RESUMEN
The control of chylomicron output by the intestine is a complex process whose outlines have only recently come into focus. In this review we will cover aspects of chylomicron formation and prechylomicron vesicle generation that elucidate potential control points. Substrate (dietary fatty acids and monoacylglycerols) availability is directly related to the output rate of chylomicrons. These substrates must be converted to triacylglycerol before packaging in prechylomicrons by a series of endoplasmic reticulum (ER)-localized acylating enzymes that rapidly convert fatty acids and monoacylglycerols to triacylglycerol. The packaging of the prechylomicron with triacylglycerol is controlled by the microsomal triglyceride transport protein, another potential limiting step. The prechylomicrons, once loaded with triacylglycerol, are ready to be incorporated into the prechylomicron transport vesicle that transports the prechylomicron from the ER to the Golgi. Control of this exit step from the ER, the rate-limiting step in the transcellular movement of the triacylglycerol, is a multistep process involving the activation of PKCζ, the phosphorylation of Sar1b, releasing the liver fatty acid binding protein from a heteroquatromeric complex, which enables it to bind to the ER and organize the prechylomicron transport vesicle budding complex. We propose that control of PKCζ activation is the major physiological regulator of chylomicron output.
Asunto(s)
Quilomicrones/metabolismo , Mucosa Intestinal/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Humanos , Transporte de Proteínas , Vesículas Transportadoras/metabolismoRESUMEN
Chylomicron output by the intestine is proportional to intestinal phosphatidylcholine (PC) delivery. Using five different variations of PC delivery to the intestine, we found that lyso-phosphatidylcholine (lyso-PC), the absorbed form of PC, concentrations in the cytosol (0 to 0.45 nM) were proportional to the input rate. The activity of protein kinase C (PKC)ζ, which controls prechylomicron output rate by the endoplasmic reticulum (ER), correlated with the lyso-PC concentration suggesting that it may be a PKCζ activator. Using recombinant PKCζ, the Km for lyso-PC activation was 1.49 nM and the Vmax 1.12 nM, more than the maximal lyso-PC concentration in cytosol, 0.45 nM. Among the phospholipids and their lyso derivatives, lyso-PC was the most potent activator of PKCζ and the only one whose cytosolic concentration suggested that it could be a physiological activator because other phospholipid concentrations were negligible. PKCζ was on the surface of the dietary fatty acid transport vesicle, the caveolin-1-containing endocytic vesicle. Once activated, PKCζ, eluted off the vesicle. A conformational change in PKCζ on activation was suggested by limited proteolysis. We conclude that PKCζ on activation changes its conformation resulting in elution from its vesicle. The downstream effect of dietary PC is to activate PKCζ, resulting in greater chylomicron output by the ER.
Asunto(s)
Sistema Biliar/metabolismo , Dieta , Intestinos/efectos de los fármacos , Intestinos/enzimología , Fosfatidilcolinas/farmacología , Proteína Quinasa C/metabolismo , Animales , Antígenos CD36/metabolismo , Caveolina 1/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Activación Enzimática/efectos de los fármacos , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestinos/citología , Lisofosfatidilcolinas/farmacología , Masculino , Proteínas de Unión al GTP Monoméricas/metabolismo , Ácido Oléico/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Conformación Proteica , Proteína Quinasa C/química , Ratas , Ratas Sprague-Dawley , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/metabolismoRESUMEN
How dietary fatty acids are absorbed into the enterocyte and transported to the ER is not established. We tested the possibility that caveolin-1 containing lipid rafts and endocytic vesicles were involved. Apical brush border membranes took up 15% of albumin bound (3)H-oleate whereas brush border membranes from caveolin-1 KO mice took up only 1%. In brush border membranes, the (3)H-oleate was in the detergent resistant fraction of an OptiPrep gradient. On OptiPrep gradients of intestinal cytosol, we also found the (3)H-oleate in the detergent resistant fraction, separate from OptiPrep gradients spiked with (3)H-oleate or (3)H-triacylglycerol. Caveolin-1 immuno-depletion of cytosol removed 91% of absorbed (3)H-oleate whereas immuno-depletion using IgG, or anti-caveolin-2 or -3 or anti-clathrin antibodies removed 20%. Electron microscopy showed the presence of caveolin-1 containing vesicles in WT mouse cytosol that were 4 fold increased by feeding intestinal sacs 1mM oleate. No vesicles were seen in caveolin-1 KO mouse cytosol. Caveolin-1 KO mice gained less weight on a 23% fat diet and had increased fat in their stool compared to WT mice. We conclude that dietary fatty acids are absorbed by caveolae in enterocyte brush border membranes, are endocytosed, and transported in cytosol in caveolin-1 containing endocytic vesicles.
