Impaired negative cooperativity of the semisynthetic analogues human [LeuB24]- and [LeuB25]-insulins

Several semisynthetic analogues of human insulin were prepared by enzyme-assisted coupling of synthetic octapeptides to the C-terminal of porcine desoctapeptide insulin. We report the receptor-binding and biological properties of [Leu^B24]- and [Leu^B25]-insulins, one of which has the same sequence as a “mutant” insulin recently found in a diabetic patient (Tager, H. et al.(1979) Nature $\text{28}$:121–125). [Leu^B24]- and [Leu^B25]-insulins had, respectively, 8–12% and 0.9–1.1% of the binding affinity of human insulin, and 11% and 2.7% of its potency in stimulating lipogenesis in isolated rat fat cells. Neither one was an antagonist of the biological effects of native insulin. While the ability of [Leu^B24]-insulin to induce negative cooperativity was clearly impaired, that of [Leu^B25]-insulin was almost abolished. [Leu^B25]-insulin was also a potent antagonist of the negative cooperativity induced by native insulin.     

A new procedure for enzymatic semisynthesis of human insulin by hydrolysis of single-chain des-(b-30)-lnsulin precursor with lysyl endopeptidase

It has been shown that the single-chain des-(B-30)-insulin precursor (SCI) can be converted into human insulin ester by transpeptidation using trypsin in the presence of a threonine derivative. The present study demonstrates that Achromobacter lyticus protease 1 (lysyl endopeptidase) can catalyze the transpeptidation reaction more efficiently than can trypsin. It is also shown that des-(B-30)-insulin (DAI) can be produced by hydrolysis of SCI with the lysyl endopeptidase. Since it is well known that SCI can be produced by gene technology, the following method is recommended for industrial production of human insulin ester: hydrolysis of SCI with lysyl endopeptidase followed by coupling of the resulting DAI with a threonine derivative using trypsin or lysyl endopeptidase.     

Supernormal insulin: [D-PheB24]-insulin with increased affinity for insulin receptors

[D-Phe^B24]- and [D-Phe^B25]-human insulin were semisynthesized from porcine insulin by enzyme assisted coupling method. Receptor binding ability of [D-Phe^B24]- and [D-Phe^B25]-insulin was 180% and 4%, respectively, of that of human insulin. Increased affinity of [D-Phe^B24]-insulin was ascribed to markedly decreased dissociation rate in binding to human cultured lymphocytes. Negative cooperative effect of [D-Phe^B24]insulin was also increased to twice of that of human insulin. Biological activity of these analogues was assessed by 2-deoxy-glucose uptake studies in isolated adipocytes and the ability of [D-Phe^B24]- and [D-Phe^B25]-insulin was 140% and 4%, respectively, of that of human insulin. These findings suggest that B25 L-Phe is more crucial for receptor binding and that [D-Phe^B24]-insulin is the first semisynthetic insulin to show increased affinity for insulin receptors.     

Conversion of exogenous insulin into high molecular weight forms in vivo

[¹²⁵I]-insulin, injected in rats, was converted into high molecular weight forms as judged by gel filtration of blood serum samples collected at various intervals. These forms represented 26% (10 min. after injection) to 81% (240 min. after injection) of the total immunoprecipitable radioactivity. Their molecular weights were not affected by rechromatography in 0.1 M borate buffer (pH 8) or in 8 M urea-1 M acetic acid (pH 2.4). On incubation of [¹²⁵I]-insulin with blood serum $\text{in}\text{vitro}$, no high molecular weight forms could be observed.     

