Team:ECNUAS/Description
Description
Inspiration：
The number of type 2 diabetes mellitus (T2DM) patients is increasing[1]. T2DM is characterized by relative insulin deficiency caused by β-cell dysfunction and insulin resistance. Hepatic glucose metabolism is modulated by numerous hormones including glucagon, insulin, and glucocorticoid, and the effect of glucocorticoid is usually mediated by the glucocorticoid receptor (GR)[2][3]. The increasing of glucocorticoids has the side effect of aggravating blood glucose levels, and the GR antagonists have been reported to improve blood glucose levels[4]. Therefore, the study of GR antagonists is important. But the effect and mechanism of GR are not clear, so it is important to establish an experimental platform for screening GR antagonists[5]. Our product is a platform for screening GR antagonists which might alleviate T2DM. In our study, the GR transactivation assay was established to verify the antagonistic effect of drugs on GR. Dexamethasone (Dex) as a GR agonist efficiently activated reporter gene expression, and mifepristone (a GR antagonist ) antagonized Dex-induced reporter gene stimulation. The success of such an experiment will boost research on the efficiency of GR-related medication, thus might help T2DM patients who have overproduced glucocorticoid decrease blood sugar level.
Background：
Diabetes Mellitus (DM) is a metabolic disorder characterized by increased glucose levels caused by genetic and environmental factors. According to their pathogenesis, DM is mainly composed of Type 1 Diabetes Mellitus (INSULin-dependent), T1DM, Type 2 Diabetes Mellitus (NON-insulin-dependent), T2DM, and Gestational Diabetes Mellitus (GDM)[7].
According to the IDF Diabetes Atlas. 9th Edition, 2019, approximately 463 million adults aged 20-79 worldwide will be suffering from Diabetes (1 in 11); Diabetes is expected to hit 578.4 million by 2030; The number of diabetics is expected to reach 702 million by 2045. At the same time, approximately 4.2 million people (20-79 years of age) die from diabetes or its complications, equivalent to one person every eight seconds, accounting for approximately 11.3 percent of all cause-related deaths globally.
The number of PATIENTS with T2DM accounts for more than 90% of DM patients. The main pathogenesis of T2DM is the relative lack of insulin caused by peripheral insulin resistance or functional defect of pancreatic beta cells, and blood glucose cannot be converted into energy demand of the body, leading to increased blood glucose. With the improvement of living standards, T2DM presents an increasing trend and has become a serious threat to human health.
Significance：
Glucocorticoid receptor, GR, is one of the members of the metabolically related nuclear receptors. It is expressed in many tissues and mainly mediates the physiological effects of Glucocorticoid. In diabetic model mice and diabetic patients, glucocorticoid levels were increased, and its effect on glucocorticoid receptors was a side effect of promoting the glucogenic elevation of blood glucose. In the development of anti-T2DM drugs, GR antagonists have been reported to improve blood glucose levels by reducing gluconeogenesis. Therefore, the study of GR antagonists is also one of the hot spots of anti-metabolic diseases. So far, the effect and mechanism of GR regulating glucose metabolism in vivo are not clear, so it is of great significance to establish an experimental platform for screening GR antagonist small molecules against T2DM.
Experimental skills：
Basic molecular cloning experiments:
1.plasmid construction (enzyme digestion, ligation, transformation, etc.);
2.Polymerase chain reaction (PCR);
3.Agarose gel electrophoresis;
4.Preparation of competent cell;
5.Plasmid transformation and amplification;
6.Plasmid extraction and purification.
Mammalian cell related experiments:
1.Mammalian Cell resuscitation, culture, passage paving, and freezing storage;
2.Plasmid transfection;
3.Dual-luciferase reporter gene experiment.
Reference：
[1] Priyadarshini E , Anuradha C V . Glucocorticoid Antagonism Reduces Insulin Resistance and Associated Lipid Abnormalities in High-Fructose-Fed Mice[J]. Canadian Journal of Diabetes, 2017, 41(1):41.
[2] van den Heuvel, José K, Boon M R , Van Hengel I , et al. Identification of a selective glucocorticoid receptor modulator that prevents both diet-induced obesity and inflammation.[J]. Br J Pharmacol, 2016, 173(11):1793-1804.
[3] Alexander SPH, Cidlowski JA, Kelly E, Marrion N, Peters JA, Benson HE et al. (2015). The Concise Guide to PHARMACOLOGY 2015/16: Nuclear hormone receptors. Br J Pharmacol 172: 5956–5978.
[4] Guillaume‐Gentil C, Assimacopoulos‐Jeannet F, Jeanrenaud B (1993). Involvement of non‐esterified fatty acid oxidation in glucocorticoid‐induced peripheral insulin resistance in vivo in rats. Diabetologia 36: 899–906.
[5] Kohsaka A, Laposky AD, Ramsey KM, Estrada C, Joshu C, Kobayashi Y et al. (2007). High‐fat diet disrupts behavioral and molecular circadian rhythms in mice. Cell Metab 6: 414–421.
[6] Waddell DS, Baehr LM, van den Brandt J, Johnsen SA, Reichardt HM, Furlow JD et al. (2008). The glucocorticoid receptor and FOXO1 synergistically activate the skeletal muscle atrophy‐associated MuRF1 gene. Am J Physiol Endocrinol Metab 295: E785–E797.
[7] de Guia RM, Rose AJ, Rose AJ, Herzig S. Glucocorticoid hormones and energy homeostasis. Hormone Molecular Biology and Clinical Investigation. 2014 Aug;19(2):117-128. DOI: 10.1515/hmbci-2014-0021.
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