preparation into a somatotrophic and an adipokinetichyperglycaemic agent

Transcription

1 Pediatrisk forskningsinstitutt, Rikshospitalet, Oslo THE LIPID-MOBILIZING EFFECT OF SOME PITUITARY GLAND PREPARATIONS IV. Subdivision of a human growth hormone preparation into a somatotrophic and an adipokinetichyperglycaemic agent By Olav Trygstad and Irene Foss ABSTRACT A comparison has been made of some physicochemical and metabolic characteristics of a human growth hormone preparation with somatotrophic and adipokinetic-diabetogenic activity (r.hgh), of a purified r.hgh with potent somatotrophic effect but negligible adipokinetic\x=req-\ diabetogenic effect in the fed animal (t.sth), and a human pituitary\x=req-\ hyperglycaemic and lipid-mobilizing factor (LMF) which was prepared from the crude pituitary extract prior to the preparation of t.sth and was without somatotrophic activity. The t.sth was separated into two bands on polyacrylamide gel electrophoresis, and an additional band was observed for r.hgh in the area of the single band obtained for LMF. The r.hgh, t.sth, and LMF preparations had maximum optical absorption at 278 m\g=m\up to ph 10. At a more alkaline ph the absorption spectrum of t.sth was unchanged, whereas an additional absorption peak at 290 m\g=m\appeared for r.hgh and LMF. Tentative molecular weights determined by Sephadex gel filtration were in the range of for r.hgh, for t.sth, and 5500 for LMF. The t.sth had a significantly higher growth promoting activity in the tibia test than r.hgh. LMF had no such activity in doses of 1 mg. The adipokinetic effect in fed rabbits was strong for LMF and r.hgh, but negligible for t.sth, which, however, caused an increase of nonesterified fatty acids in animals deprived of food. The in vitro lipolytic effect on human fat, obtained from fasting females, for LMF, r.hgh, and t.sth was on a per weight basis in the ratio of 100:10:1, respectively. In rabbits

2 daily injections of r.hgh and LMF, but not t.sth, induced a hyperglycaemia which continued for 9 and 23 days respectively. Rabbits given daily injections of r.hgh and LMF got a diabetic response of blood glucose after injection of adrenalin and prednisone, although an increased sensitivity to insulin. In contrast to this, a single injection of LMF gave a decreased sensitivity to insulin in the rabbit. Rabbits treated with t.sth behaved as untreated controls. Preparative ultracentrifugation of r.hgh dissolved in physiological saline gave a precipitate with the characteristics of t.sth, and a supernatant with potent hyperglycaemic-adipokinetic effect in rabbits. It was furthermore possible to subdivide r.hgh into t.sth and an adipotrophic component by acetone precipitation and Sephadex gel filtration. It is discussed whether the r.hgh is an aggregation product of t.sth and a hyperglycaemic-adipokinetic agent, or whether the r.hgh is original and made up of these two components. Raben Sc Westermeyer (1952) presented evidence for the diabetogenic factor of the pig pituitary gland being distinct from the growth hormone. The obser vation has not been adequately confirmed, and Levine 8c Luft (1964) suggested that the growth hormone is composed of two hormonal entities, a somatotrophic component promoting insulin liberation and protein synthesis, and an adipo kinetic moiety with diabetogenic effect. In a more recent paper Raben (1965) considered that human growth hormone is one biologically active substance causing both growth and adipokinesis, and that an excessive action of this hormone might take on a diabetogenic character. This disagreement stimulated the present studies on some human pituitary fractions, and a comparison was made on some biological and physicochemical characteristics of: (i) A dualistic human growth hormone prepared according to Roos et al. (1963) with somatotrophic as well as adipotrophic activity of high potency (Trygstad 1967). (ii) A human growth hormone prepared by the same method but after re moval of a crude lipid-mobilizing fraction from the pituitary gland extract. This modification gave increased somatotrophic activity on a per weight basis, and made the adipotrophic effect negligible in the fed rabbit (Trygstad 1967). (iii) A purified lipid-mobilizing factor prepared from the crude human pituitary lipid-mobilizing fraction. This preparation had no somatotrophic activity and only a trace of adrenocorticotrophic activity. The adipotrophic effect had been observed in human and rabbit fat, and a diabetogenic effect had also been observed in rabbits (Trygstad 1968 a). The influence of these pituitary polypeptides on the blood glucose level in rabbits was studied in more detail because most experimental data have sup ported the view that growth hormone itself is the major diabetogenic factor of the known hormones of the anterior pituitary gland (Engel 8c Kostyo 1964 Greenbcrg 1965).

