An Allosteric Binding Site on Sortilin Regulates the Trafficking of VLDL, PCSK9, and LDLR in Hepatocytes.

ApoB lipoproteins (apo B-Lp) are produced in hepatocytes, and their secretion requires the cargo receptor sortilin. We examined the secretion of apo B-Lp-containing very low-density lipoprotein (VLDL), an LDL progenitor. Sortilin also regulates the trafficking of the subtilase PCSK9, which when secreted binds the LDL receptor (LDLR), resulting in its endocytosis and destruction at the lysosome. We show that the site 2 binding compound (cpd984) has multiple effects in hepatocytes, including (1) enhanced Apo-Lp secretion, (2) increased cellular PCSK9 retention, and (3) augmented levels of LDLR at the plasma membrane. We postulate that cpd984 enhances apo B-Lp secretion in part through binding the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is present at higher levels on circulating VLDL form fed rats relative to after fasting. We attribute the enhanced VLDL secretion to its increased binding affinity for sortilin site 1 induced by cpd984 binding site 2. This hinders PCSK9 binding and secretion, which would subsequently prevent its binding to LDLR leading to its degradation. This suggests that site 2 is an allosteric regulator of site 1 binding. This effect is not limited to VLDL, as cpd984 augments binding of the neuropeptide neurotensin (NT) to sortilin site 1. Molecular dynamics simulations demonstrate that the C-terminus of NT (Ct-NT) stably binds site 1 through an electrostatic interaction. This was bolstered by the ability of Ct-NT to disrupt lower-affinity interactions between sortilin and the site 1 ligand PIP3. Together, these data show that binding cargo at sortilin site 1 is allosterically regulated through site 2 binding, with important ramifications for cellular lipid homeostasis involving proteins such as PCSK9 and LDLR.

Insulin dependent apolipoprotein B degradation and phosphatidylinositide 3-kinase activation with microsomal translocation are restored in McArdle RH7777 cells following serum deprivation.

Previous studies in rat hepatocytes demonstrated that insulin-dependent apolipoprotein (apo) B degradation (IDAD) is lost when cells are maintained for 3 d under enriched culture conditions. Loss of IDAD correlates with increased expression of protein tyrosine phosphatase 1B (PTP1B) known to be associated with resistance to insulin signaling in the liver. McArdle RH7777 hepatoma (McA) cells cultured in serum containing medium are resistant to IDAD; demonstrate a 30% increase in apo B secretion, and express increased levels of PTP1B protein and mRNA. In addition, insulin-stimulated Class I phosphatidylinositide 3-kinase (PI3K) activity of anti-pY immunoprecipitates is severely blunted. IDAD resistance in McA cells correlates with diminished translocation of insulin-stimulated pY-IRS1 to intracellular membranes. Incubation of McA cells with RK682, a protein tyrosine phosphatase inhibitor, is sufficient to restore IDAD in resistant McA cells. Overall, results further support the importance of Class I PI3K activity in IDAD, and suggest that loss of this activity is sufficient to cause resistance. Although other factors are involved in downstream events including sortilin binding to apo B, autophagy, and lysosomal degradation, loss of signal generation and reduced localization of Class I PI3K to intracellular membranes plays a significant role in IDAD resistance.

Phosphatidylinositol (3,4,5)-trisphosphate binds to sortilin and competes with neurotensin: Implications for very low density lipoprotein binding.

Sortilin is a multi-ligand sorting receptor that interacts with B100-containing VLDL and LDL as well as other ligands including neurotensin (NT). The current study investigates the hypothesis that phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated downstream of insulin action can directly bind to sortilin. NT binds to sortilin at a well characterized site via its carboxy terminus (C-term). Using a crystal structure of human sortilin (hsortilin), PIP3 is predicted to bind at this C-term site. Binding of PIP3 to hsortilin is demonstrated using surface plasmon resonance (SPR) flowing PIP3 nanodiscs over immobilized hsortilin. Studies were performed using SPR where dibutanoyl PIP3 is shown to compete with NT for sortilin binding. Rat VLDL and LDL were evaluated for PIP3 content immunologically using monoclonal antibodies directed against PIP3. Rat plasma VLDL contained three times more immunoreactive PIP3 than LDL per µg of protein. Because VLDL contains additional ligands that bind sortilin, to distinguish specific PIP3 binding, we used PIP3 liposomes. Liposome floatation assays were used to demonstrate PIP3 liposome binding to sortilin. Using SPR and immobilized hsortilin, the C-term NT tetrapeptide (P-Y-I-L) is shown to bind to hsortilin. A compound (cpd984) was identified with strong theoretical binding to the site on sortilin involved in NT N-terminal binding. When cpd984 is co-incubated with the tetrapeptide, the affinity of binding to sortilin is increased. Similarly, the affinity of PIP3 liposome binding increased in the presence of cpd984. Overall, results demonstrate that sortilin is a PIP3 binding protein with binding likely to occur at the C-term NT binding site. The presence of multiple ligands on B100-containing lipoproteins, VLDL and LDL, raises the interesting possibility for increased interaction with sortilin based on the presence of PIP3.

Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells.

Studies examining the relationship between cellular sortilin and VLDL-B100 secretion demonstrate inconsistent results. Current studies explore the possibility that discrepancies may be related to insulin sensitivity. McArdle RH7777 cells (McA cells) cultured under serum enriched conditions lose sensitivity to insulin. Following incubation in serum-free DMEM containing 1% BSA, McA cells become insulin responsive and demonstrate reduced apo B secretion. Current studies indicate that insulin sensitive McA cells express lower cellular sortilin that corresponds with reduction in VLDL-B100 secretion without changes in mRNA of either sortilin or apo B. When sortilin expression is further reduced by siRNA knockdown (KD), there are additional decreases in VLDL-B100 secretion. A crystal structure of human sortilin (hsortilin) identifies two binding sites on the luminal domain for the N- and C-termini of neurotensin (NT). A small organic compound (cpd984) was identified that has strong theoretical binding to the N-terminal site. Both cpd984 and NT bind hsortilin by surface plasmon resonance. In incubations with insulin sensitive McA cells, cpd984 was shown to enhance VLDL-B100 secretion at each level of sortilin KD suggesting cpd984 acted through sortilin in mediating its effect. Current results support a role for sortilin to facilitate VLDL-B100 secretion which is limited to insulin sensitive McA cells. Inconsistent reports of the relationship between VLDL-B100 secretion and sortilin in previous studies may relate to differing functions of sortilin in VLDL-B100 secretion depending upon insulin sensitivity.

Insulin suppression of apolipoprotein B in McArdle RH7777 cells involves increased sortilin 1 interaction and lysosomal targeting.

Insulin suppresses secretion of very low density lipoprotein (VLDL) apolipoprotein (apo) B in primary rodent hepatocytes (RH) by favoring the degradation of B100, the larger form of apo B, through post-endoplasmic reticulum proteolysis. Sortilin 1 (sort1), a multi-ligand sorting receptor, has been proposed as a mediator of lysosomal B100 degradation by directing B100 in pre-VLDL to lysosomes rather than allowing maturation to VLDL and secretion. The purpose of our studies was to investigate the role of sort1 in insulin-dependent degradation of apo B. Using liver derived McArdle RH7777 (McA) cells, we demonstrate that insulin suppresses VLDL B100 secretion via a phosphatidylinositide 3-kinase (PI3K) dependent process that is inhibitable by wortmannin in a fashion similar to RH. Using McA cells and in situ cross-linking, we demonstrate that insulin acutely (30min) stimulates the interaction of B100 with sort1. The insulin-induced interaction of sort1-B100 is markedly enhanced when lysosomal degradation is inhibited by Bafilomycin A1 (BafA1), an inhibitor of lysosomal acidification. As BafA1 also prevents insulin suppressive effects on apo B secretion, our results suggest that sort1-B100 interaction stimulated by insulin transiently accumulates with BafA1 and favors B100 secretion by default.

Insulin-dependent apolipoprotein B degradation is mediated by autophagy and involves class I and class III phosphatidylinositide 3-kinases.

