Keyang Chen

Recent insights into factors affecting remnant lipoprotein uptake.

Purpose of review Remnant lipoproteins that persist in the bloodstream after each meal have become increasingly important contributors to atherosclerotic vascular disease, owing to the spread of overnutrition, underexertion, obesity, insulin resistance, and type 2 diabetes. Here, we review recent work that clarified long-standing controversies over the molecular mediators of remnant clearance by the liver, as well as their dysregulation – but possible correction – during alterations in caloric balance. Recent findings Two endocytic receptors, the syndecan-1 heparan sulfate proteoglycan (HSPG) and the LDL receptor, plus one docking receptor, SR-BI, significantly contribute to normal hepatic remnant catabolism. Compelling evidence exists for dysfunction of the syndecan-1 HSPG in diabetic states. The major molecular defect identified so far in poorly controlled type 1 diabetes is impaired hepatic HSPG assembly. In contrast, the primary defect in hepatic HSPGs in type 2 diabetes appears to arise from accelerated de-sulfation, owing to the induction of a sulfatase. Moreover, short-term caloric restriction restores hepatic expression of this sulfatase towards normal. Summary Correct identification of hepatic remnant receptors has finally allowed investigations of their molecular dysregulation in diabetes and related conditions. New work points to novel therapeutic targets to correct postprandial dyslipoproteinemia and its consequent arterial damage.

Molecular mediators for raft-dependent endocytosis of syndecan-1, a highly conserved, multifunctional receptor

Background: Endocytosis via rafts remains incompletely characterized. Results: Raft-dependent endocytosis of syndecan-1 occurs in two phases, each requiring a kinase and a corresponding cytoskeletal partner. Each phase depends on MKKK, a novel, conserved motif within syndecan-1. Conclusion: These findings demonstrate the molecular choreography behind endocytosis of a raft-dependent receptor. Significance: Syndecans mediate uptake of biologically and medically important ligands. Endocytosis via rafts has attracted considerable recent interest, but the molecular mediators remain incompletely characterized. Here, we focused on the syndecan-1 heparan sulfate proteoglycan, a highly conserved, multifunctional receptor that we previously showed to undergo raft-dependent endocytosis upon clustering. Alanine scanning mutagenesis of three to five consecutive cytoplasmic residues at a time revealed that a conserved juxtamembrane motif, MKKK, was the only region required for efficient endocytosis after clustering. Endocytosis of clustered syndecan-1 occurs in two phases, each requiring a kinase and a corresponding cytoskeletal partner. In the initial phase, ligands trigger rapid MKKK-dependent activation of ERK and the localization of syndecan-1 into rafts. Activation of ERK drives the dissociation of syndecan-1 from α-tubulin, a molecule that may act as an anchor for syndecan-1 at the plasma membrane in the basal state. In the second phase, Src family kinases phosphorylate tyrosyl residues within the transmembrane and cytoplasmic regions of syndecan-1, a process that also requires MKKK. Tyrosine phosphorylation of syndecan-1 triggers the robust recruitment of cortactin, which we found to be an essential mediator of efficient actin-dependent endocytosis. These findings represent the first detailed characterization of the molecular events that drive endocytosis of a raft-dependent receptor and identify a novel endocytic motif, MKKK. Moreover, the results provide new tools to study syndecan function and regulation during uptake of its biologically and medically important ligands, such as HIV-1, atherogenic postprandial remnant lipoproteins, and molecules implicated in Alzheimer disease.

Type 2 diabetes in mice induces hepatic overexpression of sulfatase 2, a novel factor that suppresses uptake of remnant lipoproteins

