Prophylactic treatment with the c-Abl inhibitor, neurotinib, diminishes neuronal damage and the convulsive state in pilocarpine-induced mice.

The molecular mechanisms underlying seizure generation remain elusive, yet they are crucial for developing effective treatments for epilepsy. The current study shows that inhibiting c-Abl tyrosine kinase prevents apoptosis, reduces dendritic spine loss, and maintains N-methyl-d-aspartate (NMDA) receptor subunit 2B (NR2B) phosphorylated in in vitro models of excitotoxicity. Pilocarpine-induced status epilepticus (SE) in mice promotes c-Abl phosphorylation, and disrupting c-Abl activity leads to fewer seizures, increases latency toward SE, and improved animal survival. Currently, clinically used c-Abl inhibitors are non-selective and have poor brain penetration. The allosteric c-Abl inhibitor, neurotinib, used here has favorable potency, selectivity, pharmacokinetics, and vastly improved brain penetration. Neurotinib-administered mice have fewer seizures and improved survival following pilocarpine-SE induction. Our findings reveal c-Abl kinase activation as a key factor in ictogenesis and highlight the impact of its inhibition in preventing the insurgence of epileptic-like seizures in rodents and humans.

Genistein Activates Transcription Factor EB and Corrects Niemann-Pick C Phenotype.

Niemann-Pick type C disease (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood-brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in ß-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.

MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content.

MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria.

Mitochondrial GSH replenishment as a potential therapeutic approach for Niemann Pick type C disease.

Niemann Pick type C (NPC) disease is a progressive lysosomal storage disorder caused by mutations in genes encoding NPC1/NPC2 proteins, characterized by neurological defects, hepatosplenomegaly and premature death. While the primary biochemical feature of NPC disease is the intracellular accumulation of cholesterol and gangliosides, predominantly in endolysosomes, mitochondrial cholesterol accumulation has also been reported. As accumulation of cholesterol in mitochondria is known to impair the transport of GSH into mitochondria, resulting in mitochondrial GSH (mGSH) depletion, we investigated the impact of mGSH recovery in NPC disease. We show that GSH ethyl ester (GSH-EE), but not N-acetylcysteine (NAC), restored the mGSH pool in liver and brain of Npc1-/- mice and in fibroblasts from NPC patients, while both GSH-EE and NAC increased total GSH levels. GSH-EE but not NAC increased the median survival and maximal life span of Npc1-/- mice. Moreover, intraperitoneal therapy with GSH-EE protected against oxidative stress and oxidant-induced cell death, restored calbindin levels in cerebellar Purkinje cells and reversed locomotor impairment in Npc1-/- mice. High-resolution respirometry analyses revealed that GSH-EE improved oxidative phosphorylation, coupled respiration and maximal electron transfer in cerebellum of Npc1-/- mice. Lipidomic analyses showed that GSH-EE treatment had not effect in the profile of most sphingolipids in liver and brain, except for some particular species in brain of Npc1-/- mice. These findings indicate that the specific replenishment of mGSH may be a potential promising therapy for NPC disease, worth exploring alone or in combination with other options.

Reduction of Blood Amyloid-ß Oligomers in Alzheimer's Disease Transgenic Mice by c-Abl Kinase Inhibition.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of amyloid plaques, which are deposits of misfolded and aggregated amyloid-beta peptide (Aß). The role of the c-Abl tyrosine kinase in Aß-mediated neurodegeneration has been previously reported. Here, we investigated the therapeutic potential of inhibiting c-Abl using imatinib. We developed a novel method, based on a technique used to detect prions (PMCA), to measure minute amounts of misfolded-Aß in the blood of AD transgenic mice. We found that imatinib reduces Aß-oligomers in plasma, which correlates with a reduction of AD brain features such as plaques and oligomers accumulation, neuroinflammation, and cognitive deficits. Cells exposed to imatinib and c-Abl KO mice display decreased levels of ß-CTF fragments, suggesting that an altered processing of the amyloid-beta protein precursor is the most probable mechanism behind imatinib effects. Our findings support the role of c-Abl in Aß accumulation and AD, and propose AD-PMCA as a new tool to evaluate AD progression and screening for drug candidates.

