To evaluate the role of neuropeptide Y (NPY), a potent appetite stimulant, in controlling food intake and body weight, we investigated the use of antisense oligodeoxynucleotides (ODNs) to inhibit NPY gene expression in the hypothalamus. We compared the hypothalamic distribution of fluorescein-labelled ODNs administered intracerebroventricularly, and effects on food intake and NPY gene expression, of three different structural modifications of an antisense ODN sequence against NPY. Rats had either the antisense or missense ODNs (24 micrograms/day) or saline infused into the third ventricle by osmotic minipumps for 7 days. The unmodified phosphodiester ODN was not detectable in the hypothalamus after 7 days and had no effects on food intake. The phosphorothioate ODN was widely distributed throughout the hypothalamus but had nonselective effects, with similar changes in food intake and NPY mRNA levels in the antisense and missense groups, and was severely toxic. The propyl-protected ODN appeared to penetrate the hypothalamus well but had no antisense-selective effects on NPY mRNA levels or food intake. Antisense ODNs are increasingly used to inhibit gene expression in vitro and in intact animals. These negative findings underline the need for rigorous evaluation of any effects of antisense ODNs administered into the central nervous system, and raise doubts about the validity of this approach in physiological or pharmacological studies.

ABSTRACT

This study compared the extracellular calcium dependency and the enzymatic locus of that dependency for N⁶,O²′-dibutyryl cyclic AMP (dbcAMP)-, angiotensin II- and potassium-stimulated aldosterone secretion in dispersed rat glomerulosa cells. The need for extracellular calcium, calcium influx, and specifically for calcium influx through the calcium channel was examined. dbcAMP, angiotensin II and potassium, in the presence of calcium (3·5 mmol/l), significantly (P < 0·01) increased aldosterone output by at least 1·5-fold. Yet in the absence of extracellular calcium or in the presence of lanthanum (an inhibitor of calcium influx by most mechanisms) all three stimuli failed to increase aldosterone secretion. Nifedipine, a dihydropyridine calcium channel antagonist, significantly (P < 0·01) reduced angiotensin II- and potassium-stimulated aldosterone secretion, but had no effect on dbcAMP-stimulated aldosterone secretion (100 ± 14 vs 105 ± 19 pmol/10⁶ cells). Likewise nitrendipine failed to inhibit ACTH-stimulated aldosterone secretion.

Angiotension II and potassium activation of both the early aldosterone biosynthetic pathway (as reflected by pregnenolone production in the presence of cyanoketone) and also its late pathway (as reflected by the conversion of exogenous corticosterone to aldosterone in the presence of cyanoketone) were significantly (P < 0·01) inhibited by lanthanum, nifedipine and by reducing the extracellular calcium concentration. However, with dbcAMP stimulation, none of these manipulations modified pregnenolone production. Late pathway activation by dbcAMP was inhibited by lanthanum and a reduction in extracellular calcium, but not by nifedipine.

These observations suggest that: (1) the extracellular calcium dependency of dbcAMP-, angiotensin II- and potassium-stimulated aldosterone secretion reflects a need for calcium influx; (2) with dbcAMP stimulation, activation of the late pathway is dependent on calcium influx by a calcium channel-independent mechanism, whereas activation of the early pathway is not dependent on extracellular calcium or calcium influx and (3) activation of both the early and late pathway by angiotensin II and potassium is dependent on calcium influx by a calcium channel-dependent mechanism. Therefore, we conclude that the mechanism of activation of the early aldosterone biosynthetic pathway by dbcAMP is different from angiotensin II or potassium and early pathway activation is distinct from that of late pathway activation with dbcAMP stimulation.

J. Endocr. (1986) 110, 315–325

Abstract

3,5,3′-Tri-iodothyronine (T₃), 1α,25(OH)₂-vitamin D₃ (D₃) and retinoids activate related nuclear receptors which interact by heterodimerisation to regulate gene expression. Actions of each hormone are discrete and may be specified by changes in the relative concentrations of their receptors (T₃R, vitamin D receptor (VDR), retinoic acid receptor (RAR), retinoid X receptor (RXR)). T₃, D₃ and retinoids are essential for skeletal development and maintenance and we have previously shown complex interactions amongst their signalling pathways in osteosarcoma cells. In these studies we demonstrate that similar T₃R, VDR, RAR and RXR proteins are co-expressed in both osteoblast lineage cell primary cultures and osteosarcoma cells by Western blotting. We investigated whether hormone interactions in bone result from changes in receptor stoichiometry. Cells were treated with combinations of T₃, D₃, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (RA) that are known from previous studies to produce complex cell specific responses. No alteration in expression of any receptor protein was seen in response to any hormone combination in three phenotypically distinct osteosarcoma cell lines. Thus, in contrast to studies of overexpressed receptors in vitro, changes in the physiological concentrations of endogenous T₃R, VDR, RAR and RXR do not specify discrete hormone actions in osteoblastic cells. Other unidentified factors are likely to modulate hormone action in these bone cells.

