Biologic Properties of the 20K. Dalton Variant of Human Growth Hormone: A Review

Transcription

1 Endocrinol. Japon. 1987, 34(Suppl. No.1), Biologic Properties of the 20K. Dalton Variant of Human Growth Hormone: A Review U. J. LEWIS, E. MARKOFF, F. L. CULLER, A. HAYEK AND W. P. VANDERLAAN The Whittier Institute for Diabetes and Endocrinology La Jolla, California USA Summary 1. The hgh 20K variant accounts for 10-15% of the hgh in the pituitary gland and is, therefore, the second most abundant hormone in the gland. 2. Although no specific RIA exists for hgh 20K, which must be measured by a combination of electrophoretic and immunological techniques, the following conclusions appear warranted. During provocative in vivo secretion, the ratio of hgh 20K to hgh 22K in blood is similar to that found in the pituitary gland. During basal in vivo secretion of hgh, the ratio of hgh 22K to hgh 20K can be quite different. A differential secretion rate for hgh 22K and hgh 20K was noted during in vitro stimulation of pituitary cells with GHRH. A six-fold rise in hgh 20K contrasted with only a twofold rise in hgh 22K. 3. The 20K dalton variant has full growth promoting activity and stimulates production of somatomedins even though binding hgh 20K to membrane receptors for hgh is lower than that observed with hgh 22K. 4. In vitro insulin-like activity of hgh 20K on adipose tissue is either absent or at best 10-15% of that seen with hgh 22K, whereas in vivo insulin-like activity of hgh 20K is 30-40% of that observed with hgh 22K. 5. At high doses, hgh 20K exhibits about the same anti-insulin activity as does hgh 22K. The 20K dalton variant of human of small amounts of the variant from electrophoresis gels permitted tests that indicated growth hormone(hgh 20K), a lower molecular weight variant of the major form of the substance to be a bioactive, immunoreactive form of hgh(singh et al., 1974), hgh(hgh 22K), was first detected in human pituitary extracts by means of electrophoresis results which encouraged us to pursue its isolation. Once isolated, structure analysis once in molecular weights, the two forms indicated that the variant was identical to are easily resolved by gel electrophoresis in hgh 22K except that a 15 amino acid segment, residues 32-46, was missing(chapman sodium dodecyl sulfate, a technique which, in the absence of a specific assay for the et al., 1981; Lewis et al., 1980). A mechanism for the deletion of this segment was variant, provided a procedure to follow purification of the hormone(lewis et al., proposed by Wallis(1980) and later confirmed by DeNoto et al.(1981). The 1978; Chapman et al., 1981; Singh and Lewis, 1981; Closset et al., 1983). Elution process, illustrated in Fig. 1, involves an

2 74 LEWIS et al. Endocrinol. April 1987 Japon. Fig. 1. Structural relationships of hgh 22K and hgh 20K with diagramatic representation, of the alternative splicing pathways for precursor mrna to produce the two variants: The represents codons in the exons;, codons in the B intron. Details are presented in the text. alternative splicing pathway in the processing of the precursor mrna for hgh 22K. It can be seen in the production of hgh 20K that 45 nucleotides, which are normally preserved in the mature mrna of hgh 22K, are removed along with the B intron. When the shortened mrna is translated, the resulting hgh lacks residues and has a molecular weight near 20K instead of 22K. The 20K dalton variant is only one of several forms of hgh detected in pituitary extracts, but still an explanation for the need for the multiple forms is not apparent. There is a tendency to think that because these are minor components compared to the major 22K-dalton form they have minor significance. The complicating fact is that the amount of hgh in the pituitary gland is markedly out of proportion to other pituitary hormones. This is shown in Table 1.

