In Pursuit of Genes of Glucose Metabolism

If you don't know where you're going, you might wind up someplace else—Yogi Berra My professional journey, once I accepted the fact that I would not play first base for the Chicago Cubs, was not accomplished in a straight line. I entered medical school intending to become a family physician but soon became interested in research. I took an internal medicine residency at the University of California, San Francisco (UCSF), then a long research fellowship in the nascent field of molecular biology at the National Institutes of Health (NIH), and followed that with training in clinical endocrinology at the University of Wisconsin. I spent the next fifteen years dividing my time between the clinic and laboratory at the University of Iowa. Finally, I decided to concentrate on research and spent the next twenty-three years at Vanderbilt University. For the past eleven years, I have been at the University of Iowa, where I helped establish a new diabetes research center. My research journey also had several different directions. Upon reflection, I guess I ended up “someplace else” by choice, and it certainly has been an interesting trip. It is one I unreservedly recommend. About half of the sixteen students in my high school class in a small Iowa town were destined to go to college. A rather amazing collection of teachers recognized this and gave us a de facto college preparatory education. I often regret that I did not ask the school superintendent whether this was accidental or deliberate. I certainly had plenty of opportunities to inquire about this, as he was my father. I suspect it was not accidental. My mother was also a schoolteacher, so spending a lifetime in various educational environments was probably predetermined. Enrolled as an undergraduate student at the University of Iowa, I was a bit intimidated by the prospect of competing with students from schools with student bodies of one thousand or more. However, when I found that my freshman inorganic chemistry book was the same one used in our high school, I thought that college might not be so difficult after all. I relaxed a bit more when we got our first “unknown” inorganic compound to identify. Several years earlier, I had received a Gilbert chemistry set for Christmas, which was recently listed as one of the most dangerous “toys” ever sold. This was no surprise, as my favorite creations (first experiments?) were fireworks. I learned to identify several metals by the color of the flames they produced when heated, and so I found the “unknowns” to be old friends. The undergraduate years were fun, especially when a letter of admission to medical school at the University of Iowa arrived in 1957. My intention was to become a primary care physician. Medical school was generally challenging, but Nicholas (Nick) S. Halmi was easily the most demanding and rigorous teacher in the first year. He nominally taught histology, but this was really a remedial course for all he thought that had been omitted or taught erroneously during the year, particularly in his area of special interest, endocrinology. Two classmates and I learned that he recruited two students from each class to help him do research, which he conducted only during the three months of summer “vacation.” I was selected to ask whether he would consider taking all three of us. To our delight, he agreed, and this led to a life-changing experience. Nick studied thyroid iodide concentration and had perfected a technique of studying the transport of radioactive iodide from plasma across the thyroid membrane. He designed experiments that never seemed to fail. He initiated our education in the elements of experimental design, conduct, and interpretation. When he decided the story was complete, he wrote a draft in longhand in an evening, his secretary typed it, and he sent it to a journal, all in 24 h. In a few days, a note would come back saying the article was accepted for publication. I thought this was how it always worked. Toward the end of that summer, Nick approached me with an unexpected opportunity. The United States Public Health Service had announced a two-year program that allowed second-year medical students to split half-time academic studies with half-time