Impaired endocytosis and accumulation in early endosomal compartments defines herpes simplex virus-mediated disruption of the nonclassical MHC class I-related molecule MR1.

Presentation of metabolites by the major histocompatibility complex class I-related protein 1 (MR1) molecule to mucosal-associated invariant T cells is impaired during herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections. This is surprising given these viruses do not directly synthesise MR1 ligands. We have previously identified several HSV proteins responsible for rapidly downregulating the intracellular pool of immature MR1, effectively inhibiting new surface antigen presentation, while preexisting ligand-bound mature MR1 is unexpectedly upregulated by HSV-1. Using flow cytometry, immunoblotting, and high-throughput fluorescence microscopy, we demonstrate that the endocytosis of surface MR1 is impaired during HSV infection and that internalized molecules accumulate in EEA1-labeled early endosomes, avoiding degradation. We establish that the short MR1 cytoplasmic tail is not required for HSV-1-mediated downregulation of immature molecules; however it may play a role in the retention of mature molecules on the surface and in early endosomes. We also determine that the HSV-1 US3 protein, the shorter US3.5 kinase and the full-length HSV-2 homolog, all predominantly target mature surface rather than total MR1 levels. We propose that the downregulation of intracellular and cell surface MR1 molecules by US3 and other HSV proteins is an immune-evasive countermeasure to minimize the effect of impaired MR1 endocytosis, which might otherwise render infected cells susceptible to MR1-mediated killing by mucosal-associated invariant T cells.

Deep Proteome Profiling of White Adipose Tissue Reveals Marked Conservation and Distinct Features Between Different Anatomical Depots.

White adipose tissue is deposited mainly as subcutaneous adipose tissue (SAT), often associated with metabolic protection, and abdominal/visceral adipose tissue, which contributes to metabolic disease. To investigate the molecular underpinnings of these differences, we conducted comprehensive proteomics profiling of whole tissue and isolated adipocytes from these two depots across two diets from C57Bl/6J mice. The adipocyte proteomes from lean mice were highly conserved between depots, with the major depot-specific differences encoded by just 3% of the proteome. Adipocytes from SAT (SAdi) were enriched in pathways related to mitochondrial complex I and beiging, whereas visceral adipocytes (VAdi) were enriched in structural proteins and positive regulators of mTOR presumably to promote nutrient storage and cellular expansion. This indicates that SAdi are geared toward higher catabolic activity, while VAdi are more suited for lipid storage. By comparing adipocytes from mice fed chow or Western diet (WD), we define a core adaptive proteomics signature consisting of increased extracellular matrix proteins and decreased fatty acid metabolism and mitochondrial Coenzyme Q biosynthesis. Relative to SAdi, VAdi displayed greater changes with WD including a pronounced decrease in mitochondrial proteins concomitant with upregulation of apoptotic signaling and decreased mitophagy, indicating pervasive mitochondrial stress. Furthermore, WD caused a reduction in lipid handling and glucose uptake pathways particularly in VAdi, consistent with adipocyte de-differentiation. By overlaying the proteomics changes with diet in whole adipose tissue and isolated adipocytes, we uncovered concordance between adipocytes and tissue only in the visceral adipose tissue, indicating a unique tissue-specific adaptation to sustained WD in SAT. Finally, an in-depth comparison of isolated adipocytes and 3T3-L1 proteomes revealed a high degree of overlap, supporting the utility of the 3T3-L1 adipocyte model. These deep proteomes provide an invaluable resource highlighting differences between white adipose depots that may fine-tune their unique functions and adaptation to an obesogenic environment.

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

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.

Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate.

Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes.

Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2.

Sphingolipid dysregulation is often associated with insulin resistance, while the enzymes controlling sphingolipid metabolism are emerging as therapeutic targets for improving insulin sensitivity. We report herein that sphingosine kinase 2 (SphK2), a key enzyme in sphingolipid catabolism, plays a critical role in the regulation of hepatic insulin signaling and glucose homeostasis both in vitro and in vivo. Hepatocyte-specific Sphk2 knockout mice exhibit pronounced insulin resistance and glucose intolerance. Likewise, SphK2-deficient hepatocytes are resistant to insulin-induced activation of the phosphoinositide 3-kinase (PI3K)-Akt-FoxO1 pathway and elevated hepatic glucose production. Mechanistically, SphK2 deficiency leads to the accumulation of sphingosine that, in turn, suppresses hepatic insulin signaling by inhibiting PI3K activation in hepatocytes. Either reexpressing functional SphK2 or pharmacologically inhibiting sphingosine production restores insulin sensitivity in SphK2-deficient hepatocytes. In conclusion, the current study provides both experimental findings and mechanistic data showing that SphK2 and sphingosine in the liver are critical regulators of insulin sensitivity and glucose homeostasis.

