Biomedical subjects

C Iehlé

Publications and source records attributed to C Iehlé.

5 recordsLinked to original sources

PubMed2002

PPARgamma-dependent and PPARgamma-independent effects on the development of adipose cells from embryonic stem cells.

Peroxisome proliferator-activated receptor (PPAR) gamma was shown to be required for adipocyte formation both in vivo and in vitro. However, the role of PPARgamma in the initial steps of adipose cell development was not distinguished from its role in the terminal steps. We now show that PPARgamma is expressed early in embryoid bodies (EBs) derived from embryonic stem cells and in E.8.5 mouse embryos. Addition of a specific ligand for PPARgamma in developing EBs over-expressing PPARgamma did not commit stem cells towards the adipose lineage. In differentiated PPARgamma(-/-) EBs, only markers characteristic of preadipocytes were found to be expressed. PPARdelta is present in EBs but did not compensate for the lack of PPARgamma in terminal differentiation. Taken together, these results favor a critical PPARgamma-independent phase culminating in preadipocyte formation that precedes a PPARgamma-dependent phase in the development of adipose cells from pluripotent stem cells.

Adipocytes↗

PubMed1999

Differences in steroid 5alpha-reductase iso-enzymes expression between normal and pathological human prostate tissue.

We studied the expression level and cell-specific expression patterns of 5alpha-reductase (5alpha-R) types 1 and 2 iso-enzymes in human hyperplastic and malignant prostate tissue by semi-quantitative RT-PCR and in situ hybridisation analyses. In situ hybridisation established that 5alpha-R1 mRNA is preferentially expressed by epithelial cells and little expressed by stromal cells whereas 5alpha-R2 mRNA is expressed by both epithelium and stroma. Semi-quantitative RT-PCR has been performed on total RNA from different zones of normal prostate, BPH tissues and liver. We found that 5alpha-R1 and 5alpha-R2 mRNAs expression was near the same in all zones of normal prostate. In BPH tissue, 5alpha-R1 and 5alpha-R2 mRNAs expression was slightly but significantly increased, when it was compared to the levels recorded for normal prostate. In cancer samples, 5alpha-R1 mRNA expression was higher than in normal and hyperplastic prostate but the level of 5alpha-R2 mRNA was not statistically different from that observed in the different zones of normal prostate. In liver, 5alpha-R2 mRNA level was similar to that measured in BPH but 5alpha-R1 mRNA expression was ten times higher. The increase observed in 5alpha-R isoenzymes expression in BPH tissue could play an important role in the pathogenesis and/or maintenance of the disease.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1995

Human prostatic steroid 5 alpha-reductase isoforms--a comparative study of selective inhibitors.

The present study describes the independent expression of the type 1 and 2 isoforms of human 5 alpha-reductase in the baculovirus-directed insect cell expression system and the selectivity of their inhibition. The catalytic properties and kinetic parameters of the recombinant isozymes were consistent with published data. The type 1 isoform displayed a neutral (range 6-8) pH optimum and the type 2 isoform an acidic (5-6) pH optimum. The type 2 isoform had higher affinity for testosterone than did the type 1 isoform (Km = 0.5 and 2.9 microM, respectively). Finasteride and turosteride were selective inhibitors of the type 2 isoform (Ki (type 2) = 7.3 and 21.7 nM compared to Ki (type 1) = 108 and 330 nM, respectively). 4-MA and the lipido-sterol extract of Serenoa repens (LSESr) markedly inhibited both isozymes (Ki (type 1) = 8.4 nM and 7.2 micrograms/ml, respectively; Ki (type 2) = 7.4 nM and 4.9 micrograms/ml, respectively). The three azasteroids were competitive inhibitors vs substrate, whereas LSESr displayed non-competitive inhibition of the type 1 isozyme and uncompetitive inhibition of the type 2 isozyme. These observations suggest that the lipid component of LSESr might be responsible for its inhibitory effect by modulating the membrane environment of 5 alpha-reductase. Partially purified recombinant 5 alpha-reductase type 1 activity was preserved by the presence of lipids indicating that lipids can exert either stimulatory or inhibitory effects on human 5 alpha-reductase.

Animals↗

PubMed1994

Inhibition of the activity of 'basic' 5 alpha-reductase (type 1) detected in DU 145 cells and expressed in insect cells.

The purpose of this study was 2-fold: (1) to identify the 5 alpha-reductase (5 alpha-R) isozyme(s) present in DU 145 cells, a human cell-line of low androgen sensitivity derived from a cerebral metastasis of an epithelial prostate cancer; and (2) to compare the inhibitory potencies of three compounds on the 'basic' 5 alpha-R isozyme expressed in a baculovirus-directed insect cell system. Conversion of testosterone (T) into 5 alpha-dihydrotestosterone (DHT) in DU 145 cells was measured by HPLC coupled to a Flo-one HP radioactivity detector. DU 145 cells exhibited 5 alpha-R activity (21 pmol DHT/min/mg protein) at pH 7.4 which disappeared at pH 5.5 suggesting that, of the two genomically distinct human isozymes identified so far, type 1 5 alpha-R is expressed in DU 145 cells. This was confirmed by at least two observations: first, 5 alpha-R activity in DU 145 cells was inhibited with much higher potency by 4-MA than by finasteride which is known to be a very poor competitor of the 'basic' enzyme (IC50s = 2.8 +/- 0.2 and 264 +/- 55 nM, respectively). Second, only the type 1 5 alpha-R cDNA and not type 2 5 alpha-R cDNA hybridized with DU 145 RNA. A high potency differential was also recorded for the inhibition of 'basic' type 1 5 alpha-R expressed in a baculovirus-directed-insect cell system by these two compounds, 4-MA being considerably more active than finasteride (Ki = 8.4 +/- 2.3 and 330 +/- 9 nM, respectively). This inhibition was competitive. On the other hand, inhibition by an n-hexane lipid/sterol extract of Serenoa repens (LSESr) was non-competitive and, when expressed in terms of recommended therapeutic doses, was 3-fold greater for LSESr than for finasteride. These studies suggest that LSESr might exert a regulatory inhibitory activity due to its specific lipid/sterol composition.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1993

Baculovirus-directed expression of human prostatic steroid 5 alpha-reductase 1 in an active form.

In the human prostate, the enzyme steroid 5 alpha-reductase (h5 alpha R) catalyses the conversion of testosterone into the more potent androgen, dihydrotestosterone. Two distinct cDNAs coding for h 5 alpha R in the human prostate have been previously characterized. Enzyme h5 alpha R1 shows a maximum activity at basic pH whereas h5 alpha R2 has an acidic pH optimum activity. We report here the expression of the human steroid h5 alpha R1 in a eukaryotic expression system: the baculovirus-directed-insect cell expression system. The full length cDNA was inserted into the Autographa californica nuclear polyhedrosis virus genome and expressed in Spodoptera frugiperda, Sf9, insect cells. Sf9 cells were infected with the recombinant baculovirus and homogenates used in h5 alpha R activity assays by high pressure liquid chromatography showed that a catalytically active enzyme was produced. The recombinant enzyme showed an apparent Km for testosterone of 2.07 microM and a V(max) of 10.1 nmol of dihydrotestosterone/ min/mg of protein. Recombinant h 5 alpha R1 activity was inhibited by specific h 5 alpha R inhibitors such as 4-MA (Ki = 2.6 nM). Subcellular distribution in Sf9 cells demonstrated that the enzyme was associated with the nuclear membrane.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