Makoto Iwasaki

Cloning, characterization and overexpression of a Streptococcus pyogenes gene encoding a new type of mitogenic factor

A new type of mitogenic factor, termed MF, has been found in the culture supernatant of Streptococcus pyogenes and its N‐terminal amino acid sequence has been determined. On the basis of this sequence, an S. pyogenes gene encoding MF was cloned and its nucleotide sequence was determined. The MF gene includes a long, open reading frame with 813 nucleotides capable of encoding the MF precursor protein with 271 amino acids. Removal of the putative 43 residues as a signal peptide results in the mature MF protein with 228 amino acids. The molecular mass of the mature MF is calculated as 25,363 which is consistent with the previously determined value of 25,370 for MF secreted from S. pyogenes. Neither nucleotide nor amino acid sequence homology was found between the mature MF and other streptococcal pyrogenic exotoxins, such as SPE A, SPE B and SPE C. The mature MF was recombinantly overexpressed as a fusion protein with glutathione S‐transferase in Escherichia coli. The recombinant protein showed mitogenic activity in rabbit peripheral blood lymphocytes and immunoreactivity with the rabbit antiserum raised against the secreted MF from S. pyogenes. These data indicate that a unique gene encoding MF was cloned from S. pyogenes.

Alteration of Insulin-Receptor Kinase Activity by High-Fat Feeding

It has been demonstrated in in vivo and in vitro experiments that high-fat (HF) feeding causes insulin resistance. To elucidate the mechanism for this effect, we have measured the kinase activity of the insulin receptor purified from livers of HF-fed rats that showed impaired insulin action in isolated rat adipocytes. In adipocyte experiments, HF feeding led to a 65% decrease in the maximal response stimulated by insulin in a 2-deoxyglucose uptake study. Although insulin binding to adipocytes of HF-fed rats also decreased to 50% of control due to decreased binding affinity, the postbinding defect should be accounted for by decreased insulin action in view of the presence of spare receptor. In contrast to adipocytes, insulin binding to the lectin-purified insulin receptor from livers showed no difference in receptor-binding affinity between HF-fed and control rats. Insulin-stimulated phosphorylation of the β-subunit of the insulin receptor was decreased to almost 50% throughout the entire dose-response curve. The study of glutamine-tyrosine (4:1) phosphorylation by the insulin-receptor kinase showed results similar to those of the autophosphorylation study. These results suggest that an HF diet causes insulin resistance by affecting insulin-receptor kinase, which plays an important role in transmembrane signaling between insulin binding and insulin action.

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

Abstract [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.

Mitogenic factor secreted by Streptococcus pyogenes is a heat-stable nuclease requiring His122 for activity

The gene encoding a mitogenic factor, termed MF, was cloned from Streptococcus pyogenes and the recombinant MF was overexpressed in Escherichia coli. Both the natural and recombinant MF had heat-resistant nuclease activity. The nuclease activity of MF was characterized using the recombinant protein. MF showed endonuclease activity, digesting ssDNA, dsDNA and tRNA. The optimal pH for the DNase activity of MF was 9.5. The DNase activity was enhanced approximately tenfold by the simultaneous presence of two divalent cations, Mg2+ and Ca2+, compared to either alone and was inhibited by EDTA or NaCl. The heat stability of MF was biphasic; the DNase activity was heat-stable from 0 to 50 degrees C over 80 degrees C but very unstable at around 60 degrees C. DNA digested by MF possessed 5-phosphorylated and 3-hydroxylated termini, identical to those obtained by digestion of DNA by pancreatic deoxyribonuclease I. A mutant clone revealed that His122 was a residue essential to the nuclease activity.

Receptor binding and negative cooperativity of a mutant insulin, [LeuA3]-insulin

[LeuA3]-insulin, the third mutant insulin, was semisynthesized and was studied for receptor binding and negative cooperative effects. Receptor binding and biological effects of the mutant insulin were 0.3-0.5% of normal, the lowest among three mutant insulins. However, negative cooperative effects of the mutant insulin were almost normal at higher concentration (greater than 10(-6) M). Monoclonal anti-insulin antibody binding studies revealed that carboxyterminal region of B chain was relatively unchanged. These results suggest that N-terminal region of A-chain extending to A3 is important for receptor binding and confirm that A3 does not play an important role for negative cooperativity.

A new potentiator of insulin action: Post-receptor activation in vitro

A new potentiator of insulin action, 5‐[4‐(1‐methylcyclohexylmethoxy)‐benzyl]thiazolidine‐2,4‐dione, was tested for activation of insulin action in vitro. The agent (50 mg.kg−1.day−1) was orally administered to rats for 14 days and adipocytes from treated rats were used to assess insulin‐binding, glucose uptake and glucose oxidation. In obese rats, the agent increased glucose uptake and oxidation without any change in insulin binding, whereas in lean or streptozotocin‐treated rats it failed to increase glucose metabolism. Fat tissues were cultured with the agent for 24 h and were tested for insulin action. In the presence of insulin (10 ng/ml) in the culture media, the agent increased glucose oxidation in these cells without any change in insulin binding. However, without insulin in the culture media the agent did not increase glucose oxidation. Thus, the agent appeared to potentiate insulin action at the post receptor process.

