Hepatic very low density lipoprotein-ApoB overproduction is associated with attenuated hepatic insulin signaling and overexpression of protein-tyrosine phosphatase 1B in a fructose-fed hamster model of insulin resistance

A fructose-fed hamster model of insulin resistance was previously documented to exhibit marked hepatic very low density lipoprotein (VLDL) overproduction. Here, we investigated whether VLDL overproduction was associated with down-regulation of hepatic insulin signaling and insulin resistance. Hepatocytes isolated from fructose-fed hamsters exhibited significantly reduced tyrosine phosphorylation of the insulin receptor and insulin receptor substrates 1 and 2. Phosphatidylinositol 3-kinase activity as well as insulin-stimulated Akt-Ser473 and Akt-Thr308 phosphorylation were also significantly reduced with fructose feeding. Interestingly, the protein mass and activity of protein-tyrosine phosphatase-1B (PTP-1B) were significantly higher in fructose-fed hamster hepatocytes. Chronic ex vivo exposure of control hamster hepatocytes to high insulin also appeared to attenuate insulin signaling and increase PTP-1B. Elevation in PTP-1B coincided with marked suppression of ER-60, a cysteine protease postulated to play a role in intracellular apoB degradation, and an increase in the synthesis and secretion of apoB. Sodium orthovanadate, a general phosphatase inhibitor, partially restored insulin receptor phosphorylation and significantly reduced apoB secretion. In summary, we hypothesize that fructose feeding induces hepatic insulin resistance at least in part via an increase in expression of PTP-1B. Induction of hepatic insulin resistance may then contribute to reduced apoB degradation and enhanced VLDL particle assembly and secretion.     

Tumor necrosis factor-alpha directly stimulates the overproduction of hepatic apolipoprotein B100-containing VLDL via impairment of hepatic insulin signaling

Insulin-resistant states are commonly associated with both increased circulating levels of tumor necrosis factor (TNF)-alpha and hepatic overproduction of very low density lipoproteins (VLDL). Here, we provide evidence that increased TNF-alpha can directly stimulate the hepatic assembly and secretion of apolipoprotein B (apoB) 100-containing VLDL(1), using the Syrian golden hamster, an animal model that closely resembles humans in hepatic VLDL-apoB100 metabolism. In vivo TNF-alpha infusion for 4 h in chow-fed hamsters induced whole-body insulin resistance on the basis of euglycemic hyperinsulinemic clamp studies. Immunoprecipitation and immunoblotting analysis of livers from TNF-alpha-treated hamsters indicated decreased tyrosine phosphorylation of insulin receptor (IR)-beta, IR substrate-1 (Tyr), Akt (Ser(473)), p38, ERK1/2, and JNK but increased serine phosphorylation of IRS-1 (Ser(307)) and Shc. TNF-alpha infusion also significantly increased hepatic production of total circulating apoB100 and VLDL-apoB100 in both fasting and postprandial (fat load) states. Ex vivo experiments, using cultured primary hepatocytes from hamsters, also showed TNF-alpha-induced VLDL-apoB100 oversecretion, an effect that was blocked by TNF receptor 2 antibody. Unexpectedly, TNF-alpha decreased the sterol regulatory element-binding protein-1c mass and mRNA levels but significantly increased microsomal triglyceride transfer protein mass and mRNA levels in primary hepatocytes. In summary, these data provide direct evidence that TNF-alpha induces whole-body insulin resistance and impairs hepatic insulin signaling accompanied by overproduction of apoB100-containing VLDL particles, an effect likely mediated via TNF receptor 2.     