Neuronal gene repression in Niemann-Pick type C models is mediated by the c-Abl/HDAC2 signaling pathway.

BACKGROUND: Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of free cholesterol in lysosomes. There are currently no effective FDA-approved treatments for NPC, although in the last years the inhibition of histone deacetylases (HDACs) has emerged as a potential treatment for this disease. However, the molecular mechanisms that deregulate HDAC activity in NPC disease are unknown. Previously our group had shown that the proapoptotic tyrosine kinase c-Abl signaling is activated in NPC neurons. Here, we demonstrate that c-Abl activity increases HDAC2 levels inducing neuronal gene repression of key synaptic genes in NPC models. RESULTS: Our data show that: i) HDAC2 levels and activity are increased in NPC neuronal models and in Npc1(-/-) mice; ii) inhibition of c-Abl or c-Abl deficiency prevents the increase of HDAC2 protein levels and activity in NPC neuronal models; iii) c-Abl inhibition decreases the levels of HDAC2 tyrosine phosphorylation; iv) treatment with methyl-ß-cyclodextrin and vitamin E decreases the activation of the c-Abl/HDAC2 pathway in NPC neurons; v) in vivo treatment with two c-Abl inhibitors prevents the increase of HDAC2 protein levels in the brain of Npc1(-/-) mice; and vi) c-Abl inhibition prevents HDAC2 recruitment to the promoter of neuronal genes, triggering an increase in their expression. CONCLUSION: Our data show the involvement of the c-Abl/HDAC2 signaling pathway in the regulation of neuronal gene expression in NPC neuronal models. Thus, inhibition of c-Abl could be a pharmacological target for preventing the deleterious effects of increased HDAC2 levels in NPC disease.

Recent insights on the role of cholesterol in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of hepatic histopathological changes ranging from non-inflammatory intracellular fat deposition to non-alcoholic steatohepatitis (NASH), which may progress into hepatic fibrosis, cirrhosis, or hepatocellular carcinoma. NAFLD hallmark is the excessive hepatic accumulation of neutral lipids that result from an imbalance between lipid availability and lipid removal. Recent data suggest that disturbed hepatic cholesterol homeostasis and liver free cholesterol (FC) accumulation are relevant to the pathogenesis of NAFLD/NASH. Hepatic FC accumulation in NAFLD results from alterations in intracellular cholesterol transport and from unbalanced cellular cholesterol homeostasis characterized by activation of cholesterol biosynthetic pathways, increased cholesterol de-esterification and attenuation of cholesterol export and bile acid synthesis pathways. FC accumulation leads to liver injury through the activation of intracellular signaling pathways in Kupffer cells (KCs), Stellate cells (HSCs) and hepatocytes. The activation of KCs and HSCs promotes inflammation and fibrogenesis. In addition, FC accumulation in liver mitochondria induces mitochondrial dysfunction, which results in increasing production of reactive oxygen species, and triggers the unfolded protein response in the endoplasmic reticulum (ER) causing ER stress and apoptosis. These events create a vicious circle that contributes to the maintenance of steatosis and promotes ongoing hepatocyte death and liver damage, which in turn may translate into disease progression. In the present review we summarize the current knowledge on dysregulated cholesterol homeostasis in NAFLD and examine the cellular mechanisms of hepatic FC toxicity and its contribution to ongoing liver injury in this disease. The therapeutic implications of this knowledge are also discussed.

c-Abl stabilizes HDAC2 levels by tyrosine phosphorylation repressing neuronal gene expression in Alzheimer's disease.

In Alzheimer's disease (AD), there is a decrease in neuronal gene expression induced by HDAC2 increase; however, the mechanisms involved are not fully elucidated. Here, we described how the tyrosine kinase c-Abl increases HDAC2 levels, inducing transcriptional repression of synaptic genes. Our data demonstrate that (1) in neurons, c-Abl inhibition with Imatinib prevents the AßO-induced increase in HDAC2 levels; (2) c-Abl knockdown cells show a decrease in HDAC2 levels, while c-Abl overexpression increases them; (3) c-Abl inhibition reduces HDAC2-dependent repression activity and HDAC2 recruitment to the promoter of several synaptic genes, increasing their expression; (4) c-Abl induces tyrosine phosphorylation of HDAC2, a posttranslational modification, affecting both its stability and repression activity; and (5) treatment with Imatinib decreases HDAC2 levels in a transgenic mice model of AD. Our results support the participation of the c-Abl/HDAC2 signaling pathway in the epigenetic blockade of gene expression in AD pathology.

