Chronic propranolol treatment causes desensitization of the steroidogenic response in testicular interstitial cells but does not alter protein kinase C.

We investigated effects of chronic propranolol treatment on the secretory response of rat testicular interstitial cells (testosterone secretion) to subsequent in vitro stimulation with activators of protein kinase-C (PK-C) (L-propranolol, phorbol 12, 13-dibutyrate (PDBu), LHRH) or activators of protein kinase A (PK-A), (hCG or dibutyryl cAMP (dbcAMP)). We determined [3H]PDBu binding and PK-C activity in these cells. Treatment of rats with propranolol (Inderal 500 mg/L of water for 5 weeks) reduced by 48%, 50% and 29% the L-propranolol-, LHRH- or PDBu-induced testosterone secretion, respectively, when compared to cells from controls. This desensitization in testosterone secretion in vitro was also present when the testicular interstitial cells were stimulated with hCG or dbcAMP (secretion decreased by 65%/57%, respectively, when compared to cells from control rats). Challenging the cells originated from rats that received propranolol chronically with the addition in vitro of propranolol resulted in an additional reduction of the hCG/dbcAMP-stimulated testosterone secretion. Chronic propranolol-induced desensitization was not associated with a loss in [3H]PDBu binding or a decrease in PK-C activity. Chronic propranolol-induced desensitization can be uncoupled from down-regulation of protein kinase C. The effector responsible for the desensitization could be distal to the protein kinase C and protein kinase A.

ATP inhibits insulin-degrading enzyme activity.

We studied the ability of ATP to inhibit in vitro the degrading activity of insulin-degrading enzyme. The enzyme was purified from rat skeletal muscle by successive chromatographic steps. The last purification step showed two bands at 110 and 60 kDa in polyacrylamide gel. The enzyme was characterized by its insulin degradation activity, the substrate competition of unlabeled to labeled insulin, the profile of enzyme inhibitors, and the recognition by a specific antibody. One to 5 mM ATP induced a dose-dependent inhibition of insulin degradation (determined by trichloroacetic acid precipitation and insulin antibody binding). Inhibition by 3 mM adenosine 5'-diphosphate, adenosine 5'-monophosphate, guanosine 5'-triphosphate, pyrophosphate, beta-gamma-methyleneadenosine 5'-triphosphate, adenosine 5'-O-(3 thiotriphosphate), and dibutiryl cyclic adenosine 5'-monophosphate was 74%, 4%, 38%, 46%, 65%, 36%, and 0%, respectively, of that produced by 3 mM ATP. Kinetic analysis of ATP inhibition suggested an allosteric effect as the plot of 1/v (insulin degradation) versus ATP concentration was not linear and the Hill coefficient was more than 1 (1.51 and 2.44). The binding constant for allosteric inhibition was KiT = 1.5 x 10(-7) M showing a decrease of enzyme affinity induced by ATP. We conclude that ATP has an inhibitory effect on the insulin degradation activity of the enzyme.

Propranolol stimulates histone phosphorylation by a putative PK-C in partially purified homogenate of rat testicular interstitial cells. A possible mechanism for increased testosterone secretion by propranolol.

The beta-adrenoceptor blocker propranolol stimulated testosterone secretion by rat testicular interstitial cells (Leydig cell-enriched preparation) in vitro at concentrations ranging from 10(-5) M to 10(-4) M. Treatment of these cells with H7 (20 microM), an inhibitor of protein kinase C, reduced the stimulatory effect of L-propranolol on testosterone secretion by about 5-fold. At concentrations ranging from 31.25 microM to 1000 microM, L-propranolol reduced [3H]phorbol 12,13-dibutyrate binding (IC50 = 75 microM) to rat testicular interstitial cells. At similar concentrations, L-propranolol displaced the binding of [3H]phorbol 12,13-dibutyrate to the homogenate of these cells by only 5%. These findings suggest that the effect of L-propranolol on [3H]phorbol 12,13-dibutyrate binding could be indirect, possibly by increasing the concentration of a chemical mediator interacting with the regulatory domain of protein kinase C. At even lower concentrations (10(-9) M to 10(-7) M), propranolol added directly to the reaction mixture with protein kinase C partially purified from rat testicular interstitial cells increases the phosphorylation of histone. This phosphorylation was comparable to that obtained with (25 microg/ml) phosphatidylserine. The D- and L-stereoisomers of propranolol were equally active. A complete reversal of this propranolol effect on histone phosphorylation was achieved with (20 microM) H-7. In the absence of Ca2+, propranolol was not able to phosphorylate the histone. Taken together, these results suggest that protein kinase C could be the putative kinase involved in this reaction and that its activation by propranolol may be due to interaction of the drug with the regulatory domain of the enzyme at a site differing from the site of interaction with phorbol 12,13-dibutyrate. The ability of propranolol to activate the putative protein kinase C could be related to its stimulatory effect on testosterone secretion by Leydig cells.

