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ABSTRACT: Carboxylesterases (CES) are a well recognized, yet incompletely characterized family of proteins that catalyze neutral lipid hydrolysis. Some CES have well-defined roles in xenobiotic clearance, pharmacologic pro-drug activation, and narcotic detoxification. In addition, emerging evidence suggests other CES may have roles in lipid metabolism. Humans have six CES genes, while mice have 20 Ces genes grouped into five isoenzyme classes. Perhaps due to the high sequence similarity shared by the mouse Ces genes, the tissue-specific distribution of expression for these enzymes has not been fully addressed. Therefore, we performed studies to provide a comprehensive tissue distribution analysis of mouse Ces mRNAs. These data demonstrated that while the mouse Ces family 1 is highly expressed in liver and family 2 in intestine, many Ces genes have a wide and unique tissue distribution defined by relative mRNA levels. Furthermore, evaluating Ces gene expression in response to pharmacologic activation of lipid and xenobiotic-sensing nuclear hormone receptors (NHR) showed differential regulation. Finally, specific shifts in Ces gene expression were seen in peritoneal macrophages following LPS treatment and in a steatotic liver model induced by high-fat feeding, two model systems relevant to disease. Overall these data show that each mouse Ces gene has its own distinctive tissue expression pattern and suggest that some CES may have tissue-specific roles in lipid metabolism and xenobiotic clearance.
Drug metabolism and disposition: the biological fate of chemicals 09/2012; · 3.74 Impact Factor
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ABSTRACT: An injection of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) to mice lacking Niemann Pick type C (NPC) protein results in delayed neurodegeneration, decreased inflammation, and prolonged lifespan. Changes in sterol balance observed in Npc1(-/-) mice 24 h after HP-β-CD administration suggest that HP-β-CD facilitates the release of accumulated lysosomal cholesterol, the molecular hallmark of this genetic disorder. Current studies were performed to evaluate the time course of HP-β-CD effects. Within 3 h after HP-β-CD injection, decreases in cholesterol synthesis rates and increases in cholesteryl ester levels were detected in tissues of Npc1(-/-) mice. The levels of RNAs for target genes of sterol-sensing transcription factors were altered by 6 h in liver, spleen, and ileum. Despite the cholesterol-binding capacity of HP-β-CD, there was no evidence of increased cholesterol in plasma or urine of treated Npc1(-/-) mice, suggesting that HP-β-CD does not carry sterol from the lysosome into the bloodstream for ultimate urinary excretion. Similar changes in sterol balance were observed in cultured cells from Npc1(-/-) mice using HP-β-CD and sulfobutylether-β-CD, a variant that can interact with sterol but not facilitate its solubilization. Taken together, our results demonstrate that HP-β-CD works in cells of Npc1(-/-) mice by rapidly liberating lysosomal cholesterol for normal sterol processing within the cytosolic compartment.
The Journal of Lipid Research 08/2012; 53(11):2331-42. · 5.56 Impact Factor
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ABSTRACT: Cholesterol 7α-hydroxylase (CYP7A1) is the initiating and rate-limiting enzyme in the neutral pathway that converts cholesterol to primary bile acids (BA). CYP7A1-deficient (Cyp7a1(-/-)) mice have a depleted BA pool, diminished intestinal cholesterol absorption, accelerated fecal sterol loss, and increased intestinal cholesterol synthesis. To determine the molecular and physiological effects of restoring the BA pool in this model, adult female Cyp7a1(-/-) mice and matching Cyp7a1(+/+) controls were fed diets containing cholic acid (CA) at modest levels [0.015, 0.030, and 0.060% (wt/wt)] for 15-18 days. A level of just 0.03% provided a CA intake of ~12 μmol (4.8 mg) per day per 100 g body wt and was sufficient in the Cyp7a1(-/-) mice to normalize BA pool size, fecal BA excretion, fractional cholesterol absorption, and fecal sterol excretion but caused a significant rise in the cholesterol concentration in the small intestine and liver, as well as a marked inhibition of cholesterol synthesis in these organs. In parallel with these metabolic changes, there were marked shifts in intestinal and hepatic expression levels for many target genes of the BA sensor farnesoid X receptor, as well as genes involved in cholesterol transport, especially ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG8. In Cyp7a1(+/+) mice, this level of CA supplementation did not significantly disrupt BA or cholesterol metabolism, except for an increase in fecal BA excretion and marginal changes in mRNA expression for some BA synthetic enzymes. These findings underscore the importance of using moderate dietary BA levels in studies with animal models.