Clarification of Signaling Pathways Mediated by Insulin and Insulin-Like Growth Factor I Receptors in Fibroblasts From Patients With Specific Defect in Insulin Receptor

Receptor binding and biological action of insulin and insulin-like growth factor I (IGF-I) were studied in fibroblasts from a patient with leprechaunism and a patient with type A syndrome of insulin resistance. Insulin binding was reduced to 18.8 and 27.7% of control value, respectively. In contrast, IGF-I binding was normal in both patients. In competitive binding studies, IGF-I had 0.2% of the ability of insulin to compete with 125I-labeled insulin binding, and insulin had 0.1% of the ability of IGF-I to compete with 125I-labeled IGF-I binding in control subjects and patient fibroblasts. The dose-response curves of insulin stimulation assessed by glucose incorporation and α-aminoisobutyric acid uptake showed normal responsiveness, and ED50 was significantly shifted to the right in fibroblasts from both patients. However, normal responsiveness and sensitivity were observed in thymidine incorporation studies. For IGF-I, dose-response curves of glucose incorporation, a-aminoisobutyric acid uptake, and thymidine incorporation were all normal in both patients. These results indicate that 1) the defect is specific to the insulin-receptor binding in these patients, 2) insulin and IGF-I activate glucose incorporation and α-aminoisobutyric acid uptake mainly through their own specific receptors, but 3) the IGF-I receptor appears to have a more important role in stimulating thymidine incorporation than the insulin receptor in physiological condition or, alternatively, an unknown postreceptor process with cascade signal transmission may overcome the decreased insulin-receptor binding to produce a normal dose-response curve.

Immunoreactivity and biologic activity of semisynthetic [LeuB-30]-insulin: potential value in the treatment of insulin antibody-mediated insulin resistance

Insulin analogues with different amino acids, including threonine, alanine, L-leucine, D-leucine, L-leucine amide, phenylalanine, tri-alanine, or desalanine, at the B-30 position were semisynthesized from pork insulin by the new enzymatic method. The order of ability of the insulin analogues to bind to anti-insulin sera was [AlaB−30] > desalanine > [ThrB−30] > [Ala-Ala-AlaB−30] > [D-LeuB−30], [Leu-NH2B−30], [PheB−30] > desoctapep-tide > [LeuB−30]. The ability of insulin analogues with different amino acids at B-30 to bind to receptors, as well as their biologic potency tested with glucose uptake in isolated rat adipocytes, was comparable among the analogues. These results suggest that [LeuB−30]-insulin demonstrated the least immunoreactivity and has full activity in receptor binding and biologic effect, and that it may be useful for treatment of anti-insulin antibody-mediated insulin resistance.

Induction of reduced endothelial permeability to horseradish peroxidase by factor(s) of human astrocytes and bladder carcinoma cells: detection in multi-well plate culture

Endothelial permeability to horseradish peroxidase (HRP) was assayed in multi-well plate culture. Confluent cobblestone-monolayers of endothelial cells were incubated with HRP, and the amounts of HRP that permeated the monolayer and reached the bottom substrate were estimated by extraction of HRP with deoxycholate and following addition of chromogenic substrate of HRP into the extracts. Using this in situ assay method, we detected the reduced permeability to HRP in aortic and brain microvascular endothelial cells after 4-5 days culture with conditioned media of human astrocytes or bladder carcinoma cells. Preliminary characterization of the active factors(s) released from these cells was performed. This method will be useful for monitoring impermeable endothelial cell monolayers and identifying the active factor(s).

Insulin binding to differentiating muscle cell line L6

We studied insulin binding to cultured differentiating muscle cell line L6. Insulin binding to the cells reached a plateau after incubation with 125I-insulin for 4 h at 22 degrees C, and was at an optimum at pH 7.8. Preincubation with 10 microM of hydrocortisone for 36 h at 37 degrees C resulted in significantly increased insulin binding (1.73 +/- 0.12 ng/mg protein for treated cells vs. 1.13 +/- 0.025 ng/mg protein for control cells, mean +/- SD, P less than 0.001). Preincubation with 1 microM of hydrocortisone or 1 microM of dexamethasone also led to increased binding. The number of insulin-binding sites per cell increased 2.5-fold in glucocorticoid-treated cells (9.7 X 10(3) sites/cell for treated vs. 3.8 X 10(3) sites/cell for control cells). Preincubation with trifluoperazine (5 microM), a calmodulin inhibitor, did not affect insulin binding to the cells. These results indicate that glucocorticoid might have some important role in regulating the number of insulin receptors in L6 muscle cells.