The hypocholesterolemic agent LY295427 reverses suppression of sterol regulatory element-binding protein processing mediated by oxysterols.

The sterol LY295427 reduces plasma cholesterol levels in animals by increasing the expression of hepatic low density lipoprotein (LDL) receptors. Here we trace the hypocholesterolemic activity of LY295427 to an ability to reverse oxysterol-mediated suppression of sterol regulatory element-binding protein (SREBP) processing. Micromolar concentrations of LY295427 induced the metabolism of LDL in oxysterol-treated cultured cells and inhibited the stimulation of cholesteryl ester synthesis mediated by oxysterols. cDNA microarray and RNA blotting experiments revealed that LY295427 increased levels of the LDL receptor mRNA and those of other SREBP target genes. The compound stimulated the accumulation of SREBPs in the nuclei of cells grown in the presence of oxysterols within 4-6 h of addition to the medium. Induction required components of the normal SREBP-processing pathway, including the SREBP cleavage-activating protein and the Site 1 protease. LY295427 overcame the suppression of SREBP processing mediated by several oxysterols but not by LDL-derived cholesterol. We conclude that LY295427 achieves a therapeutically desirable end point by an unique mechanism of action.

Inhibition of gene expression inside cells by peptide nucleic acids: effect of mRNA target sequence, mismatched bases, and PNA length.

Genome sequencing has revealed thousands of novel genes, placing renewed emphasis on chemical approaches for controlling gene expression. Antisense oligomers designed directly from the information generated by sequencing are one option for achieving this control. Here we explore the rules governing the inhibition of gene expression by peptide nucleic acids (PNAs) inside cells. PNAs are a DNA/RNA mimic in which the phosphate deoxyribose backbone has been replaced by uncharged linkages. Binding to complementary sequences is not hindered by electrostatic repulsion and is characterized by high rates of association and elevated affinities. Here we test the hypothesis that the favorable properties of PNAs offer advantages for recognition of mRNA and antisense inhibition of gene expression in vivo. We have targeted 27 PNAs to 18 different sites throughout the 5'-untranslated region (5'-UTR), start site, and coding regions of luciferase mRNA. PNAs were introduced into living cells in culture as PNA-DNA-lipid complexes, providing a convenient high throughput method for cellular delivery. We find that PNAs targeted to the terminus of the 5'-UTR are potent and sequence-specific antisense agents. PNAs fifteen to eighteen bases in length were optimal inhibitors. The introduction of one or two mismatches abolished inhibition, and complementary PNAs targeted to the sense strand were also inactive. In striking contrast to effective inhibition by PNAs directed to the terminal region, PNAs complementary to other sites within the 5'-UTR do not inhibit gene expression. We also observe no inhibition by PNAs complementary to the start site or rest of the coding region, nor do we detect inhibition by PNAs that are highly C/G rich and possess extremely high affinities for their target sequences. Our results suggest that PNAs can block binding of the translation machinery but are less able to block the progress of the ribosome along mRNA. The high specificity of antisense inhibition by PNAs emphasizes both the promise and the challenges for PNAs as antisense agents and provides general guidelines for using PNAs to probe the molecular recognition of biological targets inside cells.

Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta.

LXRalpha and -beta are nuclear receptors that regulate the metabolism of several important lipids, including cholesterol and bile acids. Previously, we have proposed that LXRs regulate these pathways through their interaction with specific, naturally occurring oxysterols, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Using a ligand binding assay that incorporates scintillation proximity technology to circumvent many of the problems associated with assaying extremely hydrophobic ligands, we now demonstrate that these oxysterols bind directly to LXRs at concentrations that occur in vivo. To characterize further the structural determinants required for potent LXR ligands, we synthesized and tested a series of related compounds for binding to LXRs and activation of transcription. These studies revealed that position-specific monooxidation of the sterol side chain is requisite for LXR high-affinity binding and activation. Enhanced binding and activation can also be achieved through the use of 24-oxo ligands that act as hydrogen bond acceptors in the side chain. In addition, introduction of an oxygen on the sterol B-ring results in a ligand with LXRalpha-subtype selectivity. These results support the hypothesis that naturally occurring oxysterols are physiological ligands for LXRs and show that a rational, structure-based approach can be used to design potent LXR ligands for pharmacologic use.

The LXRs: a new class of oxysterol receptors.

The liver X receptors (LXRs) are a family of transcription factors that were first identified as orphan members of the nuclear receptor superfamily. The identification of a specific class of oxidized derivatives of cholesterol as ligands for the LXRs has been crucial to helping understand the function of these receptors in vivo and first suggested their role in the regulation of lipid metabolism. Confirmation of this role has come from the recent analysis of LXR-deficient mice, which has demonstrated the essential function of one of these receptors in the liver as a major sensor of dietary cholesterol.

Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR alpha.

We demonstrate that mice lacking the oxysterol receptor, LXR alpha, lose their ability to respond normally to dietary cholesterol and are unable to tolerate any amount of cholesterol in excess of that which they synthesize de novo. When fed diets containing cholesterol, LXR alpha (-/-) mice fail to induce transcription of the gene encoding cholesterol 7alpha-hydroxylase (Cyp7a), the rate-limiting enzyme in bile acid synthesis. This defect is associated with a rapid accumulation of large amounts of cholesterol in the liver that eventually leads to impaired hepatic function. The regulation of several other crucial lipid metabolizing genes is also altered in LXR alpha (-/-) mice. These results demonstrate the existence of a physiologically significant feed-forward regulatory pathway for sterol metabolism and establish the role of LXR alpha as the major sensor of dietary cholesterol.

An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha.

