Molecular Mechanism Of Anti-Apoptotic Actions Of Esculentin-2Cha On BRIN-BD11 Cells

Introduction to Diabetes Mellitus

Diabetes mellitus is a characterized as a chronic metabolic disorder, which leads to elevation of blood glucose level in the body. The reason behind this metabolic syndrome relies on either inability of the pancreas to produce enough insulin or inability of the body cells to respond properly to the insulin secreted in the blood, which is characterized as insulin intolerance. Based on the causes, there are three key types of diabetes. The Type 1 diabetes develops due to the failure of pancreas to produce enough insulin, which is characterized as an autoimmune disease. In this case, body cells become unable to secrete insulin due to the loss of autoimmune destruction of pancreatic beta cells. Thus, it is known as the ‘insulin-dependent diabetes mellitus or juvenile diabetes. On the other hand, Type 2 diabetes is related to insulin resistance, where body cells fail to respond to insulin effectively. With the progression of the disease insulin deficiency is also encountered. This form is thus known as the ‘non insulin-dependent diabetes mellitus’ (American Diabetes Association 2012). On the other hand, another type of diabetes is common in pregnant women, which is known as the Gestational diabetes, which is characterized as high blood sugar level during pregnancy and disappears after delivery.

Diabetes mellitus has become one of the major complications, affecting young children, adults as well as aged people. This is one of the key disorders, associated with several other health complications including hypertension, cardiovascular disorder, vision problem and musculoskeletal problems. It has also been revealed from studies that there is a significant link between cognitive deficit and diabetes. It has been seen that people, who are experiencing diabetes, have 1.2 to 1.5 fold greater rate of cognitive function decline (Ashcroft and Rorsman 2012). In older people, it is also associated with fall risk and mobility issues.

Till 2016, 422 million people are living with diabetes worldwide, which have increased from 382 million people during 2013. On the other hand, the prevalence of diabetes is 8.5 % among adults, which has been doubled from the rate in 1980 (4.7 %) (Chen et al. 2012). In 2012, WHO estimated 1.5 million deaths due to diabetes mellitus, which makes it 8^th leading cause of death. On the other hand, in 2014, the International Diabetes Federation estimated that total 4.9 million deaths worldwide are registered, which are directly caused due to diabetes or indirectly caused by the enhanced risk of some fatal consequences related to diabetes. In UK, till 2015, 3.3 million people have been estimated to suffer from diabetes. The increased incident rate is related to the enhanced health care cost and financial burdens of treating the disease. As a result of the enhanced rate of the disease complications, financial burden s well as the inefficiency of the conventional therapies are promoting the research for novel antidiabetic agents.

Types and Prevalence

The treatment of diabetes includes management of the disease, medication as well as changes in lifestyle. Till date no cure is known for diabetes, thus the management of the disease is concerned about modifying the disease severity. Management of the disease is focused upon keeping the blood sugar level at near to normal level. It can be accomplished by changes in lifestyle, i.e. healthy diet, exercise, weight loss and using appropriate medication for keeping blood glucose level near the normal range. Medications are needed for patients who have limited chances to change their lifestyle or patients at advanced stage. On the other hand, it has been revealed that combination of lifestyle modification and medications can play a significant role in treatment (Qaseem et al. 2012). The lifestyle changes also include cessation of smoking, alcohol and junk food consumption. Medications are mainly administered for reducing the blood sugar level and keeping it at normal level.

There are different classes of medications, fall into the anti-diabetic group of medication. In this context, metformin is recommended as the first line treatment for type 2 diabetes, which makes the liver to produce less glucose, thereby reducing the mortality rate. Another drug is sulfonylureas, which cause the closure of ATP-sensitive potassium channels, leading to the changes in membrane potential; this in turn leads to a downward cascade stimulating insulin release. For reducing the blood glucose level, amylin analogues are recommended often, which are toxic to beta cells due to fibrillary proteins. In type 1 diabetes patients, it is used for reducing blood glucose level. Other important peptide-based drug include GLP-1 receptor agonist and incretin, which regulate glucose metabolism with low levels of hypoglycaemia (Meier 2012).

