Phospholipid Transfer Protein Plays a Major Role in the Initiation of Apolipoprotein B-containing Lipoprotein Assembly in Mouse Primary Hepatocytes

In this study, we tested the hypothesis that phospholipid transfer protein (PLTP) is a plausible mediator of phospholipid (PL) transfer to the N-terminal 1000 residues of apoB (apoB:1000) leading to the initiation of apoB-containing lipoprotein assembly. To this end, primary hepatocytes from wild type (WT) and PLTP knock-out (KO) mice were transduced with adenovirus-apoB:1000 with or without co-transduction with adenovirus-PLTP, and the assembly and secretion of apoB:1000-containing lipoproteins were assessed. PLTP deficiency resulted in a 65 and 72% reduction in the protein and lipid content, respectively, of secreted apoB:1000-containing lipoproteins. Particles secreted by WT hepatocytes contained 69% PL, 9% diacylglycerol (DAG), and 23% triacylglycerol (TAG) with a stoichiometry of 46 PL, 6 DAG, and 15 TAG molecules per apoB:1000. PLTP absence drastically altered the lipid composition of apoB:1000 lipoproteins; these particles contained 46% PL, 13% DAG, and 41% TAG with a stoichiometry of 27 PL, 10 DAG, and 23 TAG molecules per apoB:1000. Reintroduction of Pltp gene into PLTP-KO hepatocytes stimulated the lipidation and secretion of apoB:1000-containing lipoproteins by ∼3-fold; the lipid composition and stoichiometry of these particles were identical to those secreted by WT hepatocytes. In contrast to the WT, apoB:1000 in PLTP-KO hepatocytes was susceptible to intracellular degradation predominantly in the post-endoplasmic reticulum, presecretory compartment. Reintroduction of Pltp gene into PLTP-KO hepatocytes restored the stability of apoB:1000. These results provide compelling evidence that in hepatocytes initial recruitment of PL by apoB:1000 leading to the formation of the PL-rich apoB-containing initiation complex is mediated to a large extent by PLTP.     

Phospholipid transfer protein deficiency protects circulating lipoproteins from oxidation due to the enhanced accumulation of vitamin E

Vitamin E is a lipophilic anti-oxidant that can prevent the oxidative damage of atherogenic lipoproteins. However, human trials with vitamin E have been disappointing, perhaps related to ineffective levels of vitamin E in atherogenic apoB-containing lipoproteins. Phospholipid transfer protein (PLTP) promotes vitamin E removal from atherogenic lipoproteins in vitro, and PLTP deficiency has recently been recognized as an anti-atherogenic state. To determine whether PLTP regulates lipoprotein vitamin E content in vivo, we measured alpha-tocopherol content and oxidation parameters of lipoproteins from PLTP-deficient mice in wild type, apoE-deficient, low density lipoprotein (LDL) receptor-deficient, or apoB/cholesteryl ester transfer protein transgenic backgrounds. In all four backgrounds, the vitamin E content of very low density lipoprotein (VLDL) and/or LDL was significantly increased in PLTP-deficient mice, compared with controls with normal plasma PLTP activity. Moreover, PLTP deficiency produced a dramatic delay in generation of conjugated dienes in oxidized apoB-containing lipoproteins as well as markedly lower titers of plasma IgG autoantibodies to oxidized LDL. The addition of purified PLTP to deficient plasma lowered the vitamin E content of VLDL plus LDL and normalized the generation of conjugated dienes. The data show that PLTP regulates the bioavailability of vitamin E in atherogenic lipoproteins and suggest a novel strategy for achieving more effective concentrations of anti-oxidants in lipoproteins, independent of dietary supplementation.     

