RESUMO
Obesity increases the risk of metabolic diseases, including insulin resistance and type 2 diabetes, as well as cardiovascular disease. In addition to lipid accumulation in adipose tissue, obesity is associated with increased lipid storage in ectopic tissues, such as skeletal muscle and liver. Furthermore, lipid accumulation in the heart may result in cardiac dysfunction and heart failure. It has recently been demonstrated that intracellular lipid accumulation in ectopic tissues leads to pathological responses and impaired insulin signalling. Here, we will review the current understanding of how lipid storage and lipid droplet physiology affect the risk of developing metabolic diseases.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Fígado Gorduroso/metabolismo , Resistência à Insulina , Metabolismo dos Lipídeos , Lipogênese , Lipoproteínas/metabolismo , Miocárdio/metabolismo , Obesidade/metabolismo , Animais , Proteínas de Transporte/metabolismo , Diabetes Mellitus Tipo 2/etiologia , Insuficiência Cardíaca/metabolismo , Humanos , Imunidade Inata , Inflamação/metabolismo , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Doenças Metabólicas/metabolismo , Músculo Esquelético/metabolismo , Obesidade/complicações , Perilipina-1 , Fosfoproteínas/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Triglicerídeos/metabolismo , alfa-2-Glicoproteína-HS/metabolismoRESUMO
In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSPs fused with green fluorescent protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343-bp proximal part and the 150-bp proximal part) and two variants (in forward and reverse orientation) of a previously characterized synthetic promoter derived from elements of transcription factor genes. We transduced a number of cell lines and primary cells in vitro. In addition, hematopoietic stem cells were transduced with MSPs and transferred into lethally irradiated recipient mice. Fluorescence activated cell sorting analysis was performed to determine the GFP expression in different cell populations both in vitro and in vivo. We showed that MSPs can efficiently be used for lentiviral gene delivery and that the 150-bp proximal part of the CD68 promoter provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.
Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Macrófagos/metabolismo , Regiões Promotoras Genéticas , Animais , Linhagem Celular , Dosagem de Genes , Expressão Gênica , Ordem dos Genes , Terapia Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Especificidade de Órgãos/genética , Transdução Genética , TransgenesRESUMO
This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This abstract has been retracted at the request of Jan Borén, co-author, because of conscious fabrication, corruption or suppression of basic material and conscious preparation and presentation of falsified results in the abstract by one of the authors.
RESUMO
AIMS/HYPOTHESIS: Overproduction of VLDL(1) seems to be the central pathophysiological feature of the dyslipidaemia associated with type 2 diabetes. We explored the relationship between liver fat and suppression of VLDL(1) production by insulin in participants with a broad range of liver fat content. METHODS: A multicompartmental model was used to determine the kinetic parameters of apolipoprotein B and TG in VLDL(1) and VLDL(2) after a bolus of [(2)H(3)]leucine and [(2)H(5)]glycerol during a hyperinsulinaemic-euglycaemic clamp in 20 male participants: eight with type 2 diabetes and 12 control volunteers. The participants were divided into two groups with low or high liver fat. All participants with diabetes were in the high liver-fat group. RESULTS: The results showed a rapid drop in VLDL(1)-apolipoprotein B and -triacylglycerol secretion in participants with low liver fat during the insulin infusion. In contrast, participants with high liver fat showed no significant change in VLDL(1) secretion. The VLDL(1) suppression following insulin infusion correlated with the suppression of NEFA, and the ability of insulin to suppress the plasma NEFA was impaired in participants with high liver fat. A novel finding was an inverse response between VLDL(1) and VLDL(2) secretion in participants with low liver fat: VLDL(1) secretion decreased acutely after insulin infusion whereas VLDL(2) secretion increased. CONCLUSIONS/INTERPRETATION: Insulin downregulates VLDL(1) secretion and increases VLDL(2) secretion in participants with low liver fat but fails to suppress VLDL(1) secretion in participants with high liver fat, resulting in overproduction of VLDL(1). Thus, liver fat is associated with lack of VLDL(1) suppression in response to insulin.
