A MED13-dependent skeletal muscle gene program controls systemic glucose homeostasis and hepatic metabolism.

The Mediator complex governs gene expression by linking upstream signaling pathways with the basal transcriptional machinery. However, how individual Mediator subunits may function in different tissues remains to be investigated. Through skeletal muscle-specific deletion of the Mediator subunit MED13 in mice, we discovered a gene regulatory mechanism by which skeletal muscle modulates the response of the liver to a high-fat diet. Skeletal muscle-specific deletion of MED13 in mice conferred resistance to hepatic steatosis by activating a metabolic gene program that enhances muscle glucose uptake and storage as glycogen. The consequent insulin-sensitizing effect within skeletal muscle lowered systemic glucose and insulin levels independently of weight gain and adiposity and prevented hepatic lipid accumulation. MED13 suppressed the expression of genes involved in glucose uptake and metabolism in skeletal muscle by inhibiting the nuclear receptor NURR1 and the MEF2 transcription factor. These findings reveal a fundamental molecular mechanism for the governance of glucose metabolism and the control of hepatic lipid accumulation by skeletal muscle. Intriguingly, MED13 exerts opposing metabolic actions in skeletal muscle and the heart, highlighting the customized, tissue-specific functions of the Mediator complex.

Cross-Laboratory Standardization of Preclinical Lipidomics Using Differential Mobility Spectrometry and Multiple Reaction Monitoring.

Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics' technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950-Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231-Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.

Evaluating a targeted multiple reaction monitoring approach to global untargeted lipidomic analyses of human plasma.

RATIONALE: The Lipidyzer platform was recently updated on a SCIEX QTRAP 6500+ mass spectrometer and offers a targeted lipidomics assay including 1150 different lipids. We evaluated this targeted approach using human plasma samples and compared the results against a global untargeted lipidomics method using a high-resolution Q Exactive HF Orbitrap mass spectrometer. METHODS: Lipids from human plasma samples (N = 5) were extracted using a modified Bligh-Dyer approach. A global untargeted analysis was performed using a Thermo Orbitrap Q Exactive HF mass spectrometer, followed by data analysis using Progenesis QI software. Multiple reaction monitoring (MRM)-based targeted analysis was performed using a QTRAP 6500+ mass spectrometer, followed by data analysis using SCIEX OS software. The samples were injected on three separate days to assess reproducibility for both approaches. RESULTS: Overall, 465 lipids were identified from 11 lipid classes in both approaches, of which 159 were similar between the methods, 168 lipids were unique to the MRM approach, and 138 lipids were unique to the untargeted approach. Phosphatidylcholine and phosphatidylethanolamine species were the most commonly identified using the untargeted approach, while triacylglycerol species were the most commonly identified using the targeted MRM approach. The targeted MRM approach had more consistent relative abundances across the three days than the untargeted approach. Overall, the coefficient of variation for inter-day comparisons across all lipid classes was ∼ 23% for the untargeted approach and ∼ 9% for the targeted MRM approach. CONCLUSIONS: The targeted MRM approach identified similar numbers of lipids to a conventional untargeted approach, but had better representation of 11 lipid classes commonly identified by both approaches. Based on the separation methods employed, the conventional untargeted approach could better detect phosphatidylcholine and sphingomyelin lipid classes. The targeted MRM approach had lower inter-day variability than the untargeted approach when tested using a small group of plasma samples. These studies highlight the advantages in using targeted MRM approaches for human plasma lipidomics analysis.

High-Mobility Group Box 1 Disrupts Metabolic Function with Cigarette Smoke Exposure in a Ceramide-Dependent Manner.

