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3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes
Kim, Yeonhee
Rajagopalan, Padmavathy
PLoS ONE
Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.
Related Articles
The Design of In Vitro Liver Sinusoid Mimics Using Chitosan–Hyaluronic Acid Polyelectrolyte Multilayers
Kim, Yeonhee
Larkin, Adam L.
Davis, Richey M.
Rajagopalan, Padmavathy
Tissue Engineering. Part A
Interactions between hepatocytes and liver sinusoidal endothelial cells (LSECs) are essential for the development and maintenance of hepatic phenotypic functions. We report the assembly of three-dimensional liver sinusoidal mimics comprised of primary rat hepatocytes, LSECs, and an intermediate chitosan–hyaluronic acid polyelectrolyte multilayer (PEM). The height of the PEMs ranged from 30 to 55 nm and exhibited a shear modulus of ∼100 kPa. Hepatocyte–PEM cellular constructs exhibited stable urea and albumin production over a 7-day period, and these values were either higher or similar to cells cultured in a collagen sandwich. This is of significance because the thickness of a collagen gel is ∼1000-fold higher than the height of the chitosan–hyaluronic acid PEM. In the hepatocyte–PEM–LSEC liver-mimetic cellular constructs, LSEC phenotype was maintained, and these cultures exhibited stable urea and albumin production. CYP1A1/2 activity measured over a 7-day period was significantly higher in the hepatocyte–PEM–LSEC constructs than in collagen sandwich cultures. A 16-fold increase in CYP1A1/2 activity was observed for hepatocyte–PEM–10,000 LSEC samples, thereby suggesting that interactions between hepatocytes and LSECs are critical in enhancing the detoxification capability in hepatic cultures in vitro.
Related Articles
Biosynthesis of bile acids in cerebrotendinous xanthomatosis. Relationship of bile acid pool sizes and synthesis rates to hydroxylations at C-12, C-25, and C-26.
Salen, G
Shefer, S
Tint, G S
Nicolau, G
Dayal, B
Batta, A K
Journal of Clinical Investigation
To examine the defect in side-chain oxidation during the formation of bile acids in cerebrotendinous xanthomatosis, we measured in vitro hepatic microsomal hydroxylations at C-12 and C-25 and mitochondrial hydroxylation at C-26 and related them to the pool size and synthesis rates of cholic acid and chenodeoxycholic acid as determined by the isotope dilution technique. Hepatic microsomes and mitochondria were prepared from seven subjects with cerebrotendinous xanthomatosis and five controls. Primary bile acid synthesis was markedly reduced in cerebrotendinous xanthomatosis as follows: cholic acid, 133 +/- 30 vs. 260 +/- 60 mg/d in controls; and chenodeoxycholic acid, 22 +/- 10 vs. 150 +/- 30 mg/d in controls. As postulated for chenodeoxycholic acid synthesis, mitochondrial 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol was present in all specimens and was 30-fold more active than the corresponding microsomal 25-hydroxylation. However, mean mitochondrial 26-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol was less active in cerebrotendinous xanthomatosis than in controls: 59 +/- 17 compared with 126 +/- 21 pmol/mg protein per min. As for cholic acid synthesis, microsomal 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol was substantially higher in cerebrotendinous xanthomatosis and control preparations (620 +/- 103 and 515 +/- 64 pmol/mg protein per min, respectively) than the corresponding control mitochondrial 26-hydroxylation of the same substrate (165 +/- 25 pmol/mg protein per min). Moreover in cerebrotendinous xanthomatosis, mitochondrial 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol-26-hydroxylase activity was one-seventh as great as in controls. Hepatic microsomal 12 alpha-hydroxylation, which may be rate-controlling for the cholic acid pathway, was three times more active in cerebrotendinous xanthomatosis than in controls: 1,600 vs. 500 pmol/mg protein per min. These results demonstrate severely depressed primary bile acid synthesis in cerebrotendinous xanthomatosis with a reduction in chenodeoxycholic acid formation and pool size disproportionately greater than that for cholic acid. The deficiency of chenodeoxycholic acid can be accounted for by hyperactive microsomal 12 alpha-hydroxylation that diverts precursors into the cholic acid pathway combined with decreased side-chain oxidation (mitochondrial 26-hydroxylation). However, side-chain oxidation in cholic acid biosynthesis may be initiated via microsomal 25-hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol was substantially lower in control and cerebrotendinous xanthomatosis liver. Thus, separate mechanisms may exist for the cleavage of the cholesterol side chain in cholic acid and chenodeoxycholic acid biosynthesis.
Related Articles
Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7 alpha-hydroxylase.
Princen, H M
Huijsmans, C M
Kuipers, F
Vonk, R J
Kempen, H J
Journal of Clinical Investigation
In cultured hepatocytes conversion of [4-14C]cholesterol into bile acids was dose dependently reduced by the antimycotic drug ketoconazole, giving half-maximal inhibition at 10 microM ketoconazole in rat hepatocytes and at 1 microM in human hepatocytes. No change was observed in the ratio of produced cholic, beta-muricholic, and chenodeoxycholic acid with increasing amounts of the drug. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of bile acid pathway, to bile acids was not affected by ketoconazole. These results together with kinetic studies with rat liver microsomes, demonstrating noncompetitive inhibition (Ki = 0.4 microM), indicate that cholesterol 7 alpha-hydroxylase is the main site of inhibition. In bile-diverted rats a single dose of ketoconazole (50 mg/kg) dramatically impaired bile flow and biliary bile acid output (92% inhibition). A similar blockade was observed using [4-14C]cholesterol as precursor for bile acid synthesis. Therefore, treatment of patients with this drug may inhibit bile acid synthesis, resulting in a reduction of the bile acid pool size after long-term ketoconazole therapy.
Related Articles
Liver Dysfunction and Phosphatidylinositol-3-Kinase Signalling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis
Recknagel, Peter
Gonnert, Falk A.
Westermann, Martin
Lambeck, Sandro
Lupp, Amelie
Rudiger, Alain
Dyson, Alex
Carré, Jane E.
Kortgen, Andreas
Krafft, Christoph
Popp, Jürgen
Sponholz, Christoph
Fuhrmann, Valentin
Hilger, Ingrid
Claus, Ralf A.
Riedemann, Niels C.
Wetzker, Reinhard
Singer, Mervyn
Trauner, Michael
Bauer, Michael
PLoS Medicine
Experimental studies in a rat model of fecal peritonitis conducted by Michael Bauer and colleagues show that in this model, changes in liver function occur early in the development of sepsis, with potential implications for prognosis and development of new therapeutic approaches.
Hepatic dysfunction and jaundice are traditionally viewed as late features of sepsis and portend poor outcomes. We hypothesized that changes in liver function occur early in the onset of sepsis, yet pass undetected by standard laboratory tests.
Methods and Findings
In a long-term rat model of faecal peritonitis, biotransformation and hepatobiliary transport were impaired, depending on subsequent disease severity, as early as 6 h after peritoneal contamination. Phosphatidylinositol-3-kinase (PI3K) signalling was simultaneously induced at this time point. At 15 h there was hepatocellular accumulation of bilirubin, bile acids, and xenobiotics, with disturbed bile acid conjugation and drug metabolism. Cholestasis was preceded by disruption of the bile acid and organic anion transport machinery at the canalicular pole. Inhibitors of PI3K partially prevented cytokine-induced loss of villi in cultured HepG2 cells. Notably, mice lacking the PI3Kγ gene were protected against cholestasis and impaired bile acid conjugation. This was partially confirmed by an increase in plasma bile acids (e.g., chenodeoxycholic acid [CDCA] and taurodeoxycholic acid [TDCA]) observed in 48 patients on the day severe sepsis was diagnosed; unlike bilirubin (area under the receiver-operating curve: 0.59), these bile acids predicted 28-d mortality with high sensitivity and specificity (area under the receiver-operating curve: CDCA: 0.77; TDCA: 0.72; CDCA+TDCA: 0.87).
Liver dysfunction is an early and commonplace event in the rat model of sepsis studied here; PI3K signalling seems to play a crucial role. All aspects of hepatic biotransformation are affected, with severity relating to subsequent prognosis. Detected changes significantly precede conventional markers and are reflected by early alterations in plasma bile acids. These observations carry important implications for the diagnosis of liver dysfunction and pharmacotherapy in the critically ill. Further clinical work is necessary to extend these concepts into clinical practice.
Please see later in the article for the Editors' Summary
Editors' Summary
Sepsis (blood poisoning)—a life-threatening condition caused by an inappropriate immune response to an infection—is a major global cause of death. Normally, when bacteria or other microbes enter the human body, the immune system efficiently destroys the invaders. In sepsis the immune system goes into overdrive, and the chemicals it releases into the blood to combat the infection trigger widespread inflammation (swelling). This leads to the formation of small blood clots and leaky blood vessels that block the flow of blood to vital organs such as the kidneys and liver. In the most severe cases, multiple organs fail and the patient dies. Anyone can get sepsis, but people with weakened immune systems, the very young, and the elderly are most vulnerable. Symptoms of sepsis include fever, chills, rapid breathing, a fast heart rate, and confusion. In its early stages, sepsis can be treated with antibiotics alone, but people with severe sepsis need to be admitted to an intensive care unit where the vital organs can be supported while the infection is treated.
Why Was This Study Done?
Thirty to fifty percent of people who develop severe sepsis die. If sepsis could be diagnosed in its early stages, it might be possible to save more people. Unfortunately, the symptoms of sepsis mimic those of other conditions, and, because sepsis tends to develop very quickly, it is often not diagnosed until it is too late to save the patient's life. The development of liver (hepatic) dysfunction and jaundice are both regarded as late features of sepsis (jaundice is yellowing of the skin and eyes caused by a build-up of bilirubin in the blood). However, the researchers hypothesized that changes in liver function occur early in sepsis and could, therefore, be used to improve the diagnosis and management of sepsis.
What Did the Researchers Do and Find?
