- Email: [email protected]
Section on peptides with physiologic and pharmacologic activity: Fat-mobilizing pituitary peptides
Summary of the Discussion Section on Peptides with Physiologic and Pharmacologic Activity: Fat-mobilizing Pituitary Peptides Participants: E. B. Asrwoon, H. FRIESE:\", D. T. GISH, K. HOFMANN, H. E. LEBOVITZ, M. S. RABEN, D. RumrAN AND D. STEINBERG.
R. LEBOVITZ agreed that direct assay of lipase would be desirable.
D
However, he pointed out that measurement of net FFA production is probably adequate for comparison of the potencies of closely related peptide structures. TIle assays are reproducible and the shapes of the dose-response curves are very similar. Dr. Hudman described in detail experiments from his laboratory employing a modified assay procedure in which the adipose tissue is incubated in an albumin-free medium. Under these conditions, the FFA produced cannot be discharged from the tissue. Instead they accumulate within' the tissue, reaching a plateau concentration that varies with the concentration of lipolytic hormone added to the medium. \Vhen such a plateau concentration of tissue FFA is reached, presumably the rates of degradation and reesterification are balanced at a new steady-state level. Using the plateau FFA concentration as a measure of response to hormone, it is found that the data can be interpreted according to the Clark-Stetten model of hormone-receptor interaction, If response is plotted against logarithm of dose, the hcight of the curve at its peak or the slope of the curve at its inflection point can be interpreted to reflect the number of hormone receptor sites, while the displacement of the curve to the left along the abscissa reflects a greater association constant between hormone and binding sites. Dr. Rudman's data show that according to this analysis, the number of binding sites is the same for all of the lipolytic pep tides studied and for the catecholamines. On the other hand, the affinities of the several hormones for the binding sites diller according to their structure, and this is postulated to be in part the basis for their different potencies. Another important factor stressed by Dr. Hudman' is the capacity of the adipose tissue to degrade or inactivate the peptide hormones. For example, rat adipose tissue contains an active peptidase system that inactivates pituitary peptides, whereas rabbit and guinea pig tissue contain little or no such peptidase activity. Thus, not only the capacity of the hormone to stimulate lipase activity but also the capacity of the tissue to inactivate the hormone can contribute to "potency" as determined by study of the overall response of the intact tissue. The question of whether the "lipolytic" hormones actually increase lipolysis or whether they instead influence oxidation of fat or reesterification of fatty acids was raised by Dr. Gish. Dr. Friesen and Dr. Steinberg cited the now quite convincing evidence that the lipolytic activity of hormone-treated adi1273
1274
DISCUSSION
pose tissue is increased. This is shown in the intact tissue by the accelerated rate of glycerol formation, a good index of lipolysis since glycerol is poorly metabolized by adipose tissue and thus tends to accumulate in the system. It has also been shown directly in a number of laboratories by demonstrating increased lipase activity in homogcnates of adipose tissue previously exposed to the action of hormones. Dr. Hofmann reported preliminary studies of the structure of Peptide I, made available to him by Dr. Astwood. Dr. Lerner had shown that the compound possessed some melanocyte-expanding activity. Dr. Hofmann split the peptide into two fragments using Dr. Witkop's cyanogen bromide procedure and found that one of the fragments had exactly the same amino acid composition as that of pig ,B·},ISH. Like pig ,B-MSH, it had a C-terminal aspartic acid. Dr. Raben pointed out that epinephrine increases oh)'gen consumption of the isolated perfused heart under conditions where no extra FFA are available. Dr. Steinberg agreed that effects on cardiac work, respiratory work, gastrointestinal function and on other mechanical and secretory systems, would certainly account for some of the calorigenic effect of epinephrine. However, these are not large enough effects to explain the magnitude of the total body response. In addition, there seems to be a true metabolic effect and this is what appears to be closely related to increased fat mobilization'. lIe went on to state that in tissue slices of liver, heart or skeletal muscle, it has not been possible to effect an increase in O~ consumption by raising medium FFA concentration, even though the rate of FFA utilization does increase. Only in perfused tissues has an increment in O~ consumption been observed. Dr. Astwood pointed out that rapid mobilization of FFA induced by pituitary fat-mobilizing preparations does not seem to be accompanied by -increased Oz consumption or ketosis. Dr. Hudman suggested that in addition to FFA mobilization, it may be necessary to stimulate at the same time the processes leading to their utilization'. Some hormones may combine these effects and others not.
