Updates from the Metabolism Community

This invited perspective explores how light exposure worsens glucose homeostasis by inhibiting brown adipose tissue heat production and maps the neuronal regulation of this process. These findings could provide a mechanistic explanation for the long observed worsened glucose regulation with constant light exposure.

In this invited review, Fernandez-Fuente et al. examine the emerging regulatory functions of the citrate/acetyl-CoA pathway and the specific role of the endoplasmic reticulum (ER) acetylation machinery in the maintenance of intracellular crosstalk and homeostasis. These functions are analyzed in the context of associated human diseases and specific mouse models of dysfunctional ER acetylation and citrate/acetyl-CoA flux. A primary objective of this review is to highlight the complex yet integrated response of compartment- and organelle-specific Nε-lysine acetylation to the intracellular availability and flux of acetyl-CoA, linking this important post-translational modification to cellular metabolism. A major conclusion of this article is that citrate and acetyl-CoA should not only be seen as metabolic substrates of intermediate metabolism but also as signaling molecules that direct functional adaptation of the cell to both intracellular and extracellular messages.

This comprehensive study of genetic and phenotypic diversity the genus Saccharomyces showed that cytoplasmic genetic elements, such as plasmids and the mitochondrial genome, were much more prone to gene flow and introgression. Several metabolic traits, such as galactose metabolism, have been strongly influenced by gene flow and selection.

Key techniques performed in Madison:

• Genome sequencing
• High-throughput phenotyping using growth curves
• Computational analyses

Pyruvate dehydrogenase complex (PDHC) and oxoglutarate dehydrogenase complex (OGDC) are important enzymes belong to the mitochondrial α-ketoacid dehydrogenase complex family. They catalyze key reactions controlling the influx from major substrates (glucose and glutamine) into TCA cycle. This work by Seim et. al. identified a novel mechanism for regulating PDHC and OGDC via a series of hitherto unknown covalent modifications-- Reactive nitrogen species can profoundly inhibit PDHC and OGDC by S-modifying the catalytic lipoic arm on their E2 subunits. Coenzyme A, the natural substrate of the E2 subunit, delivers the RNS-driven modifications to the lipoic arm in a targeted manner via forming SNO-CoA, making this mechanism highly efficient and specific. This regulation mechanism is partially reversible and has significant biological impacts during classical activation of macrophages.

Reactive nitrogen species can profoundly inhibit mitochondrial α-ketoacid dehydrogenase complexes by causing a series of covalent S-modifications of the catalytic lipoic arm on their E2 subunit. Coenzyme A can efficiently deliver these modifications.

Key techniques performed in Madison:

• LCMS metabolomics
• Biochemical assays for enzyme activity and modifications
• Isotopic tracing

The ESCRT machinery, critical for remodeling cellular membranes, downregulates signaling pathways by sequestering receptors inside multivesicular endosomes, including receptors involved in metabolic regulation. Here, using in vitro biochemistry, light and electron microscopy, as well as CRISPR/Cas9-mediated genome editing, we showed that the ESCRT regulator Lgd/CC2D1 is required for efficient receptor degradation through its role in recruiting/stabilizing ESCRT components on the endosomal membrane.

Key techniques performed in Madison:

• In Vitro Biochemistry
• Light and Electron Microscopy
• CRISPR/Cas9-mediated genome editing

The economic success of advanced cellulosic biorefineries that produce biofuels is tied to capturing as much carbon as possible from the initial plant material and converting it into valuable bioproducts. One way to do this is to valorize the carbon in the residue that is typically part of the biorefinery waste stream: the conversion residue, which is the residue remaining after the associated biofuel has been removed. We characterized the conversion residue from a biorefinery that converts switchgrass hydrolysate to bioethanol and showed that diverse collections of Streptomyces and yeast species could capture this waste carbon for growth. This is an important first step toward developing microbial chassis that capture waste-based carbon and convert it to valuable bioproducts, improving the economic success of biorefineries.

Key techniques performed in Madison:

• HPLC analysis of metabolites
• Bioreactor and batch culture growth assays
• Phylogenetic analysis
• Chemical Oxygen Demand analysis
• 16S rRNA community analysis

We measured mitochondrial membrane potential, oxygen consumption, extracellular acidification, metabolite levels, and the optical redox ratio in colon cancer cells. The goal of this study was to characterize our optical redox ratio imaging with respect to standard techniques, using a novel BCL2 inhibitor.

Key techniques performed in Madison:

• Two-photon fluorescence lifetime imaging
• TMRE imaging of mitochondrial membrane potential
• Seahorse analyzer

In this paper, Rigby et al. generated a transgenic mouse with neuron-specific overexpression of the mitochondria citrate transporter SLC25A1. Behavioral assessment using a combination of social-dependent paradigms demonstrated an autistic-like phenotype. Magnetic resonance imaging (MRI) of the brain reveled changes in white matter organization, which was also manifested by electron microscopy (EM) analysis of brain sections. Electrophysiology-based approaches, both at the brain slice and neuronal culture level, reveled significant defects in synaptic plasticity with underlying neuronal hyper-excitability. Finally, ultrastructural analysis of the dendritic network revealed changes in spine morphology. Mass Spectrometry (MS)-based approaches demonstrated significant changes both at the proteome and acetyl-proteome level with impact on the dynamics of the secretory pathway and spine assembly/activity. Similar findings were observed in mice with overexpression of the plasma membrane citrate transporter SLC13A5 (see Rigby et al. Brain Commun 2022) and strengthen the genetic association between gene duplication events of SLC25A1 and SLC13A5 with autism spectrum disorder (ASD) and intellectual disability in humans.

Key techniques performed in Madison:

• Generation of transgenic mice
• Behavioral assessment
• MRI
• Electrophysiology
• Neuronal morphology and electron microscopy
• Mass spectrometry for the analysis of the proteome and acetylproteome

Milk synthesis is an energy-demanding anabolic process, and ergo dependent in part on mTORC1 activity. As is well known, activators of mTORC1 include both insulin and amino acids like leucine and methionine. However, how these factors interact in vivo in lactating animals is still up for debate. Here, we assessed how insulin and gastrically-infused amino acids alter mammary metabolism in the lactating cow.

Key techniques performed in Madison:

• Carotid artery transposition
• Abomasal (gastric) infusion of nutrients
• Hyperinsulinemic clamp
• Mass spectrometry analysis of amino acids
• Metabolite profiling

We conducted a large genetic screen for genes that regulate nutrient-induced insulin secretion. One of these genes is the transcription factor Zfp148. We discovered that it is a potent negative regulator of insulin secretion. The stimulation of insulin secretion is accompanied by the induction of oscillations in calcium abundance in the beta cells. This provides a very sensitive measure of stimulation with high time resolution. In this study we measured calcium oscillations in pancreatic islets from Zfp148-knockout mice. The beta cells showed a remarkable left shift in their response to glucose and to amino acids in the calcium oscillations, revealing that this transcription factor affects the sensitivity of the beta cells to nutrient stimuli.

Key techniques performed in Madison:

• Calcium measurements – Imaging core at SMPH

Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells. We recently revealed that nutrient availability has a profound impact on gene essentiality. Guided by that study, this manuscript describes a detailed methodology for using CRISPR/Cas9-based loss-of-function screens to ask how gene essentiality in human cell lines varies with medium composition.

Folding and trafficking of nascent polypeptide chains as they exit the ribosome are inherently problematic, as thousands of polypeptide chains with differing sequences need to be accommodated. Lee, Ziegelhoffer et al. use in vivo site-specific crosslinking to provide molecular level insight into how the fungal Hsp70 chaperone system – the Ssb1:Ssz1:Zuo1 triad – assists the folding process for the nascent peptide chain emerging from the ribosome tunnel. The in vivo site-specific crosslinking method was used in yeast in this report, but it has recently been developed by others for use in metazoan cells.

Key techniques performed in Madison:

• Site-specific in vivo Bpa crosslinking analysis.
• Mass spectrometry analysis to determine the residue of site-specific in vitro crosslinking.
• Yeast genetic suppressor analysis

In this recent publication, through a series of metabolic flux analysis studies, Britt et al uncovered that upon activation, neutrophils rapidly switch from glycolysis-dominant metabolism to a unique metabolic mode—cyclic pentose phosphate pathway with reversed upper glycolysis to achieve ultra-high NADPH yield. They further found this rapid switch is quantitatively and specifically coupled with the oxidative burst, a critical neutrophil function that requires a large amount of NADPH to produce reactive oxygen species for pathogen killing, and that the adoption of cyclic pentose phosphate pathway is essential for neutrophil extracellular trap release and host defense, using various pharmacological and genetical perturbations. This work reveals the remarkable metabolic flexibility that is crucial for neutrophil effector functions and the biochemical mechanism that enables such metabolic flexibility.

Key techniques performed in Madison:

• Isotopic tracing based metabolic flux analysis
• Metabolomics
• Neutrophil functional assays including assays for oxidative burst, neutrophil extracellular traps, in vitro and in vivo pathogen killing

We show that hormonal FABP4 forms a functional hormone complex with adenosine kinase (ADK) and nucleoside diphosphate kinase (NDPK) to regulate extracellular ATP and ADP levels. We identify a substantial effect of this hormone on beta cells. Antibody-mediated targeting of this hormone complex improves metabolic outcomes, enhances beta-cell function and preserves beta-cell integrity to prevent both type 1 and type 2 diabetes.

