A mechanobiological mathematical model of liver metabolism

The liver plays a complex role in metabolism and detoxification, and better tools are needed to understand its function and to develop liver-targeted therapies. In this study, we establish a mechanobiological model of liver transport and hepatocyte biology to elucidate the metabolism of urea and albumin, the production/detoxification of ammonia, and consumption of oxygen and nutrients. Since hepatocellular shear stress (SS) can influence the enzymatic activities of liver, the effect of SS on the urea and albumin synthesis are empirically modeled through the mechanotransduction mechanisms. The results demonstrate that the rheology and dynamics of the sinusoid flow can significantly affect liver metabolism. We show that perfusate rheology and blood hematocrit can affect urea and albumin production by changing hepatocyte mechanosensitive metabolism. The model can also simulate enzymatic diseases of the liver such as hyperammonemia I, hyperammonemia II, hyperarginemia, citrollinemia, and argininosuccinicaciduria, which disrupt the urea metabolism and ammonia detoxification. The model is also able to predict how aggregate cultures of hepatocytes differ from single cell cultures. We conclude that in vitro perfusable devices for the study of liver metabolism or personalized medicine should be designed with similar morphology and fluid dynamics as patient liver tissue. This robust model can be adapted to any type of hepatocyte culture to determine how hepatocyte viability, functionality, and metabolism are influenced by liver pathologies and environmental conditions.     

Longitudinal study of Pex1-G844D NMRI mouse model: A robust pre-clinical model for mild Zellweger spectrum disorder

Zellweger spectrum disorders (ZSD) are inborn errors of metabolism caused by mutations in PEX genes that lead to peroxisomal biogenesis disorder (PBD). No validated treatment is able to modify the dismal progression of the disease. ZSD mouse models used to develop therapeutic approaches are limited by poor survival and breeding restrictions. To overcome these limitations, we backcrossed the hypomorphic Pex1 p.G844D allele to NMRI background. NMRI mouse breeding restored an autosomal recessive Mendelian inheritance pattern and delivered twice larger litters. Mice were longitudinally phenotyped up to 6 months of age to make this model suitable for therapeutic interventions. ZSD mice exhibited growth retardation and relative hepatomegaly associated to progressive hepatocyte hypertrophy. Biochemical studies associated with RNA sequencing deciphered ZSD liver glycogen metabolism alterations. Affected fibroblasts displayed classical immunofluorescence pattern and biochemical alterations associated with PBD. Plasma and liver showed very long-chain fatty acids, specific oxysterols and C₂₇ bile acids intermediates elevation in ZSD mice along with a specific urine organic acid profile. With ageing, C₂₆ fatty acid and phytanic acid levels tended to normalize in ZSD mice, as described in patients reaching adulthood. In conclusion, our mouse model recapitulates a mild ZSD phenotype and is suitable for liver-targeted therapies evaluation.     

Liver repopulation with hepatocyte transplantation: new avenues for gene and cell therapy

Liver-directed gene therapy is appropriate for many conditions. Recent work established that liver repopulation with transplanted cells can be effective in treating genetic disorders. Although hepatocytes express therapeutic genes with considerable efficiency, correction of genetic disorders is constrained by limitations in permanent gene transfer into hepatocytes and repopulation of the liver with transplanted cells. Adenoviral vectors are highly efficient for hepatic gene transfer but the onset of deleterious host immune responses against adenoviral vectors, along with clearance of transduced hepatocytes have caused problems. Nonetheless, recent work concerning engraftment and proliferation of transplanted hepatocytes in the liver has provided significant new information, which should refocus interest in hepatocyte-based therapies. Moreover, hepatocyte transplantation systems offer creative tools for defining critical mechanisms in gene regulation and survival of transduced cells.     

Mechanisms controlling the integrity of replicating chromosomes in budding yeast

Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.     

Mechanisms Controlling the Integrity of Replicating Chromosomes in Budding Yeast

Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.     

