Characterisation of 11beta-hydroxysteroid dehydrogenase 1 in human orbital adipose tissue: a comparison with subcutaneous and omental fat.

Glucocorticoids (GCs) have a profound effect on adipose biology increasing tissue mass causing central obesity. The pre-receptor regulation of GCs by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) that activates cortisol from cortisone has been postulated as a fundamental mechanism underlying the metabolic syndrome mediating adipocyte hyperplasia and hypertrophy in the omental (OM) depot. Orbital adipose tissue (OF) is the site of intense inflammation and tissue remodelling in several orbital inflammatory disease states. In this study, we describe features of the GC metabolic pathways in normal human OF depot and compare it with subcutaneous (SC) and OM depots. Using an automated histological characterisation technique, OF adipocytes were found to be significantly smaller (parameters: area, maximum diameter and perimeter) than OM and SC adipocytes (P<0 x 001). Although immunohistochemical analyses demonstrated resident CD68+ cells in all three whole tissue adipose depots, OF CD68 mRNA and protein expression exceeded that of OM and SC (mRNA, P<0 x 05; protein, P<0 x 001). In addition, there was higher expression of glucocorticoid receptor (GR)alpha mRNA in the OF whole tissue depot (P<0 x 05). Conversely, 11beta-HSD1 mRNA together with the markers of late adipocyte differentiation (FABP4 and G3PDH) were significantly lower in OF. Primary cultures of OF preadipocytes demonstrated predominant 11beta-HSD1 oxo-reductase activity with minimal dehydrogenase activity. Orbital adipocytes are smaller, less differentiated, and express low levels of 11beta-HSD1 but abundant GRalpha compared with SC and OM. OF harbours a large CD68+ population. These characteristics define an orbital microenvironment that has the potential to respond to sight-threatening orbital inflammatory disease.

Characterisation of the prereceptor regulation of glucocorticoids in the anterior segment of the rabbit eye.

The prereceptor regulation of glucocorticoids (GCs) by 11beta-hydroxysteroid dehydrogenase type-1 (11beta-HSD1), a bidirectional isozyme that interconverts active (cortisol) and inactive (cortisone) GCs, is an established determinant of GC function in tissues such as liver, adipose and bone. Although the therapeutic use of GCs is abundant in ophthalmic practice, where GC interactions with nuclear receptors modulate gene transcription, the prereceptor regulation of endogenous cortisol is not well described in ocular tissues. Recent descriptive studies have localised 11beta-HSD1 to the human corneal epithelium and non-pigmented epithelium (NPE) of the ciliary body, indicating a link to corneal epithelial physiology and aqueous humour production. In this study, we characterise the functional aspects of the autocrine regulation of GCs in the anterior segment of the rabbit eye. Using our in-house generated primary antibody to human 11beta-HSD1, immunohistochemical analyses were performed on paraffin-embedded sections of whole New Zealand white albino rabbits, (NZWAR) eyes. As in human studies, 11beta-HSD1 was localised to the corneal epithelium and the NPE. No staining was seen in the albino 'pigmented' ciliary epithelium. Specific enzyme assays for oxo-reductase (cortisone-->cortisol) and dehydrogenase (cortisol-->cortisone) activity indicated predominant 11beta-HSD1 oxo-reductase activity from both the intact ciliary body tissue (n=12, median 2.1 pmol/mg per h and range 1.25-2.8 pmol/mg per h; P=0.006) and primary cultures of corneal epithelial cells (n=12, median 3.0 pmol/mg per h and range 1.0-7.4 pmol/mg per h, P=0.008) compared with dehydrogenase activity (median 1.0 pmol/mg per h and range 0.5-2.0 pmol/mg per h; median 0.5 pmol/mg per h and range 0.25-1.9 pmol/mg per h respectively). These findings were supported by expression of 11beta-HSD1 protein as visualised by Western blotting of ciliary body tissue and immunocytochemistry of corneal epithelial cells. Reduction of corneal epithelial cell proliferation was seen after primary cultures were co-incubated with cortisol and cortisone. 11beta-HSD1 activity was not demonstrated in naïve conjunctival fibroblasts or corneal stromal keratocytes. Our results indicate that the distribution of 11beta-HSD1 in the rabbit resembles that of the human eye and activates cortisone to cortisol in both corneal and uveal tissues. The NZWAR provides a suitable in vivo model for the further evaluation of 11beta-HSD1 activity in the eye, especially its role in corneal epithelial and ciliary body physiology.

Androgen receptor-mediated regulation of the alpha-subunit of the epithelial sodium channel in human kidney.

Rodents studies suggest that androgens are involved in sex-specific differences in blood pressure. In humans, there is no difference in blood pressure between boys and girls, but after puberty, blood pressure increases more in men than in women. We investigated androgen-dependent regulation of the alpha-subunit of the epithelial sodium channel (alphaEnaC) in human kidney and in the human renal cell line immortalized human renal proximal tubular cell line (HKC-8). We used microarray technique to analyze androgen-dependent gene regulation and performed quantitative RT-PCR for verification. Promoter constructs for human alphaENaC were used in transfection studies to analyze the regulation by testosterone. We investigated the in vivo effect of testosterone on alphaENaC in a rat model and used the mouse collecting duct cell line M-1 for transepithelial electrophysiological measurements. The androgen receptor (AR) was expressed in male kidney and HKC-8 cells. AlphaENaC mRNA expression increased 2- to 3-fold after treatment with testosterone in HKC-8 cells. The induction by testosterone was completely blocked by adding the AR antagonist flutamide. Analysis of the alphaENaC promoter sequence identified a putative AR response element (ARE) located 140 nucleotides upstream from the transcription start site. HKC-8 cell transfection studies showed that testosterone directly upregulated gene expression via this ARE. In vivo, testosterone treatment of orchiectomized rats resulted in an increased renal alphaENaC mRNA expression. In testosterone-treated mouse M-1 cells, amiloride caused a significant stronger decrease in short circuit current than in control cells. These data show that alphaENaC expression is directly regulated by androgens in vitro and in vivo and highlight a potential mechanism explaining the reported gender differences in blood pressure.