Assuntos
Tecido Adiposo/anatomia & histologia , Diabetes Mellitus Tipo 2/fisiopatologia , Resistência à Insulina/fisiologia , Insulina/fisiologia , Lipoproteínas VLDL/metabolismo , Fígado/anatomia & histologia , Abdome , Tecido Adiposo/fisiologia , Adulto , Apolipoproteínas B/sangue , Índice de Massa Corporal , Diabetes Mellitus Tipo 2/sangue , Ácidos Graxos não Esterificados/metabolismo , Humanos , Insulina/sangue , Cinética , Lipoproteínas VLDL/antagonistas & inibidores , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Valores de ReferênciaRESUMO
AIMS/HYPOTHESIS: We determined whether hepatic fat content and plasma adiponectin concentration regulate VLDL(1) production. METHODS: A multicompartment model was used to simultaneously determine the kinetic parameters of triglycerides (TGs) and apolipoprotein B (ApoB) in VLDL(1) and VLDL(2) after a bolus of [(2)H(3)]leucine and [(2)H(5)]glycerol in ten men with type 2 diabetes and in 18 non-diabetic men. Liver fat content was determined by proton spectroscopy and intra-abdominal fat content by MRI. RESULTS: Univariate regression analysis showed that liver fat content, intra-abdominal fat volume, plasma glucose, insulin and HOMA-IR (homeostasis model assessment of insulin resistance) correlated with VLDL(1) TG and ApoB production. However, only liver fat and plasma glucose were significant in multiple regression models, emphasising the critical role of substrate fluxes and lipid availability in the liver as the driving force for overproduction of VLDL(1) in subjects with type 2 diabetes. Despite negative correlations with fasting TG levels, liver fat content, and VLDL(1) TG and ApoB pool sizes, adiponectin was not linked to VLDL(1) TG or ApoB production and thus was not a predictor of VLDL(1) production. However, adiponectin correlated negatively with the removal rates of VLDL(1) TG and ApoB. CONCLUSIONS/INTERPRETATION: We propose that the metabolic effect of insulin resistance, partly mediated by depressed plasma adiponectin levels, increases fatty acid flux from adipose tissue to the liver and induces the accumulation of fat in the liver. Elevated plasma glucose can further increase hepatic fat content through multiple pathways, resulting in overproduction of VLDL(1) particles and leading to the characteristic dyslipidaemia associated with type 2 diabetes.
Assuntos
Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Lipoproteínas VLDL/biossíntese , Adulto , Idoso , Apolipoproteínas B/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Lipoproteínas VLDL/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
Apolipoprotein (apo) B exists in two forms apoB100 and apoB48. ApoB100 is present on very low-density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and LDL. ApoB100 assembles VLDL particles in the liver. This process starts by the formation of a pre-VLDL, which is retained in the cell unless converted to the triglyceride-poor VLDL2. VLDL2 is secreted or converted to VLDL1 by a bulk lipidation in the Golgi apparatus. ApoB100 has a central role in the development of atherosclerosis. Two proteoglycan-binding sequences in apoB100 have been identified, which are important for retaining the lipoprotein in the intima of the artery. Retention is essential for the development of the atherosclerotic lesion.
Assuntos
Apolipoproteínas B/metabolismo , Arteriosclerose/etiologia , Apolipoproteína B-100 , Apolipoproteína B-48 , Apolipoproteínas B/química , Arteriosclerose/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Hiperlipidemias/metabolismo , Hiperlipidemias/prevenção & controle , Lipoproteínas VLDL/química , Lipoproteínas VLDL/metabolismo , Modelos Biológicos , Proteoglicanas/metabolismo , Fatores de Risco , Triglicerídeos/metabolismoRESUMO
The role of ADP-ribosylation factor 1 (ARF-1) in the assembly of very low density lipoproteins (VLDL) was investigated by expressing dominant-negative mutants in McA-RH7777 cells. Transient expression of ARF-1(T31N), a GDP-restrictive mutant, significantly inhibited apolipoprotein B-100 (apoB-100) VLDL production without influencing the biosynthesis of apoB-100 low density lipoproteins or total apoB production (indicating that it inhibited the second step of VLDL assembly) and without altering total protein production or biosynthesis of transferrin, phosphatidylcholine, or triglycerides. These effects were confirmed in stable inducible transfectants. In contrast, expression of an ARF-1 mutant lacking the N-terminal 17 amino acids, which has no myristoylation site and cannot interact with the microsomal membrane, did not affect VLDL assembly. Thus, active ARF-1 is needed for the second step of the process. To further explore these observations, we developed a cell-free system based on the postnuclear supernatant isolated