RESUMO
We recently provided evidence suggesting that mitochondrial aquaporin-8 (mtAQP8), a channel protein able to conduct H2O2, is involved in the modulation of hepatocyte cholesterogenesis. To expand that study, we cultured human hepatocyte-derived Huh-7 cells in medium with lipoprotein-deficient serum (LPDS) to induce the de novo synthesis of cholesterol and fatty acids. We found that LPDS induced mtAQP8 expression and that AQP8 gene silencing significantly down-regulated the LPDS-induced synthesis of cholesterol and fatty acids as well as the expression of the corresponding key biosynthetic enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase and fatty acid synthase. Our data further support a regulatory role of mtAQP8 in hepatocyte lipid homeostasis.
Assuntos
Aquaporinas/genética , Aquaporinas/metabolismo , Hepatócitos/metabolismo , Lipogênese/fisiologia , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Colesterol/biossíntese , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/biossíntese , Inativação Gênica , Homeostase , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Lipoproteínas/deficiênciaRESUMO
In insects, lipids are stored in the fat body mainly as triacylglycerol. Lipids can be directly provided by digestion and incorporated from the hemolymph, or synthesized de novo from other substrates such as carbohydrates and amino acids. The first step in de novo lipid synthesis is catalyzed by acetyl-CoA carboxylase (ACC), which carboxylates acetyl-CoA to form malonyl-CoA. Rhodnius prolixus is a hematophagous insect vector of Chagas disease and feeds exclusively on large and infrequent blood meals. Adult females slowly digest the blood and concomitantly accumulate lipids in the fat body. In this study, we investigated the regulation of R. prolixus ACC (RhoprACC) expression and de novo lipogenesis activity in adult females at different nutritional and metabolic conditions. A phylogenetic analysis showed that insects, similar to other arthropods and unlike vertebrate animals, have only one ACC gene. In females on the fourth day after a blood meal, RhoprACC transcript levels were similar in the anterior and posterior midgut, fat body and ovary and higher in the flight muscles. In the fat body, gene expression was higher in fasted females and decreased after a blood meal. In the posterior midgut it increased after feeding, and no variation was observed in the flight muscle. RhoprACC protein content analysis of the fat body revealed a profile similar to the gene expression, with higher protein contents before feeding and in the first two days after a blood meal. Radiolabeled acetate was used to follow de novo lipid synthesis in the fat body and it was incorporated mainly into triacylglycerol, diacylglycerol and phospholipids. This lipogenic activity was inhibited by soraphen A, an ACC inhibitor, and it varied according to the insect metabolic status. De novo lipogenesis was very low in starved females and increased during the initial days after a blood meal. The flight muscles had a very low capacity to synthesize lipids when compared to the fat body. Radiolabeled leucine was also used as a substrate for de novo lipogenesis and the same lipid classes were formed. In conclusion, our results indicate that the blood meal induces the utilization of diet-derived amino acids by de novo lipogenesis in the fat body, and that the control of this activity does not occur at the RhoprACC gene or protein expression level.