research/teaching in a discipline that led to a master's degree. (There were no formal M.D./Ph.D. programs at that time.) I was really intrigued by the summer research experience and had just gotten married, so I readily accepted this offer. I spent the next two years taking medical and graduate school classes, serving as a teaching assistant in the gross anatomy and histology laboratories, and doing research. By the time I went back to being a full-time medical student, I was seriously considering a career that involved research. Then I was offered a second surprising opportunity. Nick decided to take a one-year sabbatical and suggested that I (a) teach the endocrinology part of his histology/endocrinology course to first-year medical students, (b) keep the laboratory functioning and productive, and (c) not overspend his grant. Lecturing is never easy, but it never has been as difficult as it was facing 120 medical students for the first time. The laboratory worked well. With the assistance of several classmates who served as able technicians and with occasional directions from Nick in the form of handwritten letters (no phone calls, E-mails, or computers), we completed three projects concerning how intracellular iodide influences the transport process (1Granner D.K. Curtis S.J. Scranton J.R. Halmi N.S. Differences in thyroid function between triiodothyronine-treated and hypophysectomized rats: blocked glands.Endocrinology. 1962; 71: 816-821Crossref PubMed Scopus (2) Google Scholar, 2Granner D.K. Curtis S.J. Scranton J.R. Halmi N.S. Differences in thyroid function between triiodothyronine-treated and hypophysectomized rats: binding glands.Endocrinology. 1963; 72: 100-105Crossref PubMed Scopus (4) Google Scholar, 3Granner D.K. Scranton J.R. Curtis S.J. Kinetic analysis of thyroidal iodine concentration in hypophysectomized rats fed high or low iodine diets.Endocrinology. 1963; 72: 503-504Crossref PubMed Scopus (4) Google Scholar). All of this was accomplished while I was a full-time third-year medical student, and it had the blessing of the department chairman. (As far as I know, the dean knew nothing about this!) This was my first experience with real mentoring. I was entrusted with a variety of responsibilities, given a great opportunity to achieve, and certainly learned a bit about multitasking. After completing medical school, I was accepted for medical residency at UCSF, and I was there from July 1962 to July 1964. During the spring of 1964, one of my attending physicians, Mort Myers, an Iowa graduate and an internist in Berkeley, brought Gordon Tomkins (then at the NIH) to ward rounds one morning. This was my first exposure to Gordon's incredible intellect. Later that day, he gave a resident's conference during which he discussed the newly emerging field of molecular biology, with emphasis on the regulation of the Escherichia coli lac operon. I mentioned to him afterward that I was going to be joining the NIH as a research associate. He invited me to stop by his laboratory after I arrived. I arrived at the NIH in the summer of 1964, and a few months later, I visited Gordon, who was chief of the Laboratory of Molecular Biology. I mentioned being unhappy in my assigned laboratory and asked him for advice. He unexpectedly said he had space (a trait I later found was recurring), much to the surprise of Beverly Peterkofsky and Shin-ichi Hayashi, who became my lab mates. Both were exceptionally kind, generous, and patient as they initiated my training in biochemistry and molecular biology. Gordon had visited Paris in the early 1960s and was completely familiar with the evolving story of the structure and regulation of the E. coli lac operon. He was convinced that similar work could be done using cultured mammalian cells. Brad Thompson, a fellow United States Public Health Service research associate, eagerly accepted this challenge. He began by using the ascites form of a rat hepatoma (7288c) to establish the hepatoma tissue culture (HTC) cell line. Brad then found that the addition of dexamethasone, a synthetic glucocorticoid, to HTC cells resulted in a 10-fold induction of tyrosine transaminase activity. The effect occurred after a lag period of about two hours and required ongoing protein and RNA