from McA-RH7777 cells. In this system, 10-15% of the apoB-100 pool was converted to VLDL in a time- and temperature-dependent way. The assembly process was highly dependent on a heat-stable factor in the d > 1.21 g/ml infranatant of fetal calf serum; this factor was not present in low density lipoproteins or VLDL. Brefeldin A inhibited VLDL assembly in this system, as did a synthetic peptide (corresponding to N-terminal amino acids 2-17 of ARF-1) that displaces ARF-1 from the membrane. Thus, active ARF-1 is also needed for cell-free assembly of VLDL. Guanosine 5'-3-O-(thio)triphosphate also inhibited VLDL assembly in this system, indicating that the process requires ongoing hydrolysis of GTP. 1-Butanol, which inhibits the formation of phosphatidic acid (PA) and instead gives rise to phosphatidylbutanol, inhibited VLDL assembly, whereas 2-butanol, which does not inhibit PA formation, failed to do so. Thus, phospholipase D (PLD)-catalyzed formation of PA from phosphatidylcholine is essential for VLDL assembly. In support of this conclusion, exogenous PLD prevented brefeldin A from inhibiting the assembly process. Our results indicate that ARF-1 participates in the second step of VLDL assembly through a process that involves activation of PLD and production of PA.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Lipoproteínas VLDL/biossíntese , Fosfolipase D/metabolismo , 1-Butanol/farmacologia , Apolipoproteína B-100 , Apolipoproteínas B/metabolismo , Butanóis/farmacologia , Células Cultivadas , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Trifosfato/metabolismo , Humanos , Ácidos Fosfatídicos/biossíntese , Transferrina/metabolismoRESUMO
We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.
Assuntos
Apolipoproteínas B/biossíntese , Lipoproteínas VLDL/biossíntese , Microssomos Hepáticos/metabolismo , Microssomos/metabolismo , Animais , Apolipoproteína B-48 , Apolipoproteínas B/química , Apolipoproteínas B/isolamento & purificação , Fracionamento Celular , Glicerol/isolamento & purificação , Glicerol/metabolismo , Cinética , Lipoproteínas VLDL/química , Lipoproteínas VLDL/isolamento & purificação , Neoplasias Hepáticas Experimentais , Modelos Biológicos , Fosfatidilcolinas/isolamento & purificação , Fosfatidilcolinas/metabolismo , Ratos , Triglicerídeos/metabolismo , Células Tumorais CultivadasRESUMO
The assembly of very low-density lipoproteins (VLDL) occurs in two major steps. The first step is the co-and post-translational lipidation of apoB, forming pre-VLDL in the rough endoplasmic reticulum. The microsomal triglyceride transfer protein catalyzes this step. In the second step pre-VLDL is converted to bona fide VLDL in a smooth membrane compartment. This step depends on ADP-ribosylation factor 1 and its activation of phospholipase D.
Assuntos
Apolipoproteínas B/metabolismo , Retículo Endoplasmático Rugoso/metabolismo , Retículo Endoplasmático Liso/metabolismo , Hiperlipidemias/metabolismo , Lipoproteínas VLDL/metabolismo , Apolipoproteína B-48 , Apolipoproteínas B/sangue , Humanos , Hipertrigliceridemia/sangue , Lipoproteínas VLDL/sangueRESUMO
The assembly of lipoproteins containing apolipoprotein B is a complex process that occurs in the lumen of the secretory pathway. The process consists of two relatively well-identified steps. In the first step, two VLDL precursors are formed simultaneously and independently: an apolipoprotein B-containing VLDL precursor (a partially lipidated apolipoprotein B) and a VLDL-sized lipid droplet that lacks apolipoprotein B. In the second step, these two precursors fuse to form a mature VLDL particle. The apolipoprotein B-containing VLDL precursor is formed during the translation and concomitant translocation of the protein to the lumen of the endoplasmic reticulum. The VLDL precursor is completed shortly after the protein is fully synthesized. The process is dependent on the microsomal triglyceride transfer protein (MTP). Although the mechanism by which the lipid droplets are formed is unknown, recent observations indicate that the process is dependent on MTP. The fusion of the two precursors is not dependent on MTP, but the mechanism remains to be elucidated. The conversion of the apolipoprotein B-containing precursor to VLDL seems to be dependent on the ADP ribosylation factor 1 (ARF 1) and its activation of phospholipase D. During their assembly, nascent apolipoprotein B chains undergo quality control and are sorted to degradation. Such sorting, which occurs cotranslationally during the formation of the apolipoprotein B-containing precursor, involves cytosolic chaperons and ubiquitination that targets apolipoprotein B to proteasomal degradation. Other levels of sorting occur in the secretory pathway. Thus, lysosomal enzymes are involved as well as the LDL receptor.