R. LEBOVITZ agreed that direct assay of lipase would be desirable.
D
However, he pointed out that measurement of net FFA production is probably adequate for comparison of the potencies of closely related peptide structures. TIle assays are reproducible and the shapes of the dose-response curves are very similar. Dr. Hudman described in detail experiments from his laboratory employing a modified assay procedure in which the adipose tissue is incubated in an albumin-free medium. Under these conditions, the FFA produced cannot be discharged from the tissue. Instead they accumulate within' the tissue, reaching a plateau concentration that varies with the concentration of lipolytic hormone added to the medium. \Vhen such a plateau concentration of tissue FFA is reached, presumably the rates of degradation and reesterification are balanced at a new steady-state level. Using the plateau FFA concentration as a measure of response to hormone, it is found that the data can be interpreted according to the Clark-Stetten model of hormone-receptor interaction, If response is plotted against logarithm of dose, the hcight of the curve at its peak or the slope of the curve at its inflection point can be interpreted to reflect the number of hormone receptor sites, while the displacement of the curve to the left along the abscissa reflects a greater association constant between hormone and binding sites. Dr. Rudman's data show that according to this analysis, the number of binding sites is the same for all of the lipolytic pep tides studied and for the catecholamines. On the other hand, the affinities of the several hormones for the binding sites diller according to their structure, and this is postulated to be in part the basis for their different potencies. Another important factor stressed by Dr. Hudman' is the capacity of the adipose tissue to degrade or inactivate the peptide hormones. For example, rat adipose tissue contains an active peptidase system that inactivates pituitary peptides, whereas rabbit and guinea pig tissue contain little or no such peptidase activity. Thus, not only the capacity of the hormone to stimulate lipase activity but also the capacity of the tissue to inactivate the hormone can contribute to "potency" as determined by study of the overall response of the intact tissue. The question of whether the "lipolytic" hormones actually increase lipolysis or whether they instead influence oxidation of fat or reesterification of fatty acids was raised by Dr. Gish. Dr. Friesen and Dr. Steinberg cited the now quite convincing evidence that the lipolytic activity of hormone-treated adi1273
1274
DISCUSSION
pose tissue is increased. This is shown in the intact tissue by the accelerated rate of glycerol formation, a good index of lipolysis since glycerol is poorly metabolized by adipose tissue and thus tends to accumulate in the system. It has also been shown directly in a number of laboratories by demonstrating increased lipase activity in homogcnates of adipose tissue previously exposed to the action of hormones. Dr. Hofmann reported preliminary studies of the structure of Peptide I, made available to him by Dr. Astwood. Dr. Lerner had shown that the compound possessed some melanocyte-expanding activity. Dr. Hofmann split the peptide into two fragments using Dr. Witkop's cyanogen bromide procedure and found that one of the fragments had exactly the same amino acid composition as that of pig ,B·},ISH. Like pig ,B-MSH, it had a C-terminal aspartic acid. Dr. Raben pointed out that epinephrine increases oh)'gen consumption of the isolated perfused heart under conditions where no extra FFA are available. Dr. Steinberg agreed that effects on cardiac work, respiratory work, gastrointestinal function and on other mechanical and secretory systems, would certainly account for some of the calorigenic effect of epinephrine. However, these are not large enough effects to explain the magnitude of the total body response. In addition, there seems to be a true metabolic effect and this is what appears to be closely related to increased fat mobilization'. lIe went on to state that in tissue slices of liver, heart or skeletal muscle, it has not been possible to effect an increase in O~ consumption by raising medium FFA concentration, even though the rate of FFA utilization does increase. Only in perfused tissues has an increment in O~ consumption been observed. Dr. Astwood pointed out that rapid mobilization of FFA induced by pituitary fat-mobilizing preparations does not seem to be accompanied by -increased Oz consumption or ketosis. Dr. Hudman suggested that in addition to FFA mobilization, it may be necessary to stimulate at the same time the processes leading to their utilization'. Some hormones may combine these effects and others not.