Key techniques performed in Madison:

• Immunophenotyping
• Microscale thermophoresis (MST) and crystallography
• Immunoprecipitation and Mass Spec
• Kinase activity assays
• Mouse and human islet studies
• Monoclonal antibody neutralization

This is a review article that summarized recent advances in type 1 diabetes field.

Calorie restriction (CR) promotes healthy ageing in many species. While the effects of a CR diet on health and longevity have been attributed to the reduction in calories, it has recently been realized that the altered eating pattern of CR-fed animals – which rapidly consume their food when fed once per day – may have an impact on the metabolic response to CR. We find that a prolonged fast between meals is necessary for many of the key metabolic, molecular and geroprotective effects of a CR diet. Using a series of feeding regimens, we dissected the effects of calories and fasting, and find that fasting alone recapitulates many of the physiological and molecular effects of CR. Our results shed new light on how both when and how much we eat regulate metabolic health and longevity, and demonstrate that the prolonged fast between meals – and not solely reduced calorie intake - is necessary for the metabolic and geroprotective benefits of a CR diet.

Key techniques performed in Madison:

• Behavioral analysis
• Body composition analysis
• Epigenetic profiling
• Frailty assessment
• Glucose/insulin/pyruvate tolerance tests
• Lifespan determination
• Metabolic chambers to assess energy balance
• Metabolomics
• Transcriptional profiling

There is a growing realization that “a calorie is not just a calorie” – and that the source of the calories we eat matters. Low protein diets are associated with a reduced risk of age-related diseases and mortality in humans, and extend the lifespan of mice. However, this work has primarily taken place in young C57BL/6J mice – and there is growing realization that sex, genetic background and age are key factors in the response to diet. Using multiple strains and both sexes of mice, we find that improvements in metabolic health in response to reduced dietary protein strongly depend on sex and strain. Using a multi-omics approach, we integrated transcriptional, metabolomic, lipidomic, and phenotypic data to provide molecular insight into the differential response to protein restriction. We identified a sex-specific role for the hormone FGF21 in the response to protein restriction. Our study highlights that sex, genetic background, and age impact the response to dietary protein level, and highlight the need for a personalized medicine approach to dietary interventions.

Key techniques performed in Madison:

• Body composition analysis
• Glucose/insulin/pyruvate tolerance tests
• Lipidomic profiling
• Metabolic chambers to assess energy balance
• Multi-omics analysis
• Transcriptional profiling

There are over 500 identified lipids in circulating plasma, many of these lipids are known to causally regulate diseases including type 2 diabetes and cardiovascular disease. Despite their disease relevance, it is difficult to determine where these plasma lipids are produced and how they are functioning. To address these questions, the Simcox lab used global lipidomics on plasma and nine other tissues of cold exposed mice to identify the tissue contributors of the plasma lipid pool. Using machine learning to model tissue contribution based on lipid structure, this study identified novel regulation of circulating acylcarnitines, a lipid species known to induce insulin resistance in type 2 diabetes, through the kidney and intestine. The study also determined a tissue regulatory axis for circulating ceramides, which are known to regulate insulin sensitivity. In sum, the work in this study generates new targets for the study and functional characterization of circulating lipids in acute cold exposure and a computational resource for other investigators to explore multi-tissue lipidome remodeling during cold exposure.

Gut microbiomes are thought to exert significant changes in host physiology and metabolism by modulating a range of services such as nutrient provisioning. Using a dairy cow model, Cox et al. conducted whole-rumen contents exchange through a cannula between pairs of high- and low-efficient milk producers. They show that the exchange of rumen microbial communities induced short-term changes in milk production (i.e. high-efficient cows became low-efficient and vice versa, before reverting to their pre-exchange states) and that these changes corresponded to shifts in the bacterial portion of the ruminal microbiome but not the fungal component. This work demonstrates that the rumen microbiome can drive milk production efficiency in dairy cows but is also highly stable and resistant to change.

Key techniques performed in Madison:

• Animal Sampling and milk efficiency measurements
• 16S rRNA and ITS sequencing
• Rumen chemistry and volatile fatty acid analysis

Human pluripotent stem cell-derived cardiomyocytes provide a promising regenerative cell therapy for cardiovascular patients and an important model system to accelerate drug discovery. Qian et al. recently developed an autofluorescence imaging method to predict cardiomyocyte differentiation outcome as early as differentiation day 1. This label-free method potentially can streamline cardiomyocyte biomanufacturing from stem cells.

Key techniques performed in Madison:

• Optical metabolic imaging
• Human pluripotent stem cell differentiation into cardiomyocytes
• Establishing machine learning model to predict cardiomyocyte differentiation

The human oral cavity is teeming with microorganisms, some of which have pathogenic influence on the host. To tease out pathogenic microbes and their microenvironment, multi-omics analysis was performed on oral plaques derived from diabetic and periodontal disease patients. Correlation analysis across 'omes revealed host-specific proteins and associated lipids that were elevated in plaques from periodontal disease patients, and also led to the finding that oral community member Lautropia mirabilis synthesizes the lipid mono-methyl phosphatidylethanolamine in high abundance - an uncommon trait in oral microbiota.

Key techniques performed in Madison:

• LC-MS/MS metaproteomics
• GC-MS metabolomics
• LC-MS/MS lipidomics
• Multi-omics data integration

In recent work published in eLife by Adams et al., the functional importance of pancreatic islet architecture was tested using a mouse model where Robo1 and Robo2 are selectively deleted in islet β cells (Robo βKO) resulting in islet endocrine tissue disorganization. Using a novel live imaging platform, they found that Robo βKO mice show disruptions in the synchronous processes that control insulin secretion in vivo. They further show that this functional disruption is not due to β cell-intrinsic Robo-mediated defects, misexpression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for normal islet function. This work will help inform strategies for in vitro generation of islets from stem cells for the treatment of diabetes, which currently do not recapitulate the tissue architecture nor insulin secretory profile of native islets.

Key techniques performed in Madison:

• In vitro single cell calcium imaging
• Intravital calcium imaging
• Intravital glucose uptake assays
• Confocal microscopy

Body temperature regulation is an energy demanding process that requires rapid mobilization of stored fuels. Mitochondria in thermogenic adipocytes play an important role in regulating energy expenditure through producing and processing lipids such as cardiolipins, free fatty acids, and acylcarnitines. These lipids play a number of roles including regulating protein stability, transcription, cell signaling, and serving as an energy substrate. This review explores the recent advances in the field including the work of the Simcox lab which uses mass spectrometry based lipidomics to identify interorgan communication through lipid signaling.

Nε-lysine acetylation of nascent proteins within the lumen of the endoplasmic reticulum (ER) is part of the “quality control” machinery that ensures protein homeostasis (proteostasis) within the secretory pathway. Specifically, it regulates both the engagement of the secretory pathway by correctly folded glycoproteins and the disposal of toxic protein aggregates through ER-specific autophagy (also referred to as reticulophagy or ER-phagy). Autophagy is an essential component of the cell degradation system. Malfunction of autophagy contributes to the progression of many diseases across lifespan, whereas increased levels of autophagy can be beneficial in mouse models of diseases characterized by increased accumulation of toxic protein aggregates.

The acetyltransferase reaction within the ER lumen is carried out by ATase1 and ATase2, the only two ER-based acetyl-CoA:lysine acetyltransferases (KAT). In this study, we report the generation of Atase1−/− and Atase2−/− mice and show that these two ER-based acetyltransferases play different roles in the regulation of reticulophagy and macroautophagy. We also show that knockout of Atase1 alone results in activation of reticulophagy and alleviated proteotoxicity in a mouse model of Alzheimer’s disease. Furthermore, loss of Atase1 or Atase2 results in widespread adaptive changes in the cell acetylome and acetyl-CoA metabolism. Overall, this study supports a divergent role of Atase1 and Atase2 in cellular biology, emphasizing ATase1 as a valid translational target for diseases characterized by toxic protein aggregation in the secretory pathway.

Key techniques performed in Madison:

• Generation of KO mice
• Mass spec-based proteomics
• Mass spec-based acetylomics
• High definition live imaging

Nε-lysine acetylation in the Endoplasmic Reticulum (ER) has emerged as an essential component of the quality control (QC) machinery that maintains protein homeostasis (proteostasis) within the ER. Dysfunctional ER acetylation, as caused by gene mutation or duplication events, results in severe disease phenotypes. Key to ER acetylation is AT-1, an ER-membrane antiporter that transfers acetyl-CoA from the cytosol to the ER lumen in exchange for free CoA.

In this study, we investigated the outcomes of dysregulated cytosol-to-ER acetyl-CoA flux in mouse models of increased (AT-1sTg) and decreased (AT-1S113R/+) AT-1 activity. We found that dysregulation of the cytosol-to-ER transport of acetyl-CoA causes significant reorganization of the secretory pathway resulting in changes in the overall quality of secreted glycoproteins (globally referred to as the secretome). Collectively this study indicates that ER acetylation of nascent glycoproteins is essential to maintain proper organization and engagement of the secretory pathway.

