Role of hepatic phospholipids in development of liver injury in Mdr2 (Abcb4) knockout mice

Background/Aims: Multidrug resistance protein 2 (Abcb4) gene knockout mice (Mdr2^?/?) lack phosphatidylcholine (PC) excretion into bile and spontaneously develop sclerosing cholangitis, biliary fibrosis and hepatocellular carcinomas. We therefore aimed to test whether formation and hepatic retention of abnormal PC metabolites contribute to the pathogenesis of liver injury in Mdr2^?/? mice. Methods: Mdr2 ^?/? mice were either fed a diet supplemented with soybean lecithin 2.5% w/w [phosphatidylcholine‐enriched diet (PCD), to increase hepatic PC content] or a choline‐deficient diet (CDD, to reduce hepatic PC content) for 4 weeks; controls received chow with energy and nutrient content equivalent to PCD and CDD. Serum liver tests, liver histology, markers of fibrosis, cholangiocyte activation, cell proliferation and thin‐layer chromatography for phospholipid (PL) composition were carried out. Results: PCD decreased serum alkaline phosphatase and total bilirubin levels compared with controls, while liver histology as well as hepatic hydroxyproline content as markers of liver fibrosis did not differ among groups. Both PCD and CDD decreased hepatocellular proliferation compared with controls. Hepatic, serum and biliary PLs remained unchanged despite dietary manipulations and no potentially toxic PL metabolites were detected. Conclusions: Mdr2 ^?/? mice maintain stable hepatic, serum and biliary PL metabolism in response to dietary PC manipulations. Our findings therefore suggest that liver injury in Mdr2^?/? mice is not due to formation of toxic PL metabolites.     

Inducible nitric oxide synthase is required in alcohol-induced liver injury: studies with knockout mice

BACKGROUND & AIMS : Oxidative stress contributes to early alcohol-induced liver injury, and superoxide (O(2)*-) production from NADPH oxidase plays a key role. However, the production of the free radical nitric oxide (NO*) by inducible nitric oxide synthase (iNOS) could also be involved. METHODS : To test this hypothesis, iNOS knockout (B6.129P2-Nos2 (tm1 Lau)) and wild-type mice were fed high-fat control or ethanol-containing diets for 4 weeks. RESULTS : Mean body weight gains were not significantly different between treatment groups, and average urine ethanol concentrations were similar in wild-type and iNOS knockout mice. After 4 weeks, serum alanine aminotransferase (ALT) levels were increased significantly about 4-fold over control values (29 +/- IU/L) by enteral ethanol (113 +/- 20) in wild-type mice; this effect of ethanol was significantly blunted in iNOS knockout mice (50 +/- 9). Similar protective effects against liver damage were observed if wild-type mice were treated with the iNOS inhibitor N -(3-aminomethyl)benzyl-acetamindine (1400W). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver in wild-type mice but had no effect in iNOS knockout mice. The accumulation of 4-hydroxynonenal (lipid peroxidation) and 3-nitrotyrosine (reactive nitrogen species formation) protein adducts caused by alcohol was completely blocked in iNOS knockout mice. CONCLUSIONS : These data strongly support the hypothesis that iNOS is required for the pathogenesis of early alcohol-induced hepatitis by production of nitric oxide-derived pro-oxidants (e.g., peroxynitrite).     

Bid is upstream of lysosome-mediated caspase 2 activation in tumor necrosis factor alpha-induced hepatocyte apoptosis

BACKGROUND & AIMS: During tumor necrosis factor alpha-mediated hepatocyte cytotoxicity, cathepsin B is released from lysosomes and contributes to apoptosis by indirectly promoting mitochondrial dysfunction. How this lysosomal pathway mediates mitochondrial dysfunction is unclear. Because Bcl-2 family proteins and caspase 2 have been implicated in proximal apoptosis-signaling pathways, we examined the role of these proteins in tumor necrosis factor alpha-induced lysosomal permeabilization and cathepsin B-mediated mitochondrial dysfunction. METHODS: Studies were performed in primary hepatocytes from wild-type cathepsin B knockout, Bid knockout, and caspase 2 knockout mice and in the rat hepatoma cell line McArdle7777 by using tumor necrosis factor alpha/actinomycin D. RESULTS: Studies in wild-type and Bid knockout hepatocytes showed that tumor necrosis factor alpha-mediated lysosomal permeabilization is Bid dependent. After tumor necrosis factor alpha/actinomycin D treatment, caspase 2 activity increased severalfold in wild-type hepatocytes, whereas minimal activity was observed in hepatocytes from cathepsin B knockout mice or in hepatoma cells treated with a cathepsin B inhibitor. In contrast, Bax was activated independently of cathepsin B. Pharmacological, genetic, or small interfering RNA-mediated inhibition of caspase 2 attenuated tumor necrosis factor alpha-mediated mitochondrial dysfunction, downstream caspase activation, and hepatocyte apoptosis. CONCLUSIONS: These data suggest that tumor necrosis factor alpha triggers Bid-dependent lysosomal permeabilization, followed by release of cathepsin B into the cytosol and activation of caspase 2. Caspase 2 then facilitates efficient mitochondrial cytochrome c release and apoptosis.     

