The low density lipoprotein receptor is not necessary for maintaining mouse brain polyunsaturated fatty acid concentrations

The brain cannot synthesize n-6 or n-3 PUFAs de novo and requires their transport from the blood. Two models of brain fatty acid uptake have been proposed. One requires the passive diffusion of unesterified fatty acids through endothelial cells of the blood-brain barrier, and the other requires the uptake of lipoproteins via a lipoprotein receptor on the luminal membrane of endothelial cells. This study tested whether the low density lipoprotein receptor (LDLr) is necessary for maintaining brain PUFA concentrations. Because the cortex has a low basal expression of LDLr and the anterior brain stem has a relatively high expression, we analyzed these regions separately. LDLr knockout (LDLr(-/-)) and wild-type mice consumed an AIN-93G diet ad libitum until 7 weeks of age. After microwaving, the cortex and anterior brain stem (pons and medulla) were isolated for phospholipid fatty acid analyses. There were no differences in phosphatidylserine, phosphatidylinositol, ethanolamine, or choline glycerophospholipid esterified PUFA or saturated or monounsaturated fatty acid concentrations in the cortex or brain stem between LDLr(-/-) and wild-type mice. These findings demonstrate that the LDLr is not necessary for maintaining brain PUFA concentrations and suggest that other mechanisms to transport PUFAs into the brain must exist.     

The effect of calcium concentration on ACTH stimulation of steroidogenesis in mouse adrenal tumor cells

Calcium is required for ACTH stimulated steroidogenesis in adrenal tumor cells in tissue culture. In the absence of calcium, the dose of ACTH required to induce half maximum steroidogenesis was increased 30 fold. In contrast to intact adrenal glands or isolated adrenal cells, high doses of ACTH (50 mU/ml) maximally stimulated steroidogenesis in the absence of calcium. Growth for up to six days in medium with low calcium did not affect basal or ACTH induced steroidogenesis. The addition of calcium to cells incubated with ACTH produced a maximum steroidogenic response in 15 minutes. In contrast to intact adrenal glands, calcium is not required for adenosine-3′,5′-cyclic monophosphate (cyclic AMP) stimulated steroidogenesis in adrenal tumor cells. These experiments support the concept that calcium is important at the level of ACTH-membrane receptor site interaction or activation of adenyl cyclase in adrenal tumor cells.     

Lipoprotein receptors and steroidogenesis in adrenocortical cells.

Steroid-producing tissues require a continuous supply of cholesterol for hormone synthesis. In the majority of the steroidogenic tissues the cholesterol is imported via the receptor-mediated uptake of lipoproteins, and therefore the influence on the lipoprotein receptors provides an additional level for the regulation of hormone synthesis. Hormones regulating the adrenocortical activity exert both short- and long-term action, and thus they may control the interactions of the major cholesterol delivery particles--low- (LDLs) and high-density lipoproteins (HDLs)--and their receptors in short- and long-term action, possibly modulating the signal transduction in the former case and the number and distribution in the latter. The LDL and HDL pathway and the signal transduction mechanism is briefly reviewed. Data are discussed concerning short- and long-term action of hormones (alpha-MSH and ACTH, respectively) on the HDL3 receptors of isolated adrenocortical cells. Short-term treatment with alpha-MSH and long-term treatment with ACTH increased the binding of HDL3 to zona glomerulosa and fasciculata cells, respectively, while both treatments increased the hormone production in the presence of HDL. The lipoprotein receptors were frequently found on the microvilli of adrenocortical cell membranes.     

