Effect of insulin on transport of several hexoses and pentoses into cells of muscle and brain.

PARK, C. R., L. H. JOHNSON, J. H. WRIGHT, JR. AND H, BATSEL. Eject uj insulin on tmnsport uj several hexoses and pentuses into cells of mtiscle and brain. Am. J. Physiol. IQI(I): 13-18. q57.-The effect of insulin on transport of n-glucose, D-mannose, D-fructose, D-xylose, L-arabinose and mribose through the cell membrane has been studied. Each sugar was infused into eviscerated rats with or without insulin for I or 2 hours. The accumulation of free sugar within the cell was determined in the diaphragm, heart, gastrocnemius and brain. Intracellular sugar was estimated by subtracting the amount of sugar in the extracellular fluid space, as determined with mannitol-r-C 14, from the total tissue sugar. In muscle virtually no intracellular free sugar was detected in the absence of insulin whereas, in the presence of insulin, large amounts were found except in the case of D-ribose. Since these sugars do not arise by intracellular metabolism, it was concluded that insulin accelerated their transport through the cell membrane. These and other observations support the view that membrane transport in muscle is a site of insulin action. The transport process is distinct from phosphorylation and presumably involves combination of the sugar with a constituent of the cell membrane. In brain, no effect of insulin was observed. D-Mannitol, D-ribose and D-fructose did not pass the blood-brain barrier to a measurable extent. This explains the inefficacy of fructose in relieving the symptoms of hypoglycemia. WEVINE and associates (3-5) first observed L that insulin increased the volume of distribution of several poorly metabolized hexoses and pentoses in the body water of eviscerated-nephrectomiaed dogs. This effect was at tributed to accelerated transfer of these sugars into tissue cells. The authors indicated that transfer was accelerated only in those instances where the sugar had the same configuration as glucose in the first three carbon atoms. They proposed, by analogy, that insulin might also accelerate the entrance of glucose itself into certain cells. We have reported more direct evidence that Received for publication March 28, 1957. 1 Supported by grants from the National Science Foundation, Public Health Service and Eli Lilly and Company. 2 Part of the present data has been presented earlier in preliminary form ( 1, 2). 3 Present address : Medical Research Laboratory, Fort Knox, Ky. insulin accelerates the transport4 of glucose itself. It was found in experiments with rat muscle in vitro (6) and ipz vivo (7) that the hormone caused a rise in intracellular free glucose, accompanied by an increase in glucose utilization. These and other observations using galactose led to the following conclusions. a) In muscle, transport is the rate limiting step for the uptake and utilization of glucose-in the absence of insulin. b) The transport of glucose and galactose in muscle is accelerated by insulin. Glucose thereby becomes available in larger amounts for phosphorylation and its over-all utilization is increased c) The transport process precedes glucose phosphorylation. d) Transport in brain is probably not accelerated by insulin. In the present study, observations have been 4 The term transport will be used to describe the process by which sugars pass through the cell membrane. by 10.0.33.1 on Jne 5, 2017 http://ajple.physiology.org/ D ow nladed fom x4 PARK, JOHNSON, WRIGHT AND BATSEL made on the effect of insulin on the transport Samples between 200 and 800 mg were placed of several other hexoses and pentoses in the in mortars containing 5-8 ml of boiling 80% muscle and brain of eviscerated rats. An ethanol and were minced with scissors. The acceleration of transport was detected by the mortars were then removed from the bath and accumulation of free intracellular sugar in these left in the air stream at the edge of a hood until tissues. Intracellular sugar was estimated by the alcohol was evaporated. They were dried the difference between the total free sugar of further &z z~acu~ for 18 hours. The dry the tissue and the extracellular sugar. The material was ground to a fine powder with latter could be estimated from the extracellular sand, extracted with 6 ml of water and treated concentration, which was taken to be the same with 0.5-1.0 ml each of 0.3 N Ba(OH)z and as in the blood serum, and the extracellular ZnS04. After centrifugation, the supernatants volume, as measured with mannitol-I-C14. were then shaken with a small amount of a 3: I mixture of moist Duolite A-4, hydroxyl METHODS form, and Dowex 50, hydrogen form. The resins General Procedure. Rats of the Spraguewere very effective in reducing the background Dawley strain weighing 100-150 gm were color in the case of fructose and pentose fasted about 18 hours before use. They were analyses. eviscerated and in most instances nephrectoAliquots of blood serum were diluted mized under Nembutal or urethane anesthesia appropriately, precipitated with Ba(OH)z and immediately before use in order to eliminate &SO4 and deionized as above. the release of endogenous insulin and reduce The following analytical methods were metabolic transformation of the sugars. employed for the above solutions: fructose Immediately following operation, the rat according to Roe (8) or Kendrick (9), pentose was infused for 2 hours at a constant rate according to Mejbaum (IO) as modified by through the tail vein with the test sugar in I Horecker and Smyrniotis (I I>, glucose by the ml of 0.9% NaCl/roo gm of rat. The concenmicroenzymatic procedure using yeast hexotration of sugar was adjusted to give approxikinase and glucose-&phosphate dehydrogenase mately the same range of final blood serum (I 2), mannose and galactose by paper chroma.= levels in the control and hormone-treated tography. For the last, the solutions were evayseries. Insulin-treated animals received IOO orated to dryness in conical centrifuge tubes. pg/roo gm of glucagon-poor insulin intraThe residues were taken up in o.15 ml of water venously (Eli Lilly, lot no. 466368) at the start of the infusion, and 20 pug in the infusion TABLE I. MANNITOL SPACE OF MUSCLE AND BRAIN mixture. The rectal temperature was mainIN THE EVISCERATED-NEPHRECTOMIZED RAT tained between 30 and 3z”. Fifteen minutes was allowed at the end of the infusion for mixing. Blood was then collected by decapiInsulin tation and samples of the heart, diaphragm, gastrocnemius and brain were excised as ~rapidly as possible and placed in 0.9 % NaCl at 0