Hoppe-Seyler's Z. Physiol. Chem. Bd. 360, S. 161 9-1632, November 1979

Synthesis and Biological Activity of Seventeen Analogues of Human Insulin Fritz MÄRKI*, Marc DE GASPARO*, Karel EISLER, Bruno KAMBER, Bernhard RINIKER, Werner RITTEL and Peter SIEBER Chemische und * Biologische Forschungslaboratoiien Ciba-Geigy AG, Basel

(Received 27 August 1979)

when IleA2 or the half-cystines A6, A7, AI l or B7 were modified. Replacement of the invariant GlnAS by alanine only reduced potency slightly. All the analogues are full agonists. The effects of the analogues on glucose oxidation and lipolysis are correlated, supporting the view that they are mediated by a common receptor on the fatcell membrane. Hypoglycaemic potencies in the rat were similar to potencies in vitro. As expected, no correlation was demonstrable between antiserum binding — measured in the radioimmunoassay — and biological activity. Several results of this investigation are difficult to reconcile with the current view regarding the structure-activity relationship of insulin which appears to require further refinement.

Summary: We synthesized seventeen analogues of human insulin, applying the principle of stepwise, selective formation of the disulphide bonds. Most of these analogues only differ from human insulin in the replacement of a single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A chain and 5, 7, 13 and 16 of the B-chain. The influence of these modifications on the physicochemical properties of the analogues is discussed. Eight analogues could be crystallized. All the analogues produce the same biological effects as insulin, but differ markedly in their potency. In isolated fat cells in vitro, [HisA8]insulin showed a relative potency of 2.46 in stimulating glucose oxidation (human insulin = 1), whereas [D-CysA6>A1 ^insulin had a potency of only 0.00027. Very low potency was observed

Synthese und biologische Aktivität von siebzehn Analogen von Humaninsulin Zusammenfassung: In Anwendung des früher für die Synthese von Humaninsulin ausgearbeiteten Prinzips der stufenweisen und selektiven Bildung der Cystinbrücken wurden siebzehn Insulinanaloge hergestellt. Die Mehrzahl unterscheidet sich von Humaninsulin nur durch Austausch einer einzelnen Aminosäure in den Positionen 2, 5, 6, 7, 8 und 11 der A-Kette und 5, 7, 13 und 16 der Abbreviations: Acm = acetamidomethyl; Boc = tert-butoxycarbonyl; succinimide ester; Trt = triphenylmethyl.

B-Kette. Der Einfluß dieser Modifikationen auf die physikalisch-chemischen Eigenschaften wird diskutiert. Unter Bedingungen, bei denen Insulin als Hexameres kristallisiert, konnten acht Analoge in Kristallform erhalten werden. Die biologische Wirkung der Analogen unterscheidet sich qualitativ nicht von derjenigen von Insulin. Sie stimulieren in vitro an isolierten Fettf

= tert-butyl; OBu' = tert-butoxy; ONSu = ./V-hydroxy-

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F. Märki, M. de Gasparo, K. Eisler, B. Kamber, B. Riniker, W. Rittel and P. Sieber

zellen die Glucoseoxidation, hemmen die Lipolyse, und senken in vivo den Blutzucker bei der Ratte. Alle erreichen die volle Wirkung des Insulins, unterscheiden sich aber in bezug auf die Wirkungsstärke (Potenz) sehr stark. So besitzt [HisA8] Insulin in vitro (Glucoseoxidation) eine relative Potenz von 2.46 (bezogen auf Humaninsulin = 1), jp-Cys A6)A1 ^Insulin dagegen von nur 0.00027. Sehr niedrige Aktivität wurde festgestellt bei Analogen mit Modifikation von IleA2 oder der Halb-Cystine A6, A7, AI l, 7, während Ersatz der invarianten Aminosäure Gln A5 durch Alanin nur zu einem geringen Aktivitätsabfall

Bd. 360 (1979)

führte. Eine strenge Korrelation der Wirkung der Analogen auf Glucoseoxidation und Lipolyse stützt die These, daß Insulin diese beiden Stoffwechselprozesse über einen gemeinsamen Rezeptor an der Fettzellmembran beeinflußt. Die Antiserumbindung — bestimmt im Radioimmunoassay - korrelierte, wie erwartet, nicht mit der biologischen Aktivität. Es zeigt sich, daß die derzeitigen Vorstellungen über die Struktur-Wirkungsbeziehung beim Insulin für das Verständnis einzelner Ergebnisse der vorliegenden Arbeit unzureichend sind und der Erweiterung bedürfen.

Key words: Insulin analogues, physicochemical properties, biological activity, antibody binding, structure-activity relationship.

In previous papers we described a new approach to the total synthesis of insulin, in which the three disulphide bridges are formed at different chemical steps of the synthesis^1·2!. This approach was made possible by the use of a novel combination of protecting groups with selective chemical reactivities. It allows an unequivocal structural assignment for the product of the synthesis, and at the same time avoids the necessity of combining the separate insulin chains, a step in which low yields of insulin and complex mixtures of by-products are obtained. The same technique has already been utilized in the synthesis of two isomers of human insulin with unnatural disulphide-bond pairing^3!. A further application, reported here, is the synthesis of new analogues of human insulin, most of which differ from the natural hormone only in the replacement of one single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A and 5, 7, 13 and 16 of

the B chain (Fig. 1). The analysis of the physicochemical properties, the biological activity and the antibody-binding characteristics of these analogues has revealed some unexpected results, which may afford new insights into the intriguing structure-activity relationship of insulin.

Methods Syntheses The seventeen analogues of insulin shown in Table 1 were prepared according to the general methods of synthesis described in earlier papers' 1 > 2 > 4 ~ 7 1. For the ten analogues with modifications in the A chain, the intermediate A14-21/B1-30 (ll 2 ! (Fig. 2)) served as the common amino component in the last fragment condensation; it was coupled to the ten different fragments Al-13 (analogues of 2) as carboxyl component. Fragments Al-13 were built up as outlined in refJ 4 ^, the naturally occurring amino acids of 2 being replaced by

H-Gly-Ue-Val-Glu-Gln-Cys-Cys-Thr-Ser-lle-C^s-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH •-Ile-Cys 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 201 21

H-Phe-Val-Asn-Gln-His-Leu-(^-Gly-Ser-His-Leu^^ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 » Fig. 1. Amino acid sequence of human insulin. The residues modified in the analogues are underlined.