Type 2 diabetes mellitus (T2DM) impairs hepatic clearance of atherogenic postprandial remnant lipoproteins. Our work and that of others have identified syndecan‐1 heparan sulfate proteoglycans (HSPGs) as remnant lipoprotein receptors. Nevertheless, defects in the T2DM liver have not been molecularly characterized, and neither has the correction that occurs upon caloric restriction. We used microarrays to compare expression of proteoglycan‐related genes in livers from control db/m mice; obese, T2DM db/db littermates fed ad libitum (AL); and db/db mice pair‐fed to match the intake of db/m mice. Surprisingly, the arrays identified only one gene whose dysregulation by T2DM would disrupt HSPG structure: the heparan sulfate glucosamine‐6‐O‐endosulfatase‐2 (Sulf2). SULF2 degrades HSPGs by removing 6‐O sulfate groups, but had no previously known role in diabetes or lipoprotein biology. Follow‐up quantitative polymerase chain reaction assays revealed a striking 11‐fold induction of Sulf2 messenger RNA in the livers of AL T2DM mice compared with controls. Immunoblots demonstrated induction of SULF2 in AL livers, with restoration toward normal in livers from pair‐fed db/db mice. Knockdown of SULF2 in cultured hepatocytes doubled HSPG‐mediated catabolism of model remnant lipoproteins. Notably, co‐immunoprecipitations revealed a persistent physical association of SULF2 with syndecan‐1. To identify mechanisms of SULF2 dysregulation in T2DM, we found that advanced glycosylation end products provoked a 10‐fold induction in SULF2 expression by cultured hepatocytes and an approximately 50% impairment in their catabolism of remnants and very low‐density lipoprotein, an effect that was entirely reversed by SULF2 knockdown. Adiponectin and insulin each suppressed SULF2 protein in cultured liver cells and in murine livers in vivo, consistent with a role in energy flux. Likewise, both hormones enhanced remnant lipoprotein catabolism in vitro. Conclusion: SULF2 is an unexpected suppressor of atherogenic lipoprotein clearance by hepatocytes and an attractive target for inhibition. (HEPATOLOGY 2010;.)

Inhibition of hepatic sulfatase‐2 In Vivo: A novel strategy to correct diabetic dyslipidemia

Type 2 diabetes mellitus (T2DM) impairs hepatic clearance of atherogenic postprandial triglyceride‐rich lipoproteins (TRLs). We recently reported that livers from T2DM db/db mice markedly overexpress the heparan sulfate glucosamine‐6‐O‐endosulfatase‐2 (SULF2), an enzyme that removes 6‐O sulfate groups from heparan sulfate proteoglycans (HSPGs) and suppresses uptake of TRLs by cultured hepatocytes. In the present study, we evaluated whether Sulf2 inhibition in T2DM mice in vivo could correct their postprandial dyslipidemia. Selective second‐generation antisense oligonucleotides (ASOs) targeting Sulf2 were identified. Db/db mice were treated for 5 weeks with Sulf2 ASO (20 or 50 mg/kg per week), nontarget (NT) ASO, or phosphate‐buffered saline (PBS). Administration of Sulf2 ASO to db/db mice suppressed hepatic Sulf2 messenger RNA expression by 70%‐80% (i.e., down to levels in nondiabetic db/m mice) and increased the ratio of tri‐ to disulfated disaccharides in hepatic HSPGs (P < 0.05). Hepatocytes isolated from db/db mice on NT ASO exhibited a significant impairment in very‐low‐density lipoprotein (VLDL) binding that was entirely corrected in db/db mice on Sulf2 ASO. Sulf2 ASO lowered the random, nonfasting plasma triglyceride (TG) levels by 50%, achieving nondiabetic values. Most important, Sulf2 ASO treatment flattened the plasma TG excursions in db/db mice after corn‐oil gavage (iAUC, 1,500 ± 470 mg/dL·h for NT ASO versus 160 ± 40 mg/dL·h for Sulf2 ASO\P < 0.01). Conclusions: Despite extensive metabolic derangements in T2DM mice, inhibition of a single dys‐regulated molecule, SULF2, normalizes the VLDL‐binding capacity of their hepatocytes and abolishes postprandial hypertriglyceridemia. These findings provide a key proof of concept in vivo to support Sulf2 inhibition as an attractive strategy to improve metabolic dyslipidemia. (HEPATOLOGY 2012;55:1746–1753)

The role of pathway-selective insulin resistance and responsiveness in diabetic dyslipoproteinemia.

