Team:NUDT CHINA/Description

Description

Introduction

     Type two diabetic mellitus (T2D) is a common and serious chronic metabolic syndrome comprises a cluster of diseases associated with excess nutrition and insufficient physical activity. To date, it is estimated that more than 352 million people around the globe are at risk of developing such a chronic and painful disease, particularly in low – and middle-income countries1 (Figure 1a). In China, around 10% of its population have diabetes, with nearly 60% exposed to the T2D related risk factors2. Due to its undiminished importance, diabetic mellitus has been widely studied in the past two decades.

 

Figure 1. Regional distribution of Diabetic patients1.

     Though pathological mechanism of T2D is still yet to be further understood, peripheral insulin resistance, impaired regulation of hepatic glucose production, and islet β-cell dysfunction are thought to be related to the development of T2D3. Current treatment also mainly focused on improving peripheral insulin resistance or increasing insulin secretion by lifestyle intervention or pharmaceutical approaches4. Notably, synthetic biology approaches have also been developed to provide “smart” glycemic control over the T2D patients by inducing insulin or GLP-1 production5-7. However, it is worth noticing that although the hyperglucagonemia syndrome has been discovered on T2D patients for years, research focusing on glucagon pathophysiology and related therapeutic approaches has historically been dwarfed by research on beta cells and insulin. Glucagon is a 29-amino acid peptide hormone secreted from the pancreatic alpha cells with a powerful stimulatory effect on hepatic glucose production acting to increase plasma glucose levels as a counter-regulatory hormone to insulin8. Though the mechanisms underlying hyperglucagonemia in patients with type 2 diabetes are not clear. It seemed quite reasonable to propose a brand-new glycemic control approach targeting glucagon pathway (Figure 2a).

     Glucagon stimulates hepatic glucose production mainly via hepatic glucagon receptor (GCGR), a 62 kDa G-protein coupled receptor9. Liver specific disruption of GCGR has also shown reductions in fasting blood glucose and improvements in insulin sensitivity and glucose tolerance compared with wild-type controls under normal chow diet and high fat diet condition10-12. These results further implied that GCGR might serve as a decent drug target for glycemic control. However, as is suggested by our HP interview and several literatures, uncontrolled depletion of GCGR may result in lethal hypoglycemia and disrupted islet alpha cell function11,13. An improved approach for “smart” GCGR blockage is thus needed.

 

Figure 2. Schematic representation of the project. (a). General glycemic control approach; (b). Trim21 based protein degradation module; (c). ChREBP based glucose sensing module.

     For such matter, inspired by our project in iGEM 2018, we further expanded the application of previously reported Trim-away system14 by shifting the antibody-antigen interaction into ligand-receptor interaction, thus providing us a new approach to degrade endogenous receptors possibly within ER or Trans-Golgi Network (Figure 2b). Our results suggested that our new protein degradation approach can effectively degrade GCGR in HepG2 cells. Impaired cellular glucagon response and glucose production were also observed. Furthermore, to achieve the feedback control of the GCGR degradation system, we also incorporated Carbohydrate-response element-binding protein (CHREBP) pathway, the endogenous hepatic glucose sensing pathway15, into our system by in-silico-assisted16,17 designing of a CHREBP-activating hybrid promoter (Figure 2c). As expected, fluorescent reporter assay showed a dose-dependent increase of fluorescent intensity under the stimulation of different amount of glucose. Taking one step forward, we also successfully demonstrated that the GCGR degradation system we designed can be easily packaged into adenovirus-based delivery system and remain functional in primary hepatocytes after virus transfection. Our modeling also showed that by conjugating the glucose sensing module and the GCGR degrading module, our circuit can reestablish glycemic homeostasis of simulated T2D patient into a much healthier level without inducing the risk of hypoglycemia.

     By the meantime, our HP members also set foot into the society and hospitals, investigating their understanding on diabetes prevention and treatment, as well as their opinions on virus delivered gene therapy. The feedbacks from local society, medical doctors and experts not only help us to reshape our project, but also pushed us forward to launch a few public awareness programs related to T2D and gene therapy.

     In general, our project in iGEM 2019 not only included a brand-new protein degradation system into the synthetic biology toolkit, but also developed a new synthetic glucose sensing approach that has not yet being reported. More importantly, we provided a promising close-loop, virus-based, protein-targeting, synthetic biology inspired approach for glycemic control in T2D.

Reference

1  Cho, N. H. et al. IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Research & Clinical Practice 138, 271 (2018).

2  WHO. China. World Health Organization Diabetes country profiles (2016).

3  Mahler, R. J. & Adler, M. L. Clinical review 102: Type 2 diabetes mellitus: update on diagnosis, pathophysiology, and treatment. J Clin Endocrinol Metab 84, 1165-1171, doi:10.1210/jcem.84.4.5612 (1999).

4  Hays, N. P., Galassetti, P. R. & Coker, R. H. Prevention and treatment of type 2 diabetes: Current role of lifestyle, natural product, and pharmacological interventions. Pharmacology & Therapeutics 118, 181-191 (2008).

5  Xie, M. et al. β-cell-mimetic designer cells provide closed-loop glycemic control. Science 354, 1296-1301 (2016).

6  Xue, S. et al. A Synthetic-Biology-Inspired Therapeutic Strategy for Targeting and Treating Hepatogenous Diabetes. Molecular Therapy the Journal of the American Society of Gene Therapy 25, 443 (2017).

7  Shao, J. et al. Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice. Science Translational Medicine 9, eaal2298 (2017).

8  Asger, L., Bagger, J. I., Mikkel, C., Knop, F. K. & Tina, V. L. Glucagon and type 2 diabetes: the return of the alpha cell. Current Diabetes Reports 14, 555 (2014).

9  Lok, S., . et al. The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization. Gene 140, 203-209 (1994).

10  Rivero-Gutierrez, B. et al. Deletion of the glucagon receptor gene before and after experimental diabetes reveals differential protection from hyperglycemia. Molecular metabolism 17, 28-38, doi:10.1016/j.molmet.2018.07.012 (2018).

11  Winzell, M. S. et al. Glucagon receptor antagonism improves islet function in mice with insulin resistance induced by a high-fat diet. Diabetologia 50, 1453-1462 (2007).

12  Christine, L. et al. Liver-specific disruption of the murine glucagon receptor produces α-cell hyperplasia: evidence for a circulating α-cell growth factor. Diabetes 62, 1196-1205 (2013).

13  Vuguin, P. M. & Charron, M. J. Novel insight into glucagon receptor action: lessons from knockout and transgenic mouse models. Diabetes Obesity & Metabolism 13, 144-150 (2011).

14  Clift, D. et al. A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell 171, 1692-1706.e1618, doi:10.1016/j.cell.2017.10.033 (2017).

15  Hiromi Yamashita, M. T. M. S. R. K. B. W. J. H. W. S. D. A. K. U. A glucose-responsive transcription factor that regulates carbohydrate metabolism in the liver.

16  Naravat, P. et al. Genome-Wide Analysis of ChREBP Binding Sites on Male Mouse Liver and White Adipose Chromatin. Endocrinology 156, 1982-1994 (2015).

17  Yun-Seung, J. et al. Integrated expression profiling and genome-wide analysis of ChREBP targets reveals the dual role for ChREBP in glucose-regulated gene expression. Plos One 6, e22544 (2011).