The researchers induced sepsis in rats by injecting bacteria into the peritoneal cavity (the gap between the abdominal wall and the abdominal organs), separated the infected animals into predicted survivors and non-survivors based on their heart stroke volume measured using cardiac ultrasound, and then examined their liver function. The expression of genes encoding proteins involved in "biotransformation" and "hepatobiliary transport" (the processes that convert waste products and toxic chemicals into substances that can be conjugated to increase solubility and then excreted) was down-regulated within six hours of sepsis induction in the predicted non-survivors compared to the predicted survivors. Functional changes such as bilirubin and bile acid accumulation in the liver (cholestasis), poor excretion of xenobiotics (molecules not usually found in the body such as antibiotics), and disturbed bile acid conjugation were also seen in predicted non-survivors but not in survivors. Moreover, phosphatidylinositol-3-kinase (PI3K) signaling (which is involved in several immune processes) increased soon after sepsis induction in non-survivor but not in survivor animals. Notably, mice lacking the PI3Kγ gene did not develop cholestasis or show impaired bile acid conjugation after induction of sepsis. Finally, in human patients, plasma bile acids were increased in 48 patients on the day that severe sepsis was diagnosed, and these increases accurately predicted death in these patients.
What Do These Findings Mean?
These findings show that liver dysfunction is an early event in animal models of sepsis and that PI3K signalling plays a crucial role in the development of liver dysfunction. They show that all aspects of liver biotransformation are affected during sepsis and suggest that outcomes are related to the severity of these changes. The limited clinical data included in this study also support the hypothesis that changes in liver function occur early in sepsis, although these data need confirming and extending. Taken together, these findings suggest that liver function tests might aid early diagnosis of sepsis and might also provide information about likely outcomes. They also have important implications for the use of drugs in patients who are critically ill with sepsis, in that some of the drugs routinely administered to such patients may not be adequately detoxified and may, therefore, contribute to organ injury. Finally, these findings suggest that inhibition of PI3Kγ may alleviate sepsis-associated cholestasis.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001338.
This study is further discussed in a PLOS Medicine Perspective by John Marshall
The US National Institute of General Medical Sciences has a fact sheet on sepsis
The UK National Health Service Choices website has information about sepsis and about jaundice
The Surviving Sepsis Campaign, which was developed to improve the management, diagnosis, and treatment of sepsis, provides basic information about sepsis
The Sepsis Alliance, a US not-for-profit organization, also provides information about sepsis for patients and their families, including personal stories about sepsis
The not-for profit UK Sepsis Trust is another useful source of information about sepsis that includes patient stories
MedlinePlus provides links to additional resources about sepsis and jaundice (in English and Spanish)
Related Articles
Three-dimensional co-culture of hepatocytes and stellate cells
Abu-Absi, Susan Fugett
Hansen, Linda K.
Hu, Wei-Shou
Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts and sinusoids formed by endothelial cells. Reproducing 3-D co-culture in vitro could provide a means to develop a more complex tissue-like structure. Stellate cells participate in revascularization after liver injury by excreting between hepatocytes a laminin trail that endothelial cells follow to form sinusoids. In this study we investigated co-culture of rat hepatocytes and a rat hepatic stellate cell line, HSC-T6. HSC-T6, which does not grow in serum-free spheroid medium, was able to grow under co-culture conditions. Using a three-dimensional cell tracking technique, the interactions of HSC-T6 and hepatocyte spheroids were visualized. The two cell types formed heterospheroids in culture, and HSC-T6 cell invasion into hepatocyte spheroids and subsequent retraction was observed. RT-PCR revealed that albumin and cytochrome P450 2B1/2 expression were better maintained in co-culture conditions. These three-dimensional heterospheroids provide an attractive system for in vitro studies of hepatocyte-stellate cell interactions.
3-dimentional culture; Co-culture; Hepatocytes; Stellate cell
Related Articles
A Comparative Study of Genome-Wide Transcriptional Profiles of Primary Hepatocytes in Collagen Sandwich and Monolayer Cultures
Kim, Yeonhee
Lasher, Christopher D.
Milford, Logan M.
Murali, T.M.
Rajagopalan, Padmavathy
Tissue Engineering. Part C, Methods
Two commonly used culture systems in hepatic tissue engineering are the collagen sandwich (CS) and monolayers of cells. In this study, genome-wide gene expression profiles of primary hepatocytes were measured over an 8-day period for each cell culture system using Affymetrix GeneChips and compared via gene set enrichment analysis to elicit biologically meaningful information at the level of gene sets. Our results demonstrate that gene expression in hepatocytes in CS cultures steadily and comprehensively diverges from that in monolayer cultures. Gene sets up-regulated in CS cultures include several associated with liver metabolic and synthesis functions, such as metabolism of lipids, amino acids, carbohydrates, and alcohol, and synthesis of bile acids. Monooxygenases such as Cytochrome-P450 enzymes do not show any change between the culture systems after 1 day, but exhibit significant up-regulation in CS cultures after 3 days in comparison to hepatocyte monolayers. These data provide insights into the up- and down-regulation of several liver-critical gene sets and their subsequent effects on liver-specific functions. These results provide a baseline for further explorations into the systems biology of engineered liver mimics.
Related Articles
Designing a Multicellular Organotypic 3D Liver Model with a Detachable, Nanoscale Polymeric Space of Disse
Larkin, Adam L.
Rodrigues, Richard R.
Murali, T.M.
Rajagopalan, Padmavathy
Tissue Engineering. Part C, Methods
The design of in vitro models that mimic the stratified multicellular hepatic microenvironment continues to be challenging. Although several in vitro hepatic cultures have been shown to exhibit liver functions, their physiological relevance is limited due to significant deviation from in vivo cellular composition. We report the assembly of a novel three-dimensional (3D) organotypic liver model incorporating three different cell types (hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells) and a polymeric interface that mimics the Space of Disse. The nanoscale interface is detachable, optically transparent, derived from self-assembled polyelectrolyte multilayers, and exhibits a Young's modulus similar to in vivo values for liver tissue. Only the 3D liver models simultaneously maintain hepatic phenotype and elicit proliferation, while achieving cellular ratios found in vivo. The nanoscale detachable polymeric interfaces can be modulated to mimic basement membranes that exhibit a wide range of physical properties. This facile approach offers a versatile new avenue in the assembly of engineered tissues. These results demonstrate the ability of the tri-cellular 3D cultures to serve as an organotypic hepatic model that elicits proliferation and maintenance of phenotype and in vivo-like cellular ratios.
Related Articles
Overexpression of Cholesterol 7α-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis
Li, Tiangang
Matozel, Michelle
Boehme, Shannon
Kong, Bo
Nilsson, Lisa-Mari
Guo, Grace
Ellis, Ewa
Chiang, John Y. L.
Hepatology (Baltimore, Md.)
We reported previously that mice overexpressing Cyp7a1 (Cyp7a1-tg) are protected against high fat diet-induced hypercholesterolemia, obesity and insulin resistance (1). Here we investigated the underlying mechanism of bile acid signaling in maintaining cholesterol homeostasis in Cyp7a1-tg mice. Cyp7a1-tg mice had 2-fold higher Cyp7a1 activity and bile acid pool than wild type mice. Gallbladder bile acid composition changed from predominantly cholic acid (57%) in wild type to chenodeoxycholic acid (54%) in Cyp7a1-tg mice. Cyp7a1-tg mice had higher biliary and fecal cholesterol and bile acid secretion rates than wild type mice. Surprisingly, hepatic de novo cholesterol synthesis was markedly induced in Cyp7a1-tg mice but intestine fractional cholesterol absorption in Cyp7a1-tg mice remained the same as wild type mice despite increased intestine bile acids. Interestingly, hepatic but not intestinal expression of several cholesterol (ABCG5/G8, SR-B1) and bile acid (ABCB11) transporters were significantly induced in Cyp7a1-tg mice. Treatment of mouse or human hepatocytes with a farnesoid X receptor (FXR) agonist GW4064 or bile acids induced hepatic Abcg5/g8 expression. A functional FXR binding site was identified in the Abcg5 gene promoter. Study of tissue-specific Fxr knockout mice demonstrated that loss of the Fxr gene in the liver attenuated bile acid induction of hepatic Abcg5/g8 and gallbladder cholesterol content, suggesting a role of FXR in the regulation of cholesterol transport. In summary, this study revealed a new mechanism by which increased Cyp7a1 activity expands the hydrophobic bile acid pool, stimulating hepatic cholesterol synthesis and biliary cholesterol secretion without increasing intestinal cholesterol absorption. This study demonstrated that Cyp7a1 plays a critical role in maintaining cholesterol homeostasis and underscores the importance of bile acid signaling in regulating overall cholesterol homeostasis.
