Abstract

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)₂D), acts on intestinal, renal, and bone cells to regulate skeletal and mineral metabolism. 1,25(OH)₂D also induces 24-hydroxylase activity in these target cells. The 24-hydroxylase hydroxylates 1,25(OH)₂D to 1,24,25-trihydroxyvitamin D and 25(OH)D to 24,25-dihydroxyvitamin D. The production of 1,24,25-trihydroxyvitamin D is thought to be the first step in the inactivation of 1,25(OH)₂D by its target tissues. Previous studies have characterized the induction of the 24-hydroxylase by 1,25(OH)₂D in clonal cell lines from intestine and bone. The purpose of these studies was to characterize the induction of the 24-hydroxylase by 1,25(OH)₂D in the kidney, using the clonal rat renal cell line NRK-52E. 1,25(OH)₂D (10⁻⁷ m) increased the mRNA levels for the cytochrome P450 component of the 24-hydroxylase (P450cc24) by sevenfold after 36 h in NRK-52E cells. 1,25(OH)₂D increased P450cc24 mRNA levels in a dose-dependent manner with an EC₅₀ of 10⁻⁸ m. In parallel experiments, 1,25(OH)₂D significantly increased 24-hydroxylase enzyme activity after 48–72 h. The increase in P450cc24 mRNA induced by 1,25(OH)₂D required on-going transcription and translation and was inhibited by H-7, a protein kinase C inhibitor. Tetradecanoyl phorbol acetate markedly increased the magnitude of the tissue responsiveness to 1,25(OH)₂D by a protein kinase C-dependent pathway. These studies demonstrate that 1,25(OH)₂D increases P450cc24 mRNA levels in NRK-52E cells by a mechanism requiring new protein synthesis and involving protein kinase C. This is in contrast to the action of 1,25(OH)₂D in intestinal cells, which does not require new protein synthesis, and in osteoblastic cells, which does not involve protein kinase C.

Journal of Endocrinology (1997) 153, 199–205

In the present study, the effects of postnatal hypoxemia on the AT1 angiotensin receptor-mediated activities in the rat carotid body were studied. Angiotensin II (Ang II) concentration-dependently increased the chemoreceptor afferent activity in the isolated carotid body. Single- or pauci-fiber recording of the sinus nerve revealed that the afferent response to Ang II was enhanced in the postnatally hypoxic carotid body. To determine whether the increased sensitivity to Ang II is mediated by changes in the functional expression of Ang II receptors in the carotid body chemoreceptors, cytosolic calcium ([Ca2+]i) was measured by spectrofluorimetry in fura-2 acetoxymethyl ester-loaded type I cells dissociated from carotid bodies. Ang II (25-100 nM) concentration-dependently increased [Ca2+]i in the type I cells. The proportion of clusters of type I cells responsive to Ang II was higher in the postnatally hypoxic group than in the normoxic control (89 vs 66%). In addition, the peak [Ca2+]i response to Ang II was enhanced 2- to 3-fold in the postnatally hypoxic group. The [Ca2+]i response to Ang II was abolished by pretreatment with losartan (1 microM), an AT1 receptor antagonist, but not by PD-123177 (1 microM), an AT(2) antagonist. Double-labeling immunohistochemistry confirmed that an enhanced immunoreactivity for AT1 receptor was co-localized to the lobules of type I cells in the hypoxic group. In addition, RT-PCR analysis of subtypes of AT1 receptors showed an up-regulation of AT1a but a down-regulation of AT1b receptors, indicating a differential regulation of the expression of AT1 receptor subtypes by postnatal hypoxia in the carotid body. These data suggest that postnatal hypoxemia is associated with an increased sensitivity of peripheral chemoreceptors in response to Ang II and an up-regulation of AT1a receptor-mediated [Ca2+]i activity of the chemoreceptors. This modulation may be important for adaptation of carotid body functions in the hypoxic ventilatory response and in electrolyte and water homeostasis during perinatal and postnatal hypoxia.