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Analogues of Human Insulin

Bd. 360(1979)

those of the analogues. The analogues with Ala, D-Cys, and Gin in positionsB5, B7 and B13, respectively, were prepared by an alternative route, in which the A chain was completed first. Fragment 3al 7 l was coupled to the three different peptides Bl-16 (analogues of 4al 8 ') as carboxyl components. The des-Tyr BI6 and the endo-Tyr B16a analogues were synthesized through condensation of 3al 7 l with 4b' 8 ' and of 3b with 4a' 8 l, respectively. Analogue 3b had to be prepared from 3a by coupling an additional Tyr(Bu f ) residue to the free amino terminal in position B17. This was done by reaction of Trt-Tyr(Bu f )-ONSu with 3a, followed by selective acidolytic cleavage of the trityl group. [D-Tyr B16 ]lnsulin was obtained as a by-product in the original synthesis of human insulinf 2 !. It originated from a partial racemization of the C-terminal Tyr B16 during the preparation of 1 by condensation of 4a with the A14-21/B17-30 fragment. [Cys(Acm) A 7 ' B 7 ] Insulin is the synthetic precursor of unmodified human insulin, from which the latter is produced by oxidation with iodine. It was obtained from the protected peptide (compound VII i n ^ 2 b by reaction with 95% trifluoroacetic acid for l h at 30 °C, followed by precipitation with ether. This derivative had a strong tendency to associate in aqueous solutions

Aon But But I I I Boc-Gly-1 le-Val-Glu-Glrv-Cys-Cys-Thr-Ser-1 le-Cys-Ser-Leu-OH

1621

in the form of fibrils. It was therefore dissolved (0.5% in H2 ) immediately before use, without further purification. All the other analogues were purified by counter current distribution in the same solvent system (K values see Table 1). Special aspects of the synthesis and some physicochemical properties of the five analogues containing D-Cys in positions A6, A7, All or B7 are described in a separate communication' 7 '. Crystallization experiments were performed by making up 0.1 to 0.5 % solutions of the insulin analogues in 0.095M sodium citrate buffer (pH6.0) containing 0.03MNaCl, 0.012M ZnCl2,and 5% of tert-butanol. Analytical methods Amino acid analyses were performed after total hydrolysis with 6M HC1, containing 0.1% of phenol, for 27 h at 110°C. Oxidations with performic acid to the cysteic acid derivatives of the separate chains were carried out according to Hirs^ 9 ' during 15 min at 0°C. Thin-layer chromatography and electrophoresis were performed as described earlier 2 '. Because of the large variations in the absolute migration distances, the values in Table 1 are given relative to human insulin as a reference. Isoelectric points were determined by isoelectric focusing in flat-bed polyacrylamide gels containing carrier ampholytes (Pharmacia, Uppsala, Sweden), pH range 3 — 10, and 8M urea.

I l l , H -Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OBut

13

Λ

Acm I

BocBut

Boo-Phe-Val-Asn-Gln-His-Leu^ys-Gly-Ser-^^

1

30

Aon Bu1 Bu!

OBut

Bu'

Boc-Gly-lle-VaWlu-GlrMiys-Cys-Thr-Ser-lle-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-AsrHTyr-Cys-Asrv-OBu1 1

I

Acm I

Bu I

t

OBu I

But Βϋ*

Boc-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-R

1

21

1

15

17

ut

30

4a R . Tyr(But)-OH

3a R .

H

4b

3b

H- T

R = OH

6oc

R-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-OBu1

R.

Fig. 2. Protected intermediates used in the synthesis of the insulin analogues.

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F.Märki, M. de Caspar o, K. Eisler, B. Kamber, B. Riniker, W. Rittel and P. Sieber

Bd. 360(1979)

Table 1. Physicochemical data of human insulin and analogues. Electrophoresis 3 pH8.6 pH1.9 Human insulin [Pro A2 ]Insulin [D-allo-Ile A2 ]Insulin [Ala A 5 ]Insulin [D-Cys A6 ]Insulin [D-Cys A 6 ' A 1 1 ]Insulin [D-Cys A7 lInsulin [His A8 ]Insulin [Lys A8 [Insulin [Phe A 8 jInsulin [D-Cys A11 ]Insulin [Ala B 5 )Insulin (D-Cys B 7 )Insulin [Gln B l 3 ]Insulin [D-Tyr B 1 6 ]Insulin Des-Tyr B16 -insulin Endo-Tyr B16a -insulin [Cys(Acm) A 7 > B 7 lInsulin

1.0 1.02 0.99 0.99 0.88 0.79 0.92 1.07 1.11 0.95 0.79 0.68 0.87 1.03 0.91 0.87 0.62 0.81

1.0 0.98 0.98 0.98 0.98 0.96 0.98 1.02 0.91 0.96 0.96 0.97 0.98 0.67 0.87 0.92 0.72 0.88

Isoelectric Thin-layer chromatography point Insulin Oxidized Oxidized analogues15 A chain 0 B chain c 5.7 5.65 5.6 5.7 5.6 5.55 5.6 6.15 6.6 5.7 5.55 5.0 5.6 6.3 5.6 5.5 5.5 5.5

I

1.0 0.90 1.44 1.16 1.59 2.04 1.52 0.63 0.45 1.31 2.02 1.90 1.60 0.93 1.10 2.27 1.90 2.14

1.0 0.71 1.18 1.08 0.95 0.92 1.01 0.95 0.84 1.16 0.95 1.0 1.0 1.0 1.0 1.0 1.0

1.0 .0 .0 1.0 .0 1.0 1.0 1.0 1.0 1.0 1.0 1.05 1.02 0.97 0.97 0.97 1.01

Countercurrent distribution, K values d

1

0.84 0.65 1.09 0.84 0.96 1.34 1.0 0.56 0.4 1.27 1.36 1.0 1.05 1.1 1.25 1.7 1.86

j*b Mobilities relative to human insulin on cellulose acetate foils at pH 1.9 (90 min) and 8.6 (120 min), 20 V/cm. Migration distances on cellulose relative to human insulin. Solvent system: 1-pentanol/pyridine/water/methyl ethyl ketone/formic acid 40: 28:15 :11: 5 (V/V). c Migration distances on cellulose relative to the corresponding chain from human insulin. Solvent system: 1-butanol/1-pentanol/pyridine/methyl ethyl ketone/acetic acid/formic acid/water 20:15 :25 :10: 3: 3: 25 (V/V). d Solvent system: l-butanol/pyridine/0.1% acetic acid 5 : 3:11 (V/V).

Biological assays and statistical evaluation The methods used for the assay of glucose oxidation and lipolysis in isolated fat cells in vitro, hypoglycaemia in the rat in vivo, and anti-insulin serum binding in vitro have been described previously 13l Statistical significance was evaluated by Student's t test. The 95% fiducial limits of the relative potencies reported in Table 2 were computed on a Hewlett-Packard desk calculator Model 9815A, using a programme based on the confidence set for the ratio of means^ 10 '.

