- Email: [email protected]
AKT and MAPK pathways
Regulatory Toxicology and Pharmacology 110 (2020) 104544
Contents lists available at ScienceDirect
Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Berberine decreases insulin resistance in a PCOS rats by improving GLUT4: Dual regulation of the PI3K/AKT and MAPK pathways
T
Ning Zhang∗,1, Xiaoyan Liu1, Lili Zhuang, Xuemei Liu, Huishan Zhao, Yinghua Shan, Zhenteng Liu, Fenghua Li, Yilin Wang, Jianye Fang Reproductive Medicine Center, Yantai Yuhuangding Hospital, Yantai, 264000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Berberine Insulin resistance (IR) Polycystic ovary syndrome (PCOS) Glucose transporter 4 (GLUT4) PI3K/AKT pathway MAPK pathway
Berberine has been found to exhibit an array of pharmacological activities relating to the lowering of blood glucose and the treatment of polycystic ovarian syndrome (PCOS). The mechanism underlying these activites, however, is poorly understood. In the present study, female Sprague-Dawley (SD) rats were given oral letrozole to establish a model of insulin-resistant PCOS, and animals were then randomized into untreated or berberinetreated groups (400, 200, or 100 mg/kg). After 28 days, we measured homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) values in these animals. We further conducted H&E staining of ovarian tissues, assessed mRNA expression of glucose transporter 4 (GLUT4) via real time PCR, and used Western blotting to measure GLUT4 and PI3K/AKT and MAPK pathway protein levels. Berberine treatment was able to help restore HOMA-IR and ISI values to normal levels while simultaneously bolstering the expression of GLUT4. Normal ovarian morphology was also restored upon berberine treatment. We further found that 400 mg/kg berberine treatment was associated with activation of PI3K/AKT signaling and suppression of the MAPK pathway. In conclusion, berberine has the potential to reduce PCOS pathology and IR values in a rat model system through a mechanism linked to GLUT4 upregulation via PI3K/AKT activation and MAPK pathway suppression.
1. Introduction Polycystic ovarian syndrome (PCOS) is a disease that affects many women, and yet current treatment strategies are often unsatisfactory. Patients affected by PCOS commonly suffer from both insulin resistance (IR) as well as hyperandrogenism, and are at a higher risk for developing conditions such as heart disease, diabetes, and endometrial cancer (Shah and Patel, 2016; Moulana, 2019). Owing to the clear evidence of altered glucose metabolism in women with PCOS, there has been substantial research regarding the abnormal endometrial expression of glucose 4 (GLUT4) in women with this condition (Li et al., 2015; Ezeh et al., 2019). There is currently an important need to thoroughly assess whether directly altering endometrial insulin receptor signaling in women with PCOS can modulate GLUT4 expression. Berberine is an isoquinoline compound derived from many different plants that has been used in traditional Chinese medicine for years as a means of treating a diverse array of conditions including metabolic disorders and infertility (Wu et al., 2018; Jin et al., 2018). More recently, traditional herbal preparations designed to improve fertility
have been used in combination with cyproterone acetate (CPA) and clomiphene (CC) in an effort to enhance efficacy (Wang et al., 2015; Yin et al., 2008). There is recent evidence indicating that berberine offers promise for treating PCOS-associated IR (Li et al., 2018). Berberine treatment of hyperglycemic/hypercholesterolemic rats has also been shown to increase GLUT4 expression through uncertain mechanisms (Zhang et al., 2008). As such, it remains unclear whether and how berberine can alter PCOS IR pathology via modulation of GLUT4 expression. Herein, we first sought to assess how berberine affected the expression of GLUT4 in PCOS model rats exhibiting IR. We then explored the effects of berberine on the PI3K/AKT and MAPK signaling pathways, both of which are important mediators of insulin receptor signaling. Through this work, we hope to provide evidence that will support therapeutic efforts to treat PCOS-IR using berberine.
∗
Corresponding author. E-mail address: [email protected] (N. Zhang). 1 These authors contributed equally to this work and should be considered first authors. https://doi.org/10.1016/j.yrtph.2019.104544 Received 21 August 2019; Received in revised form 26 October 2019; Accepted 24 November 2019 Available online 26 November 2019 0273-2300/ © 2019 Elsevier Inc. All rights reserved.
Regulatory Toxicology and Pharmacology 110 (2020) 104544
N. Zhang, et al.
Fig. 2. Effects of berberine on ovarian histology. General observation, PCOS model rats exhibited a generally pale ovarian tissue with envelope thickening and many cystic follicles. Berberine (400 and 200 mg/kg) improved these pathological lesions. A) Control, B) PCOS models, C) Berberine, 400 mg/kg, D) Berberine, 200 mg/kg, and E) Berberine, 100 mg/kg.
Fig. 1. Effects of berberine on HOMA-IR and ISI. Berberine treatment altered the insulin sensitivity of model rats. A) A normal HOMA-IR value was restored in treated PCOS model rats treated using, B) PCOS model animals exhibited a lower ISI value, and this effect was counteracted by berberine. **p < 0.01 vs. control, ##, #p < 0.01, p < 0.05. vs. PCOS. BBR, berberine; PCOS, polycystic ovarian syndrome.
assessment of insulin resistance (HOMA-IR) approach (Wang et al., 2017), using the following formulas to calculate this index: HOMAIR=FBG (mmol/L) × FINS (mU/L)/22.5. Those PCOS model animals with a HOMA-IR value > 2.8 were deemed to be exhibiting IR (Coniglio et al., 2012), and these animals were randomized into the following groups (n = 12/group): a vehicle control group, or 3 berberine intragastrical administration groups (400, 200, and 100 mg/ kg.Test substance was dissolved in 0.5% CMC-Na). Untreated animals were selected as control groups, and were treated with the same volum of 0.5% CMC-Na. Animals were treated for 28 days in total. We additionally measured the insulin sensitivity index (ISI) value in these rats as follows: ISI = ln(1/FBG × FINS) (Li and Pan, 1993).
2. Materials and methods 2.1. Animals We obtained 60 female Sprague-Dawley rats from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China). Animals had free access to water and to conventional food pellets, and were housed at 25 °C, with 50% humidity and a 12-h light/dark cycle. Procedures involved in the use of laboratory animals were in accordance with the Guidelines of the Animal Care set by the Association of Laboratory Animal Science and the Center for Laboratory Animal Science.
2.3. Rat ovarian morphology 2.2. PCOS model generation At study endpoints, we excised ovarian tissue from all animals and formalin fixed/paraffin embedded the tissue, which was then cut into 4–6 μm sections and stained using hematoxylin and eosin (H&E). Two blinded pathologists then assessed ovarian morphology in these tissue sections.