Insulin acutely stimulates the degradation of apolipoprotein B (apo B) which decreases very low density lipoprotein (VLDL) secretion by liver. Insulin-dependent apo B degradation (IDAD) occurs following phosphatidylinositide 3-kinase (PI3K) activation and involves lysosomal degradation. Insulin suppression of apo B secretion is blocked by over-expression of phosphatase and tensin homologue (PTEN) in McArdle RH7777 (McA) cells suggesting the importance of Class I PI3K generated PI (3,4,5) triphosphate (PIP3) in IDAD. Classical autophagy inhibitors including 3-methyladenine, L-asparagine and bafilomycin A1 also blocked the ability of insulin to suppress apo B secretion by rat hepatocytes (RH) suggesting that IDAD occurs through an autophagy-related mechanism. IDAD is also blocked following over-expression in McA cells of a dominant negative kinase-defective Vps34, a class III PI3K that generates PI 3-monophosphate required for autophagy. Vps34 inhibition of IDAD occurs without altering insulin-dependent S473 phosphorylation of Akt indicating PI3K/PIP3/Akt signaling is intact. Cellular p62/SQSTM1, an inverse indicator of autophagy, is increased with insulin treatment consistent with the known ability of insulin to inhibit autophagy, and therefore the role of insulin in utilizing components of autophagy for apo B degradation is unexpected. Thapsigargan, an inducer of endoplasmic reticulum (ER) stress, and a recently demonstrated autophagy inhibitor, blocked apo B secretion which contrasted with other autophagy inhibitors and mutant Vps34 results which were permissive with respect to apo B secretion. Pulse chase studies indicated that intact B100 and B48 proteins were retained in cells treated with thapsigargan consistent with their accumulation in autophagosomal vacuoles. Differences between IDAD and ER stress-coupled autophagy mediated by thapsgargin suggest that IDAD involves an unique form of autophagy. Insulin action resulting in hepatic apo B degradation is novel and important in understanding regulation of hepatic VLDL metabolism.

Selective hepatic insulin resistance, VLDL overproduction, and hypertriglyceridemia.

Insulin plays a central role in regulating energy metabolism, including hepatic transport of very low-density lipoprotein (VLDL)-associated triglyceride. Hepatic hypersecretion of VLDL and consequent hypertriglyceridemia leads to lower circulating high-density lipoprotein levels and generation of small dense low-density lipoproteins characteristic of the dyslipidemia commonly observed in metabolic syndrome and type 2 diabetes mellitus. Physiological fluctuations of insulin modulate VLDL secretion, and insulin inhibition of VLDL secretion upon feeding may be the first pathway to become resistant in obesity that leads to VLDL hypersecretion. This review summarizes the role of insulin-related signaling pathways that determine hepatic VLDL production. Disruption in signaling pathways that reduce generation of the second messenger phosphatidylinositide (3,4,5) triphosphate downstream of activated phosphatidylinositide 3-kinase underlies the development of VLDL hypersecretion. As insulin resistance progresses, a number of pathways are altered that further augment VLDL hypersecretion, including hepatic inflammatory pathways. Insulin plays a complex role in regulating glucose metabolism, and it is not surprising that the role of insulin in VLDL and lipid metabolism will prove equally complex.

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

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.

Metabolic regulation of APOBEC-1 complementation factor trafficking in mouse models of obesity and its positive correlation with the expression of ApoB protein in hepatocytes.

APOBEC-1 Complementation Factor (ACF) is an RNA-binding protein that interacts with apoB mRNA to support RNA editing. ACF traffics between the cytoplasm and nucleus. It is retained in the nucleus in response to elevated serum insulin levels where it supports enhanced apoB mRNA editing. In this report we tested whether ACF may have the ability to regulate nuclear export of apoB mRNA to the sites of translation in the cytoplasm. Using mouse models of obesity-induced insulin resistance and primary hepatocyte cultures we demonstrated that both nuclear retention of ACF and apoB mRNA editing were reduced in the livers of hyperinsulinemic obese mice relative to lean controls. Coincident with an increase in the recovery of ACF in the cytoplasm was an increase in the proportion of total cellular apoB mRNA recovered in cytoplasmic extracts. Cytoplasmic ACF from both lean controls and obese mouse livers was enriched in endosomal fractions associated with apoB mRNA translation and ApoB lipoprotein assembly. Inhibition of ACF export to the cytoplasm resulted in nuclear retention of apoB mRNA and reduced both intracellular and secreted ApoB protein in primary hepatocytes. The importance of ACF for modulating ApoB was supported by the finding that RNAi knockdown of ACF reduced ApoB secretion. An additional discovery from this study was the finding that leptin is a suppressor ACF expression. Dyslipidemia is a common pathology associated with insulin resistance that is in part due to the loss of insulin controlled secretion of lipid in ApoB-containing very low density lipoproteins. The data from animal models suggested that loss of insulin regulated ACF trafficking and leptin regulated ACF expression may make an early contribution to the overall pathology associated with very low density lipoprotein secretion from the liver in obese individuals.

Overindulgence and metabolic syndrome: is FoxO1 a missing link?

