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Aim: To explore the importance of B9 and B10 amino acid residues in the insulin molecule., Methods: The [B9Glu, B10Asp] insulin (E,D-insulin) receptor binding activity, glucose upta ke activity, and lipogenesis activity were measured in isolated adipocytes. The translocation of glucose transporter 4 (Glut4) and the phosphorylation of insulin receptor (IR) stimulated by E, D-insulin were determined by Western blotting., Results: Compared with native insulin, the receptor binding activity of E,D-insulin was 31 %; the stimulating activity of E,D-insulin in glucose transport and lipogenesis were 45 % and 40 % respectively; the stimulations of Glut4 translocation and insulin receptor autophosphorylation of E,D-insulin were about 58 % and 46 % respectively., Conclusion: B9-serine residue is crucial for insulin actions in glucose metabolism.

The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form., (Copyright 2001 John Wiley & Sons, Inc.)

Binding of porcine 125I-insulin (0.15 nM) to hagfish red blood cells was time-dependent, reaching equilibrium after 1 hr at 10 degrees. The specific 125I-insulin binding to hagfish red blood cells was reversible, and unlabeled insulin accelerated the dissociation of 125I-insulin bound to receptors from a T1/2 of 60 min in cells suspended in medium alone to 23 min in medium containing 8 microM nonradioactive insulin. Porcine insulin and desoctapeptide insulin competed for specific binding of 125I-insulin in a dose-dependent manner, whereas glucagon and somatostatin did not. For porcine insulin, Scatchard analysis produced a curvilinear plot, suggesting multiple affinity binding sites with high-affinity and low-affinity association constants (Ka) 0.2 x 10(9) M-1 and 0.27 x 10(7) M-1, respectively. A total of 2090 binding sites per hagfish red blood cell was calculated. Sixty-two percent of the bound 125I-insulin was found to be internalized into the hagfish red blood cells. Less degradation of 125I-insulin was observed by Sephadex G-50 chromatography compared to human red blood cells.

Insulin, following binding to its receptor, produces a dose- and time-dependent stimulation of entry of 2-deoxy-D-[U-14C] glucose and glycogen synthesis from D-[U-14C] glucose in cultured mouse astrocytes following differentiation. Maximal stimulation of both glucose entry (217% above basal) and of glycogen synthesis (209% above basal) was observed at an insulin concentration of 1.7 x 10(-8) M. Insulin also stimulates the incorporation of leucine into astrocytic proteins with maximal stimulation (156% above basal) at an insulin concentration of 1.7 x 10(-7) M, but no effect on leucine uptake was observed at an insulin concentration of 1.7 x 10(-6) M. These results, together with a previous demonstration that insulin and certain insulin analogues stimulate pyrimidine nucleoside incorporation into nucleic acid, indicate that insulin has diverse actions on biomacromolecular metabolism in cultured mouse astrocytes.

Insulin receptor binding and effects upon uridine incorporation into nucleic acid were studied and compared to 3 insulin analogues, DPI, DPI-amide and DOI, in primary cultures of mouse astrocytes. From half-maximal inhibition of [125I]monoiodoinsulin binding (0.14 nM), the relative binding affinities of DPI, DPI-amide and DOI were 20%, 80% and 4% as compared with the native insulin, respectively. IC50 values were 158.5 nM for insulin, 199.5 nM for DPI-amide, 794.3 nM for DPI and 3980 nM for DOI. The corresponding relative potencies in stimulating [14C]uridine incorporation into TCA-insoluble material were 9% for DPI, 100% for DPI-amide and 1.1% for DOI. These findings are commensurate with data for these analogues in other insulin-sensitive tissues. It is concluded that the astrocyte insulin receptor has similar structural requirements to receptor in tissues outside the CNS, at least in terms of the involvement of the C-terminus of the insulin B-chain.

[Trp]B1 analogs to porcine and bovine insulins have been obtained through selective reaction with Msc-ONSu, and followed by Edman degradation, condensation with Msc-Trp-ONp and deprotection. Through cellulose acetate electrophoresis, amino acid composition analysis, UV absorption spectrum and N-terminal analysis, it has been proved that the products are of purified [Trp]B1 insulin. Mouse convulsion assay shows that the biological activities of porcine and bovine [Trp]B1 insulins are 18 i.u./mg and 17 i.u./mg respectively, corresponding to about 70% of the original activity of native porcine insulin. Their structural details are under investigation in our laboratory.

