Fructose Mediated Non-Alcoholic Fatty Liver Is Attenuated by HO-1-SIRT1 Module in Murine Hepatocytes and Mice Fed a High Fructose Diet

Background

Oxidative stress underlies the etiopathogenesis of nonalcoholic fatty liver disease (NAFLD), obesity and cardiovascular disease (CVD). Heme Oxygenase-1 (HO-1) is a potent endogenous antioxidant gene that plays a key role in decreasing oxidative stress. Sirtuin1 (SIRT1) belongs to the family of NAD-dependent de-acyetylases and is modulated by cellular redox.

Hypothesis

We hypothesize that fructose-induced obesity creates an inflammatory and oxidative environment conducive to the development of NAFLD and metabolic syndrome. The aim of this study is to determine whether HO-1 acts through SIRT1 to form a functional module within hepatocytes to attenuate steatohepatitis, hepatic fibrosis and cardiovascular dysfunction.

Methods and Results

We examined the effect of fructose, on hepatocyte lipid accumulation and fibrosis in murine hepatocytes and in mice fed a high fructose diet in the presence and absence of CoPP, an inducer of HO-1, and SnMP, an inhibitor of HO activity. Fructose increased oxidative stress markers and decreased HO-1 and SIRT1 levels in hepatocytes (p<0.05). Further fructose supplementation increased FAS, PPARα, pAMPK and triglycerides levels; CoPP negated this increase. Concurrent treatment with CoPP and SIRT1 siRNA in hepatocytes increased FAS, PPARα, pAMPK and triglycerides levels suggesting that HO-1 is upstream of SIRT1 and suppression of SIRT1 attenuates the beneficial effects of HO-1. A high fructose diet increased insulin resistance, blood pressure, markers of oxidative stress and lipogenesis along with fibrotic markers in mice (p<0.05). Increased levels of HO-1 increased SIRT1 levels and ameliorated fructose-mediated lipid accumulation and fibrosis in liver along with decreasing vascular dysfunction (p<0.05 vs. fructose). These beneficial effects of CoPP were reversed by SnMP.

Conclusion

Taken together, our study demonstrates, for the first time, that HO-1 induction attenuates fructose-induced hepatic lipid deposition, prevents the development of hepatic fibrosis and abates NAFLD-associated vascular dysfunction; effects that are mediated by activation of SIRT1 gene expression.     

Methylation and acetylation of 15-hydroxyanandamide modulate its interaction with the endocannabinoid system

The biological activity of endocannabinoids like anandamide (AEA) and 2-arachidonoylglycerol (2-AG) is subjected in vivo to a “metabolic control”, exerted mainly by catabolic enzymes. AEA is inactivated by fatty acid amide hydrolase (FAAH), that is inhibited competitively by hydroxyanandamides (HAEAs) generated from AEA by lipoxygenase activity. Among these derivatives, 15-HAEA has been shown to be an effective (K_i ∼0.6 μM) FAAH inhibitor, that blocks also type-1 cannabinoid receptor (CB1R) but not other components of the “endocannabinoid system (ECS)”, like the AEA transporter (AMT) or CB2R. Here, we extended the study of the effect of 15-HAEA on the AEA synthetase (NAPE-PLD) and the AEA-binding vanilloid receptor (TRPV1), showing that 15-HAEA activates the former (up to ∼140% of controls) and inhibits the latter protein (down to ∼70%). We also show that 15-HAEA halves the synthesis of 2-AG and almost doubles the transport of this compound across the membrane. In addition, we synthesized methyl and acetyl derivatives of 15-HAEA (15-MeOAEA and 15-AcOAEA, respectively), in order to check their ability to modulate FAAH and the other ECS elements. In fact, methylation and acetylation are common biochemical reactions in the cellular environment. We show that 15-MeOAEA, unlike 15-AcOAEA, is still a powerful competitive inhibitor of FAAH (K_i ∼0.7 μM), and that both derivatives have negligible interactions with the other proteins of ECS. Therefore, 15-MeOAEA is a FAAH inhibitor more selective than 15-HAEA. Further molecular dynamics analysis gave clues to the molecular requirements for the interaction of 15-HAEA and 15-MeOAEA with FAAH.     

