scholarly journals Dual-mode of insulin action controls GLUT4 vesicle exocytosis

2011 ◽  
Vol 193 (4) ◽  
pp. 643-653 ◽  
Author(s):  
Yingke Xu ◽  
Bradley R. Rubin ◽  
Charisse M. Orme ◽  
Alexander Karpikov ◽  
Chenfei Yu ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Insulin Signaling ◽  
Insulin Action ◽  
Fusion Pore ◽  
Dual Mode ◽  
Vesicle Traffic ◽  
Storage Vesicles ◽  
Vesicle Exocytosis ◽  
Single Vesicle ◽  
Pore Expansion

Insulin stimulates translocation of GLUT4 storage vesicles (GSVs) to the surface of adipocytes, but precisely where insulin acts is controversial. Here we quantify the size, dynamics, and frequency of single vesicle exocytosis in 3T3-L1 adipocytes. We use a new GSV reporter, VAMP2-pHluorin, and bypass insulin signaling by disrupting the GLUT4-retention protein TUG. Remarkably, in unstimulated TUG-depleted cells, the exocytic rate is similar to that in insulin-stimulated control cells. In TUG-depleted cells, insulin triggers a transient, twofold burst of exocytosis. Surprisingly, insulin promotes fusion pore expansion, blocked by acute perturbation of phospholipase D, which reflects both properties intrinsic to the mobilized vesicles and a novel regulatory site at the fusion pore itself. Prolonged stimulation causes cargo to switch from ∼60 nm GSVs to larger exocytic vesicles characteristic of endosomes. Our results support a model whereby insulin promotes exocytic flux primarily by releasing an intracellular brake, but also by accelerating plasma membrane fusion and switching vesicle traffic between two distinct circuits.

scholarly journals Spatiotemporal Regulators for Insulin-Stimulated GLUT4 Vesicle Exocytosis

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Xiaoxu Zhou ◽  
Ping Shentu ◽  
Yingke Xu
Keyword(s):  
Insulin Signaling ◽  
Signaling Molecules ◽  
Regulatory Proteins ◽  
Peripheral Insulin Resistance ◽  
Spatiotemporal Regulation ◽  
Storage Vesicles ◽  
Vesicle Exocytosis ◽  
And Storage ◽  
Adipose Cells

Insulin increases glucose uptake and storage in muscle and adipose cells, which is accomplished through the mobilization of intracellular GLUT4 storage vesicles (GSVs) to the cell surface upon stimulation. Importantly, the dysfunction of insulin-regulated GLUT4 trafficking is strongly linked with peripheral insulin resistance and type 2 diabetes in human. The insulin signaling pathway, key signaling molecules involved, and precise trafficking itinerary of GSVs are largely identified. Understanding the interaction between insulin signaling molecules and key regulatory proteins that are involved in spatiotemporal regulation of GLUT4 vesicle exocytosis is of great importance to explain the pathogenesis of diabetes and may provide new potential therapeutic targets.

scholarly journals Synaptotagmin-1 Utilizes Membrane Bending and SNARE Binding to Drive Fusion Pore Expansion

2008 ◽  
Vol 19 (12) ◽  
pp. 5093-5103 ◽  
Author(s):  
Kara L. Lynch ◽  
Roy R.L. Gerona ◽  
Dana M. Kielar ◽  
Sascha Martens ◽  
Harvey T. McMahon ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Protein Complexes ◽  
Snare Complex ◽  
Fusion Pore ◽  
Membrane Insertion ◽  
Loss Of Function ◽  
Fluorescent Peptide ◽  
Vesicle Exocytosis ◽  
Pore Opening ◽  
Pore Expansion

In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca2+ is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca2+ also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca2+-dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca2+-dependent SNARE binding or in Ca2+-dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of different-sized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca2+-dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca2+-dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca2+-dependent membrane insertion and SNARE binding to drive fusion pore expansion.

scholarly journals Membrane Binding of α-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore

2021 ◽  
Vol 9 ◽  
Author(s):  
Ryan Khounlo ◽  
Brenden J. D. Hawk ◽  
Tung-Mei Khu ◽  
Gyeongji Yoo ◽  
Nam Ki Lee ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Binding ◽  
Snare Complex ◽  
Fusion Pore ◽  
Fusion Inhibitors ◽  
Important Regulator ◽  
Fusion Pores ◽  
Single Vesicle ◽  
Pore Expansion

SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.

scholarly journals Stabilization of the SNARE Core by Complexin-1 Facilitates Fusion Pore Expansion

2021 ◽  
Vol 8 ◽  
Author(s):  
Josh Pierson ◽  
Yeon-Kyun Shin
Keyword(s):  
Neurotransmitter Release ◽  
Fluorescence Probe ◽  
Mechanistic Model ◽  
Reciprocal Relationship ◽  
Fusion Pore ◽  
Vesicle Recycling ◽  
Binding Core ◽  
Fusion Pores ◽  
Single Vesicle ◽  
Pore Expansion