synthesis (4Thompson E.B. Tomkins G.M. Curran J.F. Induction of tyrosine α-ketoglutarate transaminase by steroid hormones in a newly established tissue culture cell line.Proc. Natl. Acad. Sci. U.S.A. 1966; 56: 296-303Crossref PubMed Scopus (412) Google Scholar). This landmark observation was the formal beginning of studies of gene regulation by hormones in cultured mammalian cells. I arrived at the Tomkins laboratory just as Brad was completing the studies described above. Concomitantly, Robert Schimke and colleagues, then at the NIH, were establishing the conceptual framework for mechanistic studies of enzyme induction, a topic discussed in detail in a recent review (41Granner D.K. Wang J.-C. Yamamoto K.R. Regulatory actions of glucocorticoid hormones: from organisms to mechanisms.in: Wang J.-C. Harris C. Glucocorticoid Signaling: From Molecules to Mice to Man. Springer-Verlag, New York2015: 3-31Crossref Scopus (33) Google Scholar). They postulated that an increased amount of an enzyme could result from an increased rate of synthesis, a decreased rate of degradation, or some combination of both (principles I was about to employ and would revisit years later when studying mRNA regulation). These investigators presented experimental evidence of both mechanisms in studies of the regulation of tryptophan oxygenase in rat liver (5Schimke R.T. Sweeney E.W. Berlin C.M. The roles of synthesis and degradation in the control of rat liver tryptophan pyrrolase.J. Biol. Chem. 1965; 240: 322-331Abstract Full Text PDF PubMed Google Scholar). Shin-ichi, my new laboratory mate, was purifying tyrosine transaminase to make the specific antibody required for studies of its regulation in HTC cells. Shin-ichi knew he had to leave in a few months to accept a position in Japan, so we worked very hard to purify the enzyme, establish its kinetic properties, and prepare a highly specific antibody directed against the enzyme, which we renamed tyrosine aminotransferase (TAT) (6Hayashi S. Granner D.K. Tomkins G.M. Tyrosine aminotransferase: purification and characterization.J. Biol. Chem. 1967; 242: 3998-4006Abstract Full Text PDF PubMed Google Scholar). My first task upon developing the anti-TAT antibody was to validate its specificity with the intention of directly testing many possible features of enzyme induction that could not be addressed in animals. This was new experimental territory, so I performed tests that showed that the purified enzyme and the induced and basal enzymes from rat liver and HTC cells had identical antigenicity; thus, the antibody could be reliably used to study induction of the enzyme in the cultured cells (7Granner D.K. Hayashi S. Thompson E.B. Tomkins G.M. Stimulation of tyrosine aminotransferase synthesis by dexamethasone phosphate in cell culture.J. Mol. Biol. 1968; 35: 291-301Crossref PubMed Scopus (84) Google Scholar). Antigenic and catalytic activities of TAT change in parallel in a variety of experimental conditions, so induction is not the result of the conversion of a cross-reacting material to an active enzyme. A double-isotope experiment showed that the increase in enzyme amount results from an enhanced rate of synthesis, not an inhibition of degradation (7Granner D.K. Hayashi S. Thompson E.B. Tomkins G.M. Stimulation of tyrosine aminotransferase synthesis by dexamethasone phosphate in cell culture.J. Mol. Biol. 1968; 35: 291-301Crossref PubMed Scopus (84) Google Scholar). A subsequent study showed that a lag period of ∼60 min occurs before the rate of synthesis begins to increase, and this reaches its maximal level in 4–6 h (8Granner D.K. Thompson E.B. Tomkins G.M. Dexamethasone phosphate induced synthesis of tyrosine aminotransferase in hepatoma tissue culture cells. Studies of the early phases of induction and of the steroid requirement for maintenance of the induced rate of synthesis.J. Biol. Chem. 1970; 245: 1472-1478Abstract Full Text PDF PubMed Google Scholar). The induced steady-state synthesis of TAT is maintained as long as dexamethasone remains in the culture medium, but removal of the inducer results in a rapid decline to the basal level (8Granner D.K. Thompson E.B. Tomkins G.M. Dexamethasone phosphate induced synthesis of tyrosine aminotransferase in hepatoma tissue culture cells. Studies