This study was performed to compare the effects of three well-known phytoestrogens such as genistein, resveratrol, and quercetin on steroidogenesis in MA-10 mouse tumor Leydig cells. Addition of genistein or resveratrol to MA-10 cells resulted in decreases in the cAMP-stimulated progesterone secretion, but quercetin had an opposite response. Steroidogenic acute regulatory (StAR) mRNA expression and StAR promoter activity in transiently transfected MA-10 cells were significantly reduced by genistein or resveratrol, but increased by quercetin. Genistein was found to inhibit MA-10 cell proliferation, while resveratrol and quercetin had no effect. Quercetin-induced increase in cAMP-stimulated progesterone secretion was reversed by ICI 182,780, an estrogen receptor (ER) antagonist. However, ICI 182,780 had no effect on cAMP plus quercetin-stimulated StAR promoter activity. To examine whether non-ER factors are associated with quercetin-stimulated progesterone production, we treated MA-10 cells with EGTA to deprive them of extracellular Ca²⁺. We found that EGTA inhibited quercetin-plus cAMP-stimulated progesterone secretion and StAR promoter activity. Blocking of Ca²⁺ influx through L- or T-type voltage-gated Ca²⁺ channels with verapamil or mibefradil respectively, attenuated quercetin-stimulated progesterone secretion, while they had no effect on quercetin-plus cAMP-stimulated StAR promoter activity. Blocking of intracellular Ca²⁺ efflux by sodium orthovanadate, a Ca²⁺-pump inhibitor, blocked quercetin- plus cAMP-stimulated progesterone secretion and StAR promoter activity in MA-10 cells. Finally, EGTA or vanadate reduced quercetin and cAMP-increased in StAR mRNA expression in MA-10 cells, while ICI 182,780 had no effect. Taken together, these results indicate that phytoestrogens have differential effects on steroidogenesis in MA-10 cells.

Pathological changes associated with obesity are thought to contribute to GH deficiency. However, recent observations suggest that impaired GH secretion relative to excess calorie consumption contributes to progressive weight gain and thus may contribute to the development of obesity. To clarify this association between adiposity and GH secretion, we investigated the relationship between pulsatile GH secretion and body weight; epididymal fat mass; and circulating levels of leptin, insulin, non-esterified free fatty acids (NEFAs), and glucose. Data were obtained from male mice maintained on a standard or high-fat diet. We confirm the suppression of pulsatile GH secretion following dietary-induced weight gain. Correlation analyses reveal an inverse relationship between measures of pulsatile GH secretion, body weight, and epididymal fat mass. Moreover, we demonstrate an inverse relationship between measures of pulsatile GH secretion and circulating levels of leptin and insulin. The secretion of GH did not change relative to circulating levels of NEFAs or glucose. We conclude that impaired pulsatile GH secretion in the mouse occurs alongside progressive weight gain and thus precedes the development of obesity. Moreover, data illustrate key interactions between GH secretion and circulating levels of insulin and reflect the potential physiological role of GH in modulation of insulin-induced lipogenesis throughout positive energy balance.

ABSTRACT

We investigated whether human granulosa-luteal (GL) cells exhibited lipopolysaccharide (LPS)-binding protein, and the response of follicular aspirate cells to LPS in vitro. Follicular aspirates taken from a human in-vitro fertilization and gamete intrafallopian-tube transfer programme were subjected to Percoll gradients in order to isolate an enriched population of GL cells. GL cells exhibited specific LPS-binding protein, detected by autoradiography of the cellular lysate on SDS-PAGE after the cells were specifically labelled with a radioiodinated, photoactivable and reducible LPS derivative. LPS binding to the cells was also detected by the appearance of immunofluorescence associated with the cellular membrane when incubated with a fluorescent conjugated LPS receptor antibody. Ninety-four per cent of the cells exhibiting immunofluorescent LPS-binding protein were also positive for the steroidogenic enzyme 3β-hydroxysteroid dehydrogenase, as detected by cytochemistry. In order to detect a response to LPS, the enriched population of GL cells were cultured in vitro in the presence or absence of LPS; after 16 h of culture, tumour necrosis factor-α (TNF) mRNA was detected by reverse transcription-polymerase chain reaction and Southern blot analysis of the amplified cDNA. The expression of TNF mRNA was enhanced when the cells were cultured in the presence of LPS, which also significantly enhanced TNF secretion into the media during the 16-h period. These results reveal that GL cells exhibit LPS-binding protein and thus increased TNF secretion occurs in response to LPS in follicular aspirate cells. The source of ovarian TNF may be leukocytes, macrophages and/or GL cells.

