|
Senior Physiologists' News |
|
|
|
Letter to Martin Frank Julian B. Marsh writes: “In the spirit of a Scientific American feature- 50 years ago-and as a personal memoir- I wish to tell the inside story of the discovery of the existence of the insulin receptor in the laboratory of Dr. William C. Stadie at the University of Pennsylvania in 1948. “In the 1940’s, Stadie, chairman of a small department of Research Medicine at Penn, was one of a number of distinguished scientists pursuing the holy grail of physiology and biochemistry of the time- the mechanism of hormone action, especially of insulin. William Stadie had a distinguished record of research, including the role of the liver in bile production, the mechanism of action of carbonic anhydrase in the kidney, and work on the B-oxidation of fatty acids and ketogenesis. As a young medical student at Penn in 1945, I had become interested in kidney disease. I did some research in the laboratory of Dr. David L. Drabkin, famous for his crystallization of human hemoglobin, the determination of the spectrophotometric constants of chromoproteins, and the cyanmethemoglobin method for hemoglobin estimation, still in use today. He suggested I learn the Warburg manometric method of measuring oxygen uptake of cells in Stadie’s lab, which I did. After completing my internship in 1947, I became a US Public Health postdoctoral fellow in Biochemistry with Dr. Stadie. “Every Monday morning, Dr. Stadie held a conference with his laboratory people to discuss progress in his research. In 1948, the main individuals were Niels Haugaard, about to receive his PhD, a graduate student—Ella Schwartzman (about to become Ella Haugaard)—and A. Gorman Hills, an internist interested in research. After a few weeks, it was obvious to me that the only topic of interest was insulin. I listened. The problem was that while insulin effects were easy to demonstrate in intact tissues, once the tissue was homogenized the effects were lost. However, Nobel Laureate Carl Cori’s laboratory had published work showing that in a cell-free muscle preparation, insulin relieved the inhibition of hexokinase by anterior pituitary and adrenal cortical extracts. Niels Haugaard tried to repeat some of this work and failed. Nevertheless, the hexokinase theory of insulin action received much attention, as one might expect given the fact that it came from Cori’s lab. When the negative results from Stadie’s lab were published, from that day forward no paper from the Cori lab ever referenced any related work from Stadie’s lab, to the best of my knowledge. “I soon realized that my kidney metabolism ideas were leading nowhere, and I too caught the insulin fever. At that time, the rat diaphragm method of Gemmill was the main research tool since addition of insulin to one of the hemi-diaphragms allowed one to measure an increase in glucose uptake and glycogen synthesis over the hour or so of exposure to insulin. One could add various compounds to the incubation medium and see what they did to the effect of insulin on glycogen synthesis. One Monday morning I had a bright idea (the best I’ve ever had in more than 50 years of research). “I asked everyone if they knew how long it took insulin to act, let alone how it ever, as a protein, got inside the cell. The answer was no one knew, but the insulin effect could not be measured in less than at least 30 or 40 minutes since the increment in glycogen or the difference between starting and ending glucose levels would be too small to measure. I said I had an idea, and would it be OK if I did some experiments during the week. They said sure, go ahead, and you can have all the rats you want. “My idea was to incubate one of the diaphragms with insulin in oxygenated medium for an hour, then wash it off, and then incubate at 37° for an hour and compare glycogen levels. I tried this, making sure the washing period had plenty of volume so that only insulin which had, I thought, penetrated the cell, could be present. The experiment worked. I next tried decreasing the time of initial incubation from one hour to 30 minutes, then to three minutes, then to 10 seconds, and it worked every time. “You can imagine how excited I was to report these results over the next two weeks. Dr. Stadie turned to Niels and said you had better repeat these experiments. I was delighted, knowing Niels to be a superb investigator. Niels proceeded to do so, and you can imagine how I felt when he reported his results were completely negative—there was no insulin effect he could find. “The question now was why, or indeed whether, I had succeeded. Niels immediately said it must be the insulin; maybe I used a different batch containing an impurity or maybe his batch was bad. We tested both batches in the usual way; mine was active and his was not. It turned out that his batch had been placed in a glass vessel which had been used in a