Fasting and postprandial overproduction of intestinally derived lipoproteins in an animal model of insulin resistance. Evidence that chronic fructose feeding in the hamster is accompanied by enhanced intestinal de novo lipogenesis and ApoB48-containing lipoprotein overproduction

Insulin-resistant states are characterized by hypertriglyceridemia, predominantly because of overproduction of hepatic very low density lipoprotein particles. The additional contribution of intestinal lipoprotein overproduction to the dyslipidemia of insulin-resistant states has not been previously appreciated. Here, we have investigated intestinal lipoprotein production in a fructose-fed hamster model of insulin resistance previously documented to have whole body and hepatic insulin resistance, and hepatic very low density lipoprotein overproduction. Chronic fructose feeding for 3 weeks induced significant oversecretion of apolipoprotein B48 (apoB48)-containing lipoproteins in the fasting state and during steady state fat feeding, based on (a) in vivo Triton WR1339 studies of apoB48 production as well as (b) ex vivo pulse-chase labeling of intestinal enterocytes from fasted and fed hamsters. ApoB48 particle overproduction was accompanied by increased intracellular apoB48 stability, enhanced lipid synthesis, higher abundance of microsomal triglyceride transfer protein mass, and a significant shift toward the secretion of larger chylomicron-like particles. ApoB48 particle overproduction was not observed with short-term fructose feeding or in vitro incubation of enterocytes with fructose. Secretion of intestinal apoB48 and triglyceride was closely linked to intestinal enterocyte de novo lipogenesis, which was up-regulated in fructose-fed hamsters. Inhibition of fatty acid synthesis by cerulenin, a fatty acid synthase inhibitor, resulted in a dose-dependent decrease in intestinal apoB48 secretion. Overall, these findings further suggest that intestinal overproduction of apoB48 lipoproteins should also be considered as a major contributor to the fasting and postprandial dyslipidemia observed in response to chronic fructose feeding and development of an insulin-resistant state.     

Ameliorated hepatic insulin resistance is associated with normalization of microsomal triglyceride transfer protein expression and reduction in very low density lipoprotein assembly and secretion in the fructose-fed hamster

To determine whether reduction of insulin resistance could ameliorate fructose-induced very low density lipoprotein (VLDL) oversecretion and to explore the mechanism of this effect, fructose-fed hamsters received placebo or rosiglitazone for 3 weeks. Rosiglitazone treatment led to normalization of the blunted insulin-mediated suppression of the glucose production rate and to a approximately 2-fold increase in whole body insulin-mediated glucose disappearance rate (p < 0.001). Rosiglitazone ameliorated the defect in hepatocyte insulin-stimulated tyrosine phosphorylation of the insulin receptor, IRS-1, and IRS-2 and the reduced protein mass of IRS-1 and IRS-2 induced by fructose feeding. Protein-tyrosine phosphatase 1B levels were increased with fructose feeding and were markedly reduced by rosiglitazone. Rosiglitazone treatment led to a approximately 50% reduction of VLDL secretion rates (p < 0.05) in vivo and ex vivo. VLDL clearance assessed directly in vivo was not significantly different in the FR (fructose-fed + rosiglitazone-treated) versus F (fructose-fed + placebo-treated) hamsters, although there was a trend toward a lower clearance with rosiglitazone. Enhanced stability of nascent apolipoprotein B (apoB) in fructose-fed hepatocytes was evident, and rosiglitazone treatment resulted in a significant reduction in apoB stability. The increase in intracellular mass of microsomal triglyceride transfer protein seen with fructose feeding was reduced by treatment with rosiglitazone. In conclusion, improvement of hepatic insulin signaling with rosiglitazone, a peroxisome proliferator-activated receptor gamma agonist, is associated with reduced hepatic VLDL assembly and secretion due to reduced intracellular apoB stability.     

Cinnamon extract inhibits the postprandial overproduction of apolipoprotein B48-containing lipoproteins in fructose-fed animals