Semisynthesis and properties of some insulin analogs

The trypsin-catalyzed coupling of bovine (Boc)₂-desoctapeptide (B23-B30)-insulin with synthetic octapeptides, H-Gly-X₂-X₃-X₄-Thr-Pro-Lys(Boc)-Thr-OH (X₂ = Phe or Ala, X₃ = Phe or Ala, X₄ = Tyr or Ala), followed by deprotection and purification produced the [Ala^B24, Thr^B30]-, [Ala^B25, Thr^B30]-, and [Ala^B26, Thr^B30]-analogs of bovine insulin in yields of 32, 35, and 32%, respectively. The biological activity of these analogs decreased in the order, normal insulin ([Thr^B30]-bovine insulin) = Ala^B26-insulin > Ala^B25-insulin > Ala^B24-insulin, as assayed for receptor binding and some other biological effects, in contrast with the corresponding Leu-analogs of human insulin, in which the activity decreased in the order, normal insulin > Leu^B24-insulin > Leu^B25-insulin. The affinity to insulin antibodies greatly diminished in both Ala^B24-insulin and Leu^B24-insulin but not in the B25-substituted analogs. The CD spectra of the Leu- and the Ala-analogs were compared with those of normal insulins to show that no apparent correlation seems to exist between the decrease in biological activity and the conformational changes observed in solution. The effects of organic solvents on the peptide-bond equilibrium and on the stability of trypsin are also discussed.     

Tubulin-like protein from Aspergillus nidulans

[³⁵S] labeled extracts of the fungus $\text{Aspergillus nidulans}$ were copolymerized with purified porcine brain tubulin. The [³⁵S] $\text{A. nidulans}$ protein which copurified with porcine microtubules was found to be similar to [³H] chick tubulin when the two were coelectrophoresed on several polyacrylamide gel electrophoresis systems. These results strongly suggest the presence in $\text{A. nidulans}$ of a tubulin-like protein.     

Cytochalasin B inhibition and temperature dependence of 3-O-methylglucose transport in fat cells

The transport of $\text{3-O-}\text{methylglucose}$ in white fat cells was measured under equilibrium exchange conditions at $\text{3-O-}\text{methylglucose}$ concentrations up to 50 mM with a previously described method (Vinten, J., Gliemann, J. and Østerlind, K. (1976) J. Biol. Chem. 251, 794–800). Under these conditions the main part of the transport was inhibitable by cytochalasin B. The inhibition was found to be of competitive type with an inhibition constant of about 2.5 · 10^?7 M, both in the absence and in the presence of insulin (1μM). The cytochalasin B-insensitive part of the $\text{3-O-}\text{methylglucose}$ permeability was about 2 · 10^?9 cm · s^?1, and was not affected by insulin. As calculated from the maximum transport capacity, the half saturation constant and the volume/ surface ratio, the maximum permeability of the fat cell membrane to $\text{3-O-}\text{methylglucose}$ at 37°C and in the presence of insulin was 4.3 · 10^?6 cm · s^?1. From the temperature dependence of the maximum transport capacity in the interval 18–37°C and in the presence of insulin, an Arrhenius activation energy of $\text{14.8 ± 0.44}\text{kcal/mol}$ was found. The corresponding value was $\text{13.9 ± 0.89}$ in the absence of insulin. The half saturating concentration of $\text{3-O-}\text{methylglucose}$ was about 6 mM in the temperature interval used, and it was not affected by insulin, although this hormone increased the maximum transport capacity about ten-fold to 1.7 mmol · s^?1 per 1 intracellular water at 37°C.     

A new test of peripheral insulin sensitivity invivo using artificial beta cell

A new $\text{in}$$\text{vivo}$ test of insulin sensitivity is described. It utilizes closed-loop insulin delivery device (GCIIS, Biostator^®) capable of infusing glucose and insulin according to preselected algorithms. In euglycemic patients, insulin was infused by GCIIS to maintain euglycemia in the face of challenges with gradually increasing doses of intravenously administered glucose. Under the described experimental conditions, the endogenous insulin release was minimized as evidenced by serum C-peptide levels of less than 2 ng/ml, and thus the peripheral disposal of glucose should have depended entirely on the exogenous insulin. The amount of the insulin infused was considered to be a measure of peripheral insulin sensitivity. The test was applied to normal and non diabetic obese individuals, and to diabetics, both insulin dependent and independent. Significant insulin resistance was demonstrated in the obese and diabetic patients. In two obese females, the test was repeated after a prolonged period of starvation, and showed marked increase in insulin sensitivity. In two poorly controlled insulin dependent diabetics, marked increase in insulin sensitivity was also observed, here following a prolonged period of euglycemia (48 hours). It is concluded that the GCIIS controlled insulin sensitivity test is a simple, reliable test of peripheral insulin sensitivity, most convenient for clinical and experimental studies $\text{in}$$\text{vivo}$     

