Downregulation of hepatic glucose-6-phosphatase-α in patients with hepatic steatosis

Glucose-6-phosphate transporter (G6PT) and microsomal glucose-6-phosphatase-α (G6Pase-α) perform the terminal step in glycogenolysis and gluconeogenesis. Deficiency of these proteins leads to glycogen storage diseases. Partial inhibition of G6Pase in rats results in increased hepatic triglyceride content and de novo lipogenesis leading to hepatic steatosis. Hepatic steatosis represents hepatic manifestation of the metabolic syndrome. We investigated molecular mechanisms that may explain the relationship between fatty liver and G6Pase-α in humans in detail. A total of 27 patients (11 men, 16 women) underwent liver biopsy. Histological diagnosis identified nonfatty liver in seven patients and nonalcoholic fatty liver in 20 patients. We quantified G6Pase-α and G6PT mRNA expression by real-time PCR. Anthropometric measurements and analysis of plasma lipids and liver enzymes were performed. Patients with fatty liver showed no significant differences in age, HOMA(IR) (homeostasis model assessment of insulin resistance), BMI, liver enzymes or waist-to-hip ratio compared to those with nonfatty liver, but total plasma cholesterol levels and liver fat content were higher in patients with fatty liver (P < 0.05). G6Pase-α and G6PT mRNA expressions were significantly downregulated in fatty compared to histologically normal liver (P < 0.05). G6Pase-α and G6PT mRNA expressions correlated positively (R(2) = 0.406 P < 0.05). Both expressions did not correlate with age, BMI, aspartate transaminase, alanine transaminase, alkaline phosphatase, γ-glutamyl transferase, triglycerides or glucose levels. Our data suggest that expression of hepatic G6Pase-α and G6PT are closely interlinked. Downregulation of G6Pase-α in fatty liver might be associated with hepatic fat accumulation and pathogenesis of hepatic steatosis.     

Wnt/��-catenin signaling in hepatic organogenesis

Wnt/β-catenin signaling has come to the forefront of liver biology in recent years. This pathway regulates key pathophysiological events inherent to the liver including development, regeneration and cancer, by dictating several biological processes such as proliferation, apoptosis, differentiation, adhesion, zonation and metabolism in various cells of the liver. This review will examine the studies that have uncovered the relevant roles of Wnt/β-catenin signaling during the process of liver development. We will discuss the potential roles of Wnt/β-catenin signaling during the phases of development, including competence, hepatic induction, expansion and morphogenesis. In addition, we will discuss the role of negative and positive regulation of this pathway and how the temporal expression of Wnt/β-catenin can direct key processes during hepatic development. We will also identify some of the major deficits in the current understanding of the role of Wnt/β-catenin signaling in liver development in order to provide a perspective for future studies. Thus, this review will provide a contextual overview of the role of Wnt/β-catenin signaling during hepatic organogenesis.Key words: liver development, liver cancer, liver regeneration, Wnt signaling, proliferation, differentiationThe Wnt/β-catenin pathway is an evolutionarily well-conserved pathway that has proven to be essential to normal cellular processes such as development, growth, survival, regeneration and self-renewal.^1–5 Its diverse functions also include the initiation and progression of cancer.⁶ In fact, one area in which this pathway has been extensively studied is in liver cancer.Mutations of Wnt/β-catenin pathway members in hepatocarcinogenesis are common. For example, 90–100% of hepatoblastomas contain mutations in adenomatous polyposis coli (APC), CTNNB1 and/or Axin1/2, which causes cytoplasmic and nuclear localization of β-catenin.^7–9 Axin1 and β-catenin mutations have also been identified in approximately 25% of hepatocellular carcinomas,^10–12 while overexpression of the frizzled-7 receptor¹³ and glycogen synthase kinase-3 (GSK-3) inactivation¹⁴ can also lead to aberrant β-catenin pathway activation. The dysregulation of this pathway in hepatic cancers makes it an attractive target for potential therapies, and experimental treatment in vivo has shown promising results. For example, inhibiting β-catenin expression by siRNA or R-Etodolac decreases proliferation and survival of human hepatoma cell lines.^15,16 Since cancer recapitulates development, determining the timing of β-catenin activation during hepatogenesis will help us to better understand the inappropriate activation of this pathway in hepatocarcinogenesis.Recent work has elucidated the role of β-catenin signaling in the liver, and has highlighted its essential role in liver health and disease.¹⁷ In addition, emerging evidence suggests that this pathway plays a key role in liver organogenesis.     