Propranolol stimulates testicular interstitial fluid formation and testosterone secretion in rats.

We investigated the effect of intratesticularly injected propranolol on testicular interstitial fluid (TIF) formation and on testosterone levels in the TIF of intact adult male Wistar rats (4-9 rats per group). dl-propranolol at doses of 0.6, 1.2, or 6.0 mg/kg was injected into the left (L) testis whereas the right (R) testis (control testis) received vehicle. dl-propranolol (6.0 mg/kg) caused a significant increase in both TIF volume (329%) and TIF levels of testosterone (257%) in the L testis but not in the R (control) testis 3 h post-injection. In rats treated simultaneously with human chorionic gonadotropin (hCG, 5 IU/rat, sc) the same dose or propranolol (6.0 mg/kg) significantly increased the stimulatory effect of hCG on testosterone secretion by 1.8-fold, but hCG did not modify the stimulatory effect of propranolol on TIF volume. These results demonstrate a direct stimulatory effect of propranolol on TIF volume and testosterone secretion, both under basal and hCG-stimulated conditions.

Propranolol stimulates testicular interstitial fluid formation and testosterone secretion in rats

We investigated the effect of intratesticularly injected propranolol on testicular interstitial fluid (TIF) formation and on testosterone levels in the TIF of intact adult male Wistar rats (4-9 rats per group). D1-propranolol at doses of 0.6, 1.2 or 6.0 mg/kg was injected into the left (L) testis whereas the right (R) testis (control testis) received vehicle. d1-propranolol (6.0 mg/kg) caused a significant increase in both TIF volume (329 per cent) and TIF levels of testosterone (257 per cent) in the L testis but not in the R (control) testis 3 h post-injection. In rats treated simultaneously with human chorionic gonadotropin (hCG, 5 IU/rat, sc) the same dose of propranolol (6.0 mg/kg) significantly increased the stimulatory effect of hCG on testosterone secretion by 1.8-fold, but hCG did not modify the stimulatory effect of propranolol on TIF volume. These results demonstrate a direct stimulatory effect of propranolol on TIF volume and testosterone secretion, both under basal and hCG-stimulated conditions.

Inhibitory action of in vitro ethanol and acetaldehyde exposure on LHRH-and phorbol ester-stimulated testosterone secretion by rat testicular interstitial cells.

Ethanol and acetaldehyde have been shown to inhibit testicular steroidogenesis. However the mechanism(s) of signal transduction involved in their action is still unclear. We examined the possible involvement of phospholipid-sensitive, calcium-dependent protein kinase (protein Kinase C, PK-C) in the intracellular mechanism of action of ethanol and acetaldehyde by stimulating testosterone production in rat testicular interstitial cells with LHRH and the phorbol ester PDBu, both of which activate PK-C at receptor (LHRH) and post-receptor (PDBu) sites. Ethanol (2000 mg %) inhibited 10(-7) M LHRH and 200 nM PDBu-stimulated testosterone production by 81 +/- 4.7% and 60 +/- 20.4%, respectively. Acetaldehyde (20 mg %) reduced the amount of testosterone produced by 10(-7) M LHRH and 200 nM PDBu by 59.4 +/- 1.2% and 52.5 +/- 5.4% respectively. Basal testosterone levels were unaffected by ethanol and reduced by acetaldehyde. However, the functional test of cell viability by preincubating cells with these doses of ethanol and acetaldehyde did not decrease their ability to respond appropriately to subsequent stimulation with LHRH, demonstrating that cell viability was unaffected by incubation with these drugs. The data presented here suggest that direct ethanol and acetaldehyde exposure results in a reduced ability of the testicular interstitial cells to respond to stimulation of PK-C pathway.

