Cholesterol is an important regulator of cell signaling, both through direct impacts on cell membranes and through oxy-metabolites that activate specific receptors (steroids, hydroxy-cholesterols, bile acids). Cholesterol moves slowly through and between cell membranes with the assistance of specific binding proteins and transfer processes. The prototype cholesterol regulator is the Steroidogenesis Acute Regulatory (STAR), which moves cholesterol into mitochondria, where steroid synthesis is initiated by cytochrome P450 11A1 in multiple endocrine cell types. CYP27A1 generates hydroxyl cholesterol metabolites that activate LXR nuclear receptors to control cholesterol homeostatic and transport mechanisms. LXR regulation of cholesterol transport and storage as cholesterol ester droplets is shared by both steroid-producing cells and macrophage. This cholesterol signaling which is crucial to brain neuron regulation by astrocytes and microglial macrophage, is mediated by ApoE and is sensitive to disruption by β-amyloid plaques. sm-FISH delivers appreciable insights into signaling in single cells, by resolving single RNA molecules as mRNA and by quantifying pre-mRNA at gene loci. sm-FISH has been applied to problems in physiology, embryo development and cancer biology, where single cell features have critical impacts. sm-FISH identifies novel features of STAR transcription in adrenal and testis cells, including asymmetric expression at individual gene loci, delayed splicing and 1:1 association of mRNA with mitochondria. This may represent a functional unit for the translation-dependent cholesterol transfer directed by STAR, which integrates into mitochondrial fusion dynamics. Similar cholesterol dynamics repeat with different players in the cycling of cholesterol between astrocytes and neurons in the brain, which may be abnormal in neurodegenerative diseases.
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Colin R Jefcoate and Jinwoo Lee
Laura de Miguel-Santos, Elisa Fernández-Millán, María Ángeles Martín, Fernando Escrivá and Carmen Álvarez
Replication, neogenesis, and apoptosis play a main role in neonatal endocrine pancreas remodeling. IGFs are major contributors to β-cell growth and function and are highly sensitive to nutritional status. We previously showed that maternal malnutrition caused an increase in β-cell mass in fetuses related to the stimulation of β-cell proliferation due to increased pancreatic IGF-1. At 4 days of life, the β-cell mass was decreased in undernourished neonates and persisted until adult age. To clarify whether undernutrition disrupts islet remodeling, we quantified β-cell mass, neogenesis, replication, and apoptosis on days 4, 14, and 23. To determine the impact of food restriction on IGF ontogeny and the consequences for β-cell growth, we measured IGF-1/-2 protein content in pancreas and liver and pancreatic IGF-1 receptor (IGF-1R)-signaling pathway at the same days. Our results indicate that undernutrition alters the timing and intensity of neonatal β-cell ontogeny. However, although malnutrition causes β-cell deficiency in neonates, an active process of β-cell neogenesis and a lower incidence of β-cell apoptosis maintain the regenerative capacity of the endocrine pancreas. Interestingly, our data provide evidence that local production of IGFs seems to be instrumental in these processes. In particular, increased pancreatic IGF-2 in undernourished rats may contribute to the partial suppression of the developmental wave of β-cell apoptosis probably through the inhibition of glycogen synthase kinase-3. In addition, decreased pancreatic levels of IGFBP-1/-2/-3 in undernourished neonates could enhance IGF availability for interacting with IGF-1R/IR.
D Boerboom, DL Russell, JS Richards and J Sirois
One member of a new family of metalloproteinases, a disintegrin and metalloproteinase with thrombospondin-like motifs-1 (ADAMTS-1), has been found to be expressed and hormonally induced in granulosa cells of ovulating rodent follicles. Furthermore, the targeted disruption of the ADAMTS-1 gene resulted in ovarian defects associated with severely impaired fertility. While these data demonstrate the importance of ADAMTS-1 in rodent ovarian physiology, the potential role of ADAMTS-1 in the ovulatory process of monoovulatory species remains unknown. The objectives of this study were to clone the equine ADAMTS-1 primary transcript and to study its regulation during human chorionic gonadotropin (hCG)-induced ovulation. A 3573 bp follicular cDNA library clone was isolated and found to encode a nearly complete, highly conserved ADAMTS-1 homologue. Real-time RT-PCR analysis detected this transcript in diverse tIssues, including previously unreported sites of ADAMTS-1 expression such as the male reproductive tract, the follicular theca interna and the mature corpus luteum. The tIssue distribution of the progesterone receptor (PR), a known regulator of ADAMTS-1 expression in rodent preovulatory follicles, was found to overlap that of ADAMTS-1 in some tIssues. A study of the regulation of follicular ADAMTS-1 and PR mRNAs during the hCG-induced ovulatory process revealed distinct patterns of regulation in granulosa cells and in theca interna. In granulosa cells, ADAMTS-1 mRNA was found to be induced at 12 h post-hCG (P<0.05), followed by a return to basal levels by 30 h and a re-increase at 33-39 h (P<0.05). A concomitant increase in PR mRNA (P<0.05) was observed at 12 h post-hCG. In theca interna, abundant ADAMTS-1 mRNA was detected at all timepoints, and levels increased transiently at 33 h post-hCG (P<0.05), whereas no significant change was observed in PR mRNA. Together, these data demonstrate for the first time the hormonally regulated ovarian expression of ADAMTS-1 in a monoovulatory species, and identify a novel biphasic regulation of ADAMTS-1 in granulosa cells and a regulated expression in theca interna that were not previously observed in rodents.