In the neuron, neurotransmitter release is an essential function that must be both consistent and tightly regulated. The continuity of neurotransmitter release is dependent in large part on vesicle recycling. However, the protein factors that dictate the vesicle recycling pathway are elusive. Here, we use a single vesicle-to-supported bilayer fusion assay to investigate complexin-1 (cpx1)’s influence on SNARE-dependent fusion pore expansion. With total internal reflection (TIR) microscopy using a 10 kDa polymer fluorescence probe, we are able to detect the presence of large fusion pores. With cpx1, however, we observe a significant increase of the probability of the formation of large fusion pores. The domain deletion analysis reveals that the SNARE-binding core domain of cpx1 is mainly responsible for its ability to promote the fusion pore expansion. In addition, the results show that cpx1 helps the pore to expand larger, which results in faster release of the polymer probe. Thus, the results demonstrate a reciprocal relationship between event duration and the size of the fusion pore. Based on the data, a hypothetical mechanistic model can be deduced. In this mechanistic model, the cpx1 binding stabilizes the four-helix bundle structure of the SNARE core throughout the fusion pore expansion, whereby the highly curved bilayer within the fusion pore is stabilized by the SNARE pins.

scholarly journals Lumenal Protein within Secretory Granules Affects Fusion Pore Expansion

2014 ◽  
Vol 107 (1) ◽  
pp. 26-33 ◽  
Author(s):  
Annita Ngatchou Weiss ◽  
Arun Anantharam ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Secretory Granules ◽  
Fusion Pore ◽  
Pore Expansion

Does impaired mitochondrial function affect insulin signaling and action in cultured human skeletal muscle cells?

2008 ◽  
Vol 294 (1) ◽  
pp. E97-E102 ◽  
Author(s):  
Audrey E. Brown ◽  
Matthias Elstner ◽  
Stephen J. Yeaman ◽  
Douglass M. Turnbull ◽  
Mark Walker
Keyword(s):  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Insulin Signaling ◽  
Mitochondrial Respiration ◽  
Respiratory Function ◽  
Insulin Action ◽  
Human Skeletal Muscle ◽  
Human Skeletal Muscle Cells ◽  
Basal Glucose Uptake

Insulin-resistant type 2 diabetic patients have been reported to have impaired skeletal muscle mitochondrial respiratory function. A key question is whether decreased mitochondrial respiration contributes directly to the decreased insulin action. To address this, a model of impaired cellular respiratory function was established by incubating human skeletal muscle cell cultures with the mitochondrial inhibitor sodium azide and examining the effects on insulin action. Incubation of human skeletal muscle cells with 50 and 75 μM azide resulted in 48 ± 3% and 56 ± 1% decreases, respectively, in respiration compared with untreated cells mimicking the level of impairment seen in type 2 diabetes. Under conditions of decreased respiratory chain function, insulin-independent (basal) glucose uptake was significantly increased. Basal glucose uptake was 325 ± 39 pmol/min/mg (mean ± SE) in untreated cells. This increased to 669 ± 69 and 823 ± 83 pmol/min/mg in cells treated with 50 and 75 μM azide, respectively (vs. untreated, both P < 0.0001). Azide treatment was also accompanied by an increase in basal glycogen synthesis and phosphorylation of AMP-activated protein kinase. However, there was no decrease in glucose uptake following insulin exposure, and insulin-stimulated phosphorylation of Akt was normal under these conditions. GLUT1 mRNA expression remained unchanged, whereas GLUT4 mRNA expression increased following azide treatment. In conclusion, under conditions of impaired mitochondrial respiration there was no evidence of impaired insulin signaling or glucose uptake following insulin exposure in this model system.

scholarly journals Endothelial insulin receptors differentially control insulin signaling kinetics in peripheral tissues and brain of mice

2017 ◽  
Vol 114 (40) ◽  
pp. E8478-E8487 ◽  
Author(s):  
Masahiro Konishi ◽  
Masaji Sakaguchi ◽  
Samuel M. Lockhart ◽  
Weikang Cai ◽  
Mengyao Ella Li ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Endothelial Cells ◽  
Food Intake ◽  
Insulin Signaling ◽  
Insulin Action ◽  
Insulin Receptors ◽  
Brain Regions ◽  
Peripheral Tissues ◽  
Systemic Insulin Resistance

Insulin receptors (IRs) on endothelial cells may have a role in the regulation of transport of circulating insulin to its target tissues; however, how this impacts on insulin action in vivo is unclear. Using mice with endothelial-specific inactivation of the IR gene (EndoIRKO), we find that in response to systemic insulin stimulation, loss of endothelial IRs caused delayed onset of insulin signaling in skeletal muscle, brown fat, hypothalamus, hippocampus, and prefrontal cortex but not in liver or olfactory bulb. At the level of the brain, the delay of insulin signaling was associated with decreased levels of hypothalamic proopiomelanocortin, leading to increased food intake and obesity accompanied with hyperinsulinemia and hyperleptinemia. The loss of endothelial IRs also resulted in a delay in the acute hypoglycemic effect of systemic insulin administration and impaired glucose tolerance. In high-fat diet-treated mice, knockout of the endothelial IRs accelerated development of systemic insulin resistance but not food intake and obesity. Thus, IRs on endothelial cells have an important role in transendothelial insulin delivery in vivo which differentially regulates the kinetics of insulin signaling and insulin action in peripheral target tissues and different brain regions. Loss of this function predisposes animals to systemic insulin resistance, overeating, and obesity.