Assuntos
Corpo Adiposo/metabolismo , Lipogênese , Rhodnius , Acetil-CoA Carboxilase/genética , Animais , Sistema Digestório/metabolismo , Comportamento Alimentar , Feminino , Expressão Gênica , Genes de Insetos , Lipogênese/efeitos dos fármacos , Lipogênese/fisiologia , Macrolídeos/farmacologia , Músculos/metabolismo , Filogenia , Rhodnius/genética , Rhodnius/metabolismo , Rhodnius/fisiologia , Triglicerídeos/metabolismoRESUMO
Rhodnius prolixus, a vector of Chagas disease, is a hematophagous insect that feeds exclusively on blood. Each blood meal is digested within the first fourteen days after feeding, providing substrates for lipid synthesis for storage and egg production. These events are precisely regulated and emerging evidence points to a key function of insulin-like peptides (ILPs) in this control. Here we investigated the role of insulin receptor in the regulation of nutrient metabolism in fed adult females. The expression of insulin receptor (RhoprIR) gene was determined in adult organs, and it was highest in ovaries and previtellogenic follicles. We generated insects with RNAi-mediated knockdown of RhoprIR to address the physiological role of this receptor. RhoprIR deficiency improved longevity and reduced triacylglycerol storage in the fat body, whereas blood digestion remained unchanged for seven days after blood meal. The lower lipid content was attributable to decreased de novo lipogenesis as well as reduced incorporation of hemolymph-derived fatty acids into newly synthesized lipids within this organ. Consistent with that, fat bodies from RhoprIR-deficient insects exhibited decreased gene expression levels of lipophorin receptor (RhoprLpR), glycerol-3-phosphate acyltransferase 1 and 4 (RhoprGpat1 and RhoprGpat4), and carnitine palmitoyltransferase 1 (RhoprCpt1). Although hemolymph lipid profile was not affected by RhoprIR disruption, the concentration of circulating vitellogenin was increased. In line with these changes, RhoprIR-deficient females exhibited smaller ovaries and a marked reduction in oviposition. Taken together, these findings support a key role of insulin receptor in nutrient homeostasis, lipid synthesis and egg production following a blood meal.
Assuntos
Proteínas de Insetos/deficiência , Insetos Vetores/fisiologia , Oogênese/genética , Receptor de Insulina/deficiência , Rhodnius/fisiologia , Animais , Sangue , Doença de Chagas/parasitologia , Doença de Chagas/transmissão , Corpo Adiposo/metabolismo , Comportamento Alimentar , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Hemolinfa/química , Humanos , Proteínas de Insetos/genética , Insetos Vetores/parasitologia , Gotículas Lipídicas/metabolismo , Lipogênese/fisiologia , Modelos Animais , Ovário/metabolismo , Coelhos , Receptor de Insulina/genética , Rhodnius/parasitologia , Triglicerídeos/análise , Triglicerídeos/metabolismoRESUMO
Non-pharmacological early weaning (NPEW) induces liver damage in male progeny at adulthood; however, pharmacological early weaning (PEW) does not cause this dysfunction. To elucidate this difference in liver dysfunction between these two models and determine the phenotype of female offspring, de novo lipogenesis, ß-oxidation, very low-density lipoprotein (VLDL) export, and gluconeogenesis in both sexes were investigated in the adult Wistar rats that were weaned after a normal period of lactation (control group) or early weaned either by restriction of access to the dams' teats (NPEW group) or by reduction of dams' milk production with bromocriptine (PEW group). The offspring received standard diet from weaning to euthanasia (PN180). NPEW males had higher plasma triglycerides and TyG index, liver triglycerides, and cholesterol by de novo lipogenesis, which leads to intracellular lipids accumulation. As expected, hepatic morphology was preserved in PEW males, but they showed increased liver triglycerides. The only molecular difference between PEW and NPEW males was in acetyl-CoA carboxylase-1 (ACC-1) and stearoyl-CoA desaturase-1 (SCD-1), which were lower in PEW animals. Both early weaning (EW) females had no changes in liver cholesterol and triglyceride contents, and the hepatic cytoarchitecture was preserved. The expression of microsomal triglyceride transfer protein was increased in both the female EW groups, which could constitute a protective factor. The changes in hepatic lipid metabolism in EW offspring were less marked in females. EW impacted in the hepatic cytoarchitecture only in NPEW males, which showed higher ACC-1 and SCD-1 when compared to the PEW group. As these enzymes are lipogenic, it could explain a worsened liver function in NPEW males.
