Researches from different fields around the world are searching for both new sources of biomaterials and potential hepatocytes in order to supply drug tests, cell therapies, and cell transplantation as alternative therapeutic support to liver diseases and injuries. Placenta may be eligible as a new model in tissue engineering due to its rich extracellular matrix (ECM) and availability after birth. Placental scaffolds were produced by decellularization with 0.01, 0.1 and 1% SDS, and 1% Triton X-100 which were valued by means of structure and composition. Afterwards, placental scaffolds were co-cultured with mouse embryonic fibroblasts in a tridimensional (3D) rotating system. Placental scaffolds presented a well-preserved acellular ECM containing 9.42 ± 5.2 ng dsDNA per mg of ECM. Weak collagen I of the natives clearly appears in decellularized ECM while the collagen III, once well observed in native placenta, it was absent on scaffolds. This interesting observation may have been due to the solubilization SDS-induced of the collagen III fibrils during decellularization. Fibronectin was well-observed in placental scaffolds whereas laminin and collagen IV were strongly stained. Recellularized with fibroblasts by a 3D culture system, placental scaffolds showed potential for repopulation, with cells adhered throughout its acellular ECM. Placental scaffolds were then newly recellularized, aiming now for differentiation of mouse embryonic stem cells into hepatic cells. In a protocol of 23 days, it was simulated major events of liver embryonic development by adding growth factors. As result, a high index of cells adhered, proliferated and migrated throughout outer and inner scaffolds ECM surface. Absence of Oct4 and Nanog showed that Activin A and Wnt3a (d0-6) induced primitive endoderm fate, and negative label for Foxa2 and Sox17 representing BMP4 and FGF2 (d6-10) differentiation-induced generating definitive endoderm cells. Also, FGF1, FGF4 and FG8b (d10-14) induced hepatoblast phenotype cells, that were observed positive for AFP and CK7 markers. Finally, HGF and FS-288 (d14-23) induced to hepatocyte-like cells, positive for CK18 and Alb markers. The hepatocyte-like cells functional aspects were observed by glycogen storage. Though a heterogeneous cell hepatic lineage was confirmed, mouse placental scaffolds shown a useful model to support recellularization with simultaneous differentiation into hepatic fate simulating phases of embryonic development. / Pesquisas de diferentes campos ao redor do Mundo estão em busca de novas fontes tanto de biomateriais, quanto de potenciais hepatócitos, a fim de suprir testes de drogas, terapias celulares e transplante de células, como suporte terapêutico alternativo para doenças e lesões hepáticas. Placentas podem ser elegíveis como um novo modelo em Engenharia Tecidual em decorrência de sua rica matriz extracelular (ECM), e disponibilidade após o nascimento. Os scaffolds placentários foram produzidos por decelularização com SDS 0,01, 0,1 e 1% e Triton X-100 1%, os quais foram avaliados por meio da estrutura e composição. Posteriormente, os scaffolds placentários foram co-cultivados com fibroblastos embrionários de camundongos em um sistema rotativo tridimensional (3D). Os scaffolds placentários apresentaram uma MEC acelular bem conservada, contendo 9,42 ± 5,2 ng/dsDNA/mg/MEC. O fraco colágeno I nos nativos aparece claramente na MEC descelularizada, enquanto o colágeno III bem visível na placenta nativa estava ausente nos scaffolds. Esta observação interessante pode decorrido da solubilização das fibrilas de colágeno III, induzida pelo SDS durante a decelularização. A fibronectina foi bem observada nos scaffolds placentários, enquanto a laminina e o colágeno IV estiveram fortemente marcados. Recelularizados com fibroblastos por um sistema de cultura 3D, os scaffolds placentários mostraram potencial para repovoamento, com células aderidas ao longo de sua MEC acelular. Os scaffolds placentários foram então novamente recelularizados, visando agora a diferenciação de células tronco-embrionárias de camundongos em células hepáticas. Em um protocolo de 23 dias, foram simulados os grandes eventos do desenvolvimento embrionário do fígado, pela adição de fatores de crescimento. Como resultado, um alto índice de células aderiu, proliferou e migrou através das superfícies externa e interna dos scaffolds. A ausência de Oct4 