Ornithine transcarbamylase and arginase I deficiency are responsible for diminished urea cycle function in the human hepatoblastoma cell line HepG2

A possible cell source for a bio-artificial liver is the human hepatblastoma-derived cell line HepG2 as it confers many hepatocyte functions, however, the urea cycle is not maintained resulting in the lack of ammonia detoxification via this cycle. We investigated urea cycle activity in HepG2 cells at both a molecular and biochemical level to determine the causes for the lack of urea cycle expression, and subsequently addressed reinstatement of the cycle by gene transfer. Metabolic labelling studies showed that urea production from 15N-ammonium chloride was not detectable in HepG2 conditioned medium, nor could 14C-labelled urea cycle intermediates be detected. Gene expression data from HepG2 cells revealed that although expression of three urea cycle genes Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase was evident, Ornithine Transcarbamylase and Arginase I expression were completely absent. These results were confirmed by Western blot for arginase I, where no protein was detected. Radiolabelled enzyme assays showed that Ornithine Transcarbamylase functional activity was missing but that Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase were functionally expressed at levels comparable to cultured primary human hepatocytes. To restore the urea cycle, HepG2 cells were transfected with full length Ornithine Transcarbamylase and Arginase I cDNA constructs under a CMV promoter. Co-transfected HepG2 cells displayed complete urea cycle activity, producing both labelled urea and urea cycle intermediates. This strategy could provide a cell source capable of urea synthesis, and hence ammonia detoxificatory function, which would be useful in a bio-artificial liver.     

Metabolic pre-conditioning of cultured cells in physiological levels of insulin: generating resistance to the lipid-accumulating effects of plasma in hepatocytes

Understanding the regulation of hepatocyte lipid metabolism is important for several biotechnological applications involving liver cells. During exposure of hepatocytes to plasma, as is the case in extracorporeal bioartificial liver assist devices, it has been reported that hepatic-specific functions, e.g., albumin and urea synthesis and diazepam removal, are dramatically compromised and hepatocytes progressively accumulate cytoplasmic lipid droplets. We hypothesized that the composition of hepatocyte culture medium significantly affects lipid metabolism during subsequent plasma exposure. Rat hepatocytes were cultured in medium containing either physiological (50 microU/mL) or supra-physiological (500 mU/mL) insulin levels for 1 week and then exposed to human plasma supplemented with or without amino acids. We found that insulin's anabolic effects, such as stimulation of triglyceride storage, were carried over from the pre-conditioning to the plasma exposure period. While hepatocytes cultured in high insulin medium accumulated large quantities of triglycerides during subsequent plasma exposure, culture in low insulin medium largely prevented lipid accumulation. Urea and albumin secretion, as well as the ammonia removal rate, were largely unaffected by insulin but increased with amino acid supplementation. Thus, hepatocyte metabolism during plasma exposure can be modulated by medium pre-conditioning and supplements added to plasma.     

Nutritional influences on the distribution of the urea cycle: intermediates in isolated hepatocytes

After the urea cycle was proposed, considerable efforts were put forth to identify critical intermediates. This was then followed by studies of dietary and nutritional control of urea cycle enzyme activity and allosteric effectors of urea cycle enzymes. Correlation of urea cycle enzyme activity with isolated cell experiments indicated conditions where enzyme activity would be rate limiting. At physiological levels of ammonia the activation of carbamoyl-phosphate synthetase (EC 6.3.4.16) by N-acetylglutamate (NAG) is important. Various levels of NAG corresponded well with changes in the rate of citrulline and urea synthesis. Arginine was found to be an allosteric activator of N-acetylglutamate synthetase (EC 2.3.1.1). Therefore, it was possible that the rate of carbamoyl phosphate synthesis was dependent on the level of urea cycle intermediates, particularly arginine. Evidence for arginine in the regulation of NAG synthesis is not as clear as for NAG on carbamoyl phosphate synthetase I. The concentration of hepatic arginine is not necessarily an indication of the mitochondrial concentration. Only mitochondrial arginine stimulates the N-acetylglutamate synthetase. Recent studies indicate that the mitochondrial concentration of arginine is higher than the cytosolic concentration and is well above the Ka for N-acetylglutamate synthetase. Therefore, it appears that changes in arginine concentration are not physiologically important in regulating levels of NAG. However, it is possible that responses to the effector may vary with time after eating, and it may be this responsiveness that controls the level of NAG and thereby urea synthesis.     