Vitamin E dietary supplementation improves neurological symptoms and decreases c-Abl/p73 activation in Niemann-Pick C mice.

Niemann-Pick C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of free cholesterol in lysosomes. We have previously reported that oxidative stress is the main upstream stimulus activating the proapoptotic c-Abl/p73 pathway in NPC neurons. We have also observed accumulation of vitamin E in NPC lysosomes, which could lead to a potential decrease of its bioavailability. Our aim was to determine if dietary vitamin E supplementation could improve NPC disease in mice. NPC mice received an alpha-tocopherol (α-TOH) supplemented diet and neurological symptoms, survival, Purkinje cell loss, α-TOH and nitrotyrosine levels, astrogliosis, and the c-Abl/p73 pathway functions were evaluated. In addition, the effect of α-TOH on the c-Abl/p73 pathway was evaluated in an in vitro NPC neuron model. The α-TOH rich diet delayed loss of weight, improved coordination and locomotor function and increased the survival of NPC mice. We found increased Purkinje neurons and α-TOH levels and reduced astrogliosis, nitrotyrosine and phosphorylated p73 in cerebellum. A decrease of c-Abl/p73 activation was also observed in the in vitro NPC neurons treated with α-TOH. In conclusion, our results show that vitamin E can delay neurodegeneration in NPC mice and suggest that its supplementation in the diet could be useful for the treatment of NPC patients.

Disruption in connexin-based communication is associated with intracellular Ca²âº signal alterations in astrocytes from Niemann-Pick type C mice.

Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1⁻/⁻) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1⁻/⁻ hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1⁻/⁻ astrocytes also showed more intracellular Ca²âº signal oscillations mediated by functional connexin 43 hemichannels and P2Y1 receptors. Therefore, Npc1⁻/⁻ astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.

Increased copper levels in in vitro and in vivo models of Niemann-Pick C disease.

Niemann-Pick type C disease (NPC) is a hereditary neurovisceral atypical lipid storage disorder produced by mutations in the NPC1 and NPC2 genes. The disease is characterized by unesterified cholesterol accumulation in late endosomal/lysosomal compartments and oxidative stress. The most affected tissues are the cerebellum and the liver. The lysotropic drug U18666A (U18) has been widely used as a pharmacological model to induce the NPC phenotype in several cell culture lines. It has already been reported that there is an increase in copper content in hepatoma Hu7 cells treated with U18. We confirmed this result with another human hepatoma cell line, HepG2, treated with U18 and supplemented with copper in the media. However, in mouse hippocampal primary cultures treated under similar conditions, we did not find alterations in copper content. We previously reported increased copper content in the liver of Npc1 (-/-) mice compared to control animals. Here, we extended the analysis to the copper content in the cerebella, the plasma and the bile of NPC1 deficient mice. We did not observe a significant change in copper content in the cerebella, whereas we found increased copper content in the plasma and decreased copper levels in the bile of Npc1(-/-) mice. Finally, we also evaluated the plasma content of ceruloplasmin, and we found an increase in this primary copper-binding protein in Npc1 (-/-) mice. These results indicate cell-type dependence of copper accumulation in NPC disease and suggest that copper transport imbalance may be relevant to the liver pathology observed in NPC disease.

Cholinergic abnormalities, endosomal alterations and up-regulation of nerve growth factor signaling in Niemann-Pick type C disease.