AJP Gastrointestinal and Liver Physiology 05/2012; 303(2):G263-74. · 3.43 Impact Factor
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ABSTRACT: Previous work has demonstrated that the peptide hormone ghrelin raises blood glucose. Such has been attributed to ghrelin's ability to enhance GH secretion, restrict insulin release, and/or reduce insulin sensitivity. Ghrelin's reported effects on glucagon have been inconsistent. Here, both animal- and cell-based systems were used to determine the role of glucagon in mediating ghrelin's effects on blood glucose. The tissue and cell distribution of ghrelin receptors (GHSR) was evaluated by quantitative PCR and histochemistry. Plasma glucagon levels were determined following acute acyl-ghrelin injections and in pharmacological and/or transgenic mouse models of ghrelin overexpression and GHSR deletion. Isolated mouse islets and the α-cell lines αTC1 and InR1G9 were used to evaluate ghrelin's effects on glucagon secretion and the role of calcium and ERK in this activity. GHSR mRNA was abundantly expressed in mouse islets and colocalized with glucagon in α-cells. Elevation of acyl-ghrelin acutely (after sc administration, such that physiologically relevant plasma ghrelin levels were achieved) and chronically (by slow-releasing osmotic pumps and as observed in transgenic mice harboring ghrelinomas) led to higher plasma glucagon and increased blood glucose. Conversely, genetic GHSR deletion was associated with lower plasma glucagon and reduced fasting blood glucose. Acyl-ghrelin increased glucagon secretion in a dose-dependent manner from mouse islets and α-cell lines, in a manner requiring elevation of intracellular calcium and phosphorylation of ERK. Our study shows that ghrelin's regulation of blood glucose involves direct stimulation of glucagon secretion from α-cells and introduces the ghrelin-glucagon axis as an important mechanism controlling glycemia under fasting conditions.
Molecular Endocrinology 06/2011; 25(9):1600-11. · 4.54 Impact Factor
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ABSTRACT: While unesterified cholesterol (C) is essential for remodeling neuronal plasma membranes, its role in certain neurodegenerative disorders remains poorly defined. Uptake of sterol from pericellular fluid requires processing that involves two lysosomal proteins, lysosomal acid lipase, which hydrolyzes C esters, and NPC1 (Niemann-Pick type C1). In systemic tissues, inactivation of either protein led to sterol accumulation and cell death, but in the brain, inactivation of only NPC1 caused C sequestration and neurodegeneration. When injected into the CNS of the npc1(-/-) mouse, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a compound known to prevent this C accumulation, diffused throughout the brain and was excreted with a t(½) of 6.5 h. This agent caused suppression of C synthesis, elevation of C esters, suppression of sterol regulatory-binding protein 2 (SREBP2) target genes, and activation of liver X receptor-controlled genes. These findings indicated that HP-β-CD promoted movement of the sequestered C from lysosomes to the metabolically active pool of C in the cytosolic compartment of cells in the CNS. The ED(50) for this agent in the brain was ∼0.5 mg/kg, and the therapeutic effect lasted >7 d. Continuous infusion of HP-β-CD into the ventricular system of npc1(-/-) animals between 3 and 7 weeks of age normalized the biochemical abnormalities and completely prevented the expected neurodegeneration. These studies support the concept that neurons continuously acquire C from interstitial fluid to permit plasma membrane turnover and remodeling. Inactivation of NPC1 leads to lysosomal C sequestration and neurodegeneration, but this is prevented by the continuous, direct administration of HP-β-CD into the CNS.