3 MATERIALS r.hgh, human growth hormone prepared by the method of Roos et al. (1963). t.sth, prepared from human pituitary glands by the same method after removal of a lipid-mobilizing fraction (Trygstad 1967), this preparation was designated somatotrophic hormone (STH), distinctive from r.hgh. LMF, human pituitary lipid-mobilizing factor, identical to the previously described d.lmf-1 (Trygstad 1968 b). ACTH, adrenocorticotrophic hormone, 'Ferring' (Malmö). prepared from pig pituitary glands and containing per cent of the 1-39-ACTH peptide. The pituitary polypeptides employed were stored lyophilized at -20 C. Roman numerals indicate batch number of material prepared in this institute (r.hgh, t.sth, and LMF). Insulin Actrapid, 'Novo' (Copenhagen) contains less than per cent glucagon bv weight. The solution employed contained four international units (IU) of insulin per ml. Injectabile adrenalini intracardiale 0.01 /o, (Formula Norvegica) was conserved with 0.1 per cent methyli p-oxibenzoas and was diluted with 9 parts of physiolo gical saline before use. Prednison, 'Leo' (Copenhagen), pro injeclione, 10 mg per ml. METHODS Metabolic studies Evaluation of somatotrophic effect (rats) Hypophysectomized female albino Wistar rats, obtained from one supplier, were used, and the growth promoting effect was determined by the tibia test of Greenspan et al. (1949). Evaluation of adipotrophic-hyperglycaemic effect (human fat pads and intact rabbits) Human subcutaneous fat was obtained from two normal females, 24 and 36 years of age, and from a pregnant diabetic, aged 30 years. All were under barbital anaesthesia during performance of an explorative laparotomy for barrenness in the normal females who had fasted overnight, and of a Caesarean section in the pregnant diabetic who got glucose and insulin before the operation. Rabbits obtained from one breeder, of either sex, and of body weight of about 4 kg were kept on rabbit chow pellets ad libitum in the laboratory stall for at least four weeks before and usually throughout the experiments. For 14 hours preceding the in sulin and adrenalin tolerance tests and during these, they received no food. A group of rabbits fasted also 16 hours prior to and 7 hours following an injection of t.sth because Raben Se Hollenberg (1959) observed that the adipokinetic effect of somato trophin was suppressed by food. The determination of non-esterified fatty acids (NEFA) by the Dole procedure and the employed techniques for in vivo and in vitro evaluation of adipotrophic activity are described elsewhere (Trygstad 1967). The different materials administered subcutaneously were dissolved in 5 ml physio logical saline, and injected between the shoulder blades of the rabbit. Previous to this

4 a blood sample for determination of serum NEFA and/or blood glucose (Hultman 1959) had been obtained from the marginal ear vein. For evaluation of hyperglycaemic effect the pituitary polypeptides were given sub cutaneously daily for at least 13 days or until the blood glucose level was normalized. The r.hgh and t.sth preparations were given in doses of 2.0 mg and 0.5 mg to groups of three rabbits. Four rabbits were given LMF in a dosage of 0.1 mg, which is equivalent to 0.5 mg r.hgh on a molecular basis (Fig. 8). In order to exclude a hyperglycaemic effect of an ACTH-like contamination of LMF, four rabbits were treated with 0.01 mg ACTH, which corresponds to more than 1 IU ACTH. Previously the adrenocorticotrophin-like activity of LMF was observed to be less than 0.2 IU per mg (Trygstad 1968 a). Carbohydrate tolerance tests were done on untreated rabbits and on all animals treated with the pituitary polypeptides. Oral glucose tolerance tests had been performed in a preliminary study on rabbits treated with LMF for several days, but the blood glucose increase was similar to that for untreated controls (unpublished observations). This test was therefore not repeated in this work. Furthermore the test was unpleasant because the glucose had to be given by a stomach tube. For the insulin tolerance test a dose of 2 IU insulin was infused in the marginal ear vein. The adrenalin and prednisone tolerance tests were performed by subcutaneous injection of mg adrenalin and 8 mg prednisone respectively. The latter was given on the last day of treatment with 0.5 mg r.hgh, 0.5 mg t.sth, or 0.1 mg LMF. The insulin and adrenalin tolerance tests were done at time marked i and a in Fig. 1. Figs. 2, 3, and 4 give the number of controls performed in each test, and the intervals between the injections and the blood samples obtained for determination of blood glucose concentration (the abscisses). The insulin tolerance test was furthermore carried out in five rabbits which were given a single injection of 0.1 mg LMF subcutaneously 24 hours prior to the insulin infusion. Physicochemical studies The methods for polyacrylamide gel disc-electrophoresis, quantitation of protein by absorbancy measurements, and the calculation of tentative molecular weights by Sephadex G-50 gel filtration have recently been described elsewhere (Trygstad 1967, 1968 a). Technique for subdivision of r.hgh Acetone precipitation was done according to the preparation procedure lor LMF (Trygstad 1968 a). Fifty mg r.hgh was dissolved in 50 ml of 0.1 M phosphate buffer at ph 8.5, added 1 HCI until ph 4.5 and cold acetone until 55 /o saturation (v/v). After centrifugation more acetone was added to the supernatant until 90% saturation (v/v). This precipitate was dissolved in 0.05 m trishydrochloride buffer ph 7.5 con taining 0.1 M potassium chloride, and filtrated through the Sephadex G-50 gel column used for determination of molecular weights. Preparative ultracentrifugation was performed in a Beckman model L ultracentrifuge with three different r.hgh preparations dissolved in physiological saline (1 mg-' ml) at rpm. for 48 hours at 4 C.