Key techniques performed in Madison:

• Generation of KI and transgenic mice
• Mass spec-based proteomics
• Mass spec-based acetylomics
• Mass spec-based glycoproteomics
• High-definition live imaging

Pancreatic b-cells couple nutrient metabolism with appropriate insulin secretion. In a paper recently published in Cell Reports, Sdao et al.demonstrate an important role for CDK2 in the insulin secretory pathway that is independent of its role in the cell cycle. Genetic deletion of CDK2 in adult β-cells enhanced insulin secretion from isolated islets and improved glucose tolerance in vivo. This phenotype was driven by the loss of inhibitory KATP channels, which lowered the setpoint for membrane depolarization in response to activation of the phosphoenolpyruvate cycle with mitochondrial fuels.

Key techniques performed in Madison:

• Electrophysiological recordings of KATP channel activity and insulin exocytosis from single pancreatic b-cells
• Live-cell imaging of calcium, ATP/ADP, mitochondrial membrane potential, and lactate.
• Collaboration with the Blum lab on islet immunofluorescence studies.

There is a growing realization that “a calorie is not just a calorie” – and that the source of the calories we eat matters. Low protein diets are associated with a reduced risk of age-related diseases and mortality in humans, and extend the lifespan of mice. Previous work by our lab found that feeding young mice a diet with reduced levels of the three amino acids leucine, isoleucine, and valine – collectively known as the branched-chain amino acids, or BCAAs – had similar effects to a low protein diet on metabolic health. Here, we have extended these studies to examine how reducing levels of dietary BCAAs by 2/3rds affects metabolic health, frailty, and aging in mice. A low BCAA diet had similar metabolic benefits in both males and females. However, we find that beginning a low BCAA diet early in life reduced age-associated increases in frailty and extended the lifespan of male mice, but had no effect on these phenotypes in female mice. This may be due to a sex-specific effect of a low BCAA diet on signaling through the protein kinase mTORC1, which is reduced by a low BCAA diet in males but not females. If humans and mice respond to dietary BCAAs in the same way, lowering the amount of BCAAs in the diet – or pharmaceuticals that mimic the effects of a low BCAA diet – could be an effective way to promote healthy aging.

Key techniques performed in Madison:

• Body composition analysis
• Electrocardiography
• Frailty assessment
• Glucose/insulin/pyruvate tolerance tests
• Grip strength
• Metabolic chambers to assess energy balance
• Rotarod
• Transcriptional profiling

We have previously demonstrated that the metabolic benefits of low protein diets observed in mice and humans can be recapitulated by specifically reducing dietary levels of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine. However, the contribution of each BCAA to these effects was unknown. We show that each BCAA has distinct metabolic effects, and that specifically reducing isoleucine is necessary and sufficient to obtains the metabolic benefits of low protein diets in lean or obese mice. In contrast, reducing leucine does not have these metabolic benefits. Finally, using data from the Survey of Health of Wisconsin (SHOW) we find that variation in dietary isoleucine levels helps explain body mass index differences in humans. Our results reveal that isoleucine is a key regulator of metabolic health, and that reducing dietary isoleucine may be a new approach to treating and preventing obesity and diabetes.

Key techniques performed in Madison:

• Body composition analysis
• Epidemiology
• Glucose/insulin/pyruvate tolerance tests
• Metabolic chambers to assess energy balance
• Lipidomics
• Transcriptional profiling

Iron is an important micronutrient needed to support energy metabolism and many other growth processes during development. Unfortunately, iron deficiency remains common worldwide, believed to impact up to 2 billion adults and young children. Iron is also an essential component of diet consumed by most animals. Research conducted at UW–Madison demonstrated that even a short period of iron deficiency during infancy can affect brain development. Early detection is critical because deferring iron treatment until after the anemia was diagnosed did entirely correct the effect on brain growth.

Key techniques performed in Madison:

• Mouse Breeding Colony – Harlow Primate Laboratory
• Neuroimaging Core – Waisman Center

Most forward genetic screens in human cells are performed in vitro using media with little relevance to human physiology. Rossiter et al. reveal the profound impact of medium composition on gene essentiality by performing CRISPR screens of human cancer cells in conventional versus human plasma-like medium (HPLM). Analysis of conditional gene essentiality reveals gene-nutrient interactions that are linked to metabolites uniquely defined in HPLM.

Key techniques performed in Madison:

• CRISPR-based genetic screens
• Unbiased metabolite profiling and quantification
• Isotopic tracer experiments
• MS-based enzyme kinetics
• Single-cell live sorting
• Immunopurification of recombinant proteins from mammalian cells

Mammalian skin impacts metabolic efficiency system-wide, controlling the rate of heat loss and consequent heat production. Kasza et al compared the unique adipose depots associated with mouse and human skin, and found that, irrespective of body site, human subcutaneous depots mobilize in response to thermogenic cues, where mouse depots do not, they behave instead as an insulator. This is important information for the accurate modeling of thermogenesis in rodents and implies that human skin-associated fat could be a major contributor to the maintenance of body temperature.

Key techniques performed in Madison:

• Cycling environmental temperatures at the Biotron
• Sourcing of human skin-associated fat from surgical procedures: Gibson, Xu and Siebert, together with the BioBank (Department of Pathology)
• FLIR thermal imaging (Warren Porter, iBio)
• Confocal imaging (Optical Imaging Core)

The precise positioning of endocrine cells within the islets of Langerhans is required for optimal blood glucose homeostasis, however, the mechanisms underlying islet development remain unclear. We have previously identified a role for the Robo family of receptors in endocrine cell sorting. We further demonstrate the requirement of Slit-Robo signaling in islet development with Gilbert et al. We show that the canonical Robo-binding ligands Slit2 and Slit3 are expressed in non-endocrine tissue, and that simultaneous loss of both ligands results in islet fragmentation. Further, we find that Slit2/3 can prevent endocrine cell migration. Our data suggests that Slit2/3 interact with Robo receptors on the surface of islet endocrine cells to promote islet morphogenesis and cell sorting. These findings may benefit ongoing work to generate bona fide islets from stem cells in vitro.

Key techniques performed in Madison:

• Confocal imaging
• Cell migration assays
• Glucose tolerance tests

New antifungal drugs are urgently needed to address the emergence and transcontinental spread of infectious fungal diseases. Principal among these pathogens is the urgent-threat pandrug-resistant Candida auris. By employing cutting-edge metabolomics and genomic tools, we capitalized on the microbiomes of marine animals and their vast chemical diversity to identify antifungal molecules with promising in vivo efficacies. The most exciting lead, turbinmicin, displays potent in vitro and in vivo activities against not only C. auris, but also Aspergillus fumigatus. Turbinmicin shows excellent mouse-model efficacy against multiple-drug–resistant fungal pathogens and exhibits a wide safety index. Data strongly support the hypothesis that turbinmicin functions through a fungal-specific mode of action, targeting Sec14 of the vesicular trafficking pathway. The efficacy, safety, and a mode of action distinct from other antifungal drugs make turbinmicin a highly promising antifungal drug lead to help address devastating global fungal pathogens such as C. auris and A. fumigatus.

Key techniques performed in Madison:

• Hierarchical Component Analysis (HCA) and Principal Component Analysis (PCA) applied to the metabolomes of > 1000 microbial strains of marine origin.
• Nuclear Magnetic Resonance (NMR) and X-ray crystallographic structure elucidation of turbinmicin.
• In vitro microbroth antifungal assays employing multidrug-resistant pathogens.
• In vivo efficacy determination using a U.S. Food and Drug Administration (FDA) standard fungal model
• Chemical genetics using a Saccharomyces cerevisiae DNA-barcoded knockouts/knockdowns
• Fluorescence imaging of vesicle-mediated trafficking

Important advances in the field of metabolic engineering have been guided by our understanding of the structure and mechanism of enzymes that catalyze novel reactions. ObiH, an L-threonine transaldolase, is a recently discovered enzyme that catalyzes a stereoselective C-C bond formation reaction using simple metabolic precursors to form diverse β-hydroxy amino acids. We have carried out detailed mechanistic and structural characterization of ObiH to decipher its unique reactivity. Strikingly, ObiH forms a remarkably persistent glycyl quinonoid intermediate, whose unusual stability enables its reactivity with diverse aldehydes to form a palette of β-hydroxy amino acids. These results explain the basis for the unique reactivity of ObiH and provide a foundation for targeted protein engineering towards developing ObiH as a biocatalyst.

Key techniques performed in Madison:

• UV-vis spectroscopic analysis of catalytic intermediates
• Enzymatic synthesis of non-natural amino acids
• Protein crystallography
• MD simulations

Dr. Carey was invited to submit a graphical review as part of a special issue that celebrates the centennial of the 1920 Nobel Prize in Physiology or Medicine awarded to August Krogh, who is considered a foundational leader in the field of comparative physiology. Hibernation is an ideal topic for this special issue, as it beautifully fits with Krogh’s famous comment that “for a large number of problems there will be some animal of choice, or a few such animals, on which it can be most conveniently studied”, and especially to reinforce his appreciation of animals that display “mechanisms and adaptations of exquisite beauty”. Dr. Carey invited three former trainees to develop this review with her to highlight their work on hibernation biology that was carried out in the Carey Lab. They were fortunate to have the assistance of an excellent scientific illustrator – Rush Dhillon, a former member of the UW–Madison Metabolism community - who generated our terrific figures.