We have reported previously that a cinnamon extract (CE), high in type A polyphenols, prevents fructose feeding-induced decreases in insulin sensitivity and suggested that improvements of insulin sensitivity by CE were attributable, in part, to enhanced insulin signaling. In this study, we examined the effects of CE on postprandial apolipoprotein (apo) B-48 increase in fructose-fed rats, and the secretion of apoB48 in freshly isolated intestinal enterocytes of fructose-fed hamsters. In an olive oil loading study, a water-soluble CE (Cinnulin PF, 50 mg/kg body weight, orally) decreased serum triglyceride (TG) levels and the over production of total- and TG-rich lipoprotein-apoB48. In ex vivo ³⁵S labeling study, significant decreases were also observed in apoB48 secretion into the media in enterocytes isolated from fructose-fed hamsters. We also investigated the molecular mechanisms of the effects of CE on the expression of genes of the insulin signaling pathway [insulin receptor (IR), IR substrate (IRS)1, IRS2 and Akt1], and lipoprotein metabolism [microsomal TG transfer protein (MTP), sterol regulatory element-binding protein (SREBP1c) in isolated primary enterocytes of fructose-fed hamsters, using quantitative real-time polymerase chain reaction. The CE reversed the expression of the impaired IR, IRS1, IRS2 and Akt1 mRNA levels and inhibited the overexpression of MTP and SREBP1c mRNA levels of enterocytes. Taken together, our data suggest that the postprandial hypertriglycerides and the overproduction of apoB48 can be acutely inhibited by a CE by a mechanism involving improvements of insulin sensitivity of intestinal enterocytes and regulation of MTP and SREBP1c levels. We present both in vivo and ex vivo evidence that a CE improves the postprandial overproduction of intestinal apoB48-containing lipoproteins by ameliorating intestinal insulin resistance and may be beneficial in the control of lipid metabolism.     

High-carbohydrate diets affect the size and composition of plasma lipoproteins in hamsters (Mesocricetus auratus)

High-carbohydrate diets reduce plasma low-density lipoprotein (LDL)-cholesterol but also provoke the appearance of an atherogenic lipoprotein profile (ALP). Characterized by high plasma triglyceride, small dense LDL, and reduced high-density lipoprotein (HDL) cholesterol, an ALP is associated with insulin resistance. Despite extensive use of the fructose-fed hamster as a model of insulin resistance, little is known about changes that occur in the physical properties of circulating lipoproteins. Therefore, we investigated the metabolic and physical properties of lipoproteins in hamsters fed high-carbohydrate diets of varying complexity (60% carbohydrate as chow, cornstarch, or fructose) for 2 wk. Hamsters fed the high-fructose diet showed significantly increased very- low-density lipoprotein (VLDL)-triglyceride (92.3%), free cholesterol (68.6%), and phospholipid (95%), whereas apolipoprotein B levels remained unchanged. Median diameter of circulating VLDL was larger in fructose-fed hamsters (63 nm) than in cornstarch-fed hamsters. Fructose feeding induced a 42.5% increase LDL-triglyceride concurrent with a 20% reduction in LDL-cholesteryl ester. Compositional changes were associated with reduced LDL diameter. In contrast, fructose feeding caused elevations in all HDL fractions. The physical properties of apolipoprotein-B-containing lipoprotein fractions are similar between fructose-fed hamsters and humans with ALP. However, metabolism of high-density lipoprotein appears to differ in the 2 species.     

Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice

In addition to its role in the uptake of apolipoprotein B (apoB)-containing lipoproteins, apoE promotes hepatic very low density lipoprotein-triglyceride (VLDL-TG) production in animal models. However, it is not known if apoE increases the amount of TG per VLDL particle or the number of VLDL particles secreted. VLDL-apoB production is a measure of the rate of VLDL particle secretion. We determined the effects of apoE deficiency and apoE overexpression on VLDL-apoB production in mice. [(35)S]methionine was injected into endogenously label VLDL-apoB and Triton WR-1339 was simultaneously injected to block the catabolism of VLDL. Compared with wild-type mice, the VLDL-apoB production rate was decreased by 33% in apoE-deficient mice. Conversely, VLDL-apoB production was increased by 48% in mice overexpressing apoE compared with controls. Nascent VLDL, obtained from post-Triton plasma, had a decreased, not increased, content of TG per apoB in the apoE-overexpressing group compared with the control group. This study demonstrates that hepatic apoE expression increases the output of VLDL triglyceride by increasing the production rate of VLDL-apoB, suggesting that hepatic apoE influences the number of VLDL particles secreted by the liver.     

