We have examined the regulation of apolipoprotein A-I (apoA-I) gene expression in response to glucose and insulin. In Hep G2 cells, endogenous apoA-I mRNA was suppressed by one-half or induced 2-fold following 48 h of exposure to high concentrations of glucose (22.4 mM) or insulin (100 microunits/ml), respectively, compared with control. Transcriptional activity of the rat apoA-I promoter (؊474 to ؊7) in Hep G2 cells paralleled endogenous mRNA expression, and this activity was dependent on the dose of glucose or insulin. Deletional analysis showed that a 50-base pair fragment spanning ؊425 to ؊376 of the promoter mediated the effects of both insulin and glucose. Within this DNA fragment there is a motif (؊411 to ؊404) that is homologous to a previously identified insulin response core element (IRCE). Mutation of this motif abolished not only the induction of the promoter by insulin but also abrogated its suppression by glucose. Electrophoretic mobility shift assay analysis of nuclear extracts from Hep G2 cells revealed IRCE binding activity that formed a duplex with radiolabeled probe. The IRCE binding activity correlated with insulin induction of apoA-I expression. In summary, our data show that glucose decreases and insulin increases apoA-I promoter activity. This effect appears to be mediated by a single cis-acting element.

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To examine the effects of glucocorticoid (GC) on growth hormone (GH)-releasing hormone (GRH) receptor gene expression, a highly-sensitive and quantitative reverse-transcribed polymerase chain reaction (RT-PCR) method was used in this study. Rat anterior pituitary cells were isolated and cultured for 4 days. The cultured cells were treated with dexamethasone for 2, 6, and 24 h. GRH receptor mRNA levels were determined by competitive RT-PCR using a recombinant RNA as the competitor. Dexamethasone significantly increased GRH receptor mRNA levels at 5 nM after 6- and 24 h-incubations, and the maximal effect was found at 25 nM. The GC receptor-specific antagonist, RU 38486 completely eliminated the dexamethasone-induced enhancement of GRH receptor mRNA levels. Dexamethasone did not alter the mRNA levels of beta-actin and prolactin at 5 nM for 24 h, whereas GH mRNA levels were significantly increased by the same treatment. The GH response to GRH was significantly enhanced by the 24-h incubation with 5 nM dexamethasone. These findings suggest that GC stimulates GRH receptor gene expression through the ligand-activated GC receptors in the rat somatotrophs. The direct effects of GC on the GRH receptor gene could explain the enhancement of GRH-induced GH secretion.

The aim of the present study was to assess potential brain sites of stimulation by peripheral interleukin (IL)-6 of the hypothalamo-pituitary-adrenal (HPA) axis in the rat, using c-fos protein as a marker of cellular activation. Involvement of prostaglandins in IL-6-induced ACTH secretion and c-fos expression was also investigated. IL-6 was infused continuously (40 ng/min) for 90 min to conscious male rats. Blood samples were taken before the infusion and at 30 and 90 min for measurement of plasma ACTH. Expression of c-fos in the brain was examined by immunohistochemistry. Administration of IL-6 significantly elevated plasma ACTH levels at 30 min (495 ± 105 vs. 117 ± 17 pg/ml in controls, p < 0.05). Elevated levels were still present at 90 min (596 ± 139 vs. 113 ± 20 pg/ml in controls, p < 0.05). Infusion of IL-6 (3.6 µg/rat) markedly triggered c-fos expression in hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), as well as in the central amygdaloid nucleus (CeA), the nucleus tractus solitarius and the locus coeruleus. Pretreatment with the cyclooxygenase inhibitor indomethacin (10 mg/kg, i.v.) suppressed the ACTH response induced by IL-6. The number of IL-6-induced immunoreactive cells in the PVN was significantly reduced by indomethacin pretreatment (p < 0.01), but the number of IL-6-induced c-fos-positive cells in the SON and CeA remained unchanged. These findings suggest that circulating IL-6 may exert central actions by acting directly or indirectly on brain neurons. In addition, the ability of IL-6 to activate the HPA axis may depend upon the release of prostaglandins, probably in the brain.

Utilizing immunohistochemistry for the c-fosprotein to detect neuronal activity, we examined the effects of continuous intravenous and intracerebroventricular infusion of interleukin (IL)-1βin the rat brain, and the involvement of prostaglandins (PGs) in IL-1β-induced c-fosexpression. Continuous intravenous infusion of IL-1β(10 ng/min) markedly augmented c-fosexpression in the paraventricular (PVN) and the supraoptic (SON) nuclei of the hypothalamus as well as in the central amygdaloid nucleus (CeA). The number of IL-1β-induced c-fos-positive cells in the PVN and SON was significantly lower in rats pretreated with indomethacin than in vehicle-treated rats. However, the number of IL1β-induced c-fos-positive cells in the CeA remained unchanged. c-fosprotein was induced after intracerebroventricular infusion of IL-1β(200 ng) in the PVN, SON and arcuate nuclei of the hypothalamus, and in the CeA. The induction of c-fosimmunoreactivity by central administration of IL-1βwas blocked by indomethacin (500µg/rat), except in the CeA. These findings suggest that PGs are involved in the complex transmission of signals from circulating or central IL-1βto hypothalamic neurons.