Loss of the inactive myotubularin-related phosphatase Mtmr13 leads to a Charcot-Marie-Tooth 4B2-like peripheral neuropathy in mice

Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by slowed nerve conduction velocity, axon loss, and distinctive myelin outfolding and infolding. CMT4B is caused by recessive mutations in either myotubularin-related protein 2 (MTMR2; CMT4B1) or MTMR13 (CMT4B2). Myotubularins are phosphoinositide (PI) 3-phosphatases that dephosphorylate phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns(3,5)P(2), two phosphoinositides that regulate endosomal-lysosomal membrane traffic. Interestingly, nearly half of the metazoan myotubularins are predicted to be catalytically inactive. Both active and inactive myotubularins have essential functions in mammals and in Caenorhabditis elegans. MTMR2 and MTMR13 are active and inactive PI 3-phosphatases, respectively, and the two proteins have been shown to directly associate, although the functional significance of this association is not well understood. To establish a mouse model of CMT4B2, we disrupted the Mtmr13 gene. Mtmr13-deficient mice develop a peripheral neuropathy characterized by reduced nerve conduction velocity and myelin outfoldings and infoldings. Dysmyelination is evident in Mtmr13-deficient nerves at 14 days and worsens throughout life. Thus, loss of Mtmr13 in mice leads to a peripheral neuropathy with many of the key features of CMT4B2. Although myelin outfoldings and infoldings occur most frequently at the paranode, our morphological analyses indicate that the ultrastructure of the node of Ranvier and paranode is intact in Mtmr13-deficient nerve fibers. We also found that Mtmr2 levels are decreased by approximately 50% in Mtmr13-deficient sciatic nerves, suggesting a mode of Mtmr2 regulation. Mtmr13-deficient mice will be an essential tool for studying how the loss of MTMR13 leads to CMT4B2.     

Gastric parietal cell acid secretion in mice can be regulated independently of H/K ATPase endocytosis

BACKGROUND & AIMS: Gastric parietal cells secrete acid into the lumen of the stomach. They express a proton pump, the gastric H(+)/K(+) ATPase, the activity of which is tightly regulated. The H(+)/K(+) ATPase traffics between an intracytoplasmic compartment (tubulovesicles) in quiescent parietal cells and the apical plasma membrane in activated cells. These trafficking events are considered to contribute to the control of acid secretion by modulating access to apical K(+) and Cl(-) conductances that are required for transmembrane H(+) ion transport by the H(+)/K(+) ATPase. Here, we have determined whether the control of acid secretion in vivo requires membrane trafficking of the H(+)/K(+) ATPase. METHODS: We developed mice that only express an H(+)/K(+) ATPase beta subunit in which a putative tyrosine-based endocytosis motif in the cytoplasmic tail is mutated. Location of the H(+)/K(+) ATPase and parietal cell ultrastructure and gastric acid secretion were then examined. RESULTS: Parietal cells of these mice lacked a tubulovesicular compartment, and the H(+)/K(+) ATPase was resident exclusively on the apical plasma membrane. Despite the inability of the H(+)/K(+) ATPase to be endocytosed, the gastric acid secretory response to histamine or an antagonist was very similar to that of wild-type mice, indicating that control of H(+)/K(+) ATPase activity can occur independently of intracellular trafficking. CONCLUSIONS: We were able to dissociate the regulation of H(+)/K(+) ATPase activity from intracellular trafficking of the protein. Thus, it is likely that direct regulation of apical K(+) and Cl(-) conductances are sufficient to control gastric acid secretion.     