Inhibitory action of in vitro ethanol and acetaldehyde exposure on LHRH-and phorbol ester-stimulated testosterone secretion by rat testicular interstitial cells.

Ethanol and acetaldehyde have been shown to inhibit testicular steroidogenesis. However the mechanism(s) of signal transduction involved in their action is still unclear. We examined the possible involvement of phospholipid-sensitive, calcium-dependent protein kinase (protein Kinase C, PK-C) in the intracellular mechanism of action of ethanol and acetaldehyde by stimulating testosterone production in rat testicular interstitial cells with LHRH and the phorbol ester PDBu, both of which activate PK-C at receptor (LHRH) and post-receptor (PDBu) sites. Ethanol (2000 mg

) inhibited 10(-7) M LHRH and 200 nM PDBu-stimulated testosterone production by 81 +/- 4.7

and 60 +/- 20.4

, respectively. Acetaldehyde (20 mg

) reduced the amount of testosterone produced by 10(-7) M LHRH and 200 nM PDBu by 59.4 +/- 1.2

and 52.5 +/- 5.4

respectively. Basal testosterone levels were unaffected by ethanol and reduced by acetaldehyde. However, the functional test of cell viability by preincubating cells with these doses of ethanol and acetaldehyde did not decrease their ability to respond appropriately to subsequent stimulation with LHRH, demonstrating that cell viability was unaffected by incubation with these drugs. The data presented here suggest that direct ethanol and acetaldehyde exposure results in a reduced ability of the testicular interstitial cells to respond to stimulation of PK-C pathway.

Fluoride and phosphatidylserine induced inhibition of cytosolic insulin-degrading activity.

Cytosol (C) (100,000 x g/60 min, supernatant) from liver, brain and testis (Wistar male rats) are shown to contain insulin degrading activity (C-IDA). The regulation of C-IDA in these fractions by ligands that activate G protein and PKC were examined C-IDA from liver, brain and testis was inhibited 76%; 64% and 50% by 50 mM F- respectively. Chromatography of C fraction from liver on Sephadex G-50 in presence of 1 M (NH4)2SO4 and 20% (v/v) glycerol (experimental condition to remove guanine nucleotides from G proteins) decreased in about 3-fold aluminum fluoride effect on C-IDA. Mg++ (from 5mM to 10 mM) enhanced fluoride effects by inhibiting fully C-IDA. Phosphatidylserine in presence of ATP completely inhibited C-IDA; this inhibition was 31.3% mediated by a phosphorylation reaction. It is concluded that cytosol from different tissues contain proteins capable to associate ligands as aluminum fluoride and PS to regulate C-IDA. It is proposed a mechanism of protein-protein interaction to modulate C-IDA.

Fluoride and phosphatidylserine induced inhibition of cytosolic insulin-degrading activity.

Cytosol (C) (100,000 x g/60 min, supernatant) from liver, brain and testis (Wistar male rats) are shown to contain insulin degrading activity (C-IDA). The regulation of C-IDA in these fractions by ligands that activate G protein and PKC were examined C-IDA from liver, brain and testis was inhibited 76

; 64

and 50

by 50 mM F- respectively. Chromatography of C fraction from liver on Sephadex G-50 in presence of 1 M (NH4)2SO4 and 20

(v/v) glycerol (experimental condition to remove guanine nucleotides from G proteins) decreased in about 3-fold aluminum fluoride effect on C-IDA. Mg++ (from 5mM to 10 mM) enhanced fluoride effects by inhibiting fully C-IDA. Phosphatidylserine in presence of ATP completely inhibited C-IDA; this inhibition was 31.3

mediated by a phosphorylation reaction. It is concluded that cytosol from different tissues contain proteins capable to associate ligands as aluminum fluoride and PS to regulate C-IDA. It is proposed a mechanism of protein-protein interaction to modulate C-IDA.

Fluoride and phosphatidylserine induced inhibition of cytosolic insulin-degrading activity.

