Prevention of diabetic alterations in transgenic mice overexpressing Myc in the liver.

Recent studies have demonstrated that the overexpression of the c-myc gene in the liver of transgenic mice leads to an increase in both utilization and accumulation of glucose in the liver, suggesting that c-Myc transcription factor is involved in the control of liver carbohydrate metabolism in vivo. To determine whether the increase in c-Myc might control glucose homeostasis, an intraperitoneal glucose tolerance test was performed. Transgenic mice showed lower levels of blood glucose than control animals, indicating that the overexpression of c-Myc led to an increase of blood glucose disposal by the liver. Thus, the increase in c-Myc might counteract diabetic hyperglycemia. In contrast to control mice, transgenic mice treated with streptozotocin showed normalization of concentrations of blood glucose, ketone bodies, triacylglycerols and free fatty acids in the absence of insulin. These findings resulted from the normalization of liver metabolism in these animals. While low glucokinase activity was detected in the liver of diabetic control mice, high levels of both glucokinase mRNA and enzyme activity were noted in the liver of streptozotocin-treated transgenic mice, which led to an increase in intracellular levels of glucose 6-phosphate and glycogen. The liver of these mice also showed an increase in pyruvate kinase activity and lactate production. Furthermore, normalization of both the expression of genes involved in the control of gluconeogenesis and ketogenesis and the production of glucose and ketone bodies was observed in streptozotocin-treated transgenic mice. Thus, these results suggested that c-Myc counteracted diabetic alterations through its ability to induce hepatic glucose uptake and utilization and to block the activation of gluconeogenesis and ketogenesis.

High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease.

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.

Human fatty acid synthase: properties and molecular cloning.

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS.

Older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease.

Increases in plasma cholesterol are associated with progressive increases in the risk of atherosclerotic cardiovascular disease. In humans plasma cholesterol is contained primarily in apolipoprotein B-based low density lipoprotein (LDL). Cells stop making the high-affinity receptor responsible for LDL removal as they become cholesterol replete; this slows removal of LDL from plasma and elevates plasma LDL. As a result of this delayed uptake, hypercholesterolemic individuals not only have more LDL but have significantly older LDL. Oxidative modification of LDL enhances their atherogenicity. This study sought to determine whether increased time spent in circulation, or aging, by lipoprotein particles altered their susceptibility to oxidative modification. Controlled synchronous production of distinctive apolipoprotein B lipoproteins (yolk-specific very low density lipoproteins; VLDLy) with a single estrogen injection into young turkeys was used to model LDL aging in vivo. VLDLy remained in circulation for at least 10 days. Susceptibility to oxidation in vitro was highly dependent on lipoprotein age in vivo. Oxidation, measured as hexanal release from n-6 fatty acids in VLDLy, increased from 13.3 +/- 5.5 nmol of 2-day-old VLDLy per ml, to 108 +/- 17 nmol of 7-day-old VLDLy per ml. Oxidative instability was not due to tocopherol depletion or conversion to a more unsaturated fatty acid composition. These findings establish mathematically describable linkages between the variables of LDL concentration and LDL oxidation. The proposed mathematical models suggest a unified investigative approach to determine the mechanisms for acceleration of atherosclerotic cardiovascular disease risk as plasma cholesterol rises.

Transformation of mammalian cells by overexpressing H2O2-generating peroxisomal fatty acyl-CoA oxidase.

Peroxisome proliferators induce qualitatively predictable pleiotropic responses, including development of hepatocellular carcinomas in rats and mice despite the inability of these compounds to interact with and damage DNA directly. In view of the nongenotoxic nature of peroxisome proliferators, it has been postulated that hepatocarcinogenesis by this class of chemicals is due to a receptor-mediated process leading to transcriptional activation of H2O2-generating peroxisomal fatty acyl-CoA oxidase (ACOX) in liver. To test this hypothesis, we overexpressed rat ACOX in African green monkey kidney cells (CV-1 cells) under control of the cytomegalovirus promoter. A stably transfected CV-1 cell line overexpressing rat ACOX, designated CV-ACOX4, when exposed to a fatty acid substrate (150 microM linoleic acid) for 2-6 weeks, formed transformed foci, grew efficiently in soft agar, and developed adenocarcinomas when transplanted into nude mice. These findings indicate that sustained overexpression of H2O2-generating ACOX causes cell transformation and provide further support for the role of peroxisome proliferation in hepatocarcinogenesis induced by peroxisome proliferators.