Purpose of review Type 2 diabetes mellitus (T2DM) and related syndromes exhibit a deadly triad of dyslipoproteinemia, which leads to atherosclerosis, hyperglycemia, which causes microvascular disease, and hypertension. These features share a common, but unexplained, origin – namely, pathway-selective insulin resistance and responsiveness (SEIRR). Here, we review recent work on hepatic SEIRR indicating that deranged insulin signaling may have a remarkably simple molecular basis. Recent findings Comprehensive examination of a set of 18 insulin targets revealed that T2DM liver in vivo exhibits a specific defect in the ability of the NAD(P)H oxidase 4 (NOX4) to inactivate protein tyrosine phosphatase gene family members after stimulation with insulin, and that impairment of this single molecule, NOX4, in cultured hepatocytes recapitulates all features of hepatic SEIRR in vivo. These features include insulin-stimulated generation of an unusual monophosphorylated form of AKT at Thr308 (pT308-AKT) with only weak phosphorylation at Ser473, impaired insulin-stimulated pathways for lowering plasma levels of lipids and glucose, but continued lipogenic pathways and robust extracellular signal-regulated kinase activation. This new study, in combination with important prior work, provides clues to several long-standing mysteries, such as how AKT might regulate lipid-lowering and glucose-lowering pathways that become insulin-resistant but also lipogenic pathways that remain insulin-responsive, as well as a potential role for NOX4 in insulin-stimulated generation of oxysterol ligands for LXR, a key lipogenic factor. Summary These findings suggest a unified molecular explanation for fatty liver, atherogenic dyslipoproteinemia, hyperglycemia, and hence accelerated atherosclerosis and microvascular disease in T2DM, obesity, and related syndromes of positive caloric imbalance.

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

An oxide transport chain essential for balanced insulin signaling

Patients with overnutrition, obesity, the atherometabolic syndrome, and type 2 diabetes mellitus exhibit imbalanced insulin action, also called pathway-selective insulin resistance. To control glycemia, they require hyperinsulinemia that then overdrives ERK and hepatic de-novo lipogenesis. We recently reported that NADPH oxidase-4 regulates balanced insulin action. Here, we show that NADPH oxidase-4 is part of a new limb of insulin signaling that we abbreviate “NSAPP” after its five major proteins. The NSAPP pathway is an oxide transport chain that begins when insulin stimulates NADPH oxidase-4 to generate . NADPH oxidase-4 hands to superoxide dismutase-3 for conversion into H2O2. The pathway ends when aquaporin-3 channels H2O2 across the membrane to inactivate PTEN. Disruption of any component of the NSAPP chain, from NADPH oxidase-4 up to PTEN, leaves PTEN persistently active, thereby producing the same deadly pattern of imbalanced insulin action seen clinically. Unraveling the molecular basis for NSAPP dysfunction in overnutrition has now become a top priority.

Suppression of Hepatic FLOT1 (Flotillin-1) by Type 2 Diabetes Mellitus Impairs the Disposal of Remnant Lipoproteins via Syndecan-1

Objective— Type 2 diabetes mellitus (T2DM) and the atherometabolic syndrome exhibit a deadly dyslipoproteinemia that arises in part from impaired hepatic disposal of C-TRLs (cholesterol- and triglyceride-rich remnant apoB [apolipoprotein B] lipoproteins). We previously identified syndecan-1 as a receptor for C-TRLs that directly mediates endocytosis via rafts, independent from coated pits. Caveolins and flotillins form rafts but facilitate distinct endocytotic pathways. We now investigated their participation in syndecan-1–mediated disposal of C-TRLs and their expression in T2DM liver. Approach and Results— In cultured liver cells and nondiabetic murine livers, we found that syndecan-1 coimmunoprecipitates with FLOT1 (flotillin-1) but not with CAV1 (caveolin-1). Binding of C-TRLs to syndecan-1 on the surface of liver cells enhanced syndecan-1/FLOT1 association. The 2 molecules then trafficked together into the lysosomes, implying limited if any recycling back to the cell surface. The interaction requires the transmembrane/cytoplasmic region of syndecan-1 and the N-terminal hydrophobic domain of FLOT1. Knockdown of FLOT1 in cultured liver cells substantially inhibited syndecan-1 endocytosis. Livers from obese, T2DM KKAy mice exhibited 60% to 70% less FLOT1 protein and mRNA than in nondiabetic KK livers. An adenoviral construct to enhance hepatic expression of wild-type FLOT1 in T2DM mice normalized plasma triglycerides, whereas a mutant FLOT1 missing its N-terminal hydrophobic domain had no effect. Moreover, the adenoviral vector for wild-type FLOT1 lowered plasma triglyceride excursions and normalized retinyl excursions in T2DM KKAy mice after a corn oil gavage, without affecting postprandial production of C-TRLs. Conclusions— FLOT1 is a novel participant in the disposal of harmful C-TRLs via syndecan-1. Low expression of FLOT1 in T2DM liver may contribute to metabolic dyslipoproteinemia.