Endocrine (2019) 64:349–366 https://doi.org/10.1007/s12020-019-01891-3 ORIGINAL ARTICLE Liraglutide modulates adipokine expression during adipogenesis, ameliorating obesity, and polycystic ovary syndrome in mice 1 2 3 1 2 Anusha Singh ● Joseph R. D. Fernandes ● Gagan Chhabra ● Amitabh Krishna ● Arnab Banerjee Received: 9 October 2018 / Accepted: 5 March 2019 / Published online: 23 March 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract Purpose The incidence of obesity is increasing among all age groups throughout the world and it is highly associated with numerous other metabolic disorders, such as insulin resistance, polycystic ovarian syndrome (PCOS) etc. Methods and Results Using in vitro and in vivo approach, this study investigated the adipokine profile after liraglutide on differentiated murine 3T3-L1 pre-adipocytes. Effect of liraglutide on DHEA-induced PCOS mice were investigated. This study showed Liraglutide treatment resulted in up-regulation of adiponectin and IL-6 along with down-regulation of ICAM 1 in differentiated 3T3-L1 cells. Liraglutide in absence of other differentiating factors, significantly increased glucose, lipid uptake and PPARγ, C/EBPα expression in the adipocytes suggesting its ability to solely promote pre-adipocyte differ- 1234567890();,: 1234567890();,: entiation into mature adipocyte. Liraglutide treatment showed increased adiponectin expression and decreased number of cystic follicles, body weight, circulating glucose, triglyceride and testosterone levels in comparison to the PCOS induced mice. Conclusion This study suggests that adiponectin may act as a link between metabolic disorders and PCOS and that liraglutide might be a promising therapeutic agent for the treatment of PCOS in addition to obesity and insulin resistance. Highlights ● The study shows the impact of GLP-1 analogue, liraglutide’s impact on adipokine secretion in 3T3L1 adipocytes. ● For the first time, showing that liraglutide in the absence of other differentiating factors (IBMX, insulin, and dexamethasone), significantly increased glucose, lipid uptake and PPARγ, C/EBPα expression in the adipocytes suggesting its ability to solely promote pre-adipocyte differentiation into mature adipocyte. ● Also, for the first time it shows that liraglutide through the up-regulation of adiponectin manages PCOS in mice. Keywords Liraglutide ● Adipocyte ● Adiponectin ● PCOS These authors contributed equally: Anusha Singh, Joseph R. D. Fernandes * Arnab Banerjee 2 Department of Biological Sciences, BITS Pilani KK Birla Goa [email protected] Campus, Goa 403726, India 3 Department of Dermatology, University of Wisconsin-Madison, 1 Department of Zoology, Banaras Hindu University, Madison, WI, USA Varanasi 221005, India 350 Endocrine (2019) 64:349–366 Introduction Both liraglutide and GLP-1 share, some of the glucor- egulatory functions including enhancement of glucose- Obesity constitutes a serious health concern worldwide. It is dependent insulin secretion, inhibition of postprandial glu- alarming to observe obesity among all age groups in most cagon secretion, inhibition of gastric emptying, and reduc- countries including India [1]. It is a major characteristic of tion of food intake [16]. In animal studies, liraglutide metabolic syndrome and is a risk factor for type II diabetes maintenance of β-cell mass in diabetes, by inhibiting both and cardiovascular diseases [2]. Earlier anti-obesity drugs or cytokine and free fatty acid (FFA)-induced apoptosis [17]. anorexins have poor safety records. A better knowledge of Recently, a study showed that liraglutide prevented the physiology of adipogenesis and fat cell development in hypoadiponectinemia-induced increase in plasma insulin obesity is important to identify new biomarkers and ther- [18]. Liraglutide injection into high fat diet fed mice apeutic targets for the development of novel anti-obesity induced in vivo adipocyte differentiation by regulating cell drugs with improved safety profile. proliferation and apoptosis of pre-adipocytes [19]. In type II Factors responsible for obesity include those responsible diabetes patients, 14 weeks of liraglutide treatment sig- for the expansion of white adipose tissue via the mechanism nificantly reduced hemoglobin A1c (HbA1c), body mass of hypertrophy of pre-existing adipocytes and hyperplasia index, and total body fat mass, lowered insulin resistance resulting from the adipogenesis of pre-adipocytes [3]. and increased visfatin and resistin levels [20]. A recent However, changes in adipocyte turnover rate, differentia- study showed that liraglutide facilitates weight loss, tion, and apoptosis could all contribute to changes in fat improving bleeding pattern, increased SHBG levels, and mass underlying obesity. Studies have