Links between enhanced fatty acid flux, protein kinase C and NFkappaB activation, and apoB-lipoprotein production in the fructose-fed hamster model of insulin resistance

In the current study, we show evidence, in a fructose-fed hamster model of insulin resistance, that free fatty acid (FFA) can induce hepatic insulin resistance in part via PKC activation leading to increased production of atherogenic apoB100-containing lipoproteins. Interestingly, IκB-kinase β (IKKβ)-dependent NF-κB was activated in hepatocytes from the fructose-fed hamster as an indication for PKC activation. Treatment of hepatocytes with oleate for 16 h showed the activation of the PKC isoforms, PKCα/βII, in a dose dependent manner. Strikingly, the general PKC inhibitor, bisindolylmaleimide-I, Bis-I (5 μM) was found to ameliorate fructose-induced insulin resistance, restoring the phosphorylation status of PKB and suppressing apoB100 overproduction in ex vivo and in vivo. The data suggest that hepatic PKC activation, induced by increased circulating FFA may be an important factor in the development of insulin resistance and dyslipidemia seen in the fructose-fed hamster model.     

Proteomic profiling of intestinal prechylomicron transport vesicle (PCTV)-associated proteins in an animal model of insulin resistance (94 char)

Intestinal overproduction of apolipoprotein B (apoB)-48-containing chylomicrons is increasingly recognized as an underlying factor in metabolic dyslipidemia commonly observed in insulin-resistant states. Enhanced chylomicron assembly and secretion has been documented in animal models of insulin resistance, but the underlying mechanistic factors are unknown. Chylomicron assembly occurs through a series of complex vesicular interactions involving prechylomicron transport vesicles (PCTVs), which transport lipids from the endoplasmic reticulum (ER) to the Golgi. We report proteomic profiles of PCTVs isolated from the enteric ER in the small intestine of the fructose-fed hamster, an established model of diet-induced insulin resistance. Using 2D gel electrophoresis and tandem mass spectrometry, PCTVs were characterized and proteomic profiles of PCTV-associated proteins from insulin-resistant and control enterocytes were developed, with the intention of identifying proteins involved in insulin signaling attenuation and lipoprotein overproduction. A number of PCTV-associated proteins were found to be differentially expressed including microsomal triglyceride transfer protein (MTP), apoB-48, Sar1 and VAMP7. We postulate that altered expression of Sar1 and MTP may contribute to increased chylomicron assembly in the fructose-fed hamster. These findings have increased our understanding of the intracellular assembly and transport of nascent chylomicrons and potential cellular factors responsible for lipoprotein overproduction in insulin-resistant states.     

Intracellular mechanisms regulating apoB-containing lipoprotein assembly and secretion in primary hamster hepatocytes

We studied the biogenesis of apolipoprotein B (apoB) in primary hepatocytes isolated from hamster liver, an animal model with striking resemblance to humans in lipoprotein metabolism. Hamster hepatocytes were found to assemble and secrete apoB-containing lipoproteins at a density of VLDL. Intracellular mechanisms of apoB biogenesis were investigated in both intact and permeabilized hamster hepatocytes. Translocational status of hamster apoB-100 was examined using trypsin protection assays in permeabilized cells as well as isolated microsomes which revealed that 27-42% of newly synthesized apoB was trypsin accessible as opposed to a control protein, transferrin, which was found to be essentially insensitive to exogenous trypsin. Subcellular fractionation of membrane and lumenal apoB pools indicated, however, that only a minor fraction of hamster apoB was associated with the microsomal membrane. Approximately 40% of newly synthesized apoB was found to be degraded post-translationally in a process sensitive to MG132. Immunoblotting analysis of apoB immunoprecipitates revealed ubiquitination of hamster apoB suggesting the involvement of the proteasome in its intracellular turnover. In addition to MG132, o-phenanthroline, a metalloprotease inhibitor, was also effective in stabilizing hamster apoB. Experiments in permeabilized hamster hepatocytes further confirmed post-translational instability of hamster apoB which was degraded over a 3-h chase generating proteolytic fragments including 167, 70, 57, and 46 kDa intermediates. Of these only the 70 kDa fragment was ALLN sensitive. Oleate treatment of hamster hepatocytes provided protection against intracellular apoB degradation, but did not stimulate its extracellular secretion. ApoB was assembled in the microsomal lumen into lipoprotein particles with densities of LDL and VLDL which were subsequently secreted as VLDL with a minor fraction forming HDL-like particles. In summary, hamster hepatocytes appear to efficiently assemble and secrete apoB-containing VLDL, although a significant pool of newly synthesized apoB is retained intracellularly and becomes sensitive to proteasome-mediated degradation as well as other proteases in the secretory pathway, generating specific degradative intermediates.     

