BACKGROUND. Mirabegron is a β3-adrenergic receptor (β3-AR) agonist approved only for the treatmen... more BACKGROUND. Mirabegron is a β3-adrenergic receptor (β3-AR) agonist approved only for the treatment of overactive bladder. Encouraging preclinical results suggest that β3-AR agonists could also improve obesity-related metabolic disease by increasing brown adipose tissue (BAT) thermogenesis, white adipose tissue (WAT) lipolysis, and insulin sensitivity. METHODS. We treated 14 healthy women of diverse ethnicities (27.5 ± 1.1 years of age, BMI of 25.4 ± 1.2 kg/m 2) with 100 mg mirabegron (Myrbetriq extended-release tablet, Astellas Pharma) for 4 weeks in an open-label study. The primary endpoint was the change in BAT metabolic activity as measured by [ 18 F]-2-fluoro-d-2-deoxy-d-glucose (18 F-FDG) PET/CT. Secondary endpoints included resting energy expenditure (REE), plasma metabolites, and glucose and insulin metabolism as assessed by a frequently sampled intravenous glucose tolerance test. RESULTS. Chronic mirabegron therapy increased BAT metabolic activity. Whole-body REE was higher, without changes in body weight or composition. Additionally, there were elevations in plasma levels of the beneficial lipoprotein biomarkers HDL and ApoA1, as well as total bile acids. Adiponectin, a WAT-derived hormone that has antidiabetic and antiinflammatory capabilities, increased with acute treatment and was 35% higher upon completion of the study. Finally, an intravenous glucose tolerance test revealed higher insulin sensitivity, glucose effectiveness, and insulin secretion. CONCLUSION. These findings indicate that human BAT metabolic activity can be increased after chronic pharmacological stimulation with mirabegron and support the investigation of β3-AR agonists as a treatment for metabolic disease. TRIAL REGISTRATION. Clinicaltrials.gov NCT03049462.
Significant ambiguity exists in the scientific community with regard to the nomenclature of 26-hy... more Significant ambiguity exists in the scientific community with regard to the nomenclature of 26-hydroxylated oxysterols. Oxysterols constitute an important class of compounds that have biological roles in the regulation of cholesterol synthesis and as endogenous selective estrogen receptor modulators (SERMs). The ambiguity is attributable to deviations from clearly stated IUPAC rules and is likely to increase as more biologically active oxysterols are identified. This review provides a uniform approach to the naming of 26-hydroxylated sterols for those of current interest and for those on the horizon such as oxysterols of lanosterol that retain the unsaturation at C-24 and C-25 such as (E)-26-hydroxylanosterol. Using this molecule as a starting point, this review hopes to establish a common language to keep all investigators on the same page.
In 1959, Ivar Sperber contrasted bile formation with that of urine and proposed that water flow i... more In 1959, Ivar Sperber contrasted bile formation with that of urine and proposed that water flow into the canalicular conduit is in response to an osmotic not a hydrostatic gradient. Early attempts to support the hypothesis using a bile acid, sodium taurocholate, and the hormone secretin to stimulate bile flow led to conflicting data and a moratorium on attempts to further develop the initial proposal. However, current data amplify the initial proposal and indicate both paracellular and transcellular water flow into hepatic ductules and the canalicular conduit in response to an osmotic gradient. Also, the need to further modify the initial proposal became apparent with the recognition that bile acid aggregates (micelles) which form in the canalicular conduit generate lecithin-cholesterol vesicles that contain water unrelated to an osmotic
Phosphorylation of translation initiation factor 2a (eIF2a) coordinates a translational and trans... more Phosphorylation of translation initiation factor 2a (eIF2a) coordinates a translational and transcriptional program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. A screen for small molecule activators of the ISR identified two related compounds that also activated sterol-regulated genes by blocking cholesterol biosynthesis at the level of CYP51. Ketoconazole, a known CYP51 inhibitor, had similar effects, establishing that perturbed flux of precursors to cholesterol activates the ISR. Surprisingly, compound-mediated activation of sterol-regulated genes was enhanced in cells with an ISR-blocking mutation in the regulatory phosphorylation site of eIF2a. Furthermore, induction of the ISR by an artificial drug-activated eIF2a kinase reduced the level of active sterol regulatory element binding protein (SREBP) and sterol-regulated mRNAs. These findings suggest a mechanism by which interactions between sterol metabolism, the ISR, and the SREBP pathway affect lipid metabolism during ER stress.