Journal of Neuroscience 06/2011; 31(25):9404-13. · 7.11 Impact Factor
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ABSTRACT: Lipoprotein cholesterol taken up by cells is processed in the endosomal/lysosomal (E/L) compartment by the sequential action of lysosomal acid lipase (LAL), Niemann-Pick C2 (NPC2), and Niemann-Pick C1 (NPC1). Inactivation of NPC2 in mouse caused sequestration of unesterified cholesterol (UC) and expanded the whole animal sterol pool from 2,305 to 4,337 mg/kg. However, this pool increased to 5,408 and 9,480 mg/kg, respectively, when NPC1 or LAL function was absent. The transport defect in mutants lacking NPC2 or NPC1, but not in those lacking LAL, was reversed by cyclodextrin (CD), and the ED₅₀ values for this reversal varied from ~40 mg/kg in kidney to >20,000 mg/kg in brain in both groups. This reversal occurred only with a CD that could interact with UC. Further, a CD that could interact with, but not solubilize, UC still overcame the transport defect. These studies showed that processing and export of sterol from the late E/L compartment was quantitatively different in mice lacking LAL, NPC2, or NPC1 function. In both npc2(-/-) and npc1(-/-) mice, the transport defect was reversed by a CD that interacted with UC, likely at the membrane/bulk-water interface, allowing sterol to move rapidly to the export site of the E/L compartment.
The Journal of Lipid Research 02/2011; 52(4):688-98. · 5.56 Impact Factor
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ABSTRACT: Cholesterol homeostasis in the enterocyte is regulated by the interplay of multiple genes that ultimately determines the net amount of cholesterol reaching the circulation from the small intestine. The effect of deleting these genes, particularly acyl CoA:cholesterol acyl transferase 2 (ACAT2), on cholesterol absorption and fecal sterol excretion is well documented. We also know that the intestinal mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1) increases in Acat2(-/-) mice. However, none of these studies has specifically addressed how ACAT2 deficiency impacts the relative proportions of esterified and unesterified cholesterol (UC) in the enterocyte and whether the concurrent loss of ABCA1 might result in a marked buildup of UC. Therefore, the present studies measured the expression of numerous genes and related metabolic parameters in the intestine and liver of ACAT2-deficient mice fed diets containing either added cholesterol or ezetimibe, a selective sterol absorption inhibitor. Cholesterol feeding raised the concentration of UC in the small intestine, and this was accompanied by a significant reduction in the relative mRNA level for Niemann-Pick C1-like 1 (NPC1L1) and an increase in the mRNA level for both ABCA1 and ABCG5/8. All these changes were reversed by ezetimibe. When mice deficient in both ACAT2 and ABCA1 were fed a high-cholesterol diet, the increase in intestinal UC levels was no greater than it was in mice lacking only ACAT2. This resulted from a combination of compensatory mechanisms including diminished NPC1L1-mediated cholesterol uptake, increased cholesterol efflux via ABCG5/8, and possibly rapid cell turnover.
AJP Gastrointestinal and Liver Physiology 11/2010; 299(5):G1012-22. · 3.43 Impact Factor
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ABSTRACT: Niemann-Pick type C1 (NPC1) disease arises from a mutation inactivating NPC1 protein that normally moves unesterified cholesterol from the late endosomal/lysosomal complex of cells to the cytosolic compartment for processing. As a result, cholesterol accumulates in every tissue of the body causing liver, lung, and CNS disease. Treatment of the murine model of this disease, the npc1 mouse, s.c. with β-cyclodextrin (4000 mg/kg) one time each week normalized cellular cholesterol metabolism in the liver and most other organs. At the same time, the hepatic dysfunction seen in the untreated npc1 mouse was prevented. The severity of cerebellar neurodegeneration also was ameliorated, although not entirely prevented, and the median lifespan of the animals was doubled. However, in contrast to these other organs, lung showed progressive macrophage infiltration with development of lipoid pneumonitis. These studies demonstrated that weekly cyclodextrin administration overcomes the lysosomal transport defect associated with the NPC1 mutation, nearly normalizes hepatic and whole animal cholesterol pools, and prevents the development of liver disease. Furthermore, this treatment slows cerebellar neurodegeneration but has little or no effect on the development of progressive pulmonary disease.