Asunto(s)
Caveolina 1/metabolismo , Grasas de la Dieta/metabolismo , Endocitosis/fisiología , Enterocitos/metabolismo , Absorción Intestinal/fisiología , Ácido Oléico/metabolismo , Animales , Caveolina 1/genética , Citosol/metabolismo , Grasas de la Dieta/farmacología , Enterocitos/citología , Humanos , Microdominios de Membrana/genética , Microdominios de Membrana/metabolismo , Ratones , Ratones Noqueados , Microvellosidades/genética , Microvellosidades/metabolismo , Ácido Oléico/farmacología , RatasRESUMEN
The absorption of dietary fat is of increasing concern given the rise of obesity not only in the United States but throughout the developed world. This review explores what happens to dietary fat within the enterocyte. Absorbed fatty acids and monoacylglycerols are required to be bound to intracellular proteins and/or to be rapidly converted to triacylglycerols to prevent cellular membrane disruption. The triacylglycerol produced at the level of the endoplasmic reticulum (ER) is either incorporated into prechylomicrons within the ER lumen or shunted to triacylglycerol storage pools. The prechylomicrons exit the ER in a specialized transport vesicle in the rate-limiting step in the intracellular transit of triacylglycerol across the enterocyte. The prechylomicrons are further processed in the Golgi and are transported to the basolateral membrane via a separate vesicular system for exocytosis into the intestinal lamina propria. Fatty acids and monoacylglycerols entering the enterocyte via the basolateral membrane are also incorporated into triacylglycerol, but the basolaterally entering lipid is much more likely to enter the triacylglycerol storage pool than the lipid entering via the apical membrane.
Asunto(s)
Quilomicrones/biosíntesis , Metabolismo de los Lípidos/fisiología , Animales , Grasas de la Dieta/metabolismo , Retículo Endoplásmico/metabolismo , Ácidos Grasos/metabolismo , Humanos , Lípidos/biosíntesis , Vesículas Transportadoras/metabolismoRESUMEN
Native cytosol requires ATP to initiate the budding of the pre-chylomicron transport vesicle from intestinal endoplasmic reticulum (ER). When FABP1 alone is used, no ATP is needed. Here, we test the hypothesis that in native cytosol FABP1 is present in a multiprotein complex that prevents FABP1 binding to the ER unless the complex is phosphorylated. We found on chromatography of native intestinal cytosol over a Sephacryl S-100 HR column that FABP1 (14 kDa) eluted in a volume suggesting a 75-kDa protein complex that contained four proteins on an anti-FABP1 antibody pulldown. The FABP1-containing column fractions were chromatographed over an anti-FABP1 antibody adsorption column. Proteins co-eluted from the column were identified as FABP1, Sar1b, Sec13, and small VCP/p97-interactive protein by immunoblot, LC-MS/MS, and MALDI-TOF. The four proteins of the complex had a total mass of 77 kDa and migrated on native PAGE at 75 kDa. When the complex was incubated with intestinal ER, there was no increase in FABP1-ER binding. However, when the complex member Sar1b was phosphorylated by PKCζ and ATP, the complex completely disassembled into its component proteins that migrated at their monomer molecular weight on native PAGE. FABP1, freed from the complex, was now able to bind to intestinal ER and generate the pre-chylomicron transport vesicle (PCTV). No increase in ER binding or PCTV generation was observed in the absence of PKCζ or ATP. We conclude that phosphorylation of Sar1b disrupts the FABP1-containing four-membered 75-kDa protein complex in cytosol enabling it to bind to the ER and generate PCTV.
