Publications by authors named "David E James"

209 Publications

The aetiology and molecular landscape of insulin resistance.

Nat Rev Mol Cell Biol 2021 Jul 20. Epub 2021 Jul 20.

Internal Medicine Research Unit, Pfizer Inc., Cambridge, MA, USA.

Insulin resistance, defined as a defect in insulin-mediated control of glucose metabolism in tissues - prominently in muscle, fat and liver - is one of the earliest manifestations of a constellation of human diseases that includes type 2 diabetes and cardiovascular disease. These diseases are typically associated with intertwined metabolic abnormalities, including obesity, hyperinsulinaemia, hyperglycaemia and hyperlipidaemia. Insulin resistance is caused by a combination of genetic and environmental factors. Recent genetic and biochemical studies suggest a key role for adipose tissue in the development of insulin resistance, potentially by releasing lipids and other circulating factors that promote insulin resistance in other organs. These extracellular factors perturb the intracellular concentration of a range of intermediates, including ceramide and other lipids, leading to defects in responsiveness of cells to insulin. Such intermediates may cause insulin resistance by inhibiting one or more of the proximal components in the signalling cascade downstream of insulin (insulin receptor, insulin receptor substrate (IRS) proteins or AKT). However, there is now evidence to support the view that insulin resistance is a heterogeneous disorder that may variably arise in a range of metabolic tissues and that the mechanism for this effect likely involves a unified insulin resistance pathway that affects a distal step in the insulin action pathway that is more closely linked to the terminal biological response. Identifying these targets is of major importance, as it will reveal potential new targets for treatments of diseases associated with insulin resistance.
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http://dx.doi.org/10.1038/s41580-021-00390-6DOI Listing
July 2021

Akt phosphorylates insulin receptor substrate to limit PI3K-mediated PIP3 synthesis.

Elife 2021 Jul 13;10. Epub 2021 Jul 13.

Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia.

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.
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http://dx.doi.org/10.7554/eLife.66942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8277355PMC
July 2021

Global phosphoproteomics reveals DYRK1A regulates CDK1 activity in glioblastoma cells.

Cell Death Discov 2021 Apr 16;7(1):81. Epub 2021 Apr 16.

Charles Perkins Centre and School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW, 2006, Australia.

Both tumour suppressive and oncogenic functions have been reported for dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Herein, we performed a detailed investigation to delineate the role of DYRK1A in glioblastoma. Our phosphoproteomic and mechanistic studies show that DYRK1A induces degradation of cyclin B by phosphorylating CDC23, which is necessary for the function of the anaphase-promoting complex, a ubiquitin ligase that degrades mitotic proteins. DYRK1A inhibition leads to the accumulation of cyclin B and activation of CDK1. Importantly, we established that the phenotypic response of glioblastoma cells to DYRK1A inhibition depends on both retinoblastoma (RB) expression and the degree of residual DYRK1A activity. Moderate DYRK1A inhibition leads to moderate cyclin B accumulation, CDK1 activation and increased proliferation in RB-deficient cells. In RB-proficient cells, cyclin B/CDK1 activation in response to DYRK1A inhibition is neutralized by the RB pathway, resulting in an unchanged proliferation rate. In contrast, complete DYRK1A inhibition with high doses of inhibitors results in massive cyclin B accumulation, saturation of CDK1 activity and cell cycle arrest, regardless of RB status. These findings provide new insights into the complexity of context-dependent DYRK1A signalling in cancer cells.
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http://dx.doi.org/10.1038/s41420-021-00456-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052442PMC
April 2021

BET inhibition blocks inflammation-induced cardiac dysfunction and SARS-CoV-2 infection.

Cell 2021 04 16;184(8):2167-2182.e22. Epub 2021 Mar 16.

QIMR Berghofer Medical Research Institute, Brisbane 4006, QLD, Australia.