Pediatric Research 10/2010; 68(4):309-15. · 2.70 Impact Factor
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Jen-Chieh Chuang,
Huxing Cui,
Brittany L Mason,
Melissa Mahgoub,
Angie L Bookout,
Hana G Yu,
Mario Perello,
Joel K Elmquist, Joyce J Repa,
Jeffrey M Zigman,
Michael Lutter
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ABSTRACT: Several psychiatric disorders increase the risk of cardiovascular disease, including posttraumatic stress disorder and major depression. While the precise mechanism for this association has not yet been established, it has been shown that certain disorders promote an unfavorable lipid profile. To study the interaction of stress and lipid dysregulation, we utilized chronic social defeat stress (CSDS), a mouse model of chronic stress with features of posttraumatic stress disorder and major depression. Following exposure to CSDS, mice were given access to either regular chow or a Western-style diet high in fat and cholesterol (HFD). The combination of social stress and HFD resulted in significant perturbations in lipid regulation, including two key features of the metabolic syndrome: increased plasma levels of non-HDL cholesterol and intrahepatic accumulation of triglycerides. These effects were accompanied by a number of changes in the expression of hepatic genes involved in lipid regulation. Transcriptional activity of LXR, SREBP1c, and ChREBP were significantly affected by exposure to HFD and CSDS. We present CSDS as a model of social stress induced lipid dysregulation and propose that social stress alters lipid metabolism by increasing transcriptional activity of genes involved in lipid synthesis.
The Journal of Lipid Research 06/2010; 51(6):1344-53. · 5.56 Impact Factor
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Takeshi Ogihara,
Jen-Chieh Chuang,
George L. Vestermark,
James C. Garmey,
Robert J. Ketchum,
Xiaolun Huang,
Kenneth L. Brayman,
Michael O. Thorner, Joyce J. Repa,
Raghavendra G. Mirmira,
Carmella Evans-Molina
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ABSTRACT: Recent studies in rodent models suggest that liver X receptors (LXRs) may play an important role in the maintenance of glucose
homeostasis and islet function. To date, however, no studies have comprehensively examined the role of LXRs in human islet
biology. Human islets were isolated from non-diabetic donors and incubated in the presence or absence of two synthetic LXR
agonists, TO-901317 and GW3965, under conditions of low and high glucose. LXR agonist treatment enhanced both basal and stimulated
insulin secretion, which corresponded to an increase in the expression of genes involved in anaplerosis and reverse cholesterol
transport. Furthermore, enzyme activity of pyruvate carboxylase, a key regulator of pyruvate cycling and anaplerotic flux,
was also increased. Whereas LXR agonist treatment up-regulated known downstream targets involved in lipogenesis, we observed
no increase in the accumulation of intra-islet triglyceride at the dose of agonist used in our study. Moreover, LXR activation
increased expression of the genes encoding hormone-sensitive lipase and adipose triglyceride lipase, two enzymes involved
in lipolysis and glycerolipid/free fatty acid cycling. Chronically, insulin gene expression was increased after treatment
with TO-901317, and this was accompanied by increased Pdx-1 nuclear protein levels and enhanced Pdx-1 binding to the insulin
promoter. In conclusion, our data suggest that LXR agonists have a direct effect on the islet to augment insulin secretion
and expression, actions that should be considered either as therapeutic or unintended side effects, as these agents are developed
for clinical use.
Journal of Biological Chemistry 02/2010; 285(8):5392-5404. · 4.77 Impact Factor
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Takeshi Ogihara,
Jen-Chieh Chuang,
George L Vestermark,
James C Garmey,
Robert J Ketchum,
Xiaolun Huang,
Kenneth L Brayman,
Michael O Thorner, Joyce J Repa,
Raghavendra G Mirmira,
Carmella Evans-Molina
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ABSTRACT: Recent studies in rodent models suggest that liver X receptors (LXRs) may play an important role in the maintenance of glucose homeostasis and islet function. To date, however, no studies have comprehensively examined the role of LXRs in human islet biology. Human islets were isolated from non-diabetic donors and incubated in the presence or absence of two synthetic LXR agonists, TO-901317 and GW3965, under conditions of low and high glucose. LXR agonist treatment enhanced both basal and stimulated insulin secretion, which corresponded to an increase in the expression of genes involved in anaplerosis and reverse cholesterol transport. Furthermore, enzyme activity of pyruvate carboxylase, a key regulator of pyruvate cycling and anaplerotic flux, was also increased. Whereas LXR agonist treatment up-regulated known downstream targets involved in lipogenesis, we observed no increase in the accumulation of intra-islet triglyceride at the dose of agonist used in our study. Moreover, LXR activation increased expression of the genes encoding hormone-sensitive lipase and adipose triglyceride lipase, two enzymes involved in lipolysis and glycerolipid/free fatty acid cycling. Chronically, insulin gene expression was increased after treatment with TO-901317, and this was accompanied by increased Pdx-1 nuclear protein levels and enhanced Pdx-1 binding to the insulin promoter. In conclusion, our data suggest that LXR agonists have a direct effect on the islet to augment insulin secretion and expression, actions that should be considered either as therapeutic or unintended side effects, as these agents are developed for clinical use.
