Differential Regulation of Lipoprotein and Hepatitis C Virus Secretion by Rab1b

Secretory cells produce diverse cargoes, yet how they regulate concomitant secretory traffic remains insufficiently explored. Rab GTPases control intracellular vesicular transport. To map secretion pathways, we generated a library of lentivirus-expressed dominant-negative Rab mutants and used it in a large-scale screen to identify regulators of hepatic lipoprotein secretion. We identified several candidate pathways, including those mediated by Rab11 and Rab8. Surprisingly, inhibition of Rab1b, the major regulator of transport from the endoplasmic reticulum to the Golgi, differently affected the secretion of the very-low-density lipoprotein components ApoE and ApoB100, despite their final association on mature secreted lipoprotein particles. Since hepatitis C virus (HCV) incorporates ApoE and ApoB100 into its virus particle, we also investigated infectious HCV secretion and show that its regulation by Rab1b mirrors that of ApoB100. These observations reveal differential regulation of hepatocyte secretion by Rab1b and advance our understanding of lipoprotein assembly and lipoprotein and HCV secretion

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. © 2013 Andreo et al

Effects of insulin on apoB100 degradation and VLDL-apoB100 secretion are blunted in autophagy-deficient mouse primary hepatocytes.

<p>Primary hepatocytes were isolated from mice with floxed alleles of <i>Atg5</i> (<i>Atg5^+/+</i>) or <i>Atg5^f/f x Alb-Cre</i> (<i>Atg5^−/−</i>) mice (i.e., mice with hepatic deficiency of Atg5), and cultured in serum-free conditions for 16 h before insulin addition. A) A western-blot for LC3 was performed with lysates of primary hepatocytes from <i>Atg5^+/+</i> or <i>Atg5^−/−</i> mice; “(+)” represents the condition in which lysosomal degradation has been blocked (20 mM NH₄Cl + 10 µM E64D) to increase LC3 recovery and “(-)” represents untreated cells. When autophagy is active, LC3 (“LC3-I”) is lipidated to form LC3-II. GAPDH was used as the loading control. B) Primary hepatocytes from <i>Atg5^−/−</i> or <i>Atg5^+/+</i> mice were incubated in media with (INS) or without insulin (CONT) and pulse-labeled for 15 min with [³⁵S]-protein labeling mix, and were then chased for 30 and 120 min in non-radioactive medium with the treatments maintained. Total apoB100 recovery and quantification were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057590#pone-0057590-g001" target="_blank">Figure 1</a>. The histogram (mean±SEM) represents the results from 2 independent experiments, each one performed in triplicate; ** indicates P<0.01. C) Primary hepatocytes from <i>Apobec1^−/−</i> mice were labeled with [³⁵S]-protein labeling mix for 4 h in the presence (+ Insulin) or absence (− Insulin) of 100 nM insulin. Conditional media samples were collected and lipoproteins were separated by density gradient ultracentrifugation. ApoB100 in individual fractions was immunoprecipitated, resolved by SDS-PAGE, and quantified by densitometry after bands were detected by a phosphorImager. The statistical significance of the comparisons between the density profiles is based on 3 independent replicate experiments. D) An experiment similar to the one in panel C was performed, but using Atg5-deficent primary hepatocytes prepared from <i>Apobec1^−/−</i> mice.</p

Insulin stimulates class II PI-3 kinase activity in mouse primary hepatocytes.

<p>Primary hepatocytes from <i>Apobec1^−/−</i> mice were cultured in serum free conditions for 16 h before the addition of insulin to a final concentration of 100 nM. At the indicated times, the cells were harvested in ice cold 0.5 M TCA, and acidic lipids were extracted. PI(3,4)P₂ concentrations were measured by dot blotting using anti-PI(3,4)P₂ antibody (n = 3 at each time point). Data represent the mean±SEM.</p

Insulin-stimulated apoB100 degradation in mouse primary hepatocytes is dependent on class II PI3-kinase gamma.

<p>Primary hepatocytes from <i>Apobec1^−/−</i> mice were transfected with control (scrambled) siRNA or class II PI3-kinase (PIK3C2γ) specific siRNA. After a total of 48 h after transfection, (A) PIK3C2γ, (B) PIK3C2α, and (C) PIK3C2β mRNA levels were assessed by two-step qRT-PCR, and their abundance was normalized to 28S rRNA. The histogram (mean±SEM) represents the results from 2 independent experiments, each one performed in triplicate. D) Control or PIK3C2γ siRNA transfected primary hepatocytes from <i>Apobec1^−/−</i> mice were incubated in medium with (INS) or without (CONT) insulin, pulse-labeled for 15 min with [³⁵S]-protein labeling mix, and then chased for 30 and 120 min with the treatments maintained. Total apoB100 recovery and quantification were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057590#pone-0057590-g001" target="_blank">Figure 1</a>. The histogram (mean±SEM) represents the results from 2 independent experiments, each one performed in triplicate; ** and *** indicate P<0.01 and 0.001, respectively. E. Representative primary data of the experiments summarized in panel D.</p

Class I PI3-kinase activity is dispensable for insulin-stimulated apoB100 degradation in mouse primary hepatocytes.