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined but could be through direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory "cytokine-storm", a cocktail of interferon gamma, interleukin 1β, and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids (hCOs) and hearts of SARS-CoV-2-infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCOs and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression, and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the Food and Drug Administration (FDA) breakthrough designated drug, apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.
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http://dx.doi.org/10.1016/j.cell.2021.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962543PMC
April 2021

Corrigendum to 'ABHD15 regulates adipose tissue lipolysis and hepatic lipid accumulation' [Molecular Metabolism 25 (2019) p.83-94].

Mol Metab 2021 Jun 26;48:101219. Epub 2021 Mar 26.

Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, 2006, Australia; Sydney Medical School, University of Sydney, Sydney, NSW, 2006, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.molmet.2021.101219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042374PMC
June 2021

Signaling Heterogeneity is Defined by Pathway Architecture and Intercellular Variability in Protein Expression.

iScience 2021 Feb 29;24(2):102118. Epub 2021 Jan 29.

Charles Perkins Centre, The University of Sydney, Sydney, NSW 2006, Australia.

Insulin's activation of PI3K/Akt signaling, stimulates glucose uptake by enhancing delivery of GLUT4 to the cell surface. Here we examined the origins of intercellular heterogeneity in insulin signaling. Akt activation alone accounted for ~25% of the variance in GLUT4, indicating that additional sources of variance exist. The Akt and GLUT4 responses were highly reproducible within the same cell, suggesting the variance is between cells (extrinsic) and not within cells (intrinsic). Generalized mechanistic models (supported by experimental observations) demonstrated that the correlation between the steady-state levels of two measured signaling processes decreases with increasing distance from each other and that intercellular variation in protein expression (as an example of extrinsic variance) is sufficient to account for the variance in and between Akt and GLUT4. Thus, the response of a population to insulin signaling is underpinned by considerable single-cell heterogeneity that is largely driven by variance in gene/protein expression between cells.
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http://dx.doi.org/10.1016/j.isci.2021.102118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892930PMC
February 2021

Proteomics analysis of adipose depots after intermittent fasting reveals visceral fat preservation mechanisms.

Cell Rep 2021 Mar;34(9):108804

Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Camperdown, NSW, Australia. Electronic address:

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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http://dx.doi.org/10.1016/j.celrep.2021.108804DOI Listing
March 2021

PhosR enables processing and functional analysis of phosphoproteomic data.

Cell Rep 2021 Feb;34(8):108771

School of Mathematics and Statistics, The University of Sydney, Sydney, NSW, Australia; Computational Systems Biology Group, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia; Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia. Electronic address:

Mass spectrometry (MS)-based phosphoproteomics has revolutionized our ability to profile phosphorylation-based signaling in cells and tissues on a global scale. To infer the action of kinases and signaling pathways in phosphoproteomic experiments, we present PhosR, a set of tools and methodologies implemented in a suite of R packages facilitating comprehensive analysis of phosphoproteomic data. By applying PhosR to both published and new phosphoproteomic datasets, we demonstrate capabilities in data imputation and normalization by using a set of "stably phosphorylated sites" and in functional analysis for inferring active kinases and signaling pathways. In particular, we introduce a "signalome" construction method for identifying a collection of signaling modules to summarize and visualize the interaction of kinases and their collective actions on signal transduction. Together, our data and findings demonstrate the utility of PhosR in processing and generating biological knowledge from MS-based phosphoproteomic data.
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http://dx.doi.org/10.1016/j.celrep.2021.108771DOI Listing
February 2021

A co-receptor that represses beta-cell insulin action.

Nat Metab 2021 Feb;3(2):126-127

Charles Perkins Centre, University of Sydney, Sydney, New South Wales, Australia.

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http://dx.doi.org/10.1038/s42255-021-00352-4DOI Listing
February 2021

Plasma Bile Acids More Closely Align With Insulin Resistance, Visceral and Hepatic Adiposity Than Total Adiposity.