R S Guenette, H B Corbeil, J Léger, K Wong, V Mézl, M Mooibroek and M Tenniswood
After weaning, the mammary gland ceases lactation and involutes. The wet weight of the gland decreases by 70% within 4 days of weaning. This involves significant tissue remodelling as the ducts regress and return to the resting state. The presence of apoptotic bodies in the luminal epithelial compartment 2 to 3 days after weaning provides clear evidence that a substantial proportion of the regression is attributable to the induction of active cell death (ACD) of the epithelial cells.
These changes in the architecture of the gland were found to be mirrored by changes in gene expression. The steady-state level of β-casein mRNA decreased rapidly after weaning from the high levels seen during lactation to undetectable levels by 8 days after weaning. The steady-state levels of expression of a number of genes associated with ACD, including TRPM-2, tissue transglutaminase (TGase) and poly(ADP-ribose) polymerase (PARP), increased transiently during this time-frame. The steady-state level of TRPM-2 mRNA increased 2 days after weaning, reaching a peak on day 4, and decreasing to undetectable levels by day 8 after weaning. The steady-state levels of two other mRNAs, TGase and PARP, showed very similar kinetics. In contrast, the mRNA for Hsp 27, which has been shown to be induced during prostate regression, was not significantly induced in the regressing mammary gland. In-situ hybridization demonstrated that the TRPM-2, TGase and PARP genes were expressed predominantly in the luminal epithelial cells of the ducts. These cells expressed β-casein mRNA during lactation, and underwent ACD after weaning.
While the ultrastructural changes in the mammary gland after weaning, and the induction of TRPM-2, TGase and PARP mRNAs, are reminiscent of apoptosis in the prostate, several features of the process are different. Most notably, the disruption of the secretory processes and the lack of increased expression of Hsp 27 in the regressing mammary gland suggest that there may be a number of important events in ACD that are not common to all cells.
Ewa Harasim-Symbor, Karolina Konstantynowicz-Nowicka and Adrian Chabowski
Synthetic and natural glucocorticoids are able to highly modify liver lipid metabolism, which is possibly associated with nonalcoholic fatty liver disease development. We have assessed the changes in lipid and sphingolipid contents in hepatocytes, lipid composition and saturation status as well as the expression of proteins involved in fatty acid transport after both dexamethasone and palmitate treatments. The experiments were conducted on primary rat hepatocytes, incubated with dexamethasone and/or palmitic acid during short (16 h) and prolonged (40 h) exposure. Intracellular and extracellular lipid and sphingolipid contents were assessed by gas liquid chromatography and high-performance liquid chromatography, respectively. The expression of selected proteins was estimated by Western blotting. Short and prolonged exposure to dexamethasone combined with palmitic acid resulted in increased expression of fatty acid transporters, which was subsequently reflected by excessive intracellular accumulation of triacylglycerols and ceramide. The expression of microsomal transfer protein and cassette transporter was also significantly increased after dexamethasone and palmitate treatment, which was in accordance with elevated extracellular lipid and sphingolipid contents. Our data showed additive effects of dexamethasone and palmitate on protein-dependent fatty acid uptake in primary hepatocytes, resulting in the increased accumulation of triacylglycerols and sphingolipids. Moreover, the combined treatment altered fatty acid composition and diminished triacylglycerols desaturation index. Importantly, we observed that additive effects on both increased microsomal transport protein expression as well as elevated export of triacylglycerols, which may be relevant as a liver protective mechanism.