Mechanism of colchicine-induced steroidogenesis in rat adrenocortical cells

Conflicting data for the effects of colchicine on cholesterol transport and steroidogenesis raise the question of the role of microtubules in cholesterol transport from the lipid droplet to mitochondria in steroidogenic cells. In this study, using corticosterone radioimmunoassay and immunofluorescence microscopy, we re-evaluated the effects of colchicine on hormone production and morphological changes of lipid droplets' and studied the signaling pathway involved in colchicine-induced steroidogenesis. Colchicine stimulated steroid production in a dose- and time-dependent manner. The structural integrity of both the microtubules and the lipid droplet capsule was destroyed by colchicine treatment. Disruption of the lipid droplet capsule occurred later than microtubule depolymerization. After cessation of colchicine treatment and a 3 h recovery in fresh medium, capsular protein relocated to the droplet surface before the cytoplasmic microtubule network was re-established. beta-lumicolchicine, an inactive analogue of colchicine, disrupted the capsule and increased hormone production without affecting microtubular structure. Thus, microtubule depolymerization is not required for the increase in steroid production and capsular disruption. To explore the signaling pathway involved in colchicine-induced steroidogenesis, we measured intracellular cAMP levels. Unlike ACTH, colchicine did not increase cAMP levels, suggesting that the cAMP-PKA system is not involved. Colchicine and ACTH had additive effects on corticosterone production, whereas colchicine and PMA did not, implying that part of the PKC signaling mechanism may be involved in colchicine-induced steroidogenesis. Cycloheximide, a protein synthesis inhibitor, completely inhibited colchicine-induced steroidogenesis and capsular disruption. These results demonstrate that the steroid production and lipid droplet capsule detachment induced by colchicine are both protein neosynthesis-dependent and microtubule-independent.     

Ubiquinone is necessary for mouse embryonic development but is not essential for mitochondrial respiration

Ubiquinone (UQ) is a lipid found in most biological membranes and is a co-factor in many redox processes including the mitochondrial respiratory chain. UQ has been implicated in protection from oxidative stress and in the aging process. Consequently, it is used as a dietary supplement and to treat mitochondrial diseases. Mutants of the clk-1 gene of the nematode Caenorhabditis elegans are fertile and have an increased life span, although they do not produce UQ but instead accumulate a biosynthetic intermediate, demethoxyubiquinone (DMQ). DMQ appears capable to partially replace UQ for respiration in vivo and in vitro. We have produced a vertebrate model of cells and tissues devoid of UQ by generating a knockout mutation of the murine orthologue of clk-1 (mclk1). We find that mclk1-/- embryonic stem cells and embryos accumulate DMQ instead of UQ. As in the nematode mutant, the activity of the mitochondrial respiratory chain of -/- embryonic stem cells is only mildly affected (65% of wild-type oxygen consumption). However, mclk1-/- embryos arrest development at midgestation, although earlier developmental stages appear normal. These findings indicate that UQ is necessary for vertebrate embryonic development but suggest that mitochondrial respiration is not the function for which UQ is essential when DMQ is present.     

The carboxyl terminus of the hamster beta-adrenergic receptor expressed in mouse L cells is not required for receptor sequestration

The structural basis for agonist-mediated sequestration and desensitization of the beta-adrenergic receptor (beta AR) was examined by oligonucleotide-directed mutagenesis of the hamster beta AR gene and expression of the mutant genes in mouse L cells. Treatment of these cells with the agonist isoproterenol corresponded to a desensitization of beta AR activity. A mutant receptor that bound agonist but did not couple to adenylate cyclase showed a dramatically reduced sequestration response to agonist stimulation. In contrast, beta AR mutants in which the C-terminus was truncated and/or in which two regions that have been proposed as phosphorylation substrates for cAMP-dependent protein kinase were removed showed normal sequestration responses. These results demonstrate that agonist-mediated sequestration of the beta AR can occur in the absence of the C-terminus of the protein and reveal a strong correlation between effective coupling to Gs and sequestration.     

Rounding and steroidogenesis of enzyme-and ACTH-treated Y-1 mouse adrenal tumor cells

Cultured Y-1 mouse adrenal tumor cells treated with ACTH (0.5 U/ml) rounded, formed filopodia and numerous thin microvilli, and produced steroids. Rounding, filopodia and bleb formation occurred for trypsin (0.01%), and hyaluronidase (0.1%), treated cells; but neither affected control or ACTH-stimulated steroidogenesis. Neuraminidase treatment (20 mU/ml) caused rounding, thin microvilli, bleb formation, slightly increased steroid production and prevented subsequent ACTH effects. Neuraminidase appeared to alter a carbohydrate-containing ACTH receptor preventing ACTH binding. We conclude rounding and steroidogenesis are not always associated.     