TGF-β from the Porcine Intestinal Cell Line IPEC-J2 Induced by Porcine Circovirus 2 Increases the Frequency of Treg Cells via the Activation of ERK (in CD4+ T Cells) and NF-κB (in IPEC-J2)

Porcine circovirus 2 (PCV2) causes immunosuppression. Piglets infected with PCV2 can develop enteritis. Given that the gut is the largest immune organ, however, the response of the gut’s immune system to PCV2 is still unclear. Here, IPEC-J2 cells with different treatments were co-cultured with PBMC or CD4⁺ T cells (Transwell). Flow cytometry and Western blotting revealed that PCV2-infected IPEC-J2 increased the frequency of CD4⁺ T cells among piglets’ peripheral blood mononuclear cells (PBMCs) and caused CD4⁺ T cells to undergo a transformation into Foxp3⁺ regulatory T cells (Treg cells) via activating CD4⁺ T ERK. Cytokines production and an inhibitor assay showed that the induction of Tregs by PCV2-infected IPEC-J2 was dependent on TGF-β induced by PCV2 in IPEC-J2, which was associated with the activation of NF-κB. Taken together, PCV2-infected IPEC-J2 activated NF-κB to stimulate the synthesis of TGF-β, which enhanced the differentiation of CD4⁺ T cells into Treg cells through the activation of ERK in CD4⁺ T cells. This information sheds light on PCV2′s function in the intestinal immune system and suggests a potential immunosuppressive mechanism for PCV2 infection.     

The truncated ghrelin receptor polypeptide (GHS-R1b) is localized in the endoplasmic reticulum where it forms heterodimers with ghrelin receptors (GHS-R1a) to attenuate their cell surface expression

The ghrelin receptor (GHS-R1a) is remarkable amongst G-protein-coupled receptors for its high degree of constitutive activity, and this agonist-independent activity may be important for its physiological function in the control of food intake and body weight. Ghrelin receptors form heterodimers with the truncated ghrelin receptor polypeptide (GHS-R1b), which has a dominant-negative effect on ghrelin receptor function. Here we show that GHS-R1b has an intracellular localization distinct from ghrelin receptors, being primarily localized in the endoplasmic reticulum. Immunocytochemical studies suggest that GHS-R1b decreases the plasma membrane expression of ghrelin receptors, but the overall distribution profile of ghrelin receptors in isolated subcellular fractions is unaffected by GHS-R1b. Using bioluminescence resonance energy transfer methods, we have shown that while ghrelin receptor homodimers are evenly distributed in all subcellular fractions, GHS-R1a/GHS-R1b heterodimers are concentrated within the endoplasmic reticulum and these results suggest that GHS-R1b traps ghrelin receptors within the endoplasmic reticulum by the process of oligomerization. Furthermore, ghrelin receptors constitutively activated extracellular signal-regulated kinases 1/2 in the endoplasmic reticulum, but this small response was not affected by GHS-R1b and its physiological relevance is uncertain. Taken together, these results suggest that ghrelin receptors can be retained in the endoplasmic reticulum by heterodimerization with GHS-R1b, and constitutive activation of phospholipase C is attenuated due to decreased cell surface expression of ghrelin receptors. However, sufficient ghrelin receptor homodimers can still be expressed on the cell surface for maximal responses to agonist stimulation.     

Endogenous opioids modulate the growth of the biliary tree in the course of cholestasis

BACKGROUND & AIMS: There is poor knowledge on the factors that modulate the growth of cholangiocytes, the epithelial cell target of cholangiopathies, which are diseases leading to progressive loss of bile ducts and liver failure. Endogenous opioids are known to modulate cell growth. In the course of cholestasis, the opioidergic system is hyperactive, and in cholangiocytes a higher expression of opioid peptide messenger RNA has been described. This study aimed to verify if such events affect the cholangiocyte proliferative response to cholestasis. METHODS: The presence of the delta opioid receptor (OR), muOR, and kappaOR was evaluated. The effects on cholangiocyte proliferation of the in vitro and in vivo exposure to their selective agonists, together with the intracellular signals, were then studied. The effects of the OR antagonist naloxone on cell growth were also tested both in vivo and in vitro. RESULTS: Cholangiocytes express all 3 receptors studied. deltaOR activation strongly diminished the proliferative and functional response of cholangiocytes to cholestasis, whereas muOR resulted in a slight increase in cell growth. The deltaOR signal is mediated by the IP3/CamKIIalpha/PKCalpha pathway, which inhibits the cAMP/PKA/ERK1/2/AKT cascade. In contrast, muOR activation stimulates the cAMP/PKA/ERK1/2/AKT cascade but does not affect the IP3/CamKIIalpha/PKCalpha pathway. The blockage of endogenous opioid peptides by naloxone further enhanced cholangiocyte growth both in vivo and in vitro. CONCLUSIONS: The increase in opioid peptide synthesis in the course of cholestasis aims to limit the excessive growth of the biliary tree in the course of cholestasis by the interaction with the deltaOR expressed by cholangiocytes.     

