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CW1 7007 BMS Lab Report on Diabetes mellitus glp1 and its relevence to disease

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Coventry University

   

Research Techniques in Pharmacology and Drug Discovery (7007BMS)

   

Added on  2023-04-04

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This is a lab report that discusses diabetes mellitus and GLP-1, a drug target for type 2 diabetes and obesity. The report explains the mechanisms of insulin production and insulin resistance, as well as the different types of medication used to treat diabetes. The report also describes the materials and methods used in a cell culture experiment to measure intracellular cAMP levels and the toxicity of certain compounds. Overall, the report highlights the potential of GLP-1 as a drug target for treating diabetes mellitus.

CW1 7007 BMS Lab Report on Diabetes mellitus glp1 and its relevence to disease

   

Coventry University

   

Research Techniques in Pharmacology and Drug Discovery (7007BMS)

   Added on 2023-04-04

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CW1
LAB REPORT
CW1 7007 BMS Lab Report on Diabetes mellitus glp1 and its relevence to disease_1
1. INTRODUCTION
DIABETES MELLITUS GLP1 AND ITS RELEVENCE TO DISEASE
Diabetes Mellitus is a metabolic disorder resulting from a defect in insulin
secretion, insulin action, or both. Lack of insulin causes chronic hyperglycemia
and abnormalities in the metabolism of proteins, fats, and carbohydrates (Bastaki
S., 2005). The significance of insulin as an anabolic hormone leads to metabolic
abnormalities in carbohydrates, lipids, and proteins. These metabolic abnormalities
are brought on by insufficient insulin levels to produce an adequate response
and/or insulin resistance of target tissues, primarily skeletal muscles, adipose
tissue, and to a lesser extent, liver, at the level of insulin receptors, signal
transduction system, and/or effect or enzymes or genes (Kharroubi & Darwish,
2015). Diabetes Mellitus are categorized into two types are Type 1 and type 2. To
prolong life and treat symptoms, medications are generally employed. The
prevention of long-term diabetic problems and the improvement of longevity
through the elimination of various risk factors are secondary goals (Bastaki S.,
2005). Insulin replacement therapy is typically necessary for type I diabetes, which
is caused by the immunologically mediated destruction of the pancreatic beta cells.
Type II diabetes seems to be brought on by both changes in insulin sensitivity and
insulin secretion. Although it rarely needs exogenous insulin, it can be managed
with food therapy or oral hypoglycemic medications (Flier et al., 1987).
A clinically validated drug target for type 2 diabetes and obesity is the glucagon-
like peptide-1 receptor (GLP-1R) (Cong et al., 2021). GLP1 has several actions. In
addition to an increase in insulin production, the glucokinase enzyme, and glucose
transporters, functional effects in the pancreas include the glucose-dependent
release of insulin. The two main drawbacks of GLP-1 are its relatively
narrow therapeutic window, where nausea acts as the dose-limiting factor, and
have very short half-life for native peptide. The GLP-1 receptor is a part of the G
protein-coupled receptors' glucagon-secretin B family (GPCRs) (Knudsen et al.,
2007). The most well-known impact of GLP-1R activation is increased cAMP
synthesis, which, together with cell membrane depolarization (Koole et al., 2015).
Drugs used to treat diabetes mellitus are being developed with the GLP1 receptor
in consideration as a potential target.
CW1 7007 BMS Lab Report on Diabetes mellitus glp1 and its relevence to disease_2
When diet, exercise, weight loss, and oral medicines are unable to regulate blood
glucose levels in type 2 DM, insulin becomes important. Type 2 DM can also be
treated with oral hypoglycemic medications. Sulphonylureas, biguanides, alpha
glucosidase inhibitors, analogues of meglitinide, and thiazolidenediones are
examples of oral hypoglycemic medications. The primary goal of these
medications is to treat the underlying metabolic problem, such as insulin resistance
and insufficient insulin production. They ought to be prescribed along with dietary
modifications and lifestyle adjustments that are appropriate. Dietary and lifestyle
modifications are intended to lower body weight, improve glycemic control, and
lower the risk of cardiovascular problems (Bastaki S., 2005).
2. MATERIALS AND METHODS
a). Cell culture:
Cell culture is the most widely used laboratory approach.. Cell culture was done in
an aseptic condition using a cell culture hood. Spray 70% ethanol on each item put
within the cell culture hood, then rinse it off. Before adding trypsin, the entire
medium has to be removed from the flask. The cells were treated with trypsin for 3
to 5 minutes, and then centrifuged at 1200 rpm for 5 minutes. The mixture was
then transferred to a hemocytometer for cell counting after 20 ml of trypan blue
and cell suspension were added. An inverted microscope was used to count the
number of viable cells.
b). Cell signaling assay:
A membrane protein known as adenylcyclase works as an enzyme. It turns ATP
into cAMP. After conducting cell treatment, the samples were diluted, and the
LANCE-cAMP kit was used to measure the intracellular cAMP level. The LANCE
cAMP test is a homogeneous time-resolved fluorescence resonance energy transfer
immunoassay designed to measure cAMP generated upon stimulation of adenyl
cyclase activity by GPCRs.
The media was removed from the cell plates, the stimulating buffer was added, and
the cells were then allowed to incubate for 30 minutes. In this assay, the drugs
adrenaline, adrenaline+ propranolol, and froskolin were used. For these drugs,
CW1 7007 BMS Lab Report on Diabetes mellitus glp1 and its relevence to disease_3
serial dilutions were created. Using a multichannel pipette to activate the cells, the
medicines were added to cell plates and incubated at 37°C for 30 minutes. The
media was discarded, put on ice for 10 minutes, and then 5ul of lysate was added.
U light cAMP antibody was used to prepare the assay plate. The lysate was then
put to the assay plate and wrapped with aluminum foil. To measure signalling, a
fluorimeter was used.
c). Toxicity assay:
The materials used for the toxicity assay included Compound 2, 48-well cell
culture plates, Trypan blue, hemocytometer, cell culture media, Eppendorfs, and
pipettes. Liver cells were added in a 24 well plates containing 5000 cells. A series
of dilutions were prepared for Compound X from 10-3 to 10-10. 600μl of cell culture
media was added to each well of the 24-well plate containing liver cells.60μl
compound X added to triplicates at each concentration and incubated the cells for 1
hour at 37°C. In the same way positive and negative controls were used.
After incubation, the cells were transferred to eppendorf tubes. Samples were
collected into the corresponding eppendorf tube after trypsin was applied to the
wells to loosen adherent cells. Then the eppendorf tube was centrifuged at
1600rpm for 5 minutes, the supernatant was collected, and the cell pellets were
then resuspended in 100μl of cell media. After that, cells were collected and mixed
with trypan blue in a 1:1 ratio before being loaded in 20μl into haemotocytometer
for cell counting. Then Total number of cell was counted.
The total number of cells was calculated by
Cells/ml = average count per square*dilution factor*104
Total cells =cell/ml*total volume of cells
To determine the drug toxicity, EC50 and LC50 can be calculated and dose
response curve was plotted.
CW1 7007 BMS Lab Report on Diabetes mellitus glp1 and its relevence to disease_4

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