334 



M. WOODS, K. WIGHT, J. HUNTER, D. BURK 



VOL. 12 (1953) 



in stimulating acid production as was regular crystalline zinc-insulin. That HGF played 

 no significant role in producing the observed effects was further evidenced by the fact 

 that a preparation of purified factor (contained only 0.05 unit insulin per mg) at 2 y 

 per ml had no significant effect on (^co^ i^ two experiments (0.9%) ; whereas HGF-free 

 insulin in the same experiments gave 45% and 46% stimulation respectively. The 

 remainder of this paper presents in detail the results obtained with preservative-free 

 insulin, and/or varying concentrations of inorganic zinc. 



Effect of glucose concentration 



Glucose concentration had a marked effect on both the rate and the extent of 

 response to insulin. This is illustrated by the results of an experiment shown graphically 



in Fig. 4. At a glucose level of 0.125' 



Control 



150 Mmutei 



Fig. 4. The influence of glucose concentra- 

 tion and insulin on anaerobic acid produc- 

 tion by S-91 melanoma slices in Hank- 

 Simms medium (i.o ml Hank-Simms plus- 

 0.25 ml HgO ± insulin). A, B and C show 

 (by summation) microliters of COj produ- 

 ced in time (solid line minus insulin, broken 

 line plus 4 units crystalline zinc-insulin) by 

 100 mg wet weight of slices (left ordinate): 

 A, with 0.125% dextrose, B, with 0.225% 

 dextrose, and C with 0.625% dextrose. 

 Curves A', B' and C (right ordinate) repre- 

 sent non-summative plots of the percentage 

 difference between the control and insulin- 

 treated tissue (at the 3 glucose levels A, B 

 and C respectively) for the time periods 

 between successive points (heavy trian- 

 gles). Gas phase 95% Ng and 5% COg. 



3 (equivalent to 312 /xl lactic acid) the rate with 

 insulin aiter 45 minutes was below that of the 

 control (Fig. 4, A, A'), presumably due in part to 

 glucose depletion. At the intermediate level of 

 0.225 % glucose (Fig. 4, B,B') the rate with insulin 

 remained above that of the control for over 90 

 minutes although there was a steady decline in 

 the per cent stimulation. On the other hand at 

 0.625% glucose (Fig. 4, C,C') the per cent sti- 

 mulation above the control was maximal and 

 remained approximately constant for 60 minutes. 

 A number of other experiments established the 

 fact that under anaerobic conditions a high level 

 of glucose was required to give maximal Q^q^ val- 

 ues, and maximal responses to insulin. BuRK et 

 al.^ have shown that in other tissues the glyco- 

 lytic system is stabilized under anaerobic condi- 

 tions by glucose. In the present study, varying 

 the glucose concentration from 0.125% to 0.625% 

 had little effect (less than 5%o) on the Qq^ of brain 

 or melanoma slices although (Jco was increased, 

 particularly in brain. Because of these results 

 0.625% glucose was used routinely in the fol- 

 lowing experiments. 



Effect of insulin concentration 



Crystalline zinc-insulin added in distilled 

 water as the vehicle medium (0.25 ml water 

 added per i.o ml Krebs-Ringer-bicarbonate, or 

 Hank-Simms) was equally effective at final con- 

 centrations of 4.0 and 0.8 units per ml {ca. 0.15 

 to 0.03 mg insulin per ml). For example, in 3 

 experiments with melanoma from mice exposed 

 to 35° C (see next section) the average (2c6. 

 without added insulin was 7.41 (range 6.0 to 

 9.4) ; with 4 units of crystalline zinc-insulin 



References p. 346. 



