Olatz Fresnedo

HEDGEHOG SIGNALING IS CRITICAL FOR NORMAL LIVER REGENERATION AFTER PARTIAL HEPATECTOMY IN MICE

Distinct mechanisms are believed to regulate growth of the liver during fetal development and after injury in adults, because the former relies on progenitors and the latter generally involves replication of mature hepatocytes. However, chronic liver injury in adults increases production of Hedgehog (Hh) ligands, developmental morphogens that control progenitor cell fate and orchestrate various aspects of tissue construction during embryogenesis. This raises the possibility that similar Hh‐dependent mechanisms also might regulate adult liver regeneration. The current analysis of murine liver regeneration after 70% partial hepatectomy (PH), an established model of adult liver regeneration, demonstrated that PH induced production of Hh ligands and activated Hh signaling in liver cells. Treatment with a specific Hh signaling inhibitor interfered with several key components of normal liver regeneration, significantly inhibiting progenitor responses, matrix remodeling, proliferation of hepatocytes and ductular cells, and restoration of liver mass. These global inhibitory effects on liver regeneration dramatically reduced survival after PH. Conclusion: Mechanisms that mediate liver organogenesis, such as Hh pathway activation, are retained and promote reconstruction of adult livers after injury. Hepatology 2010

Profiling and Imaging of Lipids on Brain and Liver Tissue by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Using 2-Mercaptobenzothiazole as a Matrix

2-Mercaptobenzothiazole (MBT) is employed for the first time as a matrix for the analysis of lipids from tissue extracts using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We demonstrate that the performance of MBT is superior to that of the matrixes commonly employed for lipids, due to its low vapor pressure, its low acidity, and the formation of small crystals, although because of the strong background at low m/z, it precludes detection of species below approximately 500 Da. This inconvenience can be partly overcome with the formation of Cs adducts. Using a polymer-based dual calibration, a mass accuracy of approximately 10 ppm in lipid extracts and of approximately 80 ppm in tissues is achieved. We present spectra from liver and brain lipid extracts where a large amount of lipid species is identified, in both positive and negative ion modes, with high reproducibility. In addition, the above-mentioned special properties of MBT allow its employment for imaging mass spectrometry. In the present work, images of brain and liver tissues showing different lipid species are presented, demonstrating the advantages of the employment of MBT.

Matrix-assisted laser desorption ionization imaging mass spectrometry in lipidomics

The relevant structural, energetics, and regulatory roles of lipids are universally acknowledged. However, the high variability of lipid species and the large differences in concentrations make unraveling the role played by the different species in metabolism a titanic task. A recently developed technique, known as imaging mass spectrometry, may shed some light on the field, as it enables precise information to be obtained on the location of lipids in tissues. A review of the state of the art of the technique is presented in this manuscript, including detailed analysis of sample-preparation steps, data handling, and the identification of the species mapped so far.

EFFECT OF NITROGEN STARVATION ON THE BIOCHEMISTRY OF PHORMIDIUM LAMINOSUM (CYANOPHYCEAE)1

Cells of the non‐N2‐fixing cyanobacterium Phormidium laminosum (Agardh) Gomont (strain OH‐1‐pCl1) showed doubling times of 24 h in media containing nitrate and 120 h in media without a nitrogen source. Nitrogen starvation resulted in a drastic decrease in the cellular content of chlorophyll, phycobiliproteins (phycocyanin and allophycocyanin), and other soluble proteins, although the total protein of cells was unchanged. N‐starved cells showed an exocellular layer of mucilage that rapidly increased with starvation time. The appearance of N deficiency symptoms was strongly dependent on culture conditions, and it was faster under the optimal conditions used for cell growth. The relative content of C and N of nitrate‐grown cells remained more or less constant during all growth phases (C/N ratio of ca. 5) but diminished at different rates in N‐starved cells. Cells subjected to N starvation for 48 h had a C/N ratio of more than 10. N starvation also resulted in the selective degradation of soluble poly‐peptides of masses lower than 20 kDa (which include those constituting phycobiliproteins), whereas the relative content of soluble polypeptides of greater size increased.