Assuntos
Lipogênese/fisiologia , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Acetiltransferases/análise , Acetiltransferases/metabolismo , Animais , Bromocriptina/administração & dosagem , Modelos Animais de Doenças , Feminino , Antagonistas de Hormônios/administração & dosagem , Humanos , Lactação/efeitos dos fármacos , Lactação/fisiologia , Lipoproteínas VLDL/metabolismo , Fígado/enzimologia , Fígado/crescimento & desenvolvimento , Masculino , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Oxirredução , Prolactina/antagonistas & inibidores , Prolactina/metabolismo , Ratos , Ratos Wistar , Fatores Sexuais , Estearoil-CoA Dessaturase/análise , Estearoil-CoA Dessaturase/metabolismo , Fatores de Tempo , Triglicerídeos/análise , Triglicerídeos/metabolismo , DesmameRESUMO
Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) particularly among chronic consumers of added sugar-rich diets. However, the impact of early consumption of such diets on NAFLD onset and progression is unclear. Thus, this study sought to characterise metabolic factors involved in NAFLD progression in young mice fed with a high-sucrose diet (HSD). Male Swiss mice were fed HSD or regular chow (CTR) from weaning for up to 60 or 90 days. Obesity development, glucose homeostasis and serum biochemical parameters were determined at each time-point. At day 90, mice were euthanised and white adipose tissue (WAT) collected for lipolytic function assessment and liver for histology, gene expression and cytokines quantification. At day 60, HSD mice presented increased body mass, hypertriglyceridemia, peripheral insulin resistance (IR) and simple steatosis. Upon 90 days on diet, WAT from HSD mice displayed impaired insulin sensitivity, which coincided with increased fasting levels of glucose and free fatty acids (FFA), as well as NAFLD progression to NASH. Transcriptional levels of lipogenic genes, particularly stearoyl-CoA desaturase-1, were consistently increased, leading to hepatic leukocyte infiltration and pro-inflammatory cytokines spillover. Therefore, our dataset supports IR triggering in the WAT as a major factor for dysfunctional release of FFA towards portal circulation and consequent upregulation of lipogenic genes and hepatic inflammatory onset, which decisively concurred for NAFLD-to-NASH progression in young HSD-fed mice. Notwithstanding, this study forewarns against the early introduction of dietary sugars in infant diet, particularly following breastfeeding cessation.
Assuntos
Tecido Adiposo Branco/fisiopatologia , Sacarose Alimentar/efeitos adversos , Lipogênese/fisiologia , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Tecido Adiposo Branco/metabolismo , Animais , Criança , Fenômenos Fisiológicos da Nutrição Infantil , Pré-Escolar , Modelos Animais de Doenças , Progressão da Doença , Humanos , Lactente , Resistência à Insulina/fisiologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , DesmameRESUMO
Gangliosides are constituents of the mammalian cell membranes and participate in the inflammatory response. However, little is known about the presence and enzymatic activity of ganglioside sialyltransferases at the cell surface of macrophages, one of the most important immune cells involved in the innate inflammatory process. In the present study, using biochemical and fluorescent microscopy approaches, we found that endogenous ST8Sia-I is present at the plasma membrane (ecto-ST8Sia-I) of murine macrophage RAW264.7 cells. Moreover, ecto-ST8Sia-I can synthetize GD3 ganglioside at the cell surface in lipopolysaccharide (LPS)-stimulated macrophages even when LPS-stimulated macrophages reduced the total ST8Sia-I expression levels. Besides, cotreatment of LPS with an inhibitor of nitric oxide (NO) synthase recovered the ecto-ST8Sia-I expression, suggesting that NO production is involved in the reduction of ST8Sia-I expression. The diminution of ST8Sia-I expression in LPS-stimulated macrophages correlated with a reduction of GD3 and GM1 gangliosides and with an increment of GD1a. Taken together, the data supports the presence and activity of sialyltransferases at the plasma membrane of RAW264.7 cells. The variations of ecto-ST8Sia-I and ganglioside levels in stimulated macrophages constitutes a promissory pathway to further explore the physiological role of this and others ganglioside metabolism-related enzymes at the cell surface during the immune response.