The triple threat to nascent apolipoprotein B. Evidence for multiple, distinct degradative pathways

We previously showed that Omega-3 fatty acids reduce secretion of apolipoprotein B (apoB) from cultured hepatocytes by stimulating post-translational degradation. In this report, we now characterize this process, particularly in regard to the two known processes that degrade newly synthesized apoB, endoplasmic reticulum (ER)-associated degradation and re-uptake from the cell surface. First, we found that Omega-3-induced degradation preferentially reduces the secretion of large, assembled apoB-lipoprotein particles, and apoB polypeptide length is not a determinant. Second, based on several experimental approaches, ER-associated degradation is not involved. Third, re-uptake, the only process known to destroy fully assembled nascent lipoproteins, was clearly active in primary hepatocytes, but Omega-3-induced degradation of apoB continued even when re-uptake was blocked. Cell fractionation showed that Omega-3 fatty acids induced a striking loss of apoB100 from the Golgi, while sparing apoB100 in the ER, indicating a post-ER process. To determine the signaling involved, we used wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, which blocked most, if not all, of the Omega-3 fatty acid effect. Therefore, nascent apoB is subject to ER-associated degradation, re-uptake, and a third distinct degradative pathway that appears to target lipoproteins after considerable assembly and involves a post-ER compartment and PI3K signaling. Physiologic, pathophysiologic, and pharmacologic regulation of net apoB secretion may involve alterations in any of these three degradative steps.     

Anti-inflammatory effects of phospholipid transfer protein (PLTP) deficiency in mice

We previously reported that phospholipid transfer protein-deficient (PLTP KO) mice exhibit a lower rate of atherosclerosis. We proposed two possible mechanisms: a reduction in hepatic apoB secretion (Nat Med 7 (2001) 847) and induction of lipoprotein anti-oxidation activity (J Biol Chem 277 (2002) 31850). We now hypothesized that PLTP KO mice may exhibit an anti-inflammatory state per se. First, we found that PLTP KO mice have significantly lower IL-6 levels than wild type (WT) mice. Secondly, we found that IL-6 treatment increased plasma TNFalpha levels in WT mice, but not in PLTP KO mice. Thirdly, we used flow cytometric analyses to measure the mean fluorescence intensity of I-A(b), a MHC-class II molecule, on peripheral monocytes and found that IL-6 treatment significantly increased the I-A(b)-positive cell levels in WT mice, whereas no changes were observed in the cell levels in PLTP KO mice. The results of our experiments demonstrated an anti-inflammatory effect of PLTP deficiency as a further aspect of its proatherogenic potency.     

Regulation of ApoB secretion by the low density lipoprotein receptor requires exit from the endoplasmic reticulum and interaction with ApoE or ApoB

Apolipoprotein B (apoB) is required for the hepatic assembly and secretion of very low density lipoprotein (VLDL). The LDL receptor (LDLR) promotes post-translational degradation of apoB and thereby reduces VLDL particle secretion. We investigated the trafficking pathways and ligand requirements for the LDLR to promote degradation of apoB. We first tested whether the LDLR drives apoB degradation in an endoplasmic reticulum (ER)-associated pathway. Primary mouse hepatocytes harboring an ethyl-nitrosourea-induced, ER-retained mutant LDLR secreted comparable levels of apoB with LDLR-null hepatocytes, despite reduced secretion from cells expressing the wild-type LDLR. Additionally, treatment of cells with brefeldin A inhibited LDLR-dependent degradation. However, this rescue was reversible, and degradation of apoB occurred upon removal of brefeldin A. To characterize the lipoprotein reuptake pathway of degradation, we employed an LDLR mutant defective in constitutive endocytosis and internalization of apoB. This mutant was as effective in reducing apoB secretion as the wild-type LDLR. However, the effect was dependent on apolipoprotein E (apoE) as only the wild-type LDLR, and not the endocytic mutant, reduced apoB secretion in apoE-null cells. Treatment with heparin rescued a pool of apoB in cells expressing the endocytic mutant, indicating that reuptake of VLDL via apoE still occurs with this mutant. Finally, an LDLR mutant defective in binding apoB but not apoE reduced apoB secretion in an apoE-dependent manner. Together, these data suggest that the LDLR directs apoB to degradation in a post-ER compartment. Furthermore, the reuptake mechanism of degradation occurs via internalization of apoB through a constitutive endocytic pathway and apoE through a ligand-dependent pathway.     

