26-Hydroxycholesterol: synthesis, metabolism, and biologic activities

Cholest-5-ene-3 beta,26-diol (26-hydroxycholesterol) is synthesized by a mitochondrial P-450 enzyme that appears to be widely distributed in tissues. Together with other C-27 steroid intermediates it is transported to the liver and metabolized to bile acids. Although 26-hydroxycholesterol is transported in plasma lipoproteins mostly as the fatty acid ester, neither its assembly and orientation within lipoproteins nor its mechanism of transport across the sinusoidal liver membrane is known. Cell culture studies indicate that 26-hydroxycholesterol can inhibit both cholesterol synthesis and low density lipoprotein (LDL) receptor activity. Inhibition of DNA synthesis also occurs and may not be related to the reduction in HMG-CoA reductase activity. The relationship of these in vitro activities to the physiologic role(s) of 26-hydroxycholesterol remains to be clarified. A clue to its biologic role is the knowledge that markedly decreased 26-hydroxylase activity appears to be the molecular basis of cerebrotendinous xanthomatosis, an inborn error of metabolism characterized by a significant decrease in 26-hydroxycholesterol and bile acid synthesis and an increase in cholesterol synthesis.     

26-Hydroxycholesterol disulfate: metabolism and excretion in the normal neonate

Deuterated 26-hydroxycholesterol disulfate has been given in a tracer amount to neonates to evaluate the pool size, metabolism and excretion of the endogenously occurring compound in meconium. In a group of 5 normal neonates excretion of endogenous 26-hydroxycholesterol during the initial 72 h of life ranged from 327 to 1096 micrograms. Recovery of administered isotope during the same period was 66-98%. Only trace amounts of 26-hydroxycholesterol were recovered in urine. The findings indicate that relative to bile acid pool size, the normal neonate has a small intestinal pool of 26-hydroxycholesterol which, for the most part, is rapidly excreted and does not contribute significantly to bile acid synthesis. The techniques developed during the course of the study provide an approach to non invasive metabolic studies that give insights on the normal transition from fetal to neonatal life.     

25-Hydroxycholesterol and inflammation in Lovastatin-deregulated mevalonate pathway

Mevalonate pathway deregulation has been observed in several diseases, including Mevalonate kinase deficiency (MKD). MKD is a hereditary auto-inflammatory disorder, due to mutations at mevalonate kinase gene (MVK), encoding mevalonate kinase (MK) enzyme. MVK mutations have been reported as associated with impairment of mevalonate pathway with consequent decrease of protein prenylation levels, defective autophagy and increase of IL-1β secretion, followed by cell death. Since 25-hydroxycholesterol (25-HC), a metabolite of cholesterol, can suppress IL-1β production, thus reducing inflammation, we evaluated the effect of 25-HC in an in vitro model of mevalonate pathway alteration, obtained using Lovastatin. Human glioblastoma cell line (U87-MG) was chosen to mimic, at least in part, the central nervous system impairment observed in MKD; 25-HC effects were evaluated aimed at disclosing if this compound could be considered as novel potential drug for MKD.Our results showed that 25-HC is able to reduce inflammation but it is ineffective to restore autophagy flux and to decrease apoptosis levels, both caused by lower protein prenylation; so, in spite of its anti-inflammatory action it is not useful to rescue defective prenylation/autophagy impairment-driven apoptosis in Lovastatin impaired mevalonate pathway.We hypothesize the presence in the mevalonate pathway of alternative mechanisms acting between inflammation and apoptotic autophagy impairment.     

