METHOD TO MEASURE THE METABOLIC RATE OR RATE OF GLUCOSE CONSUMPTION OF CELLS OR TISSUES WITH HIGH SPATIOTEMPORAL RESOLUTION USING A GLUCOSE NANOSENSOR

• US 20130111611A1
• Filed: 06/30/2010
• Published: 05/02/2013
• Est. Priority Date: 06/30/2010
• Status: Abandoned Application

• Current Assignee: Carnegie Institution of Washington, Centro De Estudios Clentificos De Valdivia
• Original Assignee: Carnegie Institution of Washington, Centro De Estudios Clentificos De Valdivia
• Inventors: Barros Almedo, Luis Felipe, Bittner Hofmann, Carla Ximena
• Application Number:
  US13/807,150
• Publication Number:
  US 20130111611A1
• Time in Patent Office: Days
• US Class Current: 800/3
• CPC Class Codes: C12Q 1/025 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics antimicrobial activity C12Q1/18
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose

Abstract

A method to measure the metabolic rate or rate of glucose consumption of cells or tissues with high spatiotemporal resolution using a glucose nanosensor is disclosed. The method generally includes providing single cells or cell populations, cells in suspension or adherent, in a cell culture, a tissue culture, a mixed cell culture, a tissue explant, or in animal tissues in vivo for the measurement of glucose metabolic rate; expressing a glucose sensor in individual cells; calibrating the sensor in controlled conditions; disrupting the steady-state of glucose entering the cell; recording the output from the sensor and calculating the corresponding glucose concentration at different times; and determining the glucose metabolic rate. The method can be applied for the screening of molecules with pharmacological potential, determination of glucose rate of cancerous cells, tissue physiology and biochemistry research.

Claims

1. A method for the measurement of glucose metabolic rate with high temporal resolution, comprising the steps of:

a. providing single cells or cell populations, cells in suspension or adherent, in a cell culture, a tissue culture, a mixed cell culture, a tissue explant, or in animal tissues in vivo for the measurement of glucose metabolic rate;

b. expressing a glucose sensor in individual cells;

c. calibrating the sensor in controlled conditions;

d. disrupting the steady-state of glucose entering the cell;

e. recording the output from the sensor and calculating the corresponding glucose concentration at different times; and

f. determining the glucose metabolic rate.

2. The method of claim 1, wherein the glucose sensor of step (b) is a fluorescent sensor.

3. The method of claim 2, wherein the glucose sensor is FLIPglu600μ

Δ

11.

4. The method of claim 1, wherein the calibrating of step (c) is performed by pre-treating the cells for 30 minutes with 0.5 mM iodoacetic acid to block glycolysis.

5. The method of claim 4, further comprising:

exposing the cells to increasing concentrations of glucose and monitoring a signal given by the sensor; and

plotting the sensor signal versus glucose concentration to estimate saturation parameters for each different cell type.

6. The method of claim 5, further comprising:

measuring the signal transiently in cells in the absence of glucose to form a zero reading; and

transforming the sensor signal with the zero reading and saturation parameters into glucose concentration.

7. The method of claim 1, wherein the disruption of the glucose steady-state is achieved by lowering extracellular glucose to a concentration lower than the intracellular concentration during the initial steady-state.

8. The method of claim 1, wherein the disruption of the glucose steady-state is achieved by adding a glucose transporter inhibitor to the extracellular space.

9. The method of claim 8, wherein the glucose transporter inhibitor comprises cytochalasin B, phloretin, genistein, parachloromercurybenzoate, or anti-GLUT antisera.

10. The method of claim 8, wherein the glucose transporter inhibitor is cytochalasin B and is added to the extracellular space in a final concentration between 5 μ

M and 20 μ

M.

11. The method of claim 1, wherein the output of the glucose sensor is recorded at intervals of time from 100 ms to 1 minute.

12. The method of claim 1, wherein determining the glucose metabolic rate comprises fitting a monoexponential function to the time course of decay and computing the instantaneous slope.

13. A method for the determination of cancer staging, comprising determining the glucose metabolic rate of a sample of cancerous tissue according to the method of claim 1.

14. The method of claim 13, wherein the cancerous tissue is breast cancer, bladder cancer, colon cancer, glioblastoma, lung cancer, hepatocellular carcinoma, gastric cancer, melanoma, thyroid cancer, endometrial cancer, kidney cancer, cervix cancer, pancreatic cancer, esophagus cancer, prostate cancer, brain cancer, ovary cancer, small cell lung cancer, non small cell lung cancer, head and neck cancer, mesothelioma, sarcoma, pediatric malignancies, cholangiocarcinoma.

15. A method for screening potential drugs, comprising determining the glucose metabolic rate of a model cell culture exposed to the potential drug according to the method of claim 1.

16. The method of claim 15, wherein the model cell culture is a culture of 3T3-L1 fibroblast and adipocytes, C2C12 myoblasts and myocytes.

17. The method of claim 15, wherein the model cell culture is a culture of HeLa cells, Neuro 2A, Caco2, C6, A549, MCF7, PC-3, and AGS.