Assuntos
Apolipoproteínas B/metabolismo , Lipoproteínas VLDL/biossíntese , Lipoproteínas VLDL/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Humanos , Metabolismo dos Lipídeos , Triglicerídeos/metabolismoRESUMO
The liver plays a primary role in lipid metabolism. Important functions include the synthesis and incorporation of hydrophobic lipids, triacylglycerols and cholesteryl esters into the core of water-miscible particles called lipoproteins and the secretion of these particles into the circulation for transport to distant tissues. In this article, we present a brief overview of one aspect of the assembly process of very low density lipoproteins, namely, possible mechanisms for combining core lipids with apolipoprotein B. This is a complex process in which apolipoprotein B interacts with core lipids to form very low density lipoproteins by a two-step process that can be dissociated biochemically.
Assuntos
Apolipoproteínas B/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas VLDL/biossíntese , Animais , Apolipoproteína B-48 , Apolipoproteínas B/química , Brefeldina A/farmacologia , Proteínas de Transporte/metabolismo , Humanos , Lipoproteínas VLDL/química , Microssomos Hepáticos/metabolismo , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
Biosynthetic studies on the human MUC5AC mucin were performed by immunoprecipitations with antisera recognizing only the non-O-glycosylated apomucin in the colon adenocarcinoma cell line LS 174T. Pulse-chase studies and subcellular fractionations showed that MUC5AC formed dimers in the rough endoplasmic reticulum within 15 min of the initiation of biosynthesis. No non-O-glycosylated species larger than dimers were identified. The dimerization was N-glycosylation-dependent, because tunicamycin treatment significantly lowered the rate of dimerization. When the biosynthesis of MUC5AC apomucin was compared with that of MUC2 apomucin, also produced in the LS 174T cell line, both apomucins were assembled in similar ways with respect to their rates of dimerization with and without inhibition of N-glycosylation. No heterodimerization was observed between the human MUC5AC and the MUC2 apomucins despite the extensive sequence similarities in the positions of the cysteine residues in the C-termini proposed to be involved in mucin dimerization.
Assuntos
Retículo Endoplasmático Rugoso/metabolismo , Mucinas/química , Mucinas/metabolismo , Sequência de Aminoácidos , Animais , Bioquímica/métodos , Carcinoma/química , Carcinoma/metabolismo , Neoplasias do Colo/química , Neoplasias do Colo/metabolismo , Reações Cruzadas , Dimerização , Mucinas Gástricas/imunologia , Mucinas Gástricas/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Soros Imunes , Dados de Sequência Molecular , Mucina-5AC , Mucina-2 , Mucinas/imunologia , Coelhos , Frações Subcelulares , Células Tumorais Cultivadas , Tunicamicina/farmacologia , UltracentrifugaçãoRESUMO
Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using an O-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition of N-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Mucinas/metabolismo , Proteínas de Neoplasias/metabolismo , Antibacterianos/farmacologia , Transporte Biológico Ativo , Biomarcadores Tumorais , Dimerização , Retículo Endoplasmático/efeitos dos fármacos , Mucinas Gástricas/biossíntese , Glicosilação , Complexo de Golgi/efeitos dos fármacos , Humanos , Mucina-2 , Frações Subcelulares/metabolismo , Células Tumorais Cultivadas , Tunicamicina/farmacologia , UltracentrifugaçãoRESUMO
In cells in which the lipoprotein assembly process had been inactivated by brefeldin A (BFA), membrane-associated apoB-100 disappeared without forming lipoproteins or being secreted, indicating that it was degraded. Reactivation of the assembly process by chasing the cells in the absence of BFA, gave rise to a quantitative recovery of the membrane-associated apoB-100 in the very low density lipoprotein (VLDL) fraction in the medium. These results indicate that the membrane-associated apoB-100 can be converted to VLDL. A new method was developed by which the major amount (88%) of microsomal apoB-100 but not integral membrane proteins could be extracted. The major effect of this method was to increase the recovery of apoB-100 that banded in the LDL and HDL density regions, suggesting that the membrane-associated form of apoB-100 is partially lipidated. We also investigated the role of the microsomal triglyceride transfer protein (MTP) in the assembly of apoB-100 VLDL using a photoactivatable MTP inhibitor (BMS-192951). This compound strongly inhibited the assembly and secretion of apoB-100 VLDL when present during the translation of the protein. To investigate the importance of MTP during the later stages in the assembly process, the cells were preincubated with BFA (to reversibly inhibit the assembly of apoB-100 VLDL) and pulse-labeled (+BFA) and chased (+BFA) for 30 min to obtain full-length apoB-100 associated with the microsomal membrane. Inhibition of MTP after the 30-min chase blocked assembly of VLDL. This indicates that MTP is important for the conversion of full-length apoB-100 into VLDL. Results from experiments in which a second chase (-BFA) was introduced before the inactivation of MTP indicated that only early events in this conversion of full-length apoB-100 into VLDL were blocked by the MTP inhibitor. Together these results indicate that there is a MTP-dependent "window" in the VLDL assembly process that occurs after the completion of apoB-100 but before the major amount of lipids is added to the VLDL particle. Thus the assembly of apoB-100 VLDL from membrane-associated apoB-100 involves an early MTP-dependent phase and a late MTP-independent phase, during which the major amount of lipid is added.