We have previously found that cigarette smoke disrupts metabolic function, in part, by increasing muscle ceramide accrual. To further our understanding of this, we sought to determine the role of the cytokine high-mobility group box 1 (HMGB1), which is increased with smoke exposure, in smoke-induced muscle metabolic perturbations. To test this theory, we determined HMGB1 from lungs of human smokers, as well as from lung cells from mice exposed to cigarette smoke. We also treated cells and mice directly with HMGB1, in the presence or absence of myriocin, an inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in ceramide biosynthesis. Outcomes included assessments of insulin resistance and muscle mitochondrial function. HMGB1 was significantly increased in both human lungs and rodent alveolar macrophages. Further testing revealed that HMGB1 treatment elicited a widespread increase in ceramide species and reduction in myotube mitochondrial respiration, an increase in reactive oxygen species, and reduced insulin-stimulated Akt phosphorylation. Inhibition of ceramide biosynthesis with myriocin was protective. In mice, by comparing treatments of HMGB1 injections with or without myriocin, we found that HMGB1 injections resulted in increased muscle ceramides, especially C16 and C24, which were necessary for reduced muscle mitochondrial respiration and compromised insulin and glucose tolerance. In conclusion, HMGB1 may be a necessary intermediate in the ceramide-dependent metabolic consequences of cigarette smoke exposure.

A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.

Intracellular lipids and their synthesis contribute to the mechanisms and complications of obesity-associated diseases. We describe an NMR approach that provides an abbreviated lipidomic analysis with concurrent lipid biosynthetic fluxes. Following deuterated water administration, positional isotopomer analysis by deuterium NMR of specific lipid species was used to examine flux through de novo lipogenesis (DNL), FA elongation, desaturation, and TG-glycerol synthesis. The NMR method obviated certain assumptions regarding sites of enrichment and exchangeable hydrogens required by mass isotope methods. The approach was responsive to genetic and pharmacological gain or loss of function of DNL, elongation, desaturation, and glyceride synthesis. BDF1 mice consuming a high-fat diet (HFD) or matched low-fat diet for 35 weeks were examined across feeding periods to determine how flux through these pathways contributes to diet induced fatty liver and obesity. HFD mice had increased rates of FA elongation and glyceride synthesis. However DNL was markedly suppressed despite insulin resistance and obesity. We conclude that most hepatic TGs in the liver of HFD mice were formed from the reesterification of existing or ingested lipids, not DNL.

Up-regulated PLA2G10 in cancer impairs T cell infiltration to dampen immunity.

T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.

Development of a targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry based lipidomics platform applied to a coronavirus disease severity study.

The importance of lipids seen in studies of metabolism, cancer, the recent COVID-19 pandemic and other diseases has brought the field of lipidomics to the forefront of clinical research. Quantitative and comprehensive analysis is required to understand biological interactions among lipid species. However, lipidomic analysis is often challenging due to the various compositional structures, diverse physicochemical properties, and wide dynamic range of concentrations of lipids in biological systems. To study the comprehensive lipidome, a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)-based screening method with 1200 lipid features across 19 (sub)classes, including both nonpolar and polar lipids, has been developed. HILIC-MS/MS was selected due to its class separation property and fatty acyl chain level information. 3D models of class chromatographic retention behavior were established and evaluations of cross-class and within-class interferences were performed to avoid over-reporting these features. This targeted HILIC-MS/MS method was fully validated, with acceptable analytical parameters in terms of linearity, precision, reproducibility, and recovery. The accurate quantitation of 608 lipid species in the SRM 1950 NIST plasma was achieved using multi-internal standards per class and post-hoc correction, extending current databases by providing lipid concentrations resolved at fatty acyl chain level. The overall correlation coefficients (R2) of measured concentrations with values from literature range from 0.64 to 0.84. The applicability of the developed targeted lipidomics method was demonstrated by discovering 520 differential lipid features related to COVID-19 severity. This high coverage and targeted approach will aid in future investigations of the lipidome in various disease contexts.

Lipid hydroperoxides promote sarcopenia through carbonyl stress.

