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

Hepatic very-low-density lipoprotein and apolipoprotein B production are increased following in vivo induction of betaine–homocysteine S-methyltransferase

We have previously reported a positive correlation between the expression of BHMT (betaine–homocysteine S-methyltransferase) and ApoB (apolipoprotein B) in rat hepatoma McA (McArdle RH-7777) cells [Sowden, Collins, Smith, Garrow, Sparks and Sparks (1999) Biochem. J. 341, 639–645]. To examine whether a similar relationship occurs in vivo, hepatic BHMT expression was induced by feeding rats a Met (L-methionine)-restricted betaine-containing diet, and parameters of ApoB metabolism were evaluated. There were no generalized metabolic abnormalities associated with Met restriction for 7 days, as evidenced by control levels of serum glucose, ketones, alanine aminotransferase and L-homocysteine levels. Betaine plus the Met restriction resulted in lower serum insulin and non-esterified fatty acid levels. Betaine plus Met restriction induced hepatic BHMT 4-fold and ApoB mRNA 3-fold compared with Met restriction alone. No changes in percentage of edited ApoB mRNA were observed on the test diets. An increase in liver ApoB mRNA correlated with an 82% and 46% increase in ApoB and triacylglycerol production respectively using in vivo Triton WR 1339. Increased secretion of VLDL (very-low-density lipoprotein) with Met restriction plus betaine was associated with a 45% reduction in liver triacylglycerol compared with control. Nuclear run-off assays established that transcription of both bhmt and apob genes was also increased in Met-restricted plus betaine diets. No change in ApoB mRNA stability was detected in BHMT-transfected McA cells. Hepatic ApoB and BHMT mRNA levels were also increased by 1.8- and 3-fold respectively by betaine supplementation of Met-replete diets. Since dietary betaine increased ApoB mRNA, VLDL ApoB and triacylglycerol production and decreased hepatic triacylglycerol, results suggest that induction of apob transcription may provide a potential mechanism for mobilizing hepatic triacylglycerol by increasing ApoB available for VLDL assembly and secretion