Cholesterol 7α-hydroxylase; bile acid synthesis; nuclear receptors; cholesterol transport; ABCG5/G8; SR-BI
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Morphological and Functional Analysis of Hepatocyte Spheroids Generated on Poly-HEMA-Treated Surfaces under the Influence of Fetal Calf Serum and Nonparenchymal Cells
Acikgöz, Ali
Giri, Shibashish
Cho, Man-Gi
Bader, Augustinus
Poly (2-hydroxyethyl methacrylate) (HEMA) has been used as a clinical material, in the form of a soft hydrogel, for various surgical procedures, including endovascular surgery of liver. It is a clear liquid compound and, as a soft, flexible, water-absorbing material, has been used to make soft contact lenses from small, concave, spinning molds. Primary rat hepatocyte spheroids were created on a poly-HEMA-coated surface with the intention of inducing hepatic tissue formation and improving liver functions. We investigated spheroid formation of primary adult rat hepatocyte cells and characterized hepatic-specific functions under the special influence of fetal calf serum (FCS) and nonparencymal cells (NPC) up to six days in different culture systems (e.g., hepatocytes + FCS, hepatocytes – FCS, NPC + FCS, NPC – FCS, co-culture + FCS, co-culture – FCS) in both the spheroid model and sandwich model. Immunohistologically, we detected gap junctions, Ito cell/Kupffer cells, sinusoidal endothelial cells and an extracellular matrix in the spheroid model. FCS has no positive effect in the sandwich model, but has a negative effect in the spheroid model on albumin production, and no influence in urea production in either model. We found more cell viability in smaller diameter spheroids than larger ones by using the apoptosis test. Furthermore, there is no positive influence of the serum or NPC on spheroid formation, suggesting that it may only depend on the physical condition of the culture system. Since the sandwich culture has been considered a "gold standard" in vitro culture model, the hepatocyte spheroids generated on the poly-HEMA-coated surface were compared with those in the sandwich model. Major liver-specific functions, such as albumin secretion and urea synthesis, were evaluated in both the spheroid and sandwich model. The synthesis performance in the spheroid compared to the sandwich culture increases approximately by a factor of 1.5. Disintegration of plasma membranes in both models was measured by lactate dehydrogenase (LDH) release in both models. Additionally, diazepam was used as a substrate in drug metabolism studies to characterize the differences in the biotransformation potential with metabolite profiles in both models. It showed that the diazepam metabolism activities in the spheroid model is about 10-fold lower than the sandwich model. The poly-HEMA-based hepatocyte spheroid is a promising new platform towards hepatic tissue engineering leading to in vitro hepatic tissue formation.
diazepam; fetal calf serum; poly-HEMA; rat hepatocyte; nonparencymal cells; sandwich model; spheroid model
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HepatoNet1: a comprehensive metabolic reconstruction of the human hepatocyte for the analysis of liver physiology
Gille, Christoph
Bölling, Christian
Hoppe, Andreas
Bulik, Sascha
Hoffmann, Sabrina
Hübner, Katrin
Karlstädt, Anja
Ganeshan, Ramanan
König, Matthias
Rother, Kristian
Weidlich, Michael
Behre, Jörn
Holzhütter, Herrmann-Georg
Molecular Systems Biology
We present HepatoNet1, a manually curated large-scale metabolic network of the human hepatocyte that encompasses >2500 reactions in six intracellular and two extracellular compartments.Using constraint-based modeling techniques, the network has been validated to replicate numerous metabolic functions of hepatocytes corresponding to a reference set of diverse physiological liver functions.Taking the detoxification of ammonia and the formation of bile acids as examples, we show how these liver-specific metabolic objectives can be achieved by the variable interplay of various metabolic pathways under varying conditions of nutrients and oxygen availability.
The liver has a pivotal function in metabolic homeostasis of the human body. Hepatocytes are the principal site of the metabolic conversions that underlie diverse physiological functions of the liver. These functions include provision and homeostasis of carbohydrates, amino acids, lipids and lipoproteins in the systemic blood circulation, biotransformation, plasma protein synthesis and bile formation, to name a few. Accordingly, hepatocyte metabolism integrates a vast array of differentially regulated biochemical activities and is highly responsive to environmental perturbations such as changes in portal blood composition (Dardevet et al, 2006). The complexity of this metabolic network and the numerous physiological functions to be achieved within a highly variable physiological environment necessitate an integrated approach with the aim of understanding liver metabolism at a systems level. To this end, we present HepatoNet1, a stoichiometric network of human hepatocyte metabolism characterized by (i) comprehensive coverage of known biochemical activities of hepatocytes and (ii) due representation of the biochemical and physiological functions of hepatocytes as functional network states. The network comprises 777 metabolites in six intracellular (cytosol, endoplasmic reticulum and Golgi apparatus, lysosome, mitochondria, nucleus, and peroxisome) and two extracellular compartments (bile canaliculus and sinusoidal space) and 2539 reactions, including 1466 transport reactions. It is based on the manual evaluation of >1500 original scientific research publications to warrant a high-quality evidence-based model. The final network is the result of an iterative process of data compilation and rigorous computational testing of network functionality by means of constraint-based modeling techniques. We performed flux-balance analyses to validate whether for >300 different metabolic objectives a non-zero stationary flux distribution could be established in the network. Figure 1 shows one such functional flux mode associated with the synthesis of the bile acid glycochenodeoxycholate, one important hepatocyte-specific physiological liver function. Besides those pathways directly linked to the synthesis of the bile acid, the mevalonate pathway and the de novo synthesis of cholesterol, the flux mode comprises additional pathways such as gluconeogenesis, the pentose phosphate pathway or the ornithine cycle because the calculations were routinely performed on a minimal set of exchangeable metabolites, that is all reactants were forced to be balanced and all exportable intermediates had to be catabolized into non-degradable end products. This example shows how HepatoNet1 under the challenges of limited exchange across the network boundary can reveal numerous cross-links between metabolic pathways traditionally perceived as separate entities. For example, alanine is used as gluconeogenic substrate to form glucose-6-phosphate, which is used in the pentose phosphate pathway to generate NADPH. The glycine moiety for bile acid conjugation is derived from serine. Conversion of ammonia into non-toxic nitrogen compounds is one central homeostatic function of hepatocytes. Using the HepatoNet1 model, we investigated, as another example of a complex metabolic objective dependent on systemic physiological parameters, how the consumption of oxygen, glucose and palmitate is affected when an external nitrogen load is converted in varying proportions to the non-toxic nitrogen compounds: urea, glutamine and alanine. The results reveal strong dependencies between the available level of oxygen and the substrate demand of hepatocytes required for effective ammonia detoxification by the liver.
Oxygen demand is highest if nitrogen is exclusively transformed into urea. At lower fluxes into urea, an intriguing pattern for oxygen demand is predicted: oxygen demand attains a minimum if the nitrogen load is directed to urea, glutamine and alanine with relative fluxes of 0.17, 0.43 and 0.40, respectively (Figure 2A). Oxygen demand in this flux distribution is four times lower than for the maximum (100% urea) and still 77 and 33% lower than using alanine and glutamine as exclusive nitrogen compounds, respectively. This computationally predicted tendency is consistent with the notion that the zonation of ammonia detoxification, that is the preferential conversion of ammonia to urea in periportal hepatocytes and to glutamine in perivenous hepatocytes, is dictated by the availability of oxygen (Gebhardt, 1992; Jungermann and Kietzmann, 2000). The decreased oxygen demand in flux distributions using higher proportions of glutamine or alanine is accompanied by increased uptake of the substrates glucose and palmitate (Figure 2B). This is due to an increased demand of energy and carbon for the amidation and transamination of glutamate and pyruvate to discharge nitrogen in the form of glutamine and alanine, respectively. In terms of both scope and specificity, our model bridges the scale between models constructed specifically to examine distinct metabolic processes of the liver and modeling based on a global representation of human metabolism. The former include models for the interdependence of gluconeogenesis and fatty-acid catabolism (Chalhoub et al, 2007), impairment of glucose production in von Gierke's and Hers' diseases (Beard and Qian, 2005) and other processes (Calik and Akbay, 2000; Stucki and Urbanczik, 2005; Ohno et al, 2008). The hallmark of these models is that each of them focuses on a small number of reactions pertinent to the metabolic function of interest embedded in a customized representation of the principal pathways of central metabolism. HepatoNet1, currently, outperforms liver-specific models computationally predicted (Shlomi et al, 2008) on the basis of global reconstructions of human metabolism (Duarte et al, 2007; Ma and Goryanin, 2008). In contrast to either of the aforementioned modeling scales, HepatoNet1 provides the combination of a system-scale representation of metabolic activities and representation of the cell type-specific physical boundaries and their specific transport capacities. This allows for a highly versatile use of the model for the analysis of various liver-specific physiological functions. Conceptually, from a biological system perspective, this type of model offers a large degree of comprehensiveness, whereas retaining tissue specificity, a fundamental design principle of mammalian metabolism. HepatoNet1 is expected to provide a structural platform for computational studies on liver function. The results presented herein highlight how internal fluxes of hepatocyte metabolism and the interplay with systemic physiological parameters can be analyzed with constraint-based modeling techniques. At the same time, the framework may serve as a scaffold for complementation of kinetic and regulatory properties of enzymes and transporters for analysis of sub-networks with topological or kinetic modeling methods.
We present HepatoNet1, the first reconstruction of a comprehensive metabolic network of the human hepatocyte that is shown to accomplish a large canon of known metabolic liver functions. The network comprises 777 metabolites in six intracellular and two extracellular compartments and 2539 reactions, including 1466 transport reactions. It is based on the manual evaluation of >1500 original scientific research publications to warrant a high-quality evidence-based model. The final network is the result of an iterative process of data compilation and rigorous computational testing of network functionality by means of constraint-based modeling techniques. Taking the hepatic detoxification of ammonia as an example, we show how the availability of nutrients and oxygen may modulate the interplay of various metabolic pathways to allow an efficient response of the liver to perturbations of the homeostasis of blood compounds.
computational biology; flux balance; liver; minimal flux
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Increased Bile Acids in Enterohepatic Circulation by Short-term Calorie Restriction in Male Mice
Fu, Zidong Donna
Klaassen, Curtis D.
Toxicology and applied pharmacology
Previous studies showed glucose and insulin signaling can regulate bile acid (BA) metabolism during fasting or feeding. However, limited knowledge is available on the effect of calorie restriction (CR), a well-known anti-aging intervention, on BA homeostasis. To address this, the present study utilized a "dose-response" model of CR, where male C57BL/6 mice were fed 0, 15, 30, or 40% CR diets for one month, followed by BA profiling in various compartments of the enterohepatic circulation by UPLC-MS/MS technique. This study showed that 40% CR increased the BA pool size (162%) as well as total BAs in serum, gallbladder, and small intestinal contents. In addition, CR "dose-dependently" increased the concentrations of tauro-cholic acid (TCA) and many secondary BAs (produced by intestinal bacteria) in serum, such as tauro-deoxycholic acid (TDCA), DCA, lithocholic acid, ω-muricholic acid (ωMCA), and hyodeoxycholic acid. Notably, 40% CR increased TDCA over 1000% (serum, liver, and gallbladder). Interestingly, 40% CR increased the proportion of 12α-hydroxylated BAs (CA and DCA), which correlated with improved glucose tolerance and lipid parameters. The CR-induced increase in BAs correlated with increased expression of BA-synthetic (Cyp7a1) and conjugating enzymes (BAL), and the ileal BA-binding protein (Ibabp). These results suggest that CR increases BAs in male mice possibly through orchestrated increases in BA synthesis and conjugation in liver as well as intracellular transport in ileum.
