Isolation of GLUT4 Storage Vesicles

Kandror, Konstantin V.; Pilch, Paul F.

doi:10.1002/0471143030.cb0320s30

Cited by 5 publications

(4 citation statements)

References 34 publications

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“…We turned to a detergent-free, immunoadsorption method to probe the effect of GIP on the localization of the GIPR. This method has been well established in studies of GLUT4 glucose transporter trafficking in adipocytes (Bruno et al, 2016; Kandror and Pilch, 2006). Briefly, cells are broken in the absence of detergent to preserve membranes with their embedded proteins.…”

Section: Resultsmentioning

confidence: 99%

Downregulation of a GPCR by β-Arrestin2-Mediated Switch from an Endosomal to a TGN Recycling Pathway

et al. 2016

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Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in nutrient homeostasis. GIP receptor (GIPR) is constitutively internalized and returned to the plasma membrane, atypical behavior for a G protein-coupled receptor (GPCR). GIP promotes GIPR downregulation from the plasma membrane by inhibiting recycling without affecting internalization. This transient desensitization is achieved by altered intracellular trafficking of activated GIPR. GIP stimulation induces a switch in GIPR recycling from a rapid endosomal to a slow TGN pathway. GPCR kinases and β-arrestin2 are required for this switch in recycling. A coding sequence variant of GIPR, which has been associated with metabolic alterations, has altered post-activation trafficking characterized by enhanced downregulation and prolonged desensitization. Downregulation of the variant requires β-arrestin2 targeting to the TGN but is independent of GPCR kinases. The single amino acid substitution in the variant biases the receptor to promote GIP stimulated β-arrestin2 recruitment without receptor phosphorylation, thereby enhancing downregulation.

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Section: Resultsmentioning

confidence: 99%

Downregulation of a GPCR by β-Arrestin2-Mediated Switch from an Endosomal to a TGN Recycling Pathway

et al. 2016

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“…After excess uranyl acetate was removed with filter paper, the grids were examined under a transmission electron microscope (Hitachi H7600, Hitachi, Tokyo, Japan) at 80 kV. For immuno-EM, we used a modified whole-mount immuno-EM method 29 . Briefly, purified exosomes were incubated with anti-PD-L1 (or PD-L1 isotype) antibody in blocking buffer (PBS containing 1% bovine serum albumin [BSA]) for 2 h. Then, 5 μg of the exosome suspensions was applied to formvar/carbon-coated nickel grids (200 mesh) for 3 min.…”

Section: Methodsmentioning

confidence: 99%

Exosomal PD-L1 promotes tumor growth through immune escape in non-small cell lung cancer

et al. 2019

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Programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) pathway blockade is a promising new cancer therapy. Although PD-1/PD-L1 treatment has yielded clinical benefits in several types of cancer, further studies are required to clarify predictive biomarkers for drug efficacy and to understand the fundamental mechanism of PD-1/PD-L1 interaction between host and tumor cells. Here, we show that exosomes derived from lung cancer cells express PD-L1 and play a role in immune escape by reducing T-cell activity and promoting tumor growth. The abundance of PD-L1 on exosomes represented the quantity of PD-L1 expression on cell surfaces. Exosomes containing PD-L1 inhibited interferon-gamma (IFN-γ) secretion by Jurkat T cells. IFN-γ secretion was restored by PD-L1 knockout or masking on the exosomes. Both forced expression of PD-L1 on cells without PD-L1 and treatment with exosomes containing PD-L1 enhanced tumor growth in vivo. PD-L1 was present on exosomes isolated from the plasma of patients with non-small cell lung cancer, and its abundance in exosomes was correlated with PD-L1 positivity in tumor tissues. Exosomes can impair immune functions by reducing cytokine production and inducing apoptosis in CD8+ T cells. Our findings indicate that tumor-derived exosomes expressing PD-L1 may be an important mediator of tumor immune escape.