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

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 (a) adipocyte respiration is principally fueled from nonglucose sources; (b) there is a disconnect between respiration and oxidative stress, whereby mitochondrial oxidant levels do not rise with increased respiration unless glucose is present; and (c) 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.

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

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 Ser474; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser474 phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser474 phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser474 phosphorylation in 3T3-L1 adipocytes by preventing Ser474 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 bona fide Akt substrates (TSC2, PRAS40, FOXO1/3a, and AS160) was reduced by ∼50% in the absence of Ser474 phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser474 phosphorylation. We propose a model where Ser474 phosphorylation is required for maximal Akt2 kinase activity in adipocytes.

Autoencoder-based cluster ensembles for single-cell RNA-seq data analysis.

BACKGROUND: Single-cell RNA-sequencing (scRNA-seq) is a transformative technology, allowing global transcriptomes of individual cells to be profiled with high accuracy. An essential task in scRNA-seq data analysis is the identification of cell types from complex samples or tissues profiled in an experiment. To this end, clustering has become a key computational technique for grouping cells based on their transcriptome profiles, enabling subsequent cell type identification from each cluster of cells. Due to the high feature-dimensionality of the transcriptome (i.e. the large number of measured genes in each cell) and because only a small fraction of genes are cell type-specific and therefore informative for generating cell type-specific clusters, clustering directly on the original feature/gene dimension may lead to uninformative clusters and hinder correct cell type identification. RESULTS: Here, we propose an autoencoder-based cluster ensemble framework in which we first take random subspace projections from the data, then compress each random projection to a low-dimensional space using an autoencoder artificial neural network, and finally apply ensemble clustering across all encoded datasets to generate clusters of cells. We employ four evaluation metrics to benchmark clustering performance and our experiments demonstrate that the proposed autoencoder-based cluster ensemble can lead to substantially improved cell type-specific clusters when applied with both the standard k-means clustering algorithm and a state-of-the-art kernel-based clustering algorithm (SIMLR) designed specifically for scRNA-seq data. Compared to directly using these clustering algorithms on the original datasets, the performance improvement in some cases is up to 100%, depending on the evaluation metric used. CONCLUSIONS: Our results suggest that the proposed framework can facilitate more accurate cell type identification as well as other downstream analyses. The code for creating the proposed autoencoder-based cluster ensemble framework is freely available from https://github.com/gedcom/scCCESS.

Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation.

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.

High dietary fat and sucrose results in an extensive and time-dependent deterioration in health of multiple physiological systems in mice.

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 ß-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.

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction.

Akt is a key node in a range of signal transduction cascades and play a critical role in diseases such as cancer and diabetes. Fluorescently-tagged Akt reporters have been used to discern Akt localisation, yet it has not been clear how well these tools recapitulate the behaviour of endogenous Akt proteins. Here, we observed that fusion of eGFP to Akt2 impaired both its insulin-stimulated plasma membrane recruitment and its phosphorylation. Endogenous-like responses were restored by replacing eGFP with TagRFP-T. The improved response magnitude and sensitivity afforded by TagRFP-T-Akt2 over eGFP-Akt2 enabled monitoring of signalling outcomes in single cells at physiological doses of insulin with subcellular resolution and revealed two previously unreported features of Akt biology. In 3T3-L1 adipocytes, stimulation with insulin resulted in recruitment of Akt2 to the plasma membrane in a polarised fashion. Additionally, we observed oscillations in plasma membrane localised Akt2 in the presence of insulin with a consistent periodicity of 2 min. Our studies highlight the importance of fluorophore choice when generating reporter constructs and shed light on new Akt signalling responses that may encode complex signalling information.This article has an associated First Person interview with the first author of the paper.