5 RESULTS Metabolic studies Somatotrophic activity (rats), Table 1 The hypophysectomized rats treated with 500 and 1000 ßg had a similar mean width of uncalcified tibial epiphyseal cartilage as controls given physio logical saline. The r.hgh and t.sth preparations were considerably more potent than a USP bovine growth hormone reference standard used for com parison. Doses of 40 and 80 µg t.sth were significantly more potent than the same doses of r.hgh. The somatotrophic activity of LMF was less than 1 cent because 10 µg t.sth caused a significant increase of the tibial width. Adipotrophic activity In vivo studies (rabbits), Table 2. Subcutaneous injection of 5 ml physio logical saline v/as given to six fed controls and to four rabbits which had fasted for 16 hours. These fed controls had no increase of serum NEFA, and the slight increase in the fasted animals was considered to be due to the con- per Table 1. Assay of growth promoting activity by the tibia test of two human growth hormone preparations (r.hgh and t.sth) and of a human pituitary lipid-mobilizing factor (LMF), compared with U. S. P. standard of bovine growth hormone. Material and dosage Number of rats Mean tibial width ± standard error (µ ) of the mean Physiological saline 2 ml ± 4 LMF U. S. P. standard r.hgh t.sth 500 (tg 1000 µg 20 µ% 40 µß 80 µ 40 µ * 80 pg** 10 / g 20 µ 40 µ8"' 80 pg** ± 3 ± 8 ± 9 ±11 ± 2 ± 6 ± 8 The significance probability for a and t.sth: * = < ** = : difference in the somatotrophic effect of r.hgh

6 Table 2. The net increase of serum non-esterified fatty acids (NEFA) in fed rabbits following subcutaneous injection of two different human growth hormone preparations (r.hgh and t.sth), of the residue and supernatant obtained by ultracentrifugation of r.hgh (three different preparations), and of a human pituitary lipid-mobilizing factor (LMF). The t.sth was given also to rabbits which had fasted for 16 hours prior to and 7 hours after the injection. Preparation and dosage Fed rabbits: Physiological saline t.sth r.hgh r.hgh residue r.hgh supernatant LMF 5 ml 2 mg 2 mg 2 mg 0.1 mg 0.1 mg 0.01 mg Number of rabbits Mean increase of serum NEFA, meq./l ± standard error of the mean 0.01 ± ** ± * 0.35* ± 0.10 ± * ± * ± * ± 0.03 Fasted rabbits: Physiological saline 5 ml 4 t.sth 2 mg ± * ± 0.04 = < 0.001, compared with the controls. = : , compared with the controls. tinued fasting. The doses employed of the human pituitary polypeptides studied in Table 2 caused a significant elevation of serum NEFA which was at maxi mum in the 2-hour sample. The adipokinetic effect of t.sth and the residue formed by ultracentrifugation of r.hgh was negligible compared with the adipokinetic effect of r.hgh in fed rabbits. The increase of serum NEFA induced by 2 mg of the two first mentioned peptides was even smaller than that of 0.01 mg of LMF. However, the t.sth had a considerable adipokinetic effect in rabbits which had fasted. The protein from the supernatant formed by the ultracentrifugation of r.hgh induced an increase of serum NEFA in the same range as LMF. Daily injections of the pituitary polypeptides did not re duce the body weight of the rabbit. In vitro studies (human fat) Table 3. The net release of NEFA from human fat pads obtained from fasting normal females were apparently equivalent for 0.1 µ% t.sth, 0.01 µg r.hgh and /g LMF per 1.1 ml incubation buffer. The lipolysis in fat pads from the pregnant diabetic was not influenced by addition of 0.1 ßg t.sth per 1.1 ml, although in this instance r.hgh and LMF caused a considerably greater lipolysis than in the normal fat pads.