Although it is recognized that more biomarkers are needed to evaluate the progression of liver di... more Although it is recognized that more biomarkers are needed to evaluate the progression of liver disease and response to medications, plasma alkaline phosphatase and conjugated bilirubin are a focus of clinical trials and utilized by physicians in practice. Conjugated bilirubin is a surrogate marker for the status of bile acid transport and the lowering of plasma levels in response to administration of ursodeoxycholic acid indicates that a choleretic effect has occurred. Plasma alkaline phosphatase levels are affected by hepatic bile acid composition and flow and the status of the cholangioles. Medications that reduce endogenous bile acid pool size can augment the effect of ursodeoxycholic acid by expanding its proportion in those undergoing enterohepatic circulation. In addition, they can lower the elevated steady state concentration of hepatocellular bile acids and retard progression to cirrhosis.
Journal of the American College of Cardiology, 1995
Values are presented as the mean ± SD of at least three different experiments. Asterisks represen... more Values are presented as the mean ± SD of at least three different experiments. Asterisks represent a p < 0.05 of the effect of carnitine vs control. These data show that L-carnitine stimulates PDH activity by 50%. However, the flux of acetyl-CoA and glucose through Kreb's cycle was significantly decreased by this compound. These results suggest that L-carnitine enhances the removal of acetyl-CoA produced from glucose metabolism out of the mitochondrial matriX, thereby, activating the PDH complex.
Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ra... more Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7a/ 7b-hydroxycholesterol and 5a,6a/5b,6b-epoxycholesterol as well as the 7a-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds.
Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects... more Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects of oxysterols on the expression of steroidogenic acute regulatory protein (StAR), which increases the delivery of cholesterol to sterol-metabolizing P450s in the mitochondria. 22(R)-hydroxycholesterol (22(R)-OHC), 25-OHC, and 27-OHC each increased steroidogenic factor-1 (SF-1)-mediated StAR gene transactivation by ϳ2fold in CV-1 cells. In contrast, cholesterol, progesterone, and the 27-OHC metabolites, 27-OHC-5-3-one and 7␣,27-OHC, had no effect. Unlike our findings in CV-1 cells, SF-1-dependent StAR promoter activity was not augmented by 27-OHC in COS-1 cells, Y-1 cells, BeWo choriocarcinoma cells, Chinese hamster ovary (CHO) cells, and human granulosa cells. Studies examining the metabolism of 27-OHC indicated that CV-1 cells formed a single polar metabolite, 3-OH-5-cholestenoic acid from radiolabeled 27-OHC. However, this metabolite inhibited StAR promoter activity in CV-1, COS-1 and CHO cells. Because 7␣,27-OHC was unable to increase SF-1dependent StAR promoter activity, we examined 27-OHC 7␣-hydroxylase in COS-1 and CHO cells. COS-1 cells contained high 7␣-hydroxylase activity, whereas the enzyme was undetectable in CHO cells. The hypothesis that oxysterols act in CV-1 cells to increase StAR promoter activity by reducing nuclear levels of sterol regulatory element binding protein was tested. This notion was refuted when it was discovered that sterol regulatory element binding protein-1a is a potent activator of the StAR promoter in CV-1, COS-1, and human granulosa cells. Human granulosa and theca cells, which express endogenous SF-1, contained more than 5-fold more StAR protein following addition of 27-OHC, whereas StAR mRNA levels remained unchanged. We conclude that 1) there are cell-specific effects of oxysterols on SF-1-dependent transactivation; 2) the ability to increase transactivation is limited to certain oxysterols; 3) there are cell-specific pathways of oxysterol metabolism; and 4) oxysterols elevate StAR protein levels through posttranscriptional actions.