Journal of Endocrinology (1992) 135, 571–578

Graves' disease (GD) is a common organ-specific autoimmune disease with the prevalence between 0.5 and 2% in women. Several lines of evidence indicate that the shed A-subunit rather than the full-length thyrotropin receptor (TSHR) is the autoantigen that triggers autoimmunity and leads to hyperthyroidism. We have for the first time induced GD in female rhesus monkeys, which exhibit greater similarity to patients with GD than previous rodent models. After final immunization, the monkeys injected with adenovirus expressing the A-subunit of TSHR (A-sub-Ad) showed some characteristics of GD. When compared with controls, all the test monkeys had significantly higher TSHR antibody levels, half of them had increased total thyroxine (T₄) and free T₄, and 50% developed goiter. To better understand the underlying mechanisms, quantitative studies on subpopulations of CD4+T helper cells were carried out. The data indicated that this GD model involved a mixed Th1 and Th2 response. Declined Treg proportions and increased Th17:Treg ratio are also observed. Our rhesus monkey model successfully mimicked GD in humans in many aspects. It would be a useful tool for furthering our understanding of the pathogenesis of GD and would potentially shorten the distance toward the prevention and treatment of this disease in human.

ABSTRACT

A sensitive and specific double-antibody enzyme-linked immunoassay, using a synthetic analogue of human relaxin for standard and immunogen, was developed for the measurement of human relaxin (hRLX) in serum and plasma. No cross-reactivity was observed for human insulin, human insulin-like growth factor-I, hGH, human chorionic gonadotropin, hFSH, hLH or human prolactin. The assay was used to monitor RLX concentrations in samples from men, non-pregnant and pregnant women, and in pregnant rhesus monkeys infused with hRLX. RLX was not detected in serum from men nor from non-pregnant women, while a concentration of 600 ng/l was measured in pooled sera from two pregnant women (pregnancies achieved by in-vitro fertilization). Immunoreactive RLX (1·1 μg/g) was found in human corpora lutea taken from ectopic pregnancies at 7 weeks. In an experiment with a pregnant rhesus monkey infused with human RLX analogue, less than 1·5% of the maternal concentration was measured in the fetal circulation. Even though preliminary, these data suggest a low level of transfer of human analogue relaxin across the placenta in a rhesus monkey. Further studies of the physiology of RLX in human pregnancy will be facilitated by the availability of this immunoassay.

Journal of Endocrinology (1989) 120, 449–457

For centuries, Berberine has been used in the treatment of enteritis in China, and it is also known to have anti-hyperglycemic effects in type 2 diabetic patients. However, as Berberine is insoluble and rarely absorbed in gastrointestinal tract, the mechanism by which it works is unclear. We hypothesized that it may act locally by ameliorating intestinal barrier abnormalities and endotoxemia. A high-fat diet combined with low-dose streptozotocin was used to induce type 2 diabetes in male Sprague Dawley rats. Berberine (100 mg/kg) was administered by lavage to diabetic rats for 2 weeks and saline was given to controls. Hyperinsulinemia and insulin resistance improved in the Berberine group, although there was no significant decrease in blood glucose. Berberine treatment also led to a notable restoration of intestinal villi/mucosa structure and less infiltration of inflammatory cells, along with a decrease in plasma lipopolysaccharide (LPS) level. Tight junction protein zonula occludens 1 (ZO1) was also decreased in diabetic rats but was restored by Berberine treatment. Glutamine-induced glucagon-like peptide 2 (GLP2) secretion from ileal tissue decreased dramatically in the diabetic group but was restored by Berberine treatment. Fasting insulin, insulin resistance index, plasma LPS level, and ZO1 expression were significantly correlated with GLP2 level. In type 2 diabetic rats, Berberine treatment not only augments GLP2 secretion and improves diabetes but is also effective in repairing the damaged intestinal mucosa, restoring intestinal permeability, and improving endotoxemia. Whether these effects are mechanistically related will require further studies, but they certainly support the hypothesis that Berberine acts via modulation of intestinal function.