A serum fucose-binding lectin (DlFBL) from adult Dicentrarchus labrax is expressed in larva and juvenile tissues and contained in eggs

The purification, cloning, sequencing, molecular properties and expression of a fucose-binding lectin from the serum of Dicentrarchus labrax (DlFBL) have been previously reported. We now describe the distribution and expression of DlFBL during fish ontogeny. Immunohistochemistry and in situ hybridization assays were carried out at various developmental stages (from 10 days post-hatching larvae to juveniles). Another fucose-binding lectin, similar to DlFBL in biochemical, immunochemical and agglutinating properties, was extracted and purified from eggs and appeared to be localized in the embryo yolk sack residual. DlFBL was found in columnar and goblet cells of the intestinal epithelium of larvae (from 20 days post-hatching) and juveniles and in parenchymal tissue of juveniles. DlFBL mRNA and protein were detected in the intestinal epithelium and in hepatocytes. An amplification product from degenerate primers indicates that lectin isotypes with DlFBL epitopes are expressed in eggs and embryos. Whether the lectin fraction isolated from eggs and embryos includes DlFBL of maternal origin remains unclear.     

Biodegradation of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis> diethylaniline in a contaminated aquifer: laboratory- and field-scale evidences

The effectiveness of biosparging to mitigate N,N diethylaniline in aquifer was evaluated by measuring the time course of decrease in concentration of the aforementioned compound in aerobic microcosm experiments. The first-order kinetic constant for N,N diethylaniline aerobic biodegradation was estimated from microcosm data (0.037 ± 0.004 d⁻¹), and the value was consistent with the best-fitting value in the transport and reaction model of the aquifer (0.020 d⁻¹). Furthermore, the biodegradability of the compound was evaluated under anaerobic condition in microcosm experiments, which was supported by field modelling. There was no significant degradation in the anaerobic microcosm experiments, confirming the recalcitrance of N,N diethyl aniline under the aforementioned aquifer condition.     

Structural long-term changes at mushroom body input synapses

How does the sensory environment shape circuit organization in higher brain centers? Here we have addressed the dependence on activity of a defined circuit within the mushroom body of adult Drosophila. This is a brain region receiving olfactory information and involved in long-term associative memory formation. The main mushroom body input region, named the calyx, undergoes volumetric changes correlated with alterations of experience. However, the underlying modifications at the cellular level remained unclear. Within the calyx, the clawed dendritic endings of mushroom body Kenyon cells form microglomeruli, distinct synaptic complexes with the presynaptic boutons of olfactory projection neurons. We developed tools for high-resolution imaging of pre- and postsynaptic compartments of defined calycal microglomeruli. Here we show that preventing firing of action potentials or synaptic transmission in a small, identified fraction of projection neurons causes alterations in the size, number, and active zone density of the microglomeruli formed by these neurons. These data provide clear evidence for activity-dependent organization of a circuit within the adult brain of the fly.     

The Arabidopsis BET bromodomain factor GTE4 regulates the mitotic cell cycle

Proteins containing bromodomains are capable of binding to acetylated histone tails and have a role in recognizing and deciphering acetylated chromatin. Plant BET proteins contain one bromodomain. Twelve BET-encoding genes have been identified in the Arabidopsis genome. Two of these genes have been functionally characterized, one shows a role in seed germination, the other is involved in the establishment of leaf shape. Recently, we characterized a third AtBET gene, named GTE4. We demonstrated that GTE4 is involved in the activation and maintenance of cell division in the meristems and by this controls cell numbers in differentiated organs. Moreover, the quiescent center (QC) identity is partially lost in the apex of the primary root of gte4 mutant, and there is a premature switch from mitosis to endocycling. Genes involved in the retinoblastoma (RB)-E2F pathway, which is important for coupling cell division and cell differentiation in plants and animals, were either up or downregulated in the gte4 mutant. In this report we also show that the defect in germination observed in gte4 mutant seeds is not rescued by the action of GA₃. Further the root pole of the mutant embryo shows irregular cytokinesis in the procambial stem cells, and the QC of the lateral root shows a partial, but not transient, loss of QC identity. These additional results reinforce the importance of GTE4 in the control of cell proliferation.Key words: arabidopsis, BET bromodomain, cell cycle, E2F, germination     