The cellular enzyme SIRT6 maintains metabolic homeostasis through the control of specific gene expression. SIRT6 represses genes by deacetylation of chromatin proteins that wrap DNA. This study describes the molecular details by which SIRT6 binds to nucleosomes, the basic repeating unit of chromatin. SIRT6 uses multivalent interactions with nucleosomes that couple productive binding to efficient deacetylation of histones. This work provides fundamental knowledge that will help explain how mutations in SIRT6 lead to human disease, or alternatively, to increased lifespan.

Key techniques performed in Madison:

• Fluorescence-based assays
• Use of reconstituted nucleosomes
• Nuclease sensitivity and electrophoretic mobility shift assays
• Protein NMR

Type 2 diabetes occurs when the pancreatic beta-cells are unable to fully compensate for peripheral insulin resistance by increasing their insulin secretion capacity, replication rate, or both. G protein coupled receptors (GPCRs) are integral membrane proteins that transmit extracellular signals to intracellular signaling partners to elicit biological changes. Many GPCRs are well-known to be critical regulators of beta-cell function and mass. In Schaid et al., we elucidate a previously unknown signal transduction mechanism mediated by Prostaglandin EP3 receptor (EP3), a receptor for the arachidonic acid metabolite, prostaglandin E2 (PGE2), in the beta-cell’s ability to compensate for insulin resistance in a mouse model of obesity and type 2 diabetes. EP3 was found to be solely coupled to the unique inhibitory G protein, Gz, down-regulating beta-cell cyclic AMP production and hormone-potentiated insulin secretion even in the absence of enhanced PGE2 availability. In overexpression experiments in primary pancreatic islets, we found the EP3ɣ variant is nearly fully constitutively active, explaining these disparate results. Our findings provide a strong premise for further study of EP3/Gz signaling pathway in developing new therapies for the beta-cell dysfunction of type 2 diabetes.

Key techniques performed in Madison:

• Employment of a diet-induced mouse model of type 2 diabetes and phenotypic characterization
• Live-cell imaging of beta-cell calcium and cAMP in response to GPCR ligands
• Real-time measurement of insulin release from intact islets in response to GPCR ligands
• Quantification of beta-cell replication and mass by immunofluorescence

This review examines advances in research to understand the sexual cycles of Coccidia parasites, especially Eimeria spp. and Toxoplasma gondii. The molecular basis of sex in these pathogens has been significantly unraveled by new findings in parasite differentiation along with transcriptional analysis of T. gondii and Eimeria spp. pre-sexual and sexual stages. Focusing on the metabolic networks, analysis of these transcriptome datasets shows enrichment for several different metabolic pathways. Specifically, glycolytic enzymes are consistently more abundant in T. gondii cat infection stages than the asexual stages and Eimeria spp. pre-sexual and gamete stages compared to sporozoites. As the cyst stages produced from these Coccidian sexual cycles are highly infectious, analyzing these sexual cycles will allow us to develop new methods to stop the spread of these parasites.

Lipids are crucial actors in metabolism, but comprehensive annotation of their variety of identities and genetic basis remain bottleneck challenges to biological interpretation. We used mass spectrometry–based discovery lipidomics to globally survey plasma and liver lipids of 384 diversity outbred mice, quantifying 3,283 molecular features. These known and unknown lipid features were mapped to 5,622 genetic loci, creating a large-scale genome-lipid association resource, termed LipidGenie. Harnessing this resource, we identified gangliosides through their association with B4galnt1, found evidence for a potentially novel group of sex-specific phosphatidylcholines, and suggested acyl-chain-specific functions for proteins of the ABHD family.

Key techniques performed in Madison:

• Discovery lipidomics by mass spectrometry
• Mouse husbandry and biopsies
• Cell transfection for protein overexpression
• Western blot analysis
• Biostatistic and bioinformatic analysis

Cancer cells are dependent on several amino acids for their growth and survival. We have demonstrated that lysine oxidase, an enzyme that depletes lysine and generates hydrogen peroxide, kills breast tumor cells by causing oxidative stress, which is buffered by the thioredoxin antioxidant pathway. Notably, the combination of lysine oxidase and auranofin, an inhibitor of thioredoxin reductase, results in enhanced tumor cell death. Hence, lysine oxidase primes tumor cells to respond to thioredoxin reductase inhibitors, pointing to this combination as a novel metabolic strategy for breast cancer.

Key techniques performed in Madison:

• Cellular ROS assay
• Reduced glutathione

(GSH)/oxidized glutathione (GSSG) assay

• Thioredoxin reductase assay

All cells must respond to stressful cellular situations, including those that result from toxins, cellular defect, or disease. How cells coordinate a multi-cellular response to environmental stress is not fully understood, in part because less is known about the upstream signaling networks activated by stress. This work, a collaboration between the Gasch and Coon labs at UW–Madison, used quantitative phospho-proteomics and regulatory network inference to understand the upstream signaling response in yeast activated by the reducing agent dithiothreitol (DTT). They also compared the implicated signaling network to the phosphoproteomic response to salt stress, revealing surprising similarities and differences in how cells respond to different types of stress. The results provide new information about how eukaryotic cells respond to suboptimal conditions with common versus specific responses.

Key techniques performed in Madison:

• phospho-proteomic mass spec in the Coon lab
• molecular biology.

Inhibition of mTOR (mechanistic Target Of Rapamycin) signaling by rapamycin promotes healthspan and longevity in mice, but “off-target” inhibition of mTOR Complex 2 (mTORC2) activity by rapamycin results in many negative side effects that have precluded its use as an anti-aging intervention in humans. In 2014, we discovered that genetic inhibition of hepatic mTORC2 (mTOR Complex 2) specifically reduces the lifespan of males, but not females. We tested the role of sex hormones in this response using gonadectomy, and determined that the sex-specific impact of reduced hepatic mTORC2 is not reversed by depletion of sex hormones. Ovariectomy unexpectedly promoted the midlife survival of female mice lacking hepatic mTORC2, significantly increasing the survival of those mice that do not develop cancer. In addition to identifying a sex hormone-dependent role for hepatic mTORC2 in female longevity, our results demonstrate that metabolic health is not inextricably linked to lifespan in mammals, as ovariectomized females having improved survival despite paradoxically having increased adiposity and decreased control of blood glucose levels.

Key techniques performed in Madison:

• Gonadectomy surgery
• Body composition analysis
• Metabolic chambers for analysis of food consumption, activity and energy expenditure
• Determination of mouse lifespan
• Glucose and insulin tolerance tests

Pancreatic b-cells couple nutrient metabolism with appropriate insulin secretion. In a pair of papers recently published in Cell Metabolism, Lewandowski et al. demonstrate that pyruvate kinase, rather than oxidative phosphorylation, is the ATP/ADP generator that closes β-cell KATP channels to initiate insulin secretion. Small-molecule activators of pyruvate kinase were shown to potently amplify insulin secretion from human and rodent islets by switching mitochondria from oxidative phosphorylation to anaplerotic phosphoenolpyruvate biosynthesis. In the companion paper, Abulizi et al. show in preclinical models of diabetes that pyruvate kinase activators improve glucose homeostasis in vivo, by increasing insulin secretion and insulin sensitivity, decreasing gluconeogenesis, increasing red blood cell glycolysis, and reducing hepatic steatosis. Together, these papers suggest that targeting pyruvate kinase may aid type 2 diabetes treatment.

Key techniques performed in Madison:

• Single-channel recordings of the ATP-sensitive K+ channel (KATP) from pancreatic b-cells
• Patch-clamp measurements of insulin exocytosis
• Live-cell imaging of calcium, ATP/ADP, mitochondrial membrane potential, mitochondrial pH, glutamate, lactate, and pyruvate kinase activity
• Simultaneous measurements of insulin and glucagon release from intact islets

Summary: The function of a T cell depends on its subtype and activation state. Here, we show that imaging the NAD(P)H lifetime of quiescent and activated T cells can be used to classify the cells. T cells isolated from human peripheral blood and activated in culture using tetrameric antibodies against the surface ligands CD2, CD3 and CD28 showed specific activation-state-dependent patterns of NAD(P)H lifetime. Logistic regression models and random forest models classified T cells according to activation state with 97-99% accuracy, and according to activation state (quiescent or activated) and subtype (CD3+CD8+ or CD3+CD4+) with 97% accuracy. NAD(P)H lifetime imaging can be used to non-destructively determine T-cell function.