3 Vol.34, S. R. No.1 BIOLOGIC PROPERTIES OF 20K hgh 75 Table 1. Concentrations of various hormones in the human pituitary gland and plasma the structure shown in Fig. 2. The arrow G-PRL=glycosylated prolactin indicates the location of the 15 amino acid deletion ih hgh 20K. An NH2-terminal Cys was added to permit cyclization and greater It can be seen also that there is no correlation between the plasma concentration and the amount stored in the pituitary gland. Minor forms of hgh could therefore function physiologically even though they represent only a small proportion of total hgh in the pituitary gland. This report will review what is known of hgh 20K and will show that its pattern of activities suggests that this variant may have an important and distinct metabolic role. Measurement An RIA for measurement of hgh 20K has not been developed because of difficulty in producing a specific antibody to the variant. Even when immunization is done with hgh 20K, the cross-reactivity of the antibody with hgh 22K is too great to be useful for measurement. The hgh 20K shows less immunoreactivity than hgh 22K toward polyclonal antidodies produced with hgh 22K as the antigen (Lewis et al., 1978; Hizuka et al., 1982; Gomez et al., 1984; Surowy et al., 1984; Hennen et al., 1985), suggesting a different conformation for the smaller variant; but the difference is insufficient to enable one to produce an antibody that recognizes solely hgh 20K. Attempts to produce both monoclonal and polyclonal antibodies have been made, and it is curious that antibodies that measure only hgh 22K have been produced, but the reverse situation has not been achieved. In an attempt to enhance the specificity of the immune response by limiting the antigenic epitopes, a peptide, characteristic of hgh 20K but not of hgh 22K, was synthesized by Dr. Nicholas Ling at the Salk Institute, La Jolla, and used for immunization. The peptide had exposure of the center Phe-Asn-sequence. The peptide was coupled through the tyrosine to albumin by reaction with diazotized benzidine. Unfortunately, the antibodies that were produced with this peptide still recognized hgh 22K as well as hgh 20K EA novel approach is sorely needed to generate an antibody that is specific for the variant. As will be discussed below, electrophoretic separation of hgh 22K and hgh 20K is the only procedure that has provided data on the relative concentration of the two forms in the pituitary gland and plasma. This is possible because of the lower molecular weight and/or more basic isoelectric point for hgh 20K ERIA or immunoblotting identifies the two forms as growth hormones; then on the basis of electrophoretic mobility, either hgh 22K or hgh 20K is indicated. Occurrence The hgh 20K variant is the second most abundant hormone in the pituitary gland. By electrophoresis, hgh 20K was detected in the pituitary gland of the newborn(2 day), young adult(24yr), and the elderly(70yr) (Lewis et al., 1978). Smal et al.(1984) carefully studied 12 human pituitary adenomas for hgh 20K content. They reported hgh 20K to be very nearly 10% of the hgh in 7 of the glands. It was not clear, how-

4 76 LEWIS et al. Endocrinol. April 1987 Japon. Fig. 2. Analog of a synthetic flanking peptide of hgh20k. The amino acid sequence is found in hgh20k except for the NH2-terminal *Cys. This Cys permitted cyclization which provided greater exposure of the Phe-Asn region when the peptide was coupled to albumin. ever, what the ratios of the two forms were in the other 5 glands. Regarding their conclusion that synthesis of hgh22k and hgh2ok does not appear to be altered in hgh with various secretory stimuli. Similar results were found with plasma from pathological conditions, we think many more glands must be examined before this acromegalics. These investigators used affinity chromatography to remove the hgh is established. In 5 pituitary glands from 4 patients with pituitary disease and 1 with from plasma, electrophoresis to separate the a normal gland, Baumann and MacCart forms, and RIA to detect hormone in the (1982) found that the amount of hgh 20K electrophoretic pattern. Later it was reported (Baumann et al., 1985a) that during secreted during organ culture was less than 5% of all the hgh. Talamantes et al. spontaneous secretory episodes, hgh 20K amounted to about 20% of the hgh. Even as compared to hgh22k during in vitro secretion studies with tissue from 3 cases of pituitary tumor. Using pituitary cell cultures, Markoff et al. (1986) found by immunoblotting that 30% of the secreted hgh under basal conditions was hgh 20K. Another interesting observation made by Markoff and associates was that during GHRH stimulation of the cells, there was a 6-fold rise in hgh20k and only a 2-fold rise in hgh22k. This indicates that there may be preferential release of the variant in certain situations. Measurements of the concentration of hgh20k in plasma have been made. Baumann et al. (1983) and Baumann and Stolar (1986) found hgh20k to be 7-16% of all the hgh after provocative stimulation of more interesting was that the form of hgh observed during the basal state was highly variable in normal individuals. The major 22K form was at times absent under this condition with appearance of higher and lower molecular weight substances ; and in one individual, hgh20k could be considered the major component. It appears that, during provocative or spontaneous bursts of secretion, hgh20k accounts for 5-30% of the hgh immunoreactivity ; but in the basal state there is great variability, it can be absent or appear as the major form. Bercu et al. (1986) recently reported that provocative release of hgh does not always

