GLUT4 exocytosis Stöckli, Jacqueline; Fazakerley, Daniel J; James, David E
Journal of cell science,
12/2011, Letnik:
124, Številka:
Pt 24
Journal Article
Recenzirano
Odprti dostop
GLUT4 is an insulin-regulated glucose transporter that is responsible for insulin-regulated glucose uptake into fat and muscle cells. In the absence of insulin, GLUT4 is mainly found in intracellular ...vesicles referred to as GLUT4 storage vesicles (GSVs). Here, we summarise evidence for the existence of these specific vesicles, how they are sequestered inside the cell and how they undergo exocytosis in the presence of insulin. In response to insulin stimulation, GSVs fuse with the plasma membrane in a rapid burst and in the continued presence of insulin GLUT4 molecules are internalised and recycled back to the plasma membrane in vesicles that are distinct from GSVs and probably of endosomal origin. In this Commentary we discuss evidence that this delivery process is tightly regulated and involves numerous molecules. Key components include the actin cytoskeleton, myosin motors, several Rab GTPases, the exocyst, SNARE proteins and SNARE regulators. Each step in this process is carefully orchestrated in a sequential and coupled manner and we are beginning to dissect key nodes within this network that determine vesicle-membrane fusion in response to insulin. This regulatory process clearly involves the Ser/Thr kinase AKT and the exquisite manner in which this single metabolic process is regulated makes it a likely target for lesions that might contribute to metabolic disease.
A major challenge of the post-genomics era is to define the connectivity of protein phosphorylation networks. Here, we quantitatively delineate the insulin signaling network in adipocytes by ...high-resolution mass spectrometry-based proteomics. These data reveal the complexity of intracellular protein phosphorylation. We identified 37,248 phosphorylation sites on 5,705 proteins in this single-cell type, with approximately 15% responding to insulin. We integrated these large-scale phosphoproteomics data using a machine learning approach to predict physiological substrates of several diverse insulin-regulated kinases. This led to the identification of an Akt substrate, SIN1, a core component of the mTORC2 complex. The phosphorylation of SIN1 by Akt was found to regulate mTORC2 activity in response to growth factors, revealing topological insights into the Akt/mTOR signaling network. The dynamic phosphoproteome described here contains numerous phosphorylation sites on proteins involved in diverse molecular functions and should serve as a useful functional resource for cell biologists.
Display omitted
•MS/MS identified >37,000 phosphorylation sites in adipocytes•Insulin regulates the phosphoproteome over a wide temporal timescale•Akt phosphorylates SIN1 on T86 in response to insulin•SIN1 phosphorylation activates a positive feedback loop between Akt and mTORC2
Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human ...skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPK-dependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry.
Display omitted
•Identification of the human muscle acute exercise signaling repertoire•Integrated AMPK substrate prediction in human muscle and cells•Targeted validation of exercise-regulated AMPK substrates•AKAP1 phosphorylation by AMPK that regulates mitochondrial respiration
Combining phosphoproteomics, biochemical, and bioinformatics approaches, Hoffman et al. perform a global analysis of exercise signaling in human skeletal muscle and reveal an interconnected network of kinases and AMPK substrates in response to exercise. Among these, AKAP1 is shown to regulate mitochondrial respiration via AMPK-dependent phosphorylation.