Key techniques performed in Madison:

• Two-photon fluorescence lifetime imaging

Men and women display important sexual dimorphisms in breathing (and ultimately metabolism), controlled by key areas in the central nervous system (CNS) including brainstem and cervical spinal cord regions. While neurons in these regions are well-studied, the contributions of microglia, CNS-resident immune cells that are key cellular partners to neurons, have not been studied with regard to how they may contribute to some of these sex differences in respiration. We analyzed the transcriptomes of microglia from adult male and female rat brainstem and cervical spinal respiratory neural control CNS regions, and found that while brainstem microglia generally lacked sexual dimorphisms in gene expression, cervical spinal microglia isolated from regions where phrenic motor neurons reside (which innervate the diaphragm inspiratory muscle) had hundreds of genes that were differentially expressed between the sexes. While Gene Ontology and STRING analyses failed to identify functional gene categories into which the differentially upregulated genes in female microglia fell relative to males, the differentially down-regulated genes in female cervical spinal microglia segregated into categories including cellular and protein metabolism, ribosome and proteasome function, and oxidative phosphorylation. These data suggest that male spinal microglia may have higher protein synthesis and posttranslational protein regulatory capacity, as well as higher oxidative metabolism, than female cells, perhaps contributing to sexual dimorphisms in respiratory neural control. Interestingly, bioinformatics analysis using MAGICTRICKS identified the histone demethylase PHF8 (which demethylates H3K4 to repress gene transcription) as a major driver of sexually dimorphic down-regulated genes in female cervical spinal microglia versus male, and EZH2 (a component of the polycomb repressive complex that places methyl marks on H3K27) as a primary transcriptional driver of differentially down-regulated genes in cervical spinal microglia compared to brainstem microglia, in both sexes. These observations suggest that the cellular mechanisms controlling microglial transcriptomes differ both by CNS region and by sex, underscoring the need to better understand how the local CNS microenvironment and hormonal milieu impacts microglial function as it relates to how the brain controls breathing. This work sets the stage for future studies that will begin to dissect the molecular mechanisms underlying potential histone modifications in microglia, and how differentially expressed microglial genes contribute to respiratory motor neuron function in the context of physiologic systems that are integral to metabolic regulation.

Key techniques performed in Madison:

• RNA-sequencing
• Immunomagnetic cell sorting
• Computer-controlled gas delivery system for manipulating inspired oxygen and carbon dioxide levels
• Novel MAGICTRICKS bioinformatics algorithm to identify common transcriptional gene drivers

Furan fatty acids (FuFAs), characterized by the presence of a furan ring within the molecule’s hydrophobic core, are found at low levels in many cells, and have properties that make them attractive in industrial, medical and other applications, but their biosynthetic origins are not clear. In this study, an interdisciplinary research team deployed a suite of genetic, genomics, biochemical, and high-resolution analytical techniques (GC-MS and NMR) to identify the chemical structures of intermediates in the FuFA acid biosynthetic pathway and isotopic studies to determine the source of the oxygen in the furan ring. These findings lay the foundation for detailed study of novel biosynthetic enzymes, obtaining new insights into cellular FuFA functions, and engineering cells that overproduce these fatty acids for various applications.

Quantitative mass spectrometry has emerged as a central technology for biomedical research and provides deep insights into complex biological mechanisms and disease states. Multiplexed isobaric tagging is a particularly powerful tool that enables broad investigation of many samples in a single LC-MS/MS experiment, but the cost of employing commercial tagging reagents for large-scale, discovery-based studies is often prohibitive. As a practical alternative, we previously developed our own isobaric tags, dimethylated leucine (DiLeu), that we synthesize in-house at a fraction of the cost. In this work, we devise a novel synthetic avenue to generate additional isotopic variants that allow us to greatly increase multiplexing capability, beyond that of established isobaric tags, while preserving the existing design and its advantages. The result is a new generation of DiLeu tags that permits simultaneous identification and quantification of 21 samples in parallel on mainstream Orbitrap MS platforms. As such, the 21-plex DiLeu isobaric tags offer the potential to substantially facilitate the high-throughput analyses and large-scale experiments that are increasingly being conducted in discovery-based quantitative proteomics and metabolomics workflows.

Key techniques performed in Madison:

• Novel chemical tag design and synthesis
• Stable isotope labeling of complex biological samples
• Highly multiplexed quantitative analysis by mass spectrometry

General Background:
In ruminant animals, carbohydrates are fermented in the rumen and absorbed as volatile fatty acids rather than glucose and other monomers, therefore nearly all glucose must be generated via gluconeogenesis in the liver and then transported to the mammary gland as a lactose precursor. In the lactating dairy cow, this glucose need can range from 10 to 20 lb of glucose daily and lactose is a key limiter of milk production.

Study summary:
Shifting nutrient partitioning to support the glucose and energy needs during the peripartum period in lactating dairy cows can contribute to improved feed efficiency and improved metabolic health. While lactate is a known gluconeogenic precursor, previous attempts to feed lactate sources led to rumen acidosis. Supplementation of ammoniated lactate to postpartum dairy cows resulted in improved feed efficiency and decreased incidence of hyperketonemia and hepatic lipid content. Examining hepatic gene expression and protein abundance supported a multi-faceted mechanism of increasing gluconeogenic precursors to support glucose production and increasing the TCA cycle oxidative capacity which together may have supported the improvements in feed efficiency and metabolic health.

Key techniques performed in Madison:

• Bovine liver biopsies
• Liver lipid quantification
• Real-time quantitative PCR
• Western blot analysis
• Metabolite quantification

Proteins perform their function by interacting with other proteins. Protein-protein interaction (PPI) analysis allows us to better understand the functions of individual proteins, the mechanisms of biological processes, and disease mechanisms. In many metabolic reactions, PPI is essential for electron transfer reactions (ex. electron transfer from ferredoxin to lipoic acid synthase). Information on PPI are abundant in published PubMed articles and their extraction is possible only through automated approaches. We describe the standard text-mining protocol that includes four major tasks, namely, recognizing protein mentions, normalizing protein names and aliases to unique identifiers such as gene symbol, extracting PPIs, and visualizing the PPI network using Cytoscape or other visualization tools. We show the application of KinderMiner, our recent text-mining tool on retrieving significant co-occurring protein pairs from ~30 million PubMed articles. KinderMiner retrieved known and new PPIs from PubMed articles and performed better than String and PolySearch2, the well-known PPI resources, on extracting mitochondrial PPIs. KinderMiner is a general text mining system applicable to a wide range of metabolic and other biological queries.

Key techniques performed in Madison:

• Automated approach to replace abbreviations with respective original text in ~30 million PubMed articles
• Many new features were added to KinderMiner
• For applications other than PPI using KinderMiner, we generated diseases lexicon, phenotypes lexicon, and drugs lexicon from existing resources.

Identifying the causal gene(s) that connects genetic variation to a phenotype is a challenging problem in genome-wide association studies (GWASs). Here, we develop a systematic approach that integrates mouse liver co-expression networks with human lipid GWAS data to identify regulators of cholesterol and lipid metabolism. Through our approach, we pinpoint Sestrin1 as a causal gene associated with plasma cholesterol levels in humans. Our validation studies demonstrate that Sestrin1 influences plasma cholesterol in multiple mouse models and regulates cholesterol biosynthesis. Our results highlight the power of combining mouse and human datasets for prioritization of human lipid GWAS loci and discovery of lipid genes.

Key techniques performed in Madison:

• Co-expression network analysis with the Center for High Throughput Computing (CHTC)
• Radio-label tracing of cholesterol biosynthesis

Toxoplasma gondii is an obligate intracellular parasite that is auxotrophic for several key metabolites and must scavenge these from the host. It is unclear how Toxoplasma manipulates host metabolism for its overall growth rate and non-essential metabolites. To address this question, we measured changes in the joint host-parasite metabolome over a time course of infection. These experiments led to the discovery of a Toxoplasma sedoheptulose bisphosphatase, which funnels carbon from glycolysis into ribose synthesis through an energetically driven dephosphorylation reaction. This second route for ribose synthesis resolves a conflict between the Toxoplasma tricarboxylic acid cycle and pentose phosphate pathway, which are both NADP+ dependent. Sedoheptulose bisphosphatase represents a novel step in Toxoplasma central carbon metabolism that allows Toxoplasma to satisfy its ribose demand without using NADP+. Sedoheptulose bisphosphatase is not present in humans, highlighting its potential as a drug target.

Key techniques performed in Madison:

• Discovery mass spectrometry metabolomics and RNAseq
• CRISPR-mediated genome edited Toxoplasma
• Stable isotope labeling with mass spectrometry

The opportunistic human pathogen Aspergillus fumigatus synthesizes spore specific secondary metabolites, several of which (DHN melanin, trypacidin) are virulence factors in invasive aspergillosis. This article demonstrates that DHN melanin and other spore metabolites serve as UV protectants for the fungus dependent on fungal isolate. This work supports previous hypotheses that secondary metabolites which evolved for protection against environmental stresses can serve as double edged swords in pathogenesis.