glycogen assay, which involved KOH hydrolysis. Even a trace of alkali would have inactivated insulin, which is what had happened. The next 5,000 experiments of my kind were fine (this number is calculated from the number of rats used over several years of work on the problem). “Dr. Stadie, Niels Haugaard, Gorman Hills and I all pitched in and did everything one could think of to establish that there was indeed a ‘chemical combination of insulin with muscle (rat diaphragm)’ and this was the title of the paper sent to the American Journal of Medical Sciences. It was not sent to the J. Biol. Chem—we knew Cori was sure to see it there! Except for the fact that we id not use the word ‘receptor,’ every criterion of a receptor, apart from its isolation, was met. We were sure that the reason no one could find an insulin effect in a cell-free muscle preparation was that its action was at the cell surface. “When Martha Vaughan joined Stadie’s lab, she was given the job of labeling insulin to determine the actual extent of the binding. Insulin, stable in acid, could be labeled with radioactive sulfur from sulfuric acid under conditions in which its activity was preserved. She did excellent work, but it was generally dismissed by a simple fact—binding of radioactive insulin to diaphragm occurred even when the tissue was killed by boiling. This set back the field for several years—the difference between specific and non-specific binding was not understood. It was not until the brilliant work of Cuatrecasas at the NIH that the field took off. Meanwhile, Niels and I published a 113 page monograph on insulin action in 1953 and in this work we called attention to the 1910 observation of Boehm who showed that curarine bound to frog muscle and in this form exerted its physiological action. To quote our 1953 statement, ‘It is possible to speculate that the binding of insulin occurs at specific receptor points in the cell and that the combination of insulin and receptor groups may be a phenomenon analogous to the highly specific reactions taking place between antigens and antibodies.’ “For those of you reading this and thinking about laboratory chiefs and mentors, I wish to briefly comment about the personalities of Dr. William Stadie and Dr. David Drabkin. Dr. Stadie was a perfect gentleman of the old school. He had a cheerful mein, was extremely knowledgeable, and a pleasure to work for. However, he regarded everyone in his lab as extensions of his own hands and almost always put his name as first author on all publications. His letters of recommendation for me and for Niels, when it came time for us to leave, were models of faint praise and probably prevented Niels from achieving a desired post at the NIH. David Drabkin, on the other hand, was sometimes hard to work for, often irascible, though lovable, and frequently critical. But he went out of his way to help people who had worked in his lab. I recall one individual Dr. Drabkin disliked intensely. However, he personally went to the NIH to lobby for appointment there (he got it). He offered me an appointment in his small department in the Graduate School of Medicine, which I accepted at the rank of Instructor even though his letter of recommendation for me resulted in an offer of an Assistant Professorship at Temple University. Many years later, a similar letter helped me obtain the post of Professor and Chairman of a combined department of Physiology and Biochemistry at the then Medical College of Pennsylvania. “Niels Haugaard died in January 2004. My own subsequent research career was related to my interest in kidney disease. I tried to answer the question of the hyperlipidemia of the nephrotic syndrome, which led to work on the biosynthesis of the plasma lipoproteins by rat liver and in turn, to a theory of the hyperlipidemia of nephrosis. I believe this reflects the fact that studies of clinical syndromes, genetic or otherwise, still lead the way in biomedical research.” Letter to Julio Cruz Arthur H. Smith writes: “I’ve received your letter informing me of ‘the Senior Physiologists Committee,’ and my eligibility to become associated with it on the occasion of my completion of 90 orbits of the solar system. “When I retired, Professor Charles Fuller took over the centrifuges; but I kept and office for about five years after retirement, and competed analysis of some research. Since then I’ve become somewhat disabled physically and have to use a wheeled walker to get around. As a result, I’ve become completely inactive in physiology. I do keep myself occupied in tracing my genealogy. So far I’ve completed the Glass family, my father’s maternal antecedents, and the Woodson’s, my mother’s maternal antecedents which I find includes Jesse James, the notorious post-Civil War train and bank robber (he’s my mother’s fifth cousin). Now I’m working on the Brown’s, my mother’s paternal ancestors.” |
|
|