Calorie restriction; Bile acids; Enterohepatic circulation; 12α-hydroxylated BAs; BA profiling; UPLC-MS/MS
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Deletion of mouse FXR gene disturbs multiple neurotransmitter systems and alters neurobehavior
Huang, Fei
Wang, Tingting
Lan, Yunyi
Yang, Li
Pan, Weihong
Zhu, Yonghui
Lv, Boyang
Wei, Yuting
Shi, Hailian
Wu, Hui
Zhang, Beibei
Wang, Jie
Duan, Xiaofeng
Hu, Zhibi
Wu, Xiaojun
Frontiers in Behavioral Neuroscience
Farnesoid X receptor (FXR) is a nuclear hormone receptor involved in bile acid synthesis and homeostasis. Dysfunction of FXR is involved in cholestasis and atherosclerosis. FXR is prevalent in liver, gallbladder, and intestine, but it is not yet clear whether it modulates neurobehavior. In the current study, we tested the hypothesis that mouse FXR deficiency affects a specific subset of neurotransmitters and results in an unique behavioral phenotype. The FXR knockout mice showed less depressive-like and anxiety-related behavior, but increased motor activity. They had impaired memory and reduced motor coordination. There were changes of glutamatergic, GABAergic, serotoninergic, and norepinephrinergic neurotransmission in either hippocampus or cerebellum. FXR deletion decreased the amount of the GABA synthesis enzyme GAD65 in hippocampus but increased GABA transporter GAT1 in cerebral cortex. FXR deletion increased serum concentrations of many bile acids, including taurodehydrocholic acid, taurocholic acid, deoxycholic acid (DCA), glycocholic acid (GCA), tauro-α-muricholic acid, tauro-ω-muricholic acid, and hyodeoxycholic acid (HDCA). There were also changes in brain concentrations of taurocholic acid, taurodehydrocholic acid, tauro-ω-muricholic acid, tauro-β-muricholic acid, deoxycholic acid, and lithocholic acid (LCA). Taken together, the results from studies with FXR knockout mice suggest that FXR contributes to the homeostasis of multiple neurotransmitter systems in different brain regions and modulates neurobehavior. The effect appears to be at least partially mediated by bile acids that are known to cross the blood-brain barrier (BBB) inducing potential neurotoxicity.
FXR; bile acid; neurobehavior; neurotransmitter; neurotransmission; emotion; memory; motor performance
Related Articles
Cyclical oxidation-reduction of the C3 position on bile acids catalyzed by 3 alpha-hydroxysteroid dehydrogenase. II. Studies in the prograde and retrograde single-pass, perfused rat liver and inhibition by indomethacin.
Takikawa, H
Ookhtens, M
Stolz, A
Kaplowitz, N
Journal of Clinical Investigation
[3 beta-3H, 24-14C]Lithocholic, chenodeoxycholic, and cholic acids were administered in tracer bolus doses either prograde or retrograde in the isolated perfused rat liver. Little 3H loss from cholic acid was observed, whereas with the other bile acids, 20-40% of the administered 3H was lost in a single pass from perfusate to bile. Most of the 3H loss occurred rapidly (5 min) and was recovered as [3H]water in perfusate. Excretion of bile acids was delayed with retrograde administration, and 3H loss was more extensive. In both prograde and retrograde studies, indomethacin markedly inhibited the excretion of the bolus of bile acid into bile. Indomethacin inhibited the extraction of glycocholate (50 microM) during steady state perfusion without affecting transport maximum for excretion. At lower glycocholate concentration (5 microM), indomethacin inhibited both extraction and excretion. A greater effect was seen on excretion in the latter case, which suggests that displacement of bile acid from the cytosolic protein lead to redistribution in the hepatocyte as well as reflux into the sinusoid. These data suggest that binding of bile acids to cytosolic 3 alpha-hydroxysteroid dehydrogenases occurs extensively during hepatic transit and is important in mediating the translocation of bile acids from the sinusoidal to canalicular pole of the cell.
Related Articles
Dietary fish oil-induced changes in intrahepatic cholesterol transport and bile acid synthesis in rats.
Smit, M J
Temmerman, A M
Wolters, H
Kuipers, F
Beynen, A C
Vonk, R J
Journal of Clinical Investigation
Hepatic cholesterol metabolism was studied in rats fed purified diets supplemented (9% wt/wt) with either fish oil (FO) (n-3 fatty acids) or corn oil (CO) (n-6 fatty acids) for 4 wk. Rats were equipped with permanent catheters in heart, bile duct, and duodenum to allow studies under normal feeding conditions. [3H]-cholesteryl oleate-labeled small unilamellar liposomes, which are rapidly endocytosed by hepatocytes, were intravenously injected to label intrahepatic cholesterol pools, and plasma and bile were collected. FO as compared to CO induced a lowering of plasma cholesterol levels by 38% and of triglyceride levels by 69%. This reduction in plasma lipids in FO rats was accompanied by: (a) an increased bile acid pool size (28%); (b) a fourfold increase in the ratio cholic acid/chenodeoxycholic acid in bile; (c) increased biliary excretion of cholesterol (51%); (d) accelerated excretion of endocytosed free cholesterol into bile; (e) accelerated incorporation of endocytosed cholesterol in bile acids; (f) a significant increase in the bile acid-independent fraction of bile flow; and (g) a threefold increase in hepatic alkaline phosphatase activity. The results show that FO induces changes in transport and metabolic pathways of cholesterol in the rat liver, which result in a more rapid disposition of plasma-derived cholesterol into the bile.
Related Articles
Suppression of bile acid synthesis by thyroid hormone in primary human hepatocytes
Ellis, Ewa Cristine Siljevik
World Journal of Gastroenterology : WJG
AIM: It is known that thyroid hormones alter the bile acid metabolism in humans, however the effect on individual enzymes has been difficult to elucidate. This is mainly due to the lack of human liver cell lines producing bile acids. We used cultures of primary human hepatocytes to study the effects of triiodothyronine (T3) on bile acid synthesis.
METHODS: Primary hepatocytes were isolated from liver tissue obtained from three different patients undergoing liver resection due to underlying malignancy. The hepatocytes were cultured under serum-free conditions and treated from d 1 to d 5 with culture containing 0.1 - 1000 nmol/L of T3. Bile acid formation and mRNA levels of key enzymes were analysed.
RESULTS: The lowest concentration of T3 decreased cholic acid (CA) formation to 43%-53% of controls and chenodeoxycholic acid (CDCA) to 52%-75% of controls on d 5. The highest dose further decreased CA formation to 16%-48% of controls while CDCA formation remained at 50%-117% of controls. Expression of mRNA levels of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) dose-dependently decreased. Sterol 27-hydroxylase (CYP27A1) levels also decreased, but not to the same extent.
CONCLUSION: T3 dose-dependently decreased total bile acid formation in parallel with decreased expression of CYP7A1 and CYP8B1. CA formation is inhibited to a higher degree than CDCA, resulting in a marked decrease in the CA /CDCA ratio.
Hepatocytes; Human; Bile acids; CYP7A1; CYP8B1; CYP27A1
Related Articles
Delta 4-3-oxosteroid 5 beta-reductase deficiency: failure of ursodeoxycholic acid treatment and response to chenodeoxycholic acid plus cholic acid.
Clayton, P T
Mills, K A
Johnson, A W
Barabino, A
Marazzi, M G
BACKGROUND--In some infants with liver disease, 3-oxo-delta 4 bile acids are the major bile acids in urine, a phenomenon attributed to reduced activity of the delta 4-3-oxosteroid 5 beta-reductase required for synthesis of chenodeoxycholic acid and cholic acid. These patients form a heterogeneous group. Many have a known cause of hepatic dysfunction and plasma concentrations of chenodeoxycholic acid and cholic acid that are actually greater than those of the 3-oxo-delta 4 bile acids. It is unlikely that these patients have a primary genetic deficiency of the 5 beta-reductase enzyme. AIMS--To document the bile acid profile, clinical phenotype, and response to treatment of an infant with cholestasis, increased plasma concentrations of 3-oxo-delta 4 bile acids, low plasma concentrations of chenodeoxycholic acid and cholic acid, and no other identifiable cause of liver disease. PATIENTS--This infant was compared with normal infants and infants with cholestasis of known cause. METHODS--Analysis of bile acids by liquid secondary ionisation mass spectrometry and gas chromatography-mass spectrometry. RESULTS--The plasma bile acid profile of the patient was unique. She had chronic cholestatic liver disease associated with malabsorption of vitamins D and E and a normal gamma-glutamyltranspeptidase when the transaminases were increased. The liver disease failed to improve with ursodeoxycholic acid but responded to a combination of chenodeoxycholic acid and cholic acid. CONCLUSION--Treatment of primary 5 beta-reductase deficiency requires the use of bile acids that inhibit cholesterol 7 alpha-hydroxylase.
Related Articles
Defective peroxisomal cleavage of the C27-steroid side chain in the cerebro-hepato-renal syndrome of Zellweger.