e Nanog demostraram que o Activin A e o Wnt3a (d0-6) induziram o destino endoderma primitivo, e a marcação negativa para Foxa2 e Sox17 representaram a geração de células endodermais definitivas pela diferenciação induzida por BMP4 e FGF2 (d6-10). Ainda, FGF1, FGF4 e FG8b (d10-14) induziram células do fenótipo hepatoblasto, que foram observadas positivas para os marcadores AFP e CK7. Finalmente, HGF e FS-288 (d14-23) induziram as células hepatocyte-like, positivas para os marcadores CK18 e Alb. The hepatocyte-like cells functional aspects were observed by glycogen storage. Though a heterogeneous cell hepatic lineage was confirmed, mouse placental scaffolds shown a useful model to support recellularization with simultaneous differentiation into hepatic fate simulating phases of embryonic development. Os aspectos funcionais das células hepatocyte-like foi observada pelo armazenamento de glicogênio. Embora uma linhagem hepática formada por células heterogêneas tenha sido confirmada, os scaffolds placentários de camundongos se mostraram um modelo útil para sustentar a recelularização com simultânea diferenciação em destino hepático, simulando fases do desenvolvimento embrionário.

Les récepteurs Erythropoietin-producing hepatocyte (EPH) constituent la plus grande famille de récepteurs à activité tyrosine kinase transmembranaires. Leur activité kinase peut être induite par leurs ligands, les éphrines (EFN). Une fois activés, ces récepteurs sont impliqués dans la régulation de la fonction cellulaire par transduction antérograde ou rétrograde du signal EPH-EFN. Au cours de la dernière décennie, nos études ont démontré que les EPH / EFN jouent un rôle important dans la régulation de la pression artérielle par la modulation de la contractilité des cellules musculaires lisses vasculaires (VSMC). EPHB6, EFNB1 et EFNB3 ont un effet négatif sur la contractilité des VSMC et la pression artérielle, tandis que EPHB4 et EFNB2 montrent un effet positif. La famille EPH / EFN est donc un nouveau système yin et yang qui ajuste finement l'homéostasie de la pression artérielle. Nous avons également constaté que les catécholamines urinaires de 24 h sont réduites chez les souris mâles EPHB6 knockout (KO), suggérant que l’EPHB6 régule la pression artérielle non seulement via les VSMC mais aussi par la sécrétion de catécholamine (CAT). La régulation de CAT par l’EPHB6 dépend de la testostérone car (1) les niveaux réduits de CAT ne sont pas observés chez les souris femelles EPHB6 KO ; et (2) la castration chez les souris mâles EPHB6 KO ramène la CAT à des niveaux normaux. Durant ma thèse, nous avons étudié le mécanisme impliqué dans la régulation de la sécrétion et de la synthèse des catécholamines chez les cellules chromaffines des glandes surrénales (AGCC) par la voie de signalisation de l’EPHB6. En ex vivo, la teneur totale en épinéphrine et la sécrétion d'épinéphrine déclenchée par l'acétylcholine (ACh) sont toutes deux réduites dans les glandes surrénales venant des souris KO mâles mais pas dans celles venant des femelles ou de mâles castrés. Ensuite, nous avons observé une diminution de l’afflux de Ca2+ dépendant de l'ACh dans les AGCC venant des souris mâles EPHB6 KO, ce qui découle de l'effet non-génomique de la testostérone. En appliquant le patch clamping de cellules entières sur les AGCC, nous avons démontré que la diminution d’afflux de Ca2+ dans ces cellules est causée par l’augmentation des courants de potassium à grande conductance activé par le calcium (BK). En utilisant l'enregistrement ampérométrique, nous avons constaté que la sécrétion de CAT par les AGCC est compromise en l'absence d'EPHB6. Nous avons également observé une diminution du désassemblage de la F-actine corticale dans les AGCC venant de souris mâles KO associée à une diminution de l'exocytose des vésicules contenant es catécholamines. Ces deux phénomènes n’ont pas été observés chez les femelles KO ni chez les mâles castrés. Des études complémentaires ont montré que le désassemblage défectueux de la F-actine dans les AGCC est régulé par la signalisation inverse de l'EPHB6 à l'EFNB1 via deux voies de signalisations différentes : la voie du membre A de la famille des homologues Ras (RHOA) et la voie de la tyrosine kinase proto-oncogène de la famille Src (FYN) / proto-oncogène c-ABL / la calponine monooxygénase associée aux microtubules et le domaine LIM contenant 1 (MICAL-1). En outre, nous