CCCTGAACCCTAAGGCCAACCGTGAAAA-3’ and 5’-TCTCCGG AGTCCATCACAA TGCCTGTG-3’ for β-actin, and 5’-AGTCACCATCACTTCCTGGAAGA-3’ and 5’-GGTGCAGAATCGCGAGTT-3’ for phosphoenolpyruvate carboxykinase (PEPCK).
Western blot analyses
The tissue samples were homogenized and the protein levels were determined using a BCA quantification kit (Beyotime Biotechnology, Shanghai, China). The protein lysates underwent 8% SDS-PAGE and were transferred to polyvinylidene difluoride membranes. Following this, the membranes were probed with 1:1,000 diluted primary antibodies against the insulin receptor (InsR) / phospho-InsR, AKT kinase (AKT) / phospho- AKT (Cell Signaling Technology, Beverly, MA, USA), PEPCK (Santa Cruz Biotechnology, Dallas, TX, USA); and GAPDH (Abcam, Cambridgeshire, UK) at 4 °C. The membranes were washed three times with Tris-buffered saline and then incubated with a horseradish peroxidase-labeled sheep anti-rabbit antibody for 1 h. The blots were visualized using enhanced chemiluminescence.
Statistical analysis
All data are expressed as means ± SEM. Sample size was determined using PASS (version 15.0). Statistical analysis was performed using SPSS (version 21.0). The normality of data distribution was assessed using the Shapiro-Wilk test. Then, our results were analyzed by an unpaired Student’s t-test when comparing two groups, or by ANOVA followed by Tukey’s post hoc test for more than two groups. P < 0.05 was indicated significant.
Discussion
Currently, PCOS is believed to be linked with both insulin resistance and hyperinsulinemia, in addition to symptoms such as irregular menstrual cycles and excess levels of androgen. Insulin resistance and unhealthy obesity are thought to contribute significantly to the onset and progression of PCOS. Pathogenic factors such as hypertension, dyslipidemia, hyperglycemia, obesity, and metabolic dysfunction significantly increase the risk of long-term adverse cardiovascular events and type 2 diabetes [
17] in PCOS patients. The PCOS model constructed by subcutaneous injection of DHEA showed a disordered oestrus cycle, polycystic formation of ovarian atresia follicles, regression of granulosa cells, disturbed sex hormone levels, and metabolic dysfunction, including weight gain, increased leptin levels and insulin resistance. Moreover, Ressler et al. [
18] found that high-fat-fed PCOS model rats showed more severe obesity and insulin resistance and stronger depression. All these phenotypes are extraordinarily similar to the characteristics of PCOS in humans and provide an excellent animal model for studying the ovarian and metabolic disorders of PCOS.
The presence of low-grade chronic inflammation is indicated by having consistently slightly elevated levels of CRP within the normal range, more recently has been linked to PCOS in women. Studies have shown that CRP concentrations are elevated in women with PCOS [
12]. Significantly higher serum TNF-α, IL-6, IL-18 [
19], plasminogen activator inhibitor-1, as well as higher white blood cell (WBC) counts revealed peripheral inflammation conditions in PCOS [
20]. In addition, increased WBC and CRP levels in PCOS have also been shown to be independent of obestiy [
21]. The positive correlation between CRP concentrations and HOMA-IR suggests that insulin resistance is linked to low-grade chronic inflammation. Our study found that CRP was significantly increased in both the normal diet and high-fat diet of wild-type PCOS rats, which is similar to what has been found in humans. Increased CRP concentrations may be related to vascular endothelial injury, which can activate the inflammatory response system and stimulate CRP synthesis in the liver. At the same time, CRP can activate inflammatory cells to take up low density lipoprotein so that the formation of foam cells can activate endothelial cells to increase the expression of adhesion factors and inflammatory signals, reduce the production of vascular relaxation factors such as nitric oxide (NO) [
22], and promote the proliferation of vascular smooth muscle cells [
23]. In our study, the changes in blood pressure of PCOS model rats with CRP gene knockout were not significant, while reverse in wild-type rats. This was consistent with the finding that CRP levels were correlated with hypertension [
24].