We generated PCOS model rats through oral dosing with 1 mg/kg letrozole (Jiangsu HengRui Pharmaceutical Factory, China) as described previously (Rajan et al., 2017). Rats in the control group were instead given oral Sodium salt of Caboxy Methyl Cellulose (CMCNa,0.5%). Vaginal washing and smears were conducted every other day and analyzed microscopically to assess the effects of letrozole treatment on the estrous cycle. Only rats exhibiting alterations to this cycle were used as PCOS model animals. To assess IR in study animals, rats were fasted for 12 h and then fasting blood glucose (FBG) and fasting insulin (FINS) were assessed using venous blood samples. We followed the homeostasis model
2.4. Western blotting To clarify the mechanisms whereby berberine impacted PCOS model rats, ovarian tissue was excised from control, PCOS, and 400 mg/ kg berberine gruops. Western blotting was used to measure levels of GLTU4, p-IRS, p-PI3K, p-AKT, p-ERK, p-JNK, and p-P38 protein 2
Regulatory Toxicology and Pharmacology 110 (2020) 104544
N. Zhang, et al.
Fig. 3. Changes in A) control, B) PCOS, and berberine-treated ovarian histology (C, 400 mg/kg; D, 200 mg/kg; E, 100 mg/kg), with higher magnification images of individual follicles for each group shown. Controls exhibited normal ovarian morphology, whereas there were several cystic and atretic follicles evident in PCOS model animals. Berberine improved ovarian histology, restoring them to a more normal phenotype with an increased numbers of normal follicles.
(Abcam, Cambridge, UK). For all blotting, equal amounts of protein (50 μg/sample) were loaded per lane and separated via SDS-PAGE, followed by transfer to a PVDF membrane. Membranes were blocked using 5% nonfat milk in PBST, and were then probed using primary antibodies against these proteins of interest. As secondary antibodies, peroxidaselinked IgG molecules were used. An ECL Western blotting detection kit was then used to visualize protein expression (Amersham Biosciences).
2.5. Real time quantitative PCR
Fig. 4. Effects of berberine on GLUT4 expression. Berberine treatment increased GLUT4 mRNA and protein levels in the PCOS model system in which they had been significantly down-regulated. **p < 0.01 vs. control, ##, # p < 0.01, p < 0.05. vs. PCOS. BBR, berberine; PCOS, polycystic ovarian syndrome.
Ovarian tissue was excised from all animals. TRIzol was used to isolate total RNA from samples based on provided directions. 2 μg of his RNA was then reverse transcribed to cDNA with a one-step RT-PCR kit. SYBR Green-mediated RT-PCR amplification and real time fluorescence detection were conducted based on provided protocols (7500 Fast RealTime PCR System). The 2−ΔΔCt method was used to assess relative gene expression, with GAPDH used for normalization. Primers used were as follows: GLUT4 (F) 5′- GATCGGCTCTGAAGATGGGG-3′, GLUT4 (R) 5′GGAGGAAATCATGCCACCCA-3′; GAPDH (F) 5′-GGTATCGTGGAAGGA CTCAT GAC-3′, GAPDH (R)5′-ATGCCAGTGAGCTTCCCGT TCAGC-3′ (Zhao et al., 2017).
3. Results 3.1. Berberine treatment altered HOMA-IR and ISI values in PCOS model rats We first assessed whether berberine was able to influence key IRrelated indices in our PCOS rat model system. At baseline, PCOS model animals exhibited higher HOMA-IR values than control animals, whereas berberine treatment reduced these elevated values (Fig. 1A). Similarly, PCOS model rats exhibited a lower ISI value at baseline, and berberine treatment significantly increased these values relative to uncreated PCOS model rats (Fig. 1B). This suggests that berberine is able to counteract PCOS-associated changes in HOMA-IR and ISI values in this model system.
2.6. Statistical analysis Data are means ± standard deviation. SPSS 17.0 and Graphpad Prism 6.0 were used for statistical testing. Data were compared via paired Student's t tests and one-way ANOVAs as appropriate, with P < 0.05 as the significance threshold. 3
Regulatory Toxicology and Pharmacology 110 (2020) 104544
N. Zhang, et al.
Fig. 5. Berberine restored PI3K/AKT pathway activation. **p < 0.01 vs. control,
##
p < 0.01 vs. PCOS. BBR, berberine; PCOS, polycystic ovarian syndrome.
3.2. Berberine restores normal ovarian morphology
3.3. Berberine increases GLUT4 expression
Given its influence on key ovarian parameters, we next assessed the effect of berberine on ovarian morphology in this rat model of PCOS. PCOS model rats exhibited generally pale ovarian tissue with envelope thickening and many cystic follicles. Berberine (400 and 200 mg/kg) improved these pathological lesions (Fig. 2). Control animals exhibited ovarian follicles at a variety of stages upon H&E staining, whereas PCOS model animals exhibited disordered ovarian morphology with evidence of cystic dilatation in the ovarian follicles. The oocytes within these follicles were absent, and there was a significant drop in granule cell layer numbers (Fig. 3). Following berberine (400 and 200 mg/kg) treatment of these PCOS model animals, there was a partial restoration of normal tissue morphology, with oocytes again being evident in follicles and an increase in the number of granule cell layers after treatment. This suggests that berberine treatment can reverse the negative morphological changes in ovarian tissue that occur in these PCOS model rats.
GLUT4 is a key mediator of IR in the context of PCOS. We therefore used real-time PCR and Western blotting to assess GLUT4 levels in our model animals. We observed a significant reduction in GLUT4 mRNA levels in PCOS model animals relative to healthy controls, and bereberine treatment significantly increased GLUT4 expression in these model rats (Fig. 4A). Similarly, berberine significantly increased the protein levels of GLUT4 in PCOS model animals (Fig. 4B). This therefore suggests that GLUT4 may be a key mediator of the observed ability of berberine to protect against PCOS pathology. 3.4. Berberine restores PI3K/AKT pathway activation To assess the mechanisms whereby berberine alters PCOS pathology in detail, we next measured IRS, PI3K, and AKT levels via Western blotting. Our results revealed that PCOS model animals exhibited reduced expression of the phosphorylated forms of these proteins relative 4
