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Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells.

ABSTRACT: Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel-derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal colocalization. Insulin transcytosis required dynamin but was unaffected by caveolin-1 knockdown or cholesterol depletion. Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion. Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1. This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent.

SUBMITTER: Azizi PM

PROVIDER: S-EPMC4325843 | biostudies-other | 2015 Feb

REPOSITORIES: biostudies-other

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Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells.

Azizi Paymon M PM Zyla Roman E RE Guan Sha S Wang Changsen C Liu Jun J Bolz Steffen-Sebastian SS Heit Bryan B Klip Amira A Lee Warren L WL

Molecular biology of the cell 20141224 4

Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel-derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to stud ...[more]

PMID: 25540431

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Project description:JC polyomavirus (JCPyV) establishes a persistent, lifelong, asymptomatic infection within the kidney of the majority of the human population. Under conditions of severe immunosuppression or immune modulation, JCPyV can reactivate in the central nervous system (CNS) and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease. Initiation of infection is mediated through viral attachment to α2,6-sialic acid-containing lactoseries tetrasaccharide c (LSTc) on the surface of host cells. JCPyV internalization is dependent on serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs), and entry is thought to occur by clathrin-mediated endocytosis (CME). However, the JCPyV entry process and the cellular factors involved in viral internalization remain poorly understood. Treatment of cells with small-molecule chemical inhibitors and RNA interference of 5-HT2R endocytic machinery, including β-arrestin, clathrin, AP2, and dynamin, significantly reduced JCPyV infection. However, infectivity of the polyomavirus simian virus 40 (SV40) was not affected by CME-specific treatments. Inhibition of clathrin or β-arrestin specifically reduced JCPyV internalization but did not affect viral attachment. Furthermore, mutagenesis of a β-arrestin binding domain (Ala-Ser-Lys) within the intracellular C terminus of 5-HT2AR severely diminished internalization and infection, suggesting that β-arrestin interactions with 5-HT2AR are critical for JCPyV infection and entry. These conclusions illuminate key host factors that regulate clathrin-mediated endocytosis of JCPyV, which is necessary for viral internalization and productive infection.IMPORTANCE Viruses usurp cellular factors to invade host cells. Activation and utilization of these proteins upon initiation of viral infection are therefore required for productive infection and resultant viral disease. The majority of healthy individuals are asymptomatically infected by JC polyomavirus (JCPyV), but if the host immune system is compromised, JCPyV can cause progressive multifocal leukoencephalopathy (PML), a rare, fatal, demyelinating disease. Individuals infected with HIV or taking prolonged immunomodulatory therapies have a heightened risk for developing PML. The cellular proteins and pathways utilized by JCPyV to mediate viral entry are poorly understood. Our findings further characterize how JCPyV utilizes the clathrin-mediated endocytosis pathway to invade host cells. We have identified specific components of this pathway that are necessary for the viral entry process and infection. Collectively, the conclusions increase our understanding of JCPyV infection and pathogenesis and may contribute to the future development of novel therapeutic strategies for PML.

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Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells.

Publications

Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells.

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