7 Table 3. In vitro release of non-esterified fatty acids (NEFA) from human subcutaneous fat pads obtained from two normal females, aged 36 and 24 years, and from a pregnant diabetic, aged 30 years, after incubation with two different human growth hormone preparations (r.hgh and t.sth) and with a human pituitary lipid-mobilizing factor (LMF). All assays were run in quadruple. Preparation added µgl\a ml Net release of NEFA, «mol/100 mg Normal female (.36) Normal female (24) 3 hours, mean ± s. e.' Pregnant diabetic female (30) LMF, r.hgh t.sth **± ± ± ± ± :0.06 : ± ± **± ± ± ± **± **± 0.03 * = standard error of the mean. ** = insignificant results, all the other results are significant at the 0.1 per cent level (P < 0.001). Hyperglycaemic activity (rabbits) Table 4, Fig. 1 A single injection of 0.1 mg LMF or 0.1 mg protein from the supernatant formed by ultracentrifugation of r.hgh gave a prolonged hyperglycaemia which persisted for three and four days respectively (Table 4). Subcutaneous injection of 2 mg r.hgh caused hyperglycaemia for two days, though injection of 2 mg t.sth or 2 mg residue obtained by the ultracentrifugation of r.hgh gave no convincing increase of the blood glucose concentration. Daily injections of 0.01 mg ACTH and 0.5 mg t.sth had no influence on the blood glucose level. Treatment with 2 mg r.hgh increased blood glucose concentration for 9 days (P <L 0.001), and with 2 mg t.sth a questionable in crease occurred following the first injection only (P: ), Fig. 1. Daily injections of as little as 0.5 mg r.hgh elevated the blood glucose concentra tion significantly for 9 days. The hyperglycaemia induced by LMF persisted for three weeks. In spite of continued daily injections the blood glucose level normalized, and there was not either observed increase of serum NEFA fol lowing the last injections. When LMF was injected, several weeks after the LMF treatment was stopped, no elevation of blood glucose or serum NEFA was observed in these rabbits. It was not possible to demonstrate LMF-anti-

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9 2 mg HGH (3) 2mg STH (3) mg HGH(3) 0.5mg STH (3) 0.1mg LMF (4) 0.01 mg ACTH M Days Fig. 1. The mean blood glucose concentration ± one standard error of the mean (s. e., vertical bars) in rabbits (numbers in brackets) prior to the daily injection of 0.5 mg and 2 mg HGH (i.e. r.hgh) and STH (i.e. t.sth). of 0.1 mg LMF, and of 0.01 mg ACTH 'Ferring'. The i and a indicate the day of performance of the insulin and adrenalin tolerance test respectively. bodies in sera from the rabbits, although antibodies against r.hgh and t.sth were present (Norman Sc Turter 1968), and the animals had never been in jected with these preparations. Insulin tolerance tests, Fig. 2, Table 5. The untreated controls had minimum concentration of blood glucose in the 20-minute sample, and the normal level was attained after 210 minutes. Rabbits treated with 0.01 mg ACTH and

10 Fig. 2. Flic influence of at least six daily injections of 0.01 mg ACTH 'Ferring', of 0.1 mg LMF, and of 0.5 mg and 2 mg HGH (i. e. r.hgh) and STH (i. e. t.sth) on the hypoglycaemic action of 2 IU insulin (cf. i Fig. 1) given intravenously in rabbits (numbers in brackets). The shaded area indicates the mean blood glucose concentra tion ± one standard error of the mean (s. e.) in 12 untreated controls, and the different curves give the mean blood glucose concentration in the treated animals ± s. e. (ver tical bars). In order to facilitate comparison, the blood glucose results are expressed as percentages of the initial level.

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12 0.5 mg t.sth had responses in the same range. Treatment with 2 mg t.stfl did not influence the appearance of the minimum blood glucose level, but the return to normal was delayed (Fig. 2). The rabbits injected daily with the other pituitary polypeptides had increased sensitivity to insulin with marked, per sisting fall of blood glucose, and the original levels were not attained in the 270-minute sample. On treatment with LMF the blood glucose minimum was observed in the 60-minute sample. However, a decreased sensitivity to insulin was observed if the test was performed 24 hours after a single injection of 0.1 mg LMF (Table 5). Adrenalin tolerance tests, Fig. 3. The blood glucose level was not influenced in the controls or in the rabbits treated with 0.01 mg ACTH or 0.5 mg t.sth. increase of the The rabbits given daily injections of 2 mg t.sth got a slight blood glucose concentration in the 90-minute sample only (P: ). Ani mals treated with r.hgh and LMF had increased sensitivity to the hyper glycaemic effect of adrenalin, highly significant compared with the controls (P< 0.001). Prednisone tolerance tests, Fig. 4. In untreated rabbits and rabbits given 0.5 mg t.stfl the blood glucose concentration rose to a maximum 12 hours after the injection of prednisone, and was normalized in the 72-hour sample. The groups of rabbits given 0.5 mg r.hgh or 0.1 mg LMF had a prolonged hyper glycaemic response with maximum blood glucose concentration in the 48- and 96-hour samples respectively; return to normal level was observed 6 to 7 days after the injection. Physicochemical studies Polyacrylamide gel disc-electrophoresis (Figs. 5 and 6) Electrophoresis gave two bands for t.sth, and an additional slowly moving band for r.hgh in the area of the single band for LMF, Fig. 5. The residue formed by ultracentrifugation of r.hgfi had two bands, similar to those for t.sth, and the supernatant had a distinct band located as the slowly moving band of r.hgh, besides four additional faint, fast moving bands, Fig. 6. Ultraviolet absorption spectra (Fig. 7) Both r.hgfi, t.sth, and LMF had maximum optical density at 278 µ be low ph 10, and minimum absorption at about 250 µ. In more alkaline solu tions the absorption spectrum for t.sth was unchanged, though LMF and r.hgh got maximum optical density at 290 µ, and the minimum absorption was displaced towards 270 µ; this corresponds to the shift seen in the ab sorption spectrum for tyrosine.