Regulatory Toxicology and Pharmacology 110 (2020) 104544
N. Zhang, et al.
PCOS (Adak et al., 2018). Berberine is an isoquinoline alkaloid compound that can be extracted from many different plants, and which has pharmacological activities that make it well-suited to immune modulation, lowering of glucose and cholesterol levels, and combatting cancer or microbial pathogens (N. Wang et al., 2015; Pirillo and Catapano, 2015; Cao et al., 2019; Andreazza et al., 2016). Multiple recent studies have also explored the clinical use of berberine for treating PCOS(Luo et al., 2019; Li et al., 2015). While there is work suggesting that berberine can overcome IR in vitro and in vivo (Zhao et al., 2011; Li et al., 2013), the specific associated mechanisms remain uncertain. One recent study found berberine to upregulate GLUT4 expression – an activity potentially related to its ability to control glucose metabolism(Zhang et al., 2011). In this study, we found berberine to be able to normalize both IR symptoms and ovarian GLUT4 expression in PCOS model rats, suggesting that berberine offers beneficial effects against PCOS via modulation of GLUT4 expression. This insulin signaling pathway, which depends upon signaling through a number of intermediaries including IRS, PI3K, and AKT, is a key regulator of the expression and localization of GLUT4, thereby regulating cellular glucose intake (Tremblay et al., 2001; Jeon et al., 2015). Previous work has shown that suppression of normal IRS activation and downstream PI3K/AKT signaling can compromise normal GLUT4 localization and glucose uptake by cells (Carvalho et al., 2001). Indeed, PI3K/AKT signaling has previously been reported to play a role in the pathology of PCOS, likely at least in part due to the fact that reduced GLUT4 expression (Li et al., 2017). Berberine has previously been found to increase IRS expression and thereby activate PI3K/AKT signaling (Wang et al., 2018). Consistent with this, we found that berberine-mediated effects against IR in a PCOS rat model were associated with its ability to enhance activation of PI3K/AKT signaling in these animals. Indeed, we found that berberine was able to decrease IR via increasing GLUT4 expression and activating PI3K/AKT signaling in order to help alleviate this symptom of PCOS. Many signaling pathways influence IR pathogenesis beyond the PI3K/AKT pathway, with their modulated signaling being linked to dysregulated glucose metabolism in the context of disorders such as diabetes (Choi et al., 2019). MAPK pathway activation has been shown to suppress IRS activation, thus promoting IR development (Hanai et al., 2009). MAPK signaling is a key signal transduction pathway linked to androgen biosynthesis and IR in the context of PCOS (Diamanti-Kandarakis and Dunaif, 2012). In addition, MAPK activation can lead to reduced GLUT4 expression, thereby impairing glucose transport (Zhou et al., 2018). There is also evidence that PI3K/AKT signaling can influence MAPK pathway activation (Zhou et al., 2016). To assess whether the effects of berberine on IR were linked to MAPK pathway activation, we assessed MAPK-related protein expression and observed a significant berberine-mediated suppression of p38, ERK, and JNK in PCOS model animals. Thus suggests that berberine may therefore reduce IR in part via modulation of the MAPK signaling pathway. In conclusion, our research provides novel evidence that berberine treatment is able to alleviate the pathogenesis of PCOS in a rat model system, decreasing IR through a mechanism likely linked with GLUT4 upregulation. The underlying molecular mechanisms governing this upregulation are likely linked to berberine-mediated activation of PI3K/AKT signaling and suppression of MAPK signaling. Our data thus suggest that berberine may offer clinical benefits. Previous work clearly supports cross-talk between PI3K⁄AKT and MAPK signaling (Choi et al., 2018). Our results are consistent with the existence of such cross-talk, and suggest that it may be a key mediator of PCOS-associated IR.
Fig. 6. Berberine inhibited the MAPK pathway. **p < 0.01 vs. control, ## p < 0.01 vs. PCOS. BBR, berberine; PCOS, polycystic ovarian syndrome.
to control animals (Fig. 5). Rats treated with 400 mg/kg berberine exhibited significant 46%, 30%, and 40% increases in the levels of pIRS, p-PI3K, and p-AKT, respectively. These results thus suggest that berberine may protect against PCOS-associated IR via restoring the activation of this PI3K/AKT signaling pathway. 3.5. Berberine inhibits MAPK pathway activation To confirm that the MAPK pathway is associated with the ability of berberine to mediate recovery from PCOS-associated IR, we next assessed the expression of the key MAPK signaling proteins P38, ERK, and JNK via Western blotting. We observed that PCOS model animals exhibited increased levels of these proteins relative to healthy control rats, and berberine treatment significantly reduced the expression of these proteins (Fig. 6). This suggests that the ability of berberine to influence PCOS pathology is correlated with its effects upon the MAPK pathway. 4. Discussion PCOS is a disease of the endocrine system affecting up to 15% of women of childbearing age, leading to symptoms including ovarian cysts, anovulation, and hyperandrogenism(Legro et al., 2013). PCOS is also associated with high rates of obesity, with abdominal fat in particular being a common problem among affected women(Wang et al., 2016). The infertility that affects women with PCOS is thought to be attributable, at least in part, to IR, which affects up to 70% of those with PCOS and is linked to profoundly abnormal insulin activity in the reproductive system (Morgante et al., 2018). Some medicines, such as Metformin, has been reported to have beneficial effects in patient with
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 5
Regulatory Toxicology and Pharmacology 110 (2020) 104544
N. Zhang, et al.
Acknowledgments
One 10, e0144072. Li, X., Cui, P., Jiang, H.Y., Guo, Y.R., Pishdari, B., Hu, M., Feng, Y., Billig, H., Shao, R., 2015. Reversing the reduced level of endometrial GLUT4 expression in polycystic ovary syndrome: a mechanistic study of metformin action. Am. J. Tourism Res. 7, 574–586. Li, T., Mo, H., Chen, W., Li, L., Xiao, Y., Zhang, J., Li, X., Lu, Y., 2017. Role of the PI3K-Akt signaling pathway in the pathogenesis of polycystic ovary syndrome. Reprod. Sci. 24, 646–655. Li, M.F., Zhou, X.M., Li, X.L., 2018. The effect of berberine on polycystic ovary syndrome patients with insulin resistance (PCOS-IR): a meta-analysis and systematic review. Evid. Based Complement Altern. Med. https://doi.org/10.1155/2018/2532935. Luo, R., Liao, Z., Song, Y., Yin, H., Zhan, S., Li, G., Ma, L., Lu, S., Wang, K., Li, S., 2019. Berberine ameliorates oxidative stress-induced apoptosis by modulating ER stress and autophagy in human nucleus pulposus cells. Life Sci. 228, 85–97. Morgante, G., Massaro, M.G., Di Sabatino, A., Cappelli, V., De Leo, V., 2018. Therapeutic approach for metabolic disorders and infertility in women with PCOS. Gynecol. Endocrinol. 34, 4–9. Moulana, M., 2019. Immunophenotypic profile of leukocytes in hyperandrogenemic female rat an animal model of polycystic ovary syndrome. Life Sci. 220, 44–49. Pirillo, A., Catapano, A.L., 2015. Berberine, a plant alkaloid with lipid- and glucoselowering properties: from in vitro evidence to clinical studies. Atherosclerosis 243, 449–461. Rajan, R.K., Kumar, S.S.M., Balaji, B., 2017. Soy isoflavones exert beneficial effects on letrozole-induced rat polycystic ovary syndrome (PCOS) model through anti-androgenic mechanism. Pharm. Biol. 55, 242–251. Shah, K.N., Patel, S.S., 2016. Phosphatidylinositide 3-kinase inhibition: a new potential target for the treatment of polycystic ovarian syndrome. Pharm. Biol. 54, 975–983. Tremblay, F., Lavigne, C., Jacques, H., Marette, A., 2001. Defective insulin-induced GLUT4 translocation in skeletal muscle of high fat-fed rats is associated with alterations in both Akt/protein kinase B and atypical protein kinase C (zeta/lambda) activities. Diabetes 50, 1901–1910. Wang, N., Tan, H.Y., Li, L., Yuen, M.F., Feng, Y., 2015. Berberine and Coptidis Rhizoma as potential anticancer agents: recent updates and future perspectives. J. Ethnopharmacol. 