Asunto(s)
Quilomicrones/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Precursores de Proteínas/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico Activo/fisiología , Quilomicrones/genética , Citosol/metabolismo , Retículo Endoplásmico/genética , Proteínas de Unión a Ácidos Grasos/genética , Mucosa Intestinal/metabolismo , Masculino , Proteínas de Unión al GTP Monoméricas/genética , Fosforilación , Unión Proteica , Proteína Quinasa C-epsilon/genética , Proteína Quinasa C-epsilon/metabolismo , Precursores de Proteínas/genética , Ratas , Ratas Sprague-Dawley , Vesículas SecretorasRESUMEN
The rate-limiting step in the transit of dietary fat across the intestinal absorptive cell is its exit from the endoplasmic reticulum (ER) in a specialized ER-to-Golgi transport vesicle, the prechylomicron transport vesicle (PCTV). PCTV bud off from the ER membranes and have unique features; they are the largest ER-derived vesicles (average diameter 250 nm), do not require GTP and COPII proteins for their formation, and utilize VAMP7 as a v-N-ethylmaleimide sensitive factor attachment protein receptor (SNARE). However, PCTV require COPII proteins for their fusion with the Golgi, suggesting a role for them in Golgi target recognition. In support of this, PCTV contained each of the five COPII proteins when docked with the Golgi. When PCTV were fused with the Golgi, the COPII proteins were present in greatly diminished amounts, indicating they had cycled back to the cytosol. Immuno-depletion of Sec31 from the cytosol did not affect PCTV-Golgi docking, but depletion of Sec23 resulted in a 25% decrease. Immuno-depletion of Sec24C caused a nearly complete cessation of PCTV docking activity, but on the addition of recombinant Sec24C, docking activity was restored. We conclude that the COPII proteins are present at docking of PCTV with the Golgi and that Sec24C is required for this event. Sec23 plays a less important role.
Asunto(s)
Quilomicrones/metabolismo , Aparato de Golgi/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas SNARE/metabolismoRESUMEN
Dietary lipid absorption is dependent on chylomicron production whose rate-limiting step across the intestinal absorptive cell is the exit of chylomicrons from the endoplasmic reticulum (ER) in its ER-to-Golgi transport vesicle, the prechylomicron transport vesicle (PCTV). This study addresses the composition of the budding complex for PCTV. Immunoprecipitation (IP) studies from rat intestinal ER solubilized in Triton X-100 suggested that vesicle-associated membrane protein 7 (VAMP7), apolipoprotein B48 (apoB48), liver fatty acid-binding protein (L-FABP), CD36, and the COPII proteins were associated on incubation of the ER with cytosol and ATP. This association was confirmed by chromatography of the solubilized ER over Sephacryl S400-HR in which these constituents cochromatographed with an apparent kDa of 630. No multiprotein complex was detected when the ER was chromatographed in the absence of PCTV budding activity (resting ER or PKCzeta depletion of ER and cytosol). Treatment of the ER with anti-apoB48 or anti-VAMP7 antibodies or using gene disrupted L-FABP or CD36 mice all significantly inhibited PCTV generation. A smaller complex (no COPII proteins) was formed when only rL-FABP was used to bud PCTV. The data support the conclusion that the PCTV budding complex in intestinal ER is composed of VAMP7, apoB48, CD36, and L-FABP, plus the COPII proteins.
Asunto(s)
Quilomicrones/metabolismo , Retículo Endoplásmico/metabolismo , Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Complejos Multiproteicos/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Apolipoproteína B-48/metabolismo , Transporte Biológico , Antígenos CD36/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Grasas de la Dieta/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Intestinos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Chaperonas Moleculares/metabolismo , Proteínas R-SNARE/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The causes of systemic venous hypertension (SVHT) include cardiac- and circulatory-related factors, whereas its consequences include the congestion of hepatic, splanchnic, and peripheral circulations, which contribute significantly to the clinical congestive heart failure syndrome. Based on a disequilibrium in hydrostatic and oncotic pressures, the increased filtration and formation of interstitial fluid at these sites with an accompanying increase in lymph flow mandates an increment in lymphatic drainage to protect against such congestion and the appearance of edema and ascites. However, lymph flow via the thoracic duct into systemic veins is opposed by elevations in central venous pressure. Various management strategies have the potential to prevent and/or correct SVHT. The case of a 54-year-old man with a dilated cardiomyopathy who presented with decompensated biventricular failure, expressed as anasarca and ascites, is used to illustrate the importance of SVHT.