Reactive oxygen species (ROS) accumulation is a cardinal feature of skeletal muscle atrophy. ROS refers to a collection of radical molecules whose cellular signals are vast, and it is unclear which downstream consequences of ROS are responsible for the loss of muscle mass and strength. Here, we show that lipid hydroperoxides (LOOH) are increased with age and disuse, and the accumulation of LOOH by deletion of glutathione peroxidase 4 (GPx4) is sufficient to augment muscle atrophy. LOOH promoted atrophy in a lysosomal-dependent, proteasomal-independent manner. In young and old mice, genetic and pharmacological neutralization of LOOH or their secondary reactive lipid aldehydes robustly prevented muscle atrophy and weakness, indicating that LOOH-derived carbonyl stress mediates age- and disuse-induced muscle dysfunction. Our findings provide novel insights for the role of LOOH in sarcopenia including a therapeutic implication by pharmacological suppression.

A lipidomics platform to analyze the fatty acid compositions of non-polar and polar lipid molecular species from plant tissues: Examples from developing seeds and seedlings of pennycress (Thlaspi arvense).

The lipidome comprises the total content of molecular species of each lipid class, and is measured using the analytical techniques of lipidomics. Many liquid chromatography-mass spectrometry (LC-MS) methods have previously been described to characterize the lipidome. However, many lipidomic approaches may not fully uncover the subtleties of lipid molecular species, such as the full fatty acid (FA) composition of certain lipid classes. Here, we describe a stepwise targeted lipidomics approach to characterize the polar and non-polar lipid classes using complementary LC-MS methods. Our "polar" method measures 260 molecular species across 12 polar lipid classes, and is performed using hydrophilic interaction chromatography (HILIC) on a NH2 column to separate lipid classes by their headgroup. Our "non-polar" method measures 254 molecular species across three non-polar lipid classes, separating molecular species on their FA characteristics by reverse phase (RP) chromatography on a C30 column. Five different extraction methods were compared, with an MTBE-based extraction chosen for the final lipidomics workflow. A state-of-the-art strategy to determine and relatively quantify the FA composition of triacylglycerols is also described. This lipidomics workflow was applied to developing, mature, and germinated pennycress seeds/seedlings and found unexpected changes among several lipid molecular species. During development, diacylglycerols predominantly contained long chain length FAs, which contrasted with the very long chain FAs of triacylglycerols in mature seeds. Potential metabolic explanations are discussed. The lack of very long chain fatty acids in diacylglycerols of germinating seeds may indicate very long chain FAs, such as erucic acid, are preferentially channeled into beta-oxidation for energy production.

A DMS Shotgun Lipidomics Workflow Application to Facilitate High-Throughput, Comprehensive Lipidomics.

Differential mobility spectrometry (DMS) is highly useful for shotgun lipidomic analysis because it overcomes difficulties in measuring isobaric species within a complex lipid sample and allows for acyl tail characterization of phospholipid species. Despite these advantages, the resulting workflow presents technical challenges, including the need to tune the DMS before every batch to update compensative voltages settings within the method. The Sciex Lipidyzer platform uses a Sciex 5500 QTRAP with a DMS (SelexION), an LC system configured for direction infusion experiments, an extensive set of standards designed for quantitative lipidomics, and a software package (Lipidyzer Workflow Manager) that facilitates the workflow and rapidly analyzes the data. Although the Lipidyzer platform remains very useful for DMS-based shotgun lipidomics, the software is no longer updated for current versions of Analyst and Windows. Furthermore, the software is fixed to a single workflow and cannot take advantage of new lipidomics standards or analyze additional lipid species. To address this multitude of issues, we developed Shotgun Lipidomics Assistant (SLA), a Python-based application that facilitates DMS-based lipidomics workflows. SLA provides the user with flexibility in adding and subtracting lipid and standard MRMs. It can report quantitative lipidomics results from raw data in minutes, comparable to the Lipidyzer software. We show that SLA facilitates an expanded lipidomics analysis that measures over 1450 lipid species across 17 (sub)classes. Lastly, we demonstrate that the SLA performs isotope correction, a feature that was absent from the original software.

Targeting a ceramide double bond improves insulin resistance and hepatic steatosis.