Assuntos
Membrana Celular/metabolismo , Gangliosídeo G(M1)/metabolismo , Gangliosídeos/metabolismo , Macrófagos/metabolismo , Sialiltransferases/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus/metabolismo , Lipogênese/fisiologia , Lipopolissacarídeos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Células RAW 264.7RESUMO
An imbalance in the redox state, increased levels of lipid precursors and overactivation of de novo lipogenesis determine the development of fibrosis during nonalcoholic steatohepatitis (NASH). We evaluated the modulation of NADPH-producing enzymes associated with the antifibrotic, antioxidant and antilipemic effects of nicotinamide (NAM) in a model of NASH induced by excess fructose consumption. Male rats were provided drinking water containing 40% fructose for 16 weeks. During the last 12 weeks of fructose administration, water containing NAM was provided to some of the rats for 5 h/day. The biochemical profiles and the ghrelin, leptin, lipoperoxidation and TNF-α levels in serum and the glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME) and NADP+-dependent isocitric dehydrogenase (IDP) levels, the reduced/oxidized glutathione (GSH/GSSG) and reduced/oxidized nicotinamide adenine dinucleotide (phosphate) (NAD(P)H/NAD(P)+) ratios, and the levels of various lipogenic and fibrotic markers in the liver were evaluated. The results showed that hepatic fibrosis induced by fructose consumption was associated with weight gain, hunger-satiety system dysregulation, hyperinsulinemia, dyslipidemia, lipoperoxidation and inflammation. Moreover, increased levels of hepatic G6PD and ME activity and expression, the NAD(P)H/NAD(P)+ ratios, and GSSG concentration and increased expression of lipogenic and fibrotic markers were detected, and these alterations were attenuated by NAM administration. Specifically, NAM diminished the activity and expression of G6PD and ME, and this effect was associated with a decrease in the NADPH/NADP+ ratios, increased GSH levels and decreased lipoperoxidation and inflammation, ameliorating fibrosis and NASH development. NAM reduces liver steatosis and fibrosis by regulating redox homeostasis through a G6PD- and ME-dependent mechanism.
Assuntos
Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Niacinamida/farmacologia , Animais , Antioxidantes/metabolismo , Frutose/efeitos adversos , Frutose/metabolismo , Glucose/metabolismo , Glutationa/metabolismo , Homeostase , Metabolismo dos Lipídeos/fisiologia , Lipídeos/biossíntese , Lipogênese/fisiologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Masculino , NAD/metabolismo , NADP/metabolismo , Niacinamida/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Oxirredução/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Mitosis has been traditionally considered a metabolically inactive phase. We have previously shown, however, that extensive alterations in lipids occur as the cells traverse mitosis, including increased de novo fatty acid (FA) and phosphatidylcholine (PtdCho) synthesis and decreased lysophospholipid content. Given the diverse structural and functional properties of these lipids, we sought to study their metabolic fate and their importance for cell cycle completion. Here we show that FA and PtdCho synthesized at the mitotic exit are destined to the nuclear envelope. Importantly, FA and PtdCho synthesis, but not the decrease in lysophospholipid content, are necessary for cell cycle completion beyond G2/M. Moreover, the presence of alternative pathways for PtdCho synthesis renders the cells less sensitive to its inhibition than to the impairment of FA synthesis. FA synthesis, thus, represents a cell cycle-related metabolic vulnerability that could be exploited for combined chemotherapy. We explored the combination of fatty acid synthase (FASN) inhibition with agents that act at different phases of the cell cycle. Our results show that the effect of FASN inhibition may be enhanced under some drug combinations.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ácido Graxo Sintases/antagonistas & inibidores , Ácidos Graxos/biossíntese , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Mitose/efeitos dos fármacos , Membrana Nuclear/metabolismo , Fosfatidilcolinas/biossíntese , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Etoposídeo/farmacologia , Ácido Graxo Sintases/metabolismo , Células HeLa , Humanos , Lipogênese/fisiologia , Lisofosfolipídeos/biossíntese , Lisofosfolipídeos/química , Mitose/fisiologia , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/enzimologiaRESUMO