S-Adenosylmethionine increases circulating very-low density lipoprotein clearance in non-alcoholic fatty liver disease

BACKGROUND & AIMS Very-low-density lipoproteins (VLDLs) export lipids from the liver to peripheral tissues and are the precursors of low-density-lipoproteins. Low levels of hepatic S-adenosylmethionine (SAMe) decrease triglyceride (TG) secretion in VLDLs, contributing to hepatosteatosis in methionine adenosyltransferase 1A knockout mice but nothing is known about the effect of SAMe on the circulating VLDL metabolism. We wanted to investigate whether excess SAMe could disrupt VLDL plasma metabolism and unravel the mechanisms involved. METHODS Glycine N-methyltransferase (GNMT) knockout (KO) mice, GNMT and perilipin-2 (PLIN2) double KO (GNMT-PLIN2-KO) and their respective wild type (WT) controls were used. A high fat diet (HFD) or a methionine deficient diet (MDD) was administrated to exacerbate or recover VLDL metabolism, respectively. Finally, 33 patients with non-alcoholic fatty-liver disease (NAFLD); 11 with hypertriglyceridemia and 22 with normal lipidemia were used in this study. RESULTS We found that excess SAMe increases the turnover of hepatic TG stores for secretion in VLDL in GNMT-KO mice, a model of NAFLD with high SAMe levels. The disrupted VLDL assembly resulted in the secretion of enlarged, phosphatidylethanolamine-poor, TG- and apoE-enriched VLDL-particles; special features that lead to increased VLDL clearance and decreased serum TG levels. Re-establishing normal SAMe levels restored VLDL secretion, features and metabolism. In NAFLD patients, serum TG levels were lower when hepatic GNMT-protein expression was decreased. CONCLUSIONS Excess hepatic SAMe levels disrupt VLDL assembly and features and increase circulating VLDL clearance, which will cause increased VLDL-lipid supply to tissues and might contribute to the extrahepatic complications of NAFLD.

Carotenoid composition in the cyanobacterium Phormidium laminosum Effect of nitrogen starvation

When pigments of the non‐N2‐fixing cyanobacterium Pharmidium laminosum were carefully extracted and analyzed in a completely O2‐free atmosphere, by either high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) , the presence of only two carotenoids (namely, β‐carotene and nostoxanthin) was detected. However, exposure of pigments to an air atmosphere during their manipulation led to the rapid appearance in the organic extracts of at least three additional carotenoids (identified as caloxanthin, zeaxanthin and β‐cryptoxanthin). This fact could explain in the presence in cyanobacteria of such hydroxylated acrivatives of β‐carotene widely reported in the literature. Nitrogen starvation also resulted in an important decrease on the relative β‐carotene/nostoxanthin content of cells, suggesting that this nutritional condition affects thylakoid membranes more drastically than cytoplasmic membranes.

Impaired response of VLDL lipid and apoB secretion to endotoxin in the fasted rat liver

Bacterial infection elicits hypertriglyceridemia attributed to increased hepatic production of very low-density lipoprotein (VLDL) particles and decreased peripheral metabolism. The mechanisms underlying VLDL overproduction in sepsis are as yet unclear, but seem to be fed/fasted state-dependent. To learn more about this, we investigated hepatocytes isolated from fasted rats, made endotoxic by 1 mg/kg lipopolysaccharide (LPS) injection, for their ability to secrete the VLDL protein and lipid components. The results were then related to lipogenesis markers and expression of genes critical to VLDL biogenesis. Endotoxic rats showed increased levels of serum VLDL-apoB (10-fold), -triglyceride (2-fold), and -cholesterol (2-fold), whereby circulating VLDL were lipid-poor particles. Similarly, VLDL-apoB secretion by isolated endotoxic hepatocytes was ~85% above control, whereas marginal changes in the output of VLDL-lipid classes occurred. This was accompanied by a substantial rise in apoB and a moderate rise in MTP mRNA levels, but with basal de novo formation and efficiency of secretion of triglycerides, cholesterol and cholesteryl esters. These results indicate that during periods of food restriction, endotoxin does not enhance lipid provision to accomplish normal lipidation of overproduced apoB molecules, though this does occur to a sufficient extent to pass the proteasome checkpoint and secretion of lipid-poor, type 2 VLDL takes place.

