GLUT4 exocytosis Stöckli, Jacqueline; Fazakerley, Daniel J; James, David E
Journal of cell science,
12/2011, Letnik:
124, Številka:
Pt 24
Journal Article
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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.
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.
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•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.
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.
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•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.
Mitochondrial oxidative stress, mitochondrial dysfunction, or both have been implicated in insulin resistance. However, disentangling the individual roles of these processes in insulin resistance has ...been difficult because they often occur in tandem, and tools that selectively increase oxidant production without impairing mitochondrial respiration have been lacking. Using the dimer/monomer status of peroxiredoxin isoforms as an indicator of compartmental hydrogen peroxide burden, we provide evidence that oxidative stress is localized to mitochondria in insulin-resistant 3T3-L1 adipocytes and adipose tissue from mice. To dissociate oxidative stress from impaired oxidative phosphorylation and study whether mitochondrial oxidative stress per se can cause insulin resistance, we used mitochondria-targeted paraquat (MitoPQ) to generate superoxide within mitochondria without directly disrupting the respiratory chain. At ≤10 μm, MitoPQ specifically increased mitochondrial superoxide and hydrogen peroxide without altering mitochondrial respiration in intact cells. Under these conditions, MitoPQ impaired insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation to the plasma membrane in both adipocytes and myotubes. MitoPQ recapitulated many features of insulin resistance found in other experimental models, including increased oxidants in mitochondria but not cytosol; a more profound effect on glucose transport than on other insulin-regulated processes, such as protein synthesis and lipolysis; an absence of overt defects in insulin signaling; and defective insulin- but not AMP-activated protein kinase (AMPK)-regulated GLUT4 translocation. We conclude that elevated mitochondrial oxidants rapidly impair insulin-regulated GLUT4 translocation and significantly contribute to insulin resistance and that MitoPQ is an ideal tool for studying the link between mitochondrial oxidative stress and regulated GLUT4 trafficking.
Systems genetics has begun to tackle the complexity of insulin resistance by capitalising on computational advances to study high-diversity populations. 'Diversity Outbred in Australia (DOz)' is a ...population of genetically unique mice with profound metabolic heterogeneity. We leveraged this variance to explore skeletal muscle's contribution to whole-body insulin action through metabolic phenotyping and skeletal muscle proteomics of 215 DOz mice. Linear modelling identified 553 proteins that associated with whole-body insulin sensitivity (Matsuda Index) including regulators of endocytosis and muscle proteostasis. To enrich for causality, we refined this network by focusing on negatively associated, genetically regulated proteins, resulting in a 76-protein fingerprint of insulin resistance. We sought to perturb this network and restore insulin action with small molecules by integrating the Broad Institute Connectivity Map platform and in vitro assays of insulin action using the Prestwick chemical library. These complementary approaches identified the antibiotic thiostrepton as an insulin resistance reversal agent. Subsequent validation in ex vivo insulin-resistant mouse muscle and palmitate-induced insulin-resistant myotubes demonstrated potent insulin action restoration, potentially via upregulation of glycolysis. This work demonstrates the value of a drug-centric framework to validate systems-level analysis by identifying potential therapeutics for insulin resistance.
Metabolic disease is caused by a combination of genetic and environmental factors, yet few studies have examined how these factors influence signal transduction, a key mediator of metabolism. Using ...mass spectrometry-based phosphoproteomics, we quantified 23,126 phosphosites in skeletal muscle of five genetically distinct mouse strains in two dietary environments, with and without acute in vivo insulin stimulation. Almost half of the insulin-regulated phosphoproteome was modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affected insulin signalling in a strain-dependent manner. Our data revealed coregulated subnetworks within the insulin signalling pathway, expanding our understanding of the pathway's organisation. Furthermore, associating diverse signalling responses with insulin-stimulated glucose uptake uncovered regulators of muscle insulin responsiveness, including the regulatory phosphosite S469 on Pfkfb2, a key activator of glycolysis. Finally, we confirmed the role of glycolysis in modulating insulin action in insulin resistance. Our results underscore the significance of genetics in shaping global signalling responses and their adaptability to environmental changes, emphasising the utility of studying biological diversity with phosphoproteomics to discover key regulatory mechanisms of complex traits.