Stimulation of hepatic microsomal β-glucuronidase by calcium

Hydrolysis of 3-methylumbelliferyl glucuronide by liver microsomal β-glucuronidase is enhanced about 2-fold by micromolar concentrations of Ca²⁺; half-maximal stimulation occurs with 0.35 μM Ca²⁺. Dissociation of the enzyme from microsomal membranes by various treatments increases basal β-glucuronidase activity and markedly decreases the sensitivity of the enzyme to Ca²⁺. Under similar conditions, the soluble lysosomal form of the enzyme is insensitive to Ca²⁺. Ca²⁺ stimulation was unaltered by addition of calmodulin inhibitors or exogenous calmodulin. Thus, interaction of cytosolic Ca²⁺ with membrane bound β-glucuronidase may modulate glucuronidation in intact hepatocytes via a novel, calmodulin-independent mechanism.     

iPLA2β deficiency attenuates obesity and hepatic steatosis in ob/ob mice through hepatic fatty-acyl phospholipid remodeling

PLA2G6 or GVIA calcium-independent PLA2 (iPLA2β) is identified as one of the NAFLD modifier genes in humans, and thought to be a target for NAFLD therapy. iPLA2β is known to play a house-keeping role in phospholipid metabolism and remodeling. However, its role in NAFLD pathogenesis has not been supported by results obtained from high-fat feeding of iPLA2β–null (PKO) mice. Unlike livers of human NAFLD and genetically obese rodents, fatty liver induced by high-fat diet is not associated with depletion of hepatic phospholipids. We therefore tested whether iPLA2β could regulate obesity and hepatic steatosis in leptin-deficient mice by cross-breeding PKO with ob/ob mice to generate ob/ob–PKO mice. Here we observed an improvement in ob/ob–PKO mice with significant reduction in serum enzymes, lipids, glucose, insulin as well as improved glucose tolerance, and reduction in islet hyperplasia. The improvement in hepatic steatosis measured by liver triglycerides, fatty acids and cholesterol esters was associated with decreased expression of PPARγ and de novo lipogenesis genes, and the reversal of β-oxidation gene expression. Notably, ob/ob livers contained depleted levels of lysophospholipids and phospholipids, and iPLA2β deficiency in ob/ob–PKO livers lowers the former, but replenished the latter particularly phosphatidylethanolamine (PE) and phosphatidylcholine (PC) that contained arachidonic (AA) and docosahexaenoic (DHA) acids. Compared with WT livers, PKO livers also contained increased PE and PC containing AA and DHA. Thus, iPLA2β deficiency protected against obesity and ob/ob fatty liver which was associated with hepatic fatty-acyl phospholipid remodeling. Our results support the deleterious role of iPLA2β in severe obesity associated NAFLD.     

Decorin-TGFβ axis in hepatic fibrosis and cirrhosis

Hepatic fibrosis and cirrhosis are worldwide health care problems, especially in regions with a high rate of hepatitis infection. As these diseases affect a major part of the human population, the search for antifibrotic therapies has a high priority in medical research. Transforming growth factor β1 (TGF-β1) is one of the most powerful profibrotic cytokines. Thus, blocking TGF-β1 activity by natural inhibitors represents a valid and logical strategy to combat hepatic fibrosis. One of the natural inhibitors of TGF-β1 is decorin, a small leucine-rich proteoglycan that binds with high affinity to this cytokine and prevents its interaction with pro-fibrotic receptors. Recent evidence has shown that decorin has a protective role in liver fibrogenesis insofar as its genetic ablation in mice leads to enhanced matrix deposition, impaired matrix degradation, and "activation" of hepatic stellate cells, the main producers of fibrotic tissue. Moreover, TGF-β1 exerts a stronger effect when functional decorin is absent. These data provide robust genetic evidence for a direct role of endogenous decorin in preventing and retarding hepatic fibrosis. Thus, boosting the endogenous production of decorin or systemic delivery of recombinant decorin could represent an additional therapeutic modality against hepatic fibrosis.     

Wnt/β-catenin signaling in hepatic organogenesis

Wnt/β-catenin signaling has come to the forefront of liver biology in recent years. This pathway regulates key pathophysiological events inherent to the liver including development, regeneration, and cancer, by dictating several biological processes such as proliferation, apoptosis, differentiation, adhesion, zonation and metabolism in various cells of the liver. This review will examine the studies that have uncovered the relevant roles of Wnt/β-catenin signaling during the process of liver development. We will discuss the potential roles of Wnt/β-catenin signaling during the phases of development, including competence, hepatic induction, expansion, and morphogenesis. In addition, we will discuss the role of negative and positive regulation of this pathway and how the temporal expression of Wnt/β-catenin can direct key processes during hepatic development. We will also identify some of the major deficits in the current understanding of the role of Wnt/β-catenin signaling in liver development in order to provide a perspective for future studies. Thus, this review will provide a contextual overview of the role of Wnt/β-catenin signaling during hepatic organogenesis.     