Excessive production of triglyceride-rich VLDL, which can result from dietary overindulgence, underlies metabolic syndrome--a combination of disorders including high blood pressure, obesity, high triglyceride, and insulin resistance--and places individuals at increased risk of developing cardiovascular disease and type 2 diabetes. However, the link between VLDL overproduction and insulin resistance has remained unclear. VLDL assembly in the liver is catalyzed by microsomal triglyceride transfer protein (MTP). In this issue of the JCI, Kamagate et al. investigate the events controlling hepatic MTP expression and VLDL production and secretion (see the related article beginning on page 2347). They demonstrate that MTP is a target of the transcription factor FoxO1 and that excessive VLDL production associated with insulin resistance is caused by the inability of insulin to regulate FoxO1 transcriptional activation of MTP.

Acute suppression of apo B secretion by insulin occurs independently of MTP.

Secretion of apolipoprotein (apo) B-containing lipoproteins by the liver depends mainly upon apo B availability and microsomal triglyceride transfer protein (MTP) activity and is subject to insulin regulation. Hepatic MTP mRNA expression is negatively regulated by insulin which correlates with inhibition of apo B secretion suggesting that insulin might suppress apo B secretion through an MTP-dependent mechanism. To investigate this possibility, we examined the acute effect of insulin on hepatic MTP expression and activity levels in vivo utilizing apobec-1(-/-) mice. Insulin did not significantly alter hepatic MTP mRNA levels or lipid transfer activity 2h following injection, but suppressed expression of genes important in gluconeogenesis. To study the specific role of MTP, we expressed human MTP (hMTP) in primary rat hepatocytes using adenoviral gene transfer. Increased expression of hMTP resulted in a 47.6±17.9% increase in total apo B secreted. Incubation of hepatocytes with insulin suppressed apo B secretion by 50.1±10.8% in cells over-expressing hMTP and by 53.0±12.4% in control transfected hepatocytes. Results indicate that even under conditions of increased hepatic apo B secretion mediated by MTP, responsiveness of hepatocytes to insulin to suppress apo B secretion is maintained.

ABHD5/CGI-58 facilitates the assembly and secretion of apolipoprotein B lipoproteins by McA RH7777 rat hepatoma cells.

Lipolysis of stored triacylglycerols provides lipid precursors for the assembly of apolipoprotein B (apoB) lipoproteins in hepatocytes. Abhydrolase domain containing 5 (ABHD5) is expressed in liver and facilitates the lipolysis of triacylglycerols. To study the function of ABHD5 in lipoprotein secretion, we silenced the expression of ABHD5 in McA RH7777 cells using RNA interference and studied the metabolism of lipids and secretion of apoB lipoproteins. McA RH7777 cells deficient in ABHD5 secreted reduced amounts of apoB, triacylglycerols, and cholesterol esters. Detailed analysis of liquid chromatography-mass spectrometry data for the molecular species of secreted triacylglycerols revealed that deficiency of ABHD5 significantly reduced secretion of triacylglycerols containing oleate, even when oleate was supplied in the culture medium; the ABHD5-deficient cells partially compensated by secreting higher levels of triacylglycerols containing saturated fatty acids. In experiments tracking the metabolism of [(14)C]oleate, silencing of ABHD5 reduced lipolysis of cellular triacylglycerols and incorporation of intermediates derived from stored lipids into secreted triacylglycerols and cholesterol esters. In contrast, the incorporation of exogenous oleate into secreted triacylglycerols and cholesterol esters was unaffected by deficiency of ABHD5. These findings suggest that ABHD5 facilitates the use of lipid intermediates derived from lipolysis of stored triacylglycerols for the assembly of lipoproteins.

Interleukin-6 mediates hepatic hypersecretion of apolipoprotein B.

Obesity and type 2 diabetes are associated with insulin resistance (IR), increased circulating proinflammatory cytokines, and hypertriglyceridemia, the latter being caused by overproduction of hepatic very low density lipoprotein (VLDL). One cytokine strongly linked with development of hepatic IR is interleukin-6 (IL-6). Our objective was to evaluate IL-6 effects on hepatic apolipoprotein B (apoB) and VLDL secretion and to examine possible linkages between cytokine signaling and insulin-suppressive effects on lipoprotein secretion. Of the cytokines examined, only IL-6 stimulated secretion of apoB-containing lipoproteins in a dose-dependent manner. Both B100 and B48 secretion were significantly increased in VLDL and in lipoproteins with a density >1.019 g/ml. The ability of insulin to suppress hepatic apoB secretion was maintained in hepatocytes treated with IL-6. Pulse-chase studies indicated that enhanced apoB synthesis was the primary mechanism for increased lipoprotein secretion, which corresponded with higher abundance of apoB mRNA. Because IL-6 did not alter the decay rate of apoB mRNA transcripts, results support that increased apoB mRNA levels are the result of enhanced apob gene transcription. Increased apoB-lipoprotein secretion was also detected with oncostatin M (OSM), supporting involvement of the signal-transducing protein, gp130. Increased suppressor of cytokine signaling (SOCS) 3 expression negated IL-6 and OSM effects and significantly reduced cellular apoB mRNA abundance. We conclude that IL-6 favors secretion of apoB-containing lipoproteins by increasing availability of apoB through changes in apob gene transcription. These changes may contribute to hypersecretion of VLDL associated with obesity, particularly under conditions where SOCS3 is not overexpressed to an extent capable of overcoming IL-6-stimulated apob gene transcription.