Apparent radii of the native, stable intermediates and unfolded conformers of the alpha-subunit of tryptophan synthase from E. coli, a TIM barrel protein.

The urea-induced equilibrium unfolding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli can be described by a four-state model, N right harpoon over left harpoon I1 right harpoon over left harpoon I2 right harpoon over left harpoon U, involving two highly populated intermediates, I1 and I2 [Gualfetti, P. J., Bilsel, O., and Matthews, C. R. (1999) Protein Sci. 8, 1623-1635]. To extend the physical characterization of these stable forms, the apparent radius was measured by several techniques. Size-exclusion chromatography (SEC), analytical ultracentrifugation (UC), and dynamic light scattering (DLS) experiments yield an apparent Stokes radius, R(s), of approximately 24 A for the native state of alphaTS. The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration, R(g), of 19.1 A, consistent with the value predicted from the X-ray structure and the Stokes radius. As the equilibrium is shifted to favor I1 at approximately 3.2 M and I2 at 5.0 M urea, SEC and UC show that R(s) increases from approximately 38 to approximately 52 A. Measurements of the radius by DLS and SAXS between 2 and 4.5 M urea were complicated by the self-association of the I1 species at the relatively high concentrations required by those techniques. Above 6 M urea, SEC and UC reveal that R(s) increases linearly with increasing urea concentration to approximately 54 A at 8 M urea. The measurements of R(s) by DLS and R(g) by SAXS are sufficiently imprecise that both values appear to be identical for the I2 and U states and, considering the errors, are in good agreement with the results from SEC and UC. Thermodynamic parameters extracted from the SEC data for the N right harpoon over left harpoon I1 and I1 right harpoon over left harpoon I2 transitions agree with those from the optical data, showing that this technique accurately monitors a part of the equilibrium model. The lack of sensitivity to the I2 right harpoon over left harpoon U transition, beyond a simple swelling of both species with increasing urea concentration, implies that the Stokes radii for the I2 and U states are not distinguishable. Surprisingly, the hydrophobic core known to stabilize I2 at 5.0 M urea [Saab-Rincón, G., Gualfetti, P. J., and Matthews, C. R. (1996) Biochemistry 35, 1988-1994] develops without a significant contraction of the polypeptide, i.e., beyond that experienced by the unfolded form at decreasing urea concentrations. Kratky plots of the SAXS data, however, reveal that I2, similar to N and I1, has a globular structure while U has a more random coil-like form. By contrast, the formation of substantial secondary structure and the burial of aromatic side chains in I1 and, eventually, N are accompanied by substantial decreases in their Stokes radii and, presumably, the size of their respective conformational ensembles.     

Bone weighs in on obesity

Obesity, insulin resistance, and diabetes are related disorders of energy metabolism for which therapies are suboptimal. In this issue of Cell, Lee et al. (2007) demonstrate in mice that bone regulates the insulin/glucose axis and energy metabolism, providing a new framework for approaching common disorders of bioenergetics.     

Hepatocyte heterogeneity in the metabolism of amino acids and ammonia.

