Rat serum insulin levels were determined using a radioimmunoassay (RIA) kit (Novo Biolabs, Bagsvaerd, Denmark). The assay involved incubation of sample or insulin standard with anti-insulin antibody, such that the amount of unbound antibody was inversely proportional to the initial insulin concentration. ^^®l- insulin was then introduced to the assay and was bound by the free antibody from the first stage. Thus, the amount of ^^^l-insulin-antibody complex was inversely proportional to the initial sample insulin concentration while the amount of free ^^^l-insulin was directly proportional. Free ^^®l-insulin was then separated from the bound ^^^l-insulin by precipitation of the insulin-antibody complex with ethanol.
Buffer solutions used in the assay were FAM , a 0 .0 4 M phosphate buffer, p H 7 .4 , containing 0.1 % (w /v) BSA and 0 .0 2 4 % (w /v) sodium merthiolate; and NaFAM containing 0 .6 % (w /v) NaCI and 6 % (w /v) BSA in FAM. Serum samples
were obtained by centrifuging whole blood in heparinized microfuge tubes at 10OOOxg for one minute. Serum was removed, placed into clean tubes, capped and stored at 4 0 ° C until assayed.
Rat insulin standards and serum samples were diluted as required in NaFAM. Insulin standard (50//I, 0-4ng/m l) or unknown sample was aliquotted into LP4 assay tubes on ice and 50//I of antiserum (1 :1 8 0 0 0 dilution of anti-porcine insulin guinea pig serum) added to each tube. The tubes were mixed and incubated for 2 0 -2 4 hours at 4 ° C . ^^^l-labelled porcine insulin (50/yl) was then added to the tubes followed by mixing and incubation for 4 hours at 4 ° C . To stop the reaction and precipitate out the bound ^^®l-insulin, 0.8m l of 9 5 % ethanol was added to the assay tubes, still maintained at 4 ° C , which were then vortexed and the suspension centrifuged at 2 0 0 0 x g for 10 minutes. The supernatant was transferred to a clean tube and radioactivity (^^®l) counted for 10 minutes on an LKB riagamma counter. The insulin concentrations of unknown samples were determined from a standard curve.
3 .7 Centrifugal cytochalasin B binding assay
Cytochalasin B is a fungal metabolite which binds to the mammalian glucose transporters in a D-glucose but not L-glucose inhibitable manner. Cytochalasin B also binds to actin and other cellular and membrane proteins; this binding is inhibitable by dihydrocytochalasin B and cytochalasin E, thus in binding assays an excess of cytochalasin E was included to inhibit binding to these other proteins. Binding measurements were made in the presence and absence of a saturating concentration (500m M ) of D-glucose to correct for non-specific binding of cytochalasin B to the membranes. The centrifugal cytochalasin B binding assay is used in situations where the number of binding sites is low,
and separates bound cytochalasin B from free ligand by pelleting the membrane bound cytochalasin B. To calculate trapped unbound ligand in the pellet, [^^C]- urea was included in the incubation mixture.
Cytochalasin B was dried down over P2O5 in vacuo overnight, and dissolved
in ethanol to a final concentration of 200/yM. The molarity was checked by measurement of the absorbance at 2 0 0 nm, given the molar extinction
coefficient of cytochalasin B of 2 5 0 0 0 M ‘^cm \ A 10//M [^H]-cytochalasin B stock solution was prepared by drying down under a stream of nitrogen 1 0 nmol
of unlabelled cytochalasin B (50//I), as prepared above, and lOnmol of [^H]- cytochalasin B (1 3.5Ci/m m ol; NEN, DuPont, W ilmington, U .S .A .) in a glass tube. The cytochalasin B was redissolved in 40//I ethanol and diluted to 10/yM with 1 .96m l millipore filtered, milli-Q HgO. The concentration of the solution was checked by measurement of the absorbance at 21 Onm and the solution scanned from 2 0 0 -3 0 0 n m to check that the curve was smooth and peak free. A 1:1 mix of this [^H]-cytochalasin B solution with [^"^C]-urea (32//C i/m l, NEN) in 2x concentrated 20m M Tris-HCI, Im M EDTA, 2 5 5m M sucrose, p H 7 .4 , (TES) was prepared for dilution into the binding assay. This cytochalasin B:urea mix was diluted in TES to give 4 concentrations of cytochalasin B; 0 .2 5 //M , 0 .5 //M , 1/yM and 2//M , thus giving final concentrations in the assay of 4 1 .6 7 n M , 8 3 .3 3 n M , 1 6 6 .6 7 n M and 3 3 3 .3 3 n M cytochalasin B to construct a Scatchard plot (Scatchard 1 9 4 9 ). Binding was assayed in duplicate in the presence or absence of 50 0m M D-glucose at each concentration, in 0.2m l cellulose propionate micro ultracentrifuge tubes (Beckman).
A 10//I aliquot of the appropriate cytochalasin B concentration was placed into each assay tube on a bed of ice. Membranes in TES to be assayed were incubated with 4 .6 3 //M cytochalasin E and in the presence or absence of 0 .5 9 8 M D-glucose in TES, prior to pipetting 50//I of the appropriate suspension into each of the assay tubes. Tubes were vortexed and incubated on ice for 60 minutes, after which the membranes were pelleted by centrifugation at
IBSOOOxg for 1 hour at 4 ° C , in a Beckman T L -1 0 0 bench-top ultracentrifuge. A 25/yl sample was taken from the supernatant of each tube and transferred to a mini scintillation vial containing 4ml of scintillation fluid and counted for dual isotopes (^^C/^H) on a Beckman scintillation counter.
The remainder of the supernatant was drawn off and discarded, and 50//I 0 .2 M NaOH added to the pellet, which was vortexed and incubated covered at 6 0 ° C , for 1 hour. After incubation, tubes were vortexed again and placed into mini scintillation vials containing 4ml scintillation fluid, mixed vigorously and counted on a Beckman scintillation counter for dual label. Blank samples containing only [^"^Cl-urea or [^H]-cytochalasin B were also prepared for calculation of isotope crossover and specific activity. Data was calculated by Scatchard analysis and dissociation constants and binding site concentrations determined by radial subtraction of the curve obtained in the presence of D- glucose from that obtained in its absence, by use of the LINEAR computer program.