BACKGROUND: Neurotrophins and their receptors regulate several aspects of the developing and mature nervous system, including neuronal morphology and survival. Neurotrophin receptors are active in signaling endosomes, which are organelles that propagate neurotrophin signaling along neuronal processes. Defects in the Npc1 gene are associated with the accumulation of cholesterol and lipids in late endosomes and lysosomes, leading to neurodegeneration and Niemann-Pick type C (NPC) disease. The aim of this work was to assess whether the endosomal and lysosomal alterations observed in NPC disease disrupt neurotrophin signaling. As models, we used i) NPC1-deficient mice to evaluate the central cholinergic septo-hippocampal pathway and its response to nerve growth factor (NGF) after axotomy and ii) PC12 cells treated with U18666A, a pharmacological cellular model of NPC, stimulated with NGF. RESULTS: NPC1-deficient cholinergic cells respond to NGF after axotomy and exhibit increased levels of choline acetyl transferase (ChAT), whose gene is under the control of NGF signaling, compared to wild type cholinergic neurons. This finding was correlated with increased ChAT and phosphorylated Akt in basal forebrain homogenates. In addition, we found that cholinergic neurons from NPC1-deficient mice had disrupted neuronal morphology, suggesting early signs of neurodegeneration. Consistently, PC12 cells treated with U18666A presented a clear NPC cellular phenotype with a prominent endocytic dysfunction that includes an increased size of TrkA-containing endosomes and reduced recycling of the receptor. This result correlates with increased sensitivity to NGF, and, in particular, with up-regulation of the Akt and PLC-γ signaling pathways, increased neurite extension, increased phosphorylation of tau protein and cell death when PC12 cells are differentiated and treated with U18666A. CONCLUSIONS: Our results suggest that the NPC cellular phenotype causes neuronal dysfunction through the abnormal up-regulation of survival pathways, which causes the perturbation of signaling cascades and anomalous phosphorylation of the cytoskeleton.

Molecular Mechanisms Underlying the Link between Nuclear Receptor Function and Cholesterol Gallstone Formation.

Cholesterol gallstone disease is highly prevalent in western countries, particularly in women and some specific ethnic groups. The formation of water-insoluble cholesterol crystals is due to a misbalance between the three major lipids present in the bile: cholesterol, bile salts, and phospholipids. Many proteins implicated in biliary lipid secretion in the liver are regulated by several transcription factors, including nuclear receptors LXR and FXR. Human and murine genetic, physiological, pathophysiological, and pharmacological evidence is consistent with the relevance of these nuclear receptors in gallstone formation. In addition, there is emerging data that also suggests a role for estrogen receptor ESR1 in abnormal cholesterol metabolism leading to gallstone disease. A better comprehension of the role of nuclear receptor function in gallstone formation may help to design new and more effective therapeutic strategies for this highly prevalent disease condition.

Oxidative stress activates the c-Abl/p73 proapoptotic pathway in Niemann-Pick type C neurons.

Niemann-Pick type C (NPC) is a neurodegenerative disease characterized by the intralysosomal accumulation of cholesterol leading to neuronal apoptosis. We have previously reported the activation of the c-Abl/p73 proapoptotic pathway in the cerebellum of NPC mice; however, upstream signals underlying the engagement of this pathway remain unknown. Here, we investigate the possible role of oxidative stress in the activation of c-Abl/p73 using different in vitro and in vivo NPC models. Our results indicate a close temporal correlation between the appearance of nitrotyrosine (N-Tyr; a post-translational tyrosine modification caused by oxidative stress) and the activation of c-Abl/p73 in NPC models. To test the functional role of oxidative stress in NPC, we have treated NPC neurons with the antioxidant NAC and observed a dramatic decrease of c-Abl/p73 activation and a reduction in the levels of apoptosis in NPC models. In conclusion, our data suggest that oxidative stress is the main upstream stimulus activating the c-Abl/p73 pathway and neuronal apoptosis in NPC neurons.

El síndrome metabólico: De factor agravante a principal factor de riesgo patogénico en diversas enfermedades crónicas / The metabolic syndrome: From an aggravating condition to a pathogenic risk factor for chronic diseases

In recent years, a rapidly increasing number of studies have focused on the association between metabolic syndrome and several chronic diseases. However, it is difficult to determine a well defined pathogenic relationship, due to the etiological heterogeneity and comorbidities of these diseases. Research efforts are aiming to identify the convergent biological mechanisms that mediate the effects of hyperinsulinemia, hyperglycemia, dyslipidemia, and hypertension. All these conditions define the metabolic syndrome, that increases the risk for several diseases. The knowledge of these biological mechanisms associated with this syndrome will elucidate the pathogenic association between a variety of chronic diseases, including its pathogenic link with cardiovascular diseases and the most common forms of dementia. The development of new therapeutic and preventive strategies for these diseases will be a corollary of this research.