C-25 epimeric 26-haloponasterone A: synthesis, absolute configuration and moulting activity

A convenient synthesis of inokosterone has been accomplished. Inokosterone exists as two C-25 epimers, which could be separated from each other through their diacetonide derivatives. The absolute configuration of these compounds was determined. Two C-25 epimers of 26-chloroponasterone A were synthesized from the respective C-25 epimeric inokosterone. Two epimeric 26-bromo and 26-iodoponasterone A compounds were also synthesized. Moulting activity of these compounds was evaluated using the Musca bioassay, and it was found that the (25S)-26-halo analogues were more active than the corresponding (25R)-26-halo analogues. Among the 25S series, an increase in activity with an increase in size of the halogen atom was observed, indicating that the steric factor was more important than the electronic factor in binding of these ecdysteroid analogues to the receptor. On the other hand, a decrease in activity with an increase in size of the halogen atom was noted in the 25R series, suggesting that the steric factor was less important than the electronic factor. The results indicated that the configuration at C-25 and the substituent at C-26 have significant influences on the interaction of ecdysteroids with their receptor.     

Synthesis of (25R)-26-hydroxycholesterol

We describe the synthesis of (25R)-cholest-5-en-3beta,26-diol ((25R)-26-hydroxycholesterol) from diosgenin in four steps in 58% overall, yield via a modified Clemmensen reduction followed by a Barton deoxygenation reaction.     

Cholesterol metabolism. Effect of 26-thiacholesterol and 26-aminocholesterol, analogues of 26-hydroxycholesterol, on cholesterol synthesis and low-density-lipoprotein-receptor binding.

1. The effects of 26-aminocholesterol and 26-thiacholesterol on cholesterol synthesis and LDL (low-density lipoprotein)-receptor activity were compared with naturally occurring 26-hydroxycholesterol utilizing both human fibroblasts and hepatoma (Hep G2) cells. 2. At equimolar concentrations (0.625 microM), down-regulation of LDL-receptor activity and cholesterol synthesis was greater with human fibroblasts than with Hep G2 cells. 3. At much higher concentrations (5-20 microM) the 26-thia analogue had little effect on either cholesterol synthesis or LDL-receptor activity.     

Intestinal calcium-binding protein (CaBP) and bone calcium mobilization in response to 25R,26 AND 25S,26-dihydroxycholecalciferol in intact and nephrectomized rats

Since intestinal calcium-binding protein (CaBP) can he regarded as an expression of the hormone-like action of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on the duodenal enterocyte we have investigated the potential biological activity of 25R and 25S,26-(OH)₂D₃ (two recently synthesized epimers of vitamin D₃ metabolite) to promote intestinal CaBP production as compared to bone calcium mobilization in vitamin D and calcium-deficient rats. In our assay steroids exhibited a 72 hour calcemic response. Our results show a linear relationship between CaBP synthesis and the logarithm of the dose (130–2080 pmol dose range) of either 25R or 25S epimer. The CaBP response was comparable for both epimers. Similarly bone calcium mobilization response was dose related as a linear function of the logarithm of the administered dose. Again, calcemic response was comparable for both epimers. In our model these two epimers were about as active on intestine to increase CaBP amount as on bone to elevate serum calcium level. Bilateral nephrectomy abolished CaBP response to a large dose (1040 pmol) of either 25R or 25S epimer but did not abolish it to a 130 pmol dose of 1α, 25-(OH)₂D₃.     

Synthesis of (25R)-26-hydroxy-15-ketosterols

(25R)-3beta,26-Dihydroxy-5alpha-cholest-8(14)-en-15-one (1) and (25R)-3beta,26-dihydroxy-5alpha,14beta-cholest-16-en-1 5-one (2) were synthesized from (25R)-3beta,26-dibenzoyloxy-5alpha,14alpha-chole st-16-ene (4). Oxidation of 4 with CrO3-3,5-dimethylpyrazole at -20 degrees C gave (25R)-3beta,26-dibenzoyloxy-5alpha,14alpha-chole st-16-en-15-one (5) along with (25R)-3beta,26-dibenzoyloxy-5alpha-cholest-16alpha+ ++,17alpha-epoxide (6). Oxidation of 5 with selenium dioxide afforded (25R)-3beta,26-dibenzoyloxy-5alpha-cholest-8(14),16-++ +dien-15-one (7) and (25R)-3beta,26-dibenzoyloxy-5alpha,14beta-choles t-16-en-15-one (8). Selective hydrogenation of 7 followed by hydrolysis in alcoholic potassium hydroxide yielded (25R)-3beta,26-dihydroxy-5alpha-cholest-8(14)-en-15-one (1). Hydrolysis of 5 and 8 in alcoholic potassium hydroxide provided (25R)-3beta,26-dihydroxy-5alpha,14beta-cholest-16-en-1 5-one (2).     