Steroidogenic acute regulatory protein (StAR) plays a key role in steroid hormone synthesis by en... more Steroidogenic acute regulatory protein (StAR) plays a key role in steroid hormone synthesis by enhancing the metabolism of cholesterol into pregnenolone. We determined the organization of the StAR structural gene, mapped to 8~1 1. 2. The gene spans 8 kb and consists of seven exons interrupted by six introns. The 1.3 kb of DNA upstream from the transcription start site directed expression of a luciferase reporter gene in mouse Y-1 adrenal cortical tumor cells but not in BeWo choriocarcinoma cells. Reporter gene expression in the Y-1 cells was increased more than 2-fold by 8-Br-cAMP, indicating that the 1.3 kb DNA fragment contains sequences that confer tissue-specific expression and CAMP regulation. The sequence of a related StAR pseudogene, mapped to chromosome 13, lacks introns and has an insertion, numerous substitutions, and deletions. Expression of StAR in COS-1 cells cotransfected with cholesterol 27-hydroxylase (P450c27) and adrenodoxin resulted in a 6-fold increase in formation of 3P-hydroxy-5-cholestenoic acid, demonstrating that StAR's actions are not specific to steroidogenesis but extend to other mitochondrial cholesterol-metabolizing enzymes. The rate-limiting step in steroid hormone synthesis is the formation of pregnenolone from cholesterol, catalyzed by the cholesterol side-chain cleavage enzyme (P45Oscc), which resides with its associated electron transport chain in the inner mitochondrial membranes [for review see Miller (1988)l. This first committed reaction in the biosynthesis of steroid hormones is acutely stimulated by tropic hormones (ACTH in the adrenal cortex; LH in the gonads) acting through the intermediacy of CAMP. It has been known for two decades that the acute steroidogenic response to tropic stimulation involves the translocation of cholesterol from the outer to inner mitochondrial membranes. This translocation process is believed to be mediated by a short-lived, cycloheximidesensitive protein (
1. Using a human hepatoma (Hep G2) cell line that continually synthesizes 3 beta-hydroxy-5-cholen... more 1. Using a human hepatoma (Hep G2) cell line that continually synthesizes 3 beta-hydroxy-5-cholenoic acid, lithocholic acid, chenodeoxycholic acid and cholic acid we have determined the metabolism and biological effects of 26-hydroxycholesterol and 7 alpha-hydroxycholesterol. 2. Addition of 26-hydroxycholesterol to the medium (6 microM) downregulated cholesterol and chenodeoxycholic acid synthesis. 3. The predominant metabolite of 26-hydroxycholesterol was 3 beta-hydroxy-5-cholenoic acid. 4. Cholesterol synthesis was not affected by the addition of 7 alpha-hydroxycholesterol (6 and 12 microM). The predominant metabolite of 7 alpha-hydroxycholesterol was chenodeoxycholic acid. 5. In Hep G2 cells 7 alpha-hydroxylation of 26-hydroxycholesterol is not well expressed.
Smith-Lemli-Opitz syndrome (SLOS) is attributable to mutations in the gene coding for 7-dehydroch... more Smith-Lemli-Opitz syndrome (SLOS) is attributable to mutations in the gene coding for 7-dehydrocholesterol reductase. Low to absent enzyme activity accounts for the accumulation of both 7-dehydrocholesterol and 8-dehydrocholesterol in plasma and other tissues. Since oxysterols can participate in the regulation of cholesterol homeostasis, we examined the possibility that they are formed from these dehydrocholesterol intermediates. In patients with SLOS, we found serum levels of 27-hydroxy-7-dehydrocholesterol ranging from 0.1 to 0.25 M and evidence for circulating levels of 27-hydroxy-8-dehydrocholesterol (0.04-0.51 M). Picomolar quantities of 27-hydroxy-7-dehydrocholesterol were identified in normal individuals. Biologic activities of 27-hydroxy-7-dehydrocholesterol were found to include inhibition of sterol synthesis and the activation of nuclear receptor LXR␣ but not that of LXR. These activities occurred at concentrations found in plasma and presumably at those existing in tissues. Thus, patients with SLOS have increased levels of metabolites derived from intermediates in cholesterol synthesis that are biologically active and may contribute to the regulation of cholesterol synthesis in vivo.