Notes on some African hepatic genera 6–9

The present study deals with African species ofSyzygiella Spruce,Allisoniella Hodgs. andGymnomitriaceae Klinggr. Three species ofSyzygiella Spruce,S. geminifolia (Mitt.) Steph.,S. concreta (Gott.) Spruce and one indeterminable species with orbicular leaves (known only in sterile condition) occur in Africa.Allisoniella nigra (Rodw.) Schust. represents a genus hitherto unreported for the African flora. The present knowledge ofGymnomitriaceae Klinggr. in subsaharan Africa is summarised; five species ofMarsupella Dum., one ofGymnomitrion Corda and five ofHerzogobryum Grolle are reported. Finally, some additions to the generaIsotachis Mitt.,Anastrophyllum (Spruce) Steph. andLophozia (Dum.) Dum. are presented.     

Notes on some African hepatic genera 1–5

The present study deals with five genera of hepatics in Africa, Isotachis Mitt., Anastrophyllum (Spruce) Steph., Tritomaria Schiffn. ex Loeske, Gymnocoleopsis (Schust.) Schust. and Lophozia (Dum.) Dum. All African populations of the genus Isotachis Mitt. are considered to be one species, I. aubertii (Schwaegr.) Mitt. Four species of Anastrophyllum (Spruce) Steph. (s.l.), A. auritum (Lehm.) Steph., A. piligerum (Nees) Spruce, A. subcomplicatum (Lehm. et Lindenb.) Steph. and A. minutum (Schreb.) Schust., and two species of Tritomaria Schiffn. et Loeske, T. camerunensis S. Arnell and T. exsecta (Schrad.) Schiffn. ex Loeske occur in Africa. Gymmocoleopsis multiflora (Steph.) Schust. represents a genus and species hitherto unreported for the African flora. Finally, five Lophozia (Dum.) Dum. species, L. argentina (Steph.) Schust., L. capensis S. Arnell, L. decolorans (Limpr.) Steph., L. hedbergii S. Arnell and L. tristaniana (S. Arnell) Váňa, are reported from central and southern Africa; two of these (L. argentina (Steph.) Schust. and L. decolorans (Limpr.) Steph.) represent the first reports from Africa.     

Inhibition of hepatic lipogenesis by α-cyano-4-hydroxycinnamate

In agreement with its well-known inhibition of mitochondrial carrier-mediated pyruvate transport, α-cyano-4-hydroxycinnamate elevates pyruvate and lactate levels in suspensions of isolated rat hepatocytes, whereas it lowers citrate levels and causes strongly depressed rates of fatty acid synthesis with glucose as carbon precursor. It stimulates the oxidation of exogenous fatty acids and inhibits their esterification.α-Cyano-4-hydroxycinnamate also impairs fatty acid synthesis from substrates (acetate, octanoate) that bypass mitochondrial pyruvate transport. Cholesterol synthesis from acetate, a process utilizing the same cytosolic acetyl-CoA pool as does fatty acid synthesis, is hardly affected by α-cyano-4-hydroxy-cinnamate. These observations suggest an inhibitory site of action of α-cyano-4-hydroxycinnamate located in the fatty-acid biosynthetic pathway itself. This suggestion has been confirmed by demonstrating the inhibition of purified rat-liver acetyl-CoA carboxylase by α-cyano-4-hydroxycinnamate at concentrations prevailing in the intact cell upon incubation with this compound.Maximal inhibition of purified acetyl-CoA carboxylase requires about 20 min of preincubation of the enzyme with α-cyano-4-hydroxycinnamate. Fatty acid synthesis from acetate in the intact cells is further diminished after an incubation time of 20 min.The inhibition by α-cyano-4-hydroxycinnamate of fatty acid synthesis from acetate can be partially overcome by insulin. Possible interactions of the inhibitor and the hormone at the level of acetyl-CoA carboxylase are discussed.It is concluded that α-cyano-4-hydroxycinnamate does not provide a simple and unequivocal tool to distinguish between actions of effectors on hepatic fatty acid synthesis per se and on the glycolytic pathway.     