Inhibition of fatty acid synthesis decreases very low density lipoprotein secretion in the hamster.

The hamster was developed as a model to study very low density lipoprotein (VLDL) metabolism, since, as is the case in humans, the hamster liver was found to synthesize apoB-100 and not apoB-48. The effect of inhibiting fatty acid synthesis on the hepatic secretion of VLDL triglyceride (TG) and apolipoprotein (apo) B-100 in this model was then investigated. In an in vivo study, hamsters were fed a chow diet containing 0.15% TOFA (5-tetradecyloxy-2-furancarboxylic acid), an inhibitor of acetyl-CoA carboxylase. After 6 days of treatment, plasma triglyceride and cholesterol levels were decreased by 30.2% and 11.6%, respectively. When the secretion of VLDL-TG by the liver was measured in vivo after injection of Triton WR 1339, TOFA treatment was found to decrease VLDL-TG secretion by 40%. In subsequent in vitro studies utilizing cultured primary hamster hepatocytes, incubation with 20 microM TOFA for 4 h resulted in 98% and 76% inhibition in fatty acid and triglyceride synthesis, respectively; VLDL-TG secretion was decreased by 90%. When hepatocytes were pulsed with [3H]leucine, incubation with TOFA resulted in a 50% decrease in the incorporation of radiolabel into secreted VLDL apoB-100. The results of this study indicate that inhibition of intracellular triglyceride synthesis decreases the secretion of VLDL-TG and apoB-100, and does not result in the secretion of a dense, triglyceride-depleted lipoprotein.     

Assembly and secretion of chylomicrons by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly.

To develop a cell culture model for chyclomicron (CM) assembly, the apical media of differentiated Caco-2 cells were supplemented with oleic acid (OA) together with either albumin or taurocholate (TC). The basolateral media were subjected to sequential density gradient ultracentrifugations to obtain large CM, small CM, and very low density lipoproteins (VLDL), and the distribution of apoB in these fractions was quantified. In the absence of OA, apoB was secreted as VLDL/LDL size particles. Addition of OA (>/=0.8 mM) with TC, but not with albumin, resulted in the secretion of one-third of apoB as CM. Lipid analysis revealed that half of the secreted phospholipids (PL) and triglycerides (TG) were associated with CM. In CM, TG were 7-11-fold higher than PL indicating that CM were TG-rich particles. Secreted CM contained apoB100, apoB48, and other apolipoproteins. Secretion of large CM was specifically inhibited by Pluronic L81, a detergent known to inhibit CM secretion in animals. These studies demonstrate that differentiated Caco-2 cells assemble and secrete CM in a manner similar to enterocytes in vivo. Next, experiments were performed to identify the sources of lipids used for lipoprotein assembly. Cells were labeled with [3H]glycerol for 12 h, washed, and supplemented with OA, TC, and [14C] glycerol for various times to induce CM assembly and to radiolabel nascent lipids. TG and PL were extracted from cells and media and the association of preformed and nascent lipids with lipoproteins was determined. All the lipoproteins contained higher amounts of preformed PL compared with nascent PL. VLDL contained equal amounts of nascent and preformed TG, whereas CM contained higher amounts of nascent TG even when nascent TG constituted a small fraction of the total cellular pool. These studies indicate that nascent TG and preformed PL are preferentially used for CM assembly and provide a molecular explanation for the in vivo observations that the fatty acid composition of TG, but not PL, of secreted CM reflects the composition of dietary fat. It is proposed that in the intestinal cells the preformed PL from the endoplasmic reticulum bud off with apoB as primordial particles and the assembly of larger lipoproteins is dependent on the synthesis and delivery of nascent TG to these particles.     