Insulin resistance is one of the earliest pathological features of a suite of diseases including type 2 diabetes collectively referred to as metabolic syndrome. There is a growing body of evidence ...from both pre-clinical studies and human cohorts indicating that reactive oxygen species, such as the superoxide radical anion and hydrogen peroxide are key players in the development of insulin resistance. Here we review the evidence linking mitochondrial reactive oxygen species generated within mitochondria with insulin resistance in adipose tissue and skeletal muscle, two major insulin sensitive tissues. We outline the relevant mitochondria-derived reactive species, how the mitochondrial redox state is regulated, and methodologies available to measure mitochondrial reactive oxygen species. Importantly, we highlight key experimental issues to be considered when studying the role of mitochondrial reactive oxygen species in insulin resistance. Evaluating the available literature on both mitochondrial reactive oxygen species/redox state and insulin resistance in a variety of biological systems, we conclude that the weight of evidence suggests a likely role for mitochondrial reactive oxygen species in the etiology of insulin resistance in adipose tissue and skeletal muscle. However, major limitations in the methods used to study reactive oxygen species in insulin resistance as well as the lack of data linking mitochondrial reactive oxygen species and cytosolic insulin signaling pathways are significant obstacles in proving the mechanistic link between these two processes. We provide a framework to guide future studies to provide stronger mechanistic information on the link between mitochondrial reactive oxygen species and insulin resistance as understanding the source, localization, nature, and quantity of mitochondrial reactive oxygen species, their targets and downstream signaling pathways may pave the way for important new therapeutic strategies.
Display omitted
•Mitochondrial reactive oxygen species likely play a causal role in insulin resistance.•Assaying mitochondrial reactive oxygen species is challenging.•Mitochondrial lipids provide a link between reactive species and insulin resistance.•Novel therapies in insulin resistance may target mitochondrial reactive species formation.
Insulin resistance is a major risk factor for numerous diseases, including type 2 diabetes and cardiovascular disease. These disorders have dramatically increased in incidence with modern life, ...suggesting that excess nutrients and obesity are major causes of “common” insulin resistance. Despite considerable effort, the mechanisms that contribute to common insulin resistance are not resolved. There is universal agreement that extracellular perturbations, such as nutrient excess, hyperinsulinemia, glucocorticoids, or inflammation, trigger intracellular stress in key metabolic target tissues, such as muscle and adipose tissue, and this impairs the ability of insulin to initiate its normal metabolic actions in these cells. Here, we present evidence that the impairment in insulin action is independent of proximal elements of the insulin signaling pathway and is likely specific to the glucoregulatory branch of insulin signaling. We propose that many intracellular stress pathways act in concert to increase mitochondrial reactive oxygen species to trigger insulin resistance. We speculate that this may be a physiological pathway to conserve glucose during specific states, such as fasting, and that, in the presence of chronic nutrient excess, this pathway ultimately leads to disease. This review highlights key points in this pathway that require further research effort.
The liver is a critical tissue for maintaining glucose, fatty acid, and cholesterol homeostasis. Primary hepatocytes represent the gold standard for studying the mechanisms controlling hepatic ...glucose, lipid, and cholesterol metabolism in vitro. However, access to primary hepatocytes can be limiting, and therefore, other immortalized hepatocyte models are commonly used. Here, we describe substrate metabolism of cultured AML12, IHH, and PH5CH8 cells, hepatocellular carcinoma-derived HepG2s, and primary mouse hepatocytes (PMH) to identify which of these cell lines most accurately phenocopy PMH basal and insulin-stimulated metabolism. Insulin-stimulated glucose metabolism in PH5CH8 cells, and to a lesser extent AML12 cells, responded most similarly to PMH. Notably, glucose incorporation in HepG2 cells were 14-fold greater than PMH. The differences in glucose metabolic activity were not explained by differential protein expression of key regulators of these pathways, for example glycogen synthase and glycogen content. In contrast, fatty acid metabolism in IHH cells was the closest to PMHs, yet insulin-responsive fatty acid metabolism in AML12 and HepG2 cells was most similar to PMH. Finally, incorporation of acetate into intracellular-free cholesterol was comparable for all cells to PMH; however, insulin-stimulated glucose conversion into lipids and the incorporation of acetate into intracellular cholesterol esters were strikingly different between PMHs and all tested cell lines. In general, AML12 cells most closely phenocopied PMH in vitro energy metabolism. However, the cell line most representative of PMHs differed depending on the mode of metabolism being investigated, and so careful consideration is needed in model selection.