With respect to hepatocyte heterogeneity in ammonia and amino acid metabolism, two different patterns of sublobular gene expression are distinguished: 'gradient-type' and 'strict- or compartment-type' zonation. An example for strict-type zonation is the reciprocal distribution of carbamoylphosphate synthase and glutamine synthase in the liver lobule. The mechanisms underlying the different sublobular gene expressions are not yet settled but may involve the development of hepatic architecture, innervation, blood-borne hormonal and metabolic factors. The periportal zone is characterized by a high capacity for uptake and catabolism of amino acids (except glutamate and aspartate) as well as for urea synthesis and gluconeogenesis. On the other hand, glutamine synthesis, ornithine transamination and the uptake of vascular glutamate, aspartate, malate and alpha-ketoglutarate are restricted to a small perivenous hepatocyte population. Accordingly, in the intact liver lobule the major pathways for ammonia detoxication, urea and glutamine synthesis, are anatomically switched behind each other and represent in functional terms the sequence of the periportal low affinity system (urea synthesis) and a previous high affinity system (glutamine synthesis) for ammonia detoxication. Perivenous glutamine synthase-containing hepatocytes ('scavenger cells') act as a high affinity scavenger for the ammonia, which escapes the more upstream urea-synthesizing compartment. Periportal glutaminase acts as a pH- and hormone-modulated ammonia-amplifying system in the mitochondria of periportal hepatocytes. The activity of this amplifying system is one crucial determinant for flux through the urea cycle in view of the high Km (ammonia) of carbamoylphosphate synthase, the rate-controlling enzyme of the urea cycle. The structural and functional organization of glutamine and ammonia-metabolizing pathways in the liver lobule provides one basis for the understanding of a hepatic role in systemic acid base homeostasis. Urea synthesis is a major pathway for irreversible removal of metabolically generated bicarbonate. The lobular organization enables the adjustment of the urea cycle flux and accordingly the rate of irreversible hepatic bicarbonate elimination to the needs of the systemic acid base situation, without the threat of hyperammonemia.     

The urea carboxylase and allophanate hydrolase activities of urea amidolyase are functionally independent

Urea amidolyase (UAL) is a multifunctional biotin‐dependent enzyme that contributes to both bacterial and fungal pathogenicity by catalyzing the ATP‐dependent cleavage of urea into ammonia and CO₂. UAL is comprised of two enzymatic components: urea carboxylase (UC) and allophanate hydrolase (AH). These enzyme activities are encoded on separate but proximally related genes in prokaryotes while, in most fungi, they are encoded by a single gene that produces a fusion enzyme on a single polypeptide chain. It is unclear whether the UC and AH activities are connected through substrate channeling or other forms of direct communication. Here, we use multiple biochemical approaches to demonstrate that there is no substrate channeling or interdomain/intersubunit communication between UC and AH. Neither stable nor transient interactions can be detected between prokaryotic UC and AH and the catalytic efficiencies of UC and AH are independent of one another. Furthermore, an artificial fusion of UC and AH does not significantly alter the AH enzyme activity or catalytic efficiency. These results support the surprising functional independence of AH from UC in both the prokaryotic and fungal UAL enzymes and serve as an important reminder that the evolution of multifunctional enzymes through gene fusion events does not always correlate with enhanced catalytic function.     

Oxysterols: functional significance in fetal development and the maintenance of normal retinal function

PURPOSE OF REVIEW: Recent findings extend the biologic activities of oxysterols as ligands for nuclear receptors to a role in morphogenesis during fetal development and to a role in the metabolism of photooxidation products of cholesterol in the retina. RECENT FINDINGS: A 1000-fold increase of the 27-hydroxy metabolite of 7-dehydrocholesterol in the plasma of children with Smith-Lemli-Opitz syndrome imply that intermediates in cholesterol synthesis follow alternate pathways of metabolism that generate novel oxysterols. A mouse model also finds an increase in sterol intermediates as the proximate cause of dysmorphisms. A role for oxysterols in the effects of Sonic hedgehog protein focuses on their role in normal fetal development. Both CYP27A1 and CYP46A1 are expressed in primate retina indicating that local metabolism of 7-ketocholesterol to nontoxic derivatives is important for preventing retinal degeneration. SUMMARY: Recent data expand the functional roles of oxysterols to fetal development and to the detoxification of oxidation products of cholesterol. This review shifts the focus of attention from studies of their ligand-binding activity to studies of animal models that indicate a number of important biologic effects during fetal development and during the aging process.     