Low hepatic manganese concentrations in patients with hepatic steatosis – A cohort study of copper,iron and manganese in liver biopsies

Background and aimsHepatic steatosis is the most common histopathological finding on liver biopsy, with the most prevalent etiology being NAFLD. The pathogenesis of hepatic steatosis and NAFLD is multifactorial, however, studies on the importance of manganese in NAFLD are limited. We aimed to study hepatic manganese content, and other trace elements, in relation to hepatic steatosis in patients with chronic liver diseases of different etiology, mainly NAFLD.MethodsPatients with chronically elevated liver function tests underwent a diagnostic work-up, including routine blood tests and two liver biopsies. One of the biopsies was sent for histopathological evaluation, and the other for ultra-trace elemental determinations. Steatosis was graded using conventional histopathological methodology, and fat content was also quantitated in biopsy samples by measuring the steatotic area of the section using stereological point counting (SPC). Ultra-trace elemental analysis was utilized for determining manganese, iron, and copper using inductively coupled plasma sector field mass spectrometry (ICP-SFMS).Results76 patients were included in the study. Hepatic manganese concentrations in patients with steatosis were lower than in patients without hepatic steatosis (3.8 ± 1.1 vs. 6.4 ± 1.8, P < 0.001). Similar results were seen for blood manganese levels and hepatic steatosis. We found a strong inverse correlation between steatosis grade and hepatic manganese content (ρ=-0.743, P < 0.001). Also, low levels of manganese independently predicted the presence of steatosis (aOR 0.07 [95%CI: 0.01−0.63]).ConclusionPatients with NAFLD, or other CLD and concomitant hepatic steatosis, showed lower levels of hepatic manganese content with increasing grade of steatosis.     

α-Methylspermidine protects against carbon tetrachloride-induced hepatic and pancreatic damage

The role of polyamines in carbon tetrachloride (CCl₄)-induced organ injury was studied in syngenic rats and transgenic rats with activated polyamine catabolism. In syngenic rats, administration of CCl₄ resulted in the induction of hepatic spermidine/spermine N ¹-acetyltransferase (SSAT), accumulation of putrescine, reduction in spermine level and appearance of moderate hepatic injury within 24 h. Upon treatment with CCl₄, transgenic rats overexpressing SSAT displayed induction of both hepatic and pancreatic SSAT, with subsequent accumulation of putrescine and decrease of both spermidine and spermine pools. Administration of CCl₄ in SSAT transgenic rats induced not only massive hepatic injury, but also severe acute necrotizing pancreatitis. Pretreatment of the animals with catabolically stable functional polyamine mimetic, α-methylspermidine (MeSpd) prevented pancreatic and hepatic injury in SSAT rats and markedly reduced liver damage in syngenic animals. As assessed by immunostaining of proliferating cell nuclear antigen, MeSpd increased the amount of regenerating hepatocytes in both genotypes. These results show that CCl₄ induces hepatic and pancreatic polyamine catabolism, and the extent of organ damage correlates with the degree of polyamine depletion. Furthermore, MeSpd protects against CCl₄-induced hepatic and pancreatic damage and promotes tissue regeneration.     

Isolation of EpCAM+/CD133− hepatic progenitor cells

Progenitor cell-derived hepatocytes are critical for hepatocyte replenishment. Therefore, we established a line of human hepatic progenitor (HNK1) cells and determined their biological characteristics for experimental and therapeutic applications. HNK1 cells, isolated from human noncirrhotic liver samples with septal fibrosis, showed high expression of the hepatic progenitor cell (HPC) markers EpCAM, CK7, CK19, alpha-fetoprotein (AFP), CD90 (Thy1), and EFNA1. Expression of CD133 was very low. Ductular reactions at the periphery of cirrhotic nodules were immunohistochemically positive for these HPC markers, including EFNA1. Sodium butyrate, a differentiation inducer, induced hepatocyte-like morphological changes in HNK1 cells. It resulted in down-regulation of the hepatic progenitor cell markers EpCAM, CK7, CK19, AFP, and EFNA1 and up-regulation of mature hepatocyte markers, including albumin, CK8, and CK18. Furthermore, sodium butyrate treatment and a serial passage of HNK1 cells resulted in enhanced albumin secretion, ureagenesis, and CYP enzyme activity, all of which are indicators of differentiation in hepatocytes. However, HNK1 cells at passage 50 did not exhibit anchorage-independent growth capability and caused no tumors in immunodeficient mice, suggesting that they had no spontaneous malignant transformation ability. From this evidence, HNK1 cells were found to be EpCAM⁺/CD133^- hepatic progenitor cells without spontaneous malignant transformation ability. We therefore conclude that HNK1 cells could be useful for experimental and therapeutic applications.     

Diacylglycerol activation of protein kinase Cε and hepatic insulin resistance

Nonalcoholic fatty liver disease (NAFLD) is now the most frequent chronic liver disease in Western societies, affecting one in four adults in the USA, and is strongly associated with hepatic insulin resistance, a major risk factor in the pathogenesis of type 2 diabetes. Although the cellular mechanisms underlying this relationship are unknown, hepatic accumulation of diacylglycerol (DAG) in both animals and humans has been linked to hepatic insulin resistance. In this Perspective, we discuss the role of DAG activation of protein kinase Cε as the mechanism responsible for NAFLD-associated hepatic insulin resistance seen in obesity, type 2 diabetes, and lipodystrophy.