Ceramides contribute to the lipotoxicity that underlies diabetes, hepatic steatosis, and heart disease. By genetically engineering mice, we deleted the enzyme dihydroceramide desaturase 1 (DES1), which normally inserts a conserved double bond into the backbone of ceramides and other predominant sphingolipids. Ablation of DES1 from whole animals or tissue-specific deletion in the liver and/or adipose tissue resolved hepatic steatosis and insulin resistance in mice caused by leptin deficiency or obesogenic diets. Mechanistic studies revealed ceramide actions that promoted lipid uptake and storage and impaired glucose utilization, none of which could be recapitulated by (dihydro)ceramides that lacked the critical double bond. These studies suggest that inhibition of DES1 may provide a means of treating hepatic steatosis and metabolic disorders.

Glucagon Receptor Antagonism Improves Glucose Metabolism and Cardiac Function by Promoting AMP-Mediated Protein Kinase in Diabetic Mice.

The antidiabetic potential of glucagon receptor antagonism presents an opportunity for use in an insulin-centric clinical environment. To investigate the metabolic effects of glucagon receptor antagonism in type 2 diabetes, we treated Leprdb/db and Lepob/ob mice with REMD 2.59, a human monoclonal antibody and competitive antagonist of the glucagon receptor. As expected, REMD 2.59 suppresses hepatic glucose production and improves glycemia. Surprisingly, it also enhances insulin action in both liver and skeletal muscle, coinciding with an increase in AMP-activated protein kinase (AMPK)-mediated lipid oxidation. Furthermore, weekly REMD 2.59 treatment over a period of months protects against diabetic cardiomyopathy. These functional improvements are not derived simply from correcting the systemic milieu; nondiabetic mice with cardiac-specific overexpression of lipoprotein lipase also show improvements in contractile function after REMD 2.59 treatment. These observations suggest that hyperglucagonemia enables lipotoxic conditions, allowing the development of insulin resistance and cardiac dysfunction during disease progression.

Directing visceral white adipocyte precursors to a thermogenic adipocyte fate improves insulin sensitivity in obese mice.

Visceral adiposity confers significant risk for developing metabolic disease in obesity whereas preferential expansion of subcutaneous white adipose tissue (WAT) appears protective. Unlike subcutaneous WAT, visceral WAT is resistant to adopting a protective thermogenic phenotype characterized by the accumulation of Ucp1+ beige/BRITE adipocytes (termed 'browning'). In this study, we investigated the physiological consequences of browning murine visceral WAT by selective genetic ablation of Zfp423, a transcriptional suppressor of the adipocyte thermogenic program. Zfp423 deletion in fetal visceral adipose precursors (Zfp423loxP/loxP; Wt1-Cre), or adult visceral white adipose precursors (PdgfrbrtTA; TRE-Cre; Zfp423loxP/loxP), results in the accumulation of beige-like thermogenic adipocytes within multiple visceral adipose depots. Thermogenic visceral WAT improves cold tolerance and prevents and reverses insulin resistance in obesity. These data indicate that beneficial visceral WAT browning can be engineered by directing visceral white adipocyte precursors to a thermogenic adipocyte fate, and suggest a novel strategy to combat insulin resistance in obesity.

Inducible overexpression of adiponectin receptors highlight the roles of adiponectin-induced ceramidase signaling in lipid and glucose homeostasis.

OBJECTIVE: Adiponectin and the signaling induced by its cognate receptors, AdipoR1 and AdipoR2, have garnered attention for their ability to promote insulin sensitivity and oppose steatosis. Activation of these receptors promotes the deacylation of ceramide, a lipid metabolite that appears to play a causal role in impairing insulin signaling. METHODS: Here, we have developed transgenic mice that overexpress AdipoR1 or AdipoR2 under the inducible control of a tetracycline response element. These represent the first inducible genetic models that acutely manipulate adiponectin receptor signaling in adult mouse tissues, which allows us to directly assess AdipoR signaling on glucose and lipid metabolism. RESULTS: Overexpression of either adiponectin receptor isoform in the adipocyte or hepatocyte is sufficient to enhance ceramidase activity, whole body glucose metabolism, and hepatic insulin sensitivity, while opposing hepatic steatosis. Importantly, metabolic improvements fail to occur in an adiponectin knockout background. When challenged with a leptin-deficient genetic model of type 2 diabetes, AdipoR2 expression in adipose or liver is sufficient to reverse hyperglycemia and glucose intolerance. CONCLUSION: These observations reveal that adiponectin is critical for AdipoR-induced ceramidase activation which enhances hepatic glucose and lipid metabolism via rapidly acting "cross-talk" between liver and adipose tissue sphingolipids.