Cytosol (C) (100,000 x g/60 min, supernatant) from liver, brain and testis (Wistar male rats) are shown to contain insulin degrading activity (C-IDA). The regulation of C-IDA in these fractions by ligands that activate G protein and PKC were examined C-IDA from liver, brain and testis was inhibited 76

; 64

and 50

by 50 mM F- respectively. Chromatography of C fraction from liver on Sephadex G-50 in presence of 1 M (NH4)2SO4 and 20

(v/v) glycerol (experimental condition to remove guanine nucleotides from G proteins) decreased in about 3-fold aluminum fluoride effect on C-IDA. Mg++ (from 5mM to 10 mM) enhanced fluoride effects by inhibiting fully C-IDA. Phosphatidylserine in presence of ATP completely inhibited C-IDA; this inhibition was 31.3

mediated by a phosphorylation reaction. It is concluded that cytosol from different tissues contain proteins capable to associate ligands as aluminum fluoride and PS to regulate C-IDA. It is proposed a mechanism of protein-protein interaction to modulate C-IDA.

Liver cells binding with high insulin doses at 37 C.

Insulin binding and receptor mediated insulin degradation were studied in isolated rat hepatocytes under physiological conditions (37 C, 100% oxygen, Krebs improved Ringer III with glutamate, pyruvate and fumarate, 150 mg% glucose, 1% bovine albumin). 10(6) rat hepatocytes/tube were incubated with various doses of insulin. Steady state binding with low insulin doses (0.05, 0.5 and 66 ng/tube) was reached in 15 minutes, that state being kept for the rest of the experimental time (75 min). Receptor mediated degradation (Kap) at 15 minutes was 0.0479 min-1, including doses of 5 000 and 50 000 ng/tube. Direct correlation was found between degradation and low doses of insulin, being the slope value equal to Kap. Intracellular accumulation of insulin was found at pharmacological concentrations of insulin (5 000 and 50 000 ng/tube) from the first 15 minutes. That accumulation was dose and time dependent. At 75 minutes, with a 0.2 microM insulin concentration, at least 53% of insulin was estimated as insulin accumulated in the cell, since it was not filtrable with acid medium on Sephadex G 50 superfine. When Triton or dodecyl sulphate were used to solubilize the cells, insulin recovery was complete after binding. Intracellular accumulation, however, was not demonstrated at the first two minutes. Binding studies with 16.67 microM insulin in the presence of degradation inhibitors, such as 2 mM N-ethylmaleimide and 5 mM tetracaine hydrochloride, demonstrated that intracellular accumulation of the hormone occurs when degradation is blocked. On the contrary, after trypsin digestion of receptors, degradation was not observed, while increases in binding were abolished, resembling non-specific binding. Under the experimental conditions reported here, neither intracellular accumulation of insulin nor extracellular release of insulin degradation products can be demonstrated at 2 minutes; insulin accumulation is dose dependent, and it is suggested by the fact that the velocity of insulin internalization exceeds its velocity of degradation.

Insulin processing. Its correlation with glucose conversion to CO2.

Insulin-receptor binding, insulin degradation and biologic response (14C-glucose conversion into 14CO2) were studied in adipocytes of control (CG), fasted (FG-88 hr) and hyperinsulinic rats (HG-exogenous hyperinsulinism). The number of cells normalized to 3.5 X 10(5) cells/tube in all three groups. Insulin binding and degradation were studied at 5, 15, 30, 60 and 120 minutes of incubation with 3.5 X 10(-11) M, 6.66 X 10(-11) M, 1.0 X 10(-9) M, 6.66 X 10(-9) and 6.66 X 10(-6) M insulin. The net increments of 14CO2 taken into account (delta U-14C-glucose converted into 14CO2) ranged from the basal value to 10(6) microU in each case (30, 60 and 120 minutes). Quantitative analysis of results was performed with the Terris and Steiner degradation equation (formula; see text) (IR). Differences in insulin binding, comparing the three groups, lacked statistical significance, though FG data were systematically plotted above those of CG, occurring the opposite with HG. Degradation studies showed HG to have values statistically higher than the controls, while FG values were lower. HG also showed higher amounts of 14CO2, with basal levels more elevated than CG, while FG showed the inverse behavior. 14CO2 increased in the three groups along the 120-minutes incubation period (30, 60 and 120 minutes). Receptor-mediated degradation at 30 minutes, when binding is in steady state, showed a Kap value very close to that found by linear regression for the 2 and 10 microU doses (Kap min-1 CG: 0.1654, FG: 0.0824, HG: 0.5045; slope values for the 2 and 10 microU doses CG: 0.2181, FG: 0.0824, HG: 0.3718). The degradation velocity, considered as function of IR, was constant in each group at 30, 60 and 120 minutes. Since Kap values in the FG and HG indicate differences in their degradation velocities, this constant can be considered as indicative of the metabolic situations under study. At the same time, the biologic response (14C-glucose conversion into 14CO2) depends as well on the metabolic conditions. Glucose consumption and Kap value were then compared. All the groups showed linear correlation between the binding dependent velocity of degradation (Kap) and the net conversion of U-14C-glucose into 14CO2 at 30, 60 and 120 minutes, with ordinate close to zero (30 min: 0.1539; 60 min: -0.3812; 120 min: 0.1311). The slope increased along the incubation period, indicating that 14CO2 accumulation is time dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Liver cells binding with high insulin doses at 37 C.