Journal of Biological Chemistry 12/2009; 285(8):5392-404. · 4.77 Impact Factor
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ABSTRACT: A mutation in NPC1 leads to sequestration of unesterified cholesterol in the late endosomal/lysosomal compartment of every cell culminating in the development of pulmonary, hepatic, and neurodegenerative disease. Acute administration of 2-hydroxypropyl-beta-cyclodextrin (CYCLO) rapidly overcomes this transport defect in both the 7-day-old pup and 49-day-old mature npc1(-/-) mouse, even though this compound is cleared from the body and plasma six times faster in the mature mouse than in the neonatal animal. The liberated cholesterol flows into the cytosolic ester pool, suppresses sterol synthesis, down-regulates SREBP2 and its target genes, and reduces expression of macrophage-associated inflammatory genes. These effects are seen in the liver and brain, as well as in peripheral organs like the spleen and kidney. Only the lung appears to be resistant to these effects. Forty-eight h after CYCLO administration to the 49-day-old animals, fecal acidic, but not neutral, sterol output increases, whole-animal cholesterol burden is reduced, and the hepatic and neurological inflammation is ameliorated. However, lifespan is extended only when the CYCLO is administered to the 7-day-old animals. These studies demonstrate that CYCLO administration acutely reverses the cholesterol transport defect seen in the NPC1 mouse at any age, and this reversal allows the sequestered sterol to be excreted from the body as bile acid.
The Journal of Lipid Research 11/2009; 51(5):933-44. · 5.56 Impact Factor
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ABSTRACT: Niemann-Pick type C disease is largely attributable to an inactivating mutation of NPC1 protein, which normally aids movement of unesterified cholesterol (C) from the endosomal/lysosomal (E/L) compartment to the cytosolic compartment of cells throughout the body. This defect results in activation of macrophages in many tissues, progressive liver disease, and neurodegeneration. In the npc1(-/-) mouse, a model of this disease, the whole-animal C pool expands from 2,082 to 4,925 mg/kg body weight (bw) and the hepatic C pool increases from 132 to 1,485 mg/kg bw between birth and 49 days of age. A single dose of 2-hydroxypropyl-beta-cyclodextrin (CYCLO) administered at 7 days of age immediately caused this sequestered C to flow from the lysosomes to the cytosolic pool in many organs, resulting in a marked increase in cholesteryl esters, suppression of C but not fatty acid synthesis, down-regulation of genes controlled by sterol regulatory element 2, and up-regulation of many liver X receptor target genes. There was also decreased expression of proinflammatory proteins in the liver and brain. In the liver, where the rate of C sequestration equaled 79 mg x d(-1) x kg(-1), treatment with CYCLO within 24 h increased C movement out of the E/L compartment from near 0 to 233 mg x d(-1) x kg(-1). By 49 days of age, this single injection of CYCLO resulted in a reduction in whole-body C burden of >900 mg/kg, marked improvement in liver function tests, much less neurodegeneration, and, ultimately, significant prolongation of life. These findings suggest that CYCLO acutely reverses the lysosomal transport defect seen in NPC disease.
Proceedings of the National Academy of Sciences 02/2009; 106(7):2377-82. · 9.68 Impact Factor
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ABSTRACT: Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical
role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the
activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization.