Assuntos
Apolipoproteínas B/metabolismo , Proteínas de Transporte/metabolismo , Lipoproteínas VLDL/metabolismo , Microssomos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apolipoproteína B-100 , Brefeldina A , Proteínas de Transporte/antagonistas & inibidores , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Cicloeximida/farmacologia , Ciclopentanos/farmacologia , Proteínas de Membrana/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Células Tumorais CultivadasRESUMO
Permeabilized Hep G2 cells have been used to investigate the turnover of apolipoprotein B-100 (apoB-100). When such cells were chased in the presence of buffer, there was no biosynthesis of apoB-100, nor was the protein secreted from the cells. Thus the turnover of apoB-100 in these cells reflected the posttranslational degradation of the protein. Pulse-chase studies indicated that apoB-100 was degraded both when associated with the membrane and when present as lipoproteins in the secretory pathway. Neither albumin nor alpha1-antitrypsin showed any significant posttranslational intracellular degradation under the same condition. The kinetics for the turnover of apoB-100 in the luminal content differed from that of apoB-100 that was associated with the microsomal membrane. Moreover, while the degradation of the luminal apoB-100 was inhibited by N-acetyl-leucyl-leucyl-norleucinal (ALLN), this was not the case for the membrane-associated protein. Together these results suggest the existence of different pathways for the degradation of luminal apoB-100 and membrane-associated apoB-100. This was further supported by results from pulse-chase studies in intact cells, showing that ALLN increased the amount of radioactive apoB-100 that associated with the microsomal membrane during the pulse-labeling of the cells. However, ALLN did not influence the rate of turnover of the membrane-associated apoB-100. The presence of an ATP-generating system during the chase of the permeabilized cells prevented the disappearance of pulse-labeled apoB-100 from the luminal lipoprotein-associated pool. The ATP-generating system combined with cytosol protected the total apoB-100 in the system from being degraded. The cells cultured in the presence of oleic acid and chased after permeabilization in the presence of cytosol and the ATP-generating system showed an increase in the amount of apoB-100 present on dense ("high density lipoprotein-like") particles. This increase was linear during the time investigated (i. e. from 0 to 2 h chase) and independent of protein biosynthesis. Our results indicate that the dense particle was generated by a redistribution of apoB-100 within the secretory pathway and that it most likely was assembled from the membrane- associated form of apoB-100. These results indicate that the release of apoB-100 from this membrane-associated form to the microsomal lumen is dependent on cytosolic factors and a source of metabolic energy.