Expression of adiponectin receptors in mouse adrenal glands and the adrenocortical Y-1 cell line: Adiponectin regulates steroidogenesis

Obesity is frequently associated with malfunctions of the hypothalamus-pituitary-adrenal (HPA) axis and hyperaldosteronism, but the mechanism underlying this association remains unclear. Since the adrenal glands are embedded in adipose tissue, direct cross-talk between adipose tissue and the adrenal gland has been proposed. A previous study found that adiponectin receptor mRNA was expressed in human adrenal glands and aldosterone-producing adenoma (APA). However, the expression of adiponectin receptors in adrenal glands has not been confirmed at the protein level or in other species. Furthermore, it is unclear whether adiponectin receptors expressed in adrenal cells are functional. We found, for the first time, that adiponectin receptor (AdipoR1 and AdipoR2) mRNA and protein were expressed in mouse adrenal and adrenocortical Y-1 cells. However, adiponectin itself was not expressed in mouse adrenal or Y-1 cells. Furthermore, adiponectin acutely reduced basal levels of corticosterone and aldosterone secretion. ACTH-induced steroid secretion was also inhibited by adiponectin, and this was accompanied by a parallel change in the expression of the key genes involved in steroidogenesis. These findings indicate that adiponectin may take part in the modulation of steroidogenesis. Thus, adiponectin is likely to have physiological and/or pathophysiological significance as an endocrine regulator of adrenocortical function.     

Prenylation is not necessary for endogenous Ras activation in non-malignant cells

Ras monomeric GTPases are pivotal to many core cellular processes such as proliferation and differentiation. The post-translational prenylation of Ras with a farnesyl or a geranylgeranyl moiety is thought to be critical for its membrane binding and consequent signaling activity. Inhibitors of Ras prenylation have an anti-proliferative effect in some Ras-transformed cells. We present a study of the effects of prenylation inhibitors on endogenous, wild-type Ras in three renal cell types, namely primary adult human renal fibroblasts, primary adult human mesangial cells, and a primate renal fibroblast cell line (Vero cells). We have previously demonstrated that Ras is necessary for normal proliferation in these cells. Here we show that Ras is farnesylated and not geranylgeranylated in all three cell types. Furthermore, inhibiting Ras farnesylation has no effect on cell proliferation or Ras activation. Although inhibiting geranylgeranylation in these cells does inhibit proliferation, this is through an Ras-independent mechanism. Non-prenylated Ras is able to localize to the plasma membrane, bind Raf when cells are stimulated by epidermal growth factor or platelet-derived growth factor, and activate the Ras downstream effectors mitogen-activated protein kinase and phosphotidylinositol 3-kinase. We conclude that in wild-type cells, endogenous Ras does not need to be prenylated to be active.     

Glucocorticoid control of steroidogenesis in isolated rat adrenocortical cells

The role of end-product glucocorticoids in the regulation of corticosteroidogenesis in isolated adrenocortical cells was investigated. Trypsin-isolated cells from male rat adrenal glands were incubated with or without corticotropin (ACTH) and with or without corticosterone. Endogenous corticosterone production was determined by radioimmunoassay at the end of incubation. Cessation of ACTH-induced corticosterone production was apparent after 2-4 h of incubation. The suppression occurred later with lower cell concentrations. Corticosterone production was partially restored after washing the suppressed cells. Supernatant fluid from suppressed cell suspensions also suppressed steroidogenesis of a fresh population of cells. However, the suppressing property of the supernatant fluid was abolished after the removal of corticosterone by charcoal-dextran treatment, suggesting that corticosterone or other steroids caused the suppression. Exogenous corticosterone induced suppression over a wide range of ACTH concentrations, but did not change the half-maximal steroidogenic concentration of ACTH, indicating that the suppression does not change the sensitivity of the cells to ACTH. Suppression occurred within 30-60 min after corticosterone had been added to the incubation medium either at the start of incubation or while steroidogenesis was in progress. Suppression varied directly with the concentration of exogenous corticosterone. These data indicate that glucocorticoids can directly and acutely suppress corticosteroidogenesis and thus control adrenocortical function in concert with other regulators such as ACTH and Ca2+.     