Fecal bile acid excretion and messenger RNA expression levels of ileal transporters in high risk gallstone patients.

BACKGROUND: Cholesterol gallstone disease (GS) is highly prevalent among Hispanics and American Indians. In GS, the pool of bile acids (BA) is decreased, suggesting that BA absorption is impaired. In Caucasian GS patients, mRNA levels for ileal BA transporters are decreased. We aimed to determine fecal BA excretion rates, mRNA levels for ileal BA transporter genes and of regulatory genes of BA synthesis in Hispanic GS patients. RESULTS: Excretion of fecal BA was measured in seven GS females and in ten GS-free individuals, all with a body mass index < 29. Participants ingested the stool marker Cr2O3 (300 mg/day) for 10 days, and fecal specimens were collected on the last 3 days. Chromium was measured by a colorimetric method, and BA was quantitated by gas chromatography/mass spectroscopy. Intake of calories, nutrients, fiber and cholesterol were similar in the GS and GS-free subjects. Mean BA excretion levels were 520 +/- 80 mg/day for the GS-free group, and 461 +/- 105 mg/day for the GS group. Messenger RNA expression levels were determined by RT-PCR on biopsy samples obtained from ileum during diagnostic colonoscopy (14 GS-free controls and 16 GS patients) and from liver during surgery performed at 8 and 10 AM (12 GS and 10 GS-free patients operated on for gastrointestinal malignancies), all with a body mass index < 29. Messenger RNA level of the BA transporter genes for ileal lipid binding protein, multidrug resistance-associated protein 3, organic solute transporter alpha, and organic solute transporter beta were similar in GS and GS-free subjects. Messenger RNA level of Cyp27A1, encoding the enzyme 27alpha-hydroxylase, the short heterodimer partner and farnesoid X receptor remained unchanged, whereas the mRNA level of Cyp7A1, the rate limiting step of BA synthesis, was increased more than 400% (p < 0.01) in the liver of GS compared to GS-free subjects. CONCLUSION: Hispanics with GS have fecal BA excretion rates and mRNA levels of genes for ileal BA transporters that are similar to GS-free subjects. However, mRNA expression levels of Cyp7A1 are increased in GS, indicating that regulation of BA synthesis is abnormal in Hispanics with GS.

Imatinib therapy blocks cerebellar apoptosis and improves neurological symptoms in a mouse model of Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a fatal autosomal recessive disorder characterized by the accumulation of free cholesterol and glycosphingolipids in the endosomal-lysosomal system. Patients with NPC disease have markedly progressive neuronal loss, mainly of cerebellar Purkinje neurons. There is strong evidence indicating that cholesterol accumulation and trafficking defects activate apoptosis in NPC brains. The purpose of this study was to analyze the relevance of apoptosis and particularly the proapoptotic c-Abl/p73 system in cerebellar neuron degeneration in NPC disease. We used the NPC1 mouse model to evaluate c-Abl/p73 expression and activation in the cerebellum and the effect of therapy with the c-Abl-specific inhibitor imatinib. The proapoptotic c-Abl/p73 system and the p73 target genes are expressed in the cerebellums of NPC mice. Furthermore, inhibition of c-Abl with imatinib preserved Purkinje neurons and reduced general cell apoptosis in the cerebellum, improved neurological symptoms, and increased the survival of NPC mice. Moreover, this prosurvival effect correlated with reduced mRNA levels of p73 proapoptotic target genes. Our results suggest that the c-Abl/p73 pathway is involved in NPC neurodegeneration and show that treatment with c-Abl inhibitors is useful in delaying progressive neurodegeneration, supporting the use of imatinib for clinical treatment of patients with NPC disease.

Overexpression of the cholesterol-binding protein MLN64 induces liver damage in the mouse.

