Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5061—Muscle cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5067—Liver cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/507—Pancreatic cells

Definitions

• the present invention relates to the preparation of a novel insulin resistant cell based model system, to the use thereof, and to a method and device, respectively, for use in the model.
• Insulin resistance is frequently found in obese subjects and is an early hallmark in subjects prone to develop non-insulin-dependent diabetes (NIDDM). It can be defined as a diminution of the biological response to a given concentration of insulin.
• NIDDM non-insulin-dependent diabetes
• One major difficulty in attempts to study insulin resistance is that there is not yet any quantitative definition available. What is even more important is the fact that very little is known about pathogenesis of insulin resistance on molecular basis.
• a number of methods have been developed using cellular systems to study pre-diabetic or diabetic states.
• Schmitz-Peiffer, C, et al. (1999) J. Biol. Chem. 274, 24202-24210 discloses incubation of myoblasts with free fatty acids (FFA) of the concentration 0.2 to 2 mM to provide a model of lipid-induced skeletal muscle insulin resistance.
• FFA free fatty acids
• insulm resistance models often involves the measurement of glucose and fatty acid oxidation rates. Usually, this has required complicated apparatus, common experimental set-ups using cells in suspension which is not satisfactory as many of the studied cell types are of an adherent type.
• an object of the present invention to prepare a model for insulin resistance that overcomes the drawbacks of the prior art models.
• an excellent in vitro cellular model for insulin resistance in an animal may be induced by long term incubation (usually from about eight hours or longer) of cell cultures in a cell culture medium medium containing moderately elevated, compared with normo-physiological levels, concentrations of glucose and free fatty acid (FFA).
• FFA free fatty acid
• the present invention therefore provides a method of inducing insulin resistance to an animal (including human) cell culture in a cell culture medium, which method comprises incubating the cell culture in the presence of glucose and at least one fatty acid, preferably a long-chain fatty acid, wherein the concentration of glucose is in the range of about 5 to about 25 mM and the concentration of fatty acid is less than about 2 mM.
• the concentration of glucose is in the range of about 10 to 20 mM and the concentration of fatty acid is in the range of about 120 ⁇ M to about 2 mM.
• Preferred fatty acids are palmitic acid, oleic acid and linoleic acid. The most preferred fatty acid for use in the method is palmitic acid.
• the method of the invention may be applied to a variety of cells systems, including all cells affected in diabetes and obesity status.
• Exemplary cell systems are skeletal muscle cells, insulin secreting cells ( ⁇ -like cells), adipocytes and hepatocytes.
• the present invention provides the use of the method for drug/target related studies, including, for example, screening of insulin releasing, insulin sensitizing or insulm mimetic compounds, metabolic pathway analysis, differential display analysis, signaling pathway analysis etc.
• a third aspect of the invention relates to a method and device, respectively, for measuring carbohydrate and fatty acid oxidation rates by cultured cells in vitro, which method and device may be used with the model system prepared by the method of the invention as well as with other systems.
• Figure 1A is a schematic sectional view of an illustrative device for the determination of glucose and fatty acid oxidation rates.
• Figure IB is a schematic perspective view of the separate parts of a practical design of the device in Figure 1A.
• FIG. 1 is diagram showing the effects of increasing concentrations of glucose on insulin dependent glucose uptake.
• Figure 3 is a diagram showing the effect of increasing concentrations of palmitate in the presence of low glucose content (5.5 mM) on insulin stimulatable glucose uptake.
• Figure 4 is a diagram showing a comparison of glucose uptake rates under normal conditions versus insulin resistance induced conditions..
• Figure 5 is a diagram showing the effects of increasing concentration of palmitate on glucose oxidation rates.
• Figure 6 is a diagram showing the effects of increasing glucose concentrations on glucose oxidation rates, as well as the effect of the combination of different glucose concentrations with 480 ⁇ M palmitate on glucose oxidation rates.
• Figure 7 is a diagram showing an example of a practical application of established insulin resistant cell model in evaluation of effects of potential PPAR ligands on glucose uptake rates.
• the present invention is based on the fact that concentrations of glucose and circulating free fatty acids in blood from diabetic and obese patients are elevated.
• the invention resides in the provision of a cellular based model of insulin resistance obtained by incubation of cell cultures in media containing only moderately elevated concentrations of both glucose and fatty acid, such as palmitic acid, compared with normal physiological levels.
• glucose and fatty acid such as palmitic acid
• the model prepared according to the mvention permits a number of applications within the drug/target hunting area, such as metabolic pathway analysis, differential display analysis, signaling pathway analysis, as well as for screening of insulin releasers, insulin sensitizers, insulin mimetics, etc.
• Example 2 describes exclusively a skeletal muscle system