avons observé que la diminution de la teneur totale en épinéphrine dans la glande surrénale venant des souris mâles KO est causée par une expression altérée de la tyrosine hydroxylase (TH), qui est l’enzyme limitant la vitesse dans la biosynthèse des CAT. L'effet non génomique de la testostérone a également participé dans ce processus. Nous avons révélé que la signalisation inverse d'EPHB6 à EFNB1 contribue à la surexpression de TH dans les AGCC par l’augmentation de son niveau de transcription. La voie en aval de cette signalisation inverse implique la petite famille Rac GTPase 1 (RAC1) / MAP kinase kinase 7 (MKK7) / c-Jun N-terminal kinase (JNK) / proto-oncogène c-Jun / activator protein 1 (AP1) / réponse de croissance précoce 1 (EGR1). Ces travaux démontrent pour la première fois un rôle spécifique de la famille EPH / EFN dans la régulation de la biologie médullaire de la glande surrénale. La signalisation rétrograde d’EPHB6 via EFNB1 régule la synthèse et la sécrétion des catécholamines de concert avec la testostérone dans les AGCC. / Erythropoietin-producing hepatocyte (Eph) receptors are the largest family of cell surface transmembrane receptor tyrosine kinases. Their kinase activity can be activated by their ligands, ephrins (EFNs), and involved in cell function regulation through either EPH-EFN forward or reverse signaling transduction. In the last decade, we have revealed the previously unknown function of EPHs/EFNs in the regulation of blood pressure by modulating the contractility of vascular smooth muscle cells (VSMCs). EPHB6, EFNB1, and EFNB3 have a negative effect on the VSMCs contractility and blood pressure, while EPHB4 and EFNB2 show a positive effect instead. Thus, EPH/EFN family is a novel yin and yang system that finely tunes blood pressure homeostasis. EPHB6 also targets cells responsible for catecholamine (CAT) secretion in addition to the VSMCs, since we found that the 24-h urine catecholamines are reduced in male EPHB6 knockout (KO) mice. This phenotype in EPHB6 KO mice is testosterone-dependent because the reduced CAT levels are not observed in female KO mice; castration in KO male mice reverts the CAT levels to a normal range. In this research, we investigated the mechanism for the regulation of catecholamine secretion and synthesis in adrenal gland chromaffin cells (AGCCs) by EPHB6 signaling. In ex vivo, the total content of epinephrine and the acetylcholine (ACh)-triggered epinephrine secretion were both reduced in the adrenal gland from KO male but not female or castrated mice. Then, we found a reduced ACh-dependent Ca2+ influx in AGCCs from male EPHB6 KO mice, and this effect depended on the non-genomic effect of testosterone. The results of whole-cell patch clamping on AGCCs indicated that the enhanced large-conductance calcium-activated potassium (BK) currents were responsible for the reduced Ca2+ influx in these cells. Using amperometry recording, we found that CAT secretion by AGCCs was compromised in the absence of EPHB6. The cortical F-actin disassembly in AGCCs from KO male but not female or castrated mice was reduced, accompanied by decreased catecholamine vesicle exocytosis. Further study showed such defective F-actin disassembly in AGCCs was regulated by the reverse signaling from EPHB6 to EFNB1 via the Ras homolog family member A (RHOA) and proto-oncogene Src family tyrosine kinase (FYN)/proto-oncogene c-ABL/microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways. Further, we observed that the reduced total content of epinephrine in the adrenal gland from male KO mice was caused by impaired expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in CAT biosynthesis. The non-genomic effect of testosterone was also involved in this process. We revealed that the reverse signaling from EPHB6 to EFNB1 contributed to the up-regulation of TH expression in AGCCs by enhancing its transcription. The downstream pathway of this reverse signaling involved Rac family small GTPase 1 (RAC1)/MAP kinase kinase 7 (MKK7)/c-Jun N-terminal kinase (JNK)/ proto-oncogene c-Jun/activator protein 1 (AP1)/early growth response 1 (EGR1). The present research, for the first time, revealed the specific role of the EPH/EFN family on the regulation of the adrenal gland medullary biology. The EPHB6 reverse signaling through EFNB1 in concert with testosterone regulates the catecholamine synthesis and secretion in AGCCs.