FoxO1 and hepatic lipid metabolism.

PURPOSE OF REVIEW: This review summarizes recent research implicating Forkhead box (Fox)O1, a key transcription factor in glucose metabolism, in the regulation of hepatic lipid metabolism. Insulin dysregulation leading to hypertriglyceridemia is associated with increased hepatic VLDL secretion. FoxO1 is integrated in action with other regulatory factors in VLDL metabolism. The role of FoxO1 is defined in context of recent controversies. RECENT FINDINGS: FoxO1 regulates transcription of microsomal triglyceride transfer protein and apolipoprotein (apo)CIII involved in hepatic assembly and postsecretory catabolism of VLDL. Insulin activation of Akt leads to the phosphorylation of FoxO1 with nuclear exclusion and loss of transcriptional activity. Reduced insulin action increases FoxO1 activity and induces microsomal triglyceride transfer protein favoring VLDL assembly and induces apoCIII reducing peripheral triglyceride catabolism. This new mechanistic link between insulin resistance and VLDL overproduction and hypertriglyceridemia compounds effects of other known VLDL regulatory factors. SUMMARY: This review highlights recent advances in research of insulin regulation of hepatic VLDL metabolism. Formation of VLDL requires lipid, apoB structural protein, and microsomal triglyceride transfer protein. FoxO1 is a major factor in hepatic microsomal triglyceride ransfer protein regulation. A unifying hypothesis is presented linking regulation of the three necessary hepatic components for VLDL assembly with insulin action and insulin resistance.

Increased hepatic steatosis and insulin resistance in mice lacking hepatic androgen receptor.

UNLABELLED: Early studies demonstrated that whole-body androgen receptor (AR)-knockout mice with hypogonadism exhibit insulin resistance. However, details about the mechanisms underlying how androgen/AR signaling regulates insulin sensitivity in individual organs remain unclear. We therefore generated hepatic AR-knockout (H-AR(-/y)) mice and found that male H-AR(-/y) mice, but not female H-AR(-/-) mice, fed a high-fat diet developed hepatic steatosis and insulin resistance, and aging male H-AR(-/y) mice fed chow exhibited moderate hepatic steatosis. We hypothesized that increased hepatic steatosis in obese male H-AR(-/y) mice resulted from decreased fatty acid beta-oxidation, increased de novo lipid synthesis arising from decreased PPARalpha, increased sterol regulatory element binding protein 1c, and associated changes in target gene expression. Reduced insulin sensitivity in fat-fed H-AR(-/y) mice was associated with decreased phosphoinositide-3 kinase activity and increased phosphenolpyruvate carboxykinase expression and correlated with increased protein-tyrosine phosphatase 1B expression. CONCLUSION: Together, our results suggest that hepatic AR may play a vital role in preventing the development of insulin resistance and hepatic steatosis. AR agonists that specifically target hepatic AR might be developed to provide a better strategy for treatment of metabolic syndrome in men.

Hyperleptinemia without obesity in male mice lacking androgen receptor in adipose tissue.

Insulin resistance occurs through an inadequate response to insulin by insulin target organs such as liver, muscle, and adipose tissue with consequent insufficient glucose uptake. In previous studies we demonstrated that whole body androgen receptor (AR) knockout (AR(-/y)) mice develop obesity and exhibit insulin and leptin resistance at advanced age. By examining adipose tissue-specific AR knockout (A-AR(-/y)) mice, we found A-AR(-/y) mice were hyperleptinemic but showed no leptin resistance, although body weight and adiposity index of A-AR(-/y) mice were identical with those of male wild-type control mice. Hypotriglyceridemia and hypocholesterolemia found in nonobese A-AR(-/y) mice suggested a beneficial effect of high leptin levels independent of fat deposition. Further examination showed that androgen-AR signaling in adipose tissue plays a direct regulatory role in leptin expression via enhanced estrogen receptor transactivation activity due to elevated intraadipose estrogens. The present study in A-AR(-/y) mice suggests a differential tissue-specific role of AR in energy balance control in males.