INTRODUCTION AND OBJECTIVES: The objectives of this study were to investigate the underlying mechanism of PPARα, LXRα, ChREBP, and SREBP-1c at the level of gene and protein expression with high-energy diets in liver and skeletal muscle. MATERIALS AND METHODS: Metabolic changes with consumption of high fat (Hfat), high sucrose (Hsuc) and high fructose (Hfru) diets were assessed. Levels of mRNA and protein of PPARα, LXRα, ChREBP, and SREBP-1c were investigated. Body weight changes, histological structure of liver and plasma levels of some parameters were also examined. RESULTS: In Hfru group, body weights were higher than other groups (P<0.05). In liver, LXRα levels of Hsuc and Hfru groups were upregulated as 1.87±0.30 (P<0.05) and 2.01±0.29 (P<0.01). SREBP-1c levels were upregulated as 4.52±1.25 (P<0.05); 4.05±1.11 (P<0.05) and 3.85±1.04 (P<0.05) in Hfat, Hsuc, and Hfru groups, respectively. In skeletal muscle, LXRα and SREBP-1c were upregulated as 1.77±0.30 (P<0.05) and 2.71±0.56 (P<0.05), in the Hfru group. Protein levels of ChREBP (33.92±8.84ng/mg protein (P<0.05)) and SREBP-1c (135.16±15.57ng/mg protein (P<0.001)) in liver were higher in Hfru group. In skeletal muscle, LXRα, ChREBP and SREBP-1c in Hfru group were 6.67±0.60, 7.11±1.29 and 43.17±6.37ng/mg, respectively (P<0.05; P<0.01; P<0.05). The rats in Hfru group had the most damaged livers. CONCLUSION: Besides liver, fructose consumption significantly effects skeletal muscle and leads to weight gain, triggers lipogenesis and metabolic disorders.
Assuntos
Fígado Gorduroso/genética , Frutose/farmacologia , Regulação da Expressão Gênica , Receptores X do Fígado/genética , Músculo Esquelético/metabolismo , Sacarose/farmacologia , Óleo de Girassol/farmacologia , Animais , Dieta/métodos , Modelos Animais de Doenças , Ingestão de Energia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/terapia , Lipogênese/fisiologia , Fígado/metabolismo , Fígado/patologia , Receptores X do Fígado/biossíntese , Masculino , Músculo Esquelético/patologia , Ratos , Ratos WistarRESUMO
Childhood consumption of added sugars, such as sucrose, has been associated to increased risk of metabolic syndrome (MetS) and nonalcoholic fatty liver disease (NAFLD). Although the mechanisms underlying NAFLD onset are incompletely defined, recent evidence has proposed a role for the endoplasmic reticulum (ER) stress. Thus, the present study sought to investigate the metabolic outcomes of high-sucrose intake on weaned Swiss mice fed a 25% sucrose diet for 30, 60 and 90 days in comparison to regular chow-fed controls. High-sucrose feeding promoted progressive metabolic and oxidative disturbances, starting from fasting and fed hyperglycemia, hyperinsulinemia, glucose intolerance and increased adiposity at 30-days; passing by insulin resistance, hypertriglyceridemia and NAFLD onset at 60 days; until late hepatic oxidative damage at 90 days. In parallel, assessment of transcriptional and/or translational levels of de novo lipogenesis (DNL) and ER stress markers showed up-regulation of both fatty acid synthesis (ChREBP and SCD1) and oxidation (PPARα and CPT-1α), as well as overexpression of unfolded protein response sensors (IRE1α, PERK and ATF6), chaperones (GRP78 and PDIA1) and antioxidant defense (NRF2) genes at 30 days. At 60 days, fatty acid oxidation genes were down-regulated, and ER stress switched over toward a proapoptotic pattern via up-regulation of BAK protein and CHOP gene levels. Finally, down-regulation of both NRF2 and CPT-1α protein levels led to late up-regulation of SREBP-1c and exponential raise of fatty acids synthesis. In conclusion, our study originally demonstrates a temporal relationship between DNL and ER stress pathways toward MetS and NAFLD development on weaned rats fed a high-sucrose diet.