<p>With the prevalence of obesity and metabolic syndrome rising sharply world-wide, it has become increasingly important to define the molecular mechanisms underlying the pathogenesis and progression of diseases associated with lipid-induced cytotoxicity. Cardiovascular disease, type-2 diabetes mellitus, and nonalchoholic fatty liver disease (NAFLD) have all recently gained recognition as diseases that are exacerbated by lipoapoptosis. In this dissertation, we demonstrate a novel role for caspase-2 as an initiator of lipoapoptosis. Using an unbiased metabolomics approach, we discovered that the activation of caspase-2, the initiator of apoptosis in Xenopus egg extracts, is associated with an accumulation of long-chain fatty acid (LCFA) metabolites. Metabolic treatments that block the buildup of LCFAs potently inhibit caspase-2, while add-back of a saturated LCFA restores caspase activation in the extract setting. Extending these findings to mammalian cells, we show that caspase-2 is engaged and activated in response to treatment with the saturated LCFA, palmitate. Down-regulation of caspase-2 significantly impairs cell death induced by saturated LCFAs, revealing a conserved, critical role for caspase-2 in mediating LCFA-induced lipoapoptosis. </p><p> Since lipoapoptosis has been implicated as a key driver of the progression of NAFLD, we aimed to determine the therapeutic significance of our findings by evaluating the importance of caspase-2 in an in vivo model of this disease. We subjected wild-type and caspase-2 knockout mice to a diet which induces severe liver steatosis and the development nonalcoholic steatohepatitis (NASH), the most advanced stage of NAFLD characterized by liver fibrosis. Interestingly, we observed an increase in caspase-2 protein levels in the livers of wild-type mice fed a NASH-inducing diet. These findings were of particular importance, since caspase-2 expression was also significantly elevated in patients diagnosed with NASH. Most importantly, we demonstrated that caspase-2 knockout mice are protected from apoptosis and fibrosis when fed a NASH-inducing diet, suggesting that caspase-2 is major regulator of hepatocyte lipoapoptosis. Together, these findings reveal a previously unknown role for caspase-2 as an initiator of lipoapoptosis and suggest that caspase-2 may be an attractive therapeutic target for inhibiting pathological lipid-induced apoptosis.</p> / Dissertation

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

My first project was to investigate the role of Hepatocyte Nuclear Factor 1 (HNF1) and Nuclear Factor I (NFI) on alpha-fetoprotein (AFP) promoter activity during liver development. AFP is highly expressed in the fetal liver, silenced at birth, and remains at very low levels in the adult liver. A GA substitution located at -119 of the human AFP promoter is associated with hereditary persistence of AFP (HPAFP) expression in the adult liver (Hum Molec Genet, 1993, 2:379). The -120 region harbors overlapping binding sites for HNF1 and NFI. While it has been shown that the GA substitution increases HNF1 binding, the role of NFI in AFP regulation has not been investigated. This overlapping HNF1/NFI site is conserved in other mammals, including mice. In this study, I used a combination of biochemical, tissue culture, and animal studies to explore further the role of this HNF1/NFI site in AFP regulation. Transient co-transfections in Hep3B hepatoma cells indicate that HNF1 activates while NFI represses the mouse AFP promoter. EMSAs indicate that HNF1 and NF1 compete for binding to this site. Transgenes regulated by the wild-type AFP promoter are expressed at low levels in the adult liver. Transgenes with a GGAA mutation (similar to the G-A human mutation) are more active in the adult liver. My data indicate that HNF1 and NFI compete for binding to the -120 region of the AFP promoter and this competition is involved in postnatal AFP repression. My second project was to study the control of