Kase, B F
Björkhem, I
Hågå, P
Pedersen, J I
Journal of Clinical Investigation
Based on in vitro work with rat liver, we recently suggested that the peroxisomal fraction is most important for the oxidation of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid. The cerebro-hepato-renal syndrome of Zellweger is a fatal recessive autosomal disorder, the most characteristic histological feature of which is a virtual absence of peroxisomes in liver and kidneys. This disease offers a unique opportunity to evaluate the relative importance of peroxisomes in bile acid biosynthesis. A child with Zellweger syndrome was studied in the present work. In accordance with previous work, there was a considerable accumulation of THCA, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid (24-OH-THCA), 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid (C29-dicarboxylic acid), and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid in serum. In addition, a tetrahydroxylated 5 beta-cholestanoic acid with all the hydroxyl groups in the steroid nucleus was found. 3H-Labeled 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol was administered intravenously together with 14C-labeled cholic acid. There was a rapid incorporation of 3H in THCA and a slow incorporation into cholic acid. The specific radioactivity of 3H in THCA was about one magnitude higher than that in cholic acid. The conversion was evaluated by following the increasing ratio between 3H and 14C in biliary cholic acid. The rate of incorporation of 3H in cholic acid was considerably less than previously reported in experiments with healthy subjects, and the maximal conversion of the triol into cholic acid was only 15-20%. About the same rate of conversion was found after oral administration of 3H-THCA. Both in the experiment with 3H-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and with 3H-THCA, there was an efficient incorporation of 3H in the above unidentified tetrahydroxylated 5 beta-cholestanoic acid. There was only slow incorporation of radioactivity into 24-OH-THCA and into the C29-dicarboxylic acid. From the specific activity decay curve of 14C in cholic acid obtained after intravenous injection of 14C-cholic acid, the pool size of cholic acid was calculated to be 24 mg/m2 and the daily production rate to 9 mg/m2 per d. This corresponds to a reduction of approximately 85 and 90%, respectively, when compared with normal infants. It is concluded that liver peroxisomes are essential in the normal conversion of THCA to cholic acid. In the Zellweger syndrome this conversion is defective and as a consequence the accumulated THCA is either excreted as such or transformed into other metabolites by hydroxylation or side chain elongation. The accumulation of THCA, as well as the similar rate of conversion of 5 beta-cholestane-3 alpha,7 alpha.12 alpha-triol and THCA into cholic acid, support the contention that the 26-hydroxylase pathway with intermediate formation of THCA is the most important pathway for formation of cholic acid in man.
Related Articles
Role of the 26-hydroxylase in the biosynthesis of bile acids in the normal state and in cerebrotendinous xanthomatosis. An in vivo study.
Björkhem, I
Fausa, O
Hopen, G
Oftebro, H
Pedersen, J I
Skrede, S
Journal of Clinical Investigation
On the basis of different in vitro studies, we have previously suggested that the basic metabolic defect in the rare inherited disease cerebrotendinous xanthomatosis (CTX) is a lack of a hepatic mitochondrial C27-steroid 26-hydroxylase, involved in the normal biosynthesis of bile acids (1980. J. Clin. Invest. 65: 1418-1430; 1981. J. Lipid Res. 22: 191-200; 22: 632-640). In the present work, this hypothesis was tested in vivo. One patient with CTX and two control subjects received intravenously a mixture of [4-14C]7 alpha-hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha,26-dihydroxy-4-cholesten-3-one, steroids believed to be important precursors of chenodeoxycholic acid. The ratio between 14C and 3H in cholic acid and chenodeoxycholic acid isolated from bile of the CTX-patient was approximately 1/40 and 1/60 of those of the control subjects, respectively. Another patient with CTX and one control subject received a mixture of [4-14C]5 beta-cholestane-3 alpha,7 alpha-diol and [1,2-3H]5 beta-cholestane-3 alpha,7 alpha,26-triol, both possible precursors to chenodeoxycholic acid. In this case the 14C/3H ratio in cholic acid and chenodeoxycholic acid from the patient with CTX was 1/10 and 1/15, respectively, compared with that of the control subject. The most likely explanation for these findings is that very little of the 14C-precursors, i.e. without a 26-hydroxyl group, can be converted into cholic acid and chenodeoxycholic acid because of a defect of the 26-hydroxylase step. The results obtained are in accord with our previous findings in vitro. The results further underline the importance of the 26-hydroxylase pathway in the normal biosynthesis of cholic acid and chenodeoxycholic acid in man.
Related Articles
Immunoperoxidase localization of bile salts in rat liver cells. Evidence for a role of the Golgi apparatus in bile salt transport.
Lamri, Y
Roda, A
Dumont, M
Feldmann, G
Erlinger, S
Journal of Clinical Investigation
The mechanisms of intracellular transport of bile acids from the sinusoidal pole to the canalicular pole of the hepatocyte are poorly understood. There is physiological and autoradiographic evidence for a vesicular pathway. The purpose of this study was to determine the localization of natural bile acids in the liver using antibodies against cholic acid conjugates and ursodeoxycholic acid. An indirect immunoperoxidase technique was used on rat liver sections fixed either with paraformaldehyde (PF) and saponin, a membrane-permeabilizing agent that allows penetration of antibodies into the cell, or with PF alone. Retention of taurocholate in the liver after tissue processing was 26 +/- SD 15% of the bile acid initially present. When sections fixed with PF and saponin were incubated with the antibody against cholic acid conjugates, a granular cytoplasmic staining was observed by light microscopy in all hepatocytes. By electron microscopy, strong electron-dense deposits were observed mostly on vesicles of the Golgi apparatus (GA) and, sometimes, in the smooth endoplasmic reticulum (SER). After taurocholate infusion, the intensity of the reaction increased. When the liver was fixed with PF alone, almost no reaction was visible on light microscopy, but on electron microscopy the label was localized on the hepatocyte plasma membrane, mainly on the bile canalicular domain and to a lesser extent on the sinusoidal domain. With the antibody against ursodeoxycholic acid, no staining was observed in three of four livers, and a slight staining was observed in one. However, after infusion of ursodeoxycholic acid, staining of GA and SER vesicles was observed when the liver was fixed with PF and saponin. With PF alone, the reaction was intense on the canalicular membrane. These results support the view that, within the limits of the method, vesicles from the GA and possibly vesicles of the SER are involved in the intracellular transport of bile acids before canalicular secretion.
Related Articles
Regulation of Bile Salt Transport in Rat Liver
Simon, Francis R.
Sutherland, Eileen M.
Gonzalez, Manuel
Journal of Clinical Investigation
Expansion of the bile salt pool size in rats increases maximum excretory capacity for taurocholate. We examined whether increased bile salt transport is due to recruitment of centrolobular transport units or rather to adaptive changes in the hepatocyte. Daily sodium cholate (100 mg/100 g body wt) was administered orally to rats. This treatment was well tolerated for at least 4 d and produced an 8.2-fold expansion of the bile salt pool. This expanded pool consisted predominently (99%) of cholic and deoxycholic acids. Significantly increased bile salt transport was not observed until 16 h after bile acid loading, and maximum elevations of transport capacity to 2.3-fold of control required ∼2 d. In contrast, maximum sulfobromophthalein excretion rates increased 2.2-fold as early as 4 h and actually fell to 1.5-fold increase at 4 d. We studied the possibility that this adaptive increase in bile salt secretory transport was due to changes in canalicular surface membrane area, lipid composition, or increased number of putative carriers. Canalicular membrane protein recovery and the specific activities of leucine aminopeptidase, Mg++-ATPase and 5′-nucleotidase activities were unaltered by bile salt pool expansion. The content of free and esterified cholesterol and total phospholipids was unchanged in liver surface membrane fractions compared with control values. In contrast, sodium cholate administration selectively increased specific [14C]cholic acid binding sites twofold in liver surface membrane fractions. Increased numbers of [14C]cholic acid receptors (a) was associated with the time-dependent increase in bile salt transport, and (b) was selective for the taurine conjugate of cholate and (c) was reduced by chenodeoxycholate. Changes in bile acid binding sites 16 h following taurocholate and chenodeoxycholate and the lack of change with glycocholate was associated with comparable changes in bile salt transport. In conclusion, selective bile salts increase bile salt transport in the liver through an adaptive increase in the density of putative bile acid carriers in liver surface membrane.
Related Articles
Effect of crilvastatin, a new cholesterol lowering agent, on unesterified LDL-cholesterol metabolism into bile salts by rat isolated hepatocytes.
Clerc, T
Sbarra, V
Diaconescu, N
Lafont, H
Jadot, G
Laruelle, C
Chanussot, F
British Journal of Pharmacology
1. The aim of these experiments was to determine the effect of crilvastatin, a new cholesterol lowering agent, on the metabolism of unesterified low density lipoprotein (LDL)-cholesterol by rat freshly isolated hepatocytes. This preclinical model was developed as an alternative to in vivo experiments, to mimic the metabolic effects of a molecule on its target cells and to define optimal conditions for future experimentation on human hepatocytes. 2. Cells were obtained from normolipidaemic or hypercholesterolaemic rats, hypercholesterolaemia was nutritionally induced. Incubations were performed in a medium containing 600 microM taurocholate and 50 microM or 300 microM crilvastatin. 3. This molecule was shown in vitro to be carried by physiological transporters, i.e., albumin-bile salt micellar associations and LDL. Crilvastatin induced a significance increase in the synthesis and secretion by hepatocytes of bile salts resulting from the metabolism of unesterified LDL-cholesterol in both normolipidaemic and hypercholesterolaemic rats. Stimulation involved non-conjugated as well as tauro- and glyco-conjugated bile salts. These findings corroborate preliminary studies showing in vivo that crilvastatin enhances the secretion of bile acids by stimulating the uptake and incorporation of LDL-cholesterol by the liver.
Related Articles
Identification and characterization of a bile acid receptor in isolated liver surface membranes.