Oleic acid modulates mRNA expression of liver X receptor (LXR) and its target genes ABCA1 and SREBP1c in human neutrophils

Purpose: Regulation of liver X receptors (LXRs) is essential for cholesterol homeostasis and inflammation. The present study was conducted to determine whether oleic acid (OA) could regulate mRNA expression of LXRα and LXRα-regulated genes and to assess the potential promotion of oxidative stress by OA in neutrophils. Methods: Human neutrophils were treated with OA at different doses and LXR target gene expression, oxidative stress production, lipid efflux and inflammation state were analyzed. Results: We describe that mRNA synthesis of both LXRα and ABCA1 (a reverse cholesterol transporter) was induced by OA in human neutrophils. This fatty acid enhanced the effects of LXR ligands on ABCA1 and LXR expression, but it decreased the mRNA levels of sterol regulatory element-binding protein 1c (a transcription factor that regulates the synthesis of triglycerides). Although OA elicited a slight oxidative stress in the short term (15–30 min) in neutrophils, it is unlikely that this is relevant for the modulation of transcription in our experimental conditions, which involve longer incubation time (i.e., 6 h). Of physiological importance is our finding that OA depresses intracellular lipid levels and that markers of inflammation, such as ERK1/2 and p38 mitogen-activated protein kinase phosphorylation, were decreased by OA treatment. In addition, 200 μM OA reduced the migration of human neutrophils, another marker of the inflammatory state. However, OA did not affect lipid peroxidation induced by pro-oxidant agents. Conclusions: This work presents for the first time evidence that human neutrophils are highly sensitive to OA and provides novel data in support of a protective role of this monounsaturated acid against the activation of neutrophils during inflammation.

Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids.

The merozoite stage of the malaria parasite that infects erythrocytes and causes the symptoms of the disease is initially formed inside host hepatocytes. However, the mechanism by which hepatic merozoites reach blood vessels (sinusoids) in the liver and escape the host immune system before invading erythrocytes remains unknown. Here, we show that parasites induce the death and the detachment of their host hepatocytes, followed by the budding of parasite-filled vesicles (merosomes) into the sinusoid lumen. Parasites simultaneously inhibit the exposure of phosphatidylserine on the outer leaflet of host plasma membranes, which act as "eat me" signals to phagocytes. Thus, the hepatocyte-derived merosomes appear to ensure both the migration of parasites into the bloodstream and their protection from host immunity.

Imaging liver-stage malaria parasites

Plasmodium parasites, the causative agents of malaria, first invade and develop within hepatocytes before infecting red blood cells and causing symptomatic disease. Because of the low infection rates in vitro and in vivo, the liver stage of Plasmodium infection is not very amenable to biochemical assays, but the large size of the parasite at this stage in comparison with Plasmodium blood stages makes it accessible to microscopic analysis. A variety of imaging techniques has been used to this aim, ranging from electron microscopy to widefield epifluorescence and laser scanning confocal microscopy. High-speed live video microscopy of fluorescent parasites in particular has radically changed our view on key events in Plasmodium liver-stage development. This includes the fate of motile sporozoites inoculated by Anopheles mosquitoes as well as the transport of merozoites within merosomes from the liver tissue into the blood vessel. It is safe to predict that in the near future the application of the latest microscopy techniques in Plasmodium research will bring important insights and allow us spectacular views of parasites during their development in the liver.

The influence of viral genotypes and rejection episodes on the recurrence of hepatitis C after liver transplantation

Purpose. The aim of this study was to report the influence of hepatitis C virus (HCV) genotype and rejection episodes on the outcome of orthotopic liver transplantation (OLT), hepatitis recurrence, and progression to graft cirrhosis after OLT. Methods. Fifty-three patients who all had undergone OLT for end-stage liver cirrhosis were selected for this study. Hepatitis C genotype was determined. Recurrent hepatitis and rejection were diagnosed based on elevated liver function tests and a liver biopsy. Results. The patients were followed up for a mean of 51.9 +/- 34.3 months. The cumulative survival rate was no different in OLT for hepatitis C and OLT for all other liver diseases. After OLT, serum HCV RNA was detected in 93%. Histological recurrence occurred in 85% of all patients. The 1-, 3-, and 5-year recurrence rates were 48%, 77%, and 85%, respectively. Of the 41 patients with recurrent hepatitis C, 4 (10%) had cirrhosis, 18 (44%) had hepatitis with fibrosis, and 91 (46%) had hepatitis without fibrosis at the end of follow-up. A total of 32% of the patients were infected by HCV genotype 1b and 68% by other HCV genotypes. The recurrence rates were significantly higher in patients infected with genotype 1b than in those with other genotypes (p = 0.04). Twenty of 48 patients (42%) experienced acute rejection. There was a strong association between the number of rejection episodes and the incidence of HCV-related cirrhosis (p < 0.01). Conclusion. Our findings showed the genotype 1b to result in a higher recurrence rate after OLT. On the other hand, rejection episodes were associated with a more rapid progression to graft cirrhosis.