An Assessment of the Population of Cotton-Top Tamarins (Saguinus oedipus) and Their Habitat in Colombia.

Numerous animals have declining populations due to habitat loss, illegal wildlife trade, and climate change. The cotton-top tamarin (Saguinus oedipus) is a Critically Endangered primate species, endemic to northwest Colombia, threatened by deforestation and illegal trade. In order to assess the current state of this species, we analyzed changes in the population of cotton-top tamarins and its habitat from 2005 to 2012. We used a tailor-made "lure strip transect" method to survey 43 accessible forest parcels that represent 30% of the species' range. Estimated population size in the surveyed region was approximately 2,050 in 2005 and 1,900 in 2012, with a coefficient of variation of approximately 10%. The estimated population change between surveys was -7% (a decline of approximately 1.3% per year) suggesting a relatively stable population. If densities of inaccessible forest parcels are similar to those of surveyed samples, the estimated population of cotton-top tamarins in the wild in 2012 was 6,946 individuals. We also recorded little change in the amount of suitable habitat for cotton-top tamarins between sample periods: in 2005, 18% of surveyed forest was preferred habitat for cotton-top tamarins, while in 2012, 17% percent was preferred. We attribute the relatively stable population of this Critically Endangered species to increased conservation efforts of Proyecto Tití, conservation NGOs, and the Colombian government. Due to continued threats to cotton-top tamarins and their habitat such as agriculture and urban expansion, ongoing conservation efforts are needed to ensure the long-term survival of cotton-top tamarins in Colombia.

SF-1 expression in the hypothalamus is required for beneficial metabolic effects of exercise.

Exercise has numerous beneficial metabolic effects. The central nervous system (CNS) is critical for regulating energy balance and coordinating whole body metabolism. However, a role for the CNS in the regulation of metabolism in the context of the exercise remains less clear. Here, using genetically engineered mice we assessed the requirement of steroidogenic factor-1 (SF-1) expression in neurons of the ventromedial hypothalamic nucleus (VMH) in mediating the beneficial effects of exercise on metabolism. We found that VMH-specific deletion of SF-1 blunts (a) the reductions in fat mass, (b) improvements in glycemia, and (c) increases in energy expenditure that are associated with exercise training. Unexpectedly, we found that SF-1 deletion in the VMH attenuates metabolic responses of skeletal muscle to exercise, including induction of PGC-1α expression. Collectively, this evidence suggests that SF-1 expression in VMH neurons is required for the beneficial effects of exercise on metabolism.

Targeted Induction of Ceramide Degradation Leads to Improved Systemic Metabolism and Reduced Hepatic Steatosis.

Sphingolipids have garnered attention for their role in insulin resistance and lipotoxic cell death. We have developed transgenic mice inducibly expressing acid ceramidase that display a reduction in ceramides in adult mouse tissues. Hepatic overexpression of acid ceramidase prevents hepatic steatosis and prompts improvements in insulin action in liver and adipose tissue upon exposure to high-fat diet. Conversely, overexpression of acid ceramidase within adipose tissue also prevents hepatic steatosis and systemic insulin resistance. Induction of ceramidase activity in either tissue promotes a lowering of hepatic ceramides and reduced activation of the ceramide-activated protein kinase C isoform PKCζ, though the induction of ceramidase activity in the adipocyte prompts more rapid resolution of hepatic steatosis than overexpression of the enzyme directly in the liver. Collectively, our observations suggest the existence of a rapidly acting "cross-talk" between liver and adipose tissue sphingolipids, critically regulating glucose metabolism and hepatic lipid uptake.