scholarly journals Stimulation of GLUT4 (glucose transporter isoform 4) storage vesicle formation by sphingolipid depletion

2010 ◽  
Vol 427 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Zhi-Jie Cheng ◽  
Raman Deep Singh ◽  
Teng-ke Wang ◽  
Eileen L. Holicky ◽  
Christine L. Wheatley ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Fluorescent Protein ◽  
Membrane Microdomains ◽  
Glut4 Translocation ◽  
Storage Vesicles ◽  
Storage Vesicle ◽  
Mechanistic Basis ◽  
Green Fluorescent

Insulin stimulates glucose transport in fat and skeletal muscle cells primarily by inducing the translocation of GLUT4 (glucose transporter isoform 4) to the PM (plasma membrane) from specialized GSVs (GLUT4 storage vesicles). Glycosphingolipids are components of membrane microdomains and are involved in insulin-regulated glucose transport. Cellular glycosphingolipids decrease during adipocyte differentiation and have been suggested to be involved in adipocyte function. In the present study, we investigated the role of glycosphingolipids in regulating GLUT4 translocation. We decreased glycosphingolipids in 3T3-L1 adipocytes using glycosphingolipid synthesis inhibitors and investigated the effects on GLUT4 translocation using immunocytochemistry, preparation of PM sheets, isolation of GSVs and FRAP (fluorescence recovery after photobleaching) of GLUT4–GFP (green fluorescent protein) in intracellular structures. Glycosphingolipids were located in endosomal vesicles in pre-adipocytes and redistributed to the PM with decreased expression at day 2 after initiation of differentiation. In fully differentiated adipocytes, depletion of glycosphingolipids dramatically accelerated insulin-stimulated GLUT4 translocation. Although insulin-induced phosphorylation of IRS (insulin receptor substrate) and Akt remained intact in glycosphingolipid-depleted cells, both in vitro budding of GLUT4 vesicles and FRAP of GLUT4–GFP on GSVs were stimulated. Glycosphingolipid depletion also enhanced the insulin-induced translocation of VAMP2 (vesicle-associated membrane protein 2), but not the transferrin receptor or cellubrevin, indicating that the effect of glycosphingolipids was specific to VAMP2-positive GSVs. Our results strongly suggest that decreasing glycosphingolipid levels promotes the formation of GSVs and, thus, GLUT4 translocation. These studies provide a mechanistic basis for recent studies showing that inhibition of glycosphingolipid synthesis improves glycaemic control and enhances insulin sensitivity in animal models of Type 2 diabetes.

scholarly journals Sorting of GLUT4 into its insulin-sensitive store requires the Sec1/Munc18 protein mVps45

2013 ◽  
Vol 24 (15) ◽  
pp. 2389-2397 ◽  
Author(s):  
Jennifer Roccisana ◽  
Jessica B. A. Sadler ◽  
Nia J. Bryant ◽  
Gwyn W. Gould
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Vitro Assay ◽  
Protein Receptor ◽  
Storage Vesicles ◽  
Storage Vesicle ◽  
Impaired Insulin ◽  
Endosomal Pathway

Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target–soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using sub­cellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment.

scholarly journals Insulin Action on GLUT4 Traffic Visualized in Single 3T3-L1 Adipocytes by Using Ultra-fast Microscopy

2001 ◽  
Vol 12 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Varsha Patki ◽  
Joanne Buxton ◽  
Anil Chawla ◽  
Larry Lifshitz ◽  
Kevin Fogarty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
High Speed ◽  
Fluorescent Protein ◽  
Light Exposure ◽  
Neuroendocrine Cells ◽  
Half Time ◽  
Glut4 Translocation ◽  
Cytoskeletal Networks ◽  
Green Fluorescent

A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20–30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5–7 min at 37°C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.

Measuring GLUT4 translocation in mature muscle fibers

2010 ◽  
Vol 299 (2) ◽  
pp. E169-E179 ◽  
Author(s):  
Hans P. M. M. Lauritzen ◽  
Jonathan D. Schertzer
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transport ◽  
Cell Biology ◽  
Glucose Transporter ◽  
Surface Membrane ◽  
Muscle Fibers ◽  
Postprandial Glucose ◽  
Glucose Disposal ◽  
Glut4 Translocation

Skeletal muscle is the major tissue for postprandial glucose disposal. Facilitated glucose uptake into muscle fibers is mediated by increases in surface membrane levels of the glucose transporter GLUT4 via insulin- and/or muscle contraction-mediated GLUT4 translocation. However, the regulatory mechanisms controlling GLUT4 translocation in skeletal muscle have been difficult to characterize at the cell biology level due to muscle tissue complexity. Muscle cell culture models have improved our understanding of GLUT4 translocation and glucose transport regulation, but in vitro muscle models lack many of the characteristics of mature muscle fibers. Thus, the molecular and cellular details of GLUT4 translocation in mature skeletal muscle are deficient. The objective of this review is to highlight how advances in recent experimental approaches translate into an enhanced understanding of the regulation of GLUT4 translocation and glucose transport in mature skeletal muscle.