26-Hydroxycholesterol and cholest-4-en-3-one,the first metabolites of cholesterol in potato plants

Etiolated potato sprouts convert administered cholesterol-4-¹⁴C to radioactive 26-hydroxycholesterol and cholest-4-en-3-one. These two steroids must be the first products of cholesterol metabolism in potato plants.     

On sperm competition games: raffles and roles revisited

 In principle there are two approaches to modelling a trade-off between the positive and negative outcomes of a behavior: after suitably defining a value for the behavior in the absence of any trade-off, one can either multiply that value by an appropriate discount or subtract an appropriate cost. In a prospective analysis of sperm competition, Parker (Proc. Roy. Soc. Lond. B (1990) 242, 120–126) adopted the multiplicative approach to model the trade-off between the value of a mating and the cost of its acquisition. He obtained two paradoxical results. First, if two males ‘know’ whether they are first or second to mate, but these roles are assigned randomly, then sperm numbers should be the same for both males whether the ‘raffle’ for fertilization is fair or unfair. Second, if mating order is constant, then a favored male should expend less on sperm. His results are puzzling not only in terms of intuition about nature, but also in terms of his model’s consistency. In other words, they present both an external and an internal paradox. Parker assumed the fairness of the raffle to a disfavored male to be independent of how much sperm a favored male deposits. This article both generalizes Parker’s analysis by allowing fairness to decrease with sperm expenditure by the favored male and compares Parker’s results to those obtained by the additive approach. In many respects, results are similar. Nevertheless, if the costs of mating are assumed to increase with sperm expenditure but not to depend on the role in which sperm is expended, as Parker assumed, then the additive approach is more fundamentally correct. In particular, Parker’s constant-role paradox is an artifact of his approach. His random-role paradox is internally rationalized in terms of standard microeconomic theory. When fairness decreases, however slightly, with sperm expenditure by the favored male, both models demonstrate that the evolutionarily stable strategy is for more sperm to be deposited during a favored mating than during a disfavored mating. The lower the costs, the greater the divergence. Thus a possible resolution of the external paradox is that fairness is not constant in nature. Received: 7 December 1998     

Modulation of Antiviral Immunity and Therapeutic Efficacy by 25-Hydroxycholesterol in Chronically SIV-Infected,ART-Treated Rhesus Macaques

Virologica Sinica - Cholesterol-25-hydroxylase (CH25H) and its enzymatic product 25-hydroxycholesterol (25HC) exert broadly antiviral activity including inhibiting HIV-1 infection. However, their...     

RhoGDIs revisited: novel roles in Rho regulation

Small GTP-binding proteins of the Rho/Rac/Cdc42 family combine their GDP/GTP cycle, regulated by guanine nucleotide-exchange factors and GTPase-activating proteins, to a cytosol/membrane cycle, regulated by guanine nucleotide dissociation inhibitors (rhoGDIs). RhoGDIs are endowed with dual functions in the cytosol where they form soluble complexes with geranylgeranylated GDP-bound Rho proteins and at membrane interfaces where they monitor the delivery and extraction of Rho proteins to/from their site of action. They have little diversity compared with other Rho protein regulators and therefore have been regarded mostly as housekeeping regulators that distribute Rho proteins equally to any membranes. Recently, acquired data show that rhoGDIs, by interacting with candidate receptors/displacement factors or by phosphorylation, may in fact have active contributions to targeting Rho proteins to specific subcellular membranes and signaling pathways. In addition, the GDP/GTP and membrane/cytosol cycles can be uncoupled in certain cases, with Rho proteins either escaping the membrane/cytosol cycle or being regulated by rhoGDIs in their GTP-bound form. Here, we survey recent structure-function relationships and cellular studies on rhoGDIs and revisit their classical housekeeping role into novel and more specific functions. We also review their involvement in diseases.     