176, 35–48. Wang, M., Chen, S., Zhang, D., 2015. Systematic Evaluation of the randomized controlled trial system for polycystic ovary syndrome treated by berberine and metformin. Guangdong Med. J. 36, 3866–3870. Wang, Y., Fu, X., Xu, J., Wang, Q., Kuang, H., 2016. Systems pharmacology to investigate the interaction of berberine and other drugs in treating polycystic ovary syndrome. Sci. Rep. 6, 28089. Wang, Z., Zhai, D., Zhang, D., Bai, L., Yao, R., Yu, J., Cheng, W., Yu, C., 2017. Quercetin decreases insulin resistance in a polycystic ovary syndrome rat model by improving inflammatory microenvironment. Reprod. Sci. 24, 682–690. Wang, Y.Y., Tang, L.Q., Wei, W., 2018. Berberine attenuates podocytes injury caused by exosomes derived from high glucose-induced mesangial cells through TGF beta 1PI3K/AKT pathway. Eur. J. Pharmacol. 824, 185–192. Wu, J., Li, J., Li, W., Sun, B., Xie, J., Cheng, W., Zhang, Q., 2018. Achyranthis bidentatae radix enhanced articular distribution and anti-inflammatory effect of berberine in Sanmiao Wan using an acute gouty arthritis rat model. J. Ethnopharmacol. 221, 100–108. Yin, J., Gao, Z., Liu, D., Liu, Z., Ye, J., 2008. Berberine improves glucose metabolism through induction of glycolysis. Am. J. Physiol. Endocrinol. Metabol. 294, E148–E156. Zhang, W., Xu, Y.C., Guo, F.J., Meng, Y., Li, M.L., 2008. Anti-diabetic effects of cinnamaldehyde and berberine and their impacts on retinol-binding protein 4 expression in rats with type 2 diabetes mellitus. Chin. Med. J. 121, 2124–2128. Zhang, Q., Xiao, X., Feng, K., Wang, T., Li, W., Yuan, T., Sun, X., Sun, Q., Xiang, H., Wang, H., 2011. Berberine moderates glucose and lipid metabolism through multipathway mechanism. Evid. Based Complement Altern. Med. 1–10 2011. Zhao, L., Li, W., Han, F., Hou, L., Baillargeon, J.-P., Kuang, H., Wang, Y., Wu, X., 2011. Berberine reduces insulin resistance induced by dexamethasone in theca cells in vitro. Fertil. Steril. 95, 461–463. Zhao, H., Zhou, D., Chen, Y., Liu, D., Chu, S., Zhang, S., 2017. Beneficial effects of Heqi san on rat model of polycystic ovary syndrome through the PI3K/AKT pathway. Daru 25, 21. https://doi.org/10.1186/s40199-017-0188-7. Zhou, T., Meng, X., Che, H., Shen, N., Xiao, D., Song, X., Liang, M., Fu, X., Ju, J., Li, Y., 2016. Regulation of insulin resistance by multiple MiRNAs via targeting the GLUT4 signalling pathway. Cell. Physiol. Biochem. 38, 2063–2078. Zhou, D.N., Li, S.J., Ding, J.L., Yin, T.L., Yan, J., Ye, H., 2018. MIF may participate in pathogenesis of polycystic ovary syndrome in rats through MAPK signalling pathway. Curr. Med. Sci. 38, 853–860.
This work was supported by the Natural Science Foundation of Shandong Province (No. ZR2019MH117) and the Special fund for clinical research of Chinese Medical Association(No. 18010140743). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.yrtph.2019.104544 Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yrtph.2019.104544. References Adak, T., Samadi, A., Ünal, A.Z., Sabuncuoğlu, S., 2018. A reappraisal on metformin. Regul. Toxicol. Pharmacol. 92, 324–332. Andreazza, N.L., Vevert-Bizet, C., Bourg-Heckly, G., Sureau, F., Salvador, M.J., Bonneau, S., 2016. Berberine as a photosensitizing agent for antitumoral photodynamic therapy: insights into its association to low density lipoproteins. Int. J. Pharm. 510, 240–249. Cao, S., Xu, P., Yan, J., Liu, H., Liu, L., Cheng, L., Qiu, F., Kan, N., 2019. Berberrubine and its analog, hydroxypropyl-berberrubine, regulate LDLR and PCSK9 expression via the ERK signal pathway to exert cholesterol-lowering effects in human hepatoma HepG2 cells. J. Cell. Biochem. 120, 1340–1349. Carvalho, E., Jansson, P.A., Nagaev, I., Wenthzel, A.M., Smith, U., 2001. Insulin resistance with low cellular IRS-1 expression is also associated with low GLUT4 expression and impaired insulin-stimulated glucose transport. FASEB J. : Off. Publ. Feder. Am. Soc. Exp. Biol. 15, 1101–1103. Choi, J., Kim, K.J., Koh, E.J., Lee, B.Y., 2018. Gelidium elegans extract ameliorates type 2 diabetes via regulation of MAPK and PI3K/Akt signaling. Nutrients 10, 51. Choi, E., Kikuchi, S., Gao, H., Brodzik, K., Nassour, I., Yopp, A., Singal, A.G., Zhu, H., Yu, H., 2019. Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling. Nat. Commun. 10, 1473. Coniglio, R.I., Merono, T., Montiel, H., Malaspina, M.M., Salgueiro, A.M., Otero, J.C., Ferraris, R., Schreier, L., Brites, F., Rosso, L.G., 2012. HOMA-IR and non-HDL-C as predictors of high cholesteryl ester transfer protein activity in patients at risk for type 2 diabetes. Clin. Biochem. 45, 566–570. Diamanti-Kandarakis, E., Dunaif, A., 2012. Insulin resistance and the polycystic ovary syndrome revisited: an update on mechanisms and implications. Endocr. Rev. 33, 981–1030. Ezeh, U., Chen, I.Y.D., Chen, Y.H., Azziz, R., 2019. Adipocyte expression of glucose transporter 1 and 4 in PCOS: relationship to insulin-mediated and non-insulinmediated whole-body glucose uptake. Clin. Endocrinol. 90, 542–552. Hanai, Y., Adachi, S., Yasuda, I., Takai, S., Matsushima-Nishiwaki, R., Kato, H., Enomoto, Y., Akamatsu, S., Sakakibara, S., Ogura, S., 2009. Collagen-induced p38 MAP kinase activation is a biomarker of platelet hyper-aggregation in patients with diabetes mellitus. Life Sci. 85, 386–394. Jeon, H.J., Yoon, K.Y., Koh, E.J., Choi, J., Kim, K.J., Choi, H.S., Lee, B.Y.S., 2015. Dieckol improve insulin sensitivity through the regulation of the PI3K pathway in C57BL/KsJdb/db mice. J. Food Nutr. Res. 3, 648–652. Jin, F., Xie, T., Huang, X., Zhao, X., 2018. Berberine inhibits angiogenesis in glioblastoma xenografts by targeting the VEGFR2/ERK pathway. Pharm. Biol. 56, 665–671. Legro, R.S., Arslanian, S.A., Ehrmann, D.A., Hoeger, K.M., Murad, M.H., Pasquali, R., Welt, C.K., 2013. Diagnosis and treatment of polycystic ovary syndrome: an endocrine society clinical practice guideline. J. Clin. Endocrinol. Metab. 98, 4565–4592. Li, G.W., Pan, X.R., 1993. A new insulin-sensitivity index for the population-based study. Zhonghua nei ke za zhi 32, 656–660. Li, Y., Ma, H., Zhang, Y., Kuang, H., Ng, E.H., Hou, L., Wu, X., 2013. Effect of berberine on insulin resistance in women with polycystic ovary syndrome: study protocol for a randomized multicenter controlled trial. Trials 14, 226. Li, L., Li, C., Pan, P., Chen, X., Wu, X., Ng, E.H.Y., Yang, D., 2015. A single arm pilot study of effects of berberine on the menstrual pattern, ovulation rate, hormonal and metabolic profiles in anovulatory Chinese women with polycystic ovary syndrome. PLoS
6
Contents lists available at ScienceDirect
Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph
Berberine decreases insulin resistance in a PCOS rats by improving GLUT4: Dual regulation of the PI3K/AKT and MAPK pathways
T
Ning Zhang∗,1, Xiaoyan Liu1, Lili Zhuang, Xuemei Liu, Huishan Zhao, Yinghua Shan, Zhenteng Liu, Fenghua Li, Yilin Wang, Jianye Fang Reproductive Medicine Center, Yantai Yuhuangding Hospital, Yantai, 264000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Berberine Insulin resistance (IR) Polycystic ovary syndrome (PCOS) Glucose transporter 4 (GLUT4) PI3K/AKT pathway MAPK pathway
Berberine has been found to exhibit an array of pharmacological activities relating to the lowering of blood glucose and the treatment of polycystic ovarian syndrome (PCOS). The mechanism underlying these activites, however, is poorly understood. In the present study, female Sprague-Dawley (SD) rats were given oral letrozole to establish a model of insulin-resistant PCOS, and animals were then randomized into untreated or berberinetreated groups (400, 200, or 100 mg/kg). After 28 days, we measured homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity index (ISI) values in these animals. We further conducted H&E staining of ovarian tissues, assessed mRNA expression of glucose transporter 4 (GLUT4) via real time PCR, and used Western blotting to measure GLUT4 and PI3K/AKT and MAPK pathway protein levels. Berberine treatment was able to help restore HOMA-IR and ISI values to normal levels while simultaneously bolstering the expression of GLUT4. Normal ovarian morphology was also restored upon berberine treatment. We further found that 400 mg/kg berberine treatment was associated with activation of PI3K/AKT signaling and suppression of the MAPK pathway. In conclusion, berberine has the potential to reduce PCOS pathology and IR values in a rat model system through a mechanism linked to GLUT4 upregulation via PI3K/AKT activation and MAPK pathway suppression.