Key techniques performed in Madison:

• Creation of Aspergillus mutants
• Zebrafish virulence assays

Immune-mediated destruction of insulin-producing β-cells causes type 1 diabetes (T1D). However, how β-cells participate in their own destruction during the disease process is poorly understood. Here, we report that modulating the unfolded protein response (UPR) in β-cells of non-obese diabetic (NOD) mice by deleting the UPR sensor IRE1α prior to insulitis induced a transient dedifferentiation of the β-cells, resulting in substantially reduced islet immune cell infiltration and β-cell apoptosis. Single-cell and whole-islet transcriptomics analyses of immature β-cells revealed remarkably diminished expression of β-cell autoantigens, MHC class I components and upregulation of immune inhibitory markers. IRE1α-deficient mice exhibited significantly less cytotoxic CD8+ T-cells in their pancreata and adoptive transfer of their total T-cells did not induce diabetes in Rag1-/- mice. Our results indicate that inducing β-cell dedifferentiation, prior to insulitis, allows these cells to escape immune-mediated destruction and may be used as a novel preventive strategy for T1D in high-risk individuals.

Key techniques performed in Madison:

• Bulk-RNAseq
• Single cell RNAseq
• Adoptive transfer experiments
• Immunophenotyping
• Inflammation scoring
• Metabolic assays
• Islet histomorphometry

Enzymes like kinases can often catalyze many molecular events, each setting off a separate signaling cascade. However, disentangling the complex diversity of the signaling events can be a challenge. James Johnson (Burkard lab) in collaboration with Alex Hebert (Coon lab) built and tested a new chemical genetic tool. By manipulating the kinase under study, Polo-like kinase 1, he was able to reprogram it to selectively identify either serine or threonine amino acids. Next, he recoded the substrate proteins to ‘toggle’ serine-and-threonine, thereby placing desired phosphorylation events under genetic control. This was validated with deep phosphoproteomics.

Conventional cell culture media has been a staple in scientific research for over 70 years. This media has remained relatively unchanged during this time. The major goal of this media is to keep cells alive and have them grow rapidly, but is this the best way? This review published by the Cantor looks at cell culture media from a different view point and articulates the importance of understanding how cells work in the human body. Further summary.

The advent of wearable devices for health monitoring is ushering in an era of more personalized and preventive medicine. However, most consumer-grade health data is currently limited to metrics such as step count and heart rate. We imagine technology that could enable the passive and continuous monitoring of metabolic health by measuring the diverse set of metabolites in urine. There are over 4,500 metabolites in human urine that are known to be associated with more than 600 human conditions. These metabolites can further reflect lifestyle choices, such as alcohol and tobacco consumption, in addition to exercise and physical activity. In this small pilot study, we collected every urine sample from two healthy volunteers for 10 days along with biohealth data (diet, sleep, exercise) from smart phone applications. Using gas chromatography and mass spectrometry we measured the relative levels of hundreds of individual metabolites and found patterns in the urine metabolites that reflected the ebb and flow of daily life. For instance, we could see associations with nutrition, OTC drug metabolism, exercise, and sleep. We plan to expand this type of study to a larger cohort and to develop technology that could be integrated into a “smart bathroom” and achieve passive measurements of metabolic health at scale.

Key techniques performed in Madison:

• Longitudinal metabolomics analysis using gas chromatography and mass spectrometry
• Collection and integration of digital health data collected from smartphones and wearable devices.

Increased carbohydrate consumption increases hepatic de novo lipogenesis, which has been linked to the development of chronic metabolic diseases, including obesity, hepatic steatosis, and insulin resistance. Stearoyl CoA desaturase 1 (SCD1) is a critical lipogenic enzyme that catalyzes the synthesis of two monounsaturated fatty acids, oleate and palmitoleate, from the saturated fatty acids stearate and palmitate, respectively. SCD1-deficient mouse models are protected against diet-induced adiposity, hepatic steatosis, and hyperglycemia. However, the mechanism of this protection by SCD1 deficiency is unclear. Using liver-specific SCD1 knockout (LKO) mice fed a high-carbohydrate, low-fat diet, we show that hepatic SCD1 deficiency increases systemic glucose uptake. Hepatic SCD1 deficiency enhanced glucose transporter type 1 (GLUT1) expression in the liver and also up-regulated GLUT4 and adiponectin expression in adipose tissue. The enhanced glucose uptake correlated with increased liver expression and elevated plasma levels of fibroblast growth factor 21 (FGF21), a hepatokine known to increase systemic insulin sensitivity and regulate whole-body lipid metabolism. Feeding LKO mice a triolein-supplemented but not tristearin-supplemented high-carbohydrate, low-fat diet reduced FGF21 expression and plasma levels. Consistently, SCD1 inhibition in primary hepatocytes induced FGF21 expression, which was repressed by treatment with oleate but not palmitoleate. Moreover, deletion of the transcriptional coactivator PPARγ coactivator 1α (PGC-1α) reduced hepatic and plasma FGF21 and white adipocyte tissue-specific GLUT4 expression and raised plasma glucose levels in LKO mice. These results suggest that hepatic oleate regulates glucose uptake in adipose tissue either directly or partially by modulating the hepatic PGC-1α-FGF21 axis.

Key techniques performed in Madison:

• Measurements of Plasma FGF21 levels using the FGF-21 Quantikine ELISA Kit.
• in vivo 2-[3H] deoxyglucose uptake assays.
• Positron Emission Tomography-computed tomography (PET/CT) imaging analysis.

The goal of this paper was to quantify the spatial distribution of tumor cells with distinct cell metabolism using multivariate spatial statistical tools. Autofluorescence images of tumors in vivo and in 3D culture revealed spatial patterns of cell metabolism specific to drug response and resistance. This quantitative analysis framework can be used to assess the effect of drugs on cell-level heterogeneity in tumors.

Key techniques performed in Madison:

• Two photon fluorescence lifetime imaging of NAD(P)H and FAD
• Single cell segmentation
• Multivariate spatial statistical analysis

Heat production (thermogenesis) in mammalian bodies creates the so-called b-adrenergic lipolytic environment which also serves to modify disease progression and inflammatory conditions. Furthermore, stimulation of thermogenesis could be used to combat obesity. However, the total demand for heat production is offset by heat losses through skin; this manuscript therefore focuses on the key properties of skin that determine heat loss. This interdisciplinary project relied on space science engineering collaborators to calculate the total energy sink through all modes of heat transfer. We found that a large fraction of total energy expenditure in mice can be accounted for by evaporative cooling, especially for mice with genetic lesions in skin lipogenesis. These mice are also resistant to diet-induced obesity, prompting us to speculate that skin cooling is a significant player in calorie disposition.

Key techniques performed in Madison:

• Analysis of heat transfer processes in biological systems, using infrared thermography (FLIR) and assay of trans-epidermal water loss (TEWL)
• Assay of skins of genetically- and environmentally- modified mice
• Evaluation of dermal white adipose tissues by immunofluorescent stains
• Energy expenditure assays

The ability to form biofilms, multicellular communities encapsulated by an extracellular matrix, is a widespread trait in bacteria. Using time-resolved metabolomic, fluxomic, and proteomic analyses we discovered a surprisingly widespread and dynamic remodeling of both primary and secondary metabolism during biofilm development in Bacillus subtilis. This work represents the most comprehensive and systematic characterization of metabolic alterations during bacterial biofilm development ever undertaken. Our results demonstrate that dynamic remodeling of metabolism is a critical, and heretofore unreported, component of the highly coordinated response that leads to biofilm development.

Key techniques performed in Madison:

• Targeted metabolomics
• Shotgun proteomics
• ¹³C isotope tracer experiments

Type 2 diabetes occurs when pancreatic ß-cells are unable to keep up with the demand for insulin, often a consequence of insulin resistance. Human genetic studies show that most of the genetic predisposition to T2D results in a reduction of ß-cell insulin secretion or ß-cell mass. We used a genetically divers outbred population of mice to screen for genes that affect ß-cell insulin secretion. This was a heroic effort carried out by Mary Rabaglia, Kiki Schueler, Donnie Stapleton, and Shane Simonett and carefully supervised by Mark Keller. We collaborated with Gary Churchill at the Jackson Lab. The work identified multiple loci associated with insulin secretion. In three cases, we derived knockout mouse models and all showed an insulin secretion phenotype. With Josh Coon, we carried out proteomic and lipidomic analysis and have identified gene loci controlling protein and lipid abundance. With Federico Rey, we have connected the intestinal microbiome with genetic differences in serum bile acids. We now have multiple projects testing interesting candidate genes that have emerged from this screen.

Key techniques performed in Madison:

• insulin secretion measurements in isolated islets
• mass spectrometry of proteins, lipids, and metabolites
• systems biology analysis of pathways associated with metabolic disease

It has long been a puzzle why felines are the only mammal permissive of the sexual cycle of Toxoplasma gondii. Cats lack intestinal delta-6-desaturase activity and as a consequence, they have systemic increases in linoleic acid. In this study, Morgridge Metabolism Interdisciplinary (MMI) fellow Bruno Martorelli Di Genova determined that inhibition of murine delta-6-desaturase and dietary supplementation with linoleic acid allowed Toxoplasma sexual development to occur in mice.