Results The synthesis of the analogues of human insulin described here presented no special chemical problems. They were obtained after purification by counter current distribution as amorphous powders in amounts ranging from 15 to 50 mg and exhibited the expected amino acid ratios. Some of their physicochemical data are shown in Table 1 together with those of synthetic human insulin. In the presence of Zn2e-ions, under

conditions under which insulin crystallizes as hexamer, eight analogues were obtained in the crystalline state after a few hours or days. The others precipitated in amorphous form from the crystallization buffer. The photographs of the eight crystalline analogues shown in Fig. 3 were all made at the same magnification. [ProA2]-, [D-CysA6]-, [D-CysA7]-, [AlaB5]- and [Gln B13 ] insulin crystallized with a clearly rhombohedral appearance. The biological activities and the antiserum binding of the analogues were evaluated by determining the stimulation of glucose oxidation and the inhibition of lipolysis in isolated fat cells in vitro, the hypoglycaemic effect in the rat in vivo, and the binding to anti-bovine insulin serum from guinea-pigs in vitro. Qualitatively, all the analogues were found to stimulate glucose oxidation in fat cells similarly to insulin. As shown in Table 2, their potencies (relative to human insulin = 1) cover an extremely wide range, from 2.46

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Analogues of Human Insulin

Bd. 360(1979)

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Table 2. Biological activity of analogues of human insulin. Analogue

Relative potency3 Hypoglycaemia Fat cells in vitro rat, in vivo Glucose oxidation Lipolysis

Anti-insulin serum binding

Modification of A chain [Pro

A2

jlnsulin A2

[D-allo-Ile JInsulin [Ala A 5 llnsulin [D-Cys A6 ]Insulin [ I > C y s A6,AH,_

Insulin [!>CysA7 jlnsulin [His

„

llnsulin

[Lys A8 llnsulin [Phe A8 llnsulin [D-Cys A11 lInsulin

0.017 (0.014-0.022) 0.0019 (0.0018-0.0020) 0.38 (0.29-0.52) 0.0023 (0.0019-0.0028) 0.00027 (0.00024-0.00029) 0.0016 (0.0011-0.0033) 2.46 (2.06-3.03) 0.64 (0.45-1.10) 0.80 (0.64-1.06) 0.0073 (0.0049-0.0142)

0.015 (0.012-0.018)

0.49 (0.36-0.63)

0.0025 (0.0016-0.0045) 2.99 (2.03-5.07) 1.53 (0.96-2.71) 1.07 (0.55-2.13)

1.05 (0.86-1.30)

0.87 (0.65-1.09) 0.66 (0.52-0.81) 0.87 (0.58-1.16)

1.01 (0.84-1.21) 0.56 (0.50-0.62) 0.99 (0.90-1.10) 0.75 (0.57-0.93) 0.064 (0.057-0.072) 0.47 (0.40-0.53) 1.96 (1.48-2.88) 0.22 (0.18-0.25) 0.38 (0.31-0.46) 0.084 (0.074-0.096)

Modification of B chain [Ala B5 ]Insutin · [D-Cys B7 llnsulin

[Gln B13 ]lnsulin [D-Tyr B16 ]Insulin Des-TyrB16insulin Endo-Tyr Bl6a insulin

0.21 (0.16-0.31) 0.012 (0.0079-0.027) 0 18 (0.15-0.24) 0.17 (0.12-0.29) 0.00066 (0.00057-0.00078) 0.0084 (0.0064-0.012)

0.31 (0.15-0.60)

1.01 (0.76-1.58)

0.28 (0.20-0.46) 0.00075 (0.00043-0.0014) 0.0089 (0.0050-0.035)

0.85 (0.54-1.51)

0.53 (0.27-1.49) 0.13 (0.10-0.15) 0 16 (0.10-0.22) 1.08 (0.75-1.59) 0.0041 (0.0022-0.0089) 0.0091 (0.0059-0.013)

Analogue without disulphide bond A7-B7 [Cys(Acm) A7 ' B7 l- 0.0029 Insulin (0.0019-0.0060)

0.0037 (0.0024-0.0076)

0.0043 (0.0024-0.0059)

0.087 (0.076-0.100)

a Human insulin- = I; mean of at least three separate experiments (95% fiducial limits in parentheses). Absolute reference values (£€59 in vitro) for human insulin: half-maximal stimulation of glucose oxidation at 0.443 ± 0.008 ng/m/(w = 7); 50% inhibition of lipolysis at 0.452 ± 0.035 ng/m/ (« = 29); 50% displacement of [ 125 I]insulin in anti-insulin serum binding in vitro at 0.872 ± 0.026 ng/m/ (n = 16). In these tests, synthetic human insulin is equipotent with natural porcine insulin'^^'. For experimental details seel31.

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F. M rki, M. de Gasparo, K. Eisler, B. Kamber, B. Riniker, W. Rittel and P. Sieber

'

[ProA2] Insulin

:

Bd. 360 (1979)

'

\D-allo-lleA2\lnsulin

[D-CysA6]lnsulin

[D-CysA7]lnsulin

·* "./* ft .'•il · ^ Μ

Fig. 3. Crystalline analogues of human insulin. The side-length of the individual pictures corresponds to 0.2 mm.

for [HisA8]insulin to 0.00027 for [D-CysA6>A11]insulin. Despite this large quantitative difference, all of them stimulate glucose oxidation maximally, i.e to the same extent as does human insulin when added in sufficiently high concentrations. Fig. 4 shows the effects of four selected analogues:

their dose-response curves are parallel to that of insulin and reach a plateau around the same value. All the other analogues produce the same response (data not shown). Thus, the analogues studied have the same intrinsic activity as human insulin and are full agonists.

Fig. 4. Dose-response curves of human insulin and of four selected analogues in the fat-cell assay. Stimulation of glucose oxidation is expressed in per cent of the maximal stimulation obtained with human insulin at 13 ng/ ml. Maximal stimulation in different experiments varied between 3.4- and 8.3-fold basal oxidation. 104 • Human insulin, EC^o Concentration [ng/ml] A 0.44 ng/m/; ο [His ^)insulin, EC50 0.18 ng/m/; Δ [D-Tyr B16 ]insulin, EC50 2.6 ng/m/; α [D-Cys B7 ]insulin, EC 50 36 ng/m/; 0 des-TyrB16insulin, £€50 670 ng/m/.

