A. D. Landman and N. A. M. Eskin University of Manitoba Winnipeg R3T 2N2 Canada
II
Insulin Mediated '4~-~lucose hcorporation into Adipose Tissue An undergraduate biochemistry experiment
Hormones are substances of vital importance, controlling the physiological and hiochemical activity of many tissues. The functions of many hormones are well estahlished and while they are included a s a n integral part of the regular cumculum in undergraduate courses, lahoratow dealing with the endocrine system are . ex~erirnents . usually not included i n t h e laboratory periods~accompanying these courses (13). Such experiments are generally c6ns;dered tw expensive or complex for use in lahoratories a t the undergraduate level. We describe here a simple experiment demonstrating the action of the hormone insulin in uitro. This hormone is known to enhance the uptake of glucose by adipose tissue (4), thereby facilitating the incorporation of glucose into triglycerides (5). The experiment described does not require expensive materials or the use of equipment not normally found in chemistry and biochemistry departments. Equipment and Materials
The experimental procedure requires: (a) columns 10 X 300 mm in size, provided with a coarse fritted disk and Teflon stopcock, product of Coming Glass Works, Coming N.Y. (h) I-ml volume Potter-Elvehjem homogenizers, available from Arthur H. Thomas, Philadelphia, Pa. (c) insulin, which can be ohtained from most pharmaceutical and hiochemical manufacturers. The specific activity required in this experiment is 40 unitsfml. (d) 14C-glucose, 50 mCi/mmole, which can he purchased from New England Nuclear Corporation. Since the efficiency of carbon-14 counting is -8070, most p emission counters are sensitive enough and suitable for this experiment. Experimental Procedure
Starved overnight prior to their sacrifice, 250-300-g rats were snaesthetized and the epididymal adipose tissue removed. The white soft tissue covering the lower part of the peritoneal cavity was pulled and separated from the testes with which it is connected. It was washed by rinsing in 0.85% NaCl and then sliced into thin pieces with a slicer or a sharp razor blade. A rat of this size provides at least 5 g of tissue. Slices of approximately 200 mg were placed in three test tubes, followed by the addition of 2 ml Krebs-Henseleit Ringer bicarbonate buffer (KHRB) (0.118 M NaCI, 0.005 M KCI, 0.003 M CaCI2, 0.001 M KHzPO+,0.001 M MgSO,, 0.025 M NaHC03, pH 7.4), containing 2.5 mmole, 1gCi, 14Cglucose, and 50 mg/ml hovine serum albumin. To one tube was added 0.2 units of insulin (0.2 ml of 1 unit/ml solution prepared in KHRB) while to the other samples 0.2 ml of KHRB was added as a control. One of the two samples not containing insulin was placed immediately in ice, kept at 0-4'C and used as a time zem control. The other tubes were incubated in a bath-shaker at 37°C for 1 hr. Following the incubation period the KHRB medium was siphoned off with a Pasteur pipet and the slices washed with fresh KHRB at 4°C. The slices were then homogenized at high speed in 1 ml KHRB buffer using a motor driven Potter-Elvejhem homogenizer. Lipids were extracted from the homogenates by two successive treatments with 5 ml chlomfom-methanol ( 2 1 "1"). The solvent fractions fmm each sample were combined and evaporated to dryness at 40-45°C. preferably in vacuum. The residues were dissolved in 1.0 ml hexane, and 0.1 ml of this solution was
Fractionation of Fat Com~onentson a Florisil Column not apffified triglycerides diglyeerides monoglycerides
hexane 15% ether in hexanexan 50% ether in herane 2% methanol in ether 4% acetic acid in ether
FFA
115 1107 217 328 460
added to 10 ml scintillation solution (0.25% 2,5-diphenyloxazole. 0.009% p-bis(2(5-phenyloxazayl))-hen~ene,and 31% ethanol in toluene) and counted in a liquid scintillation spectrometer. The counts were converted to dpm and P C ~ units, and the yield of incorporation calculated. Ten grams of deactivated florisil (florisil obtained from Fisher Scientific Co. was deactivated by 24-hr treatment of 100 g with 7 ml water) was mixed with 50 ml hexane and poured into a column in portions. In order to obtain homogeneity within the gel, the upper layer of the settled gel was stirred with a small amount of hexane before each addition of a new portion. During all steps involved with the packing of the column and fractionation of the sample, care was taken so that the gel would not dry out. Hexane solution, 0.9 ml, ohtained from the incubation carried out in the presence of insulin, was applied on the column with a Pasteur pipet. The column was then developed by eluting sequentially with 50 ml each of: 15%ether in hexane, 50% ether in hexane, 2% methanol in ether, and 4% acetic acid in ether (6). Each of the four fractions was collected and the solvents evaporated. The residues were dissolved in 0.5 ml chloroform-methanol(21. . . v ,l v ).. and then transferred quantitatively into a scintillation vial containing 10 ml scintillation solution and counted. The table shows the count distribution of a typical fractionation. Discussion and Conclusion
Insulin is involved in several physiological mechanisms. One of these, the intercellular transport of glucose within the adipose tissue is illustrated here in conjunction with the transformation of the substrate into "fat" components. The glucose whose entry through the intercellular membrane is facilitated by the hormone, participates in the glycolytic pathway and as a result it is cleaved into the appropriate tricarhon molecul~s.Subsequently these compounds are converted to glycerol into which free fatty acids will be incorporated to form mono-, di-, and triglycerides. Startine with uniformlv or eenerallv labeled elucose. a suhsta&ial part of the"1ahel"within"the triglyceride kill ultimatelv be found on the elvcerol. However. some radioactivity will get into the fa& acid moiety since the ultimate degradation product of glucose is acetyl-CoA, and this compound is used as a building block for the synthesis of fatty acids. In the first part of the exoerimental nrocedure the ability of insulk to facilitate iransport is demonstrated. In the sample from which insulin was omitted. the glucose could he metabolized only where it is acce&hle'io the cellular matrix through membranes damaged during preparation of the tissue. As a result the amount of radioactivity incorporated into this sample will be only slightly Volume 52, Number 7, July 1975 / 479
higher than the time zero sample. The tissue slices suspended in the presence of insulin give substantially higher counts than the two controls. Preparation of KHRB and the appropriate dilutions of glucose prior to the lab period will leave the incubation as the time limiting step in the first experimental part. This part demonstrating the hormonal effect per se, can be finished within three hours. Further information can he derived from the procedure when the nature and composition of the radioactive products are analyzed. This is done in the next part. Since the successive elutions on the column are quite fast, the time limiting step here is the evaporation of the solvents. However, an appropriate evaporation technique, preferably a
480 I Journal of Chemical Education
combination of vacuum and heat, will enable completion of this lab period within an additional three hours. The procedure as a whole provides an overall picture of the involvement of the hormone in central biochemical pathways and with some other metabolic processes. Lierature Cned (11 Pritham, G. H.,"Anderaon's Labamtory Experiment8 in Biochemistry: The C. V. Mmby Co.. St. Louis. 1968. 121 Daniel, L. J., and Leslie. N. A,, "Laboratory Experiments in Biochemistry: AeademicP~ssInc.,New York. I%& 131 h n d i n a , G.. "Experimenfal Methods in Modem Biahemiatry." W. B. Saundera ""mn.n" ...y-..,, ,971 141 Bohrons. 0.K.,and Grinnan, E. L., in '"Annual Review of Biochemistry." (Edkor: Snell, E. E.I. 1969.Vol. 38, p. 96. 151 Hrmdek, S.,Lipids. 7.573(19721. 161 Carroll. K . K . . J . LipldRes., 2, 135 119611.
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