J Clin Endocrinol Metab 2021 Mar;106(3):e1131-e1139

Department of Endocrinology & Diabetes, St Vincent's Hospital, Darlinghurst, Sydney, New South Wales, Australia.

Context: The etiological mechanism of bile acid (BA) effects on insulin resistance and obesity is unknown.

Objective: This work aimed to determine whether plasma BAs are elevated in human obesity and/or insulin resistance.

Methods: This observational study was conducted at an academic research center. Seventy-one adult volunteers formed 4 groups: lean insulin-sensitive (body mass index [BMI] ≤ 25 kg/m2, Homeostatic Model Assessment of Insulin Resistance [HOMA-IR] < 2.0, n = 19), overweight/obese nondiabetic who were either insulin sensitive (Obsensitive, BMI > 25 kg/m2, HOMA-IR < 1.5, n = 11) or insulin resistant (Obresistant, BMI > 25 kg/m2, HOMA-IR > 3.0, n = 20), and type 2 diabetes (T2D, n = 21). Main outcome measures included insulin sensitivity by hyperinsulinemic-euglycemic clamp, body composition by dual energy x-ray absorptiometry, abdominal fat distribution, and liver density by computed tomography and plasma BA.

Results: In the Obresistant group, glucose infusion rate/fat-free mass (GIR/FFM, an inverse measure of insulin resistance) was significantly lower, and visceral and liver fat higher, compared to lean and Obsensitive individuals, despite similar total adiposity in Obresistant and Obsensitive. Total BA concentrations were higher in Obresistant (2.62 ± 0.333 mmol/L, P = .03) and T2D (3.36 ± 0.582 mmol/L, P < .001) vs Obsensitive (1.16 ± 0.143 mmol/L), but were similar between Obsensitive and lean (2.31 ± 0.329 mmol/L) individuals. Total BAs were positively associated with waist circumference (R = 0.245, P = .041), visceral fat (R = 0.360, P = .002), and fibroblast growth factor 21 (R = 0.341, P = .004) and negatively associated with insulin sensitivity (R = -0.395, P = .001), abdominal subcutaneous fat (R = -0.352, P = .003), adiponectin (R = -0.375, P = .001), and liver fat (Hounsfield units, an inverse marker of liver fat, R = -0.245, P = .04). Conjugated BAs were additionally elevated in T2D individuals (P < .001).

Conclusions: BA concentrations correlated with abdominal, visceral, and liver fat in humans, though an etiological role in insulin resistance remains to be verified.
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http://dx.doi.org/10.1210/clinem/dgaa940DOI Listing
March 2021

Defining the protein and lipid constituents of tubular recycling endosomes.

J Biol Chem 2021 Jan-Jun;296:100190. Epub 2021 Jan 28.

Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA; Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA. Electronic address:

Once internalized, receptors reach the sorting endosome and are either targeted for degradation or recycled to the plasma membrane, a process mediated at least in part by tubular recycling endosomes (TREs). TREs may be efficient for sorting owing to the ratio of large surface membrane area to luminal volume; following receptor segregation, TRE fission likely releases receptor-laden tubules and vesicles for recycling. Despite the importance of TRE networks for recycling, these unique structures remain poorly understood, and unresolved questions relate to their lipid and protein composition and biogenesis. Our previous studies have depicted the endocytic protein MICAL-L1 as an essential TRE constituent, and newer studies show a similar localization for the GTP-binding protein Rab10. We demonstrate that TREs are enriched in both phosphatidic acid (PA) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), supporting the idea of MICAL-L1 recruitment by PA and Rab10 recruitment via PI(4,5)P2. Using siRNA knock-down, we demonstrate that Rab10-marked TREs remain prominent in cells upon MICAL-L1 or Syndapin2 depletion. However, depletion of Rab10 or its interaction partner, EHBP1, led to loss of MICAL-L1-marked TREs. We next used phospholipase D inhibitors to decrease PA synthesis, acutely disrupt TREs, and enable monitoring of TRE regeneration after inhibitor washout. Rab10 depletion prevented TRE regeneration, whereas MICAL-L1 knock-down did not. It is surprising that EHBP1 depletion did not affect TRE regeneration under these conditions. Overall, our study supports a primary role for Rab10 and the requirement for PA and PI(4,5)P2 in TRE biogenesis and regeneration, with Rab10 likely linking the sorting endosome to motor proteins and the microtubule network.
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http://dx.doi.org/10.1074/jbc.RA120.015992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948492PMC
January 2021