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Analogues of Human Insulin

Bd. 360(1979)

Table 3. Additive effect of human insulin and [Pro A2 ]insulin on glucose oxidation in isolated fat cells. Fat cells were isolated from epididymal fat pads of rats by the collagenase technique and incubated with [l-14C]D-glucose, in the presence or absence of insulin or analogue as indicated, as described previously,[3] Glucose ο xidation Stimulation [%]b

Insulin, analogue lng/m/1

n.a.

[Pro A2 ]Insulin

10 1 0.5 25

Human insulin + (Pro A 2 lInsulin

0.5 25

Human insulin

14

C02 formed [cpm]a

1376 11133 8922 6186 5685

± 31 ± 264 ± 173 ± 307 ± 43

9017 ± 312C

100 77 45 44 78C

* Meant S.E.M. (n = 4 ) 100% stimulation = maximal increase (in presence of insulin 10 ng/m/) above control (absence of insulin or analogue). t Not significantly different from effect of human insulin 1 ng/m/ (p > 0.05). n.a. no addition.

A further indication that the analogues stimulate glucose oxidation in fat cells in the same way as does insulin, was provided by the demonstration of an additive effect of insulin and [ProA2] insulin. As shown in Table 3, combined addition of approximately equipotent, low concentrations of human insulin (0.5 ng/m/) and [ProA2] insulin (25 ng/m/) had practically the same effect as had doubling the concentration of insulin (1 ng/m/), i.e. stimulation increased from 44-45% to 77-78%. On the other hand, the effect of combined addition of insulin and [ProA2] insulin, each at maximally effective concentrations (10 and 600 ng/m/, respectively), was indistinguishable from that of either compound alone (data not shown). Finally, it was demonstrated that the stimulatory effect of insulin and [ProA2] insulin on glucose oxidation is inhibited by the addition of antiinsulin serum (Table4). Complete (97%) inhibition of the metabolic effect of 0.5 ng/m/ human insulin was obtained in the presence of antiserum at a dilution of 1: 2000, whereas a dilution of 1:200 was required to fully inhibit the effect of a biologically equipotent concentration (25 ng/ m/) of the analogue. This analogue has the same antibody-binding potency as insulin. However,

since its relative biological potency is approximately 0.02, it was used at a concentration 50 times higher than that of insulin and more antiserum was therefore needed to bind the analogue to the same extent as insulin. The inhibitory effect of eleven analogues on lipolysis in isolated fat cells, stimulated submaximally with corticotrophin-(l-24)-tetracosapeptide*, was determined as shown in Table 2. Their relative potencies were closely similar to those found in the glucose oxidation assay. In fact, a statistically highly significant correlation (p < 0.001) between the two parameters was observed (Fig. 5). We assayed the hypoglycaemic effect of six analogues with a relatively marked activity in the fatcell test in vitro, and of [Cys(Acm)A7'B7]insulin, an example of an analogue with a very weak activity, after subcutaneous injection in fasted rats. Table 2 shows that the in vitro and in vivo potencies of each analogue studied are fairly similar and always of the same order of magnitude. Antibody-binding potencies were determined by estimating the degree of competition between the analogues and [ 12S I]insulin (pork) for bindSynacthen® Ciba

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F. M rki, M. de Gasparo, K. Eisler, B. Kamber, B. Riniker, W. Rittel and P. Sieber

Bd. 360 (1979)

Table 4. Inhibition of the stimulatory effect of insulin and [Pro A2 jinsulin on glucose oxidation by antiinsulin serum. Fat cells were incubated with or without insulin, analogue and guinea-pig anti-bovine-insulin serum in the presence of [l- 14 C]D-glucose. Insulin, analogue

Anti-insulin seruma

[n g /m/| n.a. Human insulin

{Pro A2 lInsulin

n.a. n.a. n.a. 1:2000 n.a. n.a. 1 : 2000 1:200

10 0.5 0.5

1000 25 25 25

Glucose ο xidation Inhibition [%] c CO2 formed [cpmj b

14

1599 ± 13669 ± 7593 ± 1758± 13681 ± 7912 ± 7154± 1 804 ±

31 276 162 85 34 65 94 86

97

12 97

a

Same antiserum as used for determination of anti-insulin serum binding (see Table 2); the figure indicates the final dilution in the incubation mixture. b Mean ± S.E.M. (n = 4) c Difference between the control and a submaximal concentration (ca. £€59) of insulin (0.5 ng/m/) or [Pro A2 ]insulin (25 ng/m/), respectively, = 100%. rua. no addition.

A/

tο

,/

V. Λ/ AX

1

ι'°

/

/

*

^~ _, Λ p .0

1 ο U)

1 °

Δ ° /

1ΓΓ3

/

s

y = 0.06'

W1

Fig. 5. Correlation between effects of insulin analogues on glucose oxidation and lipolysis in isolated fat cells. Potencies, relative to human insulin = 1, of eleven insulin analogues and one insulin isomer are represented by the following symbols: ο human insulin, · [A6-A7, All-B7-cystine]insulin (isomer of human insulin^ 3 !), Δ six insulin analogues modified in A chain (data see Table 2), * four insulin analogues modified in Β chain (data see Table 2), α [Cys(Acm) A7 ' B7 ]insulin (analogue without disulphide bond A7-B7).

ing to anti-bovine insulin serum under the conditions used for the radioimmunoassay of insulin. They range from 1.96 for [HisA8] insulin to 0.0041 for des-TyrB16-insulin (Table 2). While some analogues, e.g. [Gln B13 ] insulin, have almost identical antibody-binding and metabolic potencies, with others, e.g. [ProA2]- and [D-allo-IleA2]insulin, there is a wide discrepancy between the two potencies. On the whole, there is no correlation between the antibody-binding and metabolic potencies of the 17 analogues (r = 0.47, ρ > 0.05). Discussion Syntheses and physicochemical properties The last step in the synthesis of the insulin analogues described here, except [Cys(Acm) A7)B7 ]insulin, was the formation of the disulphide bridge A7—B7 by oxidation of the two Acm-protected cysteine residues with iodine^ 6 !. In all cases, this reaction proceeded as easily as with insulin itself and the yields were also similar (approx. 70%). The precursor molecules lacking the disulphide bridge between A7 and B7, in the protected form, as well as after cleavage of the 19 protecting groups of the tertiary butyl type, all showed similar characteristics in the thin-layer