Elongation of very long chain fatty acids like 3 (Elovl3) in the liver by Zinc fingers and homeoboxes 2 (Zhx2). The Zhx2 gene was originally characterized in our lab based on its ability to control the developmental repression of several hepatic genes, including AFP (PNAS, 102:401). Zhx2 is a member of a small family of proteins found only in vertebrates that also includes Zhx1 and Zhx3. These proteins all contain two zinc fingers and four homeodomains, suggesting that they function as regulators of gene expression. My study shows that Zhx2 regulates Elovl3 expression in female liver. Mouse strain-specific differences in adult liver Elovl3 mRNA levels and transgenic mouse data indicate that Zhx2 activates Elovl3 expression in the female adult liver. I also demonstrate that Elovl3 is repressed in the regenerating liver and that the level of Elovl3 repression is controlled by alpha-fetoprotein regulator 2 (Afr2). In addition, I show that Elovl3 expression is reduced in liver tumors, fibrotic livers and fatty livers, raising the possibility that Elovl3 can serve as a marker for HCC and liver damage.

Mammalian skeletal muscle can regenerate after injury and this response is primarily mediated by the satellite cell, a muscle stem cell. Following injury, satellite cells are activated to myoblasts, undergo rapid proliferation, migrate towards the injury site, and subsequently differentiate into myotubes in order to facilitate functional muscle repair. Fibrosis, caused by the secretion of structural extracellular matrix (ECM) proteins such as collagen I and fibronectin, by fibroblasts, impairs complete functional repair of the muscle. In this study, the role of the microenvironment during wound conditions was assessed by analysing the effect of specific extracellular matrix and growth factors on myoblast migration. The role of the Rho/ROCK pathway as a possible mechanism in mediating the effects seen was investigated. In order to analyse wound repair in an in vitro setting, we optimised and improved a wound healing model specifically designed for skeletal muscle repair. To this end we also developed a co-culture assay using primary myoblasts and fibroblasts isolated from the same animal. The studies showed that collagen I and fibronectin both increased myoblast migration in a dose-dependent manner. Decorin displayed opposing effects on cellular movement, significantly increasing collagen I-stimulated, but not fibronectin-stimulated, migration of myoblasts. ROCK inhibitor studies revealed a significant increase in migration on uncoated plates following inhibition with Y-27632 compared to untreated control. When cells were cultured on ECM components (Matrigel, collagen I, or fibronectin), the inhibitory effect of Y-27632 on migration was reduced. Analysis of ROCK and vinculin expression, and localization at the leading front, showed that ROCK inhibition resulted in loosely packed focal adhesion complexes (matrix dependent). A reduced adhesion to the ECM could explain the increased migration rates observed upon inhibition with Y-27632. We also investigated the role of TGF-β and decorin during wound repair, as TGF-β is a known pro-fibrotic agent. TGF-β treatment decreased wound closure rates; however, the addition of decorin with TGF-β significantly increased wound closure. The addition of ECM components, Matrigel and collagen I enhanced the effect seen in response to TGF-β and decorin; however, fibronectin negated this effect, with no increase in migration seen compared to the controls. In conclusion, the importance of extracellular matrix components in regulating myoblast migration and therefore skeletal muscle wound repair was demonstrated. We emphasize that, in order to gain a better understanding of skeletal muscle wound repair, the combination of ECM and growth factors released during wounding need to be utilised in assays which mimic the in vivo environment more closely. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Many of the anabolic effects of growth hormone (GH) are indirect, occurring through GH-stimulated production of insulin-like growth factor-I (IGF-I) by the liver. As well as being GH regulated, plasma IGF-I concentrations have been demonstrated to be dependent upon protein nutrition, with low protein diets being associated with reduced plasma IGF-I concentrations. This effect cannot be reversed by GH, suggesting that liver sensitivity to GH is impaired. To investigate the mechanisms through which protein supply affects GH sensitivity, primary cultures of ovine hepatocytes were grown in defined media. In a first experiment the media contained various fractions (0.2, 1.0, 5.0) of portal vein amino acid concentrations in fed sheep. In the second 24h incubation period, unstimulated IGF-I secretion was highly sensitive the concentration of amino acids in the media, with significantly greater release of basal IGF-I in 5x compared to either 1x (P<0.05) or 0.2x amino acid containing media. In a second series of experiments the effects of specific amino acid depletions was examined. Methionine depletion of 0.2x portal amino acid concentrations ablated the GH response second 24h of culture without affecting basal IGF-I release. By comparison ³H-leucine incorporation into secreted protein, following 20 hours of culture in defined media was significantly reduced in 0.2x aa (P<0.01) and 1.0x aa (P<0.05) media compared with 5.0x aa media, however secretory protein synthesis was unaffected by methionine depletion to 0.2x portal concentrations. The results suggest that amino acid availability regulates both basal and GH stimulated IGF-I release in ovine hepatocytes. Furthermore reducing methionine concentrations in the culture media to 0.2x portal concentrations diminishes GH response without compromising protein secretion.

HIV protease inhibitors (PI) are core components of Highly Active Antiretroviral Therapy (HAART). HIV PIs are extremely effective at suppressing viral load, but have been linked to lipodystrophy and dyslipidemia, which are major risk factors for cardiovascular disease. Recent studies indicate that activation of endoplasmic reticulum (ER) stress is an important cellular mechanism underlying HIV PI-induced dysregulation of lipid metabolism. However, the exact role of ER stress in HIV PI-associated lipodystrophy and dyslipidemia remains to be identified. Hepatocytes and adipocytes are important players in regulating lipid metabolism and the inflammatory state. Dysfunction of these two cell types is closely linked to various metabolic diseases. In this dissertation research, we aimed to define the role of activation of ER stress in HIV PI-induced dysregulation of lipid metabolism in adipocytes and hepatocytes and further identifty the potential molecular mechanisms. Both cultured and primary mouse adipocytes and hepatocytes were used to examine the effect of individual HIV PIs on ER stress activation and lipid metabolism. The results indicated that HIV PIs differentially activate ER stress through depletion of ER calcium stores, activating the unfolded protein response (UPR). UPR activation further lead to an alteration of cellular differentiation through downstream transcription factor CHOP. At the same time, HIV PIs also altered adipogenesis via differential regulation of the adipogenic transcription factor PPARγ. HIV PI-induced ER stress was closely linked to dysregulation of autophagy activation through CHOP, and upstream ATF-4, signaling pathways. In hepatocytes, the integrase inhibitor raltegravir abrogated HIV PI-induced lipid accumulation by inhibiting ER stress activation and dysregulation of autophagy pathway. Our studies suggest that both ER stress and autophagy are involved in HIV PI-induced dysregulation of lipid metabolism in adipocytes and hepatocytes. The key components of ER stress and autophagy signaling pathways are potential therapeutic targets for HIV PI-induced metabolic side effects in HIV HAART-treated patients.