Multiplexed Temporal Quantification of the Exercise-regulated Plasma Peptidome.

Exercise is extremely beneficial to whole body health reducing the risk of a number of chronic human diseases. Some of these physiological benefits appear to be mediated via the secretion of peptide/protein hormones into the blood stream. The plasma peptidome contains the entire complement of low molecular weight endogenous peptides derived from secretion, protease activity and PTMs, and is a rich source of hormones. In the current study we have quantified the effects of intense exercise on the plasma peptidome to identify novel exercise regulated secretory factors in humans. We developed an optimized 2D-LC-MS/MS method and used multiple fragmentation methods including HCD and EThcD to analyze endogenous peptides. This resulted in quantification of 5,548 unique peptides during a time course of exercise and recovery. The plasma peptidome underwent dynamic and large changes during exercise on a time-scale of minutes with many rapidly reversible following exercise cessation. Among acutely regulated peptides, many were known hormones including insulin, glucagon, ghrelin, bradykinin, cholecystokinin and secretogranins validating the method. Prediction of bioactive peptides regulated with exercise identified C-terminal peptides from Transgelins, which were increased in plasma during exercise. In vitro experiments using synthetic peptides identified a role for transgelin peptides on the regulation of cell-cycle, extracellular matrix remodeling and cell migration. We investigated the effects of exercise on the regulation of PTMs and proteolytic processing by building a site-specific network of protease/substrate activity. Collectively, our deep peptidomic analysis of plasma revealed that exercise rapidly modulates the circulation of hundreds of bioactive peptides through a network of proteases and PTMs. These findings illustrate that peptidomics is an ideal method for quantifying changes in circulating factors on a global scale in response to physiological perturbations such as exercise. This will likely be a key method for pinpointing exercise regulated factors that generate health benefits.

Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes.

Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes.

Novel systems for dynamically assessing insulin action in live cells reveals heterogeneity in the insulin response.

Regulated GLUT4 trafficking is a key action of insulin. Quantitative stepwise analysis of this process provides a powerful tool for pinpointing regulatory nodes that contribute to insulin regulation and insulin resistance. We describe a novel GLUT4 construct and workflow for the streamlined dissection of GLUT4 trafficking; from simple high throughput screens to high resolution analyses of individual vesicles. We reveal single cell heterogeneity in insulin action highlighting the utility of this approach - each cell displayed a unique and highly reproducible insulin response, implying that each cell is hard-wired to produce a specific output in response to a given stimulus. These data highlight that the response of a cell population to insulin is underpinned by extensive heterogeneity at the single cell level. This heterogeneity is pre-programmed within each cell and is not the result of intracellular stochastic events.

DOC2 isoforms play dual roles in insulin secretion and insulin-stimulated glucose uptake.

AIMS/HYPOTHESIS: Glucose-stimulated insulin secretion (GSIS) and insulin-stimulated glucose uptake are processes that rely on regulated intracellular vesicle transport and vesicle fusion with the plasma membrane. DOC2A and DOC2B are calcium-sensitive proteins that were identified as key components of vesicle exocytosis in neurons. Our aim was to investigate the role of DOC2 isoforms in glucose homeostasis, insulin secretion and insulin action. METHODS: DOC2 expression was measured by RT-PCR and western blotting. Body weight, glucose tolerance, insulin action and GSIS were assessed in wild-type (WT), Doc2a (-/-) (Doc2aKO), Doc2b (-/-) (Doc2bKO) and Doc2a (-/-)/Doc2b (-/-) (Doc2a/Doc2bKO) mice in vivo. In vitro GSIS and glucose uptake were assessed in isolated tissues, and exocytotic proteins measured by western blotting. GLUT4 translocation was assessed by epifluorescence microscopy. RESULTS: Doc2b mRNA was detected in all tissues tested, whereas Doc2a was only detected in islets and the brain. Doc2aKO and Doc2bKO mice had minor glucose intolerance, while Doc2a/Doc2bKO mice showed pronounced glucose intolerance. GSIS was markedly impaired in Doc2a/Doc2bKO mice in vivo, and in isolated Doc2a/Doc2bKO islets in vitro. In contrast, Doc2bKO mice had only subtle defects in insulin secretion in vivo. Insulin action was impaired to a similar degree in both Doc2bKO and Doc2a/Doc2bKO mice. In vitro insulin-stimulated glucose transport and GLUT4 vesicle fusion were defective in adipocytes derived from Doc2bKO mice. Surprisingly, insulin action was not altered in muscle isolated from DOC2-null mice. CONCLUSIONS/INTERPRETATION: Our study identifies a critical role for DOC2B in insulin-stimulated glucose uptake in adipocytes, and for the synergistic regulation of GSIS by DOC2A and DOC2B in beta cells.