Accatino, L
Simon, F R
Journal of Clinical Investigation
It is generally assumed that hepatic transport of bile acids is a carrier-mediated process. However, the basic mechanisms by which these organic anions are translocated across the liver cell surface membrane are not well understood. Since carrier-mediated transport involved binding of the transported molecule to specific receptor sites, we have investigated the possibility that bile acid receptors are present in liver surface membranes. Isolated liver surface membranes were incubated at 4 degrees C with [14C]cholic acid and [14C]taurocholic acid, and membrane-boudn bile acid was separated from free by a rapid ultrafiltration technique through glass-fiber filters. Specific bile acid binding is rapid and reversible and represents approximately 80% of the total bile acid bound to liver surface membranes. Taurocholic acid binding is independent of the medium pH, while cholic acid binding demonstrates an optimum at pH 6.0. Analysis of equilibrium data for both cholic and taurocholic acid binding indicates that specific binding is saturable and consistent with Michaelis-Menten kinetics, while nonspecific binding is nonsaturable. Apparent maximal binding capacity and dissociation constant values indicate a large capacity system of receptors that have an affinity for bile acids comparable to that of the hepatic transport mechanism. Scatchard analysis of the saturation kinetics as well as inhibition studies suggest that bile acids bind to a single and noninteracting class of anion that competes with bile acids for hepatic uptake, also inhibits cholic acid binding. In contrast, no inhibition was demonstrated with indocyanine green and probenecid. Specific bile acid binding is enriched and primarily located in liver surface membranes and found only in tissues involved in bile acid transport. Specific bile acid binding is independnet of Na+, Ca2+, and Mg2+ and does not require metabolic energy. In addition, thiol groups and disulfide are not required for activity at the binding site. However, specific bile acid binding is markedly decreased by low concentrations of proteolytic enzymes and is also decreased by the action of neuraminidase and phospholipases A and C. These results are consistent with the existence of a homogeneous bile acid receptor protein in liver surface membranes. The primary surface membrane location of this receptor, its binding properties, and its ligand specificity suggest that bile acid binding to this receptor may represent the initial interaction in bile acid transport across liver surface membranes.
Related Articles
Hepatic Uptake of Bile Acids in Man
Angelin, Bo
Björkhem, Ingemar
Einarsson, Kurt
Ewerth, Staffan
Journal of Clinical Investigation
This investigation was undertaken in order to (a) characterize the postprandial inflow of individual bile acids to the liver and (b) determine if peripheral venous bile acid levels always adequately reflect the portal venous concentration, or if saturation of hepatic bile acid uptake can occur under physiological conditions. In five patients with uncomplicated cholesterol gallstone disease, the umbilical cord was cannulated during cholecystectomy, and a catheter was left in the left portal branch for 5 to 7 d. The serum concentrations of cholic acid, chenodeoxycholic acid, and deoxycholic acid in portal venous and systemic circulation were then determined at intervals of 15 to 30 min before and after a standardized meal. A highly accurate and specific gas chromatographic/mass spectrometric technique was used.
The sum of the fasting concentrations of the three bile acids averaged 14.04±4.13 μmol/liter in portal venous serum, and 2.44±0.31 μmol/liter in peripheral venous serum. The estimated hepatic fractional uptake of cholic acid was ∼90%, and those of chenodeoxycholic acid and deoxycholic acid were 70-80%. This resulted in an enrichment of systemic bile acids in the dihydroxy bile acid species. In response to a standardized meal, portal venous bile acid concentrations increased two- to sixfold, with a peak seen 15-60 min after the meal. The maximum postprandial portal venous bile acid concentration averaged 43.04±6.12 μmol/liter, and the corresponding concentration in peripheral serum was 5.22±0.74 μmol/liter. The estimated fractional uptakes of the individual bile acids were not affected by the increased inflow to the liver. The peripheral venous concentrations of individual as well as total bile acids were well correlated with those in portal venous serum.
The results (a) give a quantitation of postprandial bile acid inflow to the liver and (b) indicate that the hepatic uptake system for bile acids in healthy man cannot be saturated during maximal inflow of endogenous bile acids. Measurement of peripheral serum bile acids can thus give important information on the status of the enterohepatic circulation.
Related Articles
Joseph, Brigid
Kapoor, Sorabh
Schilsky, Michael L.
Gupta, Sanjeev
Hepatology (Baltimore, Md.)
Insights into disease-specific mechanisms for liver repopulation are needed for cell therapy. To understand the efficacy of pro-oxidant hepatic perturbations in Wilson disease, we studied Long-Evans Cinnamon rats with copper toxicosis under several conditions. Hepatocytes from healthy Long-Evans Agouti rats were transplanted intrasplenically into the liver. A cure was defined as lowering of copper to below 250 micrograms per gram liver, presence of atp7b mRNA in the liver and improvement in liver histology. Treatment of animals with the hydrophobic bile salt, cholic acid, or liver radiation before cell transplantation produced cure rates of 14% and 33%, respectively; whereas liver radiation plus partial hepatectomy followed bv cell transplantation proved more effective with cure in 55%, p<0.01; and liver radiation plus cholic acid followed by cell transplantation was most effective, with cure in 75%, p<0.001. As a group, cell therapy cures in rats preconditioned with liver radiation plus cholic acid resulted in less hepatic copper indicating greater extent of liver repopulation. We observed increased hepatic catalase and superoxide dismutase activities in Long-Evans Cinnamon rats, suggesting chronic oxidative stress. After liver radiation and/or cholic acid, hepatic lipid peroxidation levels increased, indicating further oxidative injury., although we did not observe overt additional cytotoxicity. This contrasted with healthy animals where liver radiation and cholic acid produced hepatic steatosis and loss of injured hepatocytes. We concluded that pro-oxidant perturbations were uniquely effective for cell therapy in Wilson disease due to the nature of pre-existing hepatic damage.
Cell transplantation; Cholic acid; Liver; Radiation; Wilson disease
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The NCJW Hannah G. Solomon Award Committee is pleased to announce
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The US Supreme Court will be a major issue in the November election campaigns. In September, NCJW brought together a panel of female experts to talk about some of the issues and cases that the Supreme Court decided in this last term. Thank you to our speakers for their insight.
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Download Jared's presentation and share it with others in your organization. Jared just asks that you don't make it available on a public site.
UIE is offering Meetup members 2 months of complimentary access to UIE's All You Can Learn Library. There are 120 seminars to explore and new seminars and conference recordings are added on a regular basis. Some of the topics include: visual design, user research, design process, information architecture, interaction design, and content strategy.
And good news. Jared is back in Denver for the UX Immersion Mobile Conference April 7-9. If creating great mobile experiences is high on your company's agenda, you won't want to miss the conference. Register with promotion code JTALK and get $200 off the price (price is going up Feb. 11, so register soon). Learn more about the conference at UXIM.com.
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Eleanora V. Mulroy, 96, homemaker in Quincy
Eleanora V. (Bonomi) Mulroy of Quincy, a homemaker, died Wednesday. She 96.
Born in Milton, Mrs. Mulroy was a participant in the Quincy Senior Olympics and received many medals. She was very proud of her dedication and hard work.
She was fluent in several languages, all of which she taught herself. She enjoyed golf, bridge, crossword puzzles, traveling to Cape Cod, and spending time with her family.
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Odd Fellows Retirement Manor Inc Retirement Homes
Reno, Nevada, 89512 USA
who are we?top
��Ron Bradley
��Ron Bradley.
��Diane Bradley.
��Helen White. FOOD
��Dee Thomas.
��Rooms 118
�� Max res 118
�� Entrance fee $765- $865
�� Avg monthly fees $0-$1,000
�� Min age 55
��Central dining
�� Housekeeping
�� Transportation
�� Social and recreational
�� Supervision of medications
�� Security
�� Library
�� Religious activities
�� Maintenance
�� Emergency call system
�� Laundry
��Private non-profit
��Odd Fellow & Rebekah Lodge of NV
Retirement Homes & Senior Housing
A place in a retirement home can be paid for on a rental basis, like an apartment, or can be bought in perpetuity on the same basis as a condominium. Some facilities provide minor help with medications as well. Senior Housing - find independent and assisted living facilities, nursing homes, retirement communities, and other elderly care. Independent living is a general name for any housing arrangement designed exclusively for seniors. Assisted living residences or assisted living facilities provide supervision or assistance with activities of daily living. Some facilities provide minor help with medications as well. Independent Living Communities enable residents to live as independently as possible, while providing certain services and social activities. An assisted living facility may be a good choice if you need more personal care services than are feasible at home or in an independent living retirement community. You have a close network of nearby family, friends, and neighbors. The housing is friendlier to older adults, more compact, with easier navigation and assistance in outside maintenance. Assisted living emerged in the 1990s as an eldercare alternative on the continuum of care for seniors for whom Independent living is no longer appropriate but who do not need the 24-hour medical care provided by a nursing home. You do not need to provide medical evidence that you need a minimum level of care. Retirement villages and retirement communities, unlike retirement homes, offer separate and autonomous homes for residents. A nursing home, convalescent home, Skilled Nursing Unit, care home or rest home provides a specific type of care for its residents. Housing arrangements for the future can be a stressful, anxiety-provoking topic for you and your family. Assisted living residences or assisted living facilities provide supervision or assistance with coordination of services by outside health careproviders.
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- Community Center
- Computer Access
- Guest Rooms
- Indoor Pool
- Laundry
- Library
- Lounges
- Mail Delivery
- Parking
- Pets Allowed
- Pool
- Private Dining Room
Languages Spoken
- Finnish
- All Religions Welcome
- Semi-private
- 1-bedroom
- 2-bedroom
- 3-bedroom
- Variety of Suite Styles
- Apartment
- Assisted Living Program
- Independent Living Program
- Secure Living Program - Dementia / Alzheimers Care
Accommodation Details
- Locks
- Storage
- Kitchen
- Kitchenettes
- Fridge
- Window Coverings
- Thermostats
- Fire Alarms
- Smoke Detector
- Sprinkler
- Air Conditioning
- Cable TV
- Bathrooms
- Gated Community
- Emergency Phone System
- Climate Control In-suite
- Wall-to-wall Broadloom
Recreational Facilities
- Pool Table
- Billiards
- Exercise Room
- Garden
- BBQ Area
- Swimming Pool
- Fitness Center
Recreational Programs
- Exercise
- Computer
- Parties
- Entertainment
- Art
- Recreational Programs
- Bingo
- Cards
Health Services
- Doctor's Office
Dining Services
- Buffet
- Cafeteria Style Dining
- Full-service restaurant
- Independent Living Dining
- Special diets accommodated
Special Diets
- Vegetarian
- Low salt
- Diabetic


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The Leading eBooks Store Online
3,667,858 members ⚫ 1,180,414 ebooks
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10 Models of Teacher EvaluationR&L Education 2014; US$ 43.99
Busy administrators lack the training and experience necessary to quickly and efficiently craft a teacher evaluation system that successfully meets the needs of their district. Questions like ?What is the best program for my district?? or ?How do I get the teachers on-board?? are being asked by school leaders across the country. This book provides... more...