Insulin binding and receptor mediated insulin degradation were studied in isolated rat hepatocytes under physiological conditions (37 C, 100

oxygen, Krebs improved Ringer III with glutamate, pyruvate and fumarate, 150 mg

glucose, 1

bovine albumin). 10(6) rat hepatocytes/tube were incubated with various doses of insulin. Steady state binding with low insulin doses (0.05, 0.5 and 66 ng/tube) was reached in 15 minutes, that state being kept for the rest of the experimental time (75 min). Receptor mediated degradation (Kap) at 15 minutes was 0.0479 min-1, including doses of 5 000 and 50 000 ng/tube. Direct correlation was found between degradation and low doses of insulin, being the slope value equal to Kap. Intracellular accumulation of insulin was found at pharmacological concentrations of insulin (5 000 and 50 000 ng/tube) from the first 15 minutes. That accumulation was dose and time dependent. At 75 minutes, with a 0.2 microM insulin concentration, at least 53

of insulin was estimated as insulin accumulated in the cell, since it was not filtrable with acid medium on Sephadex G 50 superfine. When Triton or dodecyl sulphate were used to solubilize the cells, insulin recovery was complete after binding. Intracellular accumulation, however, was not demonstrated at the first two minutes. Binding studies with 16.67 microM insulin in the presence of degradation inhibitors, such as 2 mM N-ethylmaleimide and 5 mM tetracaine hydrochloride, demonstrated that intracellular accumulation of the hormone occurs when degradation is blocked. On the contrary, after trypsin digestion of receptors, degradation was not observed, while increases in binding were abolished, resembling non-specific binding. Under the experimental conditions reported here, neither intracellular accumulation of insulin nor extracellular release of insulin degradation products can be demonstrated at 2 minutes; insulin accumulation is dose dependent, and it is suggested by the fact that the velocity of insulin internalization exceeds its velocity of degradation.

Insulin processing. Its correlation with glucose conversion to CO2.

Insulin-receptor binding, insulin degradation and biologic response (14C-glucose conversion into 14CO2) were studied in adipocytes of control (CG), fasted (FG-88 hr) and hyperinsulinic rats (HG-exogenous hyperinsulinism). The number of cells normalized to 3.5 X 10(5) cells/tube in all three groups. Insulin binding and degradation were studied at 5, 15, 30, 60 and 120 minutes of incubation with 3.5 X 10(-11) M, 6.66 X 10(-11) M, 1.0 X 10(-9) M, 6.66 X 10(-9) and 6.66 X 10(-6) M insulin. The net increments of 14CO2 taken into account (delta U-14C-glucose converted into 14CO2) ranged from the basal value to 10(6) microU in each case (30, 60 and 120 minutes). Quantitative analysis of results was performed with the Terris and Steiner degradation equation (formula; see text) (IR). Differences in insulin binding, comparing the three groups, lacked statistical significance, though FG data were systematically plotted above those of CG, occurring the opposite with HG. Degradation studies showed HG to have values statistically higher than the controls, while FG values were lower. HG also showed higher amounts of 14CO2, with basal levels more elevated than CG, while FG showed the inverse behavior. 14CO2 increased in the three groups along the 120-minutes incubation period (30, 60 and 120 minutes). Receptor-mediated degradation at 30 minutes, when binding is in steady state, showed a Kap value very close to that found by linear regression for the 2 and 10 microU doses (Kap min-1 CG: 0.1654, FG: 0.0824, HG: 0.5045; slope values for the 2 and 10 microU doses CG: 0.2181, FG: 0.0824, HG: 0.3718). The degradation velocity, considered as function of IR, was constant in each group at 30, 60 and 120 minutes. Since Kap values in the FG and HG indicate differences in their degradation velocities, this constant can be considered as indicative of the metabolic situations under study. At the same time, the biologic response (14C-glucose conversion into 14CO2) depends as well on the metabolic conditions. Glucose consumption and Kap value were then compared. All the groups showed linear correlation between the binding dependent velocity of degradation (Kap) and the net conversion of U-14C-glucose into 14CO2 at 30, 60 and 120 minutes, with ordinate close to zero (30 min: 0.1539; 60 min: -0.3812; 120 min: 0.1311). The slope increased along the incubation period, indicating that 14CO2 accumulation is time dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin binding in mouse liver cells isolated with chelating agents.