GLUT4 On the move Fazakerley, Daniel J; Koumanov, Francoise; Holman, Geoffrey D
Biochemical journal,
02/2022, Letnik:
479, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Insulin rapidly stimulates GLUT4 translocation and glucose transport in fat and muscle cells. Signals from the occupied insulin receptor are translated into downstream signalling changes in ...serine/threonine kinases within timescales of seconds, and this is followed by delivery and accumulation of the glucose transporter GLUT4 at the plasma membrane. Kinetic studies have led to realisation that there are distinct phases of this stimulation by insulin. There is a rapid initial burst of GLUT4 delivered to the cell surface from a subcellular reservoir compartment and this is followed by a steady-state level of continuing stimulation in which GLUT4 recycles through a large itinerary of subcellular locations. Here, we provide an overview of the phases of insulin stimulation of GLUT4 translocation and the molecules that are currently considered to activate these trafficking steps. Furthermore, we suggest how use of new experimental approaches together with phospho-proteomic data may help to further identify mechanisms for activation of these trafficking processes.
FGF21 improves the metabolic profile of obese animals through its actions on adipocytes. To elucidate the signaling network responsible for mediating these effects, we quantified dynamic changes in ...the adipocyte phosphoproteome following acute exposure to FGF21. FGF21 regulated a network of 821 phosphosites on 542 proteins. A major FGF21-regulated signaling node was mTORC1/S6K. In contrast to insulin, FGF21 activated mTORC1 via MAPK rather than through the canonical PI3K/AKT pathway. Activation of mTORC1/S6K by FGF21 was surprising because this is thought to contribute to deleterious metabolic effects such as obesity and insulin resistance. Rather, mTORC1 mediated many of the beneficial actions of FGF21 in vitro, including UCP1 and FGF21 induction, increased adiponectin secretion, and enhanced glucose uptake without any adverse effects on insulin action. This study provides a global view of FGF21 signaling and suggests that mTORC1 may act to facilitate FGF21-mediated health benefits in vivo.
Display omitted
•FGF21 regulates 821 phosphosites on 542 proteins in adipocytes•FGF21 activates mTORC1 and S6K independently of AKT via MAPK•FGF21-induced mTORC1 activation was not associated with insulin resistance•Rapamycin inhibits FGF21-induced UCP1, glucose uptake, and adiponectin secretion
FGF21 signaling in adipose stimulates weight loss and insulin sensitivity during obesity. Minard et al. examine the FGF21-regulated adipocyte phosphorylation network and identify mTORC1 as a key mediator of FGF21 actions, including browning, glucose uptake, and adiponectin secretion.
Mapping Insulin/GLUT4 Circuitry Rowland, Alexander F.; Fazakerley, Daniel J.; James, David E.
Traffic (Copenhagen, Denmark),
June 2011, Letnik:
12, Številka:
6
Journal Article
Recenzirano
Odprti dostop
One of the most important metabolic actions of insulin is catalysing glucose uptake into skeletal muscle and adipose tissue. This is accomplished via activation of the ...phosphatidylinositol‐3‐kinase/Akt signalling pathway and subsequent translocation of GLUT4 from intracellular storage vesicles to the plasma membrane. As such, this represents an ideal system for studying the convergence of signal transduction and protein trafficking. The GLUT4 translocation process is complex, but can be dissected into at least four discrete trafficking steps. This raises the question as to which of these is the major regulated step in insulin‐stimulated GLUT4 translocation. Numerous molecules have been reported to regulate GLUT4 trafficking. However, with the exception of TBC1D4, the molecular details of these distal signalling arms of the insulin signalling network and how they modify distinct steps of GLUT4 trafficking have not been established. We discuss the need to adopt a more global approach to expand and deepen our understanding of the molecular processes underpinning this system. Strategies that facilitate the generation of detailed models of the entire insulin signalling network will enable us to identify the critical nodes that control GLUT4 traffic and decipher emergent properties of the system that are not currently apparent.
Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. ...Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP
binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.
PDF