Kinetic Trans-omic Analysis Reveals Key Regulatory Mechanisms for Insulin-Regulated Glucose Metabolism in Adipocytes.

iScience 2020 Aug 20;23(9):101479. Epub 2020 Aug 20.

Molecular Genetics Research Laboratory, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, Japan; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan; Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address:

Insulin regulates glucose metabolism through thousands of regulatory mechanisms; however, which regulatory mechanisms are keys to control glucose metabolism remains unknown. Here, we performed kinetic trans-omic analysis by integrating isotope-tracing glucose flux and phosphoproteomic data from insulin-stimulated adipocytes and built a kinetic mathematical model to identify key allosteric regulatory and phosphorylation events for enzymes. We identified nine reactions regulated by allosteric effectors and one by enzyme phosphorylation and determined the regulatory mechanisms for three of these reactions. Insulin stimulated glycolysis by promoting Glut4 activity by enhancing phosphorylation of AS160 at S595, stimulated fatty acid synthesis by promoting Acly activity through allosteric activation by glucose 6-phosphate or fructose 6-phosphate, and stimulated glutamate synthesis by alleviating allosteric inhibition of Gls by glutamate. Most of glycolytic reactions were regulated by amounts of substrates and products. Thus, phosphorylation or allosteric modulator-based regulation of only a few key enzymes was sufficient to change insulin-induced metabolism.
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http://dx.doi.org/10.1016/j.isci.2020.101479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479629PMC
August 2020

Insulin signaling requires glucose to promote lipid anabolism in adipocytes.

J Biol Chem 2020 09 28;295(38):13250-13266. Epub 2020 Jul 28.

School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales, Australia; Charles Perkins Centre, University of Sydney, Sydney, New South Wales, Australia; Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia. Electronic address:

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 flies 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.
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http://dx.doi.org/10.1074/jbc.RA120.014907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504926PMC
September 2020

Core functional nodes and sex-specific pathways in human ischaemic and dilated cardiomyopathy.

Nat Commun 2020 06 2;11(1):2843. Epub 2020 Jun 2.

Precision Cardiovascular Laboratory, The University of Sydney, Sydney, NSW, Australia.

Poor access to human left ventricular myocardium is a significant limitation in the study of heart failure (HF). Here, we utilise a carefully procured large human heart biobank of cryopreserved left ventricular myocardium to obtain direct molecular insights into ischaemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM), the most common causes of HF worldwide. We perform unbiased, deep proteomic and metabolomic analyses of 51 left ventricular (LV) samples from 44 cryopreserved human ICM and DCM hearts, compared to age-, gender-, and BMI-matched, histopathologically normal, donor controls. We report a dramatic reduction in serum amyloid A1 protein in ICM hearts, perturbed thyroid hormone signalling pathways and significant reductions in oxidoreductase co-factor riboflavin-5-monophosphate and glycolytic intermediate fructose-6-phosphate in both; unveil gender-specific changes in HF, including nitric oxide-related arginine metabolism, mitochondrial substrates, and X chromosome-linked protein and metabolite changes; and provide an interactive online application as a publicly-available resource.
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http://dx.doi.org/10.1038/s41467-020-16584-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266817PMC
June 2020

PGC1α Controls Sucrose Taste Sensitization in Drosophila.