Asunto(s)
Hipertensión/etiología , Hipertensión/fisiopatología , Ascitis/etiología , Edema/etiología , Insuficiencia Cardíaca/complicaciones , Hemodinámica , Humanos , Hipertensión/complicaciones , Hipertensión/terapia , Sistema Linfático/patología , Sistema Linfático/fisiología , Masculino , Persona de Mediana EdadRESUMEN
The regulation of lipid homeostasis by insulin is mediated in part by the enhanced transcription of the gene encoding SREBP-1c (sterol regulatory element-binding protein-1c). Nascent SREBP-1c is synthesized and embedded in the endoplasmic reticulum (ER) and must be transported to the Golgi in coatomer protein II (COPII) vesicles where two sequential cleavages generate the transcriptionally active NH(2)-terminal fragment, nSREBP-1c. There is limited indirect evidence to suggest that insulin may also regulate the posttranslational processing of the nascent SREBP-1c protein. Therefore, we designed experiments to directly assess the action of insulin on the post-translational processing of epitope-tagged full-length SREBP-1c and SREBP-2 proteins expressed in cultured hepatocytes. We demonstrate that insulin treatment led to enhanced post-translational processing of SREBP-1c, which was associated with phosphorylation of ER-bound nascent SREBP-1c protein that increased affinity of the SREBP-1c cleavage-activating protein (SCAP)-SREBP-1c complex for the Sec23/24 proteins of the COPII vesicles. Furthermore, chemical and molecular inhibitors of the phosphoinositide 3-kinase pathway and its downstream kinase protein kinase B (PKB)/Akt prevented both insulin-mediated phosphorylation of nascent SREBP-1c protein and its posttranslational processing. Insulin had no effect on the proteolysis of nascent SREBP-2 under identical conditions. We also show that in vitro incubation of an active PKB/Akt enzyme with recombinant full-length SREBP-1c led to its phosphorylation. Thus, insulin selectively stimulates the processing of SREBP-1c in rat hepatocytes by enhancing the association between the SCAP-SREBP-1c complex and COPII proteins and subsequent ER to Golgi transport and proteolytic cleavage. This effect of insulin is tightly linked to phosphoinositide 3-kinase and PKB/Akt-dependent serine phosphorylation of the precursor SREBP-1c protein.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Hepatocitos/metabolismo , Hipoglucemiantes/farmacología , Insulina/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Vesículas Cubiertas por Proteínas de Revestimiento/genética , Línea Celular , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Hipoglucemiantes/metabolismo , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
Dietary triacylglycerols are absorbed by enterocytes and packaged in the endoplasmic reticulum (ER) in the intestinal specific lipoprotein, the chylomicron, for export into mesenteric lymph. Chylomicrons exit the ER in an ER-to-Golgi transport vesicle, the pre-chylomicron transport vesicle (PCTV), which is the rate-limiting step in the transit of chylomicrons across the cell. Here, we focus on potential mechanisms of control of the PCTV-budding step from the intestinal ER. We incubated intestinal ER with intestinal cytosol and ATP to cause PCTV budding. The budding reaction was inhibited by 60 nM of the PKC inhibitor Gö 6983, suggesting the importance of PKCzeta in the generation of PCTV. Immunodepletion of PKCzeta from the cytosol and the use of washed ER greatly inhibited the generation of PCTVs, but was restored following the addition of recombinant PKCzeta. Intestinal ER incubated with intestinal cytosol and [gamma-(32)P]ATP under conditions supporting the generation of PCTVs showed the phosphorylation of a 9-kDa band following autoradiography. The phosphorylation of this protein correlated with the generation of PCTVs but not the formation of protein vesicles and was inhibited by depletion of PKCzeta. Phosphorylation of the 9-kDa protein was restored following the addition of recombinant PKCzeta. The association of the 9-kDa protein with proteins that are important for PCTV budding was phosphorylation dependent. We conclude that PKCzeta activity is required for PCTV budding from intestinal ER, and is associated with phosphorylation of a 9-kDa protein that might regulate PCTV budding.