Mouse liver cells were isolated with Ca2+ and K+ chelating agents. Cell concentrations in all experiments ranged from 2.5 X 10(5) to 1.44 X 10(6) cells/tube. The kinetics of insulin-receptor binding was studied at 2 C and 20 C. Binding of 1.67 X 10(-11) M 125I-insulin reached equilibrium at 2 C at 180 min; Ka at 50% binding was 0.736 X 10(7) M-1 sec-1. At 20 C equilibrium occurred at 30 min; Ka at 50% binding was 7.519 X 10(7) M-1 sec-1. Non-specific binding was measured by adding 16.6 microM native insulin. Kinetics studies of association point to a pure bimolecular reaction since the constant remains unaltered at different times. In studies of bound complex dissociation, insulin release from the receptor involves first order kinetics, 50% of the bound insulin becoming released during the experimental period. Dissociation was studied at 20 C only, either by dilution or addition of 16.6 microM native insulin. Both methods yielded the same result, showing the dissociation kinetics to be a first order reaction with a half-life of 101 min and Kd: 2.5 X 10(-4) sec-1. Competitive inhibition of native insulin (1.67 X 10(-10), 3.33 X 10(-10), 1.67 X 10(-9), 3.33 X 10(-9), 1.67 X 10(-8), 3.33 X 10(-8), 1.67 X 10(-7), 3.33 X 10(-7) M) against 1.67 X 10(-11) M 125I-insulin was studied in equilibrium. Heterogeneity among active binding sites was found: one population of high affinity and low capacity (2 C: K = 4.64 X 10(7) L/M, Ro = 213 X 10(-11) M; 20 C: K = 2.90 X 10(8) L/M Ro = 28.5 X 10(-11) M) and one of low affinity and high capacity (2 C: K = 6.81 X 10(7) L/M Ro: 836 X 10(-11) M; 20 C: K = 2.63 X 10(6) L/M, Ro: 1080 X 10(-11) M). The results show the use of chelating agents in the separation of liver cells to be of value in physicochemical studies of insulin-receptor interaction.

Preparation of biologically active mono-125I-insulin of high specific activity.

Pork insulin was labeled by the chloramine T technique (phosphate buffer 0.25 M; pH 7.5; EDTA 0.001 M; chloramine T: 0.2625 mg/ml; sodium metabisulfite 2.4 mg/ml) in a reaction volume of 50 microliters, adding chloramine T every 30 seconds twice (2.1 micrograms in 1 minute) and halting the reaction with 5 microliters metabisulfite. Three fractions were separated in preparative starch gel: F1 (mono-125I-insulin contaminated with cold insulin), F2 (mono-125I-insulin free from cold insulin), and F3 (di-125I-insulin). Insulins with low and high specific activity (iodine/insulin ratios 0.1/1 and 1/1 respectively) were prepared for study purposes, and quality was assessed by means of dose-response curves with antibodies and with liver cells. Specific activity of F2 as obtained from dose-response curves utilizing Scatchard's plot was 323 and 382 mCi/mg. Specific activity of F1 varied according to the extent of contamination with cold insulin. A reduction in the F2 B/F ratio was observed upon iodination with iodine/insulin ratios of 1/1 or in the neighborhood. The mass and immunoreactivity of F3, as well as its B/F ratios were constant, regardless of specific activity. The behavior with antibodies was ratified upon observations on uptake by liver cells and glucose consumption by isolated fat cells. In conclusion, F2 labeled with 0.1/1 iodine/insulin ratios was separated from cold insulin in preparative starch gel, thus increasing its specific activity (360 mCi/mg approximately) without alteration of its immunologic or biologic properties.