Oxysterols: novel biologic roles for the 21st century

A major focus for the 21st century are the sterol intermediates in cholesterol synthesis and their metabolites. No longer considered inactive way stations in their transformation to cholesterol, both physiologic and pathophysiologic studies, though early in their development, indicate novel biologic roles for these sterols, and their oxysterol metabolites that bypass cholesterol, the expected end product. A major impetus for further inquiry is the recognition that in genetically determined errors in cholesterol synthesis such as Smith-Lemil-Opitz syndrome, the phenotypic effects on the developing fetus are not solely attributable to the lack of cholesterol but the accumulation of 7-dehydrocholesterol and its 27-hydroxy metabolite. This view is now supported by a new mouse model, the double knockout Insig1 & 2 (insulin-induced genes 1 & 2) in which lack of the protein product results in a greater production of lanosterol compared to cholesterol during fetal life with severe dysmorphic consequences. Further support can be derived from in vitro studies of the Sonic hedgehog signaling pathway, essential for normal morphogenesis in the central nervous system and perhaps other organs, which may require the local presence of oxysterols for full expression. Future studies that can delineate the specific role of a sterol intermediate or its metabolite require a paradigm shift away from the generic use of oxysterols as a class of compounds to a focus on specific sterols that can be expected in tissues and techniques for mimicking the local environment. Another class of oxysterols are those arising by photoxidation, now considered to be an expected event generated by the photons of visible blue light and therefore pari passu with normal vision. The sequence of events from peroxides of cholesterol to hydroxy and keto derivatives is the signature of singlet oxygen as opposed to free radicals and other mechanisms for generating reactive oxygen species. Perhaps surprisingly, the retina expresses CYP 27A1 and CYP 46A1, enzymes with broad substrate specificity for ring-modified sterols, implying that, in addition to a rich blood supply for disposing of potentially toxic oxysterols, they can be detoxified locally. Recognition that the retina has nuclear receptors similar to those found in other tissues raises the possibility that the sterols that are generated may function in their traditional role as ligands for modulating gene expression but other, nonligand, activities can be expected since other proteins such as the oxysterol-binding proteins exist and are considered to have biologic activities. To critically evaluate these potentially new biologic roles for oxysterols a need exists for the synthesis and utilization of the expected naturally occurring metabolites rather than available surrogates that may not be truly representative of their tissue effects and to utilize analytical techniques that can identify their existence at the expected concentrations in tissues.     

Epidermal sphingolipids: metabolism, function, and roles in skin disorders

Mammalian epidermis produces and delivers large quantities of glucosylceramide and sphingomyelin precursors to stratum corneum extracellular domains, where they are hydrolyzed to corresponding ceramide species. This cycle of lipid precursor formation and subsequent hydrolysis represents a mechanism that protects the epidermis against potentially harmful effects of ceramide accumulation within nucleated cell layers. Prominent skin disorders, such as psoriasis and atopic dermatitis, have diminished epidermal ceramide levels, reflecting altered sphingolipid metabolism, that may contribute to disease severity/progression. Enzymatic processes in the hydrolysis of glucosylceramide and sphingomyelin, and the roles of sphingolipids in skin diseases, are the focus of this review.     