1. Introduction Polycystic ovarian syndrome (PCOS) is a disease that affects many women, and yet current treatment strategies are often unsatisfactory. Patients affected by PCOS commonly suffer from both insulin resistance (IR) as well as hyperandrogenism, and are at a higher risk for developing conditions such as heart disease, diabetes, and endometrial cancer (Shah and Patel, 2016; Moulana, 2019). Owing to the clear evidence of altered glucose metabolism in women with PCOS, there has been substantial research regarding the abnormal endometrial expression of glucose 4 (GLUT4) in women with this condition (Li et al., 2015; Ezeh et al., 2019). There is currently an important need to thoroughly assess whether directly altering endometrial insulin receptor signaling in women with PCOS can modulate GLUT4 expression. Berberine is an isoquinoline compound derived from many different plants that has been used in traditional Chinese medicine for years as a means of treating a diverse array of conditions including metabolic disorders and infertility (Wu et al., 2018; Jin et al., 2018). More recently, traditional herbal preparations designed to improve fertility
have been used in combination with cyproterone acetate (CPA) and clomiphene (CC) in an effort to enhance efficacy (Wang et al., 2015; Yin et al., 2008). There is recent evidence indicating that berberine offers promise for treating PCOS-associated IR (Li et al., 2018). Berberine treatment of hyperglycemic/hypercholesterolemic rats has also been shown to increase GLUT4 expression through uncertain mechanisms (Zhang et al., 2008). As such, it remains unclear whether and how berberine can alter PCOS IR pathology via modulation of GLUT4 expression. Herein, we first sought to assess how berberine affected the expression of GLUT4 in PCOS model rats exhibiting IR. We then explored the effects of berberine on the PI3K/AKT and MAPK signaling pathways, both of which are important mediators of insulin receptor signaling. Through this work, we hope to provide evidence that will support therapeutic efforts to treat PCOS-IR using berberine.
∗
Corresponding author. E-mail address: [email protected] (N. Zhang). 1 These authors contributed equally to this work and should be considered first authors. https://doi.org/10.1016/j.yrtph.2019.104544 Received 21 August 2019; Received in revised form 26 October 2019; Accepted 24 November 2019 Available online 26 November 2019 0273-2300/ © 2019 Elsevier Inc. All rights reserved.
Regulatory Toxicology and Pharmacology 110 (2020) 104544
N. Zhang, et al.
Fig. 2. Effects of berberine on ovarian histology. General observation, PCOS model rats exhibited a generally pale ovarian tissue with envelope thickening and many cystic follicles. Berberine (400 and 200 mg/kg) improved these pathological lesions. A) Control, B) PCOS models, C) Berberine, 400 mg/kg, D) Berberine, 200 mg/kg, and E) Berberine, 100 mg/kg.
Fig. 1. Effects of berberine on HOMA-IR and ISI. Berberine treatment altered the insulin sensitivity of model rats. A) A normal HOMA-IR value was restored in treated PCOS model rats treated using, B) PCOS model animals exhibited a lower ISI value, and this effect was counteracted by berberine. **p < 0.01 vs. control, ##, #p < 0.01, p < 0.05. vs. PCOS. BBR, berberine; PCOS, polycystic ovarian syndrome.
assessment of insulin resistance (HOMA-IR) approach (Wang et al., 2017), using the following formulas to calculate this index: HOMAIR=FBG (mmol/L) × FINS (mU/L)/22.5. Those PCOS model animals with a HOMA-IR value > 2.8 were deemed to be exhibiting IR (Coniglio et al., 2012), and these animals were randomized into the following groups (n = 12/group): a vehicle control group, or 3 berberine intragastrical administration groups (400, 200, and 100 mg/ kg.Test substance was dissolved in 0.5% CMC-Na). Untreated animals were selected as control groups, and were treated with the same volum of 0.5% CMC-Na. Animals were treated for 28 days in total. We additionally measured the insulin sensitivity index (ISI) value in these rats as follows: ISI = ln(1/FBG × FINS) (Li and Pan, 1993).
2. Materials and methods 2.1. Animals We obtained 60 female Sprague-Dawley rats from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China). Animals had free access to water and to conventional food pellets, and were housed at 25 °C, with 50% humidity and a 12-h light/dark cycle. Procedures involved in the use of laboratory animals were in accordance with the Guidelines of the Animal Care set by the Association of Laboratory Animal Science and the Center for Laboratory Animal Science.
2.3. Rat ovarian morphology 2.2. PCOS model generation At study endpoints, we excised ovarian tissue from all animals and formalin fixed/paraffin embedded the tissue, which was then cut into 4–6 μm sections and stained using hematoxylin and eosin (H&E). Two blinded pathologists then assessed ovarian morphology in these tissue sections.