of the early phases of induction and of the steroid requirement for maintenance of the induced rate of synthesis.J. Biol. Chem. 1970; 245: 1472-1478Abstract Full Text PDF PubMed Google Scholar). The enthusiasm that the members of the Tomkins laboratory had for this new way of studying mammalian gene regulation was not always met with excitement by those who had been using rats for this purpose. Some investigators refused to believe that anything useful could come from analyzing regulatory processes in tumor cells. Others thought that tissue culture was useful only for determining the minimal nutritional requirements for growth of a given cell line. Finally, some conceded the above points but felt the environment in culture bore little resemblance to that in an intact animal. We therefore had to go to great lengths to address as many of these concerns as possible, all while exploiting and defining the many advantages of tissue culture. The studies described above and subsequent studies by a number of the Tomkins lab members illustrated unique advantages of cell culture systems, including the ability to perform addition-removal-addition sequences to test for an essential mRNA step in the induction process (9Peterkofsky B. Tomkins G.M. Evidence for the steroid-induced accumulation of tyrosine-aminotransferase messenger RNA in the absence of protein synthesis.Proc. Natl. Acad. Sci. U.S.A. 1968; 60: 222-228Crossref PubMed Scopus (61) Google Scholar); use synchronized cells to show that HTC cells are refractory to dexamethasone-mediated induction during certain phases of the cell cycle (10Martin Jr., D. Tomkins G.M. Granner D.K. Synthesis and induction of tyrosine aminotransferase in synchronized hepatoma cells in culture.Proc. Natl. Acad. Sci. U.S.A. 1969; 62: 248-255Crossref PubMed Scopus (86) Google Scholar); precisely manipulate the hormonal environment (11Samuels H.H. Tomkins G.M. Relation of steroid structure to enzyme induction in hepatoma tissue culture cells.J. Mol. Biol. 1970; 52: 57-74Crossref PubMed Scopus (281) Google Scholar); and select for variants, which was an early foray into mammalian cell genetics (12Thompson E.B. Granner D.K. Gelehrter T.D. Simons S.S. Hager G. Unlinked control of multiple glucocorticoid-sensitive process in spontaneous HTC cell variants.in: Sato G.H. Ross R. Hormones and Cell Culture: Sixth Cold Spring Harbor Conference on Cell Proliferation. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1979: 339-350Google Scholar). I was at the NIH from 1964 to 1968. The NIH in the 1960s was a very special place. The clinical and research associates were a bright, energetic, and happy-to-be-there group who scattered to leadership positions in academic medicine all around the country. The senior scientists in the Laboratory of Molecular Biology were an amazingly talented group, as were the postdoctoral fellows they attracted. Best of all was the experience of being around Gordon for four years. He had an infectious enthusiasm for science, a wonderful sense of humor, incredible imagination, and a legendary memory. He presented me with a wonderful opportunity, which included two extra years in his laboratory, and was a terrific mentor. I planned to study hormonal regulation of gene expression when I established my own laboratory at the University of Iowa in 1970. Unfortunately, the techniques required to extend such studies beyond the level of protein synthesis were not available until the mid-1970s. In the meantime, I engaged in an interesting and fruitful collaboration with Roger Chalkley, also at the University of Iowa. Roger was studying histone phosphorylation in rapidly dividing cells using regenerating rat liver as a model system. I suggested the use of synchronized HTC cells as a means of studying this process in precisely defined segments of the cell replication cycle (13Balhorn R. Jackson V. Granner D.K. Chalkley R. Phosphorylation of the lysine-rich histone throughout the cell cycle.Biochemistry. 