Proteomic profiling of hepatic endoplasmic reticulum-associated proteins in an animal model of insulin resistance and metabolic dyslipidemia

Hepatic insulin resistance and lipoprotein overproduction are common features of the metabolic syndrome and insulin-resistant states. A fructose-fed, insulin-resistant hamster model was recently developed to investigate mechanisms linking the development of hepatic insulin resistance and overproduction of atherogenic lipoproteins. Here we report a systematic analysis of protein expression profiles in the endoplasmic reticulum (ER) fractions isolated from livers of fructose-fed hamsters with the intention of identifying new candidate proteins involved in hepatic complications of insulin resistance and lipoprotein dysregulation. We have profiled hepatic ER-associated proteins from chow-fed (control) and fructose-fed (insulin-resistant) hamsters using two-dimensional gel electrophoresis and mass spectrometry. A total of 26 large scale two-dimensional gels of hepatic ER were used to identify 34 differentially expressed hepatic ER protein spots observed to be at least 2-fold differentially expressed with fructose feeding and the onset of insulin resistance. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-quadrupole time of flight (MALDI-Q-TOF), MALDI-TOF-postsource decay, and database mining using ProteinProspector MS-fit and MS-tag or the PROWL ProFound search engine using a focused rodent or mammalian search. Hepatic ER proteins ER60, ERp46, ERp29, glutamate dehydrogenase, and TAP1 were shown to be more than 2-fold down-regulated, whereas alpha-glucosidase, P-glycoprotein, fibrinogen, protein disulfide isomerase, GRP94, and apolipoprotein E were all found to be up-regulated in the hepatic ER of the fructose-fed hamster. Seven isoforms of ER60 in the hepatic ER were all shown to be down-regulated at least 2-fold in hepatocytes from fructosefed/insulin-resistant hamsters. Implications of the differential expression of positively identified protein factors in the development of hepatic insulin resistance and lipoprotein abnormalities are discussed.     

ApoB100 is required for increased VLDL-triglyceride secretion by microsomal triglyceride transfer protein in ob/ob mice

Microsomal triglyceride transfer protein (Mttp) is a key player in the assembly and secretion of hepatic very low density lipoproteins (VLDL). Here we determined the effects of Mttp overexpression on hepatic triglyceride (TG) and VLDL secretion in leptin-deficient (ob/ob) mice, specifically in relation to apolipoproteinB (apoB) isoforms. We crossed Apobec1(-/-) mice with congenic ob/ob mice to generate apoB100-only ob/ob mice (A-ob/ob). The obesity phenotype in both genotypes was similar, but A-ob/ob mice had greater hepatic TG content. Administration of recombinant adenovirus expressing murine Mttp cDNA (Ad-mMTP) increased hepatic Mttp content and activity and increased hepatic VLDL-TG secretion in A-ob/ob mice. However, despite equivalent overexpression of Mttp, there was no change in VLDL-TG secretion in ob/ob mice in a wild-type Apobec1 background. Metabolic labeling studies in primary hepatocytes from A-ob/ob mice demonstrated that Ad-mMTP increased triglyceride secretion without changing the synthesis and secretion of apoB100, suggesting greater incorporation of TG into existing VLDL particles rather than increased particle number. Ad-mMTP administration failed to increase hepatic VLDL secretion in lean Apobec1(-/-) mice or controls. By contrast, VLDL secretion increased and hepatic TG content decreased following Ad-mMTP administration to human APOB transgenic mice crossed into the Apobec1(-/-) line. These findings demonstrate that Ad-mMTP increases murine hepatic VLDL-TG secretion only in the apoB100 background, and even then only in situations with either increased hepatic TG accumulation or increased apoB100 expression.     