We generated PCOS model rats through oral dosing with 1 mg/kg letrozole (Jiangsu HengRui Pharmaceutical Factory, China) as described previously (Rajan et al., 2017). Rats in the control group were instead given oral Sodium salt of Caboxy Methyl Cellulose (CMCNa,0.5%). Vaginal washing and smears were conducted every other day and analyzed microscopically to assess the effects of letrozole treatment on the estrous cycle. Only rats exhibiting alterations to this cycle were used as PCOS model animals. To assess IR in study animals, rats were fasted for 12 h and then fasting blood glucose (FBG) and fasting insulin (FINS) were assessed using venous blood samples. We followed the homeostasis model
2.4. Western blotting To clarify the mechanisms whereby berberine impacted PCOS model rats, ovarian tissue was excised from control, PCOS, and 400 mg/ kg berberine gruops. Western blotting was used to measure levels of GLTU4, p-IRS, p-PI3K, p-AKT, p-ERK, p-JNK, and p-P38 protein 2
Regulatory Toxicology and Pharmacology 110 (2020) 104544
N. Zhang, et al.
Fig. 3. Changes in A) control, B) PCOS, and berberine-treated ovarian histology (C, 400 mg/kg; D, 200 mg/kg; E, 100 mg/kg), with higher magnification images of individual follicles for each group shown. Controls exhibited normal ovarian morphology, whereas there were several cystic and atretic follicles evident in PCOS model animals. Berberine improved ovarian histology, restoring them to a more normal phenotype with an increased numbers of normal follicles.
(Abcam, Cambridge, UK). For all blotting, equal amounts of protein (50 μg/sample) were loaded per lane and separated via SDS-PAGE, followed by transfer to a PVDF membrane. Membranes were blocked using 5% nonfat milk in PBST, and were then probed using primary antibodies against these proteins of interest. As secondary antibodies, peroxidaselinked IgG molecules were used. An ECL Western blotting detection kit was then used to visualize protein expression (Amersham Biosciences).
2.5. Real time quantitative PCR
Fig. 4. Effects of berberine on GLUT4 expression. Berberine treatment increased GLUT4 mRNA and protein levels in the PCOS model system in which they had been significantly down-regulated. **p < 0.01 vs. control, ##, # p < 0.01, p < 0.05. vs. PCOS. BBR, berberine; PCOS, polycystic ovarian syndrome.
Ovarian tissue was excised from all animals. TRIzol was used to isolate total RNA from samples based on provided directions. 2 μg of his RNA was then reverse transcribed to cDNA with a one-step RT-PCR kit. SYBR Green-mediated RT-PCR amplification and real time fluorescence detection were conducted based on provided protocols (7500 Fast RealTime PCR System). The 2−ΔΔCt method was used to assess relative gene expression, with GAPDH used for normalization. Primers used were as follows: GLUT4 (F) 5′- GATCGGCTCTGAAGATGGGG-3′, GLUT4 (R) 5′GGAGGAAATCATGCCACCCA-3′; GAPDH (F) 5′-GGTATCGTGGAAGGA CTCAT GAC-3′, GAPDH (R)5′-ATGCCAGTGAGCTTCCCGT TCAGC-3′ (Zhao et al., 2017).
3. Results 3.1. Berberine treatment altered HOMA-IR and ISI values in PCOS model rats We first assessed whether berberine was able to influence key IRrelated indices in our PCOS rat model system. At baseline, PCOS model animals exhibited higher HOMA-IR values than control animals, whereas berberine treatment reduced these elevated values (Fig. 1A). Similarly, PCOS model rats exhibited a lower ISI value at baseline, and berberine treatment significantly increased these values relative to uncreated PCOS model rats (Fig. 1B). This suggests that berberine is able to counteract PCOS-associated changes in HOMA-IR and ISI values in this model system.
2.6. Statistical analysis Data are means ± standard deviation. SPSS 17.0 and Graphpad Prism 6.0 were used for statistical testing. Data were compared via paired Student's t tests and one-way ANOVAs as appropriate, with P < 0.05 as the significance threshold. 3
Regulatory Toxicology and Pharmacology 110 (2020) 104544
N. Zhang, et al.
Fig. 5. Berberine restored PI3K/AKT pathway activation. **p < 0.01 vs. control,
##
p < 0.01 vs. PCOS. BBR, berberine; PCOS, polycystic ovarian syndrome.
3.2. Berberine restores normal ovarian morphology
3.3. Berberine increases GLUT4 expression
Given its influence on key ovarian parameters, we next assessed the effect of berberine on ovarian morphology in this rat model of PCOS. PCOS model rats exhibited generally pale ovarian tissue with envelope thickening and many cystic follicles. Berberine (400 and 200 mg/kg) improved these pathological lesions (Fig. 2). Control animals exhibited ovarian follicles at a variety of stages upon H&E staining, whereas PCOS model animals exhibited disordered ovarian morphology with evidence of cystic dilatation in the ovarian follicles. The oocytes within these follicles were absent, and there was a significant drop in granule cell layer numbers (Fig. 3). Following berberine (400 and 200 mg/kg) treatment of these PCOS model animals, there was a partial restoration of normal tissue morphology, with oocytes again being evident in follicles and an increase in the number of granule cell layers after treatment. This suggests that berberine treatment can reverse the negative morphological changes in ovarian tissue that occur in these PCOS model rats.
GLUT4 is a key mediator of IR in the context of PCOS. We therefore used real-time PCR and Western blotting to assess GLUT4 levels in our model animals. We observed a significant reduction in GLUT4 mRNA levels in PCOS model animals relative to healthy controls, and bereberine treatment significantly increased GLUT4 expression in these model rats (Fig. 4A). Similarly, berberine significantly increased the protein levels of GLUT4 in PCOS model animals (Fig. 4B). This therefore suggests that GLUT4 may be a key mediator of the observed ability of berberine to protect against PCOS pathology. 3.4. Berberine restores PI3K/AKT pathway activation To assess the mechanisms whereby berberine alters PCOS pathology in detail, we next measured IRS, PI3K, and AKT levels via Western blotting. Our results revealed that PCOS model animals exhibited reduced expression of the phosphorylated forms of these proteins relative 4
