We describe the use of an enzyme prodrug system based on E. coli nitroreductase (NTR) to achieve the specific ablation of adipose tissue. Transgenic mice expressing the NTR gene specifically in the adipose tissue were generated using the adipocyte specific promoter aP2. After treatment with the prodrug CB1954 these mice showed extensive cell depletion in all fat depots; this was directly correlated to both the dose of prodrug and the levels of NTR expression. Higher doses of CB1954 resulted in complete disappearance of visible adipose stores in some transgenic mice. These mice exhibited an impaired ability to thermoregulate body temperature. Lower doses of CB1954 resulted in a partial reduction of the adipose tissue leaving non-expressing cells that escape ablation. These animals show normal levels of blood glucose and triglycerides but have reduced leptin levels. After 30 days they were able to regenerate the fat depots and leptin levels returned to normal but, interestingly, no NTR-expressing cells were detectable. The present model provides a new approach to manipulate the number of adipocytes at different stages of mouse development and provides a new system for the study of fat metabolism especially in abnormal conditions such as obesity and its modulation through manipulation of the target cell population.

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) are distantly related peptides. Both act through G protein-coupled receptors on vascular smooth muscle cells to increase intracellular cAMP concentrations, causing vasorelaxation. Recent evidence suggests that both peptides bind to a common heptahelical receptor, with specificity for each peptide being determined by a receptor activity modifying protein (RAMP). This hypothesis predicts that each peptide should desensitise the cellular response to subsequent stimulation by the other. We have studied the patterns of desensitisation of ADM/CGRP receptors in rat aortic vascular smooth muscle cells. Cells were incubated for 20 min in either serum free medium (SFM), alone (control) or in SFM containing vasoactive agonist (e.g. ADM 10(-8) M, CGRP 10(-7) M, angiotensin II 10(-9) M or isoproterenol 10(-6) M). Cells were then washed and incubated for a further 20 min in SFM containing a second agonist and 1 mM isobutyryl methyl xanthine. Cells were harvested and assayed for cAMP. Pre-exposure of cells to CGRP, isoproterenol, angiotensin II or ADM, decreased cAMP generation in response to subsequent stimulation with CGRP by 84% (+/-5), 66% (+/-18), 45% (+/-5) and 60% (+/-10) respectively (mean+/-s.d.). Pre-incubation of cells with 100 nM H-89, a protein kinase A (PKA) inhibitor, abolished the desensitisation of CGRP by itself, implying that this desensitisation was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with ADM by pre-exposure to ADM and all other agonists tested. Identical results were seen with or without PKA inhibition by H-89. These results indicate that the ADM receptor does not desensitise over this time period in RAVSMCs, in contrast to the CGRP receptor, which is desensitised by pre-exposure to CGRP and other vaso-active agonists. These data also suggest that ADM and CGRP act through separate receptors in these cells.

Early descriptions of in vitro ACTH bioassays all emphasised the need to use extracted plasma samples due to interference by an unidentified component. The aim of these studies was to elucidate the effects of whole plasma on ACTH steroidogenic activity in vitro and to identify the responsible factor. A sensitive in vitro dispersed bovine adrenocortical cell bioassay was established. The addition of 10% ACTH-depleted human pooled plasma to the incubation media resulted in basal steroidogenesis equivalent to that achieved with 10(-9) M ACTH1-24 and potentiated the steroidogenic activity of 10(-9) M ACTH1-24 by 7.8-fold. This potentiation was dependent on the concentration of both ACTH and plasma in the media, but did not result from the mitogenic effect of plasma. A pituitary source was excluded and the potentiating activity was not extractable by Vycor glass. Column chromatography demonstrated two peaks of activity corresponding to molecular weights of 650 and 220x10(3) Da. These peaks did not correspond to the plasma binding of 125I-ACTH which resulted from non-specific binding to albumin. Lipoprotein-deficient serum had no effect on either basal or ACTH-stimulated steroidogenesis, but both were restored by the addition of purified lipoproteins. However, novel findings demonstrated a differential effect of low (LDL) and high (HDL) density lipoproteins on basal and ACTH-stimulated steroid production; thus, LDL exerted a greater effect on the former, whilst HDL potentiated the steroidogenic activity of added ACTH more than LDL. The addition of the lipoproteins to lipoprotein-deficient serum restored its basal and ACTH potentiating effects, the cholesterol concentrations of the chromatographic fractions exactly paralleling their ACTH potentiating effect. These findings suggest that not only are lipoproteins the plasma factor(s) which potentiates ACTH steroidogenic activity in in vitro bioassays, but also that they exert differential effects on basal and ACTH-stimulated steroid production.

Difficulty in expressing the adrenocorticotrophin (ACTH) receptor (melanocortin 2 receptor; MC2R) after transfection of various MC2R expression vectors has been experienced by many researchers. Reproducible evidence for expression has been obtained only in the Y6/OS3 corticoadrenal cell lines or in cells expressing endogenous melanocortin receptors. In order to determine the cause of this failure of expression we have undertaken the following studies. An MC2R expression plasmid was constructed in which the green fluorescent protein (GFP) coding region had been added to the C-terminus of the mature protein. Transfection of this plasmid into Y6 cells with a cAMP-responsive reporter plasmid demonstrated normal function of this receptor. Imaging of CHO cells expressing MC2R-GFP revealed perinuclear expression, although a cholecystokinin receptor (CCKR)-GFP construct was efficiently expressed at the cell surface. Y6 cells, in contrast, showed cell surface fluorescence after transfection with MC2R-GFP. Several other cell types showed a similar pattern of GFP distribution characteristic of retention in the endoplasmic reticulum. Counterstaining with an anti-KDEL antibody confirmed this location. Co-expression of the MC2R and the CCKR-GFP did not impair CCKR trafficking to the cell surface, implying a receptor-specific impairment to trafficking in the CHO cell which was absent in the Y6 cell.