Compensatory increase in hepatic lipogenesis in mice with conditional intestine-specific Mttp deficiency

Microsomal TG transfer protein (MTTP) is required for the assembly and secretion of TG (TG)-rich lipoproteins from both enterocytes and hepatocytes. Liver-specific deletion of Mttp produced a dramatic reduction in plasma very low density lipoprotein-TG and virtually eliminated apolipoprotein B100 (apoB100) secretion yet caused only modest reductions in plasma apoB48 and apoB48 secretion from primary hepatocytes. These observations prompted us to examine the phenotype following intestine-specific Mttp deletion because murine, like human enterocytes, secrete virtually exclusively apoB48. We generated mice with conditional Mttp deletion in villus enterocytes (Mttp-IKO), using a tamoxifen-inducible, intestine-specific Cre transgene. Villus enterocytes from chow-fed Mttp-IKO mice contained large cytoplasmic TG droplets and no chylomicron-sized particles within the secretory pathway. Chow-fed, Mttp-IKO mice manifested steatorrhea, growth arrest, and decreased cholesterol absorption, features that collectively recapitulate the phenotype associated with abetalipoproteinemia. Chylomicron secretion was reduced dramatically in vivo, in conjunction with an approximately 80% decrease in apoB48 secretion from primary enterocytes. Additionally, although plasma and hepatic cholesterol and TG content were decreased, Mttp-IKO mice demonstrated a paradoxical increase in both hepatic lipogenesis and very low density lipoprotein secretion. These findings establish distinctive features for MTTP involvement in intestinal chylomicron assembly and secretion and suggest that hepatic lipogenesis undergoes compensatory induction in the face of defective intestinal TG secretion.     

PPARalpha deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins.

This study investigates the importance of peroxisome proliferator activated receptor alpha (PPARalpha) for serum apolipoprotein B (apoB) levels and hepatic secretion of apoB-containing lipoproteins. Total serum apoB and VLDL-apoB levels were higher in female PPARalpha-null mice compared with female wild-type mice, but no difference was seen in male mice. Furthermore, hepatic triglyceride secretion rate, determined in vivo after Triton WR1339 injection, was 2.4-fold higher in female PPARalpha-null mice compared with female wild-type mice, but no difference was observed in male mice. However, when fed a high fat diet, male PPARalpha-null mice displayed 2-fold higher serum levels of apoB and LDL cholesterol compared with male wild-type mice, but triglyceride levels were not affected. Hepatic LDL receptor protein levels were not influenced by PPARalpha deficiency, gender, or the fat diet. Hepatocyte cultures from female PPARalpha-null mice (cultured for 4 days in serum free medium) showed 2-fold higher total apoB secretion and increased secretion of apoB-48 VLDL, as well as 2.7-fold larger accumulation of VLDL-triglycerides in the medium compared with wild-type cultures. In conclusion, PPARalpha-deficient female mice, but not males, display high serum apoB associated with VLDL and increased hepatic triglyceride secretion. Moreover, male PPARalpha-null mice show increased susceptibility to high fat diet in terms of serum apoB levels.     

Apolipoprotein B secretion and atherosclerosis are decreased in mice with phospholipid-transfer protein deficiency.

Increased secretion and levels of ApoB-containing lipoproteins (BLp) commonly occur in familial hyperlipidemia, obesity and diabetes. The plasma phospholipid-transfer protein (PLTP) is known to mediate transfer of phospholipids between BLp and HDL during their intravascular metabolism. To address a possible role of PLTP in dyslipidemia and atherogenesis, we bred mice deficient in the gene encoding PLTP (PLTP-deficient mice) using different hyperlipidemic mouse strains. In ApoB-transgenic and ApoE-deficient backgrounds, PLTP deficiency resulted in reduced production and levels of BLp and markedly decreased atherosclerosis. BLp secretion was diminished in hepatocytes from ApoB-transgenic PLTP-deficient mice, a defect that was corrected when PLTP was reintroduced in adenovirus. The studies reveal a major, unexpected role of PLTP in regulating the secretion of BLp and identify PLTP as a therapeutic target.     