Regulatory Toxicology and Pharmacology 110 (2020) 104544
N. Zhang, et al.
PCOS (Adak et al., 2018). Berberine is an isoquinoline alkaloid compound that can be extracted from many different plants, and which has pharmacological activities that make it well-suited to immune modulation, lowering of glucose and cholesterol levels, and combatting cancer or microbial pathogens (N. Wang et al., 2015; Pirillo and Catapano, 2015; Cao et al., 2019; Andreazza et al., 2016). Multiple recent studies have also explored the clinical use of berberine for treating PCOS(Luo et al., 2019; Li et al., 2015). While there is work suggesting that berberine can overcome IR in vitro and in vivo (Zhao et al., 2011; Li et al., 2013), the specific associated mechanisms remain uncertain. One recent study found berberine to upregulate GLUT4 expression – an activity potentially related to its ability to control glucose metabolism(Zhang et al., 2011). In this study, we found berberine to be able to normalize both IR symptoms and ovarian GLUT4 expression in PCOS model rats, suggesting that berberine offers beneficial effects against PCOS via modulation of GLUT4 expression. This insulin signaling pathway, which depends upon signaling through a number of intermediaries including IRS, PI3K, and AKT, is a key regulator of the expression and localization of GLUT4, thereby regulating cellular glucose intake (Tremblay et al., 2001; Jeon et al., 2015). Previous work has shown that suppression of normal IRS activation and downstream PI3K/AKT signaling can compromise normal GLUT4 localization and glucose uptake by cells (Carvalho et al., 2001). Indeed, PI3K/AKT signaling has previously been reported to play a role in the pathology of PCOS, likely at least in part due to the fact that reduced GLUT4 expression (Li et al., 2017). Berberine has previously been found to increase IRS expression and thereby activate PI3K/AKT signaling (Wang et al., 2018). Consistent with this, we found that berberine-mediated effects against IR in a PCOS rat model were associated with its ability to enhance activation of PI3K/AKT signaling in these animals. Indeed, we found that berberine was able to decrease IR via increasing GLUT4 expression and activating PI3K/AKT signaling in order to help alleviate this symptom of PCOS. Many signaling pathways influence IR pathogenesis beyond the PI3K/AKT pathway, with their modulated signaling being linked to dysregulated glucose metabolism in the context of disorders such as diabetes (Choi et al., 2019). MAPK pathway activation has been shown to suppress IRS activation, thus promoting IR development (Hanai et al., 2009). MAPK signaling is a key signal transduction pathway linked to androgen biosynthesis and IR in the context of PCOS (Diamanti-Kandarakis and Dunaif, 2012). In addition, MAPK activation can lead to reduced GLUT4 expression, thereby impairing glucose transport (Zhou et al., 2018). There is also evidence that PI3K/AKT signaling can influence MAPK pathway activation (Zhou et al., 2016). To assess whether the effects of berberine on IR were linked to MAPK pathway activation, we assessed MAPK-related protein expression and observed a significant berberine-mediated suppression of p38, ERK, and JNK in PCOS model animals. Thus suggests that berberine may therefore reduce IR in part via modulation of the MAPK signaling pathway. In conclusion, our research provides novel evidence that berberine treatment is able to alleviate the pathogenesis of PCOS in a rat model system, decreasing IR through a mechanism likely linked with GLUT4 upregulation. The underlying molecular mechanisms governing this upregulation are likely linked to berberine-mediated activation of PI3K/AKT signaling and suppression of MAPK signaling. Our data thus suggest that berberine may offer clinical benefits. Previous work clearly supports cross-talk between PI3K⁄AKT and MAPK signaling (Choi et al., 2018). Our results are consistent with the existence of such cross-talk, and suggest that it may be a key mediator of PCOS-associated IR.
Fig. 6. Berberine inhibited the MAPK pathway. **p < 0.01 vs. control, ## p < 0.01 vs. PCOS. BBR, berberine; PCOS, polycystic ovarian syndrome.
to control animals (Fig. 5). Rats treated with 400 mg/kg berberine exhibited significant 46%, 30%, and 40% increases in the levels of pIRS, p-PI3K, and p-AKT, respectively. These results thus suggest that berberine may protect against PCOS-associated IR via restoring the activation of this PI3K/AKT signaling pathway. 3.5. Berberine inhibits MAPK pathway activation To confirm that the MAPK pathway is associated with the ability of berberine to mediate recovery from PCOS-associated IR, we next assessed the expression of the key MAPK signaling proteins P38, ERK, and JNK via Western blotting. We observed that PCOS model animals exhibited increased levels of these proteins relative to healthy control rats, and berberine treatment significantly reduced the expression of these proteins (Fig. 6). This suggests that the ability of berberine to influence PCOS pathology is correlated with its effects upon the MAPK pathway. 4. Discussion PCOS is a disease of the endocrine system affecting up to 15% of women of childbearing age, leading to symptoms including ovarian cysts, anovulation, and hyperandrogenism(Legro et al., 2013). PCOS is also associated with high rates of obesity, with abdominal fat in particular being a common problem among affected women(Wang et al., 2016). The infertility that affects women with PCOS is thought to be attributable, at least in part, to IR, which affects up to 70% of those with PCOS and is linked to profoundly abnormal insulin activity in the reproductive system (Morgante et al., 2018). Some medicines, such as Metformin, has been reported to have beneficial effects in patient with
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 5
Regulatory Toxicology and Pharmacology 110 (2020) 104544
N. Zhang, et al.
Acknowledgments
One 10, e0144072. Li, X., Cui, P., Jiang, H.Y., Guo, Y.R., Pishdari, B., Hu, M., Feng, Y., Billig, H., Shao, R., 2015. Reversing the reduced level of endometrial GLUT4 expression in polycystic ovary syndrome: a mechanistic study of metformin action. Am. J. Tourism Res. 7, 574–586. Li, T., Mo, H., Chen, W., Li, L., Xiao, Y., Zhang, J., Li, X., Lu, Y., 2017. Role of the PI3K-Akt signaling pathway in the pathogenesis of polycystic ovary syndrome. Reprod. Sci. 24, 646–655. Li, M.F., Zhou, X.M., Li, X.L., 2018. The effect of berberine on polycystic ovary syndrome patients with insulin resistance (PCOS-IR): a meta-analysis and systematic review. Evid. Based Complement Altern. Med. https://doi.org/10.1155/2018/2532935. Luo, R., Liao, Z., Song, Y., Yin, H., Zhan, S., Li, G., Ma, L., Lu, S., Wang, K., Li, S., 2019. Berberine ameliorates oxidative stress-induced apoptosis by modulating ER stress and autophagy in human nucleus pulposus cells. Life Sci. 228, 85–97. Morgante, G., Massaro, M.G., Di Sabatino, A., Cappelli, V., De Leo, V., 2018. Therapeutic approach for metabolic disorders and infertility in women with PCOS. Gynecol. Endocrinol. 