excretion of coproporphyrin isomers I and III was studied in the rat. Both isomers were found to ... more excretion of coproporphyrin isomers I and III was studied in the rat. Both isomers were found to bind equally to rat plasma and liver cytosol in vitro and to disappear from plasma at equal rates after single injections in vivo. During equimolar infusions of isomers into bile fistula animals, both the I and III isomers were excreted in bile in a concentration ratio of 2: 1, respectively. Pretreatment of animals with ethinylestradiol or simultaneous infusions of phenoldibromophthalein disulfonate caused a reduction in total hepatic excretion with no change in the 2: 1 ratio in bile. As hepatic excretion fell, excretion of both isomers in urine rose, with an increase in the proportion of the I isomer. The findings mimic those reported to occur in man and can be explained by inhibition of a common carrier which requires a stereospecific configuration that statistically favors the hepatic transport of the symmetrical coproporphyrin I isomer.
After a number of years of being considered a well-established branch of biochemistry, we now hav... more After a number of years of being considered a well-established branch of biochemistry, we now have found that there is considerable less certainty about the pathways of bile acid synthesis from cholesterol, particularly in regard to human metabolism. Perhaps the major event that has occurred as part of this renaissance is the recognition that the initiation of bile acid synthesis from cholesterol can occur either by 7a-hydroxylation or by 26-hydroxylation (Anderson et al. 1972). Because there is some indication that the oxidation of the side chain of cholesterol can also occur in nonhepatic tissues, I will refer to it as the extrinsic pathway. It is the pathway for side-chain oxidation of C-27 sterols that is providing new perceptions with regard to the specific role of subcellular organelles and appears to generate intermediates that can have a regulatory role in the synthesis of cholesterol. The classical or traditional pathway of bile acid synthesis begins in the liver with cholesterol 7a-hydroxylase (Bjorkhem 1985), a microsomal P450 enzyme, and is referred to as the intrinsic pathway. There is considerable evidence in animal species that it is the rate-limiting enzyme, although it has neither been sequenced nor cloned and evidence derived from studies of liver cells in culture indicates that it is not regulated directly by bile acids (DaviS et al. 1983). The major evidence supporting its role as the rate-limiting enzyme is derived from the knowledge that cholestyrarnine treatment which induces bile acids synthesis certainly increases the activity of cholesterol 7 a-hydroxylase and if one couples this information to the more limited evidence that other enzymes in the intrinsic pathway have a higher rate of activity in the basal state which are not induced by biliary drainage, then the concept that 7a-hydroxylase is rate-limiting is reasonably sound. The enzyme is thought to have a relatively short half-life of 2-3 h and there is some evidence that the activity can be increased, at least under certain circumstances, by an increase in the cell cholesterol concentration. In addition to the relatively short half-life that provides rapid modulation in activity, experimental evidence exists for a phosphorylation-dephosphorylation mechanism and modulation by sulfhydryl compounds in the cytosol such as glutathione. The next step in the intrinsic pathway is the conversion of 7a-hydroxycholesterol to 7a-hydroxy-4-ene-3-one. An enzyme of approximately 46 000 daltons has been
The invention is directed to a pharmaceutical composi tion comprising an oxygenated cholesterol a... more The invention is directed to a pharmaceutical composi tion comprising an oxygenated cholesterol and a pene tration-enhancing agent which is useful for topical ap plication to the skin of a patient suffering from a prolif. erative skin disease characterized by geminative cells having a rapid rate of replication, e.g. psoriasis. The composition comprises an effective amount for the inhi bition of germinative cell mitosis of an oxygenated cho lesterol, e.g. 26-hydroxycholesterol, or a pharmaceuti cally effective derivative thereof e.g. an ester or ether. The invention is further directed to a method of treating a patient suffering from said skin disease comprising applying to the effected skin said therapeutic composi tion. The invention is also directed to the topical appli cation of these compositions to the skin to decrease inflammation.