Assuntos
Apolipoproteínas B/metabolismo , Digitonina , Transporte Biológico , Linhagem Celular , HumanosRESUMO
The microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of the lipoproteins containing apolipoprotein B (apoB): very low density lipoproteins and chylomicrons. Evidence indicates that the subclasses of these lipoproteins that contain apoB-48 are assembled in a distinct two-step process; first a relatively lipid-poor primordial lipoprotein precursor is produced, and then bulk neutral lipids are added to form the core of these spherical particles. To determine if either step is mediated by MTP, a series of clonal cell lines stably expressing apoB-53 and MTP was established in non-lipoprotein-producing HeLa cells. MTP activity in these cells was approximately 30%, and apoB secretion was 7-33% of that in HepG2 cells on a molar basis. Despite having robust levels of triglyceride and phospholipid synthesis, these cell lines, as exemplified by HLMB53-59, secreted >90% of the apoB-53 on relatively lipid-poor particles in the density range of 1.063-1.21 g/ml. These results suggested that coexpression of MTP and apoB only reconstituted the first but not the second step in lipoprotein assembly. To extend this observation, additional studies were carried out in McArdle RH-7777 rat hepatoma cells, in which the second step of apoB-48 lipoprotein assembly is well defined. Treatment of these cells with the MTP photoaffinity inhibitor BMS-192951 before pulse labeling with [35S]methionine/cysteine led to an 85% block of both apoB-48 and apoB-100 but not apoAI secretion, demonstrating inhibition of the first step of lipoprotein assembly. After a 30-min [35S]methioneine/cysteine pulse labeling and 120 min of chase, all of the nascent apoB-48 was observed to have a density of high density lipoproteins (1.063-1.21 g/ml), indicating that only the first step of lipoprotein assembly had occurred. The addition of oleic acid to the cell culture media activated the second step as evidenced by the conversion of the apoB-48 high density lipoproteins to very low density lipoproteins (d < 1.006 g/ml) during an extended chase period. Inactivation of MTP after completion of the first step, but before stimulation of the second step by the addition of oleic acid, did not block this conversion. Thus, inhibition of MTP did not hinder the addition of bulk core lipid to the primordial lipoprotein precursor particles, indicating that MTP is not required for the second step of apoB-48 lipoprotein assembly.
Assuntos
Apolipoproteínas B/biossíntese , Proteínas de Transporte/metabolismo , Glicoproteínas , Lipoproteínas/biossíntese , Animais , Apolipoproteína A-I/metabolismo , Proteínas de Transferência de Ésteres de Colesterol , Células HeLa , Humanos , Ratos , Células Tumorais CultivadasRESUMO
Hypophysectomized female rats were treated for 1 week with thyroxine (10 micrograms/kg.day), cortisol (400 micrograms/kg.day), and bovine GH (1 mg/kg.day) either as two daily subcutaneous injections (GH x 2) or as a continuous subcutaneous infusion (GHc) in order to mimic the male and female specific GH secretory patterns, respectively. Hepatocytes were then isolated and kept in short-term cultures. Hypophysectomy decreased the synthesis of triacylglycerol. Treatment with GH x 2 had no or small effects, while GHc normalized the effect of hypophysectomy. ApoB-100 VLDL was assembled before apoB-48 VLDL. ApoB-48 was first assembled as an HDL particle (apoB-48 "HDL"). Hypophysectomy decreased the proportion of intracellular apoB-48 that was recovered as VLDL. Moreover, the proportion of apoB-48 of total apoB in VLDL decreased. Only GHc fully restored the effect of hypophysectomy by inducing an 4-fold increase in the assembly of apoB-48 VLDL, while treatment with GH x 2 gave rise to a 1.8-fold increase. Hypophysectomy resulted in a decrease in the proportion of apoB-48 that was secreted as VLDL and a decrease in the proportion of apoB-48 of total apoB in VLDL. Only treatment with GHc fully restored the secretion of apoB-48 VLDL by inducing an almost 4-fold increase in the secretion of apoB-48 VLDL, while the corresponding value for treatment with GH x 2 was 1.7. However, GH x 2 increased the proportion of the secreted apoB-48 that was recovered in VLDL to the levels found in normal rats and in rats treated with GHc, but this finding was due to a failure of GH x 2 treatment to increase the secretion of apoB-48 "HDL". In summary, a continuous infusion of GH to hypophysectomized rats, mimicking the female secretion of GH, normalized the triacylglycerol synthesis and secretion as well as apoB-48 VLDL assembly and secretion to those levels observed in hepatocyte cultures from intact female rats.