Our finding that CRP knockout rats had higher anal temperature and oxygen consumption suggests that CRP knockout rats may have a higher basal metabolic rate. CRP knockout PCOS rats had a lower respiratory quotient, suggesting that their metabolic substrates may be more prone to fat, and their adipose tissues may be more sensitive to catecholamines. Catabolism of fat requires the stimulation of hormones such as catecholamines. Fat desensitization to catecholamines leads to a reduction in fat catabolism, resulting in obesity. Leptin can enhance the effect of catecholamines [
25]. The association of PCOS patients with adipose tissue catecholamine resistance, insulin resistance and obesity has been well documented [
26]. We speculated that the PCOS model constructed by CRP knockout rats may have a higher basal metabolic rate and a lower body weight because the loss of the CRP gene weakened catecholamine resistance in adipose tissue. In addition, studies in skeletal muscle cells have found that CRP can even directly inhibit the activation of insulin signaling. CRP disturb the phosphorylation of insulin receptor substrate 1 (IRS-1) in muscle cells and attenuates insulin signaling, thereby disrupting glucose transport [
27]. Several studies in people with impaired glucose tolerance and impaired fasting glucose have verified that CRP is positively correlated with insulin resistance, obesity, and blood triglyceride levels [
28]. By constructing the PCOS model, we found that CRP knockout rats showed better glucose disposal ability and higher insulin sensitivity than wild-type rats in GTT and ITT tests, which further reflected that CRP may be implicated in insulin resistance in PCOS.
White adipose tissue is the primary source of leptin production in humans. It’s widely accepted that leptin participates in the regulation of food intake, body mass, lipolysis, energy metabolism, proinflammatory immune responses and even reproductive functioning. After being released from adipocytes into the bloodstream, leptin crosses the blood-brain barrier (BBB) and reaches specific areas of the brain that regulate food intake, energy expenditure, and glucose metabolism [
29]. Leptin’s aberrant expression and malfunction are significant factors in the development of PCOS, with insulin being considered as the key regulator of leptin production. Prolonged hyperinsulinemia results in an increase in circulating levels of leptin. Current studies suggest that PCOS patients have hyperleptinemia, the level of which is positively correlated with serum leptin, insulin and body mass index [
9]. Indeed, we found that an increase level of leptin in PCOS model rats. In combination with the enlargement of central leptin infusion to lower glucose production and regulate insulin sensitivity in the absence of CRP, we deduce that hypothalamic inflammation may mediate the leptin resistance in PCOS.
A robust positive correlation was observed between plasma hypersensitive CRP and leptin levels in healthy people [
30], as well as obese [
31] and diabetic patients [
32]. It is believed that chronically high levels of CRP aggravate leptin resistance in insulin-resistant people. Tracing it to its cause, circulating CRP and leptin can regulate each other’s bioavailability. On the one hand, leptin can stimulate CRP synthesis in liver cells in a PI3K-dependent manner. Leptin binds to leptin receptors on endothelial cells, activating downstream leptin signaling and increasing CRP expression in the vascular endothelium. On the other hand, CRP forms five polymer molecules in vivo, which bind to leptin and directly affect the affinity between leptin and its receptors, thereby blocking the transmission of downstream leptin signals (including AMPK, AKT, and phosphorylation of endothelial nitric oxide synthase) [
33]. CRP in chronic inflammatory conditions of obesity can also damage the function of BBB and increase BBB permeability, resulting in reactive glial proliferation and affecting central nervous system function, which may be a major factor of central leptin resistance in chronic inflammation [
34]. Whether leptin was administered by peripheral or central, we found that CRP gene knockout rats have stronger insulin sensitivity to regulate hepatic glucose production. We speculated that peripheral infusion of leptin may successfully cross the blood‒brain barrier and bind to its receptors in the hypothalamus due to CRP deficiency, then improving liver glucose metabolism through the brain-liver axis. These data highlight the vital role of leptin to maintain glucose homeostasis rely on the change of CRP in PCOS.
In summary, our findings provide novel insights into the connection between CRP and leptin in the regulation of hepatic glucose homeostasis and insulin sensitivity in PCOS. This finding may reveal novel therapeutic approach targets to reduce inflammation signaling pathways to restore glucose homeostasis in PCOS.
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