13 Fig. 3. The influence of at least 10 daily injections of 0.01 mg ACTH 'Ferring', of 0.1 mg LMF, and of 0.5 mg and 2 mg HGH (i.e. r.hgh) and STH (i.e. t.sth) on the hyperglycaemic action of mg adrenalin (cf. a Fig. 1) given subcutaneously in rabbits (numbers in brackets). The shaded area indicates the mean blood glucose con centration ± one standard error of the mean (s. e.) of 9 untreated controls, and the curves give the mean blood glucose concentration ± s. e. (vertical bars) in treated animals in order to facilitate comparison, the blood glucose results are expressed as percentages of the initial level.

14 Fig. 4. The influence of 14 daily injections of 0.5 mg HGH (i. e. r.hgh), and STH (i. e. t.sth), and of 27 daily injections of 0.1 mg LMF on the hyperglycaemic action of 8 mg prednisone given subcutaneously in rabbits (numbers in brackets). The shaded area indicates the mean blood glucose concentration ± one standard error of the mean (s. e.) of 10 untreated controls, and the curves give the mean blood glucose concen tration ± s. e. (vertical bars) in treated animals. Molecular weight determinations (Fig. 8) The r.hgh and t.sth preparations were filtrated six times on the Sephadex G-50 gel column, the elution volumes were ± 0.48 ml and ± 0.33 ml (mean ± standard error of the mean, s. e.) respectively, corresponding to tentative molecular weights in the range of ± 440 and ± 235. The LMF was run four times, and the mean elution volume was ml, corresponding to a molecular weight of 5400 ± 50. The residue obtained by ultracentrifugation of r.hgh was filtrated twice,

15 Fig. 5. Fig. 6. Fig. 5. Disc-electrophoresis of 0.2 mg t.sth, r.hgh, and LMF on 7.5 /o polyacryl amide gel columns, cm, run anodally in glycine-tris buffer, ph 8.7, for two hours at 4 ma. Fig. 6. Disc-electrophoresis of 0.2 mg of the residue and of the protein in the super natant obtained by preparative ultracentrifugation of r.hgh on 7.5 /o polyacrylamide gel columns (0.7 X 8 cm), run anodally in glycine-tris buffer, ph 8.7, for two hours at 4 ma. and had elution volumes of 119 ml and 120 ml, in the same range as t.sth. The elution volume for the protein in the supernatant was 148 ml in both of of about two runs, which corresponds to a tentative molecular weight Subdivision of r.hgh into t.sth and an adipokinetic agent Acetone precipitation and Sephadex gel filtration (Fig. 9) The soluble fraction of the precipitate formed at 55 /o acetone saturation had the same metabolic and physicochemical characteristics as t.sth. Sephadex G-50 gel filtration of the precipitate formed at 90 /o acetone saturation gave a small protein peak in the void volume of the column, and a large peak in an elution volume larger than the working range of the column. Within this range there appeared two peaks with elution volumes of 124 ml and 139 ml, corresponding to molecular weights of about and

16 ' - - Tyrosine LMF ß B Wavelength mu Wavelength mu ph 12.0 HGH STH \ V/»i T Wavelength mu Wavelength mu Fig. 7. Ultraviolet absorption spectra of tyrosine, LMF, HGH (i. e. (i. e. t.sth) at ph 7.0 and ph r.hgh), and STH The Sephadex G-50 gel filtration was run twice with the same material, and both runs were identical. When tested in the rabbit with 0.1 mg protein from different fractions, adipokinetic activity was observed in the latter of the two peaks only, signified by the bars in Fig. 1. The same fractions were assayed