Microsomal triacylglycerol transfer protein is required for lumenal accretion of triacylglycerol not associated with ApoB,as well as for ApoB lipidation

The assembly of very low density lipoproteins in hepatocytes requires the microsomal triacylglycerol transfer protein (MTP). This microsomal lumenal protein transfers lipids, particularly triacylglycerols (TG), between membranes in vitro and has been proposed to transfer TG to nascent apolipoprotein (apo) B in vivo. We examined the role of MTP in the assembly of apoB-containing lipoproteins in cultured murine primary hepatocytes using an inhibitor of MTP. The MTP inhibitor reduced TG secretion from hepatocytes by 85% and decreased the amount of apoB100 in the microsomal lumen, as well as that secreted into the medium, by 70 and 90%, respectively, whereas the secretion of apoB48 was only slightly decreased and the amount of lumenal apoB48 was unaffected. However, apoB48-containing particles formed in the presence of inhibitor were lipid-poor compared with those produced in the absence of inhibitor. We also isolated a pool of apoB-free TG from the microsomal lumen and showed that inhibition of MTP decreased the amount of TG in this pool by approximately 45%. The pool of TG associated with apoB was similarly reduced. However, inhibition of MTP did not directly block TG transfer from the apoB-independent TG pool to partially lipidated apoB in the microsomal lumen. We conclude that MTP is required for TG accumulation in the microsomal lumen and as a source of TG for assembly with apoB, but normal levels of MTP are not required for transferring the bulk of TG to apoB during VLDL assembly in murine hepatocytes.     

Hepatic overexpression of microsomal triglyceride transfer protein (MTP) results in increased in vivo secretion of VLDL triglycerides and apolipoprotein B.

The microsomal triglyceride transfer protein (MTP) is essential for the hepatic secretion of apolipoprotein (apo) B-containing lipoproteins. Previous studies have indicated that inhibition of MTP results in decreased apoB plasma levels and decreased hepatic triglyceride secretion. However, the metabolic effects of overexpression of MTP have not been investigated. We constructed a recombinant adenovirus expressing MTP (AdhMTP) and used it to assess the effects of hepatic overexpression of MTP in mice. Injection of AdhMTP into C57BL/6 mice resulted in a 3-fold increase in hepatic microsomal triglyceride transfer activity compared to mice injected with Adnull. On day 4 after virus injection, AdhMTP-injected mice had significantly elevated plasma TG levels as compared to control virus (Adnull)-injected mice. Hepatic TG secretion rates were significantly greater in AdhMTP-injected mice (184 +/- 12 mg/kg/h) compared with Adnull-injected mice (65 +/- 9 mg/kg/h, P < 0.001). In addition, hepatic very low density lipoprotein (VLDL) apoB secretion in the AdhMTP-injected group was 74% higher than in the control virus group. Hepatic secretion of apoB-48 and apoB-100 contributed equally to this increase.These results provide the first data that hepatic overexpression of MTP results in increased secretion of VLDL-triglycerides as well as VLDL-apoB in vivo. These results suggest that MTP is rate-limiting for VLDL apoB secretion in wild-type mice under basal chow-fed conditions.     

Octanoate reduces very low-density lipoprotein secretion by decreasing the synthesis of apolipoprotein B in primary cultures of chicken hepatocytes

Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B (apoB) levels. To identify the mechanisms underlying the effect of octanoate on very low-density lipoprotein (VLDL) secretion, chicken primary hepatocytes were incubated with either fatty acid-bovine serum albumin (BSA) complexes or BSA alone. Addition of octanoate to culture medium significantly reduced VLDL-triacylglycerol (TG), VLDL-cholesterol and apoB secretion from hepatocytes compared to both control cultures with BSA only and palmitate treatments, but did not modulate intracellular TG accumulation. However, no differences in cellular microsomal triglyceride transfer protein levels were observed in the cultures with saturated fatty acid. In pulse-chase studies, octanoate treatment resulted in reduced apoB-100 synthesis, in agreement with its promotion of secretion. This characteristic effect of octanoate was confirmed by addition of a protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN), to hepatocyte cultures. Analysis showed that the level of apoB mRNA was lower in cultures supplemented with octanoate than in the control cultures, but no significant changes were observed in the levels of apolipoprotein A-I, fatty acid synthase and 3-hydroxy-3-methylglutaryl-CoA reductase mRNA as a result of octanoate treatment. Time-course studies indicate that a 50% reduction in apoB mRNA levels requires 12 h of incubation with octanoate. We conclude that octanoate reduced VLDL secretion by the specific down-regulation of apoB gene expression and impairment of subsequent synthesis of apoB, not by the modulation of intracellular apoB degradation, which is known to be a major regulatory target of VLDL secretion of other fatty acids.     