In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction
with 14-3-3. 14-3-3 binds an α-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3
is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for
its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to
interact with importin α. Notably, 14-3-3 appears to compete with importin α for ChREBP binding. 14-3-3β bound to a synthetic
peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 μm, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues
of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3β. These results suggest that interactions
with importin α and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions
are regulated by the ChREBP phosphorylation status.
Journal of Biological Chemistry 09/2008; 283(36):24899-24908. · 4.77 Impact Factor
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ABSTRACT: Niemann-Pick C1-like 1 (NPC1L1) facilitates the uptake of sterols into the enterocyte and is the target of the novel cholesterol absorption inhibitor, ezetimibe. These studies used the Golden Syrian hamster as a model to delineate the changes in the relative mRNA expression of NPC1L1 and other proteins that regulate sterol homeostasis in the enterocyte during and following cessation of ezetimibe treatment and also to address the clinically important question of whether the marked inhibition of cholesterol absorption alters biliary lipid composition. In hamsters fed a low-cholesterol, low-fat basal diet, the abundance of mRNA for NPC1L1 in the small intestine far exceeded that in other regions of the gastrointestinal tract, liver, and gallbladder. In the first study, female hamsters were fed the basal diet containing ezetimibe at doses up to 2.0 mg.day(-1).kg body wt(-1). At this dose, cholesterol absorption fell by 82%, fecal neutral sterol excretion increased by 5.3-fold, and hepatic and intestinal cholesterol synthesis increased more than twofold, but there were no significant changes in either fecal bile acid excretion or biliary lipid composition. The ezetimibe-induced changes in intestinal cholesterol handling were reversed when treatment was withdrawn. In a second study, male hamsters were given a diet enriched in cholesterol and safflower oil without or with ezetimibe. The lipid-rich diet raised the absolute and relative cholesterol levels in bile more than fourfold. This increase was largely prevented by ezetimibe. These data are consistent with the recent finding that ezetimibe treatment significantly reduced biliary cholesterol saturation in patients with gallstones.
AJP Gastrointestinal and Liver Physiology 09/2008; 295(4):G813-22. · 3.43 Impact Factor
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ABSTRACT: These studies investigated the role of gangliosides in governing the steady-state concentration and turnover of unesterified cholesterol in normal tissues and in those of mice carrying the NPC1 mutation. In animals lacking either GM2/GD2 or GM3 synthase, tissue cholesterol concentrations and synthesis rates were normal in nearly all organs, and whole-animal sterol pools and turnover also were not different from control animals. Mice lacking both synthases, however, had small elevations in cholesterol concentrations in several organs, and the whole-animal cholesterol pool was marginally elevated. None of these three groups, however, had changes in any parameter of cholesterol homeostasis in the major regions of the central nervous system. When either the GM2/GD2 or GM3 synthase activity was deleted in mice lacking NPC1 function, the clinical phenotype was not changed, but lifespan was shortened. However, the abnormal cholesterol accumulation seen in the tissues of the NPC1 mouse was unaffected by loss of either synthase, and clinical and molecular markers of hepatic and cerebellar disease also were unchanged. These studies demonstrate that hydrophobic interactions between cholesterol and various gangliosides do not play an important role in determining cellular cholesterol concentrations in the normal animal or in the mouse with the NPC1 mutation.
The Journal of Lipid Research 09/2008; 49(8):1816-28. · 5.56 Impact Factor
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ABSTRACT: The endocrine pancreas comprises the islets of Langerhans, tiny clusters of cells that contribute only about 2% to the total pancreas mass. However, this little endocrine organ plays a critical role in maintaining glucose homeostasis by the regulated secretion of insulin (by beta-cells) and glucagon (by alpha-cells). The rapid increase in the incidence of diabetes worldwide has spurred renewed interest in islet cell biology. Some of the most widely prescribed oral drugs for treating type 2 diabetes include agents that bind and activate the nuclear hormone receptor, peroxisome proliferator-activated receptor-gamma. As a first step in addressing potential roles of peroxisome proliferator-activated receptor-gamma and other nuclear hormone receptors (NHRs) in the biology of the endocrine pancreas, we have used quantitative real-time PCR to profile the expression of all 49 members of the mouse NHR superfamily in primary islets, and cell lines that represent alpha-cells (alphaTC1) and beta-cells (betaTC6 and MIN6). In summary, 19 NHR members were highly expressed in both alpha- and beta-cell lines, 13 receptors showed predominant expression (at least an 8-fold difference) in alpha- vs. beta-cell lines, and 10 NHRs were not expressed in the endocrine pancreas. In addition we evaluated the relative expression of these transcription factors during hyperglycemia and found that 16 NHRs showed significantly altered mRNA levels in mouse islets. A similar survey was conducted in primary human islets to reveal several significant differences in NHR expression between mouse and man. These data identify potential therapeutic targets in the endocrine pancreas for the treatment of diabetes mellitus.