5 Vol.34, S. R. No.1BIOLOGIC PROPERTIES OF 20K hgh reflect endogenous secretion of hgh; this fact must be considered when investigating the concentrations of hgh 20K in serum in relation to possible physiologic significance. Markoff et al.(1986) and Sinha et al.(1986), using immunoblotting on nitrocellulose paper to measure hgh 20K in plasma, detected hgh 20K under a variety of conditions, but it was always present at a concentration of about 10% of that of hgh 22K.Multiple forms of hgh were found in urine(baumann and Abramson, 1983), hgh 20K being among them and amounting to about 10% of all the hgh. What remains unknown, however, is how hgh 20K varies over a 24 hr period with regard to food intake, obesity, diabetes and other states that alter metabolic reactions. The expression of a hgh 20K-like protein in the human placenta was reported by Hennen et al.,(1985). This substance could be measured by an antibody that recognized both hgh 22K and hgh 20K, but the hgh-like substance was not detected by an antibody that detected only hgh 22K. The conclusion was that the material had a conformational structure more like hgh 20K than hgh 22K. Because the hgh-like substance behaved as a more basic protein than the placental lactogen, the investigators suggested that it might be a 20K-dalton variant of the hgh-v gene(seeburg, 1982). This member of the hgh family of genes, if expressed, would produce an hgh with 13 amino acid substitutions. Such a form has never been detected in the pituitary gland. Detection of a 20K dalton variant of GH in species other than man has been made only in mouse pituitary glands(sinha and Gilligan, 1984). This murine GH-like substance exhibited a similarity to mouse GH by peptide mapping but showed no immuno-crossreactivity. Although a bovine 20K dalton variant of GH has not been identified, Hampson and Rottman(1986) found an mrna for bovine growth hormone in the pituitary gland that was produced by an entirely different alternative mrna processing procedure. The D intron had not been removed during splicing and only the first 50 nucleotides of exon 5 were present. If translated, the protein would have an amino acid sequence different from bovine growth hormone from residue 125 to the COOH-terminus and the chain length would be 42 amino acids longer. The molecular weight would be 27K instead of 22K. Alternative splicing of precursor mrna may, therefore, prove to be a general procedure for production of GH variants. Receptor binding activity The binding of hgh 20K to membrane receptors is different in various tissues. If liver or mammary gland receptors for hgh are used, there is definite binding but a lower affinity as compared to hgh 22K. The lower binding has been reported to be from 10 to 60% of that observed with hgh 22K (Closset et al., 1983; Hizuka et al., 1982; Sigel et al., 1981; Wohnlich and Moore, 1982). Hughes et al.(1983) proposed a subset of receptors, with high affinity for hgh 20K but with low binding of hgh 22K. The hgh receptor of IM-9 lymphocytes presents a less complicated situation. Smal et al.(1985) found that hgh 20K binds less well than hgh 22K to this receptor, and this could be explained by a simple lower association rate and affinity. This is not the case with the hgh receptor of rat adipocytes. Here Smal et al.(1986a, 1986b) found that there was essentially no binding of hgh 20K, and the lack of binding correlated well with the almost complete absence of in vitro insulin-like activity as measured by stimulation of lipogenesis. This correlation encourages us to investigate further the significance of the lower binding affinity of hgh 20K in liver. Alteration of a postreceptor reaction as the result of lower binding may result in a yet undetected difference in biologic activity for the two forms. Because hgh 20K and hgh 22K exhibit

6 LEWIS et al. Endocrinol. April 1987 Japon. different receptor binding activities in adipose tissue, it is reasonable to predict that differences in activities will be found in other tissues. Growth promoting activity The hgh 20K variant has essentially full growth-promoting activity in hypophysectomized rats(lewis et al., 1978; Closset et al., 1983; Kostyo et al., 1986). Both body weight gain and increase in the width of tibial line were measured. Furthermore, hypophysectomized rats treated with hgh 20K showed an increase in serum somatomedin activity(spencer et al., 1981; Mosier and Lewis, 1982) similar to that seen with hgh 22K. This growth-promoting activity for hgh 20K must be reconciled with its attenuated insulin-like activity in vivo and an essential absence of stimulating insulin-like effects in adipose tissue in vitro(see below). The growth-promoting activity measurements were short term studies in rats. Examination of the long term growth promoting effects of hgh 20K in hypopituitary children would be most helpful in addressing the question of the importance of the insulinlike activity of hgh in the growth process. In vitro activities Table 2 summarizes the reports made on the in vitro insulin-like activities of hgh 20K. The first report made by Frigeri et al.