<p>A) Experiments were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057590#pone-0057590-g001" target="_blank">Figure 1</a>, but in the presence or absence of the class I specific PI3-kinase inhibitor, PIK75. The histogram (mean±SEM) represents the results from 2 independent experiments, each performed in triplicate. B) Representative primary data of the experiments summarized in panel A.</p

Insulin-stimulated apoB100 degradation in mouse primary hepatocytes is PI3-kinase- dependent.

<p>A) Primary hepatocytes from <i>Apobec1^−/−</i> mice (which only synthesize apoB100) were incubated in media containing (INS) or lacking (CONT) insulin and/or wortmannin (WORT) and were pulse labeled for 15 min with [³⁵S]-protein labeling mix and chased in non-radioactive medium for 30 and 120 min with the treatments maintained. ApoB100 was then immunoprecipitated and separated by SDS-PAGE and quantified as described in Materials and Methods. The histogram (mean±SEM) represents the results from 2 independent experiments, each performed in triplicate. B) Representative primary data of the experiments summarized in panel A; ** indicates P<0.01.</p

Analysis of Hepatitis C Virus Particle Heterogeneity in Immunodeficient Human Liver Chimeric fah-/- MiceSummary

Background & Aims: Hepatitis C virus (HCV) is a leading cause of chronic liver diseases and the most common indication for liver transplantation in the United States. HCV particles in the blood of infected patients are characterized by heterogeneous buoyant densities, likely owing to HCV association with lipoproteins. However, clinical isolates are not infectious in vitro and the relative infectivity of the particles with respect to their buoyant density therefore cannot be determined, pointing to the need for better in vivo model systems. Methods: To analyze the evolution of the buoyant density of in vivoâderived infectious HCV particles over time, we infected immunodeficient human liver chimeric fumaryl acetoacetate hydrolase-/- mice with J6/JFH1 and performed ultracentrifugation of infectious mouse sera on isopicnic iodixanol gradients. We also evaluated the impact of a high sucrose diet, which has been shown to increase very-low-density lipoprotein secretion by the liver in rodents, on lipoprotein and HCV particle characteristics. Results: Similar to the severe combined immunodeficiency disease/Albumin-urokinase plasminogen activator human liver chimeric mouse model, density fractionation of infectious mouse serum showed higher infectivity in the low-density fractions early after infection. However, over the course of the infection, viral particle heterogeneity increased and the overall in vitro infectivity diminished without loss of the human liver graft over time. In mice provided with a sucrose-rich diet we observed a minor shift in HCV infectivity toward lower density that correlated with a redistribution of triglycerides and cholesterol among lipoproteins. Conclusions: Our work indicates that the heterogeneity in buoyant density of infectious HCV particles evolves over the course of infection and can be influenced by diet. Keywords: HCV, Lipoprotein, Mouse Model, Human Liver Chimeric Mic

Huh-7 or HepG2 cells: which is the better model for studying human apolipoprotein-B100 assembly and secretion?[S]

Apolipoprotein-B100 (apoB100) is the essential protein for the assembly and secretion of very low density lipoproteins (VLDL) from liver. The hepatoma HepG2 cell line has been the cell line of choice for the study of synthesis and secretion of human apoB-100. Despite the general use of HepG2 cells to study apoB100 metabolism, they secrete relatively dense, lipid-poor particles compared with VLDL secreted in vivo. Recently, Huh-7 cells were adopted as an alternative model to HepG2 cells, with the implicit assumption that Huh-7 cells were superior in some respects of lipoprotein metabolism, including VLDL secretion. In this study we addressed the hypothesis that the spectrum of apoB100 lipoprotein particles secreted by Huh-7 cells more closely resembles the native state in human liver. We find that Huh-7 cells resemble HepG2 cells in the effects of exogenous lipids, microsomal triglyceride transfer protein (MTP)-inhibition, and proteasome inhibitors of apoB100 secretion, recovery, and degradation. In contrast to HepG2 cells, however, MEK-ERK inhibition does not correct the defect in VLDL secretion. Huh-7 cells do not appear to offer any advantages over HepG2 cells as a general model of human apoB100-lipoprotein metabolism