Adipocyte lipid chaperone AP2 is a secreted adipokine regulating hepatic glucose production.

Proper control of hepatic glucose production is central to whole-body glucose homeostasis, and its disruption plays a major role in diabetes. Here, we demonstrate that although established as an intracellular lipid chaperone, aP2 is in fact actively secreted from adipocytes to control liver glucose metabolism. Secretion of aP2 from adipocytes is regulated by fasting- and lipolysis-related signals, and circulating aP2 levels are markedly elevated in mouse and human obesity. Recombinant aP2 stimulates glucose production and gluconeogenic activity in primary hepatocytes in vitro and in lean mice in vivo. In contrast, neutralization of secreted aP2 reduces glucose production and corrects the diabetic phenotype of obese mice. Hyperinsulinemic-euglycemic and pancreatic clamp studies upon aP2 administration or neutralization demonstrated actions of aP2 in liver. We conclude that aP2 is an adipokine linking adipocytes to hepatic glucose production and that neutralizing secreted aP2 may represent an effective therapeutic strategy against diabetes.

Preliminary report of impaired oestrogen receptor-alpha expression in bone, but no involvement of androgen receptor, in male idiopathic osteoporosis.

In western countries, osteoporosis affects at least 1 in 12 of all adult males and a third of osteoporotic men have idiopathic disease (MIO). Both oestrogen and testosterone are now known to be important to the male skeleton. As normal oestrogen levels have been found in younger MIO cases, it is hypothesized that, in bone, their responses to gonadal steroids may be defective, through impaired receptor expression. This study therefore compared oestrogen receptor (ER)-alpha and androgen receptor (AR) expression, by indirect immunofluorescence and semi-quantitative image analysis, in undecalcified fresh frozen bone sections from MIO patients (33-56 years), age-matched control men (n=7), and, for reference, ovarian steroid-replete (n=7) and -deficient women (n=6). In normal men, 23%+/-SEM 6% osteoblasts and 14%+/-SEM 2% osteocytes expressed ERalpha protein, similar to hormone-replete women. Although receptor expression decreased in hormone-deficient women, loss of ERalpha protein in MIO patients was more severe (1%+/-SEM 0.5% osteocytes, 2%+/-SEM 1% osteoblasts expressed receptor). In all four groups, there was little osteocyte AR expression, but in the women, a proportion of osteoblasts were receptor-positive. Deficient osteoblast and osteocyte ERalpha protein expression could explain the bone loss in these MIO patients.

Preliminary evidence for impaired estrogen receptor-alpha protein expression in osteoblasts and osteocytes from men with idiopathic osteoporosis.

Although osteoporosis is usually associated with women, 1 in 12 men in the UK have the disease, and a third of these cases are idiopathic. Estrogen is now known to be associated with bone loss in older men, but we found, previously, that levels of this hormone were normal in younger cases of male idiopathic osteoporosis (MIO) in the age range 33-61 years. We therefore hypothesized that their estrogen responses in bone might be defective, through impaired estrogen receptor-alpha (ER)-alpha expression. Consequently, in the present study, we compared expression of ER-alpha by indirect immunofluorescence, semiquantitative image analysis, and in situ reverse transcription-polymerase chain reaction in bone sections from MIO patients (33-56 years) (N = 7); age-matched control men (N = 7); and, for reference, ovarian steroid (OS)-replete (N = 7) and OS-deficient women (N = 6). In the control men, 23 +/- 6% (mean +/- SEM) of osteoblasts and 14 +/- 2% of osteocytes expressed ER-alpha protein, similar to OS-replete women. Although receptor expression decreased in OS-deficient women, the loss of ER-alpha protein in MIO patients was more severe (1 +/- 0.5% osteocytes, 2 +/- 1% osteoblasts expressed receptor); however, ER-alpha messenger RNA (mRNA) was still expressed in controls and MIO patients. Bone loss in these patients may be due to deficient ER-alpha protein expression.

Effect of ovarian steroid deficiency on oestrogen receptor alpha expression in bone.

The mechanism by which oestrogen and hormone replacement therapy (HRT) maintain bone mass in women is still unclear. It has previously been shown that cells of osteoblast lineage in vivo, particularly osteocytes, express oestrogen receptor alpha (ERalpha). Nevertheless, it is still debatable whether oestrogen and the ovarian steroids have a direct affect on osteocytes. If they could regulate osteocyte ERalpha expression, this would be strong evidence for the involvement of these cells in the hormonal regulation of bone mass. This study therefore aimed to compare bone biopsies from women who were replete with ovarian steroids (pre-ovariectomy or post-HRT) with those from the same women when hormone-deficient (post-ovariectomy or pre-HRT) for cellular localization of ERalpha protein or mRNA expression by indirect immunofluorescence, or by in situ hybridization combined with reverse transcriptase-polymerase chain reaction (IS-RT-PCR) respectively. Image analysis showed that proportions of osteocytes positive for immunodetectable ERalpha were higher in hormone-replete than in hormone-deficient women (25+/-SEM 3 per cent, 12+/-SEM 4 per cent, respectively; n=5), with similar but non-statistically significant changes in osteoblasts. This was observed even when HRT was commenced 18 years after menopause. In contrast, grain volume/unit cell area of osteoblast mRNA signal was markedly higher when hormone-deficient (0.055+/-0.01) than when hormone-replete (0.016+/-0.004), with similar but non-significant differences in osteocytes. This preliminary study indicates up-regulation of osteocyte ERalpha protein by ovarian steroids in these patients, which is accompanied by decreased osteoblast ERalpha mRNA expression, providing further evidence for the involvement of osteocytes in the regulation of skeletal structure by ovarian steroids.