(1979) made use of a single point test(50 Đg/animal) in measurement of oxidation of glucose to CO2 in adipose tissue of hypophysectomized rats. The response at that dose was not statistically different from the control value, whereas hgh 22K produced a significant increase in CO2 production. We have repeated the experiments with hgh 20K isolated from pituitary glands recently made available for investigative work by the National Hormone and Pituitary Program. A at best we found hgh 20K to be 10% as active as hgh 22K in stimulating CO2 production from glucose. An accurate comparison of potencies for the two forms was not possible because the dose response curves were not parallel. Using essentially the same assay Goodman et al.(1986) reported a loss of 80-90% of this in vitro insulin-like activity when compared with hgh 22K. Similarly, Kostyo et al.(1986) found about an 80% loss of activity in hgh 20K. Using lipogenesis in rat adipocytes as an indication of insulin-like activity, Smal et al.(1986a and 1986b) found hgh 20K to be only 3% as active as hgh 22K. As mentioned above, this low activity correlated with loss of binding to membrane receptors. The results indicate that hgh 20K differs markedly from hgh 22K in in vitro insulinlike activities in adipose tissue. As shown below, the variant also exhibits a lower in vivo insulin-like activity with respect to the decrease in free fatty acid concentration in serum. Whether these differences are related to a preferential need for one form or the other is still unknown. Schwartz and Foster(1986) did not find a difference between hgh 22K and hgh 20K in ability to stimulate glucose oxidation in Table 2. In vitro insulin-like activity of hgh 20K

7 Vol.34, S. R. No.1 BIOLOGIC PROPERTIES OF 20K hgh 79 Table 3. In vivo insulin-like activity of hgh20k Early 1-2 h decrease in serum glucose and FFA used in all experiments. 3T3 adipocytes. This may be an indication ing FFA and about 40% as active in de- that the pathways for oxidation of creasing blood glucose. Tinsley et al.(1986) glucose in rat adipose tissue and 3T3 cells recently reported results with biosynthetic are different. Morikawa et al.(1984) found hgh20k that are only slightly different from ours. We noted a greater loss of activity for hgh20k in its effect on FFA than on glucose whereas Tinsley et al.(1986) found As was found with purified hgh22k the reverse. Nonetheless the two studies indicate that 60-70% of the in vivo insulinlike, hgh20k failed to stimulate lipolysis in rabbit adipose tissue(bowden et al., 1985). Im- activity characteristic of hgh22k is eliminated by deletion of the se- pure preparations of hgh22k did produce lipolysis, but this was attributed to contaminating substances. In vivo activities Table 3 provides a summary of the reports made on the in vive insulin-like activity of hgh20k.in the early report by Frigeri et al.(1979) no effect of hgh20k on lowering blood glucose and free fatty acids(ffa) in hypophysectomized rats was noted. In the study only a single 50 Đg dose was used. The same amount of hgh22k was quite effective in the assay. We repeated these experiments with increased doses of Đg(to be published), and, rather than showing no in vivo activity, hgh20k produced an attenuated response as compared to hgh22k. The hgh20k was about 30% as effective as hgh22k in lower- quence. In a limited study of hypopituitary children, Cutler et al.(manuscript submitted) found that a dose of 2mg of hgh20k failed to lower serum FFA whereas the same dose of hgh22k produced a 37% decline in FFA. At this dosage neither hgh22k nor hgh20k had an effect on blood glucose, an indication that at this concentration hgh has less effect on glucose metabolism than on lipid metabolism. The study was done with pituitary hgh20k, and the work had to be terminated before higher doses were tried because of the concern of possible viral contamination of the hormone. The result does support the finding that in rats hgh20k has less insulin-like activity than does hgh22k. The exact extent of the loss will have to await testing with biosynthetic material.