BACKGROUND. Mirabegron is a β3-adrenergic receptor (β3-AR) agonist approved only for the treatmen... more BACKGROUND. Mirabegron is a β3-adrenergic receptor (β3-AR) agonist approved only for the treatment of overactive bladder. Encouraging preclinical results suggest that β3-AR agonists could also improve obesity-related metabolic disease by increasing brown adipose tissue (BAT) thermogenesis, white adipose tissue (WAT) lipolysis, and insulin sensitivity. METHODS. We treated 14 healthy women of diverse ethnicities (27.5 ± 1.1 years of age, BMI of 25.4 ± 1.2 kg/m 2) with 100 mg mirabegron (Myrbetriq extended-release tablet, Astellas Pharma) for 4 weeks in an open-label study. The primary endpoint was the change in BAT metabolic activity as measured by [ 18 F]-2-fluoro-d-2-deoxy-d-glucose (18 F-FDG) PET/CT. Secondary endpoints included resting energy expenditure (REE), plasma metabolites, and glucose and insulin metabolism as assessed by a frequently sampled intravenous glucose tolerance test. RESULTS. Chronic mirabegron therapy increased BAT metabolic activity. Whole-body REE was higher, without changes in body weight or composition. Additionally, there were elevations in plasma levels of the beneficial lipoprotein biomarkers HDL and ApoA1, as well as total bile acids. Adiponectin, a WAT-derived hormone that has antidiabetic and antiinflammatory capabilities, increased with acute treatment and was 35% higher upon completion of the study. Finally, an intravenous glucose tolerance test revealed higher insulin sensitivity, glucose effectiveness, and insulin secretion. CONCLUSION. These findings indicate that human BAT metabolic activity can be increased after chronic pharmacological stimulation with mirabegron and support the investigation of β3-AR agonists as a treatment for metabolic disease. TRIAL REGISTRATION. Clinicaltrials.gov NCT03049462.
Significant ambiguity exists in the scientific community with regard to the nomenclature of 26-hy... more Significant ambiguity exists in the scientific community with regard to the nomenclature of 26-hydroxylated oxysterols. Oxysterols constitute an important class of compounds that have biological roles in the regulation of cholesterol synthesis and as endogenous selective estrogen receptor modulators (SERMs). The ambiguity is attributable to deviations from clearly stated IUPAC rules and is likely to increase as more biologically active oxysterols are identified. This review provides a uniform approach to the naming of 26-hydroxylated sterols for those of current interest and for those on the horizon such as oxysterols of lanosterol that retain the unsaturation at C-24 and C-25 such as (E)-26-hydroxylanosterol. Using this molecule as a starting point, this review hopes to establish a common language to keep all investigators on the same page.
In 1959, Ivar Sperber contrasted bile formation with that of urine and proposed that water flow i... more In 1959, Ivar Sperber contrasted bile formation with that of urine and proposed that water flow into the canalicular conduit is in response to an osmotic not a hydrostatic gradient. Early attempts to support the hypothesis using a bile acid, sodium taurocholate, and the hormone secretin to stimulate bile flow led to conflicting data and a moratorium on attempts to further develop the initial proposal. However, current data amplify the initial proposal and indicate both paracellular and transcellular water flow into hepatic ductules and the canalicular conduit in response to an osmotic gradient. Also, the need to further modify the initial proposal became apparent with the recognition that bile acid aggregates (micelles) which form in the canalicular conduit generate lecithin-cholesterol vesicles that contain water unrelated to an osmotic
Phosphorylation of translation initiation factor 2a (eIF2a) coordinates a translational and trans... more Phosphorylation of translation initiation factor 2a (eIF2a) coordinates a translational and transcriptional program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. A screen for small molecule activators of the ISR identified two related compounds that also activated sterol-regulated genes by blocking cholesterol biosynthesis at the level of CYP51. Ketoconazole, a known CYP51 inhibitor, had similar effects, establishing that perturbed flux of precursors to cholesterol activates the ISR. Surprisingly, compound-mediated activation of sterol-regulated genes was enhanced in cells with an ISR-blocking mutation in the regulatory phosphorylation site of eIF2a. Furthermore, induction of the ISR by an artificial drug-activated eIF2a kinase reduced the level of active sterol regulatory element binding protein (SREBP) and sterol-regulated mRNAs. These findings suggest a mechanism by which interactions between sterol metabolism, the ISR, and the SREBP pathway affect lipid metabolism during ER stress.