Tumor progression in hepatocellular carcinoma: Relationship with tumor stroma and parenchymal disease

Background: Encapsulation in hepatocellular carcinoma is associated with decreased invasiveness and improved survival in several series. Although active fibrogenesis by myofibroblasts has been demonstrated in the capsule, it is unclear if the capsule results from a general increase in peritumoral fibrosis, or an inherently less invasive tumor phenotype. The relationship between collagen deposition within tumor stroma, presence of cirrhosis and invasiveness also needs clarification. Methods: We performed immunohistochemistry for collagens I, III, IV and VI on sections of encapsulated and non-encapsulated hepatocellular carcinoma, arising in cirrhotic and non-cirrhotic livers. Staining was graded semi-quantitatively in tumor stromal elements and adjacent parenchymal sinusoids. The relationship of this staining with encapsulation, cirrhosis, and vascular invasion was analyzed. Results: Formation of a discrete capsular layer was associated with reduced vascular invasion, but not with a pervasive increase in peritumoral fibrosis. Increased collagen I content of tumor stroma and adjacent parenchymal sinusoids was associated with non-encapsulated tumors and vascular invasion. The presence of cirrhosis had little effect on capsule composition. Conclusions: Encapsulation of hepatocellular carcinoma reflects reduced invasiveness, rather than increased peritumoral collagen synthesis, which may instead enhance invasion. Increased intratumoral collagen I protein is also associated with increased tumor invasiveness. Pre-existing cirrhosis has little effect on tumor progression, possibly because the characteristics of cirrhosis are overwhelmed by tumor-induced changes in the adjacent parenchyma.(C) 2003 Blackwell Publishing Asia Pty Ltd.

Function and Regulation of Cytochrome P450 4V2 and the Implications in Bietti’s Crystalline Dystrophy

Thesis (Ph.D.)--University of Washington, 2016-06

IRS-1 regulation in health and disease

The global incidence of diabetes is increasing at epidemic rates. Estimates suggest there are currently 150 million people with diabetes and this number is expected to double in the next 20 years. Type 2 diabetes accounts for 95% of all cases and is characterized in part by impaired sensitivity to insulin or 'insulin resistance'. Defects in the insulin signalling pathways underpin this resistance. In the current article we discuss the regulation of Insulin Receptor Substrate-1 (IRS-1), a protein that plays a pivotal role in insulin signalling and whose function is impaired in subjects with insulin resistance. Coordination of IRS-1 function is multi-faceted, involving phosphorylation of IRS-1 at multiple serine/threonine residues. This controls many aspects of IRS-1, including its interaction with the insulin receptor and subsequent tyrosine phosphorylation, as well as its subcellular distribution and targeting for degradation by the proteasome. Such tight control ensures appropriate transduction and attenuation of the insulin signal, thereby regulating insulin action in healthy individuals. Emerging evidence indicates that `diabetogenic factors' associated with insulin resistance, such as TNFalpha and elevated circulating fatty acids, impact on insulin signalling at the level of IRS-1 serine/threonine phosphorylation. The expression and/or activity of several kinases, such as IkappaB kinase beta (IKKbeta) and salt-induced kinase 2 (SIK2), and the phosphorylation of IRS-1 at key sites, such as Ser307 and Ser789, are increased in states of insulin resistance. Identifying the pathways by which such factors activate these and other kinases, and de. ning the precise roles of specific serine/threonine phosphorylation events in IRS-1 regulation, represent important goals which may eventually provide a rationale for therapeutic intervention.

Donor pretreatment with progenipoietin-1 is superior to granulocyte colony-stimulating factor in preventing graft-versus-host disease after allogeneic stem cell transplantation

