Glucose uptake assay

GLUCOSE UPTAKE ASSAY

1. GLUCOSE UPTAKE ASSAY Sanju Kaladharan

2. Introduction  DM is a metabolic disorder characterized by abnormal carbohydrate, protein, fat metabolism caused by the complete or relative insufficiency of insulin secretion and/or insulin action.  Type-I: IDDM  Specific/complete loss of β-cell  Type-II: NIDDM  Associated with insulin resistance/obesity/hyperinsulinaemia

3. β -Cell mass β-cell growth Neogenesis /Rep l ic a tion/Hyp e r t roph y β-cell loss Ne c r o s i s / Apo p to s i s/Hypotrophy

4. Type-I: IDDM Type-1 Diabetes Mellitus Absolute lack of insulin β cell mass reduction Mechanisms for destruction of islet cells Genetic susceptibility Acute autoimmunity Environmental insult

5. Type-II: NIDDM Type 2 Diabetes Mellitus Insulin resistance Relative impairment in insulin secretion Mechanisms for development of NIDDM Defect at Insulin receptor level Defect at β-cell function Combination of both

6. 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.

7. Screening methods IDDM Chemically Induced DM Surgically Induced DM Miscellaneous Genetic model Hormone induced DM Insulin antibody induced DM DM induced by viral agent NI D DM Chemically Induced DM Normoglycemic Animal model Miscellaneous Genetic model Isolated pancreas of Rat [in vitro] In vitro assay of insulin on adipocyte Glucose uptake by isolated diaphragm from mice/rat Insulin receptor binding assay

8. Glucose uptake assay Using 3T3 L1 cells Insulin promotes glucose uptake, metabolism and storage in adipose tissue and skeletal muscle. Insulin stimulates phosphorylation ofinsulin receptor substrates (IRS) by kinase , which leads to activation of PI3 kinase , PKB and protein kinase C isoforms . Activated PKB translocate Glut4 to the cell surface and stimulate glucose transport in muscle and fat cells , whereas it phosphorylates and inhibits GSK-3 . Inhibitor of GSK-3 enhances insulin signalling thereby favouring glucose entry into the muscle cells and adipose tissue . Inactivation of GSK-3 in 3T3 L1 differentiated cells [ adipocytes ] stimulates glucose uptake which can be measured by incorporation of 2-deoxy- 14C-glucose and quantified by using scintillation counter .

9. Cell culture The cell line 3T3-L1 pre- adipocytes are cultured and made to differentiate The pre- adipocytes are then treated with 0.5 mM 3-isobutylmethylxanthine, 5 μg / mL insulin and 0.25 μM dexamethasone in 10% FBS containing DMEM for 2 days. Later, the cells are transferred to 10% FBS/DMEM containing only insulin and then to 10% FBS/DMEM without insulin for next 2 days. After ten days of differentiation , the cells are used for the experiments . Chinese hamster ovaric cells over expressing the human insulin receptor ( CHO-IR) cells are grown in Ham's F12 supplemented with 100 U/ mL penicillin; 10% fetal bovine serum; 2.5 μg / mL fungizone ; 0.5% G-418 and 100 mg/ml streptomycin in 5% CO2/humidified atmosphere at 37°C. Cells are passed two to three times a week. Confluent cells are used for the experiments .

10. Glucose uptake assay using 3T3- L1adipocytes 2-deoxy-D- [3H] glucose uptake of 3T3-L1 adipocyte is used to measure the glucose transport system38. 3T3-L1adipocyte cells cultured on 12 well microtitre plate are incubated in a transport solution containing 1 μ Ci 2-deoxy D-[3H] glucose (10 mCi / mmol ) and 0.1 mM 2- deoxy-Dglucose for 7 minutes. Uptake of glucoseis terminated by the addition of 50 mM glucose and 0.1MNaOH/ 0.1% PBS for disruption of cells. Radioactivity incorporated in the cells is determined using scintillation counter. Protein is used to standardize the glucose transport values

11. Glucose uptake by isolated diaphragm from mice & rat To study the effect of Insulin & Insulin like substance on muscle tissue To study glycogen synt h esis To study glucose transport To study glycogen synthase activity

12. 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.

13. 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.