8 80 LEWIS et al. Endocrinol. April 1987 Japon. The retention by hgh20k of 30-40% of the in vivo insulin-like activity in hgh22k is most certainly related to the lower but not complete loss of receptor binding of hgh20k by liver. The almost complete loss of binding activity in adipose tissue correlates well with the 97% loss of in vitro insulin-like activity(smal et al., 1986b). It will be important to measure in vitro activity of hgh20k in liver so a correlation of activity and binding can also be made with this tissue. To explain the lower receptor binding but unaltered growth promoting activity for hgh20k, Baumann et al.(1985b) offered the explanation of a decreased metabolic clearance rate for the variant as compared to hgh22k. The suggestion was that because hgh20k circulated longer, its contribution to growth promotion was greater. This is an interesting explanation because it would provide an answer for the equal growth-promoting response produced by the two forms, but there is still the lower in vivo insulin-like activity of hgh20k to be accounted for. Also, aggregation with lowered renal clearance was used to help explain the longer half life for hgh20k. Our experience, which has been that aggregation of hgh20k lowers growth-promoting properties, implies that the circulating hormone would lose even more of its receptor binding activity but also argues against Baumann's contention that hgh20k retains full growth promoting activity. Diabetogenic activity Obviously one cannot refer to a substance being diabetogenic in physiological dosage. On the other hand a pituitary peptide could have anti-insulin or hyperglycemic effects as part of the complex regulation of metabolism. Because" diabetogenic activity" is the accepted jargon, we use the expression, but reluctantly. There have been conflicting reports as to whether or not hgh20k is diabetogenic, that is, anti-insulin with a rest iting hyperglycemia. Examining these reports, one sees that the discrepancies are a result of differences in dosage of hormone used rather than in a conflict of data. If given in large enough quantities, both hgh22 and hgh20k produce a lowered toleranc, to glucose, but we consider these quantities to be so large as to lack physiological significance. In the dog we found that a dosage of 1mg hgh per kg per dog was needed to give a hyperglycemi response(lewis et al., 1977). Rosenfeld et al.(1982) produced glucose intolerance in man with a total of 32mg hgh in 4 days, a dosage far in excess of that required for growth promotion. Lostroh and Krahl(1976) found that 500ƒÊg hgh per day for 10 days was needed to produce glucose intolerance in the ob/ob mouse, a dosage equivalent to 3.5g hgh for a 70kg person; one fifth that amount, still an enormous dose, produced no glucose intolerance in the ob/ob mouse. Kostyo et al.(1984) found that in the ob/ob mouse 50ƒÊg hgh per day for 3 days was effective in producing glucose intolerance, but, again, this amounts to some 105mg hgh for a 70 kg man. Using the yellow obese mouse Frigeri et al.(1983) could not elicit hyperglycemia with the large dose of 200ƒÊg hgh per mouse. Louis and Conn(1972) were among the first to advance the view that another substance in the pituitary gland was the diabetogenic agent. Their conclusion followed their observation that 1mg hgh per kg failed to produce glucose intolerance in the dog. Lostroh and Krahl(1976), finding in the ob/ob mouse little diabetogenic effect from intact hgh, showed that after the hormone was partially digested with pepsin, hyperglycemic activity increased. Sonenberg et al.(1965) had already demonstrated that limited proteolytic digestion of bovine GH generated diabetogenic activity when the material was tested in diabetic patients. Intact bovine GH was inactive as had previously been shown by Lewis et

9 Vol.34, S. R. No.1BIOLOGIC PROPERTIES OF 20K hgh Table 4. Diabetogenic activity of hgh 20K * Clearance of blood glucose after glucose tolerance test. al.(1950). Our approach to this problem (Lewis et al., 1977) was first to attempt the purification from pituitary extracts of a diabetogenic factor that was more potent than hgh 22K and secondly to intentionally cleave hgh by proteolysis to produce a modified form that had greater diabetogenic activity than the starting preparation. Experiments showed that a more potent hyperglycemic fraction could be recovered from preparations of clinical grade hgh by ionexchange chromatography. It produced 100% or greater rise in blood glucose above control values when tested at 0.1 mg/kg whereas the residual purified hgh produced only a 35% rise at 1 mg/kg. Limited proteolysis with subtilisin enhanced the diabetogenic activity of the purified hgh. Our conclusion was that hgh had weak hyperglycemic activity in the dog which could be increased by proteolysis and that the highly active fraction detected in clinical grade hgh was due to proteolytically altered forms possibly related to the subtilisincleaved forms. Additional support for the idea that intact growth hormone is not the most potent diabetogenic substance in the pituitary gland comes from the work of Hart et al.(1984). They found that bovine GH with greatest growth promoting activity was not the most diabetogenic fraction isolated. Our position on the diabetogenic activity of hgh is that the intact hormone is a prohormone for a much more active fragment. As ACTH has 300 times the activity of pro-acth/endorphin(gasson, 1979) so we believe intact hgh must undergo cleavage to produce a peptide that is truly potent in diabetogenic activity. To return then to the diabetogenic activity of hgh 20K E Table 4 summarizes the results. Lewis et al.(1981) found no abnormality in glucose utilization in the dog when hgh 20K was given at 0.25 mg/kg. The same dose of hgh 22K produced a slight degree of glucose intolerance. This dose was equivalent to giving 17.5 mg of hgh to a 70 kg individual. Shaar et al.(1986), using the same type of assay in the dog, found hgh 20K to be hyperglycemic when administered at 0.5 mg/kg, a dose equivalent to the 32 mg used by Rosenfeld et al.(1982) to produce intolerance in man. Kostyo et al.(1986) reported hgh 20K to be diabetogenic when 75 Đg was given to ob/ob mice over a period of 3 days, again an enormous dose. Agajanian et al.(1986) reported diabetogenic activity for hgh 22K and hgh 20K when administered at dosages considerably less than those discussed above. The hor-

