Shaohui Huang

Dextran and polymer polyethylene glycol (PEG) coating reduce both 5 and 30 nm iron oxide nanoparticle cytotoxicity in 2D and 3D cell culture.

Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG). Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS) formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL) and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated) nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles.

The interaction of Akt with APPL1 is required for insulin-stimulated Glut4 translocation.

APPL1 (adaptor protein containing PH domain, PTB domain, and leucine zipper motif 1) is an Akt/protein kinase B-binding protein involved in signal transduction and membrane trafficking pathways for various receptors, including receptor tyrosine kinases. Here, we establish a role for APPL1 in insulin signaling in which we demonstrate its interaction with Akt2 by co-immunoprecipitation and pulldown assays. In primary rat adipocytes and skeletal muscle, APPL1 and Akt2 formed a complex that was dissociated upon insulin stimulation in both tissues. To investigate possible APPL1 function in adipocytes, we analyzed Akt phosphorylation, 2-deoxyglucose uptake, and Glut4 translocation by immunofluorescence following APPL1 knockdown by small interfering and short hairpin RNAs. We show that APPL1 knockdown suppressed Akt phosphorylation, glucose uptake, and Glut4 translocation. We also tested the effect in 3T3-L1 adipocytes of expressing full-length APPL1 or an N- or a C-terminal APPL1 construct. Interestingly, expression of full-length APPL1 and its N terminus suppressed insulin-stimulated 2-deoxyglucose uptake and Glut4 translocation to roughly the same extent (40-60%). We confirmed by cellular fractionation that Glut4 translocation was substantially blocked in 3T3-L1 adipocytes transfected with full-length APPL1. By cellular fractionation, APPL1 was localized mainly in the cytosol, and it showed a small degree of re-localization to the light microsomes and nucleus in response to insulin. By immunofluorescence, we also show that APPL1 partially co-localized with Glut4. These data suggest that APPL1 plays an important role in insulin-stimulated Glut4 translocation in muscle and adipose tissues and that its N-terminal portion may be critical for APPL1 function.

Insulin Stimulates Membrane Fusion and GLUT4 Accumulation in Clathrin Coats on Adipocyte Plasma Membranes

ABSTRACT Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by ∼4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.

Phosphatidylinositol-4,5-bisphosphate-rich plasma membrane patches organize active zones of endocytosis and ruffling in cultured adipocytes

ABSTRACT A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCδ1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.

Microparticles initiate decompression-induced neutrophil activation and subsequent vascular injuries

Progressive elevations in circulating annexin V-coated microparticles (MPs) derived from leukocytes, erythrocytes, platelets, and endothelial cells are found in mice subjected to increasing decompression stresses. Individual MPs exhibit surface markers from multiple cells. MPs expressing platelet surface markers, in particular, interact with circulating neutrophils, causing them to degranulate and leading to further MP production. MPs can be lysed by incubation with polyethylene glycol (PEG) telomere B surfactant, and the number of circulating MPs is reduced by infusion of mice with PEG or antibody to annexin V. Myeloperoxidase deposition and neutrophil sequestration in tissues occur in response to decompression, and the pattern differs among brain, omentum, psoas, and leg skeletal muscle. Both MP abatement strategies reduce decompression-induced intravascular neutrophil activation, neutrophil sequestration, and tissue injury documented as elevations of vascular permeability and activated caspase-3. We conclude that MPs generated by decompression stresses precipitate neutrophil activation and vascular damage.

PECAM-targeted delivery of SOD inhibits endothelial inflammatory response

Elevated generation of reactive oxygen species (ROS) by endothelial enzymes, including NADPH‐oxidase, is implicated in vascular oxidative stress and endothelial proinflammatory activation involving exposure of vascular cell adhesion molecule‐1 (VCAM‐1). Catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet/endothelial cell adhesion molecule 1 (PECAM‐1) bind specifically to endothelium and inhibit effects of corresponding ROS, H2O2, and superoxide anion. In this study, anti‐PECAM/SOD, but not anti‐PECAM/catalase or nontargeted enzymes, including polyethylene glycol (PEG)‐SOD, inhibited 2‐ to 3‐fold VCAM expression caused by tumor necrosis factor (TNF), interleukin‐1β, and lipopolysaccharide. Anti‐PECAM/SOD, but not nontargeted counterparts, accumulated in vascular endothelium after intravenous injection, localized in endothelial endosomes, and inhibited by 70% lipopolysaccharide‐caused VCAM‐1 expression in mice. Anti‐PECAM/SOD colocalized with EEA‐1‐positive endothelial vesicles and quenched ROS produced in response to TNF. Inhibitors of NADPH oxidase and anion channel ClC3 blocked TNF‐induced VCAM expression, affirming that superoxide produced and transported by these proteins, respectively, mediates inflammatory signaling. Anti‐PECAM/SOD abolished VCAM expression caused by poly(I:C)‐induced activation of toll‐like receptor 3 localized in intracellular vesicles. These results directly implicate endosomal influx of superoxide in endothelial inflammatory response and suggest that site‐specific interception of this signal attained by targeted delivery of anti‐PECAM/SOD into endothelial endo‐somes may have anti‐inflammatory effects.—Shuvaev, V. V., Han, J., Yu, K. J., Huang, S., Hawkins, B. J., Madesh, M., Nakada, M., and Muzykantov, V. R. PECAM‐targeted delivery of SOD inhibits endothelial inflammatory response. FASEB J. 25, 348–357 (2011). www.fasebj.org