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Analogues of Human Insulin

Bd. 360(1979;

chromatogram or in counter current distribution (data not shown). After performic acid oxidation, the two separate chains of the analogues likewise revealed no Chromatographie differences beyond the expected range. By contrast, the end-products differed much more than would have been expected from the nature of the modification introduced. The majority of the analogues are considerably more lipophilic than human insulin, as can be seen from their RF values in the thin-layer chromatogram and their K values in countercurrent distribution. This tendency towards increased lipophilic character is especially notable in des-Tyr B16 - and endo-TyrB16a-insulin and in the five analogues with D-Cys in different positions. It may be explained by the fact that when the third disulphide bridge is formed the insulin molecule is forced to assume a rather rigid conformation. A small structural change may give rise to a large change on the surface of the protein by exposing parts of the lipophilic core. This is in contrast to the conditions prevailing in a flexible, extended polypeptide chain. Biological activity in vitro Binding to a receptor on the membrane of the target cell is considered to be the first step leading to the manifestation of the biological effect of insulin and other hormones f l l l. It has indeed

been demonstrated that the receptor-binding affinity of naturally occurring insulins and many insulin analogues (e.gJ12~16l) is closely related to the potency of their biological effects (e.g. glucose oxidation or lipogenesis in isolated fat cells). Only recently, possible exceptions to this concept have come to light. There seem to be significant differences between receptor-binding affinity and biological potency in insulin analogues with an intramolecular cross-link113J, in insulin dimers with an intermolecular crosslink' 17 ' and in two disulphide-bond isomers of insulin^3!, (P. de Meyts, personal communication). Insulin from a primitive vertebrate, the Atlantic hagfish, previously reported to have different receptor-binding affinity and biological potency^18!, has been restudied and now appears to be equally potent in both tests, when they are performed in assay systems using mammalian tissues'19!*. It would be interesting to know whether discrepancies between receptor affinity and biological activity occur in our series of analogues. However, no direct receptor assays have been done so far. Indirect evidence suggests that the analogues with very low biological potency listed in Table 5 most likely also have similarly low receptor-binding affinities. This is borne out See "note added in prof".

Table 5. Inability of insulin analogues to antagonize the stimulatory effect of insulin on glucose oxidation in fat cells. Analogue

Human insulin [ng/m/]

[D-allo-IleA2]Insulin [D-CysA6]lnsulin [D-CysA11]InsuIin [D-Cys A6)A11 ]Insulin Des-TyrB16-insulin

10 10 10 10 10

a

[ng/m/1

Glucose oxidation 14 CO2 formed [cpml b

0 10 0.5 0.5 0.5 0.5 0.5 0.5

3151 ± 77 13490 ± 350 8701 ± 286 8 7 6 4 ± 83C 8824 ± l l l c 9489 ± 165C 8510 ± 219C 8444 ± 133C

a

The insulin analogues were pre-incubated with the fat cells for l h at 37 °C prior to addition of human insulin and [l-14C]D-g!ucose;for further details see Table 3. Essentially similar results were obtained when the analogues were added to the fat cells simultaneously with insulin (no pre-incubation). b Mean ± S.E.M. (n = 4) c Not significantly different (p > 0.05) from effect of human insulin (0.5 ng/m/) without addition of analogue.

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1628

F. Märki, M. de Gasparo, K. Eisler, B. Kam her, B. Riniker, W. Rittel and P. Sieber

by the fact that addition of these analogues to fat cells in a large excess (20 times higher concentration than insulin) does not interfere with the biological effect of insulin. If these analogues bound as efficiently to the receptors as does insulin, but produced a biologically less active or inactive analogue-receptor complex, this would result in competitive inhibition of the biological effect of insulin. The absence of such antagonism suggests that the analogues studied do not bind to the receptors to any appreciable degree. This result is in line with earlier observations made by Gliemann & Gammeltoft with a different series of insulin analogues^14'. These authors suggested that complexes of the receptor with insulin and with an insulin analogue exhibit the same biological activity. The results we obtained with our analogues support this conclusion. When added to fat cells at maximally effective concentrations (about 20 times EC 50 ), all 17 produced exactly the same stimulation of glucose oxidation as human insulin. These analogues are therefore full agonists with the same intrinsic activity as insulin. As indicated by the parallel dose-response curves, the four analogues shown in Fig. 4 differ from insulin only in their potency, i.e. quantitatively, but not qualitatively. Furthermore, the results listed in Table 3 demonstrate that strictly additive effects are produced by insulin and the analogue [ProA2]insulin applied together in submaximally effective concentrations, but not in maximally effective concentrations. This suggests that insulin and the analogue both act through the same mechanism. The biological activities of the analogues, as measured in the isolated fat-cell assay in vitro, cover a remarkably wide potency range extending from more than 2 to less than 0.001. Thus, modification of the chemical structure of the insulin molecule by exchange, deletion or insertion of only one single amino acid may greatly affect the biological activity of the resultant analogue. Of special interest are changes involving the cystine residues. These belong to the invariant residues that have been conserved unchanged during phylogenesis in all naturally occurring insulins. Apparently they play a decisive role in maintaining the gross tertiary structure of the insulin

Bd. 360 (1979)

molecule by fixing certain regions of the molecule through covalent bonds. In addition, they are among the residues that are assumed to compose the nonpolar core^ 20 '. One might therefore expect that modification of cystine residues would affect biological activity considerably. On the other hand, upon examination of the structural model of insulin^ 21 1 it would appear that D-half cystines could be accomodated in positions A6 and A7 without major changes of the tertiary structure. Indeed, these two analogues seem to be similar in structure to insulin, as indicated by the fact that they crystallize and by their degree of binding to anti-insulin serum. It was therefore unexpected that they should have extremely weak biological activity, like the other three analogues containing D-Cys residues. This result suggests that factors other than insulinlike conformation are essential to biological activity. If enzymatic processes should be involved in triggering the biological response to insulin, the almost total lack of activity displayed by D-Cys analogues would not be unexpected. It is consistent with this assumption that two disulphide-bond isomers of insulin show unexpectedly potent biological activity!3), although they differ markedly from insulin in their structure. A further example of cystine modification is [Cys(Acm)A7'B7]insulin, lacking the disulphide bridge between A7 and B 7. It has a very low potency of 0.0029. On the other hand, two closely related analogues were found to be much more active: [Cys(CH2CO2H)A7'B7]insulin, prepared by selective electrochemical reduction of insulin and subsequent carboxymethylation, had a potency of 40% in the mouse convulsion assay and of 100% in the rat hypoglycaemia assayl22!, whereas [Cys(SO3H)A7>B7]insulin, obtained by partial sulphitolysis in the presence of tetrathionate, was somewhat less potent (15% in the mouse and 4-10% in the rat)l23!. A considerable decrease in potency was observed when the invariant residue He in A2 was replaced by Pro or D-allo-Ile. The resultant analogues had potencies of only 0.017 and 0.0019, respectively. [D-allo-IleA2 ]insulin was clearly less potent than [Pro A2 ] insulin, although D-allo-Ile differs from lie only in the steric arrangement at the -carbon atom. It is conceivable that the low potency of our A2 analogues is related to the proximity of

