Determinants of growth-promoting activity reside in the A-domain of insulin-like growth factor I.

A two-chain, disulfide linked, insulin-like compound embodying the A-domain of insulin-like growth factor I (IGF-I) and the B-chain of insulin has been synthesized and characterized with respect to insulin-like biological activity and growth-promoting potency. The compound displays a potency of ca. 41% relative to insulin in assays for insulin-like activity (e.g., lipogenesis) but significantly higher activity than insulin, ca. 730% relative to insulin, in growth factor assays (e.g., thymidine incorporation). The compound is, however, a less potent growth factor than IGF-I itself, ca. 26.5% relative to IGF-I, and is not recognized by IGF carrier proteins. We conclude that structural features contained in the A-domain of IGF-I are primarily responsible for the growth-promoting ability displayed by IGF-I, while features in the B-domain are responsible for recognition by IGF carrier proteins.

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 1. [21-Desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin.

The C-terminal region of the A chain of insulin has been shown to play a significant role in the expression of the biological activity of the hormone. To further delineate the contribution of this segment, we have synthesized [21-desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin, in which the C-terminal amino acid residue of the A chain of insulin, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an amide and an isopropylamide, respectively. Both insulin analogues display biological activity, 14-15% for the unsubstituted amide analogue and 20-22% for the isopropylamide analogue, both relative to bovine insulin. In contrast, a [21-desasparagine-A]insulin analogue has been reported to display less than 4% of the activity of the natural hormone [Carpenter, F. (1966) Am. J. Med. 40, 750-758]. The implications of these findings are discussed, and we conclude that the A20-A21 amide bond plays a significant role in the expression of the biological activity of insulin.

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 2. [21-Asparagine diethylamide-A]insulin.

We have synthesized [21-asparagine diethylamide-A]insulin, which differs from the parent molecule in that the free carboxyl group of the C-terminal amino acid residue, asparagine, of the A chain moiety has been converted to a diethylamide group. The analogue displays equivalent potency in receptor binding and biological activity, 48% and 56%, respectively, relative to bovine insulin. In contrast, we have reported previously [Burke, G. T., Chanley, J. D., Okada, Y., Cosmatos, A., Ferderigos, N., & Katsoyannis, P. G. (1980) Biochemistry 19, 4547-4556] that [21-asparaginamide-A]insulin exhibits a divergence in these properties, ca. 60% in receptor binding and ca. 13% in biological activity. The disparity in the biological behavior of these analogues is discussed, and we ascribe the modulation of biological activity independent of receptor binding activity observed between these analogues to the difference in the negativity of the carbonyl oxygen of the A chain moiety C-terminal amino acid residue.

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 3. [21-Desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin.

We have synthesized [21-desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin, which differ from natural insulin in that the C-terminal amino residue of the A chain, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an ethylamide and a trifluoroethylamide group, respectively. [21-Desasparagine,20-cysteine ethylamide-A]insulin displayed equivalent potency in receptor binding and biological activity, ca. 12% and ca. 14%, respectively, relative to bovine insulin. In contrast, [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin displayed a divergence in these properties, ca. 13% in receptor binding and ca. 6% in biological activity. This disparity is ascribed to a difference in the electronic state of the A20-A21 amide bond in these two analogues. A model is proposed to account for the observation of divergence between receptor binding and biological activity in a number of synthetic insulin analogues and naturally occurring insulins. In this model, changes in the electronic state and/or the orientation of the A20-A21 amide bond can modulate biological activity independently of receptor binding affinity. The A20-A21 amide bond is thus considered as an important element in the "message region" of insulin.

Effect of N-methylation of selected peptide bonds on the biological activity of insulin. [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin.

Hydrogen bonding involving peptide bonds of the backbone of the insulin molecule may play an important role in insulin-receptor interaction. Our previous work suggested that the A2-A8 helical segment of the hormone molecule participates in this interaction. To investigate the possible involvement of peptide bonds of this segment in insulin-receptor interaction the [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin ([MeIle2-A]- and [MeVal3-A]insulins) were synthesized. The circular dichroic spectra of the analogues were obtained and their properties were examined in several biological assays. The circular dichroic spectra suggested that the analogues remained monomeric at concentrations at which insulin is predominantly dimeric, and that their A2-A8 helical segments are distorted. The in vitro biological activity and the receptor binding affinity of these analogues were compared with that of natural insulin. Both analogues are weak full agonists. [MeIle2-A]insulin displayed a potency of 5.4 +/- 0.3% in stimulating lipogenesis and 4.6 +/- 2.3% in receptor binding affinity in rat fat cells and rat liver plasma membranes respectively. [MeVal3-A]insulin displayed a potency of 2.1 +/- 0.2% in lipogenesis and 1.0 +/- 0.3% in receptor binding assays. In radioimmunoassays [MeIle2-A]- and [MeVal3-A]insulins exhibited potencies of 13% and 11% respectively relative to the natural hormone. The substantially decreased biological activity and receptor binding affinity of these analogues may be attributed partly to the change of conformation and partly to the loss of hydrogen bonding capacity of the A2-A8 segment brought about by N-methylation of the A1-A2 or A2-A3 peptide bonds.