The granulocyte colony-stimulating factor (G-CSF) and Fit-3 receptor agonist progenipoietin-1 (ProGP-1) has potent effects on dendritic cell (DC) expansion and may be an alternative to G-CSF for the mobilization of stem cells for allogeneic stem cell transplantation (SCT). We studied the ability of stem cell grafts mobilized with this agent to induce graft-versus-host disease (GVHD) to minor and major histocompatibility antigens in the well-described B6 --> B6D2F1 SCT model. ProGP-1, G-CSIF, or control diluent was administered to donor B6 mice. ProGP-1 expanded all cell lineages in the spleen, and unseparated splenocytes from these animals produced large amounts of interleukin 10 (IL-10) and transforming growth factor beta (TGFbeta) whereas the expression of T-cell adhesion molecules was diminished. Transplantation survival was 0%, 50%, and 90% in recipients of control-, G-CSF-, and ProGP-1-treated allogeneic donor splenocytes, respectively (P < .0001). Donor pretreatment with ProGP-1 allowed a 4-fold escalation in T-cell dose over that possible with G-CSF. Donor CD4 T cells from allogeneic SCT recipients of ProGP-1 splenocytes demonstrated an anergic response to host antigen, and cytokine production (interferon gamma [IFNγ], IL-4, and IL-10) was also reduced while CD8 T-cell cytotoxicity to host antigens remained intact. Neither CD11c(hi) DCs nor CD11c(dim)/B220(hi) DCs from ProGP-1-treated animals conferred protection from GVHD when added to control spleen. Conversely, when equal numbers of purified T cells from control-, G-CSF-, or ProGP-1-treated allogeneic donors were added to allogeneic T-cell-depleted control spleen, survival at day 60 was 0%, 15%, and 90%, respectively (P < .0001). The improved survival in recipients of ProGP-1 T cells was associated with reductions in systemic tumor necrosis factor alpha generation and GVHD of the gastrointestinal tract. We conclude that donor pretreatment with ProGP-1 is superior to G-CSIF for the prevention of GVHD after allogeneic SCT, primarily due to incremental affects on T-cell phenotype and function

Steatosis and liver cell apoptosis in chronic hepatitis C: A mechanism for increased liver injury

Steatosis is increasingly recognized as a cofactor influencing the progression of fibrosis in chronic hepatitis Q however, the mechanisms by which it contributes to liver injury remain uncertain. We studied 125 patients with chronic hepatitis C to assess the effect of steatosis on liver cell apoptosis and the expression of Bcl-2, Bd-x(L), Bax, and tumor necrosis factor alpha (TNF-alpha) and the relationship between liver cell apoptosis and disease severity. A significant increase in liver cell apoptosis was seen in liver sections with increasing grade of steatosis (r = 0.42; P < .0001). Hepatic steatosis and previous heavy alcohol consumption were the only two variables independently associated with the apoptotic index. Increasing steatosis was associated with decreased Bcl-2 mRNA levels and an increase in the proapoptotic Bax/Bcl-2 ratio (r = -0.32, P = .007; and r = 0.27, P = .02, respectively). In the absence of steatosis, increased liver cell apoptosis was not associated with stellate cell activation or fibrosis (r = 0.26, P = .11; r = 0.06, P = .71, respectively). In contrast, in the presence of steatosis, increasing apoptosis was associated with activation of stellate cells and increased stage of fibrosis (r = 0.35, P = .047; r = 0.33, P = .03, respectively), supporting the premise that the steatotic liver is more vulnerable to liver injury. In patients with hepatitis C virus genotype 3, there was a significant correlation between TNF-α mRNA levels and active caspase-3 (r = 0.54, P = .007). In conclusion, these observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C. Further investigation will be required to determine the molecular pathways responsible for the proapoptotic effect of steatosis and whether this increase in apoptosis contributes directly to fibrogenesis.

Antioxidative function of L-FABP in L-FABP stably transfected Chang liver cells

Liver fatty acid binding protein (L-FABP) contains amino acids that are known to possess antioxidant function. In this study, we tested the hypothesis that L-FABP may serve as an effective endogenous cytoprotectant against oxidative stress. Chang liver cells were selected as the experimental model because of their undetectable L-FABP mRNA level. Full-length L-FABP cDNA was subcloned into the mammalian expression vector pcDNA3.1 (pcDNA-FABP). Chang cells were stably transfected with pc-DNA-FABP or vector (pcDNA3.1) alone. Oxidative stress was induced by incubating cells with 400 mu mol/L H2O2 or by subjecting cells to hypoxia/reoxygenation. Total cellular reactive oxygen species (ROS) was determined using the fluorescent probe DCF. Cellular damage induced by hypoxia/reoxygenation was assayed by lactate dehydrogenase (LDH) release. Expression of L-FABP was documented by regular reverse transcription polyrnerase chain reaction (RT-PCR), real-time RT-PCR, and Western blot. The pcDNA-FABP-transfected cells expressed full-length L-FABP mRNA, which was absent from vector-transfected control cells. Western blot showed expression of 14-kd L-FABP protein in pcDNA-FABP-transfected cells, but not in vector-transfected cells. Transfected cells showed decreased DCF fluorescence intensity under oxidative stress (H2O2 and hypoxia/reoxygenation) conditions versus control in inverse proportion to the level of L-FABP expression. Lower LDH release was observed in the higher L-FABP-expressed cells in hypoxia/reoxygenation experiments. In conclusion, we successfully transfected and cloned a Chang liver cell line that expressed the L-FABP gene. The L-FABP-expressing cell line had a reduced intracellular ROS level versus control. This finding implies that L-FABP has a significant role in oxidative stress.