Cell Rep 2020 04;31(1):107480

The Dr. John and Anne Chong Laboratory for Functional Genomics, Charles Perkins Centre and School of Life & Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia; Molecular Evolution Laboratory, Centenary Institute, The University of Sydney, Sydney, NSW 2006, Australia. Electronic address:

Perceived palatability of food controls caloric intake. Sweet taste is the primary means of detecting the carbohydrate content of food. Surprisingly, sweet taste sensitivity is responsive to extrinsic factors like diet, and this occurs by unknown mechanisms. Here, we describe an unbiased proteomic investigation into sweet taste sensitivity in the fruit fly. We identify a dopamine/cyclic AMP (cAMP)/CREB axis acting within sweet taste neurons that controls taste perception but is largely dispensable for acute taste transduction. This pathway modulates sweet taste perception in response to both sensory- and nutrient-restricted diets and converges on PGC1α, a critical regulator of metabolic health and lifespan. By electrophysiology, we found that enhanced sucrose taste sensitivity was the result of heightened sweet taste intensity and that PGC1α was both necessary and sufficient for this effect. Together, we provide the first molecular insight into how diet-induced taste perception is regulated within the sweet taste neuron.
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http://dx.doi.org/10.1016/j.celrep.2020.03.044DOI Listing
April 2020

Quantification of exercise-regulated ubiquitin signaling in human skeletal muscle identifies protein modification cross talk via NEDDylation.

FASEB J 2020 04 5;34(4):5906-5916. Epub 2020 Mar 5.

Charles Perkins Centre, School of Life and Environmental Science, The University of Sydney, Sydney, NSW, Australia.

The maintenance of muscle function is extremely important for whole body health and exercise is essential to this process. The ubiquitin-proteasome system (UPS) is required for muscle adaptation following exercise but little is known about acute posttranslational regulation and proteome remodeling during and after high-intensity exercise. Here, we used quantitative proteomics to study ubiquitin signaling dynamics in human skeletal muscle biopsies from healthy males before, during, and after a single bout of high-intensity exercise. Exercise resulted in a marked depletion of protein ubiquitylation in the vastus lateralis muscle consistent with proteasome activation. This was also associated with acute posttranslational modification of protein abundance. Integration of these data with phosphoproteomics identified co-regulated proximal modifications suggesting a cross talk between phosphorylation and ubiquitylation. We also identified additional protein modification cross talk and showed acute activation of protein NEDDylation. In vitro experiments revealed that cAMP-dependent activation of the proteasome requires NEDD8, an ubiquitin-like protein involved in protein NEDDylation, to maintain cellular protein ubiquitylation levels. Our data reveal the complexity of ubiquitin signaling and proteome remodeling in muscle during and after high-intensity exercise. We propose a model whereby exercise and the resulting cAMP signaling activate both the proteasome and ubiquitylation via NEDDylation to rapidly remove potentially damaged proteins.
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http://dx.doi.org/10.1096/fj.202000075RDOI Listing
April 2020

Dynamic C Flux Analysis Captures the Reorganization of Adipocyte Glucose Metabolism in Response to Insulin.

iScience 2020 Feb 21;23(2):100855. Epub 2020 Jan 21.

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan; Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan; CREST, Japan Science and Technology Agency, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address:

Cellular metabolism is dynamic, but quantifying non-steady metabolic fluxes by stable isotope tracers presents unique computational challenges. Here, we developed an efficient C-tracer dynamic metabolic flux analysis (13C-DMFA) framework for modeling central carbon fluxes that vary over time. We used B-splines to generalize the flux parameterization system and to improve the stability of the optimization algorithm. As proof of concept, we investigated how 3T3-L1 cultured adipocytes acutely metabolize glucose in response to insulin. Insulin rapidly stimulates glucose uptake, but intracellular pathways responded with differing speeds and magnitudes. Fluxes in lower glycolysis increased faster than those in upper glycolysis. Glycolysis fluxes rose disproportionally larger and faster than the tricarboxylic acid cycle, with lactate a primary glucose end product. The uncovered array of flux dynamics suggests that glucose catabolism is additionally regulated beyond uptake to help shunt glucose into appropriate pathways. This work demonstrates the value of using dynamic intracellular fluxes to understand metabolic function and pathway regulation.
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http://dx.doi.org/10.1016/j.isci.2020.100855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005519PMC
February 2020

Effects of lake water level fluctuation due to drought and extreme winter precipitation on mixing and water quality of an alpine lake, Case Study: Lake Arrowhead, California.