Sterol regulatory element-binding proteins (SREBP) are transcription factors that regulate genes involved in lipid metabolism especially in the liver. Therefore, hepatic SREBP is significant regulator of systemic lipid metabolism. Evidence demonstrates that insulin and dietary unsaturated fatty acid (UFA) regulate SREBP1 expression and subsequent SREBP1-mediated gene transcription, events that in many instances result in modulation of systemic fatty acid and triglyceride (TG) homeostasis. A series of investigations was designed to uncover novel regulators of SREBP1. Dietary and exogenous addition of UFA has been shown to regulate SREBP function yet, an endogenous source of UFA capable of modulating SREBP remains elusive. Group VIA calcium-independent phospholipase A2 (iPLA2) releases UFA from the sn-2 position of glycerophospholipids. We hypothesized that iPLA2 provides UFA to suppress SREBP. iPLA2 overexpression and inhibition studies were implemented. iPLA2 inhibition increased SREBP1 expression, SREBP-mediated transcription and the expression of SREBP1 gene targets in vitro. In vivo overexpression of iPLA2 resulted in decreased expression of SREBP1 protein and plasma triglyceride. In contrast, iPLA2 overexpression attenuated SREBP1 expression, SREBP-mediated transcription and expression of SREBP1 targets genes. These data support the hypothesis that iPLA2 generates endogenous UFA that limit SREBP function. Use of a replication-deficient adenovirus 5 (Ad-5) expression vector in the iPLA2 study led to the unexpected observation of hepatic SREBP1 activation following Ad-5 infection. Because of this observation, we tested the hypothesis that replication-deficient Ad-5 might augment lipid synthesis in liver. We demonstrate that first generation Ad-5, a ubiquitous transgene expression vector, induces expression of SREBP1 and its target genes and leads to increases in fatty acid synthesis in vivo and in vitro. The phosphatidylinositol 3-kinase (PI3K) inhibitor, PX-866, suppressed Ad-5-induced SRBEP1 expression and hypertriglyceridemia implicating the PI3K/Akt pathway in Ad-5 activation of SREBP1. Use of PX-866 led to the discovery of a third mechanism of SREBP1 regulation. In vivo studies demonstrate that PX-866 modulates basal lipid metabolism in part through decreasing plasma TG, an increased trend toward decreased SREBP1 expression and a significant increase in plasma cholesterol. These studies characterize three distinct novel regulatory mechanisms of SREBP1.

Avaliaram-se os efeitos da administração do extrato seco de maracujá (Passiflora incarnata), veiculado na dieta, sobre a morfologia dos hepatócitos de juvenis de tilápias do Nilo (Oreochromis niloticus). Peixes isolados (86,50 &plusmn; 10,17 g) receberam durante 28 dias ração comercial extrusada (32% PB - 2% biomassa) contendo o extrato diluído em alginato de sódio nas doses 0 (controle), 50, 100 e 200 mg/Kg, (n = 6 peixes/tratamento), registrando-se diariamente o consumo. No início e ao final do experimento cada indivíduo foi exposto (30 minutos) à reflexão da própria imagem em espelho (presença virtual de um coespecífico - estresse social), sendo a seguir anestesiado (2-fenoxietanol 0,5 mL/L) para realização de biometria e coleta de sangue (veia caudal) para determinação dos níveis plasmáticos de glicose e cortisol. Após 28 dias todos os animais foram sacrificados para remoção do fígado e obtenção de fragmentos utilizados na contagem de células e avaliação da morfometria do citoplasma dos hepatócitos (H/E), observando-se também as reservas de glicogênio hepático (PAS). Visando a comparação dos efeitos do estresse social natural com aquele empregado no experimento, seis peixes provenientes de um grupo de 30 indivíduos foram também sacrificados e utilizados como referência (valores basais) para avaliação dos parâmetros histológicos. Os dados foram submetidos a ANOVA, utilizando-se o proc mixed SAS 8.0 (p<0,05) para os parâmetros consumo de alimento, ganho em peso e bioquímica sangüínea (cortisol e glicose) e GLM SAS 8.0 (p<0,01) para a contagem de células e morfometria dos hepatócitos. Verificou-se que os pesos e os comprimentos iniciais não diferiram, que o consumo de alimento não foi alterado