Carbonic anhydrase IX: Biochemical and crystallographic characterization of a novel antitumor target

Isoform IX of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), CA IX, is a transmembrane protein involved in solid tumor acidification through the HIF-1α activation cascade. CA IX has a very high catalytic activity for the hydration of carbon dioxide to bicarbonate and protons, even at acidic pH values (of around 6.5), typical of solid, hypoxic tumors, which are largely unresponsive to classical chemo- and radiotherapy. Thus, CA IX is used as a marker of tumor hypoxia and as a prognostic factor for many human cancers. CA IX is involved in tumorigenesis through many pathways, such as pH regulation and cell adhesion control. The X-ray structure of the catalytic domain of CA IX has been recently reported, being shown that CA IX has a typical α-CA fold. However, the CA IX structure differs significantly from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the proteoglycan (PG) domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. As all mammalian CAs, CA IX is inhibited by several main classes of inhibitors, such as the inorganic anions, the sulfonamides and their bioisosteres (sulfamates, sulfamides, etc.), the phenols, and the coumarins. The mechanism of inhibition with all these classes of compounds is understood at the molecular level, but the sulfonamides and their congeners have important applications. It has been recently shown that both in vitro, in cell cultures, as well as in animals with transplanted tumors, CA IX inhibition with sulfonamides lead to a return of the extracellular pH to more normal values, which leads to a delay in tumor growth. As a consequence, CA IX represents a promising antitumor target for the development of anticancer agents with an alternative mechanism of action.     

Asháninka medicinal plants: a case study from the native community of Bajo Quimiriki, Junín, Peru

Background

The Asháninka Native Community Bajo Quimiriki, District Pichanaki, Junín, Peru, is located only 4 km from a larger urban area and is dissected by a major road. Therefore the loss of traditional knowledge is a main concern of the local headman and inhabitants. The present study assesses the state of traditional medicinal plant knowledge in the community and compares the local pharmacopoeia with the one from a related ethnic group.

Methods

Fieldwork was conducted between July and September 2007. Data were collected through semi-structured interviews, collection of medicinal plants in the homegardens, forest walks, a walk along the river banks, participant observation, informal conversation, cross check through voucher specimens and a focus group interview with children.

Results

Four-hundred and two medicinal plants, mainly herbs, were indicated by the informants. The most important families in terms of taxa were Asteraceae, Araceae, Rubiaceae, Euphorbiaceae, Solanaceae and Piperaceae. Eighty-four percent of the medicinal plants were wild and 63% were collected from the forest. Exotics accounted to only 2% of the medicinal plants. Problems related to the dermal system, digestive system, and cultural belief system represented 57% of all the medicinal applications. Some traditional healers received non-indigenous customers, using their knowledge as a source of income. Age and gender were significantly correlated to medicinal plant knowledge. Children knew the medicinal plants almost exclusively by their Spanish names. Sixteen percent of the medicinal plants found in this community were also reported among the Yanesha of the Pasco Region.

Conclusions

Despite the vicinity to a city, knowledge on medicinal plants and cultural beliefs are still abundant in this Asháninka Native Community and the medicinal plants are still available in the surroundings. Nevertheless, the use of Spanish names for the medicinal plants and the shift of healing practices towards a source of income with mainly non-indigenous customers, are signs of acculturation. Future studies on quantification of the use of medicinal plants, dynamics of transmission of ethno-medicinal knowledge to the young generations and comparison with available pharmacological data on the most promising medicinal plants are suggested.