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.

Binding of 125I-insulin to primary cultures of differentiated mouse astrocytes was time-dependent, reaching equilibrium after 2 h at 22 degrees C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing approximately 5,000 occupied binding sites/cell. The half-life of 125I-insulin dissociation at 22 degrees C was 2 min, with an initial dissociation rate constant of 4.12 X 10(-2) s-1. Dissociation of bound 125I-insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 microM). Porcine and desoctapeptide insulins competed for specific 125I-insulin binding in a dose-dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple-affinity binding sites (high-affinity Ka = 4.92 X 10(8) M-1; low-affinity Ka = 0.95 X 10(7) M-1). After incubation with insulin (0.5 microM) for 2 h at 37 degrees C, increases above basal values of 254 +/- 23 and 189 +/- 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 microM) for 24 h at 37 degrees C, there were increases of 145 +/- 6% for [3H]thymidine uptake and 166 +/- 11% for thymidine incorporation. Basal and stimulated uridine and thymidine uptake and incorporation were inhibited by 50 microM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.

The C-terminus of the insulin B chain is essential for dimerisation and expression of negative cooperativity. In order to evaluate the possible physiological role of these phenomena, we have studied the properties in vivo and in vitro of despentapeptide insulin (B26-30 deleted), derived from beef insulin, and deshexapeptide insulin (B25-30 deleted), derived from pork insulin. These materials do not dimerise and have 15% and 0% retention of negative cooperativity respective. Lipogenesis potencies in rat adipocytes were: despentapeptide insulin 19.9 +/- 0.3%; deshexapeptide insulin 19.9 +/- 1.5%. Binding potencies in adipocytes were: despentapeptide insulin 22.6 +/- 7.8%; deshexapeptide insulin 13.2 +/- 3.3%. Metabolic clearance rates were reduced compared to insulin (insulin = 19.1 +/- 0.9; despentapeptide insulin = 9.7 +/- 0.8; deshexapeptide insulin = 6.4 +/- 0.6 ml . min-1 . kg-1 at plasma concentration 0.5 nmol/l). Hypoglycaemic potencies were reduced for both analogues (40% and 30%) when calculated on the basis of plasma concentration although both analogues and insulin were equally effective at lowering plasma glucose concentration in equimolar doses. Plasma half-disappearance time was prolonged (despentapeptide insulin = 7.3 +/- 0.5; deshexapeptide insulin = 9.1 +/- 0.2 min). Both analogues were full agonists and conformed to the general relationship between in vitro and in vivo properties seen with a wide range of modified insulins. They resemble other analogues with modifications which reduce receptor affinity without impairing dimerisation or negative cooperativity. The results do not support a physiological role for dimerisation or negative cooperativity.

Despentapeptide (B26-30)-insulinamide (B25) prepared by a semisynthetic procedure was found to have about 65% of the hypoglycaemic activity of natural insulin. In contrast, the binding of the modified insulin analogue to insulin specific receptors was markedly increased. The discrepancy between the loss of biological potency and the apparent increase in binding affinity for membrane receptors reveals that not all of the biological activity of insulin is regulated by the receptor-binding system. The tetrapeptidamide of the B-chain of insulin (Arg-Gly-Phe-Phe-NH2) was clearly shown to have both insulin-like and insulin-potentiating actions in vivo although it had no effect on insulin receptor function in vitro. Evidence suggests that the small peptide fragment of insulin may be internalized and acts at the post-binding site(s) of the glucose metabolic pathway in target tissues. The present data support the general concept that insulin may exert its complex molecular actions through internalized hormonal fragment as well as the transmembrane mediators generated from receptor binding.

The semisynthesis of a series of B23-D-Ala deshexapeptide and despentapeptide analogues with B22-arginine replaced are described. The following semisynthetic scheme is used: desnonapeptide insulin pentamethyl ester is prepared from insulin hexamethyl ester through the action of trypsin and carboxypeptidase B, the amino groups are protected with Boc-groups, the protected product is condensed with synthetic peptides, and the protecting groups are removed with trifluoroacetic acid treatment and by saponification. The biological activities of these analogues indicate that the B22-Arg is non-essential and can be replaced by Lys or even Asp without any influence on the biological activity. The activity is reduced to one half when it is replaced by valine or to a very low level when replaced by Gly or D-Arg. The reason for the low activity of guinea pig insulin is discussed.