Although it is recognized that more biomarkers are needed to evaluate the progression of liver di... more Although it is recognized that more biomarkers are needed to evaluate the progression of liver disease and response to medications, plasma alkaline phosphatase and conjugated bilirubin are a focus of clinical trials and utilized by physicians in practice. Conjugated bilirubin is a surrogate marker for the status of bile acid transport and the lowering of plasma levels in response to administration of ursodeoxycholic acid indicates that a choleretic effect has occurred. Plasma alkaline phosphatase levels are affected by hepatic bile acid composition and flow and the status of the cholangioles. Medications that reduce endogenous bile acid pool size can augment the effect of ursodeoxycholic acid by expanding its proportion in those undergoing enterohepatic circulation. In addition, they can lower the elevated steady state concentration of hepatocellular bile acids and retard progression to cirrhosis.
Journal of the American College of Cardiology, 1995
Values are presented as the mean ± SD of at least three different experiments. Asterisks represen... more Values are presented as the mean ± SD of at least three different experiments. Asterisks represent a p < 0.05 of the effect of carnitine vs control. These data show that L-carnitine stimulates PDH activity by 50%. However, the flux of acetyl-CoA and glucose through Kreb's cycle was significantly decreased by this compound. These results suggest that L-carnitine enhances the removal of acetyl-CoA produced from glucose metabolism out of the mitochondrial matriX, thereby, activating the PDH complex.
Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ra... more Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7a/ 7b-hydroxycholesterol and 5a,6a/5b,6b-epoxycholesterol as well as the 7a-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds.
Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects... more Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects of oxysterols on the expression of steroidogenic acute regulatory protein (StAR), which increases the delivery of cholesterol to sterol-metabolizing P450s in the mitochondria. 22(R)-hydroxycholesterol (22(R)-OHC), 25-OHC, and 27-OHC each increased steroidogenic factor-1 (SF-1)-mediated StAR gene transactivation by ϳ2fold in CV-1 cells. In contrast, cholesterol, progesterone, and the 27-OHC metabolites, 27-OHC-5-3-one and 7␣,27-OHC, had no effect. Unlike our findings in CV-1 cells, SF-1-dependent StAR promoter activity was not augmented by 27-OHC in COS-1 cells, Y-1 cells, BeWo choriocarcinoma cells, Chinese hamster ovary (CHO) cells, and human granulosa cells. Studies examining the metabolism of 27-OHC indicated that CV-1 cells formed a single polar metabolite, 3-OH-5-cholestenoic acid from radiolabeled 27-OHC. However, this metabolite inhibited StAR promoter activity in CV-1, COS-1 and CHO cells. Because 7␣,27-OHC was unable to increase SF-1dependent StAR promoter activity, we examined 27-OHC 7␣-hydroxylase in COS-1 and CHO cells. COS-1 cells contained high 7␣-hydroxylase activity, whereas the enzyme was undetectable in CHO cells. The hypothesis that oxysterols act in CV-1 cells to increase StAR promoter activity by reducing nuclear levels of sterol regulatory element binding protein was tested. This notion was refuted when it was discovered that sterol regulatory element binding protein-1a is a potent activator of the StAR promoter in CV-1, COS-1, and human granulosa cells. Human granulosa and theca cells, which express endogenous SF-1, contained more than 5-fold more StAR protein following addition of 27-OHC, whereas StAR mRNA levels remained unchanged. We conclude that 1) there are cell-specific effects of oxysterols on SF-1-dependent transactivation; 2) the ability to increase transactivation is limited to certain oxysterols; 3) there are cell-specific pathways of oxysterol metabolism; and 4) oxysterols elevate StAR protein levels through posttranscriptional actions.