The pro-peptide is not necessary for active renin secretion from transfected mammalian cells

Cultured mouse myeloma cells were transfected with expression vectors encoding the aspartyl proteinase, human renin. The full construct, encoding the renin precursor prorenin, allows transfected cells to secrete the enzymically inactive pro-protein. Activity is detectable only following trypsin treatment which mimics the physiological activation step. Accordingly, it appears that myeloma cells do not contain detectable levels of an appropriate activating proteinase. However, when these cells are transfected with a construct from which the pro-peptide coding sequence has been deleted, they secrete an apparently fully active enzyme which is indistinguishable from mature renin. We conclude that expression of the pro-peptide is not necessary to allow correct folding of the molecule and its passage through the secretory pathway.     

Column perifusion system for steroidogenesis in isolated rat adrenal cells

A perifusion system using a plastic column into which isolated rat adrenal cells had been installed was attempted. After ACTH or cAMP was administered to the column, the corticosterone concentration in the eluate was determined. ACTH in 10(-13) and 10(-12) M did not promote corticosterone production, whereas 10(-11) and 10(-10) M showed a dose dependent production of corticosterone. By iterative infusion of 10(-11) or 10(-9) M of ACTH, very clear responses to restimulation of ACTH were noted. Following the administrations of 10(-3) or 10(-2) M of dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP), the production of corticosterone increased dose-dependently. These results suggest that this perifusion system is effective for examining the effects of ACTH or cAMP on steroidogenesis of cells.     

The lymphotoxin-beta receptor is necessary and sufficient for LIGHT-mediated apoptosis of tumor cells

LIGHT is a tumor necrosis factor (TNF) ligand superfamily member, which binds two known cellular receptors, lymphotoxin-beta receptor (LTbetaR) and the herpesvirus entry mediator (HveA). LIGHT is a homotrimer that activates proapoptotic and integrin-inducing pathways. Receptor binding residues via LIGHT were identified by introducing point mutations in the A' --> A" and D --> E loops of LIGHT, which altered binding to LTbetaR and HveA. One mutant of LIGHT exhibits selective binding to HveA and is inactive triggering cell death in HT29.14s cells or induction of ICAM-1 in fibroblasts. Studies with HveA- or LTbetaR-specific antibodies further indicated that HveA does not contribute, either cooperatively or by direct signaling, to the death pathway activated by LIGHT. LTbetaR, not HveA, recruits TNF receptor-associated factor-3 (TRAF3), and LIGHT-induced death is blocked by a dominant negative TRAF3 mutant. Together, these results indicate that TRAF3 recruitment propagates death signals initiated by LIGHT-LTbetaR interaction and implicates a distinct biological role for LIGHT-HveA system.     

The effect of intermediate filament inhibitors on steroidogenesis and cytoskeleton in Y-1 mouse adrenal tumor cells

When Y-1 mouse adrenal tumor cells were treated with sodium orthovanadate, an agent disrupting BHK21-F cell microtubule-intermediate filament (IF) interactions, there was no change in the amount of 20-dihydroprogesterone produced. A neurofilament-microtubule perturbing agent, beta,beta-iminodipropionitrile (IDPN), enhanced the ability of Y-l cells to produce steroid in response to ACTH by acting on the plasma membrane. Electron microscopy of Y-l cells extracted with Triton X-100 revealed that both vanadate and IDPN caused the aggregation of granular structures in the perinuclear area. Based on the steroidogenic effects of IDPN, perinuclear granule aggregation may result from an altered interaction between intermediate filaments, microtubules and the plasma membrane. The reason for the ultrastructural changes caused by vanadate is unknown.     

The mouse glucocorticoid receptor DNA-binding domain is not phosphorylated in vivo

The glucocorticoid receptor is phosphorylated, but the precise location of the phosphorylated groups is unknown. We cultured AtT-20 cells in medium containing [32P]-orthophosphate and used immunoaffinity methods to isolate the intact receptor and a tryptic fragment containing the DNA binding domain. Analysis of the intact receptor, co-labeled with the affinity ligand dexamethasone-mesylate, confirmed that the receptor was phosphorylated. Isolation of the DNA binding domain by trypsinization and immunopurification showed that it was not phosphorylated. Interestingly, a non-immunoreactive phosphorylated fragment similar in size to the DNA-binding fragment was observed. Our results suggest that phosphorylation of the DNA binding domain of the glucocorticoid receptor is not essential for hormone action.