Sci Total Environ 2020 Apr 18;714:136762. Epub 2020 Jan 18.

Water Quality Solutions Inc., McGaheysville, VA, USA. Electronic address:

Lake Arrowhead, an oligotrophic alpine lake in southern California, experienced a drought period from summer 2012 to winter 2018 followed by a season of intense storms in winter 2019 resulting in lake flooding. This study investigates the effects of seasonal variations combined with 3.5 m water level fluctuation from May 2018 to April 2019, on water quality and hydrodynamics of Lake Arrowhead. In-situ measured meteorological data and water quality profiles in five different bays were used to develop and calibrate a three-dimensional lake hydrodynamic model. The mean relative errors between simulated and measured temperature and salinity profiles were 6.1% and 4.2%, respectively. Root mean square errors between the measured and simulated water temperatures were slightly larger during the stratified period. However, no specific pattern was observed in error analysis of salinity simulations. Strong thermal stratification during summer and early-fall resulted in hypoxic hypolimnetic waters with dissolved oxygen (DO) concentrations of <1 mg L. Turbulent kinetic energy (TKE) generated by convective motions in the water column due to surface heat loss was typically more than two times greater than the wind-induced mixing energy during the stratification period. The lake experienced an energetic turbulent mixing regime with TKE fluxes >1.5 m s, and Lake numbers <0.1 during the winter cooling period, resulting in a complete water column turnover and resuspension of bottom sediments. Entrainment of the hypoxic hypolimnion layers and sediment resuspension resulted in decreased DO and pH in the water column from December 2018 through mid-January 2019. Comparisons of Wedderburn and Lake numbers during different stratification conditions indicated the same trends in the strong stratification period (square of buoyancy frequency >10 s). However, in other conditions, the Lake number, considering the lake bathymetry and density profile, could better reflect vertical mixing conditions.
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http://dx.doi.org/10.1016/j.scitotenv.2020.136762DOI Listing
April 2020

Global redox proteome and phosphoproteome analysis reveals redox switch in Akt.

Nat Commun 2019 12 2;10(1):5486. Epub 2019 Dec 2.

Charles Perkins Centre, The University of Sydney, Sydney, NSW, 2006, Australia.

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.
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http://dx.doi.org/10.1038/s41467-019-13114-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889415PMC
December 2019

Estimation of water quality profiles in deep lakes based on easily measurable constituents at the water surface using artificial neural networks coupled with stationary wavelet transform.

Sci Total Environ 2019 Dec 1;694:133690. Epub 2019 Aug 1.

Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, MS, USA. Electronic address:

This study proposes a novel framework to accurately estimate water quality profiles in deep lakes based on parameters measured at the water surface, considering Boulder Basin of Lake Mead as a case study. Hourly-measured meteorological data were used to compute heat exchange between lake and atmosphere. Heat fluxes combined with every 6-hour measured water temperature, conductivity, and dissolved oxygen (DO) profiles, from the water surface to a depth of 100 m over a 48-month period, were used to train seven different artificial neural network-based methods for estimating water quality profiles. Effects of different factors influencing lake water quality, including lake-atmosphere interactions, wind-induced mixing, thermocline depth, winter turnover, oxygen depletion and other factors were investigated in different methods. A method employing stationary wavelet transform with a depth-progressive estimation of temperature, conductivity, and DO generated the smallest average relative errors of 0.52%, 0.22%, and 0.62%, respectively in the water column over a 48-month period. Abrupt changes in temperature, conductivity, and DO profiles due to thermal stratification, winter turnover, and oxygen hypoxia increased estimation errors. The largest errors occurred near the interface between the epilimnion and metalimnion, where vertical mixing intensity significantly decreased.
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http://dx.doi.org/10.1016/j.scitotenv.2019.133690DOI Listing
December 2019

Mitochondrial oxidants, but not respiration, are sensitive to glucose in adipocytes.