Cholesterol and its oxysterol congeners are important constituents of cell membranes and function as intermediates in several crucial biosynthetic pathways. These compounds autoregulate their metabolic fate by end-product repression and activation of downstream catabolism. Although end-product repression by oxysterols is relatively well understood, the mechanism by which these compounds act as positive transcription signalling molecules is unknown. Here we identify a specific group of endogenous oxysterols that activate transcription through the nuclear receptor LXR alpha. Transactivation of LXR alpha by oxysterols occurs at concentrations at which these compounds exist in vivo. The most potent activators also serve as intermediary substrates in the rate-limiting steps of three important metabolic pathways: steroid hormone biosynthesis, bile acid synthesis, and conversion of lanosterol to cholesterol. Our results demonstrate the existence of a nuclear receptor signalling pathway for oxysterols and suggest that LXR alpha may be important as a sensor of cholesterol metabolites.

Trypsin display on the surface of bacteriophage.

The gene III and VIII-encoded coat proteins (pIII and pVIII) from bacteriophage M13 have been fused to the C terminus of the serine protease, trypsin (Tsn). The genes encoding the fusions were then inserted directly into M13mp18 to create vectors which expressed both the Tsn-coat protein hybrids and the wild-type (wt) coat proteins. Immunoblot analysis confirmed that the bacteriophage express Tsn on their surface. Isolated fusion phage possess kinetic parameters which approximate those of the wt enzyme. An endogenous Escherichia coli protease inhibitor, ecotin, copurifies with the Tsn phage. Immobilized ecotin can be used to selectively bind bacteriophage which express Tsn::pIII fusion proteins.

Hypothalamic regulation of growth hormone secretion during food deprivation in the rat.

Suppressed pulsatile GH secretion in food-deprived rats has been hypothesized to be due to an increase in hypothalamic somatostatin secretion. We investigated this hypothesis and the role of GHRH in regulating GH secretion during food deprivation using two different models. In experiment one, rats were food deprived for 72h during which time they received a saline infusion (n = 5). At the same time rats were normal fed for 72h during which time they received a somatostatin infusion (5 micrograms/h, n = 7). After the 72h infusion period, all rats received two iv injections of GHRH (1 microgram/rat) at 2h intervals. GH concentrations in food-deprived rats rose from approximately 10 ng/ml to 400-800 ng/ml in response to both GHRH injections. This increase was significantly greater (p < 0.01) than the GH response (100-400 ng/ml) observed in somatostatin-infused animals. The significantly higher GH response observed in food-deprived rats as compared to somatostatin-infused, normal-fed rats suggests that somatostatin concentrations may decrease during food deprivation. In experiment two, rats were infused for 5h with either saline (n = 6) or GHRH (10 micrograms/h, n = 9) at the end of a 72h fast. GH concentrations did not change in saline-infused animals. In contrast, GH concentrations significantly increased (p < 0.01) upon initiation of the continuous GHRH infusion. Yet, this release of GH was pulsatile in nature. Pulsatile GH secretion in the presence of a constant GHRH infusion suggests that pulsatile somatostatin release from the hypothalamus is maintained during food deprivation. These studies suggest that during food deprivation in the rat 1) absolute concentrations of somatostatin decrease, but its pattern of secretion remains pulsatile, and 2) decreased GHRH release may be responsible for the absence of spontaneous GH pulses.

Effects of passive immunization of growth hormone-releasing hormone and somatostatin on growth hormone secretion under conditions of high somatostatin tone.

Pulsatile GH secretion decreases during food-deprivation in the rat. It has been hypothesized that this decrease is due to elevated hypothalamic somatostatin secretion. This is based on the observation that GH increases in food-deprived rats following removal of endogenous somatostatin using passive immunization techniques. Cognizant of the important stimulatory effects of growth hormone-releasing hormone (GHRH) on GH secretion, we sought to determine if this neuropeptide plays any role in mediating GH secretion in food-deprived rats. Male rats were prepared with indwelling venous catheters using sodium pentobarbital anesthesia seven days prior to experimentation. Animals were food-deprived for 72 h, after which control blood samples were drawn from -60 to 0 min. One group was then treated with normal rabbit serum (NRS), while a second group was treated with GHRH antiserum (GHRHab). At 55 min all animals received somatostatin antiserum (SSab). No animal exhibited any spontaneous GH peak during the one hour control period or in the subsequent one hour period following the administration of GHRHab or NRS. Absence of GH pulsatility during food-deprivation, coupled with no decrease in GH levels in food-deprived rats treated with GHRHab suggest that diminished GHRH pulsatility is likely during food-deprivation. Subsequent treatment of these animals with SSab resulted in an identical 2.5 fold increase in GH concentrations. This result suggests that GHRH is not involved in the GH rebound following somatostatin withdrawal in food-deprived rats.

Glucocorticoids: potent inhibitors and stimulators of growth hormone secretion.

Excessive glucocorticoid concentrations are well recognized inhibitors of linear growth, due in part to their suppression of GH secretion. The mechanism of this inhibition has been unclear, especially since glucocorticoids enhance the in vitro GH response of pituitary cells to GH-releasing hormone (GHRH). We investigated the possibility that hypothalamic somatostatin might be mediating these dichotomous observations by using passive immunization techniques. The GH response to GHRH was significantly blunted in rats pretreated with the synthetic glucocorticoid, dexamethasone, compared to that in normal animals. In marked contrast, the immunoneutralization of somatostatin resulted in a significantly enhanced GH response to GHRH in dexamethasone-treated animals. These results suggest that the previously described inhibitory action of glucocorticoids on GH secretion in vivo are mediated via altered hypothalamic somatostatin tone.