Ontogeny of the mammalian insulin receptor: Studies of human and rat fetal liver plasma membranes

Insulin binding to human fetal plasma liver membranes was studied in preparations segregated into three pools according to length of gestation: 15–18 weeks (Pool A), 19–25 weeks (Pool B), and 26–31 weeks (Pool C). Receptor numbers, calculated by extrapolation of Scatchard plots to the X axis, increased from 25 × 10¹⁰ sites per 100 μg protein in the youngest group (Pool A) to 46 × 10¹⁰ sites per 100 μg protein in Pool B. No further increase in receptor number was seen in Pool C. The affinity constant for insulin at tracer concentrations, $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ (“empty site”), was 1.53 × 10⁸M^?1 in Pool A and was only slightly higher than $\text{K}{}_{\mathrm{<mtext>f</mtext>}}$ (“filled site”). $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ was higher in Pool B, 1.75 × 10⁸M^?1, and in Pool C reached a value of 5.63 × 10⁸M^?1. In Pool C $\text{K}{}_{\mathrm{<mtext>f</mtext>}}$ was 2.3 × 10⁸M^?1. Insulin binding of liver plasma membranes from rat fetuses aged 14, 16, 18, and 21 (term) days and adults was also studied. Maximum binding capacity tended to increase with gestational age and was 130 × 10¹⁰ sites per 100 μg protein at term, which was in excess of that found in adult rats (89–90 × 10¹⁰). In addition, $\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ increased from 0.75 × 10⁸M^?1 at 14 days to 3.02 × 10⁸M^?1 at term, a value higher than that found in pregnant and nonpregnant adults. Dissociation of insulin in the presence of high concentrations of insulin was significantly enhanced in tissues from 18-day and term fetuses and adults, but not in membranes from fetal rats aged 14 and 16 days. These data appear to indicate that site-site interactions are not present in early fetal existence. These changes in insulin binding with increased length of gestation are not ascribable to changes in relative proportions of hematopoietic and parenchymal tissue. Human fetal plasma liver membranes demonstrated elevated insulin binding with increased gestational age, but comparison of fetal and adult liver could not be done. However, newborn human infants have been shown to have a higher capacity for binding insulin to circulating monocytes than adults. Also, human fetuses apparently lack the capability to diminish monocyte receptors in the presence of hyperinsulinemia. These experiments show that an increase in insulin receptor binding capacity and affinity also occurs in the liver of the rat fetus at term as compared to the adult rat. The reasons and mechanisms underlying enhanced capacity for insulin binding by fetal and newborn members of human and rodent species are not known.     

Insulin receptors in rat brain cortex. Kinetic evidence for a receptor subtype in the central nervous system

Binding kinetics of porcine ¹²⁵I-insulin were studied in synaptosomal and microsomal fractions of rat brain cortex. Receptor binding was temperature- and pH-dependent with optimum at 4°C and pH 8.0–8.3. At 15°C, steady state binding was heterogenous, and Scatchard analysis revealed two classes of receptors with K_d of 2 nmol/l and 40 nmol/l in amounts of 50 pmol/g and 200 pmol/g of membrane protein. Dissociation kinetics were biexponential with $\text{T}\text{1}\text{2}$ of about 5 min and 180 min, and in contrast to other cell-types, not influenced by negative cooperativity. No receptor-mediated insulin degradation was detectable at 37°C in the presence of bacitracin. Insulin analogues inhibited ¹²⁵I-insulin binding with potencies relative to porcine insulin (%): human insulin 100, rat insulin (I+II) 71, coypu insulin 47, rat multiplication stimulating activity 8, porcine proinsulin 5, among which the three last values were significantly higher than in rat liver and fat cells. No competition was observed with porcine relaxin and mouse nerve growth factor up to about 1 μmol/l. Receptors were present in all regions of central nervous system with highest concentrations in the cerebral cortex, cerebellum and olfactory bulb, and lowest in the pons, medulla oblongata and spinal cord. In conclusion, insulin receptors in rat brain cortex are functionally different from other tissues regarding the insulin specificity and the absence of negative cooperativity. It is suggested that an insulin receptor subtype in rat brain mediates the growth activity of insulin on nerve cells.     