Asunto(s)
Quilomicrones/metabolismo , Exocitosis , Proteína Quinasa C/metabolismo , Vesículas Transportadoras/enzimología , Animales , Apolipoproteína B-48/metabolismo , Citosol/efectos de los fármacos , Citosol/enzimología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/enzimología , Activadores de Enzimas/farmacología , Exocitosis/efectos de los fármacos , Proteínas de Unión a Ácidos Grasos/metabolismo , Intestinos/citología , Intestinos/enzimología , Peso Molecular , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas R-SNARE/metabolismo , Ratas , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , Vesículas Transportadoras/efectos de los fármacosRESUMEN
Research in dietary fat absorption has developed urgency because of the widely recognized epidemic of obesity in the United States. Despite its clinical importance, many controversies exist over some of the basic aspects of this process from the mechanisms of fatty acid uptake to the control of triacylglycerol export in chylomicrons. Recent advances have included the identification of a number of fatty acid transporters, the discovery of families of acyl-CoA synthetase long chains and acyltransferases, a physiological function for liver-fatty acid binding protein, and the characterization of the prechylomicron transport vesicle transporting chylomicrons from the endoplasmic reticulum to the Golgi.
Asunto(s)
Quilomicrones/fisiología , Grasas de la Dieta/metabolismo , Absorción Intestinal/fisiología , Metabolismo de los Lípidos , Animales , Quilomicrones/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Triglicéridos/biosíntesisRESUMEN
The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.
Asunto(s)
Quilomicrones/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Resinas Acrílicas/farmacología , Animales , Transporte Biológico , Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Intestinal dietary triacylglycerol absorption is a multi-step process. Triacylglycerol exit from the endoplasmic reticulum (ER) is the rate-limiting step in the progress of the lipid from its apical absorption to its basolateral membrane export. Triacylglycerol is transported from the ER to the cis Golgi in a specialized vesicle, the pre-chylomicron transport vesicle (PCTV). The vesicle-associated membrane protein 7 (VAMP7) was found to be more concentrated on PCTVs compared with ER membranes. VAMP7 has been previously identified associated with post-Golgi sites in eukaryotes. To examine the potential role of VAMP7 in PCTV trafficking, antibodies were generated that identified a 25 kDa band consistent with VAMP7 but did not crossreact with VAMP1,2. VAMP7 was concentrated on intestinal ER by immunofluorescence microscopy. Immunoelectron microscopy showed that the ER proteins Sar1 and rBet1 were present on PCTVs and colocalized with VAMP7. Iodixanol gradient centrifugation showed VAMP7 to be isodense with ER and endosomes. Although VAMP7 localized to intestinal ER, it was not present in the ER of liver and kidney. Anti-VAMP7 antibodies reduced the transfer of triacylglycerol, but not newly synthesized proteins, from the ER to the Golgi by 85%. We conclude that VAMP7 is enriched in intestinal ER and that it plays a functional role in the delivery of triacylglycerol from the ER to the Golgi.
Asunto(s)
Retículo Endoplásmico/metabolismo , Intestino Delgado/metabolismo , Proteínas R-SNARE/biosíntesis , Animales , Anticuerpos/metabolismo , Transporte Biológico/fisiología , Quilomicrones/metabolismo , Aparato de Golgi/metabolismo , Técnicas In Vitro , Intestino Delgado/citología , Proteínas R-SNARE/fisiología , Ratas , Triglicéridos/metabolismoRESUMEN
Dietary long chain fatty acids are absorbed in the intestine, esterified to triacylglycerol, and packaged in the unique lipoprotein of the intestine, the chylomicron. The rate-limiting step in the transit of chylomicrons through the enterocyte is the exit of chylomicrons from the endoplasmic reticulum in prechylomicron transport vesicles (PCTV) that transport chylomicrons to the cis-Golgi. Because chylomicrons are 250 nm in average diameter and lipid absorption is intermittent, we postulated that a unique SNARE pairing would be utilized to fuse PCTV with their target membrane, cis-Golgi. PCTV loaded with [(3)H]triacylglycerol were incubated with cis-Golgi and were separated from the Golgi by a sucrose step gradient. PCTV-chylomicrons acquire apolipoprotein-AI (apoAI) only after fusion with the Golgi. PCTV became isodense with Golgi upon incubation and were considered fused when their cargo chylomicrons acquired apoAI but docked when they did not. PCTV, docked with cis-Golgi, were solubilized in 2% Triton X-100, and proteins were immunoprecipitated using VAMP7 or rBet1 antibodies. In both cases, a 112-kDa complex was identified in nonboiled samples that dissociated upon boiling. The constituents of the complex were VAMP7, syntaxin 5, vti1a, and rBet1. Antibodies to each SNARE component significantly inhibited fusion of PCTV with cis-Golgi. Membrin, Sec22b, and Ykt6 were not found in the 112-kDa complex. We conclude that the PCTV-cis-Golgi SNARE complex is composed of VAMP7, syntaxin 5, Bet1, and vti1a.