Lysophosphatidylcholine enhances I(Ks) currents in cardiac myocytes through activation of G protein, PKC and Rho signaling pathways

Lysophosphatidylcholine (LPC) is a bioactive phospholipid that accumulates rapidly in the ischemic myocardium. In recent years, it has been shown that some of the actions of LPC are mediated through the activation of the membrane G proteins. However, the precise mechanism(s) responsible for the LPC-related intracellular signaling in the regulation of cardiac ion channels are still poorly understood. The present study was undertaken to examine whether LPC regulates the slow component of the delayed rectifier K⁺ current (I_Ks) and, if so, what intracellular signals are important for this process. Isolated guinea pig cardiac myocytes were voltage-clamped using the whole-cell configuration of the patch-clamp method. The bath application of 1-palmitoyl-lysophosphatidylcholine (LPC-16) concentration-dependently (EC₅₀ = 0.7 μM) and reversibly increased I_Ks in atrial cells, but failed to potentiate I_Ks in ventricular myocytes. In contrast, 1-oleoyl-lysophosphatidylcholine (LPC-18:1) only produced a slight I_Ks increase, and 1-caproyl-lysophosphatidylcholine (LPC-6) or the LPC-16 precursor (phosphatidylcholine) had no effect on I_Ks. Pretreatment of atrial cells with an antibody against the N-terminus of the G2A receptor significantly reduced the LPC-16-induced potentiation of I_Ks. The inhibition of heterotrimeric G protein, phospholipase C (PLC) and protein kinase C (PKC) significantly reduced LPC-16-induced enhancement of I_Ks. Moreover, the blockade of Rho and Rho-kinase by specific inhibitors also inhibited the activity of LPC-16. Immunohistochemical studies demonstrated that G2A was densely distributed in the plasma membrane of atrial myocytes. Therefore, the present study suggests that the activation of a G protein (probably Gα_q) by LPC-16 potentiates I_Ks currents through the PLC-PKC and Rho-kinase pathways.     

Mucosal adaptation to enteral nutrients is dependent on the physiologic actions of glucagon-like peptide-2 in mice

BACKGROUND & AIMS: Our understanding of the intestinotropic actions of glucagon-like peptide-2 (GLP-2)(1-33) is based on pharmacologic studies involving exogenous administration. However, the physiologic role of GLP-2 in mucosal growth and adaptation to nutritional stimulation remains poorly understood. METHODS: The properties of GLP-2(3-33), a GLP-2(1-33) metabolite, were determined in baby-hamster kidney cells transfected with the mouse GLP-2 receptor complementary DNA and in isolated murine intestinal muscle strips. To investigate the role of endogenous GLP-2(1-33) in gut adaptation, GLP-2(3-33) was administered to mice that were re-fed for 24 hours after 24 hours of fasting, and the small intestine was analyzed. GLP-2(3-33) also was injected into rats for analysis of circulating GLP-2(1-33) levels. RESULTS: GLP-2(3-33) antagonized the actions of GLP-2(1-33) in vitro and ex vivo. Fasting mice exhibited small intestinal atrophy (37% +/- 1% decrease in small intestinal weight, 19% +/- 2% decrease in crypt-villus height, and 99% +/- 35% increase in villus apoptosis, P < .05-.01). Adaptive growth in re-fed mice restored all these parameters, as well as crypt-cell proliferation, to normal control levels (P < .05 vs. fasting); these adaptive changes were prevented partially or completely by co-administration of GLP-2(3-33) to refeeding mice (by 32% +/- 19% to 103% +/- 15%, P < .05-.01 vs re-fed mice). Exogenous GLP-2(3-33) did not affect endogenous GLP-2(1-33) levels. CONCLUSIONS: These data show that endogenous GLP-2 regulates the intestinotropic response in re-fed mice through modulation of crypt-cell proliferation and villus apoptosis. GLP-2 is therefore a physiologic regulator of the dynamic adaptation of the gut mucosal epithelium in response to luminal nutrients.     

Effect of low-molecular-weight heparin on the commitment of bone marrow cells to liver sinusoidal endothelial cells in CCl(4)-induced liver injury.