LZP is required for hepatic triacylglycerol transportation through maintaining apolipoprotein B stability

The conserved zona pellucida (ZP) domain is found in hundreds of extracellular proteins that are expressed in various organs and play a variety of roles as structural components, receptors and tumor suppressors. A liver-specific zona pellucida domain-containing protein (LZP), also named OIT3, has been shown to be mainly expressed in human and mouse hepatocytes; however, the physiological function of LZP in the liver remains unclear. Here, we show that Lzp deletion inhibited very low-density lipoprotein (VLDL) secretion, leading to hepatic TG accumulation and lower serum TG levels in mice. The apolipoprotein B (apoB) levels were significantly decreased in the liver, serum, and VLDL particles of LZP-deficient mice. In the presence of LZP, which is localized to the endoplasmic reticulum (ER) and Golgi apparatus, the ER-associated degradation (ERAD) of apoB was attenuated; in contrast, in the absence of LZP, apoB was ubiquitinated by AMFR, a known E3 ubiquitin ligase specific for apoB, and was subsequently degraded, leading to lower hepatic apoB levels and inhibited VLDL secretion. Interestingly, hepatic LZP levels were elevated in mice challenged with a high-fat diet and humans with simple hepatic steatosis, suggesting that LZP contributes to the physiological regulation of hepatic TG homeostasis. In general, our data establish an essential role for LZP in hepatic TG transportation and VLDL secretion by preventing the AMFR-mediated ubiquitination and degradation of apoB and therefore provide insight into the molecular function of LZP in hepatic lipid metabolism.     

CuZn-SOD deficiency causes ApoB degradation and induces hepatic lipid accumulation by impaired lipoprotein secretion in mice

Elevated hepatic reactive oxygen species play an important role in pathogenesis of liver diseases, such as alcohol-induced liver injury, hepatitis C virus infection, and nonalcoholic steatohepatitis. In the present study, we investigated and compared the hepatic lipid metabolisms of liver-specific Sod2 (superoxide dismutase 2) knock-out (Sod2 KO), Sod1 knock-out (Sod1 KO), and Sod1/liver-specific Sod2 double knock-out mice (double KO). We observed significant increases in lipid peroxidation and triglyceride (TG) in the liver of Sod1 KO and double KO mice but not in the liver of Sod2 KO mice. We also found that high fat diet enhanced fatty changes of the liver in Sod1 KO and double KO mice but not in Sod2 KO mice. These data indicated that CuZn-SOD deficiency caused lipid accumulation in the liver. To investigate the molecular mechanism of hepatic lipid accumulation in CuZn-SOD-deficient mice, we measured TG secretion rate from liver using Triton WR1339. We found significant decrease of TG secretion in CuZn-SOD-deficient mice. Furthermore, we observed marked degradation of apolipoprotein B (apoB) in the liver and plasma of CuZn-SOD-deficient mice, indicating that degradation of apoB impairs secretion of lipoprotein from the liver. Our data suggest that oxidative stress enhances hepatic lipid accumulation by impaired lipoprotein secretion due to the degradation of apoB in liver.     