1975; 14: 2504-2511Crossref PubMed Scopus (50) Google Scholar). We also studied the role of histone acetylation in the regulation of TAT gene expression a few years before this became a topic of broad interest (14Plesko M.M. Hargrove J.L. Granner D.K. Chalkley R. Inhibition by sodium butyrate of enzyme induction by glucocorticoids and dibutyryl cAMP. A role for the rapid form of histone acetylation.J. Biol. Chem. 1983; 258: 13738-13744Abstract Full Text PDF PubMed Google Scholar). When suitable techniques became available, hormonal regulation of abundant mRNAs, not surprisingly, was studied first. Ringold et al. (15Ringold G.M. Yamamoto K.R. Tomkins G.M. Bishop M. Varmus H.E. Dexamethasone-mediated induction of mouse mammary tumor virus RNA: a system for studying glucocorticoid action.Cell. 1975; 6: 299-305Abstract Full Text PDF PubMed Scopus (157) Google Scholar) at UCSF used a very sensitive hybridization technique to demonstrate that dexamethasone, in a mechanism involving the newly discovered glucocorticoid receptor (GR), induces mammary tumor virus RNA. Chan et al. (16Chan L. Means A.R. O'Malley B.W. Rates of induction of specific translatable messenger RNAs for ovalbumin and avidin by steroid hormones.Proc. Natl. Acad. Sci. U.S.A. 1973; 70: 1870-1874Crossref PubMed Scopus (109) Google Scholar) at Baylor College of Medicine showed that estrogen and progesterone induce the mRNAs that encode avidin and ovalbumin, respectively, in chick oviduct. Subsequently, three groups, including ours, used different in vitro translation systems to demonstrate that glucocorticoid hormones cause proportionate increases in mRNATAT activity and enzyme-specific activity in rat liver (17Nickol J.M. Lee K.L. Hollinger T.G. Kenney F.T. Translation of messenger RNA specific for tyrosine aminotransferase in oocytes of Xenopus laevis.Biochem. Biophys. Res. Commun. 1976; 72: 687-693Crossref PubMed Scopus (33) Google Scholar, 18Roewekamp W.G. Hofer E. Sekeris C.E. Translation of mRNA from rat-liver polysomes into tyrosine aminotransferase and tryptophan oxygenase in a protein-synthesizing system from wheat germ. Effects of cortisol on the translatable levels of mRNA for these two enzymes.Eur. J. Biochem. 1976; 70: 259-268Crossref PubMed Scopus (71) Google Scholar, 19Diesterhaft M. Noguchi T. Hargrove J. Thornton C. Granner D.K. Translation of tyrosine aminotransferase mRNA in a modified reticulocyte system.Biochem. Biophys. Res. Commun. 1977; 79: 1015-1022Crossref PubMed Scopus (18) Google Scholar), as does dibutyryl cAMP (Bt2cAMP) (20Noguchi T. Diesterhaft M. Granner D.K. Dibutyryl cyclic AMP increases the amount of functional messenger RNA coding for tyrosine aminotransferase in rat liver.J. Biol. Chem. 1978; 253: 1332-1335Abstract Full Text PDF PubMed Google Scholar). (This compound was used because, unlike cAMP, it rapidly penetrates cells.) Marty Diesterhaft 1Unless indicated otherwise, the people named in this paper performed the research described while they were colleagues in my laboratory. and Tamio Noguchi, who started these studies in my laboratory, showed that the kinetics and magnitude of the responses to the two inducers are quite different (21Diesterhaft M. Noguchi T. Granner D.K. Regulation of rat liver tyrosine aminotransferase mRNA by hydrocortisone and by dibutyryl cAMP.Eur. J. Biochem. 1980; 108: 357-365Crossref PubMed Scopus (27) Google Scholar), which suggested that different mechanisms are involved, a topic that we and others studied in detail, as discussed below. As our ability to isolate poly(A)+ RNA improved, we were able to do similar experiments using HTC cells. Dexamethasone increased mRNATAT activity from 0.04 to 0.4% of total poly(A)+ activity in the cells, an increase that matched the increase in catalytic activity (22Olson P.S. Thompson E.B. Granner D.K. Regulation of hepatoma tissue culture cell tyrosine aminotransferase mRNA by dexamethasone.Biochemistry. 1980; 19: 1705-1711Crossref PubMed Scopus (31) Google Scholar). Proportionate changes in catalytic activity, the rate of synthesis of the protein, and mRNATAT activity were noted when various concentrations of dexamethasone were added to the culture medium. In all cases, this requires ongoing RNA synthesis (22Olson P.S. Thompson E.B. Granner D.K. Regulation of hepatoma tissue culture cell tyrosine aminotransferase mRNA by dexamethasone.Biochemistry. 