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Bd. 360(1979)

Analogues of Human Insulin

A2 and GlyA1, an invariant residue on the surface, critical for biological activity for either steric or conformational reasons^24!. Furthermore, A2 is located on the borderline of the putative receptor-binding region^25!. Two other analogues modified in position A2, [Nle A2 ]insulinl 26 l and [AlaA2]insulin(27l, were also found to be only slightly active, whereas two with valine and leucinel27! at A2 have been reported to be surprisingly potent. Replacement of the amino acids GlnAS and HisBS by alanine caused only a slight decrease in the potency of the respective analogues in vitro, while the potencies in vivo were not affected at all. These results are rather unexpected, since it has been speculated that the strictly invariant residue Gln AS may be involved in the expression of biological activityl20-24!. HisBS - invariant in all high-potency insulins — is replaced by another basic amino acid, arginine, in the insulin of certain caviomorph rodents [Myocastor coypus, Echimys (casiragua)]; the potency of these insulins is less than 10%1281. Removal of B5 (together with B1-B4) in des-(pentapeptide Bl-5)human insulin, also decreased potency to about 5%i 29 ^. Thus, the B5 residue is indeed important for high biological activity. However, as shown by the full potency of [AlaB5]insulin in vivo, there is no absolute requirement for histidine at this position. The [AlaB5]-analogue of sheep insulin has also been reported to be highly active in vivo!30!. TyrB16, also an invariant residue, is involved in dtmer formation and constitutes part of the putative receptor-binding region^25!. We therefore expected that modification of B16 would have a marked influence on biological activity. This was confirmed when B16 was deleted (des-Tyr B16 insulin) or when an additional tyrosine was inserted, as in endo-TyrB16a-insulin; both analogues have potencies of less than 1% of that of insulin. However, replacement of L- by D-tyrosine only moderately decreased the potency in vitro (0.17), without causing any significant change in vivo. It seems that the D-enantiomer can be accomodated in position B16 without seriously disturbing the contact with the biological receptor. It should be interesting to study the aggregation characteristics of these analogues by physico-

1629

chemical techniques and compare the results with their biological potencies. The only analogue in our series with an in vitro potency greater than one is [HisA8]insulin. In the isolated fat-cell (glucose oxidation and lipolysis) and the anti-insulin serum binding assays it is twice to three times as potent as human insulin. This value corresponds closely to the in vitro potency reported for chicken and turkey insulin [31-34]^ which also contain histidine in position Ag(35,36] Although the insulins from these birds differ from human and bovine insulin in 6 and 5 additional residues, respectively, it has been speculated that their greater potency might be due to His A8 l 25>36 l. This is now confirmed by our analogue. As shown by Simon et aÜ16l and Gammeltoft and Gliemannl37!, the greater biological activity of chicken insulin can be fully ascribed to an enhanced binding affinity for the receptor; most probably, this will also prove true of [HisA8]insulin, although direct receptor-binding studies with this analogue remain to be done. The elucidation of the exact nature of the interaction between histidine in A8 and other residues of the insulin molecule and/or the receptor region, leading to increased biological activity, might be a rewarding topic for future investigations. The unaugmented potency of two analogues with other bulky residues in A8, viz. lysine or phenylalanine, however, seems to exclude large size per se as a possible cause, as suggested by Pullen et al.[2Sl. Whether the effects of insulin on glucose metabolism and lipolysis are mediated by one and the same or by two distinct types of receptor on the fat-cell membrane is still a controversial issue. Renner and Hepp i38 l adduced evidence indicating that different receptors are involved; on the other hand, studies by Ellis et alJ 39 l, Green and Newsholmel40! and Thomas et alJ 41 ) argue in favour of a common receptor. The last-mentioned authors showed, for instance, that the concentrations not only of insulin, but also of seven insulin analogues required to produce halfmaximal effects on lipogenesis and lipolysis were identical. Our own results with a different and even larger series of analogues are in full agreement with those of Thomas et al. The statistically significant correlation between the effects of our analogues on glucose oxidation and lipolysis

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1630

F. M rki, M. de Gasparo, K. Eisler, B. Kamber, B. Rinikcr, W. Kittel and P. Sieber

(Fig. 5) supports the concept that these two metabolic processes are mediated by a common receptor on the fat-cell membrane. Biological activity in vivo When the biological activity of seven analogues was assayed in vivo, their hypoglycaemic effects after s.c. injection in fasted rats were found to be similar to, or at least of the same order of magnitude as their activity on the isolated fatcell assay in vitro. With four of these analogues there were statistically significant differences between the two potencies: [AlaA5]-, [AlaB5]and [o-TyrB16] insulin were more potent in vivo than in vitro, while the opposite was found with [HisAS] insulin. Similar observations have been made previously with several other analogues [13,14] [n some of those studied in detail, the differences between in vivo and in vitro potencies could be ascribed to differences in the rates of degradation of insulin and the analogues [14,42] This may or may not also apply to our analogues. The durations of their hypoglycaemic effects after s.c. administration in the rat are not noticeably different from those of equipotent doses of insulin. A similar, detailed study of the duration of action after i.v. administration is in progress. Antibody binding Binding of a modified insulin by antibody raised with bovine or porcine insulin in guinea-pigs — a measure of antigenicity — can be quantitated in vitro under the experimental conditions of the radioimrnunoassay'16·43"45! or in an immune haemolysis inhibition assay'46!. Immunogenicity can be analysed by immunizing animals such as guinea-pigs'43!, cows'47', or mice'48! with insulin or its analogues and characterizing the antibodies formed, or by inducing passive cutaneous anaphylaxis in guinea-pigs'49'. Results of such tests show the presence of an antigenic determinant in the vicinity of the TV-terminal region qf the B chain (Bl-8) and in the A10-A21 area [43,46,48,49]

Genetic factors in the animal can direct antibody production towards specific determinants on the insulin molecule'50!, and experience has shown that antisera obtained from different animals,