Analysis and Quantification of the Mitochondrial-ER Lipidome.

Mitochondria are vital organelles essential for cellular functions, but their lipid composition and response to stressors are not fully understood. Recent advancements in lipidomics reveal insights into lipid functions, especially their roles in metabolic perturbations and diseases. Previous methods have focused on the protein composition of mitochondria and mitochondrial-associated membranes. The advantage of our technique is that it combines organelle isolation with targeted lipidomics, offering new insights into the composition and dynamics of these organelles in pathological conditions. We developed a mitochondria isolation protocol for L6 myotubes, enabling lipidomics analysis of specific organelles without interference from other cellular compartments. This approach offers a unique opportunity to dissect lipid dynamics within mitochondria and their associated ER compartments under cellular stress. Key features • Analysis and quantification of lipids in mitochondria-ER fraction through liquid chromatography-tandem mass spectrometry-based lipidomics (LC-MS/MS lipidomics). • LC-MS/MS lipidomics provide precise and unbiased information on the lipid composition in in vitro systems. • LC-MS/MS lipidomics facilitates the identification of lipid signatures in mammalian cells.

Dual Tracer Test to Measure Tissue-Specific Insulin Action in Individual Mice Identifies In Vivo Insulin Resistance Without Fasting Hyperinsulinemia.

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 [14C]2-deoxyglucose ([14C]2DG) alone, followed 40 min later by injection of [3H]2DG 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.

Multi-targeted loss of the antigen presentation molecule MR1 during HSV-1 and HSV-2 infection.

The major histocompatibility complex (MHC), Class-I-related (MR1) molecule presents microbiome-synthesized metabolites to Mucosal-associated invariant T (MAIT) cells, present at sites of herpes simplex virus (HSV) infection. During HSV type 1 (HSV-1) infection there is a profound and rapid loss of MR1, in part due to expression of unique short 3 protein. Here we show that virion host shutoff RNase protein downregulates MR1 protein, through loss of MR1 transcripts. Furthermore, a third viral protein, infected cell protein 22, also downregulates MR1, but not classical MHC-I molecules. This occurs early in the MR1 trafficking pathway through proteasomal degradation. Finally, HSV-2 infection results in the loss of MR1 transcripts, and intracellular and surface MR1 protein, comparable to that seen during HSV-1 infection. Thus HSV coordinates a multifaceted attack on the MR1 antigen presentation pathway, potentially protecting infected cells from MAIT cell T cell receptor-mediated detection at sites of primary infection and reactivation.

Deletion of miPEP in adipocytes protects against obesity and insulin resistance by boosting muscle metabolism.

Mitochondria facilitate thousands of biochemical reactions, covering a broad spectrum of anabolic and catabolic processes. Here we demonstrate that the adipocyte mitochondrial proteome is markedly altered across multiple models of insulin resistance and reveal a consistent decrease in the level of the mitochondrial processing peptidase miPEP. OBJECTIVE: To determine the role of miPEP in insulin resistance. METHODS: To experimentally test this observation, we generated adipocyte-specific miPEP knockout mice to interrogate its role in the aetiology of insulin resistance. RESULTS: We observed a strong phenotype characterised by enhanced insulin sensitivity and reduced adiposity, despite normal food intake and physical activity. Strikingly, these phenotypes vanished when mice were housed at thermoneutrality, suggesting that metabolic protection conferred by miPEP deletion hinges upon a thermoregulatory process. Tissue specific analysis of miPEP deficient mice revealed an increment in muscle metabolism, and upregulation of the protein FBP2 that is involved in ATP hydrolysis in the gluconeogenic pathway. CONCLUSION: These findings suggest that miPEP deletion initiates a compensatory increase in skeletal muscle metabolism acting as a protective mechanism against diet-induced obesity and insulin resistance.