Differing roles of Akt and serum- and glucocorticoid-regulated kinase in glucose metabolism,DNA synthesis,and oncogenic activity

Serum- and glucocorticoid-regulated kinase (SGK) is a serine kinase that has a catalytic domain homologous to that of Akt, but lacks the pleckstrin homology domain present in Akt. Akt reportedly plays a key role in various cellular actions, including glucose transport, glycogen synthesis, DNA synthesis, anti-apoptotic activity, and cell proliferation. In this study, we attempted to reveal the different roles of SGK and Akt by overexpressing active mutants of Akt and SGK. We found that adenovirus-mediated overexpression of myristoylated (myr-) forms of Akt resulted in high glucose transport activity in 3T3-L1 adipocytes, phosphorylated glycogen synthase kinase-3 (GSK3) and enhanced glycogen synthase activity in hepatocytes, and the promotion of DNA synthesis in interleukin-3-dependent 32D cells. In addition, stable transfection of myr-Akt in NIH3T3 cells induced an oncogenic transformation in soft agar assays. The active mutant of SGK (D-SGK, substitution of Ser422 with Asp) and myr-SGK were shown to phosphorylate GSK3 and to enhance glycogen synthase activity in hepatocytes in a manner very similar to that observed for myr-Akt. However, despite the comparable degree of GSK3 phosphorylation between myr-Akt and d-SGK or myr-SGK, d-SGK and myr-SGK failed to enhance glucose transport activity in 3T3-L1 cells, DNA synthesis in 32D cells, and oncogenic transformation in NIH3T3 cells. Therefore, the different roles of SGK and Akt cannot be attributed to ability or inability to translocate to the membrane thorough the pleckstrin homology domain, but rather must be attributable to differences in the relatively narrow substrate specificities of these kinases. In addition, our observations strongly suggest that phosphorylation of GSK3 is either not involved in or not sufficient for GLUT4 translocation, DNA synthesis, or oncogenic transformation. Thus, the identification of substrates selectively phosphorylated by Akt, but by not SGK, may provide clues to clarifying the pathway leading from Akt activation to these cellular activities.     

Essential,deadly, enigmatic: Polyamine metabolism and roles in fungal cells

Polyamines are essential metabolites found in all organisms. Intracellular polyamine levels are tightly maintained by biosynthesis, degradation, uptake and excretion processes that involve regulatory mechanisms – such as the antizyme inhibitory protein – that are conserved across the kingdoms of life, indicating that polyamine levels are critical to cell function. Nonetheless, the biochemical roles of polyamines and their involvement in numerous fundamental cellular processes including aging, cell cycle progression and growth only become apparent when polyamine homeostasis is perturbed. Thus, while polyamines are present in most cells and essential for cell growth, their biochemical functions are largely enigmatic. Studies in fungi have contributed to our basic understanding of polyamines, and might continue to bridge knowledge gaps regarding polyamine metabolism and cell function. Moreover, when considering the impact of fungi – directly or indirectly, for good or for ill – on human society, closing gaps in our understanding of polyamine functions in fungal physiology is an important goal in itself that might lead to the discovery of new targets for enhancing beneficial fungal interactions and diminishing those detrimental to crop and human health. To facilitate progress towards this prospect, here we appraise what is known about polyamine metabolism in fungi, how prevalent polyamines impact fungal physiology and metabolism, and how the levels of each polyamine are maintained in the fungal cell – thus pointing to how they might be perturbed.     

Clemmensen reduction of diosgenin and kryptogenin: synthesis of [16,16,22,22,23,23-(2)H(6)]-(25R)-26-hydroxycholesterol

A new experimental protocol has been established for the Clemmensen reduction of diosgenin and kryptogenin with the aim to prepare deuterated isotopomers of (25R)-26-hydroxycholesterol. Uncontrolled deuteration has been achieved from diosgenin, whereas [16,16,22,22,23,23-(2)H(6)]-(25R)-26-hydroxycholesterol (1) can be synthesized from kryptogenin.