Glucose uptake is the key to understanding health and disease. As glucose plays a pivotal role in metabolic and structural demands in the human body, as well as playing a vital role in regulation. The extracellular measurement of glucose allows for the development of high throughput assays for kinetics of glucose uptake in cells, tissues, and organs of different genetic background in response to nutrients or drugs.
This assay would be a valuable tool for the discovery of therapeutics and nutrition for diabetes, cancer, degenerative diseases of the central nervous system, obesity, and all the diseases of glucose and metabolism. The assay needs an adjustment of time for starvation and stimulation that should be based on metabolic specifics for different tissues and cell cultures. Culture 3T3-L1 mouse fibroblasts by plating 10 to the 6 cells, diluted in 20 milliliters of medium 1 in 296 well plates.
Plate 100 microliters of cell suspension per well, ensuring to maintain homogeneity of this suspension by mixing. Grow cells for 48 hours in a 37 degree Celsius incubator without changing media until about 70 to 80%confluency. Maintain cells confluent and fasted by culturing them in the same medium for 48 hours.
Vary the incubation time from 24 to 72 hours, depending on the desired fasting catabolic state. Decant media from cells in the 96 well plates. Absorb the remaining fluid using sterile paper towels.
Rinse the 96 well plate with 100 microliters of PBS per well, and absorb the remaining fluid with sterile paper towels. Add 100 microliters of glucose free DMEM per well, and incubate for 40 minutes. Adjust the incubation time depending on cell type stimuli, and desired level of fasting.
Use eight replicates for each simulation condition. Therefore, prepare one milliliter for each simulation condition. Use seven simulation conditions without insulin.
FD glucose of 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter, and 0 milligram per milliliter in 196 well plate. Use seven stimulation conditions with insulin. FD glucose of 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter, and 0 microgram per milliliter in another 96 well plate.
To prepare stimulation conditions, dilute one microliter of five milligrams per milliliter FD glucose working solution, and 999 microliters of glucose free DMEM to obtain one milliliter of working stock solution. Using this working stock solution, prepare 1 to 2000, 1 to 5, 000, 1 to 10, 000, 1 to 25, 000, 1 to 50, 000, and 1 to 100, 000 delusions of FD glucose to obtain 2.5 micrograms per milliliter, 1 microgram per milliliter, 0.5 microgram per milliliter, 0.2 microgram per milliliter, 0.1 microgram per milliliter, 0.05 microgram per milliliter in two milliliter tubes immediately before experiments in a biosafety cabinet without lights. Add one microliter of insulin to each of these conditions.
After 40 minutes of incubation, decant the glucose free DMEM from each well in 96 well plates, and absorb the remaining fluid with sterile paper towels. Add 100 microliters each from the various treatment conditions described before, to wells along one column. And 100 microliters from the same treatment condition to the wells along the row in both 96 wall plates.
Label the plates that utilized the conditions, with and without insulin. Add glucose free DMEM alone to the control wells. Incubate the plate for 40 minutes in the cell culture incubator in the dark.
After the cells have been stimulated for 40 minutes. Transfer the stimulation media from both the plates into new 96 well plates, maintaining the same experimental layout. Wash the cells with PBS.
Remove PBS and decant any remaining solution on sterile paper towels. Add a protease inhibitor to the radio immunoprecipitation assay buffer to protect proteins. Place plates containing radio immunoprecipitation assay in a shaker for 30 minutes.
Using a microplate reader, measure fluorescence at excitation and emission wavelengths at 485 and 535 nanometers respectively. First in the medium containing extracellular FD glucose, and then the plate with radioimmunoprecipitation assay lized cells at the end of 30 minutes incubation. Perform BCA protein assay according to manufacturer's instructions.
Quantify the protein concentrations in each well containing radio immunoprecipitation assay lized cells. Use 10 microliters of the radio immunoprecipitation assay homogenate, and measure protein concentrations, and triplicate to increase the accuracy of measurements in 96 well plates. Quantify protein based on protein standards analyzed together with samples on each 96 well plate.
Incubate the harvested tissues or organs in a six well plate containing PBS for one minute. Handle each tissue or organ in a separate well. After one minute, place each tissue on a sterile paper towel to absorb PBS.
Transfer tissues or organs in a separate six wall plate containing 4, 000 microliters of glucose free DMEM, and incubate for two minutes. After two minutes, remove and transfer the tissues into wells of a six well plate containing 0.29 Millimolar FD glucose working solution. Incubate the six well plates containing tissues in FD glucose working solution at 37 degrees Celsius.
Collect 100 microliters of FD glucose working solution from each well after 0, 10, 20, 30, 40, 60, 90, and 120 minutes of incubation, and transfer collected 100 microliters of FD glucose working solution into 96 well plates to analyze the kinetics of extracellular FD glucose depletion. Shake before and after collection. Measure the fluorescence at excitation in emission wavelengths at 485 and 535 nanometer respectively using a microplate reader.
Dose dependency in the intracellular uptake of FD glucose was significantly increased in the presence of insulin. The insulin stimulation led to significantly decreased extracellular FD glucose levels compared to the samples without insulin stimulation. Extracellular depletion of FD glucose can measure a change in glucose uptake with comparable accuracy as intracellular FD glucose uptake.
The extracellular FD glucose was not depleted in non stimulated control visceral fat during 120 minutes of incubation. In contrast, pretreatment of mice with insulin or AAC2 before the dissection, led to significant depletion of extracellular FD glucose within a time interval of 30 minutes, and 60 minutes respectively. Extracellular FD glucose was not depleted in insulin stimulated liver explants, compared with non stimulated liver explants.
Extracellular FD glucose was significantly decreased in the time dependent manner, and liver explants pretreated with AAC2.22%depletion of glucose was observed in the extracellular medium containing the non-treated brain after 60 minutes of incubation. The medium incubated with brains from insulin treated mice have shown a 5%linear decrease in FD glucose. AAC2 stimulated brains led to a profound rapid decrease to 67.4%of the extracellular FD glucose during the first 20 minutes.
Substance involving washing are especially important for removing traces of glucose, blood, or serum within medium and, or organs, as it can interfere with the fluorescence reading of these mediums. After high throughput identification of compounds of metabolites in genes with glycemic properties, as well as the optimal conditions for their action in vivo experiments, could be validated with labeled FD glucose and PET scanning to confirm glucose accumulation in specific organs. This technique for discovery of glycemic action is both beneficial in deletion of numerous metabolites, therapeutics or their combinations, and highlights their actions in different organs.