Key techniques performed in Madison:

• Intestinal organoid culture of cat and mouse cells
• CRISPR-mediated genome edited mouse model
• High resolution microscopy for Toxoplasma sexual stages 

The mechanistic target of rapamycin (mTOR) is a conserved protein kinase that regulates growth and metabolism. Rapamycin, a drug that extends lifespan, promotes longevity through inhibition of mTORC1, but we have found that rapamycin also inhibits mTORC2. We find that hypothalamic mTORC2 signaling normally increases with age, and that mice genetically engineered to lack hypothalamic mTORC2 signaling display higher fat mass and impaired glucose homeostasis throughout life, become more frail with age, and have decreased overall survival. We conclude that hypothalamic mTORC2 is essential for the normal metabolic health, fitness, and lifespan of mice.

Key techniques performed in Madison:

• Phenotyping of glucoregulatory control, including in vivo glucose and insulin tolerance tests
• Determination of body composition, activity and energy expenditure using metabolic chambers
• Determination of lifespan and longitudinal assessment of frailty

Mitochondrial proteins are replete with phosphorylation, yet its functional relevance remains largely unclear. The presence of multiple resident mitochondrial phosphatases, however, suggests that protein dephosphorylation may be broadly important for calibrating mitochondrial activities. Here, we use CRISPR to delete the mitochondrial phosphatase Pptc7 in Mus musculus and find these mice exhibit hypoketotic hypoglycemia, elevated acylcarnitines and serum lactate, and die soon after birth. Pptc7−/− tissues have markedly diminished mitochondrial size and protein content despite normal transcript levels, and aberrantly elevated phosphorylation on select mitochondrial proteins. Overall, our data define Pptc7 as a protein phosphatase essential for proper mitochondrial function and biogenesis during the extrauterine transition.

Key techniques performed in Madison:

• Generation of a knockout mouse using CRISPR
• Acylcarnitine profiling coupled with unbiased metabolomic and lipidomic analysis of perinatal mouse tissue
• Phosphoproteomic and proteomic analysis of perinatal mouse tissue
• Electron microscopy of mitochondria in perinatal mouse tissue
• Mitochondrial import assays

AT-1/SLC33A1 is a key member of the endoplasmic reticulum (ER) acetylation machinery, transporting acetyl-CoA from the cytosol into the ER lumen where acetyl-CoA serves as the acetyl-group donor for Nε-lysine acetylation. Dysfunctional ER acetylation, as caused by heterozygous or homozygous mutations as well as gene duplication events of AT-1/SLC33A1, has been linked to both developmental and degenerative diseases. Here, we used two mouse models of AT-1 dysregulation to study how the cytosol-to-ER flux of acetyl-CoA promotes functional crosstalk between different intracellular organelles and compartments.

Key techniques performed in Madison:

• New mouse models
• Biochemistry, histology, EM and diet manipulation
• Quantitative proteomics
• Stoichiometry of acetylation
• Metabolomics
• Primary cell isolation and culture
• SIM microscopy

Growth factor signaling plays a key role in directing metabolic flux within cells. In this work, we demonstrate a mechanism by which the mammalian ESCRT machinery acts to attenuate growth factor driven changes in metabolism using a combination of CRISPR-mediated genome editing, lattice light sheet microscopy, and super resolution STED microscopy.

Key techniques performed in Madison:

• CRISPR-mediated genome editing using mammalian cell lines
• FACS sorting to identify clonal edited cell populations
• Stimulated emission depletion (STED) microscopy at the UW–Madison Optical Imaging Core
• Thin section electron microscopy in collaboration with the SMPH EM facility
• Electron tomography using the TF30 transmission electron microscope

New technology is needed to advance the characterization of protein glycosylation, which is a prevalent, chemically complex, and biologically diverse post-translational modification that plays particularly important roles at the cell surface. Here we applied a new technology we developed called activated ion electron transfer dissociation (AI-ETD) to improve the identification of intact N-glycopeptides, and we also designed new visualization approaches to capture the heterogeneity of the glycoproteome at a systems scale.

Key techniques performed in Madison:

• Mass spectrometer instrumentation modifications
• MS-based glycoproteomics
• Development of new visualization tools

In this study, GLBRC researchers identified and characterized a new type of enzyme that specifically cleaves β-aryl ether bonds. This work demonstrates that there is more variability in the bacterial pathway for cleaving the β-aryl ether bond than previously thought, and illustrates the importance of studying this pathway – as well as other pathways involved in metabolizing lignin-derived compounds – in multiple species to better understand and be able to capitalize on this diversity. Characterizing microbial strategies for lignin breakdown is important for understanding plant biomass turnover in nature, and could aid in developing industrial systems for producing commodity chemicals from this abundant renewable resource.

Key techniques performed in Madison:

• Expression and purification of proteins from recombinant hosts and cell free systems
• Kinetic analysis of wild type and mutant proteins (including determine of Km, Vmax and kcat)
• Modeling of enzyme active site
• HPLC of aromatic compounds and glutathione-aromatic conjugates
• Phylogenetic analysis of protein databases

Here the Hittinger lab showed how the genes encoding maltose transporters could undergo ectopic gene conversion to produce genes encoding transporters for the bulkier sugar maltotriose, a critical sugar in wort or the malt extract used to brew beer. This adaptive laboratory evolution experiment showed how Saccharomyces eubayanus, the wild, cold-tolerant parent of hybrid lager yeasts, could gain this industrially important function.

Key techniques performed in Madison:

• Illumina sequencing (Biotechnology Center)
• Bulk segregant analysis (Hittinger Lab)
• Adaptive laboratory evolution (Hittinger Lab)
• Phylogenetic analysis (Hittinger Lab)

The fungal secondary metabolome is both a primary source of new pharmaceuticals and an endogenous source of secondary metabolites important for fungal warfare, defense and development. This review provides up-to-date insights into how to harvest bioactive fungal secondary metabolites and to elucidate their roles in fungal ecology.

Key techniques performed in Madison:

• Fungal molecular biology (deletion, over-expression and mutation of genes)
• Culture growth and extraction of fungal metabolites
• Liquid chromatography, high resolution mass spectrometry, NMR analysis
• Bioinfomatics

Low protein diets promote metabolic health in both humans and mice, but the mechanisms that drive the physiological benefits of these diets have not been determined. Here, we find that reducing dietary protein significantly alters the taxonomic composition of the gut microbiome, but that this shift does not mediate the metabolic benefits of a low protein diet.

Key techniques performed in Madison:

• 16S rRNA profiling, RNA-seq and all gene expression analysis
• Phenotyping of glucoregulatory control, including in vivo glucose and insulin tolerance tests
• Determination of body composition, activity and energy expenditure using metabolic chambers

This study demonstrated that two tyrosine aminotransferases play key roles in tyrosine degradation and metabolism in Arabidopsis thaliana, by combining reverse genetics, metabolite profiling, and 13C-precursor time course feeding experiments.

Key techniques performed in Madison:

• 13C labeling measurement using GC-MS
• Dark respiration measurement using LI-COR LI-6400XT

Beta cell mitochondria play a central role in coupling glucose metabolism with insulin secretion. These studies identify a novel role for complex I in mediating the effects of obesity on the beta cell secretory pathway, and implicate cyclin-dependent kinase 1 (CDK1) signaling as the mechanism driving this effect. Two graduate students in the Merrins lab, Trilly Gregg and Sophie Sdao, spearheaded the project (https://www.merrinslab.org/publications).

Key techniques performed in Madison:

• Live-cell imaging of the beta cell secretory pathway, including real-time measurements of cytosolic citrate, ATP/ADP, and calcium
• 2-photon fluorescence lifetime imaging of mitochondrial NAD(P)H
• Direct measurements of electron transport chain fluxes using oxygen consumption rate measurements

Coenzyme Q is among the most hydrophobic molecules in nature, which presents challenges for its biosynthesis and delivery. In this paper, we describe how COQ9 uses accesses, binds, and presents CoQ precursors to another member of the biosynthesis machinery.

Key techniques performed in Madison:

• MS-based lipidomics with Josh Coon’s group
• Protein purification
• Crystallography with Craig Bingman and Bob Smith from the crystallography core
• Liposome flotation assays (to assess protein binding to membranes of varying lipid composition)
• Yeast growth assays

A multi-disciplinary research team has uncovered new clues about calorie restriction and how it works to delay aging and age-related diseases. The researchers used a range of molecular profiling techniques to catalog over 20,000 molecules in rhesus liver. Data were analyzed using complex statistical approaches including machine learning techniques. The team showed that CR reprogrammed metabolism by harnessing distinct control mechanisms including RNA processing.

**Special Note: This publication was highlighted on the cover of the March 6, 2018 issue of Cell Metabolism.

Key techniques performed in Madison:

The normal turnover of many integral membrane proteins is mediated by the formation of multivesicular endosomes, which sequester cargoes destined for degradation within intralumenal vesicles that bud away from the cytoplasm. In this work, we demonstrate a direct role for the ESCRT-III complex during membrane bending that is necessary to create intralumenal vesicles and define a new regulatory mechanism that preserves the continual action of the ESCRT machinery in repetitive cycles of vesicle biogenesis.

