Takao Fujimura

Transcriptional inhibition of insulin by FK506 and possible involvement of FK506 binding protein-12 in pancreatic β-cell

FK506 (tacrolimus) is a strong immunosuppressant: it has been approved as a drug for liver transplantation in Japan, the United States, and the United Kingdom. One of its main adverse effects is hyperglycemia. Thus, in this study, we investigated the mechanism and the reversibility of the hyperglycemia caused by FK506. FK506 did not affect the glucose uptake by insulin into rat strio-muscle cell line, but suppressed insulin production in rat insulinoma cells. Two-week oral administration of FK506 at 10 mg/kg/day suppressed insulin production time-dependently at the transcriptional step in pancreatic β-cells, while glucagon content in pancreatic α-cells was not affected. When FK506 administration was stopped in these rats, insulin mRNA transcription and insulin production returned to normal. This recovery indicates that the adverse effect of FK506 on the pancreas is reversible. A high content of FK506 binding protein-12 (FKBP-12) in the pancreatic β-cells was confirmed by immunostaining with anti-human FKBP-12 mAb, but the content was less in the pancreatic α-cells and almost negligible in the acinar cells. In contrast, a high content of calcineurin in the pancreatic α-cells was confirmed by using anti-calcineurin polyclonal antibody, but this content was less in the pancreatic β-cells and not found in the acinar cells. Thus, as in the case with NF-AT in T cells, these findings point to the reduction of unidentified nuclear factors for insulin mRNA transcription caused by the binding of FK506 to FKBP-12 and a subsequent inhibition of calcineurin in the β-cells.

EXPRESSION AND POSSIBLE FUNCTIONAL ROLE OF THE beta 3-ADRENOCEPTOR IN HUMAN AND RAT DETRUSOR MUSCLE

PURPOSE To investigate the presence of the beta3-adrenoceptor (beta3-AR) in human and rat detrusor muscle and the usefulness of beta3-AR agonists as drugs for the treatment of urinary frequency. MATERIALS AND METHODS FK175, ethyl [(S)-8-[(R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-6,7,8,9-tetrah ydro-5H-benzocyclohepton-2-yloxy]acetate monohydrochloride monohydrate, was used as a beta3-AR selective agonist. The expression of beta-AR subtypes (beta1-, beta2-, beta3-AR) mRNA was investigated in rat and human detrusor muscle by RT-PCR. Beta3-AR agonist induced cyclic AMP (cAMP) levels were measured in rat detrusor muscle strips. The relaxation response produced by a beta3-AR agonist was measured in a KCl induced tonic contraction model in rat detrusor muscle strips. The effect of a beta3-AR agonist on urinary bladder function was investigated by cystometry using a conscious rat model of urinary frequency. RESULTS beta3-AR mRNA was substantially expressed in both rat and human detrusor muscles. The beta3-AR agonist, FK175 (10(-7) M), increased the cAMP level by 30% in rat detrusor muscle. In isolated rat detrusor muscle strips contracted with KCl, the beta3-AR agonist, FK175 (10(-8) to 10(-4) M), produced a concentration-dependent relaxation. Moreover, although the relaxation induced with FK175 was blocked by the non-selective beta-AR antagonist, bupranolol, it was unaffected by ether the beta1-AR selective antagonist, CGP 20712A, or the beta2-AR selective antagonist, ICI 118551, suggesting that FK175 induced the relaxation via the beta3-AR. Furthermore, in the rat model, the orally administered beta3-AR agonist, FK175 (10 mg./kg.) significantly increased bladder capacity with no change of micturition pressure or threshold pressure. CONCLUSIONS These results suggest that beta3-AR agonists may be effective in the treatment of urinary frequency.

Evaluation of mRNAs encoding muscarinic receptor subtypes in human detrusor muscle

PURPOSE The present study evaluated the muscarinic receptor subtypes corresponding to m1 to m5 genes in human detrusor muscle. MATERIALS AND METHODS The mRNAs encoding m2 and m3 subtypes were assessed by reverse transcription (RT)-polymerase chain reaction (PCR). The amounts of cDNA synthesized from m2 and m3 mRNAs were measured by using subcloned plasmid DNAs. The distribution of m2 and m3 mRNAs in detrusor was estimated by comparing the amount of m2 cDNA with that of m3 cDNA. RESULTS The m2 mRNA:m3 mRNA ratio was 1.06:1.00 in human detrusor. In the cryostat sections of human detrusor, the presence of both m2 and m3 mRNAs was confirmed by in situ hybridization. However, the RT-PCR products derived from m1, m4 and m5 subtype mRNAs were not detected. CONCLUSION These results suggest that human detrusor muscle coexpresses muscarinic m2 and m3 receptors and that the populations of the 2 subtypes are not significantly different.