Asunto(s)
Quilomicrones/química , Aparato de Golgi/metabolismo , Proteínas SNARE/química , Animales , Retículo Endoplásmico/metabolismo , Intestino Delgado/metabolismo , Proteínas Qa-SNARE/química , Proteínas Qb-SNARE/química , Proteínas Qc-SNARE/química , Proteínas R-SNARE/química , Ratas , Triglicéridos/químicaRESUMEN
Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.
Asunto(s)
Apolipoproteínas A/biosíntesis , Quilomicrones/química , Intestinos/citología , Lípidos/química , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Apolipoproteínas/química , Apolipoproteínas A/fisiología , Western Blotting , Línea Celular , Pollos , Clonación Molecular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Doxiciclina/metabolismo , Doxiciclina/farmacología , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoprecipitación , Mucosa Intestinal/metabolismo , Metabolismo de los Lípidos , Lipoproteínas/metabolismo , Microscopía Electrónica , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Mutación , Ácido Oléico/química , Ácido Oléico/metabolismo , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Tetraciclina/farmacología , Activación Transcripcional , Triglicéridos/metabolismoRESUMEN
Following digestion of dietary triacylglycerol (TAG), intestinal epithelial cells absorb fatty acids and monoacylglycerols that are resynthesized into TAG by enzymes located on the endoplasmic reticulum (ER). A study in rat liver (Abo-Hashema, K. A., M. H. Cake, G. W. Power, and D. J. Clarke. 1999. Evidence for TAG synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases. J. Biol. Chem. 274: 35577-35582) showed that there is a carnitine-dependent ER lumenal synthesis of TAG. We wanted to test the hypothesis that a similar pathway was present in rat intestine by utilizing etomoxir, a specific inhibitor of carnitine palmitoyltransferase (CPT). Intraduodenal infusion of etomoxir inhibited CPT activity in the ER by 69%. Etomoxir did not affect either the uptake of intraduodenally infused [3H]glyceryltrioleate by the intestinal mucosa or the production of mucosal [3H]TAG, excluding the possibility that etomoxir interfered with TAG absorption or synthesis. Etomoxir did not inhibit protein synthesis, glucose, cholesterol or palmitate absorption or metabolism, or ATP concentrations. Etomoxir substantially (74%) diminished lymph TAG output from intralumenally infused glyceryltrioleate. In conclusion, these data strongly support the hypothesis that an ER CPT system exists and is necessary for processing dietary TAG into chylomicrons. The significant reduction in lymphatic output of chylomicron TAG on etomoxir treatment suggests that the major source of chylomicron TAG is a diacylglyceroltransferase on the lumenal surface of the ER.
Asunto(s)
Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Intestino Delgado/enzimología , Linfa/metabolismo , Triglicéridos/metabolismo , Animales , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/farmacología , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Microsomas/metabolismo , Modelos Biológicos , Ratas , Factores de TiempoRESUMEN
Many advances in the study of nutrient absorption have been made with the use of molecular and genetic techniques; however, standard in vivo studies have provided interesting and important new information. Omega-3 long-chain fatty acids have unexpected effects on lipoprotein formation and secretion in neonatal intestinal cells; this needs to be considered in the modification of infant formulas. Rexinoids affect intestinal cholesterol homeostasis via two receptors: retinoic acid receptor/liver X receptor (cholesterol efflux to lumen) and retinoic acid receptor/farnesoid X receptor (cholesterol catabolism). Absorption of the antioxidant plant polyphenol quercetin involves interaction with the glucose transporter and deglycolsylation and conjugation reactions. Cells of the polarized human colon cancer cell line, CaCo-2, take up phenylalanine by two mechanisms: passive uptake across the basolateral membrane, and temperature-dependent transcellular movement from apical to basolateral media. Absorption of vitamins A and E is markedly enhanced in normal and damaged intestine by the administration of restructured triacylglycerols derived from fish oil and medium-chain fatty acids. Surprisingly, dietary protein and phosphorus apparently have no significant effect on the efficiency of calcium absorption in adult women. Finally, many studies examined a variety of genes that regulate iron absorption and homeostasis.