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“…Insulin resistance is an earlier stage in the development of type 2 diabetes, and skeletal muscle, as the largest glucose consumption and insulin-sensitive organ in the human body, is responsible for the disposal of ~2/3 of postprandial blood glucose. 10,37 Therefore, it is of great significance to study the molecular mechanism of insulin resistance in skeletal muscle induced by statins. Although Hwang et al have reported that the TAZ/IRS1 axis mediates statin-induced skeletal muscle insulin resistance, 4 whether and how GGPP is involved in statin-induced skeletal muscle insulin resistance remains unclear.…”

Section: Discussionmentioning

confidence: 99%

“…These results strongly support the conclusion that statins cause new‐onset diabetes. Insulin resistance is an earlier stage in the development of type 2 diabetes, and skeletal muscle, as the largest glucose consumption and insulin‐sensitive organ in the human body, is responsible for the disposal of ~2/3 of postprandial blood glucose 10,37 . Therefore, it is of great significance to study the molecular mechanism of insulin resistance in skeletal muscle induced by statins.…”

Section: Discussionmentioning

confidence: 99%

Geranylgeranyl pyrophosphate depletion by statins compromises skeletal muscle insulin sensitivity

Wang

Zheng

Zhu

et al. 2022

J cachexia sarcopenia muscle

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Background Statins are widely prescribed cholesterol-lowering drugs but have been shown to increase the risk of type 2 diabetes mellitus. However, the molecular mechanisms underlying the diabetogenic effect of statins are still not fully understood. Methods The effects of geranylgeranyl transferase I and II (GGTase I and II) inhibition on insulin-stimulated glucose uptake and GLUT4 translocation, and the dependence of these effects on insulin signalling were investigated in skeletal muscle cells. The protective effects of geranylgeranyl pyrophosphate (GGPP) and its precursor geranylgeraniol (GGOH) on simvastatin-induced insulin resistance were evaluated in vitro and in vivo. The effect of GGTase II inhibition in skeletal muscle on insulin sensitivity in vivo was confirmed by adeno-associated virus serotype 9 (AAV9)-mediated knockdown of the specific subunit of GGTase II, RABGGTA. The regulatory mechanisms of GGTase I on insulin signalling and GGTase II on insulin-stimulated GLUT4 translocation were investigated by knockdown of RhoA, TAZ, IRS1, geranylgeranylation site mutation of RhoA, RAB8A, and RAB13. Results Both inhibition of GGTase I and II mimicked simvastatin-induced insulin resistance in skeletal muscle cells. GGPP and GGOH were able to prevent simvastatin-induced skeletal muscle insulin resistance in vitro and in vivo. GGTase I inhibition suppressed the phosphorylation of AKT (Ser473) (À51.3%, P < 0.01), while GGTase II inhibition had no effect on it. AAV9-mediated knockdown of RABGGTA in skeletal muscle impaired glucose disposal without disrupting insulin signalling in vivo (À46.2% for gastrocnemius glucose uptake, P < 0.001; À52.5% for tibialis anterior glucose uptake, P < 0.001; À17.8% for soleus glucose uptake, P < 0.05; À31.4% for extensor digitorum longus glucose uptake, P < 0.01). Inhibition of RhoA, TAZ, IRS1, or geranylgeranylation deficiency of RhoA attenuated the beneficial effect of GGPP on insulin signalling in skeletal muscle cells. Geranylgeranylation deficiency of RAB8A inhibited insulin-stimulated GLUT4 translocation and concomitant glucose uptake in skeletal muscle cells (À42.8% for GLUT4 translocation, P < 0.01; À50.6% for glucose uptake, P < 0.001). Conclusions Geranylgeranyl pyrophosphate regulates glucose uptake via GGTase I-mediated insulin signalling-dependent way and GGTase II-mediated insulin signalling-independent way in skeletal muscle. Supplementation of GGPP/GGOH could be a potential therapeutic strategy for statin-induced insulin resistance.

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Isolation of GLUT4 Storage Vesicles

Cited by 5 publications

References 34 publications

Downregulation of a GPCR by β-Arrestin2-Mediated Switch from an Endosomal to a TGN Recycling Pathway

Downregulation of a GPCR by β-Arrestin2-Mediated Switch from an Endosomal to a TGN Recycling Pathway

Exosomal PD-L1 promotes tumor growth through immune escape in non-small cell lung cancer

Geranylgeranyl pyrophosphate depletion by statins compromises skeletal muscle insulin sensitivity

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