J Biol Chem 2020 01 19;295(1):99-110. Epub 2019 Nov 19.

School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales 2006, Australia; Charles Perkins Centre, The University of Sydney, Sydney, New South Wales 2006, Australia; Sydney Medical School, The University of Sydney, Sydney, New South Wales 2006, Australia. Electronic address:

Insulin action in adipose tissue is crucial for whole-body glucose homeostasis, with insulin resistance being a major risk factor for metabolic diseases such as type 2 diabetes. Recent studies have proposed mitochondrial oxidants as a unifying driver of adipose insulin resistance, serving as a signal of nutrient excess. However, neither the substrates for nor sites of oxidant production are known. Because insulin stimulates glucose utilization, we hypothesized that glucose oxidation would fuel respiration, in turn generating mitochondrial oxidants. This would impair insulin action, limiting further glucose uptake in a negative feedback loop of "glucose-dependent" insulin resistance. Using primary rat adipocytes and cultured 3T3-L1 adipocytes, we observed that insulin increased respiration, but notably this occurred independently of glucose supply. In contrast, glucose was required for insulin to increase mitochondrial oxidants. Despite rising to similar levels as when treated with other agents that cause insulin resistance, glucose-dependent mitochondrial oxidants failed to cause insulin resistance. Subsequent studies revealed a temporal relationship whereby mitochondrial oxidants needed to increase before the insulin stimulus to induce insulin resistance. Together, these data reveal that () adipocyte respiration is principally fueled from nonglucose sources; () there is a disconnect between respiration and oxidative stress, whereby mitochondrial oxidant levels do not rise with increased respiration unless glucose is present; and () mitochondrial oxidative stress must precede the insulin stimulus to cause insulin resistance, explaining why short-term, insulin-dependent glucose utilization does not promote insulin resistance. These data provide additional clues to mechanistically link nutrient excess to adipose insulin resistance.
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http://dx.doi.org/10.1074/jbc.RA119.011695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952605PMC
January 2020

Growth Factor-Dependent and -Independent Activation of mTORC2.

Trends Endocrinol Metab 2020 01 4;31(1):13-24. Epub 2019 Nov 4.

Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Copenhagen, Denmark. Electronic address:

The target of rapamycin complex 2 (TORC2) was discovered in 2002 in budding yeast. Its mammalian counterpart, mTORC2, was first described in 2004. Soon thereafter it was demonstrated that mTORC2 directly phosphorylates Akt on Ser473, ending a long search for the elusive 'second' insulin-responsive Akt kinase. In this review we discuss key evidence pertaining to the subcellular localization of mTORC2, highlighting a spatial heterogeneity that relates to mTORC2 activation. We summarize current models for how growth factors (GFs), such as insulin, trigger mTORC2 activation, and we provide a comprehensive discussion focusing on a new exciting frontier, the molecular mechanisms underpinning GF-independent activation of mTORC2.
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http://dx.doi.org/10.1016/j.tem.2019.09.005DOI Listing
January 2020

Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion.

Cell Rep 2019 11;29(6):1524-1538.e6

Charles Perkins Centre, School of Life and Environmental Sciences, The University of Sydney, Camperdown, NSW, Australia; Charles Perkins Centre, Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia. Electronic address:

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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http://dx.doi.org/10.1016/j.celrep.2019.10.001DOI Listing
November 2019

Lactate production is a prioritized feature of adipocyte metabolism.

J Biol Chem 2020 01 5;295(1):83-98. Epub 2019 Nov 5.