100 Ideas for Secondary Teachers: Assessment for LearningBloomsbury Publishing 2015; US$ 15.99
100 Ideas: QUICK - EASY - INSPIRED - OUTSTANDING This brand new book develops a broader understanding of the impact and reasons for using Assessment for Learning in order to help teachers become outstanding teachers whilst their pupils also become outstanding learners. Emphasis is placed on helping pupils become autonomous and reflective learners... more...
100 Ideas for Secondary Teachers: RevisionBloomsbury Publishing 2016; US$ 14.99
The word 'revision' can strike fear into the heart of teachers and students alike! The exam cycle in today's modern education system can feel relentless, resulting in students suffering from information overload and exam fatigue. But it doesn't have to be like this! John Mitchell strongly believes that revision can be a opportunity to deepen students'... more...
500 Tips for Open and Online LearningTaylor and Francis 2008; US$ 50.95
All types of organisations have recognised the growing demand for open and flexible learning programmes. With the increased emphasis on new ways of learning, and the rapid move towards open and online courses, this practical guide will help those involved overcome the challenges they face. This guide provides user friendly advice and support for... more...
500 Tips for TutorsTaylor and Francis 2013; US$ 48.95
This book presents over 500 practical suggestions designed to help tutors establish active learning amongst their students. Divided into useful sections the tips cover the entire range of teaching and learning situations and comprise a 'start anywhere', dip-in resource suitable for both the newcomer and the old hand. Intended mainly for the university... more...
53 interesting ways to assess your studentsFrontinus Ltd 2015; US$ 30.00
For reflective practitioners in professional and higher education, this book offers practical ways to develop broad, flexible assessment repertoires. The 53 suggestions support both assessment of learning and assessment for learning. Topics include written tasks, examinations, problem-based activities, live and authentic forms of assessment, assessment... more...
8 Qualities of Successful School LeadersBloomsbury Publishing 2013; US$ 16.99
"Imagine you were cast adrift on a desert island with a school full of children in desperate need of a great headteacher. What eight qualities would you take with you to run your desert island school??? In this inspirational book, some of the UK's most successful headteachers answer this question, talking openly about what it takes to effectively... more...
A* in ExamsSummersdale Publishers Ltd 2015; US$ 8.99
This upbeat, easy-to-use guide will show you how to organise your work, revise effectively and prepare for exam day to give you the best chance of getting that A*! more...
The ABCs of EvaluationWiley 2014; US$ 70.00 US$ 60.67
Thoroughly revised and updated, the third edition of bestselling The ABCs of Evaluation offers an introduction to program evaluation. This comprehensive textbook covers topics such as stakeholder relationships, program design, data collection and analysis, reporting results, and other important steps in the evaluation process. The ABCs of Evaluation... more...
According to the BookSpringer Netherlands 2012; US$ 49.95
How are curriculum policies translated into opportunities to learn in the classroom? According to the Book presents findings from the largest cross-national study of textbooks carried out to date - the curriculum analysis of the 1995 Third International Mathematics and Science Study (TIMSS). This study included a detailed, page-by-page, inventory... more...


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Strip Club Owner Pleads Guilty to Trying to Put Hit on Arlington Mayor
In January, under pressure from the city of Arlington and the state, Ryan Walker Grant agreed to close down Flashdancer Cabaret, one of the city's last remaining strip clubs. Grant agreed, but he wasn't happy about it. Almost immediately, he set about doing what any gent as classy as himself would do when confronted with an unfavorable decision by municipal government: he set out to kill Mayor Robert Cluck and a city councilman.
The plan must have seemed like a fantastic idea, so he contacted a guy he knew and told him "I really need somebody to take a vacation up here to do a job from down south, man." Subsequent conversations made clear that "do a job" meant "assassinate," for which Grant was willing to pay $10,000 per head. The guy wound up blabbing to the FBI, who arrested Grant after getting a recording of him planning the murder.
He was indicted in July on one count of murder for hire and one count of unlawfully transferring a firearm. He initially pleaded not guilty but today he changed his mind, pleading guilty to the murder-for-hire charge. He faces a maximum sentence of 10 years in prison and a $250,000 fine. Sentencing is scheduled for Dec. 28. He's been in jail since his April arrest.
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Rent The Collected Shorter Plays of Samuel Beckett 1st edition today, or search our site for other textbooks by Samuel Beckett. Every textbook comes with a 21-day "Any Reason" guarantee. Published by Grove Press.
Need help ASAP? We have you covered with 24/7 instant online tutoring. Connect with one of our tutors now.


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Bethlehem Lutheran Church
The Heart of Bay Ridge
About Us
Pastor's Message
Good Stuff
Website Designed
Make a Website
List Your Business
Welcome to Bethlehem Lutheran Church. Our members invite you to tour our website, learn more about us, and come to one of our 10:30 a.m. Sunday services. We hold a traditional service with music from our organist and 15-member choir, and have a "family friendly" program that takes care of your children during the service.
We strive to offer an experience that makes everyone in our diverse congregation and all visitors feel welcome.
Sunday Service 10:30am
4th & Ovington Avenues
Brooklyn NY 11209
Our Mission & Welcome
To serve God and God's people. To live our faith daily through worship, and providing education, support, nourishment, compassion, good counsel, love, and service to the larger community - welcoming all who join us in this mission.
Bethlehem Lutheran Church is a Reconciling in Christ congregation. People of all sexual orientations and gender identities are welcome within the membership of this congregation and are encouraged to share in the sacramental and general life of this Christian family.
Quick Links to Bethlehem Pages:
What's our service like?
The Healing Center
Pastor's Message
Blog & News
Calendar of Events
Church History
(For Google mapping use 6999 4th Avenue)
Bethlehem Lutheran Church ® | 4th & Ovington Avenues, Brooklyn NY 11209 | 718.748.9502
A member of the Evangelical Lutheran Church in America (
Website designed by RP PR Consulting - Affordable websites for small businesses -
Bethlehem Choir on
YouTube fan page for our Choir
Read Pastor Paul's
September Message
September @ Bethlehem
9/4 @ 10:30 a.m. -- Sunday morning worship service
9/7 @ 6:30 p.m. -- BRC Community Advisory Board
9/8 @ 7:15 p.m. -- SW Brooklyn Lutheran Council @ Good Shepherd
9/11 @ 10:30 a.m. -- Sunday morning worship service (LES Opening)
9/11 @ 3:00 p.m. -- Ruth Wohltjen Memorial service
9/12 @ 6:30 p.m. -- BRC Board of Directors
9/13 @ 7:00 p.m. -- LES School Board
9/17 @ 10:00 a.m. -- BRC Annual Walkathon
9/18 @ 10:30 a.m. -- Sunday morning worship service (Rally Sunday / Bible Sunday)
9/18 @ 12:00 p.m. -- Congregation BBQ
9/20 @ 7:00 p.m. -- Church Council meeting
9/25 @ 10:30 a.m. -- Sunday morning worship service
9/25 @ 12:00 p.m. -- All Youth Planning Meeting in Joy Room
9/27 @ 7:00 p.m. -- Worship/Music Commission meeting in the parsonage


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The R.I. House of Representatives on Wednesday restored nearly a third of a $1 million cut that was jeopardizing future operations at the Office of the Health Insurance Commissioner. more
By Eli Sherman
Insurance companies have submitted their rate requests to the state Office of the Health Commissioner for 2017, and members of the public who want to weigh in can attend a Public Utilities Commission meeting on June 22. more
By Nancy Kirsch
(Updated 4:40 p.m. and 8:42 a.m.) The Lifespan nonprofit health system in fiscal 2015 reported an increase in operating revenue and operating expenses compared with the prior year, but a loss when it had previously posted a profit. Similarly, Care New England saw its 2014 gain turn into a loss in 2015. more
By Lori Stabile
Left on the cutting-room floor of the R.I. House Finance Committee last week was a proposed $1 million that without could effectively gut the Office of the Health Insurance Commissioner. more
By Eli Sherman
The state's economic momentum in April "deteriorated noticeably," said University of Rhode Island economist Leonard Lardaro in his latest Current Conditions Index. more
By Emily Gowdey-Backus
On Friday, the R.I. Attorney General and the R.I. Department of Health announced that the Expedited Review Hospital Conversion Initial Application, which was submitted by Yale-New Haven Health Services Corp. and Lawrence + Memorial Corp., LMW Healthcare Inc., d/b/a Westerly Hospital, is complete and accepted for review. more
By Nancy Kirsch
The Rhode Island Business Group on Health and the Rhode Island Medical Society launched Rhode Island's Choosing Wisely program this morning at Brown University. more
By PBN Staff
Peter J. Snyder, senior vice president and chief research officer at Lifespan, has been named chairman of the 10-member Rhode Island Neurosciences Leadership and Steering Committee. more
By PBN Staff
Provant Health Solutions Inc. has formed a partnership with Zipongo Inc., a digital nutrition company in San Francisco that specializes in healthy eating. more
By PBN Staff
The $220 million South Street Landing project will have a later occupancy for the state Nursing Education Center, the developer has announced. more
By Mary MacDonald


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Shrimp Au Gratin
Total Time
Prep 15 mins
Cook 10 mins
Since I don't care for crab I came up with this to take the place of shrimp and crab gratins.