Steroidogenic acute regulatory protein (StAR) plays a key role in steroid hormone synthesis by en... more Steroidogenic acute regulatory protein (StAR) plays a key role in steroid hormone synthesis by enhancing the metabolism of cholesterol into pregnenolone. We determined the organization of the StAR structural gene, mapped to 8~1 1. 2. The gene spans 8 kb and consists of seven exons interrupted by six introns. The 1.3 kb of DNA upstream from the transcription start site directed expression of a luciferase reporter gene in mouse Y-1 adrenal cortical tumor cells but not in BeWo choriocarcinoma cells. Reporter gene expression in the Y-1 cells was increased more than 2-fold by 8-Br-cAMP, indicating that the 1.3 kb DNA fragment contains sequences that confer tissue-specific expression and CAMP regulation. The sequence of a related StAR pseudogene, mapped to chromosome 13, lacks introns and has an insertion, numerous substitutions, and deletions. Expression of StAR in COS-1 cells cotransfected with cholesterol 27-hydroxylase (P450c27) and adrenodoxin resulted in a 6-fold increase in formation of 3P-hydroxy-5-cholestenoic acid, demonstrating that StAR's actions are not specific to steroidogenesis but extend to other mitochondrial cholesterol-metabolizing enzymes. The rate-limiting step in steroid hormone synthesis is the formation of pregnenolone from cholesterol, catalyzed by the cholesterol side-chain cleavage enzyme (P45Oscc), which resides with its associated electron transport chain in the inner mitochondrial membranes [for review see Miller (1988)l. This first committed reaction in the biosynthesis of steroid hormones is acutely stimulated by tropic hormones (ACTH in the adrenal cortex; LH in the gonads) acting through the intermediacy of CAMP. It has been known for two decades that the acute steroidogenic response to tropic stimulation involves the translocation of cholesterol from the outer to inner mitochondrial membranes. This translocation process is believed to be mediated by a short-lived, cycloheximidesensitive protein (
1. Using a human hepatoma (Hep G2) cell line that continually synthesizes 3 beta-hydroxy-5-cholen... more 1. Using a human hepatoma (Hep G2) cell line that continually synthesizes 3 beta-hydroxy-5-cholenoic acid, lithocholic acid, chenodeoxycholic acid and cholic acid we have determined the metabolism and biological effects of 26-hydroxycholesterol and 7 alpha-hydroxycholesterol. 2. Addition of 26-hydroxycholesterol to the medium (6 microM) downregulated cholesterol and chenodeoxycholic acid synthesis. 3. The predominant metabolite of 26-hydroxycholesterol was 3 beta-hydroxy-5-cholenoic acid. 4. Cholesterol synthesis was not affected by the addition of 7 alpha-hydroxycholesterol (6 and 12 microM). The predominant metabolite of 7 alpha-hydroxycholesterol was chenodeoxycholic acid. 5. In Hep G2 cells 7 alpha-hydroxylation of 26-hydroxycholesterol is not well expressed.
Smith-Lemli-Opitz syndrome (SLOS) is attributable to mutations in the gene coding for 7-dehydroch... more Smith-Lemli-Opitz syndrome (SLOS) is attributable to mutations in the gene coding for 7-dehydrocholesterol reductase. Low to absent enzyme activity accounts for the accumulation of both 7-dehydrocholesterol and 8-dehydrocholesterol in plasma and other tissues. Since oxysterols can participate in the regulation of cholesterol homeostasis, we examined the possibility that they are formed from these dehydrocholesterol intermediates. In patients with SLOS, we found serum levels of 27-hydroxy-7-dehydrocholesterol ranging from 0.1 to 0.25 M and evidence for circulating levels of 27-hydroxy-8-dehydrocholesterol (0.04-0.51 M). Picomolar quantities of 27-hydroxy-7-dehydrocholesterol were identified in normal individuals. Biologic activities of 27-hydroxy-7-dehydrocholesterol were found to include inhibition of sterol synthesis and the activation of nuclear receptor LXR␣ but not that of LXR. These activities occurred at concentrations found in plasma and presumably at those existing in tissues. Thus, patients with SLOS have increased levels of metabolites derived from intermediates in cholesterol synthesis that are biologically active and may contribute to the regulation of cholesterol synthesis in vivo.