Phospholipid transfer protein deficiency ameliorates diet-induced hypercholesterolemia and inflammation in mice

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids from triglyceride-rich lipoproteins into HDL. PLTP has been shown to be an important factor in lipoprotein metabolism and atherogenesis. Here, we report that chronic high-fat, high-cholesterol diet feeding markedly increased plasma cholesterol levels in C57BL/6 mice. PLTP deficiency attenuated diet-induced hypercholesterolemia by dramatically reducing apolipoprotein E-rich lipoproteins (-88%) and, to a lesser extent, LDL (-40%) and HDL (-35%). Increased biliary cholesterol secretion, indicated by increased hepatic ABCG5/ABCG8 gene expression, and decreased intestinal cholesterol absorption may contribute to the lower plasma cholesterol in PLTP-deficient mice. The expression of proinflammatory genes (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) is reduced in aorta of PLTP knockout mice compared with wild-type mice fed either a chow or a high-cholesterol diet. Furthermore, plasma interleukin-6 levels are significantly lower in PLTP-deficient mice, indicating reduced systemic inflammation. These data suggest that PLTP appears to play a proatherogenic role in diet-induced hyperlipidemic mice.     

Phospholipid transfer protein (PLTP) deficiency attenuates high fat diet induced obesity and insulin resistance

Increased phospholipid transfer protein (PLTP) activity has been found to be associated with obesity, and metabolic syndrome in humans. However, whether or not PLTP has a direct effect on insulin sensitivity and obesity is largely unknown. Here we analyzed the effect by using PLTP knockout (PLTP^−/−) mouse model. Although, PLTP^−/− mice have normal body-weight-gain under chow diet, these mice were protected from high-fat-diet-induced obesity and insulin resistance, compared with wild type mice. In order to understand the mechanism, we evaluated insulin receptor and Akt activation and found that PLTP deficiency significantly enhanced phosphorylated insulin receptor and Akt levels in high-fat-diet fed mouse livers, adipose tissues, and muscles after insulin stimulation, while total Akt and insulin receptor levels were unchanged. Moreover, we found that the PLTP deficiency induced significantly more GLUT4 protein in the plasma membranes of adipocytes and muscle cells after insulin stimulation. Finally, we found that PLTP-deficient hepatocytes had less sphingomyelins and free cholesterols in the lipid rafts and plasma membranes than that of controls and this may provide a molecular basis for PLTP deficiency-mediated increase in insulin sensitivity. We have concluded that PLTP deficiency leads to an improvement in tissue and whole-body insulin sensitivity through modulating lipid levels in the plasma membrane, especially in the lipid rafts.     

Effect of selenium deficiency and vitamin E deficiency on glutathione metabolism in isolated rat hepatocytes

Selenium deficiency and vitamin E deficiency both affect xenobiotic metabolism and toxicity. In addition, selenium deficiency causes changes in the activity of some glutathione-requiring enzymes. We have studied glutathione metabolism in isolated hepatocytes from selenium-deficient, vitamin E-deficient, and control rats. Cell viability, as measured by trypan blue exclusion, was comparable for all groups during the 5-h incubation. Freshly isolated hepatocytes had the same glutathione concentration regardless of diet group. During the incubation, however, the glutathione concentration in selenium-deficient hepatocytes rose to 1.4 times that in control hepatocytes. The selenium-deficient cells also released twice as much glutathione into the incubation medium as did the control cells. Total glutathione (intracellular plus extracellular) in the incubation flask increased from 47.7 +/- 8.9 to 152 +/- 16.5 nmol/10(6) selenium-deficient cells over 5 h compared with an increase from 46.7 +/- 7.1 to 92.0 +/- 17.4 nmol/10(6) control cells and from 47.7 +/- 11.7 to 79.5 +/- 24.9 nmol/10(6) vitamin E-deficient cells. This overall increase in glutathione concentration suggested that glutathione synthesis was accelerated by selenium deficiency. The activity of gamma-glutamylcysteine synthetase was twice as great in selenium-deficient liver supernatant (105,000 X g) as in vitamin E-deficient or control liver supernatant (105,000 X g). Hemoglobin-free perfused livers were used to determine the form of glutathione released and its route. Selenium-deficient livers released 4 times as much GSH into the caval perfusate as did control livers. Plasma glutathione concentration in selenium-deficient rats was found to be 2-fold that in control rats, suggesting that increased GSH synthesis and release is an in vivo phenomenon associated with selenium deficiency.     

Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins

Glucosamine-induced endoplasmic reticulum (ER) stress was recently shown to specifically reduce apolipoprotein B-100 (apoB-100) secretion by enhancing the proteasomal degradation of apoB-100. Here, we examined the mechanisms linking glucosamine-induced ER stress and apoB-lipoprotein biogenesis. Trypsin sensitivity studies suggested glucosamine-induced changes in apoB-100 conformation. Endoglycosidase H studies of newly synthesized apoB-100 revealed glucosamine induced N-linked glycosylation defects resulting in reduced apoB-100 secretion. We also examined glucosamine-induced changes in VLDL assembly and secretion. A dose-dependent (1-10 mM glucosamine) reduction was observed in VLDL-apoB-100 secretion in primary hepatocytes (24.2-67.3%) and rat McA-RH7777 cells (23.2-89.5%). Glucosamine also inhibited the assembly of larger VLDL-, LDL-, and intermediate density lipoprotein-apoB-100 but did not affect smaller HDL-sized apoB-100 particles. Glucosamine treatment during the chase period (posttranslational) led to a 24% reduction in apoB-100 secretion (P < 0.01; n = 4) and promoted post-ER apoB degradation. However, the contribution of post-ER apoB-100 degradation appeared to be quantitatively minor. Interestingly, the glucosamine-induced posttranslational reduction in apoB-100 secretion could be partially prevented by treatment with desferrioxamine or vitamin E. Together, these data suggest that cotranslational glucosamine treatment may cause defects in apoB-100 N-linked glycosylation and folding, resulting in enhanced proteasomal degradation. Posttranslationally, glucosamine may interfere with the assembly process of apoB lipoproteins, leading to post-ER degradation via nonproteasomal pathways.     

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.     

Novel roles of hepatic lipase and phospholipid transfer protein in VLDL as well as HDL metabolism

Objective

Elevated plasma phospholipid transfer protein (PLTP) expression may increase atherosclerosis in mice by reducing plasma HDL and increasing hepatic VLDL secretion. Hepatic lipase (HL) is a lipolytic enzyme involved in several aspects of the same pathways of lipoprotein metabolism. We investigated whether the effects of elevated PLTP activity are compromised by HL deficiency.

Methods and results

HL deficient mice were crossbred with PLTP transgenic (PLTPtg) mice and studied in the fasted state. Plasma triglycerides were decreased in HL deficiency, explained by reduced hepatic triglyceride secretion. In PLTPtg mice, a redistribution of HL activity between plasma and tissue was evident and plasma triglycerides were also decreased. HL deficiency mitigated or even abolished the stimulatory effect of elevated PLTP activity on hepatic triglyceride secretion. HL deficiency had a modest incremental effect on plasma HDL, which remained present in PLTP transgenic/HL^−/− mice, thereby partially compensating the decrease in HDL caused by elevation of PLTP activity. HDL decay experiments showed that the fractional turnover rate of HDL cholesteryl esters was delayed in HL deficient mice, increased in PLTPtg mice and intermediate in PLTPtg mice in an HL^−/− background.

Conclusions

HL affects hepatic VLDL. Elevated PLTP activity lowers plasma HDL-cholesterol by stimulating the plasma turnover and hepatic uptake of HDL cholesteryl esters. HL is not required for the increase in hepatic triglyceride secretion or for the lowering of HDL-cholesterol induced by PLTP overexpression.     

Selenium deficiency activates mouse liver Nrf2-ARE but vitamin E deficiency does not