Role of Akt substrate of 160 kDa in insulin-stimulated and contraction-stimulated glucose transport

2007 ◽  
Vol 32 (3) ◽  
pp. 557-566 ◽  
Author(s):  
Gregory D. Cartee ◽  
Jørgen F.P. Wojtaszewski
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Contractile Activity ◽  
Glut4 Translocation ◽  
Amp Activated Protein Kinase ◽  
Glucose Transporter Proteins ◽  
Response To Exercise

Insulin and exercise, the most important physiological stimuli to increase glucose transport in skeletal muscle, trigger a redistribution of GLUT4 glucose transporter proteins from the cell interior to the cell surface, thereby increasing glucose transport capacity. The most distal insulin signaling protein that has been linked to GLUT4 translocation, Akt substrate of 160 kDa (AS160), becomes phosphorylated in insulin-stimulated 3T3-L1 adipocytes; this is im​portant for insulin-stimulated GLUT4 translocation and glucose transport. Insulin also induces a rapid and dose-dependent increase in AS160 phosphorylation in skeletal muscle. Available data from skeletal muscle support the concepts developed in adipocytes with regard to the role AS160 plays in the regulation of insulin-stimulated glucose transport. In vivo exercise, in vitro contractions, or in situ contractions can also stimulate AS160 phosphorylation. AMP-activated protein kinase (AMPK) is likely important for phosphorylating AS160 in response to exercise/contractile activity, whereas Akt2 appears to be important for insulin-stimulated AS160 phosphorylation in muscle. Evidence of a role for AS160 in exercise/contraction-stimulated glucose uptake is currently inconclusive. The distinct signaling pathways that are stimulated by insulin and exercise/contraction converge at AS160. Although AS160 phosphorylation is apparently important for insulin-stimulated GLUT4 translocation and glucose transport, it is uncertain whether elevated AS160 phosphorylation plays a similar role with exercise/contraction.

scholarly journals Role for the microtubule cytoskeleton in GLUT4 vesicle trafficking and in the regulation of insulin-stimulated glucose uptake

2000 ◽  
Vol 352 (2) ◽  
pp. 267-276 ◽  
Author(s):  
Laura M. FLETCHER ◽  
Gavin I. WELSH ◽  
Paru B. OATEY ◽  
Jeremy M. TAVARÉ
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Microtubule Depolymerization ◽  
Microtubule Cytoskeleton ◽  
Storage Vesicles ◽  
Intracellular Vesicles ◽  
Green Fluorescent

Insulin stimulates glucose uptake into adipocytes by promoting the translocation of the glucose transporter isoform 4 (GLUT4) from intracellular vesicles to the plasma membrane. In 3T3-L1 adipocytes GLUT4 resides both in an endosomal pool, together with transferrin receptors, and in a unique pool termed ‘GLUT4 storage vesicles’(GSVs), which excludes endosomal proteins. The trafficking of GLUT4 vesicles was studied in living 3T3-L1 adipocytes by time-lapse confocal microscopy of GLUT4 tagged with green fluorescent protein. GLUT4 vesicles exhibited two types of motion: rapid vibrations around a point and short (generally less than 10µm) linear movements. The linear movements were completely blocked by incubation of the cells in the presence of microtubule-depolymerizing agents. This suggests that a subpopulation of GLUT4 vesicles can exhibit motor-driven movements along microtubules. Upon further examination, microtubule depolymerization inhibited insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane by approx. 40%, but had no effect on insulin-induced translocation of the transferrin receptor to the plasma membrane from endosomes. We propose that an intact microtubule cytoskeleton may be required for optimal trafficking of GLUT4 present in the GSV pool, but not that resident in the endosomal pool.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

scholarly journals Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Andi R. Sultan ◽  
Kirby R. Lattwein ◽  
Nicole A. Lemmens-den Toom ◽  
Susan V. Snijders ◽  
Klazina Kooiman ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Clonal Complex ◽  
Regulatory Pathways ◽  
Immune Modulator ◽  
Viability Assay ◽  
Analgesic Agents ◽  
Green Fluorescent

AbstractStaphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.

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