Adipose tissue is essential for whole-body glucose homeostasis, with a primary role in lipid storage. It has been previously observed that lactate production is also an important metabolic feature of ...adipocytes, but its relationship to adipose and whole-body glucose disposal remains unclear. Therefore, using a combination of metabolic labeling techniques, here we closely examined lactate production of cultured and primary mammalian adipocytes. Insulin treatment increased glucose uptake and conversion to lactate, with the latter responding more to insulin than did other metabolic fates of glucose. However, lactate production did not just serve as a mechanism to dispose of excess glucose, because we also observed that lactate production in adipocytes did not solely depend on glucose availability and even occurred independently of glucose metabolism. This suggests that lactate production is prioritized in adipocytes. Furthermore, knocking down lactate dehydrogenase specifically in the fat body of Drosophila flies lowered circulating lactate and improved whole-body glucose disposal. These results emphasize that lactate production is an additional metabolic role of adipose tissue beyond lipid storage and release.
Intermittent fasting is a beneficial dietary treatment for obesity. But the response of each distinct adipose depot is currently poorly defined. Here we explore the response of key adipose depots to ...every-other-day fasting (EODF) in mice using proteomics. A key change in subcutaneous white adipose tissue (scWAT) and visceral WAT (vWAT) depots is an increase in mitochondrial protein content after EODF. This effect is correlated with increased fatty acid synthesis enzymes in both WAT depots but not in brown adipose tissue. Strikingly, EODF treatment downregulates lipolysis specifically in vWAT, mediated by a large decrease in the abundance of the catecholamine receptor (ADRB3). Together, these changes are important for preservation of the visceral lipid store during EODF. Enrichment analysis highlights downregulation of inflammatory collagen IV specifically in vWAT, allowing improved insulin sensitivity. This resource for adipose-depot-specific fasting adaptations in mice is available using a web-based interactive visualization.
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•Every-other-day fasting (EODF) has depot-specific effects on the adipose proteome•EODF leads to suppression of lipolysis in vWAT by repression of ADRB3•Mitochondrial content and fatty acid synthesis enzymes are higher in WAT after EODF•EODF reduces inflammatory extracellular matrix components of vWAT
Harney et al. show that every-other-day fasting improves metabolic health in mice in the absence of weight loss and examine the proteomic response of several key adipose depots. They find depot-specific increases in mitochondria, suppression of lipolysis, increased fatty acid synthesis, and changes in extracellular matrix content.
Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput ...phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of β-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.
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•Global phosphoproteomes of 9 acute cell stressors integrated with human acute exercise•Comparing AMPK activators reveals potential AMPK substrates and aripiprazole mechanism•Co-administering β-adrenergic and calcium agonists causes extensive interactions•Global secretomes show potential links between exercise benefits and upstream signaling
Needham et al. measure phosphoproteomes of acute cell stressors targeting different aspects of exercise signaling. This reveals treatment actions and creates a resource of treatments that regulate phosphosites regulated in acute aerobic exercise in human skeletal muscle. Combining isoproterenol and thapsigargin causes extensive interactions within the phosphoproteome and secretome.
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.
The phosphoinositide 3-kinase (PI3K)-Akt network is tightly controlled by feedback mechanisms that regulate signal flow and ensure signal fidelity. A rapid overshoot in insulin-stimulated recruitment ...of Akt to the plasma membrane has previously been reported, which is indicative of negative feedback operating on acute timescales. Here, we show that Akt itself engages this negative feedback by phosphorylating insulin receptor substrate (IRS) 1 and 2 on a number of residues. Phosphorylation results in the depletion of plasma membrane-localised IRS1/2, reducing the pool available for interaction with the insulin receptor. Together these events limit plasma membrane-associated PI3K and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) synthesis. We identified two Akt-dependent phosphorylation sites in IRS2 at S306 (S303 in mouse) and S577 (S573 in mouse) that are key drivers of this negative feedback. These findings establish a novel mechanism by which the kinase Akt acutely controls PIP3 abundance, through post-translational modification of the IRS scaffold.
The ability of metabolically active tissues to increase glucose uptake in response to insulin is critical to whole-body glucose homeostasis. This report describes the Dual Tracer Test, a robust ...method involving sequential retro-orbital injection of 14C2-deoxyglucose (14C2DG) alone, followed 40 min later by injection of 3H2DG with a maximal dose of insulin to quantify both basal and insulin-stimulated 2DG uptake in the same mouse. The collection of both basal and insulin-stimulated measures from a single animal is imperative for generating high-quality data since differences in insulin action may be misinterpreted mechanistically if basal glucose uptake is not accounted for. The approach was validated in a classic diet-induced model of insulin resistance and a novel transgenic mouse with reduced GLUT4 expression that, despite ubiquitous peripheral insulin resistance, did not exhibit fasting hyperinsulinemia. This suggests that reduced insulin-stimulated glucose disposal is not a primary contributor to chronic hyperinsulinemia. The Dual Tracer Test offers a technically simple assay that enables the study of insulin action in many tissues simultaneously. By administering two tracers and accounting for both basal and insulin-stimulated glucose transport, this assay halves the required sample size for studies in inbred mice and demonstrates increased statistical power to detect insulin resistance, relative to other established approaches, using a single tracer. The Dual Tracer Test is a valuable addition to the metabolic phenotyping toolbox.
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.
Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades ...to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride–glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo. Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.
Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms ...underlying IR development remain poorly understood. Previously we showed that deficiency of coenzyme Q (CoQ) is necessary and sufficient for IR in adipocytes and skeletal muscle (Fazakerley et al., 2018). Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, CoQ deficiency, mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells result in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (mice, C57BL/6J) (under chow and high-fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.
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.
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