10 LEWIS et al. Endocrinol. April 1987 Japan. mones were given intravenously to dogs for 12 days at a rate of 20ƒÊg/kg/day which would equate to 1.4mg/day to a 70kg individual. With this dosage there was no impairment of glucose tolerance. Both forms did, however, induce a marked insulin resistance and they stimulated insulin release. A curious aspect of these results is that the exogenously administered hgh22k raised the serum concentrations of hgh22k to about 4ng/ml and this was sufficient to produce a marked insulin resistance. The normal 24h integrated concentration of hgh in children was calculated to be near 7ng/ml (Zadik et al., 1985) but this does not cause any apparent problem with insulin resistance. An explanation may be that hgh is more active in dogs than in man, or one could speculate that there is a difference between the exogenously administered hormone and the hgh secreted under physiologic control. A report by Baumann et al. (1985a) may be important here. Those investigators found a great deal of low molecular weight immunoreactive forms of hgh but little hgh22k in the basal secretory state, indicating that significant alteration of hgh occurs. Another question raised by the work of Agajanian et al. (1986) concerns the hyperinsulinemia noted when hgh was administered. Our partially purified diabetogenic factor (Lewis et al., 1977) produced both hyperglycemia and hyperinsulinemia, whereas when purified to a preparation free of immunoreactive hgh (Singh et al., 1982), hyperinsulinemia was no longer detected. Louis and Tai (1976) found similar results with their diabetogenic factor. This presents a puzzling situation, that is, hyperglycemia without concomitant hyperinsulinemia. A possible explanation is that the glucose intolerance produced by the peptide may be partially the result of inhibition of insulin secretion in addition to production of insulin resistance. This illustrates the complex nature of the metabolic effects of hgh, but by studying hghrelated peptides found in pituitary extracts, we think a better understanding of the actions of hgh will be obtained. Several investigators (Cameron et al., 1985;Schwartz and Foster, 1986;Agajanian et al., 1986) have quoted us as saying that hgh22k lacks intrinsic diabetogenic activity. One report (Kostyo et al., 1984) comments that we failed to detect intrinsic diabetogenic activity in hgh. Actually the paper by Lewis et al. (1977) reported that with a dose of 1mg/kg, a 35% increase in glucose intolerance was produced by hgh, a value that agrees well with the 26% increase noted by Shaar et al. (1986) when 0.5mg/kg of biosynthetic hgh22k was tested. It would appear that our statement that we do not think intact hgh22k is the principle diabetogenic substance of the pituitary has been misinterpreted. Our hypothesis has been that the weak diabetogenic activity detected in hgh can be enhanced by proteolysis and that, by this alteration, a potent substance is produced which, even in small quantities, is capable of causing glucose intolerance. A low molecular weight fraction that produced glucose intolerance has been recovered from clinical grade hgh, and we postulate that the active component is a fragment of hgh. With increasing availability of modified forms and fragments of hgh, these conjectures can be tested in the laboratory to give us a better understanding of the peculiar insulin-like and anti-insulin activities of growth hormone. With this will come an indication of whether the two variants of hgh exert the same magnitude of these two activities in normal physiologic situations. References Agajanian, T., M. Adel and R. N. Bergman (1986). Both recombinant DNA-derived 22K and 20K-human growth hormone(hgh) possess diabetogenic activity during chronic low