Interaction between the A2 and A19 amino acid residues is of critical importance for high biological activity in insulin: [19-leucine-A]insulin.

The replacement of tyrosine at position A19 by leucine in the insulin molecule led to an analogue, [19-leucine-A]insulin [( Leu19-A]insulin), displaying insignificant receptor binding affinity and in vitro biological activity less than 0.1 and 0.05%, respectively, compared to the natural hormone. This analogue along with the previously reported [2-glycine-A]-, [2-alanine-A]-, and [2-norleucine-A]insulins is the least potent insulin analogue we have examined. Circular dichroic studies showed that all these analogues are monomeric at concentrations at which insulin is primarily dimeric. We conclude that an aromatic ring at position A19 and the presence of the side chain of isoleucine at position A2 are each of critical importance for high biological activity in insulin. It appears that the van der Waals interaction between the side chain of isoleucine A2 and tyrosine A19, present in crystalline insulin, is among the most important determinants for high biological activity in insulin.

Critical role of the A2 amino acid residue in the biological activity of insulin: [2-glycine-A]- and [2-alanine-A]insulins.

We report the synthesis of [2-glycine-A]insulin ([ Gly2 -A]insulin) and [2-alanine-A]insulin ([ Ala2-A]insulin) in which the indicated amino acid has been substituted for isoleucine found in this position in the natural hormone. The circular dichroic (CD) spectra of the analogues were obtained, and their properties were examined in several biological assays. CD studies suggested that the analogues remain monomeric at concentrations at which insulin is partly or mostly dimeric. Both analogues are extremely weak full agonists. [ Gly2 -A]-insulin displays 0.05% of the potency of bovine insulin, whereas [Ala2-A]insulin assays at 0.4% of the activity of the natural hormone. We conclude that the presence of the side chain of isoleucine at position A2 is a critical requirement for high biological activity in insulin. The data, together with previous observations, are discussed in connection with an interaction between the side chain of isoleucine-A2 and the phenolic ring system of tyrosine-A19, which are in van der Waals contact in crystalline insulin. This interaction may be required to permit the molecule to assume a conformation consistent with dimerization and with binding to the insulin receptor.

An insulin analogue possessing higher in vitro biological activity than receptor binding affinity. [21-Proline-B]insulin.

To study the effect of an increase in the potential for beta-turn formation of the B20-B23 segment of the B-chain moiety on the biological behavior of insulin, the [21-proline-B]insulin [( Pro21-B]insulin) was synthesized. The in vitro biological activity and the receptor binding affinity of this analogue were compared with that of insulin. In stimulating labeled glucose incorporation into lipids in rat fat cells, the analogue displayed 33.2% potency relative to insulin; receptor binding affinity for the analogue was 15.9% in rat liver membranes and 17.8% in isolated fat cells. [Pro21-B]insulin is thus the first example of a modified insulin for which the biological activity exceeds the receptor binding potency. The secondary structure of this analogue was investigated by circular dichroism studies. Although no significant differences in the conformation of monomeric insulin and analogue could be discerned, their difference in behavior with respect to dimerization and biological properties indicates that these forms are not equivalent. We suggest that the intrinsic activity of receptor-bound [Pro21-B]insulin is greater than that of insulin, although the receptor displays greater affinity for insulin than for the analogue. We consider a model for the interaction between insulin and its receptor that accommodates our findings.

Lithium-induced changes in plasma amino acid levels during treatment of affective disorders.

Plasma amino acid (AA) profiles were determined in patients with bipolar affective disorders before and during treatment with lithium (Li) carbonate. After 90 days of Li intake, there were moderately (20-60%) but significantly decreased plasma levels of isoleucine, leucine, and valine. After 150 days of Li intake, the plasma lysine was also significantly decreased (12%). No significant changes in plasma levels were noted for any of the other 17 AAs that were studied. Since isoleucine, leucine, and valine are important competitors of tryptophan uptake at the blood-brain barrier, these observations suggest a possible mechanism for the therapeutic activity of Li, i.e., the enhancement of brain serotonin levels.