The v-SNARE Vti1a regulates insulin-stimulated glucose transport and Acrp30 secretion in 3T3-L1 adipocytes.

Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and trans-Golgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNA-based gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.

Rab38 targets to lamellar bodies and normalizes their sizes in lung alveolar type II epithelial cells.

Rab38 is a rat Hermansky-Pudlak syndrome gene that plays an important role in surfactant homeostasis in alveolar type II (ATII) pneumocytes. We examined Rab38 function in regulating lamellar body (LB) morphology in ATII cells. Quantitative electron microscopy revealed that LBs in ATII cells were ∼77% larger in Rab38-null fawn-hooded hypertension (FHH) than control Sprague-Dawley (SD) rats. Rab38 protein expression was restricted in lung epithelial cells but was not found in primary endothelial cells. In SD ATII cells, Rab38 protein level gradually declined during 5 days in culture. Importantly, endogenous Rab38 was present in LB fractions purified from SD rat lungs, and transiently expressed enhanced green fluorescent protein (EGFP)-tagged Rab38 labeled only the limiting membranes of a subpopulation (∼30%) of LBs in cultured ATII cells. This selective targeting was abolished by point mutations to EGFP-Rab38 and was not shared by Rab7 and Rab4b, which also function in the ATII cells. Using confocal microscopy, we established a method for quantitative evaluation of the enlarged LB phenotype temporally preserved in cultured FHH ATII cells. A direct causal relationship was established when the enlarged LB phenotype was reserved and then rescued by transiently reexpressed EGFP-Rab38 in cultured FHH ATII cells. This rescuing effect was associated with dynamic EGFP-Rab38 targeting to and on LB limiting membranes. We conclude that Rab38 plays an indispensible role in maintaining LB morphology and surfactant homeostasis in ATII pneumocytes.

Characterization of the Niemann-Pick C pathway in alveolar type II cells and lamellar bodies of the lung

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.

Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

Niemann-Pick C (NPC) disease is due to loss of NPC1 or NPC2 protein function that is required for unesterified cholesterol transport from the endosomal/lysosomal compartment. Though lung involvement is a recognized characteristic of Niemann-Pick type C disease, the pathological features are not well understood. We investigated components of the surfactant system in both NPC1 mutant mice and felines and in NPC2 mutant mice near the end of their expected life span. Histological analysis of the NPC mutant mice demonstrated thickened septae and foamy macrophages/leukocytes. At the level of electron microscopy, NPC1-mutant type II cells had uncharacteristically larger lamellar bodies (LB, mean area 2-fold larger), while NPC2-mutant cells had predominantly smaller lamellar bodies (mean area 50% of normal) than wild type. Bronchoalveolar lavage from NPC1 and NPC2 mutant mice had an approx. 4-fold and 2.5-fold enrichment in phospholipid, respectively, and an approx. 9-fold and 35-fold enrichment in cholesterol, consistent with alveolar lipidosis. Phospholipid and cholesterol also were elevated in type II cell LBs and lung tissue while phospholipid degradation was reduced. Enrichment of surfactant protein-A in the lung and surfactant of the mutant mice was found. Immunocytochemical results showed that cholesterol accumulated in the LBs of the type II cells isolated from the affected mice. Alveolar macrophages from the NPC1 and NPC2 mutant mice were enlarged compared to those from wild type mice and were enriched in phospholipid and cholesterol. Pulmonary features of NPC1 mutant felines reflected the disease described in NPC1 mutant mice. Thus, with the exception of lamellar body size, the lung phenotype seen in the NPC1 and NPC2 mutant mice were similar. The lack of NPC1 and NPC2 proteins resulted in a disruption of the type II cell surfactant system contributing to pulmonary abnormalities.