Bd. 360 (1979)

by means of the same immunization schedule, vary in their energy of reaction with antigen' 5 !1. For example, Simon et al.'16! noted that among 21 guinea-pig anti-insulin sera, there were only 3 that cross-reacted with chicken insulin. In the radioimmunoassay, the displacement of radioactive insulin by unlabelled insulin from antibody-binding sites is inversely related to the degree of iodination' 52 '. Since the labelled insulin we used always had the same specific activity (ca. 700μΟϊ/μιηο1), we may assume that the degree of iodination was the same over the entire experimental period. Further, in each experiment synthetic human insulin was included as a reference. The radioimmunochemical characterization of our analogues was performed with one antibovine insulin serum which is most probably composed of several populations of antibodies, each population directed towards one of several immunologic determinants of the insulin molecule'5 31. However, unpublished observations obtained with two anti-porcine insulin sera (Miles GP-4 and Novo LAA-78) gave similar results; with all three antisera the analogues tested ranked in the same order of relative potency. The antigenic determinant on the insulin molecule requires the A and B chains to be in a precise steric relationship to each other'46'54'55!. This is critical for both the metabolic effect and antibody binding, although distinctly different regions of the insulin molecule are involved in receptor'25! and antibody binding'43»46'48·49!. Modification of the insulin structure by replacement of the invariant amino acid isoleucine in A2 by proline or D-allo-isoleucine does not markedly affect the binding of the resultant analogues to the antibody. The same is also true of alanine replacing glutamine in AS. Indeed, A2 and A5 have not been reported to be major elements of the antigenic determinant of insulin. In our assay, B5 also appears to be unimportant in the binding reaction. This is rather unexpected because Bl—B8 is an important component of the antigenic determinant' 49 '. Moreover, splitting off the amino acid sequence Bl to B4 gave analogues with greatly reduced immunogenicity and antigenicity but almost unchanged biological activities and receptor binding characteristics Brought to you by | provisional a Unauthenticated | 129.130.252 Download Date | 7/11/14 2:16

Bd.360 (1979)

Analogues of Human Insulin

[43,56] jhe des-(pentapeptide B1-5)human insulin had likewise only 15% potency in the radioimmunoassay technique!29!. Analogues with natural cystine pairing, but with D- instead of L- half-cystine residues in A6 and A7, retain a relatively marked potency to displace labelled insulin from its binding sites. Indeed, as discussed above, the unnatural D-stereoisomer does not seem to influence the peptide backbone in these analogues, and they are still able to crystallize. In contrast, D-Cys in position AI l leads to a drastic reduction of the anti-insulin serum-binding properties; AI l is part of the described antigenie locus of the insulin molecule!48'49!. In a study of the species-specificity of anti-insulin serum, it was shown that residues 8 to 10 of the A chain constitute at least part of the antigenie site as well as of the site of reaction with the antibody! 44 ' 55 1. Substitution in A8 in our analogues produces puzzling results. In the same way as it does the biological activity, the presence of histidine in A8 doubles the potency of the molecule to displace labelled insulin, while phenylalanine or lysine markedly reduce the antibody-binding potency. No explanation for this is available yet. Replacement of L-tyrosine in B16 by the D-stereoisomer causes no change in antibody-binding potency. B16 therefore does not seem to be important for binding with anti-insulin serum. However, des-Tyr n16 - and endo-Tyr B16a -insulinhave the least antibody-binding potency of all the analogues studied. They are among the most lipophilic analogues of insulin, and show very weak biological activity. This is strong evidence that the structure of these analogues differs markedly from that of insulin. On the other hand, [Cys(Acm)A7)B7]insulin and the two disulphide-bond isomers of human insulin!3! retain relatively high antibody-binding potency (range 9—20%), despite the marked structural change. Two general conclusions can be drawn from the results of our investigation. First, there appears to be no simple relationship between physicochemical parameters (e.g. electrophoretic mobility, lipophilic character, ability

1631

to crystallize) and biological activity or antibody binding of the insulin analogues. Secondly, the current hypotheses about the receptor-binding region and the antigenic-determinant area afford reasonable explanations for the observed activities of some of our analogues; however, unexpected results obtained with others indicate that refinements or modifications of these hypotheses are needed to account for all the findings reported to date. It is hoped that the data presented here may stimulate further efforts to clarify the still elusive structure-activity relationship of insulin. Note added in proof (18 October 1979): There remains a discrepancy between biological potency and receptor-binding affinity of hagfish insulin, if the different rates at which pig and hagfish insulin reach the steady-state of receptor binding are taken into account (S. Emdin, O. Sonne & J. Gliemann, manuscript submitted to Diabetes). We thank Mrs. N. Dietrich, G. Hosteller, J. Motz, J. See· berger, V. von Arx, I. Zimmermann, Mr. /. R. Frei, H. R. Keller, R. Lindecker and E. Manz for skilful tech^nical assistance, Mr. M. Faupel and E. von Arx for the 'analytical work, Mr. K. Schul for the photographs of the crystalline analogues, Mrs. Ch. Mosch for typing the manuscript, Mr. A.M. Kirkwood for linguistic assistance, and - last, but not least - Dr. Guy Dodson and Prof. Axel Wollmer for stimulating discussions.

Literature 1 Sieber, P., Kamber, B., Hartmann, ., Johl, ., Riniker, B. & Rittel, W. (1974) Helv. Chim. Acta 57, 2617-2621. 2 Sieber, P., Kamber, B., Hartmann, A., Johl, A., Riniker, B. & Rittel, W. (1977) Helv. Chim. Acta 60, 27-37. 3 Sieber, P., Eisler, K., Kamber, B., Riniker, B., Rittel, W., Mäiki, F. & de Gasparo, M. (191%) Hoppe-Seyler's Z. Physiol. Chem. 359, 113-123. 4 Sieber, P., Kamber, B., Eisler, K., Hartmann, A., Riniker, B. & Rittel, W. (1976) Helv. Chim. Acta 59, 1489-1497. 5 Kamber, B„ Riniker, B., Sieber, P. & Rittel, W. (1976) Helv. Chim. Acta 59, 2830-2840. 6 Kamber, B. (1911) Helv. Chim. Acta 54, 927-930. 7 Eisler, K., Kamber, B., Riniker, B., Rittel, W., Sieber, P., de Gasparo, M. & Mäiki, F. (1979) Bioorg. Chem., in p>ess. 8 Riniker, B., Eisler, K., Kamber, B., Müller, H., Rittel, W. & Sieber, P. (1979) in Proc. 15th Eur. Peptide Sympos. (Siemion, I.Z., ed.) Wroclaw University Press, in press. 9 Hirs, C.H.W. (1956)7. Biol. Chem. 219,611-621.