scholarly journals Chromogranin A, the major lumenal protein in chromaffin granules, controls fusion pore expansion

2018 ◽  
Vol 151 (2) ◽  
pp. 118-130 ◽  
Author(s):  
Prabhodh S. Abbineni ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Plasma Membrane ◽  
Small Molecules ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Fusion Pore ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Chromogranin B ◽  
Tissue Plasminogen ◽  
Pore Expansion

Upon fusion of the secretory granule with the plasma membrane, small molecules are discharged through the immediately formed narrow fusion pore, but protein discharge awaits pore expansion. Recently, fusion pore expansion was found to be regulated by tissue plasminogen activator (tPA), a protein present within the lumen of chromaffin granules in a subpopulation of chromaffin cells. Here, we further examined the influence of other lumenal proteins on fusion pore expansion, especially chromogranin A (CgA), the major and ubiquitous lumenal protein in chromaffin granules. Polarized TIRF microscopy demonstrated that the fusion pore curvature of granules containing CgA-EGFP was long lived, with curvature lifetimes comparable to those of tPA-EGFP–containing granules. This was surprising because fusion pore curvature durations of granules containing exogenous neuropeptide Y-EGFP (NPY-EGFP) are significantly shorter (80% lasting &lt;1 s) than those containing CgA-EGFP, despite the anticipated expression of endogenous CgA. However, quantitative immunocytochemistry revealed that transiently expressed lumenal proteins, including NPY-EGFP, caused a down-regulation of endogenously expressed proteins, including CgA. Fusion pore curvature durations in nontransfected cells were significantly longer than those of granules containing overexpressed NPY but shorter than those associated with granules containing overexpressed tPA, CgA, or chromogranin B. Introduction of CgA to NPY-EGFP granules by coexpression converted the fusion pore from being transient to being longer lived, comparable to that found in nontransfected cells. These findings demonstrate that several endogenous chromaffin granule lumenal proteins are regulators of fusion pore expansion and that alteration of chromaffin granule contents affects fusion pore lifetimes. Importantly, the results indicate a new role for CgA. In addition to functioning as a prohormone, CgA plays an important role in controlling fusion pore expansion.

Abstract 1209: Interleukin-6 Reduces Myocardial Glucose Metabolism by Altering AMPK, Insulin Signaling, and PKC-𝛉 in Mice

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Zhiyou Zhang ◽  
Hwi Jin Ko ◽  
Dae Young Jung ◽  
Zhexi Ma ◽  
Jason K Kim
Keyword(s):  
Glucose Metabolism ◽  
Insulin Signaling ◽  
Insulin Action ◽  
Cardiac Metabolism ◽  
Negative Regulator ◽  
Saline Control ◽  
Myocardial Glucose Metabolism ◽  
Peripheral Organs

Increasing evidence implicates the role of inflammation in the pathogenesis of diabetes and complications. Inflammatory cytokines (IL-6, TNF-α) are elevated in obese diabetic subjects, and are shown to modulate glucose metabolism in peripheral organs. In this report, we examined the effects of IL-6 on cardiac metabolism and insulin action in vivo. Male C57BL/6 mice were intravenously treated with IL-6 (16 ng/hr) or saline (control) for 2 hrs, and [ 14 C]2-deoxyglucose was intravenously injected in awake mice to measure myocardial glucose metabolism (n=9~10). Hyperinsulinemic-euglycemic clamps (2.5 mU/kg/min insulin infusion) were also performed in IL-6 or saline-treated mice (n=4~5) to measure cardiac insulin action. Acute treatment with IL-6 caused a 25% increase in myocardial STAT3 activity and significantly reduced basal myocardial glucose metabolism (Fig. 1 ; * P< 0.05). IL-6 treatment also reduced insulin-stimulated glucose uptake in heart, and these effects were associated with marked decreases in AMPK activity (Thr-phosphorylation of AMPK; Fig. 2 ) and IRS-1 tyrosine phosphorylation (Fig. 3 ). Acute IL-6 treatment increased myocardial expression of PKC-𝛉, which has been shown to mediate insulin resistance in peripheral organs (Fig. 4 ). These results indicate that IL-6 is a potent negative regulator of myocardial glucose metabolism and insulin action, and the underlying mechanism may involve IL-6 mediated activation of PKC-𝛉 and defects in AMPK and insulin signaling activity. Thus, our findings suggest a potential role of IL-6 in the pathogenesis of diabetic heart failure.

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