Rapid down regulation of insulin receptors in adipocytes artifact of the incubation buffer

Rat adipocytes were incubated with 15 nM insulin in different buffers at 37°C. The cells were washed and reincubated at 16°C in the presence of 18 pM A14-[¹²⁵I]monoiodoinsulin to determine the insulin receptor concentration. After incubation for 2 h in Tris buffer the binding decreased to about 30 %, whereas no decrease was found after incubation in Hepes, phosphate or bicarbonate buffers. Binding of tracer insulin reached a constant level by 45 min in Hepes buffer at 37°C, whereas it continued to increase in Tris buffer. Washout of tracer insulin after incubation in Tris buffer at 37°C showed a large, slowly dissociable fraction. It is suggested that the rapid down regulation of insulin receptors $\text{in}\text{vitro}$ is an artifact of the Tris buffer and that the phenomenon is due to a slowly reversible occupancy of a receptor pool with unlabelled insulin.     

Action of insulin analogues on cultured human fibroblasts reflects biological potency.

The biological sensitivity of cultured human skin-derived fibroblasts was examined in terms of the ability of insulin and insulin analogues to stimulate the uptake of alpha-aminoisobutyrate in these cells. The relative biological activity measured: insulin=desalanine insulin > proinsulin > desoctapeptide insulin parallels exactly the relative activity of these compounds on fat and muscle preparations both $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$. Inactive insulin analogues do not stimulate alpha-aminoisobutyrate uptake. It is concluded that the chemical specificity of human fibroblast insulin receptors is retained in culture and that the biological responsiveness (alpha-aminoisobutyrate uptake) of such cells to insulin should prove useful for comparative studies of receptors obtained from different individuals.     

Postinhibitory overshoot of insulin release by gastric inhibitory polypeptide

Following intravenous infusion of somatostatin $\text{in vivo}$ occasionally there is a large rebound overshoot of insulin release. An $\text{in vitro}$ model to simulate this phenomenon was made by perfusing rat pancreas with gastric inhibitory polypeptide (GIP) during simultaneous perfusion with somatostatin. Adding GIP (100 ng/ml) to the perfusate for 2 minutes beginning either 3 or 9 minutes before terminating the somatostatin perfusion produced a large overshoot in insulin release. The magnitude of overshoot was greater when medium contained 300 mg/dl glucose that when it contained 150 mg/dl glucose. Perfusion with GIP for 2 minutes beginning 9 minutes before increasing the glucose concentration of the medium from 30 to 300 mg/dl elicited a large increase in both the acute and second-phase release of insulin. These suggest that post-inhibitory overshoot of insulin release after somatostatin may be produces $\text{in vitro}$ by the suppressed action of stimulatory hormones such as GIP. Prior infusion with GIP can also potentiate glucose-stimulated insulin increase.     

The amino terminal sequences of acid proteases-human pepsin and gastricsin and the protease of Rhizopus chinensis.

The amino acid sequences near the amino termini of human pepsin (34 residues) and gastricsin (24 residues) and the acid protease from $\text{Rhizopus chinensis}$ (27 residues) have been determined using automated Edman degradation. From these results three additional observations were made. First, two structural variants have been observed for human gastricsin and for the $\text{Rhizopus}$ protease. Both cases are apparently genetic in origin. Second, a stretch of sequence in the $\text{Rhizopus}$ protease, residues 14 to 26, is highly homologous to the known sequence of porcine pepsin at the region of residues 11 to 23. Third, the sequences of the NH₂-terminal region of human pepsin and gastrisin are homologous.     