RESUMEN
The budding of vesicles from endoplasmic reticulum (ER) that contains nascent proteins is regulated by COPII proteins. The mechanisms that regulate lipid-carrying pre-chylomicron transport vesicles (PCTVs) budding from the ER are unknown. To study the dependence of PCTV-ER budding on COPII proteins we examined protein and PCTV budding by using ER prepared from rat small intestinal mucosal cells prelabeled with (3)H-oleate or (14)C-oleate and (3)H-leucine. Budded (3)H-oleate-containing PCTVs were separated by sucrose density centrifugation and were revealed by electron microscopy as 142-500 nm vesicles. Our results showed the following: (1) Proteinase K treatment did not degrade the PCTV cargo protein, apolipoprotein B-48, unless Triton X-100 was added. (2) PCTV budding was dependent on cytosol and ATP. (3) The COPII proteins Sar1, Sec24 and Sec13/31 and the membrane proteins syntaxin 5 and rBet1 were associated with PCTVs. (4) Isolated PCTVs were able to fuse with intestinal Golgi. (5) Antibodies to Sar1 completely inhibited protein vesicle budding but increased the generation of PCTV; these changes were reversed by the addition of recombinant Sar1. (6) PCTVs formed in the absence of Sar1 did not contain the COPII proteins Sar1, Sec24 or Sec31 and did not fuse with the Golgi complex. Together, these findings suggest that COPII proteins may not be required for the exit of membrane-bound chylomicrons from the ER but that they or other proteins may be necessary for PCTV fusion with the Golgi.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Quilomicrones/metabolismo , Retículo Endoplásmico/metabolismo , Células Epiteliales/metabolismo , Aparato de Golgi/metabolismo , Mucosa Intestinal/metabolismo , Fusión de Membrana/genética , Animales , Anticuerpos , Apolipoproteína B-48 , Apolipoproteínas B/efectos de los fármacos , Apolipoproteínas B/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/ultraestructura , Células Cultivadas , Quilomicrones/ultraestructura , Retículo Endoplásmico/ultraestructura , Células Epiteliales/ultraestructura , Aparato de Golgi/ultraestructura , Absorción Intestinal/genética , Mucosa Intestinal/ultraestructura , Metabolismo de los Lípidos , Masculino , Microscopía Electrónica , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Transporte de Proteínas/fisiología , Ratas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte VesicularRESUMEN
To define the developmental expression of microsomal triglyceride transfer protein (MTP) large subunit mRNA and protein, samples of small intestine and liver were collected from 40-day gestation fetal, 2-day-old newborn, 3-week-old suckling, and 2-month-old weanling swine. In fetal animals, MTP mRNA expression was high in intestine and liver. Postnatally, jejunal expression paralleled the intake of a high-fat breast milk diet and declined after weaning. Ileal expression was comparable with that of jejunum in 2-day-old animals, but declined to low levels afterward. Hepatic expression declined postnatally and remained low. MTP protein expression generally paralleled mRNA expression, except in fetal intestine in which no 97 kDa protein was detected. In 2-day-old piglets, a high-triacylglycerol diet increased jejunal and ileal MTP mRNA levels, as compared to a low-triacylglycerol diet. To test the roles of glucocorticoids and fatty acids in MTP regulation, a newborn swine enterocyte cell line (IPEC-1) was used. Except at day 2 of differentiation, dexamethasone did not influence MTP expression. Fatty acids either up-regulated or down-regulated MTP expression, depending on the specific fatty acid and duration of exposure. Although programmed genetic cues regulate MTP expression during development, clearly the amount and fatty acid composition of dietary lipid also play regulatory roles.