Action Mechanisms of Metformin Combined with Exenatide and Metformin Only in the Treatment of PCOS in Obese Patients

Background Polycystic ovary syndrome (PCOS) is the most common endocrine disease in women of reproductive age, whose clinical characteristics are hyperandrogenism (HA), ovulatory dysfunction, and polycystic ovary, often accompanied by insulin resistance (IR) and metabolic abnormalities. Glucagon-like peptide (GLP)-1 receptor agonists (GLP-1Ra), such as exenatide, can bind to specific receptors on tissues such as the ovaries to improve the clinical phenotype of PCOS, while insulin-sensitizing agents, such as metformin, can also benefit to metabolic abnormalities in PCOS. Liquid chromatography-mass spectrometry (LC/MS) metabolomics revealed differences between the mechanisms of exenatide and metformin treatment of PCOS to some extent. Methods In this study, 50 obese subjects with PCOS were randomly divided into the exenatide combined with metformin group (COM group, n = 28) and the metformin group (MF group, n = 22) for 12-week treatment. Before and after, serum samples were subjected to LC/MS analysis. Results After treatment, there were 153 named differential metabolites in the COM group and 99 in the MF group. Most phosphatidylcholines (PC) and deoxycholic acid 3-glucuronide (DA3G) were significantly upregulated, while most glycerophosphoethanolamine (PE-NMe2), glycerophosphocholine (GPC), and threonine were downregulated in both groups. Only the decrease of neuromedin B, glutamate, and glutamyl groups and the increase of chenodeoxycholic acid sulfate docosadienoate (22: 2n6), and prostaglandin E2 have been observed in the COM group. In addition, salicylic acid and spisulosine increased and decanoylcarnitine decreased in the MF group. Both groups were enriched in glycerophospholipid, choline, and sphingolipid metabolism, while the COM group was especially superior in the glutamine and glutamate, bile secretion, and amino acid metabolism. Conclusion Compared with metformin alone in the treatment of PCOS, the differential metabolites of the exenatide combined with metformin group are more extensive. The COM group may act on the hypothalamic-pituitary-gonadal axis (HPO) and its bypass, regulate multiple metabolism pathways such as phospholipids, amino acids, fatty acids, carnitine, bile acids, and glucose directly or indirectly in obese PCOS patients.


Introduction
Polycystic ovary syndrome (PCOS) is a heterogeneous disease with clinical manifestations of hyperandrogenic signs, ovulation disorders, and oligomenorrhea.Meanwhile, 50%-70% of PCOS patients with reproductive and endocrine metabolic disorders, such as insulin resistance (IR) [1], and obese PCOS patients are more prone to infertility, diabetes, and cardiovascular disease [2].In addition to adjusting lifestyle, metformin, a classic hypoglycemic drug, is commonly used clinically in the treatment of PCOS patients with impaired glucose tolerance or metabolic syndrome who are inefective in simple lifestyle adjustment [3].It can inhibit hepatic gluconeogenesis, promote peripheral glucose uptake, and reduce fatty acid oxidation, thereby improving liver sensitivity to insulin [4].Exenatide is a glucagon-like peptide (GLP)-1 receptor agonist (GLP-1Ra) analog, which can inhibit appetite and gastrointestinal peristalsis [5].It can also help in weight loss, improve IR, and reduce the risk of cardiovascular disease [6].At present, many studies have confrmed the efcacy and safety of exenatide in type 2 diabetes mellitus (T2DM) [7], and recent studies have recommended exenatide alone or in combination with metformin are good options for PCOS women with obesity, glucose intolerance or high risks of CVD [8].However, there are few reports about the metabolic differences between metformin and exenatide.
Metabolomics is a helpful tool to identify small metabolite molecules.Compared to genomics and proteomics, metabolomics is directly infuenced by factors such as genetics and environment, which means it can clearly reveal the exact metabolic changes that occur in the body under a certain disease state (such as physiological or pathological stimuli, before and after administration, etc.).By studying those diferences, the biomarkers of the body in a specifc state are clarifed, which provides a new direction for clinical diagnosis and treatment [9].
Tis study intends to summarize the value and pharmacodynamics of glucagon-like peptide receptor agonists and insulin sensitizers by analyzing the metabolite changes after exenatide combined with metformin or single metformin treatment in PCOS patients who are obese.