Several studies have provided evidences for the useful actions of the esculentin-2Cha group of peptides in management of type 2 diabetes. It showed significant role in stimulating insulin secretion from BRIN-BD11 cells in the “dose-dependent and glucose-responsive manner”. This host-defence peptide has been isolated from frog skin. It has displayed antimicrobial, antitumor and immunomodulatory activities. With selected analogues, the anti-diabetic effects of the peptide have been tested. It has been revealed to stimulate insulin secretion from rat BRIN-BD11 clonal pancreatic β-cells at concentrations greater than 0.3 nM, instead of showing cytotoxicity (Ojo et al. 2015). The mechanism involved membrane depolarization and increased intracellular Ca2+. The research has also shown the attenuation of insulinotropic activity by activating K_ATP channel, inhibiting the voltage dependent calcium channels and chelating extracellular Ca2+. While analyzing the correct dosage of the peptide, it has been seen that the acute application of [L28K] esculentin-2Cha enhanced the insulin secretion and glucose tolerance in high fat fed mice with obesity and insulin resistance. The dosage showed no additional side effects upon oral or intraperitoneal administration. The peptide administration was associated with significantly low plasma and pancreatic glucagon concentration and normalization of α-cell mass. The drug administration revealed decreased circulating triglyceride concentration, without affecting cholesterol and LDL concentration. However, further investigation of the peptide related treatments are required.

Management of Diabetes Mellitus

Another host-defence peptide, isolated from frog skin is tigerinin-1R, which has also shown beneficial role in stimulating insulin secretion in vitro and improved glucose tolerance as well as insulin sensitivity in animal models of diabetes type 2. Ojo et al. (2016) studied Tigerinin-1R analogue [Arg⁴] revealed that it can significantly reduce the non-fating plasma glucose and glucagon concentration in diet-induced obese mice, whereas increased the plasma insulin twice, while administered daily for 28 days. The peptide also significantly improved oral and peritoneal glucose tolerance with enhanced secretion of insulin. The mechanism of action of this peptide showed the blockage of K_ATP channels and improvement in beta cell activity of isolated islets to a range of secretagogues. Administration of this peptide resulted in the up-regulation of functional genes in islets, which are associated with insulin secretion, including Kcnj11, Abcc8, Cacna1c. The evidences indicated that further developments of the peptide analogue are required for improved treatment and management of patients with type 2 diabetes (Srinivasan et al. 2016).

The aim of the current study is to investigate the molecular mechanism behind the insulin-releasing anti anti-apoptotic actions of esculentin-2Cha in BRIN-BD11 cells.

• To learn the laboratory skills in tissue culture and molecular biology
• To investigate the effects of 24-48 hours exposure of BRIN-BD11 cells to esculentin -2Cha on insulin secretion in the presence of increasing glucose concentration and modulators of insulin secretion
• To assess apoptosis and cell viability in BRIN-BD11 cells exposed to esculentin-2Cha for 24-48 hours
• To investigate the changes in expression of crucial genes involved in the secretion of insulin in cells exposed to esculentin-2Cha chronically
• To investigate changes in the expression of crucial genes involved in the apoptosis in cells exposed to esculentin-2Cha chronically

Reference List

American Diabetes Association, 2012. Standards of medical care in diabetes—2012. Diabetes care, 35(Supplement 1), pp.S11-S63.

Ashcroft, F.M. and Rorsman, P., 2012. Diabetes mellitus and the β cell: the last ten years. Cell, 148(6), pp.1160-1171.

Chen, L., Magliano, D.J. and Zimmet, P.Z., 2012. The worldwide epidemiology of type 2 diabetes mellitus—present and future perspectives. Nature Reviews Endocrinology, 8(4), pp.228-236.

Meier, J.J., 2012. GLP-1 receptor agonists for individualized treatment of type 2 diabetes mellitus. Nature Reviews Endocrinology, 8(12), pp.728-742.

Ojo, O.O., Srinivasan, D.K., Owolabi, B.O., McGahon, M.K., Moffett, R.C., Curtis, T.M., Conlon, J.M., Flatt, P.R. and Abdel-Wahab, Y.H., 2016. Molecular mechanisms mediating the beneficial metabolic effects of [Arg4] tigerinin-1R in mice with diet-induced obesity and insulin resistance. Biological chemistry, 397(8), pp.753-764.

Ojo, O.O., Srinivasan, D.K., Owolabi, B.O., Vasu, S., Conlon, J.M., Flatt, P.R. and Abdel-Wahab, Y.H., 2015. Esculentin-2CHa-Related Peptides Modulate Islet Cell Function and Improve Glucose Tolerance in Mice with Diet-Induced Obesity and Insulin Resistance. PloS one, 10(10), p.e0141549.

Qaseem, A., Humphrey, L.L., Sweet, D.E., Starkey, M. and Shekelle, P., 2012. Oral pharmacologic treatment of type 2 diabetes mellitus: a clinical practice guideline from the American College of Physicians. Annals of internal medicine, 156(3), pp.218-231.

Srinivasan, D.K., Ojo, O.O., Owolabi, B.O., Conlon, J.M., Flatt, P.R. and Abdel-Wahab, Y.H., 2016. [I10W] tigerinin-1R enhances both insulin sensitivity and pancreatic beta cell function and decreases adiposity and plasma triglycerides in high-fat mice. Acta diabetologica, 53(2), pp.303-315.