Direct effects of growth hormone on production and secretion of apolipoprotein B from rat hepatocytes

The aim of this study was to investigate the direct effects of growth hormone (GH) on production and secretion of apolipoprotein B (apoB)-containing lipoproteins from hepatocytes. Bovine GH (5-500 ng/ml) was given for 1 or 3 days to rat hepatocytes cultured on laminin-rich matrigel in serum-free medium. The effects of GH were compared with those of 3 nM insulin and 500 microM oleic acid. GH increased the editing of apoB mRNA, and the proportion of newly synthesized apoB-48 (of total apoB) in the cells and secreted into the medium changed in parallel. GH increased total secretion of apoB-48 (+30%) and apoB-48 in very low density lipoproteins (VLDL) more than twofold. Total apoB-100 secretion decreased 63%, but apoB-100-VLDL secretion was unaffected by GH. Pulse-chase studies indicated that GH increased intracellular early degradation of apoB-100 but not apoB-48. GH had no effect on apoB mRNA or LDL receptor mRNA levels. The triglyceride synthesis, the mass of triglycerides in the cells, and the VLDL fraction of the medium increased after GH incubation. Three days of insulin incubation had effects similar to those of GH. Combined incubation with oleic acid and GH had additive effects on apoB mRNA editing and apoB-48-VLDL secretion. In summary, GH has direct effects on production and secretion of apoB-containing lipoproteins, which may add to the effects of hyperinsulinemia and increased flux of fatty acids to the liver during GH treatment in vivo.     

An unexpected requirement for phosphatidylethanolamine N-methyltransferase in the secretion of very low density lipoproteins

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine (PC). We investigated whether there was diminished secretion of lipoproteins from hepatocytes derived from mice that lacked PEMT (Pemt(-/-)) compared with Pemt(+/+) mice. Hepatocytes were incubated with 0.75 mm oleate, the media were harvested, and triacylglycerol (TG), PC, apolipoprotein (apo) B100, and apoB48 were isolated and quantified. Compared with hepatocytes from Pemt(+/+) mice, hepatocytes from Pemt(-/-) mice secreted 50% less TG, whereas secretion of PC was unaffected. Fractionation of the secreted lipoproteins on density gradients demonstrated that the decrease in TG was in the very low density lipoprotein (VLDL)/low density lipoprotein fractions. The secretion of apoB100 was decreased by approximately 70% in VLDLs/low density lipoproteins, whereas there was no significant decrease in apoB48 secretion in any fraction. Transfection of McArdle hepatoma cells (that lack PEMT) with PEMT cDNA enhanced secretion of TG in the VLDLs. Because the levels of PC in the hepatocytes and hepatoma cells were unaffected by the lack of PEMT expression, there appears to be an unexpected requirement for PEMT in the secretion of apoB100-containing VLDLs.     

Dietary fish oils inhibit early events in the assembly of very low density lipoproteins and target apoB for degradation within the rough endoplasmic reticulum of hamster hepatocytes

Dietary fish oils inhibited secretion and stimulated intracellular degradation of apolipoprotein (apo)B in hamster hepatocytes, while dietary sunflower oils stimulated secretion and had no effect on degradation of apoB. To investigate the intracellular site at which fish oils act, we have made use of our previous observations that inhibition of degradation by N-acetyl-leucyl-leucyl-norleucinal (ALLN) results in accumulation of apoB in the trans -Golgi membrane and does not stimulate secretion, while inhibition of degradation by o-phenanthroline results in accumulation of apoB in the rough endoplasmic reticulum membrane and stimulates secretion. Thus, ALLN protects apoB which has been diverted from secretion and o -phenanthroline protects apoB which is targetted for secretion. Addition of o -phenantholine to the incubation medium of hepatocytes from fish oil-fed hamsters inhibited degradation of apoB and stimulated its secretion in particles of the density of VLDL, while addition of ALLN had no effect.These observations suggest that dietary fish oils reversibly inhibit early steps in the assembly of very low density lipoprotein precursors and target apoB for degradation in the rough endoplasmic reticulum.