Somatostatin analogs which inhibit glucagon and growth hormone more than insulin release

Three analogs of somatostatin, [$\text{D}$-Cys¹⁴] -, [Ala², $\text{D}$-Cys¹⁴] - and [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, were synthesized by the solid phase method, characterized by several means, and tested for their effects on the release of insulin, glucagon, and growth hormone. The peptides sharply suppressed the release of growth hormone $\text{in vitro}$ and glucagon $\text{in vivo}$, but had less effect on insulin secretion $\text{in vivo}$. These analogs, particularly [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, could possibly be useful for the treatment of diabetes mellitus.     

The mitogenic activity of insulin: An intrinsic property of the molecule

Higher than physiological concentrations of insulin stimulate the incorporation of [${}^3\text{H}$]thymidine into DNA in a large variety of cells in culture. In order to exclude a contaminant of insulin preparations as responsible for this mitogenic action, porcine insulin was purified by reverse phase HPLC and assayed for mitogenic activity. HPLC- purified insulin had a mitogenic activity similar to that of impure insulin. Moreover, semisynthetic and synthetic insulins as well as HPLC-purified insulin derivatives such as monodesamido- and monoarginine insulin were also biologically active.     

Specificity of the collagenolytic enzyme from the fungus Entomophthora coronata: comparison with the bacterial collagenase from Achromobacter iophagus.

The specificity of the collagenolytic enzyme from the fungus Entomophthora coronata toward some inhibitors and the B chain of oxidized insulin was investigated and compared to that of the bacterial collagenase from Achromobacter iophagus. The fungal enzyme was completely inhibited by diisopropylfluorophosphate, tosyl-l-lysine chloromethyl ketone, and tosyl-amino-2-phenylethyl chloromethyl ketone but not at all by ethylenediaminetetraacetate. This indicates that it is not a metalloenzyme like the bacterial Achromobacter collagenase. The B chain of insulin was not hydrolysed at all by the bacterial enzyme under conditions where extensive digestion was observed with the Entomophthora enzyme. The fungal enzyme cleaves preferentially the bonds $\text{Leu}\text{15}\text{-}\text{Tyr}\text{16}\text{and}\text{Leu}\text{11}\text{Val}\text{12}$ as determined by automatic sequencing; the secondary cleavages were identified by a systematic analysis of the digestion mixture; thus, the fungal collagenolytic enzyme from Entomophthora coronata differs both structurally and functionally from the bacterial Achromobacter collagenase.     

Characterization of insulin receptor subunits in brain and other tissues by photoaffinity labeling

Specific insulin receptor proteins of plasma membrane preparations from various tissues of the rat were identified using a photoreactive insulin derivative, N^εB29-mono(azidobenzoyl)insulin. Except for the brain, all tissues examined showed the specific photolabeling of two proteins of M_r～130K and ～90K. In brain tissue, only one protein, M_r～115K, was specifically labeled. Liver and adipocyte membranes of the genetic obese ($\text{ob}\text{ob}$) mice showed decreased labeling of both 130K and 90K proteins when compared to those of lean littermates. Labeling of these proteins in liver plasma membranes was abolished by trypsin, whereas neuraminidase increased their electrophoretic mobility in SDS-polyacrylamide gel. The labeling of these two proteins was inhibited by a human anti-receptor serum which also formed an immunocomplex with both proteins. The labeling of the 115K protein in brain tissue was, however, not affected by the antiserum.     

Hormonal regulation of acetyl CoA carboxylase effect of insulin and epinephrine

Acetyl CoA carboxylase in the isolated epididymal fat tissue is activated by insulin and inactivated by epinephrine and dibutyryl cAMP. Insulin activation of the enzyme occurs in the presence of inhibitors of protein synthesis and can be observed as early as 15 min. A time dependent inactivation of the enzyme occurs in the presence of ATP and Mg⁺⁺$\text{in vitro}$.