17 220 KOXYTOCIN 200 -BACITRACIN 180 -GLUCAGON NSULIN (di-mer) I.CYTOCHROME-C l-ribonuclease MYOGLOBIN -INSULIN (Iri-mer) TH-Sl o-chymotrypsinogen HGH-^lS PEPSIN OVALBUMIN l-albumin 1,000 3,000 MOLECULAR WEIGHT 10,000 20, Fig. 8. The relationship for some globular proteins' molecular weight and elution volume on filtration on Sephadex G-50 gel, column 1.6 X 110 cm, equilibrated with 0.05 M tris hydrochloride buffer ph 7.5 containing 0.1 m potassium chloride (cf. Trygstad 1968 a). The LMF, STH (i. e. t.sth), and HGH (i. e. r.hgh) were preparations filtrated on the same column, and tentative molecular weights can be calculated from their elution volumes. radioimmunologically for human growth hormone activity (kindly performed by Dr. N. Norman, Aker Hospital, Oslo) which was observed in the first of these peaks only (dotted line in Fig. 9). Preparative ultracentrifugation The metabolic and physicochemical studies of the precipitate formed by ultracentrifugation of r.hgh made evident that it was similar to t.sth. The supernatant contained a substance with adipokinetic (Table 2) and hyper glycaemic (Table 4) effect on the rabbit. Acrylamide gel disc-electrophoresis of this substance (Fig. 6) demonstrated a slowly moving band in the same area as the LMF-band.

18 - E Tubes ( 3ml ) Fig. 9. Filtration on Sephadex G-50 gel column (1.6 X 110 cm, equilibrated with 0.05 M tris hydrochloride buffer ph 7.5, containing 0.1 m potassium chloride) of the 55-90% acetone precipitate, which was obtained by addition of 1 HCI until ph 4.5, and acetone until 55 /o saturation to a solution of r.hgh in 0.1 M phosphate buffer ph 8.5, and after centrifugation further addition of acetone to the supernatant until 90 /o saturation. Upturned bars show maximum increase of serum non-esterified fatty acids (NEFA) in milligram-equivalents per litre (meq./l) in rabbits following subcutaneous injection of 0.1 mg protein lrom diflerent fractions. The dots give the radioimmuno logical human growth hormone activity compared with a STH standard preparation (t.sth:xi). Experimental combination of LMF and t.sth A mixture of 5 mg t.sth and 1.2 mg LMF was filtrated in three different runs through the Sephadex G-50 gel column, and there always appeared two protein peaks with elution volumes in the range of 118 ml and 158 ml, cor responding to those for t.sth and LMF (Fig. 8). However, a mixture of 5 mg residue and 1.2 mg protein from the supernatant obtained by ultracentrifuga tion of r.hgh gave only one protein peak on the Sephadex G-50 gel filtration, and the elution volume was ml, i. e. the same as for r.hgh. Polyacryl amide gel disc electrophoresis of the latter mixture gave the same appearance as for r.hgh.

19 DISCUSSION It has been demonstrated above that a purified human growth hormone pre paration (Roos et al. 1963) extracted from deep-frozen pituitary glands can be split up into a more potent and homogeneous growth promoting component (Table 1, Fig. 5) and an adipokinetic-hyperglycaemic component by acetone precipitation of the growth hormone extract followed by Sephadex gel filtra tion (Trygstad 1967, Fig. 9) or by ultracentrifugation of r.hgh dissolved in physiological saline (Tables 2 and 4). Polyacrylamide gel electrophoresis of the residue and supernatant obtained by the ultracentrifugation of r.hgh (Fig. 6) and of r.hgh, t.sth, and LMF (Fig. 5) indicated that r.hgh is composed of t.sth and LMF. About 20 per cent of the dissolved r.hgh preparation re mained in the supernatant after the ultracentrifugation. This would fit well with the tentative molecular weights of LMF, t.sth, and r.hgh, which are in the range of 5000, , and respectively (Fig. 8). Furthermore the ultraviolet absorption spectra (Fig. 7) would support the contention that LMF may originate from r.hgh. The metabolic studies confirmed the physicochemical observations (Tables 1-4, Figs. 1-4). The small adipokinetic and hyperglycaemic effect of 2 mg t.sth in the fed rabbit may in part be due to a contamination with LMF. A contamination in the range of 0.5 per cent, i. e mg LMF per 2 mg t.sth, may be responsible for the adipokinetic activity observed for t.sth (Table 2). It is seen that the r.hgh preparation always moves as one molecular species on Sephadex gel filtration and appears as one symmetrical peak (Roos etal. 1963; Trygstad 1967). This indicates that the adipokinetic-hyperglycaemic component of r.hgh (e. g. LMF) may be a fragment of the r.hgh molecule or that it may be a contamination which strongly aggregates with a molecule of the t.sth preparation. The former of these two possibilities would explain the high antibody titers against r.hgh and t.sth in rabbits immunized with LMF (Norman 8c Turter 1968). No antibodies towards LMF itself were demonstrated in these experiments. Radioimmunologically the LMF contained less than 0.1 per cent t.sth (Trygstad 1968 a), and should therefore not have produced high titers of antibodies with the doses employed for immunization. Laron (personal communication) observed furthermore that the in vitro lipolytic activity of t.sth:xi increased significantly by proteolytic digestion, which indicated that the human growth hormone molecule may have an adipotrophic core. This pos sibility gained support by the observation of an increased adipokinetic effect in fed rabbits following hydrolysis of t.sth:viii. Subcutaneous injection of 5 mg untreated t.stflviii gave a net increase of serum NEFA of 0.56 ± 0.06 meq./l (mean of 6 anmals ± s. e.), although 5 mg hydrolysed t.sth:viii caused a net increase of 1.54 ± 0.08 meq./l (mean of 3 animals ± s. e., un published data). It has also been suggested that the growth hormone can exert