A selective peroxisome proliferator-activated receptor γ modulator with distinct fat cell regulation properties

Adipogenesis is an important process for the improvement of insulin resistance by peroxisome proliferator-activated receptor (PPAR) γ agonists, such as rosiglitazone and pioglitazone. FK614 [3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3-Hbenzimidazole-5-carboxamide] is a structurally novel class of PPARγ agonist that improves insulin sensitivity in animal models of type 2 diabetes. Herein, we characterize FK614, a selective PPARγ modulator (SPPARM) with differential properties affecting the regulation of fat cell function. FK614 behaves as a partial agonist in inducing the interaction of PPARγ with both transcriptional coactivators, cAMP response element-binding protein-binding protein and steroid receptor coactivator-1, but as a full agonist with both PPAR-binding protein and PPAR-interacting protein, which are required for PPARγ-mediated adipogenesis. In the differentiating 3T3-L1 adipocytes, the levels of adipose fatty acid-binding protein (aP2) mRNA expression and triglyceride accumulation induced by FK614 were as efficacious as those of rosiglitazone and pioglitazone. In contrast, the effect of FK614 on aP2 gene expression in mature adipocytes was less than that of the other PPARγ agonists. Furthermore, the long-term treatment of mature adipocytes with rosiglitazone and pioglitazone reduced the expression of phosphodiesterase 3B, the down-regulation of which has an important role in the development of insulin resistance; however, FK614 had no such effect in mature adipocytes. Thus, FK614 behaves as an SPPARM with differential effects on the activation of PPARγ at each stage of adipocyte differentiation. The stage-dependent selectivity of FK614 may contribute to its enhanced insulin sensitization in differentiating adipocytes and to reduced insulin resistance at the stage of adipocyte hypertrophy.

A novel 7-β-(4-carboxybutanamido)-cephalosporanic acid acylase isolated from Pseudomonas strain C427 and its high-level production in Escherichia coli

Abstract We cloned the gene for 7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) acylase from Pseudomonas strain C427. The DNA sequence revealed an open reading frame of 2154 bp coding for 718 amino acid residues. The deduced amino acid sequence consists of 4 structural domains: (i) a signal peptide (positions 1–27), (ii) a small subunit of the acylase (positions 28–190), designated as α, (iii) a spacer peptide (positions 191–198), (iv) a large subunit (positions 199–718), designated as β. Plasmids were constructed to direct the synthesis of the acylase in Escherichia coli and the following results were obtained. The active acylase consists of two subunits which are processed from a single precursor protein, removing the spacer peptide during processing. A proportion of active acylase is secreted into the periplasm and the remainder is retained in the cytoplasm. The amount of precursor protein accumulated in the cytoplasm is greatly reduced when plasmids for the acylase lacking the signal sequence are expressed. Therefore, processing is independent of the translocation of the gene product through the cytoplasmic membrane, in contrast to the situation for penicillin G acylase. A high level of active enzyme production was achieved with a plasmid coding for an acylase in which the amino terminal sequence (positions 1–32) of native acylase is replaced by MFPTT.

Chemical modification and site-directed mutagenesis of tyrosine residues in cephalosporin C acylase from Pseudomonas strain N176

Cephalosporin C (CC) acylase from Pseudomonas strain N176 was chemically modified by tetranitromethane (TNM), causing complete loss of activity. Modification using molar excesses of TNM up to 10 resulted in complete inactivation when 1.4 mol tyrosines/mol enzyme were modified. Digestion of native and TNM-modified acylase with Achromobacter protease I (API), separation by high performance liquid phase chromatography (HPLC) and amino terminal sequencing of the resultant peptides were used to identify the modified tyrosine residues. The major difference in HPLC profile between these API digests was shown to be the peak corresponding to the peptide Ser239-Lys301 of native acylase. A portion of the peak for the peptide Ala45-Lys73 was also shifted in HPLC analysis of TNM-modified acylase. The peptides isolated from the modified acylase were shown to contain nitrated tyrosines (3-nitrotyrosine) at positions 270 and 52, respectively. These findings indicate that Tyr270 is completely modified, and Tyr52 is partially modified in the inactivated acylase. Each of the fifteen tyrosines in the acylase was altered to leucine by site-directed mutagenesis to complement the chemical modification with TNM. At pH 8.7, the mutant acylase in which tyrosine at position 270 is changed to leucine showed GL-7ACA and CC acylase activities reduced to 28.0 and 32.2% of native acylase, respectively. The results correspond to those obtained from TNM-modification. A similar reduction in activity was also obtained in the case of Tyr491 mutant, although nitration of this residue was not confirmed by chemical modification. Therefore Tyr270 and Tyr491 are important for exerting the maximum activity of the enzyme, but are not essential for catalysis. However, mutation of Tyr52 to Leu produced little change in acylase activity. The mutant acylase in which Tyr705 is changed to leucine has a lowered pH optimum for GL-7ACA, which may be useful for further improvement of the acylase.

An Approach to High-Level Production of Cephalosporin Acylase from Pseudomonas strain N176 in Escherichia coli

Abstract The cultivation conditions for N176 cephalosporin acylase production were optimized using Escherichia coli JM109 pCK305S , which encodes a highly expressed mutant, C305S, in which Cys305 is altered to Ser, under the control of the trp promoter. Processing of the precursor protein to a two-chain active form in vivo was a key factor for high-level production of the enzyme. The active acylase was produced in a high amount under the following conditions: (i) addition of Trp in the second seed medium, (ii) the use of glycerol as a carbon source, (iii) induction with IAA at 9 h, and (iv) a shift down in the cultivation temperature from 25 to 22.5°C after induction. Under these high-level production conditions, the OD at 600 nm was found to increase in proportion to the number of colony forming units (CFUs), indicating that the CFU is an important index for the expression of the active enzyme.