Obesity is associated with metabolic dysfunction, including insulin resistance and hyperinsulinemia, and with disorders such as cardiovascular disease, osteoporosis, and neurodegeneration. Typically, ...these pathologies are examined in discrete model systems and with limited temporal resolution, and whether these disorders co-occur is therefore unclear. To address this question, here we examined multiple physiological systems in male C57BL/6J mice following prolonged exposure to a high-fat/high-sucrose diet (HFHSD). HFHSD-fed mice rapidly exhibited metabolic alterations, including obesity, hyperleptinemia, physical inactivity, glucose intolerance, peripheral insulin resistance, fasting hyperglycemia, ectopic lipid deposition, and bone deterioration. Prolonged exposure to HFHSD resulted in morbid obesity, ectopic triglyceride deposition in liver and muscle, extensive bone loss, sarcopenia, hyperinsulinemia, and impaired short-term memory. Although many of these defects are typically associated with aging, HFHSD did not alter telomere length in white blood cells, indicating that this diet did not generally promote all aspects of aging. Strikingly, glucose homeostasis was highly dynamic. Glucose intolerance was evident in HFHSD-fed mice after 1 week and was maintained for 24 weeks. Beyond 24 weeks, however, glucose tolerance improved in HFHSD-fed mice, and by 60 weeks, it was indistinguishable from that of chow-fed mice. This improvement coincided with adaptive β-cell hyperplasia and hyperinsulinemia, without changes in insulin sensitivity in muscle or adipose tissue. Assessment of insulin secretion in isolated islets revealed that leptin, which inhibited insulin secretion in the chow-fed mice, potentiated glucose-stimulated insulin secretion in the HFHSD-fed mice after 60 weeks. Overall, the excessive calorie intake was accompanied by deteriorating function of numerous physiological systems.
Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, ...we performed mass spectrometry‐based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and in situ muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross‐species phosphosite responses, as well as unique model‐specific regulation. We identified > 22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK‐mediated phosphorylation of STIM1 negatively regulates store‐operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross‐species resource of exercise‐regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.
Synopsis
Exercise stimulates cellular and physiological adaptations associated with widespread health benefits, however the underlying signalling events remain unclear. Here, integrated cross‐species analysis of exercise‐regulated protein phosphorylation in the skeletal muscle uncovers conserved signaling networks including crosstalk between AMPK and intracellular calcium flux.
Phospoproteomic analysis of human, rat and mouse skeletal muscle identifies > 5,000 exercise‐regulated phosphosites.
Cross‐species integration reveals conserved pathways and a compendium of potential exercise regulators.
AMPK phosphorylates stromal interaction molecule 1 (STIM1) and inhibits skeletal muscle store‐operated calcium entry.
Inhibition of STIM1 improves exercise tolerance and delays fatigue in Drosophila.
Global analysis of contraction‐regulated protein phosphorylation in skeletal muscle uncovers signalling nodes conserved between mouse, rat and human.
Skeletal muscle and adipose tissue insulin resistance are major drivers of metabolic disease. To uncover pathways involved in insulin resistance, specifically in these tissues, we leveraged the ...metabolic diversity of different dietary exposures and discrete inbred mouse strains. This revealed that muscle insulin resistance was driven by gene-by-environment interactions and was strongly correlated with hyperinsulinemia and decreased levels of ten key glycolytic enzymes. Remarkably, there was no relationship between muscle and adipose tissue insulin action. Adipocyte size profoundly varied across strains and diets, and this was strongly correlated with adipose tissue insulin resistance. The A/J strain, in particular, exhibited marked adipocyte insulin resistance and hypertrophy despite robust muscle insulin responsiveness, challenging the role of adipocyte hypertrophy per se in systemic insulin resistance. These data demonstrate that muscle and adipose tissue insulin resistance can occur independently and underscore the need for tissue-specific interrogation to understand metabolic disease.
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•Gene-by-environment interactions drive tissue insulin action•Adipose insulin resistance is dissociated from systemic insulin resistance•Large adipocytes strongly associate with adipose insulin resistance•Muscle insulin resistance correlates with hyperinsulinemia and decreased glycolysis
Using a diverse panel of mouse strains, Nelson et al. reveal that muscle insulin resistance is most closely linked to levels of circulating insulin and muscle glycolytic enzymes. Intriguingly, certain strains had enlarged fat cells and adipose insulin resistance, but completely healthy muscle insulin response, showing tissue-specific progression toward metabolic disease.
Insulin and exercise stimulate glucose uptake into skeletal muscle via different pathways. Both stimuli converge on the translocation of the glucose transporter GLUT4 from intracellular vesicles to ...the cell surface. Two Rab guanosine triphosphatases-activating proteins (GAPs) have been implicated in this process: AS160 for insulin stimulation and its homolog, TBC1D1, are suggested to regulate exercise-mediated glucose uptake into muscle. TBC1D1 has also been implicated in obesity in humans and mice. We investigated the role of TBC1D1 in glucose metabolism by generating TBC1D1(-/-) mice and analyzing body weight, insulin action, and exercise. TBC1D1(-/-) mice showed normal glucose and insulin tolerance, with no difference in body weight compared with wild-type littermates. GLUT4 protein levels were reduced by ∼40% in white TBC1D1(-/-) muscle, and TBC1D1(-/-) mice showed impaired exercise endurance together with impaired exercise-mediated 2-deoxyglucose uptake into white but not red muscles. These findings indicate that the RabGAP TBC1D1 plays a key role in regulating GLUT4 protein levels and in exercise-mediated glucose uptake in nonoxidative muscle fibers.
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