1980; 19: 1705-1711Crossref PubMed Scopus (31) Google Scholar). At this point, the hypothesis was that an increased amount of mRNA was responsible for these effects. However, this could be due to an enhanced rate of synthesis (transcription) or a decreased rate of degradation of the mRNA. The answer to this question required the cloning of a specific cDNATAT that could then be used as a hybridization probe to quantitate the amount of mRNATAT and isolate the gene. If you come to a fork in the road, take it—Yogi Berra Yogi Berra's quote does not suggest how many options there are for the road best taken. Indeed, in the late 1970s, I had several choices. Continuing on the route to the cloning of a cDNATAT was the obvious first choice. A second, rather unexpected option was to move in the heavily traveled direction of immunoglobulin gene regulation. A final intriguing possibility involved the exploration of whether insulin regulates specific gene expression. With the encouragement of a group of enthusiastic colleagues, I chose to move in all three directions at the same time. Jim Hargrove became interested in the synthesis, degradation, and post-translational modification of TAT and performed a number of studies on these topics (23Hargrove J.L. Granner D.K. Biosynthesis and intracellular processing of tyrosine aminotransferase.in: Christian P. Metzler D.E. Transaminases. John Wiley & Sons, New York1983: 511-532Google Scholar) while another group in the lab attempted to isolate a cDNA specific for mRNATAT. Unfortunately, that effort was not successful. This is when I realized that selecting a good research project is of prime importance and knowing when to let go is equally important. In the words of Kenny Rogers, “You've got to know when to hold ‘em, know when to fold ‘em.” Because other projects were advancing, as described below, I regretfully said good-bye to TAT. Tris Parslow, an M.D./Ph.D. student, joined my lab to explore gene regulation but, seeing the challenges of working with low-abundance mRNAs, chose instead to work on immunoglobulin genes in B lymphocytes. Many groups were studying the structure and rearrangements of those genes, but few were examining how their expression is controlled. The results of his choice and hard work were remarkable. After cloning a cDNA for the κ light chain constant region, Tris showed that lipopolysaccharide treatment of the pre-B cell line 70Z/3 rapidly induced κ mRNA, accompanied by the appearance of a DNase-hypersensitive site within an intron of the gene that signified a change in chromatin structure (24Parslow T.G. Granner D.K. Chromatin changes accompany immunoglobulin kappa gene activation: a potential control region inside the gene.Nature. 1982; 299: 449-451Crossref PubMed Scopus (66) Google Scholar, 25Parslow T.G. Granner D.K. Structure of a nuclease-sensitive region inside the immunoglobulin kappa gene: evidence for a role in gene regulation.Nucleic Acids Res. 1983; 11: 4775-4792Crossref PubMed Scopus (42) Google Scholar). This was surprising because it was assumed at the time that intron sequences were functionless and that gene regulation and inducible hypersensitive sites occurred only at promoters. In sequencing the κ promoter, Tris also discovered a sequence motif (ATGCAAAT), now called the octamer, situated 70 bp upstream from the transcription start site in all immunoglobulin light chain promoters and, in the opposite orientation (ATTTGCAT), in heavy chain promoters as well (26Parslow T.G. Blair D.L. Murphy W.J. Granner D.K. Structure of the 5′ ends of immunoglobulin genes: a novel conserved sequence.Proc. Natl. Acad. Sci. U.S.A. 1984; 81: 2650-2654Crossref PubMed Scopus (335) Google Scholar). These findings sparked tremendous interest and turned out to be vital clues to the mechanisms of B cell-specific gene regulation. Other labs soon showed that the site in the intron was a new type of eukaryotic regulatory sequence called an enhancer. It became hypersensitive when bound by a novel protein complex named the nuclear factor for kappa in B cells (or NF-κB), and the octamer is the binding site for a family of proteins called the Oct proteins, whose members include key regulators of B cell and stem cell genes. I chose not to continue along this path, but it was exciting to watch the stories unfold from our “hit-and-run” foray into immunology! By the late 1970s, the general features of steroid and peptide hormone