Journal of Endocrinology (1997) 154, 63–74

Steroid receptors are key transcriptional regulators of mammary growth, development and lactation. Expression of estrogen receptors alpha (ERα) and beta (ERβ), progesterone receptor (PR), and estrogen-related receptor alpha-1 (ERRβ) have been evaluated in bovine mammary gland. The ERRα is an orphan receptor that, in other species and tissues, appears to function in the regulation of estrogen-response genes including lactoferrin and medium chain acyl-CoA dehydrogenase and in mitochondrial biogenesis. Expression of ERα, ERβ, PR and ERRα was characterized in mammary tissue obtained from multiple stages of bovine mammary gland development using quantitative real-time RT-PCR. Expression was evaluated in prepubertal heifers, primigravid cows, lactating non-pregnant cows, lactating pregnant cows and non-lactating pregnant cows (n=4 to 9 animals/stage). In addition, ERα, ERβ, PR and ERRα were mapped to chromosomes 9, 10, 15 and 29 respectively, by linkage and radiation hybrid mapping. Results indicated that expression of ERα, PR and ERRα was largely coordinately regulated and they were present in significant quantity during all physiological stages evaluated. In contrast, ERβ transcripts were present at a very low concentration during all stages. Furthermore, no ERβ protein could be detected in bovine mammary tissue by immunohistochemistry. The ERα and PR proteins were detected during all physiological states, including lactation. Our results demonstrate the presence of ERα, PR and ERRα during all physiological stages, and suggest a functional role for ERRα and a relative lack of a role for ERβ in bovine mammary gland development and lactation.

Endogenous purines including inosine have been shown to exert immunomodulatory and anti-inflammatory effects in a variety of disease models. The dosage of inosine required for protection is very high because of the rapid metabolism of inosine in vivo. The aim of this study was to determine whether a metabolic-resistant purine analogue, INO-2002, exerts anti-inflammatory effects in two animal models of type I diabetes. Type I diabetes was induced chemically with streptozotocin or genetically using the non-obese diabetic (NOD) female mouse model. Mice were treated with INO-2002 or inosine as required at 30, 100, or 200 mg/kg per day, while blood glucose and diabetes incidence were monitored. The effect of INO-2002 on the pancreatic cytokine profile was also determined. INO-2002 reduced both the hyperglycaemia and incidence of diabetes in both streptozotocin-induced and spontaneous diabetes in NOD mice. INO-2002 proved to be more effective in protecting against diabetes than the naturally occurring purine, inosine, when administered at the same dose. INO-2002 treatment decreased pancreatic levels of interleukin (IL)-12 and tumour necrosis factor-α, while increasing levels of IL-4 and IL-10. INO-2002 also reduced pancreatic levels of the chemokine MIP-1α. The inosine analogue, INO-2002, was protected more effectively than the naturally occurring purine, inosine, against development of diabetes in two separate animal models. INO-2002 exerts protective effects by changing the pancreatic cytokine expression from a destructive Th1 to a protective Th2 profile. The use of analogues of inosine such as INO-2002 should be considered as a potential preventative therapy in individuals susceptible to developing type I diabetes.

Recent studies have demonstrated photoperiodic changes in leptin sensitivity of seasonal mammals. Herein, we examined the interaction of season (long days (LD) versus short days (SD)) and recombinant ovine leptin (roleptin) on secretion of melatonin and prolactin (PRL) and on mRNA expression of suppressor of cytokine signaling-3 (SOCS-3) in the medial basal hypothalamus (MBH) in sheep. Twenty-four Polish Longwool ewes, surgically fitted with third ventricle (IIIV) cannulas, were utilized in a replicated switchback design involving 12 ewes per season. Within-season and replicate ewes were assigned randomly to one of three treatments (four ewes/treatment) and infused centrally three times at 0, 1 and 2 h beginning at sunset. Treatments were 1) control, Ringer–Locke buffer; 2) L1, roleptin, 0.5 μg/kg BW; and 3) L2, roleptin, 1.0 μg/kg BW. Jugular blood samples were collected at 15-min intervals beginning immediately before the start of infusions and continued for 6 h. At the end of blood sampling, a washout period of at least 3 days elapsed before ewes were re-randomized and treated with one of the treatments described above (four ewes/treatment). Ewes were then killed and brains were collected for MBH processing. Leptin treatments increased (P<0.001) circulating leptin concentrations compared with controls during both seasons in a dose-dependent manner. Overall, mean plasma concentrations of melatonin were greater (P<0.001) during LD than SD. However, leptin treatments increased melatonin concentrations during SD in a dose-dependent manner and decreased it during LD. Similarly, plasma concentrations of PRL were greater (P<0.001) during LD than SD. However, unlike changes in melatonin, circulating PRL decreased (P<0.001) in response to leptin during LD. Semi-quantitative PCR revealed that leptin increased (P<0.001) SOCS-3 expression in the MBH region during LD in a dose-dependent manner. Data provide evidence that secretion of photoperiodic hormones such as melatonin and PRL are inversely regulated by leptin during SD and LD. However, the increase in expression of SOCS-3 in the MBH during LD compared with SD fails to fully explain these effects.