suggested that pre- decreased testosterone levels thereby improving the sex adipocyte differentiation may be impaired in obese humans hormone levels and gonadotropin levels further ameliorat- [4, 5]. One possible avenue to reduce fat mass is to ther- ing other metabolic dysfunction in overweight women with apeutically regulate pre-adipocyte differentiation, but a PCOS. This study also assessed the ovarian morphology better understanding of the pathways controlling adipo- with three-dimensional ultrasound, however, no effect was genesis is needed. Another important aspect of adipogenesis observed in both the liraglutide and the placebo groups [21]. is the adipokines secretion by the adipose tissue, which is The study showed when women with PCOS were treated the central factor how fat mass directly influence insulin with liraglutide, the levels of fasting glucose and leptin were sensitivity and vascular injury [6]. The success of any new reduced, however, measures of insulin resistance, adipo- drug to control obesity depends on how therapeutically it nectin, and glucagon strangely did not change. This study might regulate the profile of adipokines secreted from the further showed liraglutide treatment of 26 weeks in PCOS fat mass. women resulted in significant reductions of liver fat content, Glucagon-like peptide 1 (GLP-1), is a proglucagon- VAT, and the prevalence NAFLD [22]. Another study derived peptide secreted from endocrine cells in the gut, showed that treatment with liraglutide in combination with GLP-1 is used as a therapeutic option for the pharma- metformin resulted in a significant weight loss in over- cotherapy of type II diabetes and obesity. It is responsible weight and obese women with PCOS [23]. 3T3-L1 cells for the stimulation of glucose-dependent insulin secretion treated with different doses of liraglutide, showed a sig- from pancreatic β cells, inhibition of glucagon secretion, nificant decrease in the mRNA expression of some adipo- and inhibition of food intake [7–10]. Obese individuals are kines, such as leptin, TNF-α, and IL-6, whereas, the mRNA reported to have reduced GLP-1 levels and improved GLP- expression of adiponectin and CTRP3 were increased sig- 1 levels after weight loss [11]. One of the major short- nificantly [24]. Interestingly, liraglutide treatment to type II comings in the implementation of GLP-1 is its rapid inac- diabetes patients has also been shown to significantly tivation in the body by the enzyme dipeptidyl peptidase IV decrease serum adiponectin level [25] and among Chinese (DPP-IV) and rapid renal elimination [7]. Studies have type 2 diabetes (T2DM) patients, liraglutide led to increased shown the effects of GLP-1 on cardiomyocytes involving in adiponectin and decreased resistin levels [26]. However, the the regulation of the PI3K/Akt signaling pathway [12–14]. direct effect of liraglutide on the adipokine profile of the For the gut–brain axis, GLP-1 and GLP-2 are important for differentiated pre-adipocyte is not known, knowledge of food intake, maintaining glucose homeostasis thereby con- which will not only help us to better understand the phy- trolling insulin sensitivity through the PI3K/Akt signaling siology of liraglutide action on adipocytes but also might pathway [15]. To overcome this shortcoming, a long-acting open new research pertaining liraglutide pharmacotherapy. analogue of GLP-1, liraglutide, with a 97% sequence The murine 3T3-L1 cells are one of the best in vitro homology to human GLP-1 has been increasingly used as a models to study the mechanisms involved in pre-adipocyte therapeutic agent in improving insulin resistance, glucose proliferation, differentiation, adipokine secretion, and gene/ tolerance, and obesity [7]. protein expression [2]. Liraglutide previously has been Endocrine (2019) 64:349–366 351 shown to induce differentiation in the presence of differ- # I9278) were procured from Sigma-Aldrich Chemicals Co., entiation medium consisting of insulin (IN), dexamethasone USA. Mouse Adipokine antibody array kit was obtained (DEX), and 3-isobutyl-1-methylxanthine (IBMX) [19]. from R&D Systems, USA (Catalog # ARY013). Other However, whether liraglutide alone has any effect on the chemicals of analytical grade were purchased from Sigma- differentiation of the 3T3L1 pre-adipocytes has not been Aldrich Chemicals Co., USA. studied. Antibody, cata- Host Type Source Supplier Dilution Since 1935, Stein and Leventhal [27] had recognized log number species IB/IHC obesity as a common feature of the PCOS, one of the GLUT-4 (H- Rabbit Polyclonal Human Santa Cruz Biotechnol- 1:500 common metabolic disorders of females, characterized by 61):sc-7938 ogy Inc., CA, USA hyperandrogenism and chronic oligo-anovulation [28]. Adipo R1 (H- Rabbit Polyclonal