action on specific gene expression were being elucidated; it was rapidly becoming a very crowded field. The one exception was insulin. The cell-surface insulin receptor had been described, but virtually nothing was known about its intracellular signal transduction pathway. Insulin modulates the extent of phosphorylation of a number of important cytoplasmic enzymes and thus influences several important metabolic pathways (27Larner J. Inositol, glycogen, insulin, and six Nobelists.J. Biol. Chem. 2013; 288: 12313-12324Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). But would insulin, which plays such an important role in glucose, protein, and lipid metabolism, do all this without an effect on specific genes? I doubted it, so the question was, “which gene to study?” The rate of gluconeogenesis, a critical metabolic process, is increased by glucagon (cAMP) and glucocorticoids when plasma glucose levels decline. It is normally decreased by insulin when the plasma glucose exceeds the normal limit. The activity of cytosolic phosphoenolpyruvate carboxykinase (PEPCK), 2PEPCK exists in two forms, cytosolic and mitochondrial, which are encoded by different genes. My studies dealt with the cytosolic form, so PEPCK in this paper represents that protein. a key gluconeogenic enzyme, is regulated by the aforementioned hormones in accordance with their effects on gluconeogenesis in rat liver (28Shrago E. Lardy H.A. Nordlie R.C. Foster D.O. Metabolic and hormonal control of phosphoenolpyruvate carboxykinase and malic enzyme in rat liver.J. Biol. Chem. 1963; 238: 3188-3192Abstract Full Text PDF PubMed Google Scholar) and, most importantly for our intended purpose, in H4IIE cells (29Barnett C.A. Wicks W.D. Regulation of phosphoenolpyruvate carboxykinase and tyrosine transaminase in hepatoma cell cultures.J. Biol. Chem. 1971; 246: 7201-7206Abstract Full Text PDF PubMed Google Scholar, 30Gunn J.M. Tilghman S.M. Hanson R.W. Reshef L. Ballard F.J. Effects of cyclic adenosine monophosphate, dexamethasone and insulin on phosphoenolpyruvate carboxykinase synthesis in Reuber H-35 hepatoma cells.Biochemistry. 1975; 14: 2350-2357Crossref PubMed Scopus (47) Google Scholar). Thus, the regulation of the PEPCK gene became the objective of our trek along path 3. We purified PEPCK, used it to produce a specific antibody, and began to study the effect of insulin and other hormones on PEPCK gene expression. Patrick Iynedjian and Richard Hanson, then at Temple University, had shown that Bt2cAMP increases mRNAPEPCK activity in rat liver (31Iynedjian P.B. Hanson R.W. Increase in level of functional messenger RNA coding for phosphoenolpyruvate carboxykinase (GTP) during induction by cyclic adenosine 3′:5′-monophosphate.J. Biol. Chem. 1977; 252: 655-662Abstract Full Text PDF PubMed Google Scholar). Elmus Beale and others in my laboratory confirmed this observation by showing that this agent causes a 20-fold increase in mRNAPEPCK through a process that requires RNA synthesis (32Beale E.G. Katzen C.S. Granner D.K. Regulation of rat liver phosphoenolpyruvate carboxykinase (GTP) messenger RNA activity by N6,O2-dibutyryladenosine 3′,5′-phosphate.Biochemistry. 1981; 20: 4878-4883Crossref PubMed Scopus (26) Google Scholar). They also showed that the inducer does not affect the half-life of the mRNA and concluded that it must increase activity by promoting RNA synthesis or processing. Terry Andreone performed a set of experiments, using H4IIE cells treated with various combinations of Bt2cAMP and insulin, that helped set the stage for our future research. The cyclic nucleotide causes proportionate changes in the rate of synthesis of PEPCK and mRNAPEPCK activity (33Andreone T.L. Beale E.G. Bar R.S. Granner D.K. Insulin decreases phosphoenolpyruvate carboxykinase (GTP) mRNA activity by a receptor-mediated process.J. Biol. Chem. 1982; 257: 35-38Abstract Full Text PDF PubMed Google Scholar). Physiologic concentrations of insulin prevent this induction through a receptor-mediated process and also inhibit basal mRNAPEPCK activity, an observation that provides evidence of the dominant role insulin plays in the regulation of this important enzyme. Elmus and Jim Hartley cloned cDNAPEPCK, proved its authenticity, and used it to show that the hormonally modul