Subjects and Methods
Tis study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Peking Union Medical College, Chinese Academy of Medical Sciences, and Peking Union Medical College (No. HS-2032).Informed consent forms were signed by all subjects before the start of the study.Obese PCOS patients were selected from Peking Union Medical College Hospital from July 2019 to January 2021.PCOS was diagnosed based on the presence of two out of three criteria (oligomenorrhoea, clinical or biochemical hyperandrogenism, and polycystic ovaries on ultrasound after exclusion of other endocrine causes of hyperandrogenism according to the Rotterdam criteria) [10].Tey were aged 18-40, overweight or obese (body mass index ≥ 25 kg/m 2 ), took no regular medications known efects on reproductive or metabolic function, have no other endocrine diseases, no other malignancies, no multiple endocrine neoplasia type 2 (MEN2) or personal or family history of medullary thyroid carcinoma (MTC), no history of thromboembolic disease or thrombosis, no serious or unstable physical diseases, no drug allergy, not in other clinical trials, not breastfeeding or pregnant, and nonheavy smokers or alcoholics.All the subjects had anthropometric measurements and fasting blood sampling.
A total of 70 patients were recruited to receive randomized treatment, and 50 completed the trial due to the impact of the COVID epidemic.Terein, patients in the metformin group (MF group, n � 22) received metformin 500 mg alone, three times a day; others in the exenatide combined with the metformin group (COM group, n � 28) received a subcutaneous injection of exenatide 2 mg once a week in addition to regular metformin treatment.Te waist circumference and body weight were measured before and after 12-week treatment, and the body mass index (BMI) was calculated in both groups.,A standard 75 g oral glucose tolerance test (OGTT) was performed in the morning after overnight fasting for 12 hours, respectively.At 0, 60, and 120 min, blood glucose and insulin levels were measured.Fasting blood samples taken on the 2nd or 3rd of the patients' menstrual cycle were also used to measure hemoglobin A1C (HbA1c), sex hormones (total testosterone, TT, and dehydroepiandrosterone sulfate, DHAS), blood lipids, complete blood count, C-reactive protein, and markers of liver and kidney function.During the period, patients recorded adverse reaction events in the patient diary.

Analysis of Untargeted Metabolomics.
At baseline and after 12 weeks of treatment, 2 tubes of venous blood were collected from eligible patients (n � 50) with a 4 ml vacuum blood collection tube, for a total of 8 ml.Samples were placed in a refrigerator at 4 °C for 1 hour, centrifuged at 3000 rpm for 5 minutes, and the supernatant was dispensed into 1.5 ml EP tubes and immediately stored in a −80 °C refrigerator until all specimens were collected.After serum samples were centrifuged at 13000 g at 4 °C for 15 min, the supernatant was carefully transferred to sample vials for LC-MS/MS analysis.A pooled quality control sample (QC) was fulflled by mixing equal volumes of all samples in order to monitor the stability of the analysis.
Te metabolomic analysis of the blood samples was performed using a liquid chromatography-mass spectrometer Q Exactive (Termofsher), including positive and negative ion detection modes.
(1) Liquid chromatography conditions: the column was BEH Amide (2.1 mm × 100 mm, waters, USA), the column temperature was set to 35 °C, and the fow rate was 300 µL/ min.(2) Mass spectrometry conditions: a heated electrospray ion source (HESI) was used, the temperature of the ion transfer tube was 320 °C, the temperature of the auxiliary gas was 300 °C, and the fow rates of the sheath gas and the auxiliary gas were 35 and 10 (arb), respectively.Data acquisition was performed in a lazy scanning mode (Full MS/ dd-MS2).Data acquisition was divided into positive ion mode (3500 V) and negative ion (−2800 V).
Collected data were secondarily matched to local databases (established based on chemical standards or biological samples) by Tracerfnder 3.2 (Termo Fisher, CA).Metabolites after matching and identifcation will have a secondary score (library score, LS).Only metabolites with an LS score higher than 30 are considered credible.Te higher the score, the more credible the identifed metabolites.

Data Analysis.
Metabolite data were normalized prior to statistical analysis, with peak areas normalized to the mean of the total sample area.Metabolite data were imported into SIMCA (Umetrics, Sweden) software for analysis.Principal component analysis (PCA) and supervised orthogonal partial least squares discriminant analysis (OPLS-DA), cross-validation analysis of variance (CV-ANOVA), R2Y, and the Q 2 value assessed the quality of the OPLS-DA model

Comparison of Clinical Characteristics and Biochemical
Data.Clinical characteristics and biochemical data of subjects at the baseline and after intervention are shown in Table 1.Although BMI and androgen in both groups were signifcantly decreased after treatment, there is no diference between the two groups.In terms of glucose metabolism, the 2h-OGTT and HbA1c of the COM group reduced signifcantly, while the level of the MF group did not drop compared to baseline, and the COM group had a better result in alleviating the level of FINS than the MF group.We observed changes in some indicators of lipid metabolism in each group; TG, TC, and LDL levels did not signifcantly change from baseline in any group.HDL and ApoA1 have markedly improved from baseline in both groups, and only the MF group had lower lipoprotein B (ApoB) after treatment.In our study, TT was signifcantly decreased from baseline in each treatment group and DHAS levels did not change signifcantly.