pela adição do extrato e que todos os peixes, independentemente do tratamento, cresceram significativamente. Os níveis de cortisol e de glicose também não diferiram inicialmente entre os grupos e não foram alterados pela presença do agente ou pela adição do extrato. Porém, observou-se um aumento significativo da glicose e redução dos níveis de cortisol em todos os peixes. A adição do extrato nas diferentes doses provocou aumento crescente e significativo da área citoplasmática e redução do número de células em todos os animais, com destaque para a dose 100 mg/Kg. O mesmo não ocorreu nos peixes do grupo controle, cujas áreas citoplasmáticas foram significativamente menores, em decorrência de um menor acúmulo de glicogênio hepático. Embora os efeitos do agente estressor empregado não tenham sido detectados pela análise dos parâmetros bioquímicos sangüíneos, verificou-se que este procedimento provocou alterações metabólicas que contribuíram para a depleção dos estoques de glicogênio no fígado. Este efeito parece ter sido revertido nos peixes que receberam a dieta contendo o extrato de maracujá que, por meio de mecanismos não elucidados neste trabalho, contribuiu para a manutenção ou aumento dos estoques de glicogênio hepático. Concluiu-se que o extrato de maracujá veiculado na dieta, na dose de 100 mg/Kg, protege juvenis de tilápia da depleção dos estoques de glicogênio hepático causada pelo estresse social. / Effects of Passion fruit (Passiflora incarnata) dry extract administration on hepatocyte morphometry of juveniles Nile tilapias (Oreochromis niloticus) were investigated. Male isolated fish (86,50 &plusmn; 10,17 g) were daily fed (28 days) with extruded commercial ration (32% crude protein - 2% biomass) containing the extract diluted in sodium alginate in graded doses 0 (control), 50, 100 and 200 mg/Kg (n= 6 animals/treatment), registering daily consumption. At the start and after 28 days of experiment each fish was displayed to the reflection of own mirror image (30 minutes) in order to simulate the virtual presence of a coespecific (social stress). Then the animals were anesthetized (2-fenoxietanol) for biometric measures and blood collection (caudal vein) for determination of the plasmatic levels of glucose and cortisol. After 28 days the animals were sacrificed for removal of liver fragments samples used to histological examination (cytoplasmic area and number of cells - HE stained; hepatic glycogen supplies - PAS stained). Aiming the comparison of the natural social stress with that utilized in this experiment, six fish from a group of 30 individuals were also sacrificed and used as reference (basal values) for the histological parameters evaluation. Food consumption, weight gain and sanguine biochemist parameters (cortisol and glucose) were submitted to ANOVA, SAS 8.0 proc mixed (p<0,05) and data of cells counting and hepatocyte morfometry to the same test, using SAS 8.0 GLM (p<0,01). The initial weights and lengths were similar, the food consumption was not modified by the addition of the extract and all the fish grew significantly during the experiment. Initial levels of cortisol and glucose were also similar between groups and did not modify by the stressor agent or treatments with the extract. However, a significant increase of glucose and reduction of cortisol levels were observed for all the fish. The addition of different extract doses provoked significant and noticeable increase of the cytoplasmic area and reduction of the cells number, mainly for 100 mg/Kg dose. In the control group cytoplasmatic area was significantly minor due to lesser hepatic glycogen accumulation. The stressor agent did not affect sanguine biochemists parameters but seems to lead metabolic alterations that had collaborated with the depletion of liver glycogen supplies. This effect seems to have been reverted in the fish that received the diet contend Passion fruit extract that, by means of a mechanism not elucidated in this experiment, contributed for the maintenance or increase of hepatic glycogen supplies. It was conclude that Passion fruit extract diet inclusion at 100 mg/Kg dose protects the tilapia against the hepatic glycogen depletion caused by social stress.