Glucose uptake assay

Glucose uptake assay

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  GLUCOSE UPTAKE ASSAY SanjuKaladharan
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  Introduction DM is ametabolic 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
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  β-Cell mass β-cell growth Neogenesis /Replication/Hypertroph y β-cellloss Necrosis/Apoptosi s/Hypotrophy
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  Type-I: IDDM Type-1 DiabetesMellitus • Absolute lack of insulin • β cell mass reduction Mechanisms for destruction of islet cells • Genetic susceptibility • Acute autoimmunity • Environmental insult
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  Type-II: NIDDM Type 2Diabetes 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
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  Principle • Glucose uptakeactivity 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.
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  Screening methods IDDM Chemically InducedDM Surgically Induced DM Miscellaneous Genetic model Hormone induced DM Insulin antibody induced DM DM induced by viral agent NIDDM • 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
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  Glucose uptake assayUsing 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.
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  Cell culture • Thecell 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.
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  Glucose uptake assayusing 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 μCi 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
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  Glucose uptake byisolated diaphragm from mice &rat To study the effect of Insulin & Insulin like substance on muscle tissue To study glycogen synthesis To study glucose transport To study glycogen synthase activity
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  • Glucose uptakeactivity 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.
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  • Then wetreat 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.