• the invention can, of course, be applied to other cellular systems, including all cells affected in diabetes and obesity states, such as e.g. insulin secreting cells, adipocytes and hepatocytes.
• One and each of the named cell types has its own specificity in terms of its specialized functions which in turn serve as a specific read out (insulin secretion, triglyceride synthesis, glucose production).
• the device comprises a cell culture flask, generally designated by reference numeral 1.
• a tube 2 having a plurality of holes or apertures 3 in the tubular wall and adapted to receive a rolled up (liquid-soaked) filter paper (not shown) in the apertured section thereof, such that the filter paper is in contact with the atmosphere within the flask through the apertures 3.
• the tube 2 has an end part 4 fitting through the flask opening and sealed by a septum 5.
• An aperture 6 made in the tube wall near the flask opening permits the needle of a syringe which has pierced the septum 5 to be inserted into the interior of the flask 1.
• the flask 1 contains a layer of adherent cells 7 and a culture medium 8.
• the device may be used for measuring the cellular oxidation rates of substances, or substrates, where one of the final products is carbon dioxide.
• a substrate labeled by a radioactive carbon isotope, such as ⁇ C is added to the flask containing adherent cells and culture medium.
• a filter paper soaked in a CO2-trapping agent, e.g. hyamine solution (hyamine is a strong base) is rolled up and placed in the tube 2, and after a pre-determined incubation time, the incubation is stopped by adding e.g.
• FIG. IB illustrates a practical design of the device in Fig. 1A. Corresponding parts are designated by the same reference numerals as in Fig. 1 A.
• the culture flask 1 is of standard type and has a tubular inlet part 10 with an opening 11 and an external thread 12.
• the support tube 2 for the filter paper which tube is a separate part designed to be inserted into the flask 1, has a fore part 13 slightly angled to a rear part 14 provided with a number of holes 3 and adapted to receive the rolled up hyamine-soaked filter paper (not shown).
• the fore end of the tube 2 is sealed, 15.
• the insertable tube 2 is arranged to be inserted through the flask opening 11 and kept in position by a screw cap 16 (here shown on the tube 2) engaging with the thread 12 of the inlet part 10 and acting against an o-ring (not shown) which is secured on the tube 2 and abuts the edge of the flask opening 11 so that the system is closed.
• Bovine insulin Dulbecco's Modified Eagle's medium (DMEM), Phosphate Buffered Saline (PBS), Foetal Calf Serum (FCS), Penicillin and Streptomycin (PEST) were bought from Gibco Laboratories. Tissue culture plates were purchased from Costar. Bovine Serum Albumin (BSA) and cytochalasin B were obtained from Sigma, USA. U-l ⁇ C-glucose, H-2-deoxy-glucose and U-l ⁇ C-palmitate were from Du Pont NEN, Medical Scandinavia, Sweden. Whatman no. 1 filter paper was from Kebo Lab., Sweden, and Hyamine hydroxide from ICN, USA.
• DMEM Dulbecco's Modified Eagle's medium
• PBS Phosphate Buffered Saline
• FCS Foetal Calf Serum
• PEST Penicillin and Streptomycin
• Tissue culture plates were purchased from Costar.
• Rat L6 myoblasts were grown on culture flasks in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS and 2% PEST. To initiate differentiation, the media of sub-confluent cell cultures were replaced with DMEM supplemented with 1% FCS and 0.3 ⁇ M insulin as described in Klip, A., et al. (1984) Am. J. Physiol. 247, E291-E296; and Walker, P. S., et al. (1989) J. Biol. Chem. 264, 6587-6595.
• DMEM Dulbecco's modified Eagle's medium
• Glucose uptake was measured as described by Hundal H.S., Bilan P.J., Tsakiridis T.,
• the principle of the glucose/FFA oxidation assay is based on the fact that one of the final products along metabolic pathways of these two substrates is carbon dioxide.
• the radioactivity of carbon dioxide trapped in a carbon dioxide trap is a direct measure of the metabolic activity in studied cells (Rodbell, M. (1964), J. Biol. Chem. 239, 375-380).
• the cells were cultivated until sub-confluence in T-25 Costar flasks. Prior to the experiment, the cells were deprived of serum for 6 hours in DMEM medium containing 5 mM glucose. 3 ml of medium supplemented with (U-l ⁇ Q-glucose or (U-l ⁇ C)- palmitic acid (0.2 ⁇ Ci/ml of each) were added to each flask.
• a filter paper (1.5 x 5.5 cm) soaked in hyamine solution was rolled up, blotted on a paper towel to remove excess of fluid, and placed carefully into the tube (2) of the device illustrated in Figures 1 A and IB and described above.
• the tube was mounted in the flasks, the screw caps (16) were tightened and cells were incubated for indicated time periods.
• the remaining cells were washed briefly with ice cold PBS, solubilized with 1 M KOH and the protein content was determined according to the Bradford method (Bradford, M. M. 1976, Anal. Biochem. 72, 248-254).
• the rate of substrate oxidation was obtained by correcting the observed number of disintegrations per minute for counting efficiency, milligram of protein in the culture flask, trapping interval, and a specific activity of the substrate at time zero using the following equation:
• M protein content of the cultured cell plate/flask (mg. prot.)
• Glucose oxidation rate was determined as follows. The radioactivity of a medium sample was measured (e.g. 100 ⁇ l gives approx. 40,000 dpm). Since the glucose concentration in medium was 5.5 mmol/1 the specific radioactivity was calculated to 400,000 d ⁇ m/5.5 ⁇ mol (72,727 dpm/ ⁇ mol).

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• 1999
  • 1999-12-30 SE SE9904849A patent/SE9904849D0/sv unknown
• 2000
  • 2000-12-27 AU AU27192/01A patent/AU2719201A/en not_active Abandoned
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