11 Vol.34, S. R. No.1 BIOLOGIC PROPERTIES OF 20K hgh dose infusion in dogs. Endocrinology 118 (Suppl.), 218(Abstract). Baumann, G. and J. G. MacCart(1982). Growth hormone production by pituitary glands in organ culture: Evidence for predominant secretion of the single-chain 22,000 molecular weight form (Isohormone B). J. Clin. Endocrinol. Metab. 55, Baumann, G. and E. C. Abramson(1983). Urinary growth hormone in man: Evidence for multiple molecular forms. J. Clin. Endocrinol. Metab. 56, Baumann, G., J. G. MacCart and K. Amburn (1983). The molecular nature of circulating growth hormone in normal and acromegalic man: Evidence for a principal and minor monomeric form. J. Clin. Endocrinol. Metab. 56, Baumann, G., M. W. Stolar and K. Amburn (1985a). Molecular forms of circulating growth hormone during spontaneous secretory episodes and in the basal state. J. Clin. Endocrinol. Metab. 60, Baumann, G., M. W. Stolar and T. A. Buchanan (1985b). Slow metabolic clearance rate of the 20,000-dalton variant of human growth hormone: Implications for biological activity. Endocrinology 117, Baumann, G. and M. W. Stolar(1986). Molecular forms of human growth hormone secreted in vivo: Nonspecificity of secretory stimuli. J. Clin. Endocrinol. Metab. 62, Bercu, B. B., D. Shulman, A. W. Root and B. E. Spiliotis(1986). Growth hormone(gh) provocative testing frequently does not reflect endogenous GH secretion. J. Clin. Endocrinol. Metab. 63, Bowden, C. R., K. D. White, U. J. Lewis and G. F. Tutwiler(1985). Highly purified human growth hormone fails to stimulate lipolysis in rabbit adipocytes in vitro or in rabbits in vivo. Metab. 4, Cameron, C. M., J. L. Kostyo and H. Papkoff (1985). Nonmammalian growth hormones have diabetogenic and insulin-like activities. Endocrinology 116, Chapman, G. E., K. M. Rogers, T. Brittain, R. A. Bradshaw, O. J. Bates, C. Turner, P. D. Cary and C. Crane-Robinson(1981). The 20,000 molecular weight variant of human growth hormone. Preparation and some physical and chemical properties. J. Biol. Chem. 256, Closset, J., J. Smal, F. Gomez and G. Hennen (1983). Purification of the 22,000- and 20,000- mol. wt. forms of human somatotropin and characterization of their binding to liver and mammary binding sites. Biochem. J. 214, DeNoto, F. M., D. D. Moore and H. M. Goodman(1981). Human growth hormone DNA, sequence and mrna structure: Possible alternative splicing. Nucleic Acid Res. 9, Frigeri, L. G., S. M. Peterson and U. J. Lewis (1979). The 20,000-dalton structural variant of human growth hormone: Lack of some early insulin-like effects. Biochem. Biophys. Res. Commun. 91, Frigeri, L. G., G. L. Wolff and G. Robel 1983). Impairment of glucose tolerance in yellow (AvY/A)(BALB/c X VY) F-1 hybrid mice by hyperglycemic peptide(s) from human pituitary glands. Endocrinology 113, Gasson, J. C.(1979). Steroidogenic activity of high molecular weight forms of corticotropin. Biochemistry 18, Gomez, F., G. Pirens, C. Schaus, J. Closset and G. Hennen(1984). A highly sensitive radioimmunoassay for human growth hormone using a monoclonal antibody. J. Immunol. 5, Goodman, H. M., G. Grichting and V. Coliro (1986). Growth hormone action on adipocytes. In Human Growth Hormone (S. Raiti and R. A. Tolman, eds.) Plenum Pub. Corp., New York, pp Hampson, R. K. and F. M. Rottman(1986). A potential variant of bovine growth hormone resulting from non-splicing of an intron. Fed. Proc. 45, Hart, I. C., L. A. Blake, P. M. E. Chadwick, G. A. Payne and A. D. Simmonds(1984). The heterogeneity of bovine growth hormone. Extraction from the pituitary of components with different biological and immunological properties. Biochem. J. 218, Hennen, G., F. Frankenne, J. Closset, F. Gomez, G. Pirens and N. El Khayat(1985). A human placental GH: Increasing levels during second half of pregnancy with pituitary GH suppression as revealed by monoclonal radioimmunoassays. Int. J. Fertil. 30, Hizuka, N., C. M. Hendricks, G. N. Pavlakis, D. H. Hamer and P. Gorden(1982). Properties of human growth hormone polypeptides:

12 LEWIS et al. Endocrinol. April 1987 Japan. Purified from pituitary extracts and synthesized in monkey kidney cells and bacteria. J. Crin. Endocrinol. Metab. 55, Hughes, J. P., E. Tokuhiro, J. Steven, A. Simpson and H. G. Friesen(1983). 20K is bound with high affinity by one rat and one of two rabbit growth hormone receptors. Endocrinology 113, Kostyo, J. L., S. E. Gennick and S. E. Sander (1984). Diabetogenic activity of native and biosynthetic human growth hormone in obese (ob/ob) mouse. Am. J. Physiol. 246, E350-E Kostyo, J. L., C. M. Cameron, K. C. Olson, A. J. S. Jones and R.-C. pal(1986). Biosynthetic 20-kilodaltaon methionyl-human growth hormone has diabetogenic and insulin-like activities. Proc. Natl. Acad. Sci. USA 82, Lewis, R. A., R. Klein and L. Wilkins(1950). The effect of pituitary growth hormone in dwarfism with osseous retardation and hypoglycemia and in a cretin treated with thyroid. J. Clin. Invest. 29, Lewis, U. J., R. N. P. Singh, W. P. VanderLaan and G. F. Tutwiler(1977). Enhancement of the hyperglycemic activity of human growth hormone by enzymic modification. Endocrinology 101, Lewis, U. J., J. T. Dunn, L. F. Bonewald, B. K. Seavey and W. P. VanderLaan(1978). A naturally occurring structural variant of human growth hormone. J. Biol. Chem. 253, Lewis, U. J., L. F. Bonewald and L. J. Lewis (1980). The 20,000-dalton variant of human growth hormone: Location of the amino acid deletions. Biochem. Biophys. Res. Commun. 92, Lewis, U. J., R. N. P. Singh and G. F. Tutwiler (1981). Hyperglycemic activity of the 20,000- dalton variant of human growth hormone. Endocr. Res. Commun. 8, Lostroh, A. J. and M. E. Krahl(1976). Diabetogenic peptide from human growth hormane: Partial purification from peptide digest and long term action in ob/ob mice. Proc. Natl. Acad. Sci. USA 73, Louis, L. H. and J. W. Conn(1972). Diabetogenic polypeptide from human pituitaries similar to that excreted by proteinuric diabetic patients. Metab. Clin. Exp. 21, 1-9. Louis, L. H. and T.