Immunofluorescent localization of estrogen receptor-alpha in growth plates of rabbits, but not in rats, at sexual maturity.

Estrogens are considered essential to the mechanism for closure of epiphyses in both males and females. The mechanism for this, however, is still unclear. It is likely that estrogen acts directly on growth plate chondrocytes, but the localization of the cells expressing the estrogen receptor (ER) has yet to be ascertained. Moreover, in rodents, growth plates remain open well into adult life. Whether the distribution of estrogen target cells in rodent epiphyses differs from that in other species, is also unclear. We therefore compared localization of estrogen target cells (denoted by ER-alpha protein expression) in species in which growth plates fuse, with that in rodents. Thus, we have investigated ER-alpha protein expression in femoral growth plates from male and female rabbits, just at sexual maturity (6 months), when growth plate fusion was just commencing, and in rats of equivalent developmental stage (9 weeks). ER-alpha was detected in undecalcified cryosections by immunofluorescence with 1D5 monoclonal antibody, raised to human ER-alpha; uterine sections were positive controls. ER-alpha-positive cells were localized to the proliferative/early hypertrophic zone of male and female rabbits. By contrast, cells in the similar region of the mature rat growth plate were ER-alpha-negative in both genders, although receptor could be readily detected in uteri of mature female rats. In growth plates of immature male and female rats (6 weeks), however, ER-alpha was clearly expressed by cells of the proliferative/early hypertrophic zone, but was barely detectable in uteri from immature females. Our findings support the view that estrogen may act directly on the growth plate and, in species in which there is epiphyseal fusion, may thus have a role in this process. If ER-alpha expression is lost at sexual maturity, as in rodents, growth plates may remain open into adulthood. Our results also show the changes in ER-alpha expression in growth plates of maturing rats may be opposite to that in the uterus and raise the possibility that receptor expression may be controlled differently in reproductive and skeletal tissues.

A method for immunofluorescent localization of oestrogen receptors in bone sections from an egg-laying poultry strain.

Although oestrogen has profound skeletal effects in hens, the identity of its target cells in bone is still unclear. We wished to address this by indirect immunofluorescent detection of oestrogen receptors, using monoclonal antibodies, similar to our method for mammalian bone. Avian bone, however, is prone to autofluorescence at the excitation wavelength for fluorescein isothiocyanate, and non-specific binding of mammalian antibodies. We therefore improved receptor detection by comparing three commercially available monoclonal antibodies to the human oestrogen receptor. We found that the best identification of oestrogen target cells was produced by ID5 antibody diluted 1/20, with initial binding disclosed by Cy3trade mark-conjugated immunoglobin, which has similar fluorescence to rhodamine. Clear localisation of these cells was reliably obtained in sections of both receptor positive human breast tissue and hen oviduct. Preliminary observations showed that immunofluorescence in avian oviduct and undecalcified bone cryosections was stable after 6 weeks storage and of sufficient clarity for semiquantification. Thus, in hens aged 18 weeks (first ovarian follicle), osteoblasts and 38% of osteocytes were clearly immunofluorescent. After 8 to 10 weeks egg lay, receptor-positive osteocytes decreased in structural bone to 19%; cells adjacent to medullary bone and in marrow cavities were strongly immunofluorescent.

Thyrotrophin-releasing hormone inactivation by human postmortem brain.

The mechanisms of inactivation of thyrotrophin-releasing hormone (TRH) by peptidases in several areas of normal human postmortem brain have been investigated by radioimmunoassay and high-performance liquid chromatography. Of the several brain regions studied, the cerebral cortex (Brodman's area, BA10) had the highest TRH-degrading activity in both subcellular fractions. Deamidated-TRH (TRH-OH) was the only product formed by the soluble fraction whereas the histidyl-proline diketopiperazine, cyclo(His-Pro), and a small amount of TRH-OH were formed by the particulate fraction. Several centrally acting TRH analogues showed varying degrees of resistance to degradation by the peptidases in the two fractions, the most stable analogue being RX77368 (pGlu-His-3,3'-dimethyl(ProNH2]. Areas of human postmortem brain appear to contain two of the enzymes capable of degrading TRH, a proline endopeptidase forming TRH-OH and a pyroglutamyl aminopeptidase forming cyclo(His-Pro). The use of the assay procedures in further studies on the inactivation of TRH by peptidases from brain areas of patients with neurological disorders may provide complementary information on the dynamics of TRH in these disorders. The stability of the centrally acting TRH analogues may prove useful in examining their therapeutic potential.

Direct effects of endotoxin on the microcirculation.

Endotoxin (E Coli, Difco), 0.5 mg in 10 ml saline, or whole blood, was infused over a ten-minute period in the retrograde direction into a branch of a mesenteric artery in anesthetized dogs; control infusions were physiologic saline or whole blood without endotoxin. After infusion, the animal's blood was allowed to circulate through the microcirculatory field. Changes in the microcirculation were recorded by cine and still photography. After infusion of endotoxin there was coincident margination of leukocytes and thickening of endothelial cells, then formation of fibrin thrombi in small veins, followed by lyses of the thrombi and extravasation of erythrocytes. Systemic blood pressure did not fall until after endothelial cell thickening and leukocyte margination (five to 25 minutes after infusion). The mean pressure fell to between 30 and 50 mm Hg, even though the dose of endotoxin was less than 1/100 of that required to cause this blood pressure fall when given IV. It is hypothesized that intracellular hydration of endothelial cells causes increased leakage of fluids and extravasation of erythrocytes following endotoxin. If the internal volumes of hexahedron-shaped cells increase without an increase in their surface areas they must assume a more spherical shape, thereby permitting gaps to develop between adjacent cells.