Identification of rare variants causing urea cycle disorders: A clinical,genetic, and biophysical study

Urea cycle disorders (UCDs) are a group of rare metabolic conditions characterized by hyperammonemia and a broad spectrum of phenotypic severity. They are caused by the congenital deficiency in the eight biomolecules involved in urea cycle. In the present study, five cases of UCD were recruited and submitted to a series of clinical, biochemical, and genetic analysis with a combination of high throughput techniques. Moreover, in silico analysis was conducted on the identified missense genetic variants. Various clinical and biochemical indications (including profiles of amino acids and urinary orotic acids) of UCD were manifested by the five probands. Sequence analysis revealed nine diagnostic variants, including three novel ones, which caused Argininosuccinic aciduria (ASA) in one case, Carbamoyl phosphate synthetase 1deficiency (CPS1D) in two cases, Ornithine transcarbamylase deficiency (OTCD) in one case, and Citrin deficiency in 1case. Results of in silico biophysical analysis strongly suggested the pathogenicity of each the five missense variants and provided insight into their intramolecular impacts. In conclusion, this study expanded the genetic variation spectrum of UCD, gave solid evidence for counselling to the affected families, and should facilitate the functional study on the proteins in urea cycle.     

Crystal structure of urea carboxylase provides insights into the carboxyltransfer reaction

Urea carboxylase (UC) is conserved in many bacteria, algae, and fungi and catalyzes the conversion of urea to allophanate, an essential step in the utilization of urea as a nitrogen source in these organisms. UC belongs to the biotin-dependent carboxylase superfamily and shares the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains with these other enzymes, but its carboxyltransferase (CT) domain is distinct. Currently, there is no information on the molecular basis of catalysis by UC. We report here the crystal structure of the Kluyveromyces lactis UC and biochemical studies to assess the structural information. Structural and sequence analyses indicate the CT domain of UC belongs to a large family of proteins with diverse functions, including the Bacillus subtilis KipA-KipI complex, which has important functions in sporulation regulation. A structure of the KipA-KipI complex is not currently available, and our structure provides a framework to understand the function of this complex. Most interestingly, in the structure the CT domain interacts with the BCCP domain, with biotin and a urea molecule bound at its active site. This structural information and our follow-up biochemical experiments provided molecular insights into the UC carboxyltransfer reaction. Several structural elements important for the UC carboxyltransfer reaction are found in other biotin-dependent carboxylases and might be conserved within this family, and our data could shed light on the mechanism of catalysis of these enzymes.     

Inhibition of nitric oxide synthesis in primary cultured mouse hepatocytes by alpha-lipoic acid

Recent work shows that septic or endotoxic shock is associated with lipopolysaccharide and cytokine mixture-induced nitric oxide (NO) synthesis in liver. Here we found that DL-alpha-lipoic acid inhibited but other thiol-containing antioxidants such as glutathione and N-acetylcysteine enhanced lipopolysaccharide and cytokine mixture (referred as LPS/CM)-induced NO synthesis in hepatocytes. The inhibitory action of alpha-lipoic acid on hepatocyte NO synthesis was as potent as that of NG-monomethyl-L-arginine without obvious cytotoxicity. Deletion by diethylmaleate or inhibition by buthionine sulfoximine of intracellular glutathione caused a significant decrease in hepatocyte NO synthesis, implying that increased intracellular reduced glutathione levels could not be the reason for alpha-lipoic acid inhibited NO synthesis. alpha-Lipoic acid inhibition of NO synthesis seems to be from alpha-lipoic acid improved carbohydrate metabolism in hepatocytes. Since alpha-lipoic acid is an essential compound existing naturally in physiological systems, it may serve as both a research and therapeutic agent for sepsis.