34, 4–9. Moulana, M., 2019. Immunophenotypic profile of leukocytes in hyperandrogenemic female rat an animal model of polycystic ovary syndrome. Life Sci. 220, 44–49. Pirillo, A., Catapano, A.L., 2015. Berberine, a plant alkaloid with lipid- and glucoselowering properties: from in vitro evidence to clinical studies. Atherosclerosis 243, 449–461. Rajan, R.K., Kumar, S.S.M., Balaji, B., 2017. Soy isoflavones exert beneficial effects on letrozole-induced rat polycystic ovary syndrome (PCOS) model through anti-androgenic mechanism. Pharm. Biol. 55, 242–251. Shah, K.N., Patel, S.S., 2016. Phosphatidylinositide 3-kinase inhibition: a new potential target for the treatment of polycystic ovarian syndrome. Pharm. Biol. 54, 975–983. Tremblay, F., Lavigne, C., Jacques, H., Marette, A., 2001. Defective insulin-induced GLUT4 translocation in skeletal muscle of high fat-fed rats is associated with alterations in both Akt/protein kinase B and atypical protein kinase C (zeta/lambda) activities. Diabetes 50, 1901–1910. Wang, N., Tan, H.Y., Li, L., Yuen, M.F., Feng, Y., 2015. Berberine and Coptidis Rhizoma as potential anticancer agents: recent updates and future perspectives. J. Ethnopharmacol. 176, 35–48. Wang, M., Chen, S., Zhang, D., 2015. Systematic Evaluation of the randomized controlled trial system for polycystic ovary syndrome treated by berberine and metformin. Guangdong Med. J. 36, 3866–3870. Wang, Y., Fu, X., Xu, J., Wang, Q., Kuang, H., 2016. Systems pharmacology to investigate the interaction of berberine and other drugs in treating polycystic ovary syndrome. Sci. Rep. 6, 28089. Wang, Z., Zhai, D., Zhang, D., Bai, L., Yao, R., Yu, J., Cheng, W., Yu, C., 2017. Quercetin decreases insulin resistance in a polycystic ovary syndrome rat model by improving inflammatory microenvironment. Reprod. Sci. 24, 682–690. Wang, Y.Y., Tang, L.Q., Wei, W., 2018. Berberine attenuates podocytes injury caused by exosomes derived from high glucose-induced mesangial cells through TGF beta 1PI3K/AKT pathway. Eur. J. Pharmacol. 824, 185–192. Wu, J., Li, J., Li, W., Sun, B., Xie, J., Cheng, W., Zhang, Q., 2018. Achyranthis bidentatae radix enhanced articular distribution and anti-inflammatory effect of berberine in Sanmiao Wan using an acute gouty arthritis rat model. J. Ethnopharmacol. 221, 100–108. Yin, J., Gao, Z., Liu, D., Liu, Z., Ye, J., 2008. Berberine improves glucose metabolism through induction of glycolysis. Am. J. Physiol. Endocrinol. Metabol. 294, E148–E156. Zhang, W., Xu, Y.C., Guo, F.J., Meng, Y., Li, M.L., 2008. Anti-diabetic effects of cinnamaldehyde and berberine and their impacts on retinol-binding protein 4 expression in rats with type 2 diabetes mellitus. Chin. Med. J. 121, 2124–2128. Zhang, Q., Xiao, X., Feng, K., Wang, T., Li, W., Yuan, T., Sun, X., Sun, Q., Xiang, H., Wang, H., 2011. Berberine moderates glucose and lipid metabolism through multipathway mechanism. Evid. Based Complement Altern. Med. 1–10 2011. Zhao, L., Li, W., Han, F., Hou, L., Baillargeon, J.-P., Kuang, H., Wang, Y., Wu, X., 2011. Berberine reduces insulin resistance induced by dexamethasone in theca cells in vitro. Fertil. Steril. 95, 461–463. Zhao, H., Zhou, D., Chen, Y., Liu, D., Chu, S., Zhang, S., 2017. Beneficial effects of Heqi san on rat model of polycystic ovary syndrome through the PI3K/AKT pathway. Daru 25, 21. https://doi.org/10.1186/s40199-017-0188-7. Zhou, T., Meng, X., Che, H., Shen, N., Xiao, D., Song, X., Liang, M., Fu, X., Ju, J., Li, Y., 2016. Regulation of insulin resistance by multiple MiRNAs via targeting the GLUT4 signalling pathway. Cell. Physiol. Biochem. 38, 2063–2078. Zhou, D.N., Li, S.J., Ding, J.L., Yin, T.L., Yan, J., Ye, H., 2018. MIF may participate in pathogenesis of polycystic ovary syndrome in rats through MAPK signalling pathway. Curr. Med. Sci. 38, 853–860.
This work was supported by the Natural Science Foundation of Shandong Province (No. ZR2019MH117) and the Special fund for clinical research of Chinese Medical Association(No. 18010140743). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.yrtph.2019.104544 Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yrtph.2019.104544. References Adak, T., Samadi, A., Ünal, A.Z., Sabuncuoğlu, S., 2018. A reappraisal on metformin. Regul. Toxicol. Pharmacol. 92, 324–332. Andreazza, N.L., Vevert-Bizet, C., Bourg-Heckly, G., Sureau, F., Salvador, M.J., Bonneau, S., 2016. Berberine as a photosensitizing agent for antitumoral photodynamic therapy: insights into its association to low density lipoproteins. Int. J. Pharm. 510, 240–249. Cao, S., Xu, P., Yan, J., Liu, H., Liu, L., Cheng, L., Qiu, F., Kan, N., 2019. Berberrubine and its analog, hydroxypropyl-berberrubine, regulate LDLR and PCSK9 expression via the ERK signal pathway to exert cholesterol-lowering effects in human hepatoma HepG2 cells. J. Cell. Biochem. 120, 1340–1349. Carvalho, E., Jansson, P.A., Nagaev, I., Wenthzel, A.M., Smith, U., 2001. Insulin resistance with low cellular IRS-1 expression is also associated with low GLUT4 expression and impaired insulin-stimulated glucose transport. FASEB J. : Off. Publ. Feder. Am. Soc. Exp. Biol. 15, 1101–1103. Choi, J., Kim, K.J., Koh, E.J., Lee, B.Y., 2018. Gelidium elegans extract ameliorates type 2 diabetes via regulation of MAPK and PI3K/Akt signaling. Nutrients 10, 51. Choi, E., Kikuchi, S., Gao, H., Brodzik, K., Nassour, I., Yopp, A., Singal, A.G., Zhu, H., Yu, H., 2019. Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling. Nat. Commun. 10, 1473. Coniglio, R.I., Merono, T., Montiel, H., Malaspina, M.M., Salgueiro, A.M., Otero, J.C., Ferraris, R., Schreier, L., Brites, F., Rosso, L.G., 2012. HOMA-IR and non-HDL-C as predictors of high cholesteryl ester transfer protein activity in patients at risk for type 2 diabetes. Clin. Biochem. 45, 566–570. Diamanti-Kandarakis, E., Dunaif, A., 2012. Insulin resistance and the polycystic ovary syndrome revisited: an update on mechanisms and implications. Endocr. Rev. 33, 981–1030. Ezeh, U., Chen, I.Y.D., Chen, Y.H., Azziz, R., 2019. Adipocyte expression of glucose transporter 1 and 4 in PCOS: relationship to insulin-mediated and non-insulinmediated whole-body glucose uptake. Clin. Endocrinol. 90, 542–552. Hanai, Y., Adachi, S., Yasuda, I., Takai, S., Matsushima-Nishiwaki, R., Kato, H., Enomoto, Y., Akamatsu, S., Sakakibara, S., Ogura, S., 2009. Collagen-induced p38 MAP kinase activation is a biomarker of platelet hyper-aggregation in patients with diabetes mellitus. Life Sci. 85, 386–394. Jeon, H.J., Yoon, K.Y., Koh, E.J., Choi, J., Kim, K.J., Choi, H.S., Lee, B.Y.S., 2015. Dieckol improve insulin sensitivity through the regulation of the PI3K pathway in C57BL/KsJdb/db mice. J. Food Nutr. Res. 3, 648–652. Jin, F., Xie, T., Huang, X., Zhao, X., 2018. Berberine inhibits angiogenesis in glioblastoma xenografts by targeting the VEGFR2/ERK pathway. Pharm. Biol. 56, 665–671. Legro, R.S., Arslanian, S.A., Ehrmann, D.A., Hoeger, K.M., Murad, M.H., Pasquali, R., Welt, C.K., 2013. Diagnosis and treatment of polycystic ovary syndrome: an endocrine society clinical practice guideline. J. Clin. Endocrinol. Metab. 98, 4565–4592. Li, G.W., Pan, X.R., 1993. A new insulin-sensitivity index for the population-based study. Zhonghua nei ke za zhi 32, 656–660. Li, Y., Ma, H., Zhang, Y., Kuang, H., Ng, E.H., Hou, L., Wu, X., 2013. Effect of berberine on insulin resistance in women with polycystic ovary syndrome: study protocol for a randomized multicenter controlled trial. Trials 14, 226. Li, L., Li, C., Pan, P., Chen, X., Wu, X., Ng, E.H.Y., Yang, D., 2015. A single arm pilot study of effects of berberine on the menstrual pattern, ovulation rate, hormonal and metabolic profiles in anovulatory Chinese women with polycystic ovary syndrome. PLoS
6