20 its adipokinetic effect only after the injected hormone preparation had under gone some chemical modification which separates the active core from the rest of the molecule (Weil 1965). The lipolytic mechanism is different for t.sth and LMF. Dactinomycin in hibited the in vitro release of NEFA caused by t.sth, though that of LMF was unaffected (Trygstad 1968 b). Moreover Yen 8c Allan (1967) observed re lease of NEFA from isolated fat cells of hyperglycaemic-obese mice by LMF but not by t.sth. These observations would suggest that LMF is an indepen dent molecule attached to t.sth. The disintegration of r.hgh by preparative ultracentrifugation also indicated an aggregation of LMF and t.sth. But again, pituitary proteolytic enzymes might split off an adipokinetic core from the r.hgh molecule in a saline solution at the actual ph. However, this seems unlikely because an aggregation could be re-established when the supernatant and the residue obtained by the ultracentrifugation of r.hgh were mixed. A mixture of LMF and t.sth behaved as two different polypeptides on Sephadex gel filtration. A hypocalcaemic factor is another moiety to be considered in the r.hgh preparation, because r.hgh but not t.sth or purified LMF gives hypocalcaemia in the rabbit (Trygstad 1967, 1968 b). This factor may be essen tial for an aggregation of LMF and t.sth. From Table 2 it is obvious that deprivation of food prior to administration of t.sth intensified its adipokinetic effect in rabbits. This fits well with the relatively potent in vitro lipolytic effect of t.sth in human fat obtained from fasting individuals and the absence of effect in fat from the pregnant diabetic who got glucose and insulin prior to the laparotomy (Table 3). This observa tion, of course, should be confirmed by further studies in fat from normal controls obtained following administration of glucose. However, it has been shown by others that the fat-mobilizing property of somatotrophin was ex tremely sensitive to the blood glucose level. It was most marked in fasting conditions and completely suppressed by administration of sufficient amounts of glucose (Weil 1965). The r.hgh may therefore have two different adipo kinetic properties, one attached to the somatotrophin proper active in the postabsorptive period or in hypoglycaemia (Raben Sc Hollenberg 1959) and one attached to the adipokinetic-hyperglycaemic fragment, which seems to be a more powerful lipid-mobilizer and active in the fed state as well. The hyperglycaemic effect in the rabbit of LMF, r.hgh, and the super natant after ultracentrifugation of r.hgh was obvious although t.sth lacked this effect (Table 4, Fig. 1). In addition, rabbits treated with LMF and r.hgh had a diabetic adrenalin and prednisone tolerance test (Figs. 3, 4), in contrast to animals treated with t.sth. The increased sensitivity to insulin in rabbits treated with LMF and r.hgh was not expected. Young (1938) observed de creased insulin sensitivity in rabbits after injections of pituitary gland extracts, but his rabbits got treatment for less than four days. A single injection of

21 LMF 24 hours prior to the insulin tolerance test demonstrated decreased sen sitivity to insulin also in our rabbits (Table 5). The insulin tolerance test in rabbits treated with LMF and r.hgh for several days resembled that in children with pituitary dwarfism, and the control ani mals had a test response similar to normal children (Trygstad 1965). An exo genous supply of a pituitary adipokinetic-diabetogenic factor may disturb a feed-back mechanism and its normal regulation. Krulich 8c McCann (1966) observed a decrease of the growth hormone content in pituitary glands removed from rats treated with bovine growth hormone for 11 days. Considerable variation has been found in the susceptibility of different species to the diabetogenic action of pituitary hormones (Engel 8c Kostyo 1964). Knobil Sc Greep (1959) observed that administration of a crude alkaline extract of monkey pituitary glands to hypophysectomized monkeys had a temporary diabetogenic effect only, despite continued injections. When the purified somatotrophin preparations became available this diabetogenic effect could not be duplicated in most instances. A temporary (idiohypophyseal) hyperglycaemia was induced in rabbits treated with LMF, Fig. 1. The unresponsiveness of the rabbit to the adipokinetic-hyperglycaemic effect of LMF after some days of treatment might indicate development of antibodies, but these could not be shown to be present (Norman Sc Turter 1968). However, as opposed to normal rabbit sera, serum from LMF-treated rabbits significantly inhibited the in vitro release of NEFA from normal rabbit fat pads incubated with LMF (to be published). Recent studies have indicated that the unresponsiveness to LMF in the LMF-treated rabbits could also be located in the fat tissue itself. Fat pads from the LMF-treated rabbits got a reduced in vitro release of NEFA during incubation with LMF and r.hgh compared with the release in fat pads from untreated control rabbits. Addition of homogenized fat from LMF-treated rabbits inhibited the in vitro release of NEFA occurring in normal rabbit fat pads (to be published). The preparation of LMF and r.hgh is dependent on the use of deep frozen and not acetone preserved pituitary glands. No LMF was obtained from the latter (Trygstad 1968 a). The growth hormone prepared from these by the method of Roos et al. (1963) had only the two fast moving bands on poly acrylamide gel electrophoresis, and the molecular weight was in the range of (own observations). It seems physiological to use deep frozen glands for preparation of pituitary hormones (Lepp 8c Oliner 1967). The acetone preservation may lead to ex traction of pituitary polypeptides. and a more drastic procedure seems to be needed for obtaining a good yield of somatotrophin activity (Trygstad 1968 a). This might denaturate the hormones, and offers an explanation of the growth hormone antibodies observed during treatment of pituitary dwarfism (Prader et al. 1967; Tanner 8c Whitehouse 1967). No unresponsiveness to r.hgh or