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10 Chakravarti, I.M. (1971) Biom. Z. 13, 89-94. 11 Cuatrecasas, P. (1974) Annu. Rev. Biochem. 43, 169-214. 12 Freychet, P., Roth, J. & Neville, D.M. (l97l)Proc. Nail. Acad. Sei. U.S.A. 68, 1833-1837. 13 Freychet, P., Brandenburg, D. & Wollmer, A. (1974) Diabetologia 10, 1-5. 14 Gliemann, J. & Gammeltoft, S. (1974) Diabetologia 10, 105-113. 15 Renner, R. & Kemmler, W. (1974) Hoppe-Seyler's Z. Physiol. Chem. 355, 1555-1556. 16 Simon, J., Freychet, P. & Rosselin, G. (l 974) Endocrinology 95, 1439-1449. 17 Schüttlet, A. (1979) Dissertation, MathematischNaturwissenschaftliche Fakultät der RWTH, Aachen. 18 Emdin, S., Gammeltoft, S. & Gliemann, J. (1977) / Biol. Chem. 252, 602-608. 19 Muggeo, M., Obberghen, E. van, Kahn, C.R., Roth, J., Ginsberg, H., De Meyts, P., Emdin, S.O. & Falkmer, S. (1979) Diabetes 28, 175-181. 20 Blundell,T.L., Cutfield, J.F., Cutfield, S.M., Dodson, E. J., Dodson, G.G., Hodgkin, D.C., Mercola, D. A. & Vijayan, M. (1971) Nature (London) 231, 506-511. 21 Blundell, T. L., Dodson, G., Hodgkin, D.C. & Mercola, D. A. (1972) Adv. Protein Chem. 26, 279-402. 22 Brandenburg, D., Gattner, H.-G., Weinert, M., Herbertz, L., Zahn, H. & Wollmer, A. (1971) Excerpta Med. Int. Congr. Ser. 231, 363-376. 23 Busse, W.-D. & Gattner, H.-G. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 147-155. 24 Dodson, E. J., Dodson, G.G., Hodgkin, D.C. & Reynolds, C.D. (1979) Can. J. Biochem. 57, 469-479. 25 Pullen, R.A., Lindsay, D.G., Wood, S.P., Tickle, I.J., Blundell, T. L., Wollmer, A., Krail, G., Brandenburg, D., Zahn, H., Gliemann, J. & Gammeltoft, S. (1976) Nature (London) 259, 369-373. 26 Ferderigos, N. & Katsoyannis, P.G. (l977)7. Chem. Soc. Perkin Trans. /, 1299-1305. 27 Hörnle, S., Weber, U. & Weitzel, G. (1968) HoppeSeyler's Z. Physiol. Chem. 349, 1428-1430. 28 Horuk, R., Goodwin, P., O'Connor, K., Neville, R.W.J., Lazarus, N.R. & Stone, D. (1979)Mzfure (London) 279, 439-440. 29 Schwartz, G. & Katsoyannis, P. G. (1978) Biochemistry 17, 4550-4556. 30 Weitzel, G., Weber, U., Eisele, K., Zollner, H. & Martin, J. (191 ) Hoppe-Seyler's Z. Physiol. Chem 351,263-267. 31 Kemmler, W. & Rager, K. (1968) Hoppe-Seyler's Z. Physiol. Chem. 349, 515-516. 32 Weitzel, G., Oertel, W., Rager, K. & Kemmler, W. (1969) Hoppe-Seyler's Z. Physiol. Chem. 350, 57-62.

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33 Schauder, P. & Bück, M. D. (1969) Z. Naturforsch. 24B, 1424-1428. 34 Hahn, J. & Martini, 0. (1971) Z. Naturforsch. 26B, 1378. 35 Smith, L.F. (1966) Am. J. Med. 40, 662-666. 36 Weitzel, G., Renner, R., Kemmler, W. & Rager, K. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 980-986. 37 Gammeltoft, S. & Gliemann, J. (1971) Acta Physiol. Scand. 91, 7A-7B. 38 Renner, R. & Hepp, K. D. (1974) Diabetologia 10, 384 (Abstr.). 39 Ellis, M.J., Daiby, S.C., Jones, R. H. & Sönksen, P.H. (1978) Diabetologia 15,403-410. 40 Green, A. & Newsholme, E. A. (1979) Biochem. J. 180,365-370. 41 Thomas, S. H. L, Willey, K.P. & Wisher, M. H. (1978) Diabetologia 14, 276 (Abstr.). 42 Jones, R.H., Dron, D.I., Ellis, M.J., Sönksen, P.H. & Brandenburg, D. (1976) Diabetologia 12, 601-608. 43 Kerp, L., Steinhilber, S., Kasemii, D., Henrichs, H. R., Petersen, K.G. & Geiger, R. (1975) Acta Endocrinol. (Copenhagen) Suppl. 199, 150. 44 Berson, S.A. & Yalow, R.S. (1959)/. Clin. Invest. 38,2017-2025. 45 Barth, T., Siseva, A., Sirakov, L., Slaninova, J. & Ditzov, S. (1978) Collect. Czech. Chem. Commun. 43, 2967-2972. 46 Arquilla, E.R., Bromer, W.W. & Mercola, D. (1969) Diabetes 18, 193-205. 47 Renold, A.E., Soeldner, J.S. & Steinke, J. (1964) Ciba Found. Colloq. Endocrinol. (Proc.) 15, 122-134. 48 Keck, K. (1975) Nature (London) 254, 78-79. 49 Wilson, S. (1969) Excerpta Med. Int. Congr. Ser. 172, 403-405. 50 Arquilla, E. R., Ooms, H. & Finn, J. (1966) Diabetologia 2, 1-13. 51 Berson, S.A. & Yalow, R.S. (1959)7. Clin. Invest. 38, 1996-2016. 52 Ooms, M. A. & Arquilla, E. R. (1966) in Labelled Proteins in Tracer Studies (Donato, L., Milhaud, G. & Sirchis, J., eds.) pp. 237-245, Euratom, Brüssels. 53 Arquilla, E. R., Dorio, R.J. & Brugman, T.M. (1976) Diabetes 25, 397-403. 54 Berson, S.A. & Yalow, R.S. (1961) Nature (London) 191, 1392-1393. 55 Berson, S.A. & Yalow, R. S. (1963) Science 139, 844-845. 56 Kerp, L., Steinhilber, S., Kasemir, H., Han, J., Henrichs, H. R. & Geiger, R. (1974) Diabetes 23, 651-656. 57 Märki, F. & Albrecht, W. (1977) Diabetologia 13, 293-295.

F. Märki, M. de Gasparo, K. Eisler, B. Kamber, B. Riniker, W. Rittel und P. Sieber, Forschungslaboratorien der Division Pharma der Ciba-Geigy AG, CH-4002 Basel, Schweiz.

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