Selenium (Se) and vitamin E are antioxidant micronutrients. Se functions through selenoproteins and vitamin E reacts with oxidizing molecules in membranes. The relationship of these micronutrients with the Nrf2-antioxidant response element (ARE) pathway was investigated using ARE-reporter mice and Nrf2-/- mice. Weanling males were fed Se-deficient (0 Se), vitamin E-deficient (0 E), or control diet for 16 or 22 weeks. The ARE reporter was elevated 450-fold in 0 Se liver but was not elevated in 0 E liver. Antioxidant enzymes induced by Nrf2-ARE (glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase (NQOR), and heme oxygenase-1 (HO-1)) were elevated in 0 Se livers but not in 0 E livers. Deletion of Nrf2 had varying effects on the inductions, with GST induction being abolished by it but induction of NQOR and HO-1 still occurring. Thus, Se deficiency, but not vitamin E deficiency, induces a number of enzymes that protect against oxidative stress and modify xenobiotic metabolism through Nrf2-ARE and other stress-response pathways. We conclude that Se deficiency causes cytosolic oxidative stress but that vitamin E deficiency does not. This suggests that the oxidant defense mechanisms in which these antioxidant nutrients function are independent of one another.     

Insulin-Stimulated Degradation of Apolipoprotein B100: Roles of Class II Phosphatidylinositol-3-Kinase and Autophagy

Both in humans and animal models, an acute increase in plasma insulin levels, typically following meals, leads to transient depression of hepatic secretion of very low density lipoproteins (VLDL). One contributing mechanism for the decrease in VLDL secretion is enhanced degradation of apolipoprotein B100 (apoB100), which is required for VLDL formation. Unlike the degradation of nascent apoB100, which occurs in the endoplasmic reticulum (ER), insulin-stimulated apoB100 degradation occurs post-ER and is inhibited by pan-phosphatidylinositol (PI)3-kinase inhibitors. It is unclear, however, which of the three classes of PI3-kinases is required for insulin-stimulated apoB100 degradation, as well as the proteolytic machinery underlying this response. Class III PI3-kinase is not activated by insulin, but the other two classes are. By using a class I-specific inhibitor and siRNA to the major class II isoform in liver, we now show that it is class II PI3-kinase that is required for insulin-stimulated apoB100 degradation in primary mouse hepatocytes. Because the insulin-stimulated process resembles other examples of apoB100 post-ER proteolysis mediated by autophagy, we hypothesized that the effects of insulin in autophagy-deficient mouse primary hepatocytes would be attenuated. Indeed, apoB100 degradation in response to insulin was significantly impaired in two types of autophagy-deficient hepatocytes. Together, our data demonstrate that insulin-stimulated apoB100 degradation in the liver requires both class II PI3-kinase activity and autophagy.     

A deficiency of microsomal triglyceride transfer protein reduces apolipoprotein B secretion

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent ( approximately 25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice.     

Fenofibrate reverses the decline in HDL cholesterol in mice overexpressing human phospholipid transfer protein

In humans, fibrates are used to treat dyslipidemia, because these drugs lower plasma triglycerides and raise HDL cholesterol. Treatment with fibrates lowers plasma phospholipid transfer protein (PLTP) activity in humans, but increases PLTP activity in mice, without a consistent effect on HDL-cholesterol concentration. Earlier, we found that PLTP overexpression in transgenic mice results in decreased plasma HDL levels and increased diet-induced atherosclerosis. So it seems that the interplay between fibrates, PLTP and HDL is different in mice and man, which may be important for atherosclerosis development. In the present study, we measured the effects of fibrates on PLTP expression in cultured human hepatocytes and effects of fibrate treatment on human PLTP expression, plasma PLTP activity and HDL levels in human PLTP transgenic mice. Fibrate treatment did not influence PLTP mRNA levels in human hepatocytes. Hepatic human PLTP mRNA levels and PLTP activity were both moderately elevated by fenofibrate treatment in human PLTP transgenic mice. In wild-type mice, however, feeding fenofibrate resulted in a strong induction of PLTP mRNA in the liver and a more than 4-fold increase of plasma PLTP activity. Plasma triglycerides were reduced in all mice by 48% or more by fenofibrate treatment. HDL-cholesterol concentrations were substantially increased by fenofibrate in PLTP overexpressing mice (+72%), but unaffected in wild-type mice. We conclude that fenofibrate treatment reverses the HDL-lowering effect of PLTP overexpression in human PLTP transgenic mice.