-Y. Tai(1976). A study on diabetogenic polypeptide. Preliminary report: Isolation of diabetogenic peptide from urine of proteinuric patients and its effect on glucose tolerance in rabbits and dogs. J. Formosan Med. Assoc. 75, Markoff, E., D. W. Lee, F. L. Culler, K. L. Jones and U. J. Lewis(1986). Release of the 22,000- and 20,000-dalton variants of growth hormone in vivo and in vitro by human anterior pituitary cells. J. Clin. Endocrinol. Metab. 62, Morikawa, M., H. Green and U. J. Lewis(1984). Activity of human growth hormone and related polypeptides on the adipose conversion of 3T3 cells. Molec. Cell. Biol. 4, Mosier, H. D. and U. J. Lewis(1982). The 20,000 dalton variant of human growth hormone. Effect on bioassayable somatomedin activity in serum. Hormone and Metab. Res. 14, Rosenfeld, R. G., D. M. Wilson, L. A. Dollar, A. Bennett and R. L. Hintz(1982). Both human pituitary growth hormone and recombinant DNA-derived human growth hormone cause insulin resistance at a postreceptor site. J. Clin. Endocrinol. Metab. 54, Schwartz, J. and C. M. Foster(1986). Pituitary and recombinant deoxyribonucleic acid-derived human growth hormones alter glucose metabolism in 3T3 adipocytes. J. Clin. Endocrinol. Metab. 62, Seeburg, P. H.(1982). The human growth hormone gene family: Nucleotide sequences show recent divergence and predict a new polypeptide hormone. DNA 1, Shaar, C. J., E. L. Grinnan, W. G. Short, J. G. Powell, N. Bryan, K. G. Bemis and F. C. Tinsley(1986). Hyperglycemic activity in dogs of recombinant DNA-derived 20,000 dalton variant of methionyl human growth hormone. Endocr. Res. 12, Sigel, M. B., N. A. Thorpe, M. S. Kobrin, U. J. Lewis and W. P. VanderLaan(1981). Binding characteristics of a biologically active variant of human growth hormone(20k) to growth hormone and lactogen receptors. Endocrinology 108, Singh, R. N. P., B. K. Seavey and U. J. Lewis (1974). Heterogeneity of human growth hormone. Endocr. Res. Commun 1, Singh, R. N. P. and U. J. Lewis(1981). Procedure for isolation of the 20,000 dalton variant of human growth hormone. Prep. Biochem.

13 Vol.34, S. R. No.1 BIOLOGIC PROPERTIES OF 20KhGH 11, Singh, R. N. P., L. J. Lewis, R. O' Brien, U. J. Lewis and G. F. Tutwiler(1982). Characterization of the pituitary hyperglycemic factor as a low molecular weight peptide. Endocrinology 110(Suppl.), 102(Abstract). Sinha, Y. N. and T. A. Gilligan(1984). A 20K hform of growth hormone in the murine pituitary gland. Proc. Soc. Expel. Biol. Med. 177, Sinha, Y. N., T. A. Gilligan, D. W. Lee, S. C. Baxi and W. P. VanderLaan(1986). Demonstration of 20K growth hormone in human plasma by gel electrophoretic-immunostainingautoradiographic assay(geisaa). Hormone Metab. Res. 18, Smal, J., J. Closset, A. Stevenaert g and G. Hennen (1984). Detection and quantification of 22K and 20K forms of human growth hormone in pituitary adenomas. Program of the Seventh International Congress of Endocrinology, Quebec City, Canada, p. 1456(Abstract). Smal, J., J. Closset, G. Hennen and P. DeMeyts (1985). Receptor-binding and down-regulatory properties of 22,000-Mr human growth hormone and its natural 20,000-Mr variant on IM-9 human lymphocytes. Biochem. J. 225, Smal, J., J. Closset, G. Hennen and P. DeMeyts (1986a). Receptor binding and insulin-like effects of 22K human growth hormone and its 22K variant in the rat adipocytes. Endocrinology 118(Suppl.), 1(Abstract). Smal, J., J. Closset, G. Hennen and P. De Meyts (1986b). The receptor binding properties of the 20K variant of human growth hormone explain its discrepant insulin-like and growth promoting activities. Biochem. Biophys. Res. Commun. 134, Sonenberg, M., C. A. Free, J. M. Dellacha, G. Bonadonna, A. Haymowitz and A. C. Nadler (1965). The metabolic effects in man of bovine growth hormone digested with trypsin. Metabolism 14, Spencer, E. M., L. J. Lewis and U. J. Lewis (1981). Somatomedin generating activity of the 20,000-dalton variant of human growth hormone. Endocrinology 109, Surowy, T. K., R. M. Bartholomew and W. P. VanderLaan(1984). Antigenic sites of human growth hormone and related molecules detected by monoclonal antibodies directed to human growth hormone. Molec. Immunol. 21, Talamantes, F., J. Lopez, U. J. Lewis and C. B. Wilson(1981). Multiple forms of growth hormone: Detection in medium from cultured pituitary adenoma explants. Acta Endocrinologica 98, Tinsley, F. C., E. L. Grinnan, S. H. Baker, J. G. Powell, K. G. Bemis and C. J. Shaar(1986). The 20,000 dalton structural variant of recombinant DNA-derived methionyl human growth hormone has early insulin-like effects in hypophysectomized rats. Biochem. Biophys. Res. Commun. 138, Wallis, M.(1980). Growth hormone: Deletions in the protein and introns in the gene. Na-t ure 284, 512. Wohnlich, L. and M. V. Moore(1982). Binding of a variant of human growth hormone to liver plasma membrane. Hormone Metab. Res. 14, Zadik, Z., S. A. Chalew, S. Raiti and A. A. Kowarski(1985). Do short children secrete insufficient growth hormone?. Pediatrics 76,