Molecular Endocrinology 09/2008; 22(10):2353-63. · 4.54 Impact Factor
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ABSTRACT: Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an alpha-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin alpha. Notably, 14-3-3 appears to compete with importin alpha for ChREBP binding. 14-3-3beta bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 microm, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3beta. These results suggest that interactions with importin alpha and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.
Journal of Biological Chemistry 08/2008; 283(36):24899-908. · 4.77 Impact Factor
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ABSTRACT: To characterize the effect of HIF-2alpha haploinsufficiency on retinal neovascularization and angiogenic signaling in neonatal mice.
Retinal samples were obtained from HIF-2alpha-haploinsufficient (Epas1+/-) and wild-type (Epas1+/+) neonatal mice subjected to an oxygen-induced retinopathy (OIR) protocol. Histologic and molecular studies were performed immediately, 12 hours, or 5 days after initiation of the hypoxia phase of the OIR protocol. Molecular profiling was performed in mouse brain endothelial cells maintained in normoxia or hypoxia. Transfection studies assessed the response of isolated promoter regions from proangiogenic genes to HIF-1alpha or -2alpha overexpression.
Epas1+/- mice exhibited no significant differences in retinal vasculature during normal development but had reduced retinal neovascularization in an OIR protocol. Multiple proangiogenic factors were induced during the hypoxia phase in Epas1+/+ OIR retinal samples, whereas Epas1+/- OIR retinal samples had absent or blunted induction of these same factors. Several, but not all, proangiogenic factors were induced in mouse brain endothelial cells after hypoxia. In transfection assays, most proangiogenic promoter regions were preferentially activated by HIF-2alpha relative to HIF-1alpha.
HIF-2alpha deficiency results in reduced neovascularization and blunted inducibility of multiple proangiogenic factors in the retinas of mice with OIR. The authors propose that HIF-2alpha is a master regulator of proangiogenic factors in retinal vascular endothelial cells, the predominant cell type of the retina in which HIF-2alpha is expressed. Future studies will address whether the molecular and functional roles for HIF-2alpha identified from these studies can be generalized to other pathophysiological states involving neovascularization.
Investigative Ophthalmology & Visual Science 07/2008; 49(6):2714-20. · 3.60 Impact Factor
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ABSTRACT: Niemann-Pick type C (NPC) disease is a multisystem disorder caused primarily by a mutation in the npc1 gene. These studies evaluated the effect of genetic background, deletion of additional genes, and administration of several agents on the age at death in a murine model of this disorder. Such factors as differing strain background or genetic drift within a given background in the npc1(-/-) mouse significantly altered the age at death and the degree of organ disease. Genetic deletion of Siat9 (GM3 synthetase) or Nr1h2 [liver X receptor (LXR)beta] shortened the life of the npc1(-/-) animals. Daily treatment of the npc1(-/-) mice with an LXR agonist or administration of a single dose of cyclodextrin, with or without the neurosteroid allopregnanolone, significantly slowed neurodegeneration and increased the lifespan of these animals. These data illustrate that the age at death of the npc1(-/-) mouse can be significantly influenced by many factors, including differences in strain background, other inactivating gene mutations (Siat9 and lxrbeta), and administration of agents such as LXR agonists and, particularly, cyclodextrin. It is currently not clear which of these effects is nonspecific or which might relate directly to the molecular defect present in the NPC1 syndrome.
The Journal of Lipid Research 04/2008; 49(3):663-9. · 5.56 Impact Factor
