Neu-p11 reduces clock/apelin expression in insulin-resistant mouse adipocyte model

Abstract

It is generally accepted that a major risk factor of type 2 diabetes mellitus (T2DM) is insulin resistance and is often associated with obesity [1]. Apelin (expressed in adipose tissue) appears as a beneficial adipokine with anti-diabetic and antiobesity properties [2]. Clock is a large and complex gene encoding a novel member of the basic helix-loop-helix/ Per-ARNT-Sim protein family of transcription factors. Circadian clocks can regulate metabolic homeostasis and clock disruption leads to obesity and the metabolic syndrome [3]. Melatonin (Mel) is an integral part of the homeostatic mechanism in the body. It is best known as a regulator of seasonal and circadian rhythms, with high expression level during the night and low level during the day. Interestingly, insulin levels are also adapted to day/night changes through Mel-dependent synchronization. Mel may influence diabetes and be associated with metabolic disturbances by regulating insulin secretion [4]. Luzindole is the non-specific Mel receptor antagonist that can block some functions of Mel [5]. Although Mel has a wide range of biological effects, studies have found that once it gets into human body, its metabolism can be very fast, with a half-life of 20–30 min. Therefore, apparent effects cannot be achieved from direct administration of this medicine. Besides, the extraction and synthesis of Mel are relatively difficult, and some side effects may arise if its dose is too high. Neu-P11 is a new type of non-selective agonist of Mel [6]. It has the following characteristics: it can be synthesized relatively easily in vitro; its effective time can be longer with fewer side effects; it can substitute Mel to interact with its receptors, and sequentially play a wide range of biological roles. The aim of the present study was to explore the mechanism of the Mel and its receptor agonist Neu-P11 and the change of clock/apelin in insulin-resistant 3T3-L1 adipocytes. To examine the changes of clock and apelin expression levels resulted from Neu-P11 treatment, insulin-resistant 3T3-L1 adipocytes were used in this study. Neu-P11 was provided by Neurim Pharmaceuticals Ltd. (Habarzel, Israel). A 5-mM stock solution was prepared by dissolving Neu-P11 in dimethylsulfoxide. 3T3-L1 pre-adipocytes were purchased from Institute of Basic Medicine, Chinese Academy of Medical Sciences (Beijing, China). Cells were differentiated in adipogenic cocktail by 3-isobutyl-1-methylxanthine (Sigma, St Louis, USA), dexamethasone, and insulin, and identified by oil red O staining. Insulin-resistant 3T3-L1 adipocytes were induced in Dulbecco’s modified Eagle’s medium (Gibco, Gaithersburg, USA) with high glucose/ high insulin for 24 h. Glucose consumption was detected by enzymatic method to evaluate the model [7,8].