BACKGROUND/AIMS: Recently liver regeneration by bone marrow transplantation has been proposed as an alternative source of functional liver cells. We investigate commitment of bone marrow cells (BMCs) to liver regeneration and the effect of dalteparin sodium (DS) on regeneration of the damaged liver caused by carbon tetrachloride (CCl(4)) administration in the mice. METHODS: Liver injury was produced in 8-week-old mice by treating with CCl(4) for 4 weeks. Thereafter, mice received a lethal dose of irradiation (10Gy) to whole body, followed by injection of 1x10(7) green fluorescent protein (GFP)-positive BMCs via the tail vein. DS (50IU/kg, intraperitoneally) was administered daily for 28 consecutive days starting at 1 day post-BMC transplantation. Lineage marker analysis of GFP-positive liver cells was performed immunostaining with a CD31 antibody. RESULT: Four weeks after BMC transplantation, GFP-positive cells in the CCl(4)-damaged liver could be detected in the lobule displaying a meshwork architecture extending from the periportal to pericentral regions, a pattern simulating sinusoidal lining. This localization of GFP-positive cells suggested that these cells were closely associated with sinusoidal endothelial cells. By staining the GFP-positive cells for CD31, it was confirmed that the majority of the GFP-positive cells are also positive for CD31. The GFP(+)CD31(+) cells were barely detected in the control group (1.0+/-1.2 per field). In marked contrast, a numerous number of GFP(+)CD31(+) cells were detected in the liver section obtained from the CCl(4)-induced liver damage group (3.8+/-1.3 per field, P<0.05 versus control). The number of GFP(+)CD31(+) cells in CCl(4) plus DS-treated group was further increased to 8.3+/-1.3 per field (P<0.05 versus CCl(4)-induced liver damage group). CONCLUSION: The majority of GFP-positive BMCs was committed to sinusoidal endothelial cells. DS promoted BMC differentiation into sinusoidal endothelial cells in the CCl(4)-damaged liver.     

Effect of Nicotinic acid/Laropiprant in the lipoprotein(a) concentration with regard to baseline lipoprotein(a) concentration and LPA genotype

Background

Lipoprotein(a) [Lp(a)] is a lipoprotein in which apolipoproteinB-100 is linked to apolipoprotein(a) [apo(a)]. Significant variation in Lp(a) concentration is specific to LPA gene, which codes for apo(a). Nicotinic acid (NA) is used for treatment of dyslipidemias, and the lowering effect of NA on Lp(a) has been previously reported.

Objective

To evaluate the Lp(a) lowering effect of 1 g/20 mg and 2 g/40 mg day of Nicotinic acid/Laropiprant in subjects with different baseline Lp(a) concentrations and depending on the LPA genotype.

Methods

In an open-label, 10-week study, 1 g/20 mg day of NA/Laropiprant for 4 weeks followed by 6 weeks of 2 g/40 mg day conducted at 3 centers in Spain, 82 subjects were enrolled. Patients were studied at baseline and at the end of both treatment periods and were enrolled in three groups: normal Lp(a) (< 50 mg/dL), high Lp(a) (50–120 mg/dL) and very high Lp(a) (> 120 mg/dL). The LPA genetic polymorphism was analyzed by a real-time PCR.

Results

There was a significant difference in LPA genotypes among Lp(a) concentration groups and an inverse and significant correlation between baseline Lp(a) concentration and LPA genotype was found (R = − 0.372, p < 0.001). There were a significant decrease in total cholesterol, triglycerides, LDL cholesterol, apo B and Lp(a), and a significant increase in HDL cholesterol after NA/Laropiprant treatment, without changes in BMI. However, there were no statistical differences in percentage variation of analyzed variables depending on LPA genotype.

Conclusion

LPA genotype is a major determinant of Lp(a) baseline concentration. However, the lipid lowering effect of NA is not related to LPA genotype.     

Schistosoma mansoni: gene expression of the nucleotide excision repair factor 2 (NEF2) during the parasite life cycle, and in adult worms after exposure to different DNA-damaging agents

DNA is often damaged by many environmental agents, which lead to the up-regulation of several genes involved in different repair pathways. Schistosoma mansoni has a complex life cycle, being exposed to a subset of DNA-damaging agents, such as those present in the environment and host immune response. Recently, studies showed that nucleotide excision repair (NER) is an indispensable mechanism for removing a broad spectrum of different DNA lesions. In the present report, we showed the gene expression of nucleotide excision repair factor 2 (NEF2) SmRad23 and SmRad4, in different developmental stages of S. mansoni, as well as the differential expression of these genes in S. mansoni adult worms treated with DNA-damaging agents. Furthermore, it was revealed the correlation of these genes with their orthologues in other eukaryotes. Our reports suggest that NER is an important repair pathway during the complex life cycle of S. mansoni.