Assuntos
Apolipoproteínas B/metabolismo , Hormônio do Crescimento/administração & dosagem , Lipoproteínas/biossíntese , Fígado/metabolismo , Triglicerídeos/biossíntese , Animais , Apolipoproteína B-100 , Apolipoproteína B-48 , Bovinos , Células Cultivadas , Feminino , Glicerol/metabolismo , Hormônio do Crescimento/farmacologia , Hipofisectomia , Cinética , Fígado/efeitos dos fármacos , Ácido Oleico/metabolismo , Ratos , Ratos Sprague-DawleyAssuntos
Apolipoproteínas B/metabolismo , Lipoproteínas/biossíntese , Animais , Apolipoproteína B-48 , Apolipoproteínas B/genética , Catálise , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Processamento de Proteína Pós-Traducional , Edição de RNA , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BFA inhibited in a dose dependent way the assembly of apoB-48 very low density lipoprotein (VLDL) but allowed a normal rate of biosynthesis of the apolipoprotein and of the assembly of the dense ("high density lipoprotein (HDL)-like") apoB-48 particle (apoB-48 HDL). The inhibition of the assembly of apoB-48 VLDL occurred at BFA levels that allowed a major secretion of both transferrin and apoB-48 HDL. The assembly of apoB-100 containing lipoproteins was also inhibited by BFA but could be reactivated by a 30-60 min chase in the absence of BFA, which agreed with the time that was estimated to be needed to restore the secretory pathway (approximately 60 min). Also the assembly of apoB-48 VLDL was reversible. Both apoB-48 and apoB-100 that was labeled in the presence of BFA assembled VLDL after removal of the BFA. Both apoB-100 and apoB-48 were associated with the membrane pellet of the microsomes. Virtually all (122 +/- 30%) of the membrane associated pulse-labeled apoB-48 remained in the membrane after a 180-min chase in the presence of BFA, compared to only 21 +/- 2% in normal cells (mean +/- S.D., n = 4). The corresponding figures for apoB-100 was 40 +/- 7% in BFA-treated cells and 9 +/- 7% in normal cells (mean +/- S.D., n = 4). Pulse-chase experiments with BFA offered conditions to selectively follow the turnover of membrane-associated apoB-100. Such experiments indicated that this apoB-100 pool is a precursor to VLDL.
Assuntos
Apolipoproteínas B/metabolismo , Ciclopentanos/farmacologia , Lipoproteínas VLDL/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Apolipoproteína B-100 , Apolipoproteína B-48 , Transporte Biológico , Brefeldina A , Linhagem Celular , Membranas Intracelulares/metabolismo , Microssomos/metabolismo , Triglicerídeos/biossínteseRESUMO
GH has previously been shown to regulate serum lipoprotein levels and hepatic secretion of apolipoprotein-B (apo-B) and apo-E in the rat. The aim of this investigation was to study a possible role of insulin-like growth factor-I (IGF-I) in this regulation. Adult female rats were hypophysectomized and treated with recombinant human IGF-I (1.25 mg/kg.day) as a sc continuous infusion for 7 days. The effects of IGF-I were compared with those of bovine GH, given either as a continuous sc infusion or as two daily sc injections. All hypophysectomized rats were given replacement therapy with L-T4 and cortisol. Serum IGF-I concentrations increased to similar levels as a result of treatment with bovine GH and IGF-I. There was no effect of IGF-I on serum concentrations of glucose or insulin, whereas GH, independent of its mode of administration, increased serum insulin concentrations. Food intake was not affected by treatment with IGF-I. IGF-I had no effect on serum concentrations of cholesterol or apo-E, whereas GH given twice daily decreased serum cholesterol concentrations, and a continuous infusion of GH increased serum apo-E concentrations. Serum triglyceride and apo-B concentrations increased markedly as a result of IGF-I treatment, whereas GH had no effect on serum triglycerides, but decreased serum apo-B concentrations. Hepatocytes were isolated from hypophysectomized rats treated with L-T4 and cortisol alone or in combination with IGF-I and kept in short term cultures. In this system, IGF-I had no effect on the incorporation of [3H]glycerol in triglycerides or the mass of triglycerides in the cells and medium. There was no effect of IGF-I treatment on the secretion of apo-E or apo-B. Moreover, there was no effect of IGF-I treatment on the relationship between newly synthesized and secreted apo-B 48 and apo-B 100, as determined by [35S]methionine labeling of the proteins. In conclusion, the previously observed effects of GH on serum lipoproteins and hepatic apolipoprotein secretion does not seem to be mediated via IGF-I, but IGF-I has its own unique effects on serum triglyceride and apo-B levels. The increases in serum apo-B and serum triglyceride concentrations after IGF-I treatment were not dependent on increased hepatic secretion of apo-B or triglycerides.