Application of 2-hydroxypropyl-β-cyclodextrin in the assay of acyl-CoA: cholesterol acyltransferase and neutral and acid cholesterol ester hydrolases

The utility of 2-hydroxypropyl-β-cyclodextrin for increasing the sensitivity of assays for the microsomal acyl-CoA:cholesterol acyltransferase, and the acid lysosomal and the neutral microsomal and cytosolic cholesterol ester hydrolase activity was studied in rat hepatocytes. Enzyme assays, at optimal concentrations of cyclodextrin, were validated by assessing: (i) linearity of product formation with incubation time and protein amount, and saturation with substrate, and (ii) the effect of treatments of cells or of subcellular fractions on enzyme activities. Delivery of cholesterol dissolved in 2-hydroxypropyl-β-cyclodextrin to the acyl-CoA:cholesterol acyltransferase assay mixture raised the enzyme activity more than 8-fold and was twice that measured when cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-β-cyclodextrin itself was partially effective, apparently by making endogenous cholesterol more accesible to the enzyme. Inclusion of 2-hydroxypropyl-β-cyclodextrin in cholesterol ester hydrolase assays using standard micellar substrates doubled the activity estimated in lysosome and microsome preparations and enhanced the cytosolic cholesterol esterase activity by about 50%. Differences in the catalytic activity of acyl-CoA:cholesterol acyltransferase and cholesterol ester hydrolases caused by treatment of hepatocytes with compound 58-035 or 25-hydroxycholesterol, or of subcellular fractions with NaF, were maintained when enzymes were assayed with cyclodextrin. The results indicate that 2-hydroxypropyl-β-cyclodextrin is a suitable vehicle for delivering cholesterol to acyl-CoA:cholesterol acyltransferase and enhances the sensitivity of standard assays of the enzymes governing the intrahepatic hydrolysis of cholesteryl esters.

Effect of estradiol and progesterone on cholesterol 7α-hydroxylase activity in rats subjected to different feeding conditions

The regulation of cholesterol 7 alpha-hydroxylase activity by estradiol and progesterone was investigated in liver microsomes isolated from rats fed standard diet, either ad libitum or fasted for 24 h, and diet containing the bile acid sequesterant cholestyramine. Differential effects were observed when the direct action of estradiol and progesterone on microsome preparations was examined. Cholesterol 7 alpha-hydroxylase activity was inhibited by progesterone in a dose-dependent way to almost complete abolition; similar patterns of declines were found in the three feeding groups under study. In contrast, the addition of 5 microM estradiol induced small and selective 7 alpha-hydroxylase increases in fasting and cholestryamine-fed animals, then activity declined to control values and consistent decreases were found from 20 microM. The administration of estradiol (50 micrograms) or progesterone (100 micrograms) for 21 days resulted in depressed cholesterol 7 alpha-hydroxylase activity in rats with high bile acid synthesis basal rate due to cholestyramine feeding. In rats receiving a standard diet, either ad libitum or after 24 h fasting, the hormonal effects did not reach significance. Declines in the content of free cholesterol were provoked by progesterone, not by estradiol, in liver microsomes prepared from all feeding groups. No changes in cholesterol 7 alpha-hydroxylase activity and microsomal free cholesterol were observed after administration of the sex hormones for 3 days. Rapid and transient inhibitions in 7 alpha-hydroxylase activity were found after the single injection of progesterone to fed animals. Estradiol, on the contrary, was unable to alter rapidly the hepatic 7 alpha-hydroxylase capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

A role for transcription factor E2F2 in hepatocyte proliferation and timely liver regeneration

E2F transcription factors are key regulators of the cell cycle although the relative contribution of each E2F member in regulating cellular proliferation is still poorly defined. Present evidence suggests that E2F2 may act both as a suppressor and promoter of proliferation, depending on the cellular context. We used a loss-of-function mutant mouse model to investigate the function of E2F2 in liver regeneration after partial hepatectomy, a paradigm of cell-cycle progression. Liver mass recovery and histology were examined over 9 days in 70% hepatectomized E2F2(-/-) and wild-type animals. Transcriptome analysis was performed in quiescent and 48-h regenerating liver samples. TIGR MultiExperiment Viewer was used for the statistical analysis of microarray data, significance was determined by Fischer, and P values were adjusted applying Benjamini-Hochberg multiple-testing correction. We show that E2F2 is required for adult hepatocyte proliferation and for timely liver regeneration, as disruption of the E2F2 gene in hepatocytes leads to a reduced rate of S-phase entry and to delayed liver regeneration. Transcriptome analysis followed by ontological classification of differentially expressed genes and gene-interaction network analysis indicated that the majority of genes involved in normal liver regeneration were related to biosynthetic and catabolic processes of all major biomolecules as well as cellular location and intracellular transport, confirming the complex nature of the regeneration process. Remarkably, transcripts of genes included in functional categories that are crucial for cell cycle, apoptosis and wound-healing response, and fibrosis were absent in the transcriptome of posthepatectomized E2F2(-/-) mice. Our results indicate that the transcriptional activity of E2F2 contributes to promote adult hepatocyte proliferation and liver regeneration.