excretion of coproporphyrin isomers I and III was studied in the rat. Both isomers were found to ... more excretion of coproporphyrin isomers I and III was studied in the rat. Both isomers were found to bind equally to rat plasma and liver cytosol in vitro and to disappear from plasma at equal rates after single injections in vivo. During equimolar infusions of isomers into bile fistula animals, both the I and III isomers were excreted in bile in a concentration ratio of 2: 1, respectively. Pretreatment of animals with ethinylestradiol or simultaneous infusions of phenoldibromophthalein disulfonate caused a reduction in total hepatic excretion with no change in the 2: 1 ratio in bile. As hepatic excretion fell, excretion of both isomers in urine rose, with an increase in the proportion of the I isomer. The findings mimic those reported to occur in man and can be explained by inhibition of a common carrier which requires a stereospecific configuration that statistically favors the hepatic transport of the symmetrical coproporphyrin I isomer.
After a number of years of being considered a well-established branch of biochemistry, we now hav... more After a number of years of being considered a well-established branch of biochemistry, we now have found that there is considerable less certainty about the pathways of bile acid synthesis from cholesterol, particularly in regard to human metabolism. Perhaps the major event that has occurred as part of this renaissance is the recognition that the initiation of bile acid synthesis from cholesterol can occur either by 7a-hydroxylation or by 26-hydroxylation (Anderson et al. 1972). Because there is some indication that the oxidation of the side chain of cholesterol can also occur in nonhepatic tissues, I will refer to it as the extrinsic pathway. It is the pathway for side-chain oxidation of C-27 sterols that is providing new perceptions with regard to the specific role of subcellular organelles and appears to generate intermediates that can have a regulatory role in the synthesis of cholesterol. The classical or traditional pathway of bile acid synthesis begins in the liver with cholesterol 7a-hydroxylase (Bjorkhem 1985), a microsomal P450 enzyme, and is referred to as the intrinsic pathway. There is considerable evidence in animal species that it is the rate-limiting enzyme, although it has neither been sequenced nor cloned and evidence derived from studies of liver cells in culture indicates that it is not regulated directly by bile acids (DaviS et al. 1983). The major evidence supporting its role as the rate-limiting enzyme is derived from the knowledge that cholestyrarnine treatment which induces bile acids synthesis certainly increases the activity of cholesterol 7 a-hydroxylase and if one couples this information to the more limited evidence that other enzymes in the intrinsic pathway have a higher rate of activity in the basal state which are not induced by biliary drainage, then the concept that 7a-hydroxylase is rate-limiting is reasonably sound. The enzyme is thought to have a relatively short half-life of 2-3 h and there is some evidence that the activity can be increased, at least under certain circumstances, by an increase in the cell cholesterol concentration. In addition to the relatively short half-life that provides rapid modulation in activity, experimental evidence exists for a phosphorylation-dephosphorylation mechanism and modulation by sulfhydryl compounds in the cytosol such as glutathione. The next step in the intrinsic pathway is the conversion of 7a-hydroxycholesterol to 7a-hydroxy-4-ene-3-one. An enzyme of approximately 46 000 daltons has been
The invention is directed to a pharmaceutical composi tion comprising an oxygenated cholesterol a... more The invention is directed to a pharmaceutical composi tion comprising an oxygenated cholesterol and a pene tration-enhancing agent which is useful for topical ap plication to the skin of a patient suffering from a prolif. erative skin disease characterized by geminative cells having a rapid rate of replication, e.g. psoriasis. The composition comprises an effective amount for the inhi bition of germinative cell mitosis of an oxygenated cho lesterol, e.g. 26-hydroxycholesterol, or a pharmaceuti cally effective derivative thereof e.g. an ester or ether. The invention is further directed to a method of treating a patient suffering from said skin disease comprising applying to the effected skin said therapeutic composi tion. The invention is also directed to the topical appli cation of these compositions to the skin to decrease inflammation.
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