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

Alteration of nephrocystins and IFT-A proteins causes similar ciliary phenotypes leading to Nephronophthisis

International audienceNephronophtisis (NPH) is a kidney ciliopathy often associated with extra-renal defects and for which 12 genes (NPHP1-12) have been identified. NPHP1 and NPHP4 control the ciliary access at the transition zone and the velocity of some intraflagellar transport (IFT)/BBS proteins in C.elegans. Recently, in a collaborative effort, we have identified, in families with isolated NPH, mutations in TTC21B as well as in WDR19, which encode the retrograde IFT-A proteins IFT139 and IFT144, respectively. By ciliome sequencing of 1600 candidate genes from 14 NPH patients followed by Sanger sequencing of a cohort of 52 patients, we have found respectively 8 and 7 patients carrying pathogenic missense mutations in genes coding IFT-A proteins, including WDR35, TTC21B and IFT140, which could partially affect their function. Together, these results indicate that IFT-A are involved in nephronophtisis. Moreover, alteration of cilia length was observed in patient kidney, Nphp4-/- mice kidney tubules and NPHP1 or NPHP4 knockdown IMCD3 cell lines. In these cells, primary cilia present swellings at the distal region accompanied by an accumulation of IFT-B at the base and the tip, similar to what was observed in IFT-A mutants, suggesting a possible alteration of retrograde transport. Additionally, ARL13B, a small GTPase required for proper cilium shape and IFT stability, is absent along the axoneme of NPHP4-KD-IMCD cells. By controlling the entry of ciliary components at the transition zone, NPHP1 and NPHP4 may modulate IFT-A cargos thus participating in the same pathway (i.e. Wnt/PCP), alteration of which would lead to renal lesions observed in nephronophthisis

Longitudinal assessment of the platelet transcriptome in advanced heart failure patients following mechanical unloading

Patients with heart failure (HF) and left ventricular assist devices (LVAD) have dysregulated thrombo-inflammatory responses, mediated in part by platelets. While studies of platelet activation have been undertaken in HF, changes in the platelet transcriptome in HF patients following mechanical unloading with an LVAD have not been investigated. We prospectively enrolled and longitudinally followed advanced HF patients (n = 32) for a mean of 57 months post-LVAD implantation. For comparison, healthy donors were also enrolled (n = 20). Platelets were hyperactive in HF, as evidenced by significantly increased formation of circulating platelet-monocyte aggregate formation. Platelet transcriptome interrogation by next-generation RNA-sequencing identified that the expression of numerous genes (n = 588) was significantly (FDR < 0.05) altered in HF patients prior to LVAD implantation. Differentially expressed genes were predicted to have roles in angiogenesis, immune and inflammatory responses, apoptosis, and cardiac muscle contraction. 90 days following LVAD implantation, the majority (80%) of differentially expressed genes in HF patients normalized, as compared to the platelet transcriptomes of healthy donors. In conclusion, advanced HF is associated with marked alterations in the platelet transcriptome. While LVAD implantation to off load the failing heart results in resolution in the majority of differentially expressed genes, a subset of the platelet transcriptome remains persistently altered

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