Islet-level oxidative stress has been proposed as a trigger for type 1 diabetes (T1D), and release of cytokines by infiltrating immune cells further elevates reactive oxygen species (ROS), exacerbating β cell duress. To identify genes/mechanisms involved with diabetogenesis at the β cell level, gene expression profiling and targeted follow-up studies were used to investigate islet activity in the biobreeding (BB) rat. Forty-day-old spontaneously diabetic lymphopenic BB DRlyp/lyp rats (before T cell insulitis) as well as nondiabetic BB DR+/+ rats, nondiabetic but lymphopenic F344lyp/lyp rats, and healthy Fischer (F344) rats were examined. Gene expression profiles of BB rat islets were highly distinct from F344 islets and under-expressed numerous genes involved in ROS metabolism, including glutathione S-transferase (GST) family members (Gstm2, Gstm4, Gstm7, Gstt1, Gstp1, and Gstk1), superoxide dismutases (Sod2 and Sod3), peroxidases, and peroxiredoxins. This pattern of under-expression was not observed in brain, liver, or muscle. Compared with F344 rats, BB rat pancreata exhibited lower GST protein levels, while plasma GST activity was found significantly lower in BB rats. Systemic administration of the antioxidant N-acetyl cysteine to DRlyp/lyp rats altered abundances of peripheral eosinophils, reduced severity of insulitis, and significantly delayed but did not prevent diabetes onset. We find evidence of β cell dysfunction in BB rats independent of T1D progression, which includes lower expression of genes related to antioxidative defense mechanisms during the pre-onset period that may contribute to overall T1D susceptibility.

Clinical and animal studies have shown that treatment with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists slows the progression of nephropathy in diabetes, indicating that Ang II plays an important role in its development. We have reported previously that insulin inhibits the stimulatory effect of high glucose levels on angiotensinogen (ANG) gene expression in rat immortalized renal proximal tubular cells (IRPTCs) via the mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We hypothesize that the suppressive action of insulin on ANG gene expression might be attenuated in renal proximal tubular cells (RPTCs) of rats with established diabetes. Two groups of male adult Wistar rats were studied: controls and streptozotocin (STZ)-induced diabetic rats at 2, 4, 8 and 12 weeks post-STZ administration. Kidney proximal tubules were isolated and cultured in either normal glucose (i.e. 5 mM) or high glucose (i.e. 25 mM) medium to determine the inhibitory effect of insulin on ANG gene expression. Immunoreactive rat ANG (IR-rANG) in culture media and cellular ANG mRNA were measured by a specific radioimmunoassay and reverse transcription-polymerase chain reaction assay respectively. Activation of the p44/42 MAPK signal transduction pathway in rat RPTCs was evaluated by p44/42 MAPK phosphorylation employing a PhosphoPlus p44/42 MAPK antibody kit. Insulin (10(-7) M) inhibited the stimulatory effect of high glucose levels on IR-rANG secretion and ANG gene expression and increased p44/42 MAPK phosphorylation in normal rat RPTCs. In contrast, it failed to affect these parameters in diabetic rat RPTCs. In conclusion, our studies demonstrate that hyperglycaemia induces insulin resistance on ANG gene expression in diabetic rat RPTCs by altering the MAPK signal transduction pathway.