Elevation of erythrocyte glycine levels during lithium treatment of affective disorders.

An elevation of erythrocyte (RBC) glycine was observed in a group of patients with bipolar affective disorder who were being treated with lithium (Li) carbonate. A maximal increase of 90% or more was generally attained after about 100 days of Li intake and was maintained for 2 years or longer. After 3 or more years of Li therapy, the RBC glycine had often returned to the normal range. Abrupt withdrawal of Li in such a case produced an immediate, dramatic, and paradoxical increase in RBC glycine which lasted for 100 days. Plasma glycine was not affected by Li.

[A2-Norleucine]insulin. An analog with unanticipated biological properties.

The simple replacement of the A2-isoleucine by norleucine in the insulin molecule gave an analog [A2-norleucine]insulin ([Nle A2]-insulin) which was found to possess insignificant receptor-binding affinity and in vitro biological activity, 0.6 and 0.9 percent, respectively, compared to the natural hormone. Circular dichroic studies showed that this analog is monomeric and indicated that the helical segment A2-A8 and the beta strand B24-B29 have undergone conformational changes. The reduced receptor-binding affinity of [Nle A2]insulin is attributed to the distortion of the A2-A8 helical segment which in turn may lead to the displacement of the A1, A5 and A19 residues which are putatively involved in receptor binding.

Divergence of the in vitro biological activity and receptor binding affinity of a synthetic insulin analogue, [21-asparaginamide-A]insulin.

The [21-asparaginamide-A]insulin ([Asn-(NH2)21-A]insulin) was synthesized by the procedures developed in this laboratory to investigate the contribution of the C-terminal residue, asparagine, of the A chain to the biological activity and receptor binding affinity of insulin. Its secondary structure was investigated by circular dichroism studies. The biological behavior of this analogue was compared with that of insulin in in vitro and in vivo tests and in receptor binding assays. In contrast to other naturally occurring insulins and to all insulin analogues synthesized thus far, [Asn-(NH2)21-A]insulin displays a disparity between receptor binding affinity and in vitro biological potency. In stimulating glucose oxidation and lipogenesis the analogue exhibited potencies of 12 and 14.8%, respectively, compared to insulin. In insulin receptor binding assays, however, this analogue was found to possess a relative potency at least fourfold higher than the in vitro biological activities. In rat liver membranes and in isolated fat cells the analogue exhibited affinities of ca. 63.9 and 51.4%, respectively, compared to the natural insulin. Both the synthetic analogue and the natural hormone have the same maximal activity in the in vitro assays and their dose-response curves are parallel. When assayed in vivo by the mouse convulsion test, [Asn(NH2)21-A]insulin displays a potency of ca. 72% that of the native insulin. This might indicate partial amidolysis of the analogue in vivo, resulting in conversion to the natural hormone. The implications of these observations are considered with regard to insulin-receptor interactions and the generation of the physiological response to the hormone.

Determination of erythrocyte amino acids by gas chromatography.

Erythrocyte amino acid levels were determined, by gas chromatography, in a group of 34 normal human adults. No significant sex or age correlations were noted. A method for the quantitative gas chromatographic analysis of free amino acids in erythrocytes is described. Following hemolysis and deproteinization the amino acids were isolated on a cation-exchange resin. Glutathione was removed from the amino acid mixture by adsorption on an anion-exchange resin. Following conversion to their N-acetyl-n-propyl esters, 19 amino acids were separated and quantitated by gas chromatography on a single column in 18 min. Typical reproducibility data indicate that a coefficient of variation of 2-5% is attainable.

Amino acids in bipolar affective disorders: increased glycine levels in erythrocytes.

The authors measured the concentrations of 20 amino acids in the erythrocytes and plasma of 13 female bipolar patients and 10 female normal controls. The concentration of glycine in the erythrocyte was significantly elevated in the patient group. No differences were present in the plasma. Preliminary findings indicate that the high glycine levels were present in patients who were depressed, manic, or in remission and were unaffected by electroshock therapy.

Dopamine-beta-hydroxylase: evidence for increased activity in sympathetic neurons during psychotic states.

Tritiated dopamine was infused into psychiatric patients during acute psychotic episodes and in remission. An index of the activity of dopamine-beta-hydroxylase of salivary gland sympathetic neurons was determined by measuring the distribution of tritiated metabolites in salivary fluid. Increased synthesis of norepinephrine occurred in acute schizophrenia and in the manic state of manic-depressive psychosis but not in the depressed phase.