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Glucose Uptake Assay
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Objective

 

•    To find out the glucose uptake activity of the cells.
•    To find out the effect of plant extract and other products on glucose uptake activity.

 

Principle

 

Glucose uptake activity was analyzed by measuring the rate of uptake of radioactively tagged 2-deoxy glucose in differentiated 3T3 L1 cells. After starvation the cells were treated with insulin and other plant extracts. Then these ligands will bind to the receptors on the surface of the cells. These triggered the translocation of glucose transporters to the cell surface. Then we treat it with radioactive cocktail containing10µM 2-deoxy glucose and 0.25µCi of 2 deoxy-D-(3H) – glucose. So the radioactively tagged glucose will enter the cell along with the normal glucose. By measuring this uptake rate by using liquid scintillation counter help to analyze the glucose uptake activity and the effect of plant extract on the glucose uptake activity.

 

Diabetes mellitus

 

Diabetes mellitus is commonly called diabetes which is characterized by elevated level of glucose. The word “diabetes” is adapted from the Greek word “a siphon” which means a “passer through”.  Diabetes is a condition that affects all kind of people all over the world. This is a metabolic disorder that affects pancreas, a small gland just behind the stomach.
Pancreas is a dual function gland with both exocrine and endocrine activity. It is about five inches long. Many clusters of cells with endocrine activity are spread all over the pancreas called Islets of Langerhans. Islets are made of four main type of cells called α, β, δ and PPcells. Alpha cells produce glucagon which helps to increase the glucose level in blood. Beta cells of pancreas produce a hormone called insulin which helps to absorb the glucose level in the blood. Delta cells produce a hormone called somatostatin which regulates α and β cells. The PP cells secrete pancreatic polypeptides which help to regulate the pancreatic secretion activity.  
A healthy pancreas releases a regular supply of insulin into the blood supply. When blood glucose level rises pancreas will release insulin in response to that.  The glucose in the blood reaches the pancreas and released to the beta cells from the capillaries, the beta cells will produce insulin in response to that. This insulin enters the blood stream and travels to three main destinations- the muscle, fat and liver cells. The surface of these cells contains insulin receptors. When insulin binds to these receptors it acts as a key for opening the door for glucose by sending signals to the cell which allows the special glucose transport channels on to the cell surface. These channels enable glucose to enter into the cell and thus decrease the blood glucose level. Diabetes will finally lead to heart attack, stroke, blindness, kidney failure and nerve damage.
 

Diabetes mellitus is of different types. They are type 1 diabetes mellitus, type 2 diabetes mellitus and gestational diabetes.

 

Type 1 diabetes/ Insulin dependent diabetes:

 

Type 1 diabetes is not so common. Only 5-15% of diabetes is type 1. In a person with type 1 diabetes, the pancreas will produce little or no insulin. This is other ways called juvenile diabetes, because it mainly affects childrens and young adults. This type of diabetes is considered as an auto immune disease. In the patient with this disease the pancreatic beta cells are destroyed by their own immune cells. The exact reason for this type of diabetes is still unknown. So there is no production of insulin.
 

 

Type 2 diabetes/ Non insulin dependent diabetes:

 

The most prevalent form of diabetes is type 2. This is other ways called adult onset diabetes.85- 95% of diabetes is type 2 in nature. This type of diabetes is mainly occur due to life style, food habits and sometimes hereditary. Most of the time obese persons will develop this type of diabetes. Type 2 diabetes persons have impaired secretion of insulin from pancreas and insulin resistant peripheral cells (muscle, liver and adipose). So cells need more insulin to trigger the opening of glucose transporters and finally that leads to high level of glucose in blood. This can be controlled by proper medication and healthy life style.
 

 

Gestational diabetes:

 

This type of diabetes is appeared during pregnancy. It occurs in 5- 7% of pregnancy cases. This usually disappears after child birth, but increases the risk of type 2 diabetes in future. Gestational diabetes occurs when the pancreas is not able to produce enough insulin due to changes in the hormones.
 

 

Signaling pathway

 

When high levels of glucose are detected by the beta cells in pancreas, insulin is released by the cells into the blood stream. This insulin circulates in the blood and binds to the receptors embedded in the cell membrane of the muscle, liver, adipose and brain cells. Then the receptor became autophosphorylated by adding phosphate group on its intracellular domain as well as tyrosine residues of IRS1 (Insulin receptor substrate) protein. This activation will lead to activation of a signaling cascade. The activated receptor protein then adds the phosphate group to another protein called PI3 kinase (phosphatidylinositol 3 kinase) that pass the signal to the next step in the signal pathway. This PI3 kinase will phosphorylate the PIP2 and that will activate other downstream kinases and finally the PKB became activated and this PKB will facilitate the translocation of Glut 4 (Glucose transporter) to the plasma membrane. This will allow the transport of glucose into the cells.
 

 

Insulin Resistance

 

Insulin resistance is a condition in which the body cells become resistant to insulin. When glucose level stays elevated, the pancreas will keep on secreting insulin and the glucose will be utilized by the cells. Then the excess glucose will  be transformed into glycogen and fat and stored in muscle cells and adipose cells respectively. And finally the muscle and fat cells shut themselves from the insulin. This will lead to the condition called insulin resistance. The normal level of insulin is not able to activate the cells to take up the glucose. So they want more amount of insulin for sensitization of cells and thereby the transport of GLUT 4 transporters to the cell surface. This chronically elevated glucose will lead obesity and finally that lead to the condition called type 2 diabetes.

 

 

 Liquid Scintillation Counter

 

 Liquid scintillation counter is the most widely used instrument for detection and quantification of radioactivity. This instrument is usually not portable and highly sophisticated. Radioactive samples for counting are prepared by combining with a liquid scintillation cocktail. An ionizing particle is produced by the decay of radionuclide. Part of the kinetic energy of this ionizing particle is transferred to the ‘scintillator’ which converts this energy into light photons and these light photons are detected by the LS system. These photons are directed towards either of two photomultiplier tubes (PMT’s). The PMT’s activated by photon produce measurable electrical pulses. These pulses were analyzed and converted to digital form and stored in the appropriate channel of a multichannel analyzer, corresponding to the particle energy. This data accumulated in the multichannel analyzer is used to determine the rate of radioactivity or counts per minute (cpm). The cpm is the total number of pulses in the channels of the multi channel analyzer divided by the total time in minutes for obtaining the counts.  
 

 


 

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