22 t.sth treatment has been observed in 21 patients with pituitary dwarfism treated for 1-6 years (Seip 8c Trygstad 1966, own observations). Therapeutically the t.sth preparation has been preferred because the r.hgh preparation initially gave more unwanted effects as nausea, vomiting, perspiration, palpi tation, lethargy, increase of liver size, and often fever (Dr. Moe, Bergen, per sonal communication), all symptoms which have been associated with a more potent adipokinetic effect of the r.hgh. Rubens (1965) assertion that human somatotrophin causes both growth and adipokinesis is confirmed by this study. The t.sth has potent growth pro moting effect (Table 1) and a significant, though slight lipolytic effect in the fed rabbit, but increased effect in the postabsorptive period and after hydro lysis of the polypeptide (Tables 2, 3). However, the assumption of Levine 8c Luft (1964) of the existence of a human growth hormone composed of a somatotrophic and an adipokinetic component may also be suggested by a sub division of r.hgh into t.sth and LMF, although t.sth and LMF may be two different molecules which aggregate. It is views of Raben (1965) and Levine 8c Luft concluded that the presented results can possibly make the opposed (1964) compatible. REFERENCES Engel F. L. Se Kostyo ]. L. In: Pincus G., Thimann K. V. and Astwood E. B., eds., The hormones, physiology, chemistry and applications, vol. 5, Academic Press, New York, London (1964) 113. Greenberg E.: Diabetes 14 (1965) 43. Greenspan F. S., Li C. H Simpson M. E. Se Evans H. M.: Endocrinology 45 (1949) 445. Hultman E.: Nature (Lond.) 183 (1959) 108. Knobil E. Se Greep R. O. In: Pincus G., ed., Recent Progress in Hormone Research, vol 15. Academic Press, New York, London (1959) 1. Krulich L. Sc McCann S. M.: Proc. Soc. exp. Biol. (N. Y.) 121 (1966) Lepp A. Sc Oliner L.: Endocrinology 81 (1967) 299. Levine R. Se Luft R.: Diabetes 13 (1964) 651. Norman N. Se Turter A. R.: Acta endocr. (Kbh.) JS (1968) 318. Prader., Zachmann M., Poley J. R., lllig R. Sc Szoky ].: Helv. paediat. Acta 22 (1967) 423. Raben M. S.: Diabetes 14 (1965) 374. Raben M. S. Se Hollenberg C. H.: J. clin. Invest. 38 (1959) 484. Raben M. S. Se Westermeyer V. W.: Proc. Soc. exp. Biol. (N. Y.) 80 (1952) 83. Roos P., Fevold H. R. Se Gemzell C..: Biochim. biophys Acta 74 (1963) 525. Seip M. Se Trygstad O.: Acta paediat. (Uppsala) 55 (1966) 287. Tanner J. M. Sc Whitehouse R. H.: Brit. med. J. 2 (1967) 69. Trygstad O.: Arch. Dis. Childh. 40 (1965) 508. Trygstad O.: Acta endocr. (Kbh.) 56 (1967) 626. Trygstad O.: Acta endocr. (Kbh.) 57 (1968 a) 81.

23 Trygstad O.: Acta endocr. (Kbh.) 58 (1968) 277. Weil R.: Pituitary growth hormone and intermediary metabolism, Acta endocr. (Kbh.) Suppl. 98 (1965). Yen T. T. T. Se Allan J.: Genetica 56 (1967) 598. Young F. G: Biochem. J. 32 (1938) Received on December 14th, 1967.