P R Shepherd, B T Navé and S O'Rahilly
Muscle and adipose tissue play a central role in the maintenance of glucose homeostasis as they account for the majority of insulin-mediated glucose disposal in the post-prandial state. In obese and diabetic subjects, resistance to the stimulatory effects of insulin on glucose disposal into muscle and fat are instrumental in the development of the chronic hyperglycaemic state associated with these conditions. Studies using a range of techniques including nuclear magnetic resonance studies (Rothman et al. 1992), in vivo forearm perfusion studies (Yki-Jarvinen et al. 1990), indirect calorimetry (Butler et al. 1990) and biochemical assays in isolated muscle strips (Dohm et al. 1988, Andreasson et al. 1991) have demonstrated that the insulin resistance appears to be due to the additive effects of defects in multiple aspects of insulin action in muscle. These investigations have identified defects at the level of insulin stimulation of glucose transport across the plasma membrane as
Bolander FF Jr
Prolactin (PRL) is capable of stimulating both calcium and nitric oxide (NO) accumulation in mammary epithelial cells within 15min. A calcium ionophore was also able to stimulate NO levels to an extent similar to that generated by PRL. Furthermore, maximal concentrations of PRL and the ionophore were not additive, suggesting that they were both using the same pathway, i.e. calcium. Finally, the depletion of intracellular calcium completely abrogated the effect of PRL on NO production. No other pathway known to affect NO synthase (NOS) influenced the action of PRL. Specifically, manipulations of protein phosphatase 2B, protein kinase B (PKB), protein kinase C (PKC), and arginine transport did not alter the activation of NOS by PRL. Therefore, the ability of PRL to stimulate NO production at 15min can be completely explained by its ability to elevate intracellular calcium.
P. Enyedi, T. Balla, F. A. Antoni and A. Spät
We have previously shown that arginine vasopressin (AVP) stimulates the production of aldosterone in isolated superfused adrenal glomerulosa cells by a mechanism that involves an increased turnover of phosphoinositides. In the present study we compared the characteristics of AVP- and angiotensin II (AII)-induced changes in phosphoinositide turnover and aldosterone production in the rat. Selected concentrations of the two peptides, which were equipotent in terms of the magnitude of changes induced in phosphoinositide turnover, stimulated aldosterone production to the same extent only in the initial phase of the stimulation. A sustained aldosterone response was only observed in AII-stimulated cells. On the other hand, the AVP-induced increase in incorporation of [32P]phosphate into phosphatidyl-inositol and the stimulation of inositol phosphate production were maintained during incubation. Preincubation of the cells with AVP failed to modify the effects of AII on phosphoinositide breakdown or aldosterone production. These results indicate that desensitization at the level of the receptor or at a post-receptor site is not responsible for the transient character of AVP-induced aldosterone production. Delayed activation of an inhibitory mechanism by AVP can also be excluded. Additivity of the stimulation of the phosphoinositide turnover observed at submaximally, but not maximally, effective concentrations of AII indicates that the two agonists act on the same phosphoinositide pool. We suggest that the sustained steroidogenic effect of AII involves an as yet unidentified mechanism, which is absent when the cells are stimulated with AVP.
C Massart, J Gibassier, C Lucas, F Le Gall, S Giscard-Dartevelle, J Bourdinière, M S Moukhtar and M Nicol
We studied the hormonal secretion of a human mixed follicular and medullary carcinoma. Thyroglobulin (Tg) secretion, especially by large cells and sometimes by small ones, was visualized with immunoenzymatic staining. Calcitonin (CT) was produced by small spindle-shaped cells. Moreover, immunofluorescence double staining performed on the resected thyroid tissue showed the secretion of both Tg and CT in a small number of cells. The cells lost their hormonal secretion after 2 months of culture. Hormonal secretion was modulated by different additives in the medium. Tg secretion was induced when TSH was added to the culture medium; the maximal effect was produced with the addition of 1 mU TSH/ml and 1 μm cortisol, which potentiated the effect of TSH on Tg production. A durable Tg secretion was obtained by embedding the cells in Engelbretch—Hohn—Swarn (EHS) tumour matrix. The CT production was reinduced by the addition of 4 mm Ca2+, 1 μm glucagon and 1 μm cortisol to the culture medium. These findings show that different cells are found in a mixed follicular and medullary carcinoma, some of which can secrete both CT and Tg. They can remain differentiated for a long period after being embedded in EHS tumour matrix with Ca2+ and hormonal components.
B. Quérat, A. Hardy and Y.A. Fontaine
The gonadotrophic function of the European eel (Anguilla anguilla L.) at the silver stage is very weak: gonadotrophin-releasing hormone (GnRH) secretion is deficient and, moreover, dopamine inhibition overrides GnRH action. At the silver stage, œstradiol stimulates the biosynthesis of the type-II gonadotrophin (GTH-II). To study the molecular mechanism of this activation further, we examined the effect of testosterone and œstradiol administration on pituitary levels of mRNA encoding GTH-II α and β subunits. Corresponding eel cDNA probes and Northern blot analysis were used. After 2 weeks, testosterone and œstradiol implantation resulted in a strong increase in mRNA encoding the GTH-II β subunit (7-fold and 25-fold, respectively) and in a slight, but non-significant, rise in the a subunit mRNA level (1.8-fold and 1.5-fold, respectively). Co-implantation of these two steroids suggested a potentiation of their effects on the β subunit (104-fold) while an additive effect was indicated on the α mRNA level (2.9-fold). Effects were detectable within 4 days and were maximal 4 weeks after implantation. These results indicate that in the European eel at the silver stage, gonadal steroids stimulate differentially the expression of GTH-II subunit genes at a pretranslational level.