AIM: To examine the in vivo phenotype associated with hepatic metastatic lymph node 64 (MLN64) over-expression. METHODS: Recombinant-adenovirus-mediated MLN64 gene transfer was used to overexpress MLN64 in the livers of C57BL/6 mice. We measured the effects of MLN64 overexpression on hepatic cholesterol content, bile flow, biliary lipid secretion and apoptosis markers. For in vitro studies cultured CHO cells with transient MLN64 overexpression were utilized and apoptosis by TUNEL assay was measured. RESULTS: Livers from Ad.MLN64-infected mice exhibited early onset of liver damage and apoptosis. This response correlated with increases in liver cholesterol content and biliary bile acid concentration, and impaired bile flow. We investigated whether liver MLN64 expression could be modulated in a murine model of hepatic injury. We found increased hepatic MLN64 mRNA and protein levels in mice with chenodeoxycholic acid-induced liver damage. In addition, cultured CHO cells with transient MLN64 overexpression showed increased apoptosis. CONCLUSION: In summary, hepatic MLN64 over-expression induced damage and apoptosis in murine livers and altered cholesterol metabolism. Further studies are required to elucidate the relevance of these findings under physiologic and disease conditions.

Adenovirus-mediated hepatic syndecan-1 overexpression induces hepatocyte proliferation and hyperlipidaemia in mice.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) have been involved in the regulation of cell growth, apoptosis and lipid metabolism in vitro; however, their functional role in vivo remains unknown. AIM: Here, we describe hepatic tissue and lipid metabolism changes after liver overexpression of syndecan-1 (SDC-1), the main hepatic HSPG, in mice induced by adenoviral gene transfer. RESULTS: SDC-1 overexpression was associated with marked hepatocyte proliferation, cell-isolated apoptosis and increased plasma alanine aminotransferase (ALT) levels. Additionally, SDC-1 liver overexpression significantly raised plasma cholesterol and triglyceride concentrations due to an increase in all lipoprotein particles, including the appearance of large and apolipoprotein (apo) E-enriched high-density lipoprotein (HDL) particles. Hepatic very low-density lipoprotein (VLDL) production was not affected by SDC-1 overexpression, suggesting a delayed plasma clearance of apo B lipoproteins as the underlying hyperlipidaemic mechanism. These pleotropic effects were qualitatively equivalent, even though less intense, in mice overexpressing a cytoplasmic C-terminal domain-deleted SDC-1. CONCLUSIONS: This is the first report in vivo of the biological effects induced by a specific HSPG in the liver, with potential implications in both regenerative biology and molecular lipidology.

Increased activity of hepatic microsomal triglyceride transfer protein and bile acid synthesis in gallstone disease.

UNLABELLED: A strong interrelationship exists between the regulation of bile acid (BA) metabolism and hepatic very low density lipoprotein (VLDL) production. We have recently shown that BA synthesis is increased in gallstone disease. We investigated the activity of hepatic microsomal triglyceride transfer protein (MTTP) as a surrogate of VLDL production, BA synthesis, and mRNA expression levels of proteins that regulate fatty acid (FA) metabolism in the liver of gallstone (GS) patients compared with GS-free patients. Twenty-seven volunteers subjected to elective surgery; 9 were GS-free and 18 with GS agreed to have a liver biopsy. We quantified by a fluorescence assay the activity of MTTP and by quantitative reverse-transcription PCR (RT-PCR) the mRNA content of hepatic MTTP and genes that regulate hepatic sterol and FA metabolism. Plasma was assayed for lathosterol and 7alpha-hydroxy-4-cholesten-3-one. Liver histology was normal in GS and GS-free patients. Serum VLDL triglycerides and apoB were significantly increased in GS. Hepatic triglycerides tripled in GS (P<0.001) compared with GS-free. MTTP activity increased 70% (P<0.001). Serum lathosterol and hepatic cholesterol concentrations, and mRNA expressions of MTTP, CD36, and FABP1 were similar in GS-free and GS patients. Hepatic mRNA expression of hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and 3-hydroxyl-3-methyl-glutaryl-CoA synthase (HMGS) were significantly decreased--40% and 27%, respectively--in GS. Serum 7alpha-hydroxy-4-cholesten-3-one was 75% higher, and mRNA expression of CYP7A1 was increased sevenfold (P<0.001) in GS. CONCLUSION: Hepatic MTTP activity and BA synthesis are increased in GS. Results suggest that hepatic VLDL production and trafficking of BA are increased in gallstone patients.