General Characteristics of Serum Metabolic Profling
Changes in the Two Groups.Te OPLS-DA model was also used to analyze the changes in the distribution of serum metabolites in the two groups before and after treatment.Serum metabolites in the COM and MF group separated signifcantly (Figure 1).
We also compared the change in serum metabolites in obese PCOS patients between groups.We had observed certain metabolites, including PCs, PE-NMe2s, GPC, DA3G, chenodeoxycholic acid sulfate, and certain amino acids, had similar improving trend in both groups (Figure 3).

Enrichment Analysis on Diferential Metabolites and
Diferential KEGG Pathways.KEGG pathway enrichment analysis of the diferential metabolites in the COM and MF groups showed that a total of 31 metabolic pathways were observed to be enriched in the COM group and only 12 in the MF group (Figure 4).Both groups were enriched in glycerophospholipid metabolism, choline metabolism, sphingolipid metabolism, protein digestion, and absorption.While metabolite changes in the COM group were also associated with glutamine and glutamate metabolism, bile secretion, and amino acid metabolism.Only phenylalanine metabolism showed in the MF group.

Discussion
Exenatide could signifcantly reduce body weight, insulin, testosterone, and interleukin 6 (IL-6) levels in the rat model and also improve changes in ovarian morphology, whose efect was similar to that of metformin [12].Te decrease of the body weight and central adiposity by exenatide may further explain improvements in IR, infammatory markers, and menstrual cycles [13].Some clinical studies comparing GLP-1 RA with metformin showed that the former has advantages in improving body weight, HOMA-IR, menstrual frequency, and spontaneous pregnancy rate [14].Te investigators further suggested that exenatide combined with metformin appeared to have a greater (additive) efect than exenatide or metformin in terms of menstrual cycle frequency, hormonal and metabolic disturbances [8], and remission rates for prediabetes [15].
However, most of the current articles mainly compare the efcacy of exenatide and metformin in the treatment of PCOS patients from the perspective of clinical biochemical data.Tang et al. [16] frst analyzed the metabolites after exenatide therapy in OB/OW PCOS patients and found that lipid and amino acid metabolic abnormalities and steroid and nucleotides synthesis might be improved after exenatide International Journal of Endocrinology treatment.In our study, we showed that exenatide combined with metformin have a better efect on remising OB/OW PCOS patients by improving multiple metabolisms.
Phosphatidylcholine (PC), glycerophospholipids (GPC), and phosphatidylethanolamine (PE) in PCOS patients are lower than those in healthy people [17][18][19].In our study, the COM group and MF group both signifcantly increased PC and deoxycholic acid 3-glucuronide (DA3G), while most glycerophosphoethanolamine (PE-NMe2), glycerophosphocholine (GPC), and threonine were downregulated after treatment.PC can promote cell proliferation and programmed cell death and improve insulin sensitivity [20].Blood phosphatidylcholine can be used as a chemical attractant to recruit immune cells such as macrophages and dendritic cells, thereby improving immune function [21].Tese results were consistent with the conclusion of Yao et al. [22], transplanting the brown adipose tissue in PCOS model mice.GPC/PC was signifcantly decreased in OB/OW PCOS patients, which was contrary to the conclusion of the previous study [18].
Previous studies have proved that the increase of the branched-chain amino acid (BCAA) levels afected women with PCOS due to the protein phosphatase Mg 2+ /Mn 2+dependent 1K (PPM1K) defciency.In nonobese PCOS patients, arginine, lysine, ornithine, proline, glutamate, acetone, citrate, and histidine decreased signifcantly [23], and in those with obesity, specially, branched-chain amino acids (e.g., leucine) and aromatic amino acids (e.g., phenylalanine) were signifcantly lower than in healthy people [16,18].However, we found that both groups had lower levels of many amino acids like histidine, proline, threonine, and tryptophan, especially in the COM group, which suggested that exenatide combined with metformin played an important part in the amino acid metabolism.Neurokinin B (NKB) is a peptide neurotransmitter regulated by KNDy neurons in the hypothalamus.Studies suggested that the abnormal activation of KNDy neurons could regulate the autocrine and/or paracrine of kisspeptin, NKB, and dynorphin (Dyn), which might increase the GnRH pulse frequency and resultantly LH secretion, involving the   International Journal of Endocrinology pathology of PCOS [24].At present, only Gorkem et al. [25] reported that kisspeptin in PCOS patients was signifcantly increased, and none have reported the changes of NKB in PCOS patients.We found out that NKB levels decreased after treatment only in the COM group, suggesting that exenatide may downregulate KNDy neuronal activity, namely, hypothalamic-pituitary-gonadal axis (HPO) or its passby, thereby improving the PCOS phenotype.
Besides, PCOS patients had higher n − 6 series polyunsaturated fatty acids (PUFAs) than healthy women, while n − 3 series PUFAs were lower.Our results showed that docosatrienoic acid, a kind of n − 3 series PUFAs, and prostaglandin E2 (PGE2) elevated after treatment in the COM group only.n − 3 series PUFAs have a protective efect on insulin sensitivity [26,27], inhibit the synthesis of androgens in PCOS patients, and promote the synthesis of estradiol and progesterone [28,29].PGE2 is a lipid-derived substance that has been shown to regulate the function of many cell types, including dendritic cells, macrophages, and T and B lymphocytes, leading to proinfammatory and antiinfammatory efects [30].What's more, we noticed that the (E)-Casimiroedine level, belonging to glycosylamines, was only reduced in the COM group.Glycosylamines are valuable sugar derivatives that have attracted much attention as synthetic intermediates en route to iminosugar-Cglycosyl compounds, which work as inhibitors of a wide variety of glycosidases and glycosyltransferases [31], leading to glucose metabolism disorders.
In addition, our study showed that chenodeoxycholic acid sulfate (CDCA) was increased in the COM group after treatment.Jia et al. [32] frst found that glycocholic acid, which can dissolve fat, was reduced in PCOS patients; therefore, these patients have problems with fat absorption.CDCA is the most potent bile acid (BA) for activating the farnesoid X receptor (FXR) [33].FXR not only efectively regulates bile acid synthesis and secretion and lipid and glucose metabolism in the liver and intestine [34] but also counteracts the proinfammatory and proatherosclerotic responses of cardiovascular disease, modulates liver infammation and regeneration, modulates the degree of infammatory response, and inhibits barrier function and intestinal bacterial translocation [35].When CDCA increases, PCOS phenotypes can be alleviated due to the activation of FXR.Terefore, exenatide combined with metformin could directly and indirectly improve glucose and lipid metabolism more efectively compared to the MF group.International Journal of Endocrinology  International Journal of Endocrinology