Ingredients Nutrition
- 340.19 g cooked medium shrimp
- 44.37 ml butter
- 44.37 ml onions, chopped
- 59.14 ml flour
- 2.46 ml salt
- 1.23 ml dry mustard
- 0.25 ml pepper
- 354.88 ml milk
- 0.5 ml hot sauce
- 236.59 ml grated cheddar cheese
- 14.79 ml butter
- 59.14 ml dry breadcrumbs
- Remove shells and tails from shrimp.
- Cook onion in butter until tender; stir in flour and seasonings.
- Add milk gradually and cook until thick, stirring constantly.
- Add 3/4 cup cheese and heat until melted.
- Stir in shrimp and mix well.
- Pour into 4 to 6 individual well-greased ramekins.
- Combine melted butter, bread crumbs and remaining 1/4 cup of cheese; sprinkle over top of each ramekin.
- Bake in a 400 degree oven for 10-12 minutes or until bubbly and brown.
Most Helpful
This was a lovely shrimp dish. We really enjoyed the flavors of the sauce with the succulent shrimp. The shrimp were perfectly cooked, plump and juicy while the sauce was smooth and velvety. Thank you for sharing a recipe that I will make again and again. Made for Fall Pac/12
VERY GOOD! Made as written, except I added one pound raw shrimp to the cheese sauce and immediately poured the mixture into my casserole dish, topped with the bread crumbs and cheese and baked at 400F for 15 minutes. I did not want to risk overcooking the shrimp! I loved the crunch of the bread crumbs on top! My sauce was a little thick, so next time I'll either reduce the flour a tad, or make it with 1 3/4 cups milk. Made for my adopted baby, Spring PAC 2012.


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The chicks have hatched and left, summer reading program is over, and the heat has broken (for a day or two anyway.) Late summer kid boredom is about to peak. Get them out of the house and into the library! Stop in for a board game on Tuesday! Work on a puzzle! Get a book! Here's a good one for a restless teenager:
It's about a young woman who reads a book that causes her to question everything. It's a great read by the author of The Silver Linings Playbook, on which the award-winning film was based. We just got it in along with some other new titles for young adults. Browse and see what you find.
You can also work at our new puzzle table:
If you're still bored, maybe you're just not library-ing right!


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"I don't need to be Kareem (Abdul-Jabbar) or (James) Worthy or nothing like that. I can see white and red so I'm good. I'll be fine."
Related quotes
"Have you been watching American Idol? They have Simon Cowell and Paula Abdul judgin' the singin. Paula Abdul?! Gettin' Paula Abdul to judge a singin' contest is like gettin' Christopher Reeve to judge a dance contest!"
"Who's judging American Idol? Paula Abdul? Paula Abdul judging a singing contest is like Christopher Reeve judging a dance contest!"


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About 33% of consumers who drink coffee with breakfast choose where to eat based on which coffee they like best, up from 25% in 2009, Technomic found. The researcher also found that 46% of consumers sometimes purchase breakfast during the week, up from 33% in 2009. "Breakfast is a very dynamic segment in which consumers are looking for healthier options and place a premium on convenience," said Darren Tristano, Technomic's executive vice president.
Full Story:BevIndustry.com


text: My wife and I ate there on a Tuesday evening. We ordered the $33 "create your own" entree. The appetizer came out quickly. The salad came out about 10 minutes later. the entree came out 45 minutes later. We watched groups larger than us who came in after us get seated, order, get their food and pay before we saw... More


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Team Fortress 2 Arriving on Mac Tomorrow
Port is the latest Valve game to arrive on the Mac.
Portal 2 has been delayed, but Mac owners can at least take solace in finally being able to play Team Fortress 2.
The Mac version of Valve's popular shooter will be launching on Steam this Thursday. It will join Half-Life 2 and Portal, with the rest of the Valve catalogue following at a later date.
Look for it sometime tomorrow. In the meantime, start thinking about which classes you want to use, Mac owners.
See Also
Team Fortress 2 Community Members Earn Thousands from Custom Items
Comments (21)
I excite.Posted: Jun 12, 2010 12:00AM PST by WickedCobra03
I will download this game onto my mac, and then come fall, probably delete it so it doesn't interfere with my work. Seriously, Team Fortress and Half Life 2 anywhere I go, not good. That was one good thing about buying a mac.
where is it!?!?!Posted: Jun 10, 2010 12:00AM PST by Ahiru
been waiting for over 6 hours and still nothing...
i hope this runs better than it did via Crossover games - it was great, but after seeing the Portal port, i'm a little apprehensive..(it ran better via crossover than it does running it natively)
I bought it for 9.99Posted: Jun 10, 2010 12:00AM PST by SamuraiTerry
What a steal, but agreed. I'm still waiting for the Mac version to come available.
...Posted: Jun 10, 2010 12:00AM PST by AllShamNoWow
I wonder if Mac users will have the option of wearing iPod headphones on their character. That would be hilarious.
Have fun :)Posted: Jun 10, 2010 12:00AM PST by Barlowe
Always good with more players...I'll be the Soldier running around with the Gold badge (since I bought The Orange Box and played TF2 for the first time pretty close to the initial retail date) rocketjumping myself to death somewhere silly :P
NOOOO!!!Posted: Jun 09, 2010 12:00AM PST by The_Claude
Now the evil doers have access to the glory. Is there no justice in this world?
Don't really care!Posted: Jun 09, 2010 12:00AM PST by Malicore_IV
I'm still waiting for the engi update, and I would be surpised if they ever did the update for the 360 verison that they've been talking about for over a year.
How's this gonna work?Posted: Jun 09, 2010 12:00AM PST by BananaJane
The Apple mouses only have 1 button.
Nope.Posted: Jun 09, 2010 12:00AM PST by DevourerOfTime
My macbook still has one button.
And almost any USB mouse will work on a Mac.
Actually...Posted: Jun 09, 2010 12:00AM PST by wangetti324
all mac's have the ability to set up a right click function. The mouse is touch sensitive to what side of the mouse you are clicking on. So while it has one button, it functions as two.
contemporary Macbooks don't even have a button.Posted: Jun 10, 2010 12:00AM PST by SamuraiTerry
Its touch anywhere technology. If you want to right click you just use two fingers.
It's The Best Day Ever.Posted: Jun 09, 2010 12:00AM PST by DevourerOfTime
(Best Day Ever).
FINALLY!Posted: Jun 09, 2010 12:00AM PST by SamuraiTerry
I hope it's on sale too: )
portalPosted: Jun 09, 2010 12:00AM PST by onemileperhour
my media blackout for this game is so intense that i didn't even know it was supposed to come out this year. NO SPOILERS!!!


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Try on all types of fun Halloween costumes! This girly dress-up game gives you access to tons of scary outfits. You can mix and match clothes to create a hybrid costume, or wear something traditional. Try everything from cute to terrifying!
Halloween Costumes is one of our selected Halloween Dress Up Games. Also very popular on this website right now are Yacht Wedding, Barbie Moving To Manhattan, Beautiful Journalist and Super Wedding Stylist.
Report game as broken
Cancel report of broken game


text:
It's back!! Join us for the OT100MTB on September 24
Into the woods for the OTA Fall Mega Event
Over 160 hardworking Mega volunteers relax with BBQ, prize raffle and music
Thank you, REI! $15,000 grant presented at Sept 26, 2015 Mega
Thanks Show-Me Missouri Back Country Horsemen for work on the Current River section
Nearly a mile of sweet trail built at the Spring 2015 Mega: Thanks volunteers!
It takes a lot of hard hats to protect 175 Mega trail builders
Dedicated volunteers fording a stream with trail tools
OTA volunteers at Current River State Park: National Trails Day, June 7, 2014
OTA presented with USFS 2013 Trails Group Volunteer national and Region 9 awards
OTA volunteers roughing it on the trail
Dogpacking across the Current River
Just you and your shadow on the Ozark Trail
"Is this heaven?" "It's Missouri."
Wow! Missouri "Best Trails State:" International Trails Symposium. THANK YOU!
Nothing like a blue-sky day on the trail: Current River section
Trail's-eye view of an OTA crew
Box turtles (named for their hinged shell) live in Ozark forests and brushy fields
REMINDER: Peck Ranch Closures
Make it a Father's Day you'll never forget!
Prescribed Fire Alert- Trace Creek Section
Upcoming Events
Saturday, Oct 08 2016 - 8:30amPrepping for the OTA Fall Mega
Join us as we prepare for another great Mega! We will be based out of the OTA house as we get back to the Berryman reroute we sta...
sign up
Tuesday, Oct 11 2016 - 6:00pmMonthly Meeting - Powder Valley CA
A meeting of the Volunteers who run the Ozark Trail Association gathers monthly to discuss progress made and to set out operational tas...
sign up
Saturday, Oct 15 2016 - 8:30amOTA Fall Mega on the Berryman Reroute
Join us for the 2016 Fall Mega as we set out to finish up the Berryman Reroute we started this past spring. Last spring we broke the r...
sign up
Sunday, Oct 16 2016 - 8:00amOTA Fall Mega Tool Clean Up Extravagansa
After a grand day of building trail, eating great food, and having a good night with friends and music; come join us for a some tool cleanin...
sign up
Saturday, Oct 29 2016 - 8:30amWIUIW - Fall 2016 (Whoopin' It Up In Winona!)
This promises to be a spectacular Fall outing on the Ozark Trail. A little maintenance, a bit of fun and maybe an evening of ghost stories '...
sign up
Recent Events
Mega Event, Sept. 26, 2015
Mega Event, Apr. 11, 2015
Powder Mill, Feb. 13-14, 2015
OT100 Mountain Bike Race
April 26, 2014 Mega Event
Backcountry Trail Build, 4-5-14
Whoopin' in Up in Winona, 3-22-14
Womens Day and Maintenance Outing, 3-8-14
OTA Trivia Night, Feb. 1, 2014
Backcountry Trail Build, November 2013
Join Us
Our Mission
To develop, maintain, preserve, promote, and protect the rugged natural beauty of the Ozark Trail.