School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales 2006, Australia; Charles Perkins Centre, The University of Sydney, Sydney, New South Wales 2006, Australia; Sydney Medical School, The University of Sydney, Sydney, New South Wales 2006, Australia. Electronic address:

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 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.
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http://dx.doi.org/10.1074/jbc.RA119.011178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952601PMC
January 2020

Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes.

J Biol Chem 2019 11 22;294(45):16729-16739. Epub 2019 Sep 22.

Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales 2006, Australia

The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation, Akt is phosphorylated at Ser; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser phosphorylation in 3T3-L1 adipocytes by preventing Ser phosphorylation without perturbing mTORC2 activity. This was achieved by utilizing a chemical genetics approach, where ectopically expressed S474A Akt2 was engineered with a W80A mutation to confer resistance to the Akt inhibitor MK2206, and thus allow its activation independent of endogenous Akt. We found that insulin-stimulated phosphorylation of four Akt substrates (TSC2, PRAS40, FOXO1/3a, and AS160) was reduced by ∼50% in the absence of Ser phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser phosphorylation. We propose a model where Ser phosphorylation is required for maximal Akt2 kinase activity in adipocytes.
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http://dx.doi.org/10.1074/jbc.RA119.010036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851323PMC
November 2019

A modified gas-trapping method for high-throughput metabolic experiments in .

Biotechniques 2019 09 6;67(3):123-125. Epub 2019 Aug 6.

Charles Perkins Centre, The University of Sydney, Camperdown, NSW 2006, Australia.

Metabolism is often studied in animal models, with the fruit fly model offering ease of genetic manipulation and high-throughput studies. Fly metabolism is typically studied using end-point assays that are simple but destructive, and do not provide information on the utilization of specific nutrients. To address these limitations, we adapted existing gas-trapping protocols to measure the oxidation of radiolabeled substrates (such as glucose) in multi-well plates. This protocol is cost effective, simple, and offers precise control over experimental diet and measurement time, thus being amenable to high-throughput studies. Furthermore, it is nondestructive, enabling time-course experiments and multiplexing with other parameters. Overall, this protocol is useful for merging fly genetics with metabolic studies to understand whole organism responses to different macronutrients.
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http://dx.doi.org/10.2144/btn-2019-0062DOI Listing
September 2019

Phosphoproteomics reveals conserved exercise-stimulated signaling and AMPK regulation of store-operated calcium entry.

EMBO J 2019 12 5;38(24):e102578. Epub 2019 Aug 5.

Charles Perkins Centre, School of Life and Environmental Sciences, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia.

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.
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http://dx.doi.org/10.15252/embj.2019102578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912027PMC
December 2019

Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma.

Mol Cell Proteomics 2019 09 15;18(9):1899-1915. Epub 2019 Jul 15.

‡Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia. Electronic address:

Unbiased and sensitive quantification of low abundance small proteins in human plasma ( hormones, immune factors, metabolic regulators) remains an unmet need. These small protein factors are typically analyzed individually and using antibodies that can lack specificity. Mass spectrometry (MS)-based proteomics has the potential to address these problems, however the analysis of plasma by MS is plagued by the extremely large dynamic range of this body fluid, with protein abundances spanning at least 13 orders of magnitude. Here we describe an enrichment assay (SPEA), that greatly simplifies the plasma dynamic range problem by enriching small-proteins of 2-10 kDa, enabling the rapid, specific and sensitive quantification of >100 small-protein factors in a single untargeted LC-MS/MS acquisition. Applying this method to perform deep-proteome profiling of human plasma we identify C5ORF46 as a previously uncharacterized human plasma protein. We further demonstrate the reproducibility of our workflow for low abundance protein analysis using a stable-isotope labeled protein standard of insulin spiked into human plasma. SPEA provides the ability to study numerous important hormones in a single rapid assay, which we applied to study the intermittent fasting response and observed several unexpected changes including decreased plasma abundance of the iron homeostasis regulator hepcidin.
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http://dx.doi.org/10.1074/mcp.TIR119.001562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731089PMC
September 2019
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