Conclusion
Exenatide combined with metformin had more diferential metabolites compared to metformin alone, acting positively and efectively on the HPO and its bypass, improving amino acid, glucose, and lipid metabolism directly or indirectly, thereby inhibiting androgen synthesis, improving insulin resistance, and other metabolic syndromes in obese PCOS patients.

Figure 1 :
Figure 1: General characteristics of serum metabolites in two groups.OPLS-DA score map: the frst prediction of Comp1 is mainly the decomposition degree, and the frst orthogonality of orthogonal Comp1 is the decomposition degree.Scores map of the OPLS-DA model before and after treatment to obese PCOS women in the COM group (a) (R 2 X � 0.341, R 2 Y � 0.95, Q 2 � 0.458) and in the MF group (b) (R 2 X � 0.398, R 2 Y � 0.973, Q 2 � 0.668).

Figure 2 :
Figure 2: Heatmaps of the top 30 diferential metabolites in the COM and MF groups before and after treatment.Heatmap: the color indicates the relative expression level of the metabolite in this group of samples.Te corresponding relationship between the color gradient and the numerical value is shown in the gradient color block.Te right side is the metabolite VIP bar chart.Te length of the bar represents the contribution value of the metabolite to the diference between the two groups (VIP > 1), the larger the value, the greater the diference between the two groups.Te color of the bar indicates the signifcance of the diference between the two groups of metabolites, namely, the P value.Te smaller the P value is, the darker the color is.* P < 0.05, * * P < 0.01, * * * P < 0.001.(a) Heatmap of the top 30 VIP metabolites that were diferentially expressed in the COM group.(b) Heatmap of the top 30 VIP metabolites that were diferentially expressed in the MF group.

Figure 3 :
Figure 3: Box diagram of diferential metabolites with the same change trend between groups before and after treatment.

Figure 4 :
Figure 4: KEGG pathway analysis of the diferentially expressed metabolites.Te abscissa is the P value of enrichment signifcance.Te smaller the P value, the more signifcant it is in statistics.Te ordinate is the KEGG pathway.Te size of the bubbles in the fgure represents the number of metabolites in the pathway that are enriched in the metabolic set.(a) Bubble chart in the COM group before and after treatment.(b) Bubble chart in the MF group before and after treatment.

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International Journal of Endocrinology normal distribution were analyzed by the Wilcoxon rank sum test.For all analyses, a two-tailed P ≤ 0.05 was considered to indicate statistical signifcance.

Table 1 :
Clinical characteristics and biochemical data between the COM and MF group.