Key techniques performed in Madison:

• Immunogold labeling for electron microscopy
• Super resolution (stimulated emission depletion) fluorescence microscopy
• Electron tomography

This publication describes the isolation and analysis of gain-of-function mutations that overcome the requirement for an essential, conserved molecular chaperone. Results of analyses of isolated suppressor variants point to the idea that fine-tuning of Hsp70’s interaction cycle with substrate proteins can have major, unexpected consequences in vivo.

Key techniques performed in Madison:

• Yeast gain-of-function genetic selections
• Structural analysis using NMR through National Magnetic Resonance Facility at Madison (NMRFAM)
• Interaction studies using fluorescence anisotropy

This work identifies antagonistic small molecule communication between two agricultural pathogens, the bacterium Ralstonia solanacearum and the fungus Aspergillus flavus. The fungal alkaloid product enhances fungal germination and slows bacterial growth but is inhibited in production by a bacterial lipopeptide.

Key techniques performed in Madison:

• Creation of genetic mutant strains of bacteria and fungi
• RNA-seq and all gene expression analysis
• Microbial physiology and statistical analysis

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in the blood of obese, insulin-resistant humans and rodents. In this work, we show that specifically reducing dietary BCAAs rapidly reverses diet-induced obesity and improves glucose tolerance and insulin sensitivity in diet-induced obese mice. Significantly, this occurs even in mice continuing to eat an otherwise unhealthy high-calorie, high-sugar “Western” diet. Reducing dietary levels of the BCAAs promotes leanness by increasing energy expenditure. Our results link dietary BCAAs with the regulation of metabolic health and energy balance in obese animals, and suggest that specifically reducing dietary BCAAs may represent a highly translatable option for the treatment of obesity and insulin resistance.

Key techniques performed in Madison:

• Feeding of amino acid defined diets to diet-induced obese mice
• Phenotyping of glucoregulatory control, including in vivo glucose and insulin tolerance tests, and ex vivo characterization of pancreatic islet function and metabolism
• Determination of body composition, activity and energy expenditure using metabolic chambers

This publication demonstrates that brown adipose tissue thermogenesis regulates whole body lipid homeostasis. Using mice lacking uncoupling protein-1, a protein critical for heat production, we revealed that loss of brown adipose tissue thermogenesis elevates the synthesis of monounsaturated fatty acids in white adipose tissue and causes triglyceride accumulation in the liver.

Key techniques performed in Madison:

• Analysis of fatty acid composition using gas chromatography
• in vivo lipid synthesis and export assays

In a three-paper series, we established new post-transcriptional regulatory processes for coenzyme Q (CoQ) metabolism. First, we discovered small- molecule and lipid activators for the atypical kinase-like protein COQ8 and developed an analog- sensitive version that can be selectively inhibited in vivo. Second, we used a multi-omic approach that incorporates measurements of mRNAs, proteins, lipids, and metabolites to identify endogenous targets for the mRNA binding protein, Puf3p, including the CoQ-related methyltransferase, COQ5. Finally, using a similar approach, we reveal numerous connections between mitoproteases and their biological functions, including a direct role for Oct1p in processes COQ5.

UW Collaborators: Josh Coon’s group, Marv Wicken’s group, John Markley’s group, and Craig Bingman

Further summary: https://morgridge.org/story/cracking-the-code-of-coenzyme-q-biosynthesis/

Key techniques performed in Madison:

• One-dimensional (1D) 1H nuclear magnetic resonance (NMR) ligand-affinity screen
• Mass spec-based proteomics, metabolomics, and lipidomicsa
• Liposome flotation analyses (i.e., protein interactions with liposomes)
• Molecular modeling
• Recombinant protein purification and various yeast growth assays
• RNA binding protein analyses (RNA Tagging and HITS/CLIP)

The endoplasmic reticulum (ER) serves as a platform for the packaging of most secretory proteins into conserved COPII-coated transport carriers destined for ER-Golgi intermediate compartments (ERGIC) in animal cells. In this work, we demonstrate that Trk-fused gene (TFG) simultaneously captures and concentrates COPII transport carriers at the ER/ERGIC interface to enable the rapid movement of secretory cargoes to the ERGIC.

Key techniques performed in Madison:

• CRISPR-mediated genome editing
• Super resolution (stimulated emission depletion) fluorescence microscopy
• In vitro reconstitution of vesicle tethering

This paper describes the isolation and structural characterization of the natural product ecteinamycin, the product of a marine-derived bacterium. Additionally, this paper strongly supports the hypothesis that ionophore antibiotics such as ecteinamycin and other more well established ionophores such as commonly used feed additives have tremendous potential as therapeutics for treating Clostridium difficile infections in humans. Ecteinamycin showed exceptional potency toward C. difficile, and yeast chemical genomics suggested that ecteinamycin disrupts vesicular trafficking pathways in eukaryotic cells that are required for C. difficile toxin maturation; impairment of such pathways appears to protect humans from the toxin effects of C. difficile.

Key techniques performed in Madison:

• LCMS-based metabolomics
• Yeast chemical genomics using a DNA-barcoded knockout library
• E. coli chemical genomics using DNA-barcoded knockout library
• Residual Dipolar Couplings (RDCs) to assist with configurational analysis

This study identifies WBSCR16 as a protein that co-localizes with OPA1 on the outer surface of the inner mitochondrial membrane. It also shows WBSCR16 to act as a GEF for OPA1, a dynamin-like GTPase essential to mitochondrial fusion, which is in turn essential to mitochondrial function.

Key techniques performed in Madison:

• Gene mapping and deep sequencing to identify a spontaneous mutation in the Wbscr16 gene in mice
• Mitochondrial subfractionation to localize proteins within different mitochondrial compartments
• Use of a proximity ligation assay to demonstrate close enough proximity of WBSCR16 and OPA1 in situ for direct protein-protein interactions
• GEF activity assays, in vitro and for intact mitochondria, to demonstrate a role for WBSCR16 as an intramitochondrial GEF with specific activity for OPA1

In this paper, we describe the functional identification of a kidney-specific genomic regulatory region in the mouse that controls the expression of the Cyp27b1 gene whose enzymatic product is responsible for the final synthesis of 1α,25-dihydroxyvitamin D3, the biologically active hormonal form of vitamin D3.

Key techniques performed in Madison:

• ChIP-seq analysis of genetic and epigenetic features of mouse kidney tissue to define the locations and activity of novel regulatory regions of the Cyp27b1 gene
• CRISPR/Cas9 gene-editing methods to delete potential regulatory regions in the mouse for subsequent loss-of-function analyses
• Extensive systemic, regulatory and skeletal phenotyping to describe the consequence of aberrant Cyp27b1 regulation by mineral regulating hormones

This publication describes the identification and molecular characterization of mutations in yeast that were engineering and evolved to ferment xylose, a pentose sugar that the organism does not normally metabolize. Using genetic, proteomic and metabolomic comparisons, it was determined that inactivating mutations in Fe-S cluster mitochondrial biogenesis, cAMP/Protein Kinase A and MAP Kinase signaling pathways enabled the conversion of xylose to ethanol.

Key techniques performed in Madison:

• High throughput genome resequencing (UW Biotech Center) and sequence analysis
• Yeast genetic engineering and directed evolution
• Proteome quantification
• Intracellular and extracellular metabolite quantification

This publication reveals functional attributes of Tim44 the "hub" protein of the Hsp70 chaperone-based “import motor”, which is located in the matrix of mitochondria and required for the translocation of proteins into that subcompartment from the cytosol.

Key techniques performed in Madison:

• Site-specific in vivo crosslinking using photoactivatable alternative amino acid Bpa
• in organellar mitochondria import assays
• Yeast genetic suppressor analysis

In this paper, we developed a new computational method to infer gene regulatory networks in multiple non-model species from global transcriptomic profiles. We used this approach to study how regulatory networks, associated with stress response, evolve across six yeast species.

Key techniques performed in Madison:

• Design and implementation of the multi-species network inference algorithm
• Analytical measures used to study regulatory network evolution at the structure and function level.

This publication presents a statistical method for normalizing data from single-cell RNA sequencing experiments. Normalization is a critical step required prior to downstream analysis to ensure that technical artifacts are removed and that gene expression profiles can be compared across cells.

Key techniques performed in Madison:

• Quality control, normalization, and downstream analysis of bulk and single-cell RNA-sequencing data.

This study conducted phylogeny-guided structure-function analyses of key regulatory enzymes of tyrosine biosynthesis (prephenate and arogenate dehydrogenases), discovered a single amino acid residue that confers their substrate specificity and feedback inhibition, and provided molecular basis of long-known two alternative tyrosine biosynthetic pathways.

Key techniques performed in Madison:

• Structure-guided phylogenetic analysis of proteins from distantly-related organisms.
• Prephenate and arogenate dehydrogenase assays using a plate reader and LC-fluorescence detection.
• Key amino acid residue identification by defining a precise evolutionary timing of enzyme neofunctionalization, followed by primary sequence and structure comparisons, and site-directed mutagenesis.

In this publication, we showed that more active metabolic networks also require more robust repression systems. The paper also showcases some of the metabolomic capabilities of the LBMS facility.

Key techniques performed in Madison:

• Liquid chromatography-mass spectrometry (LC-MS) assays to measure central carbon metabolites by the Laboratory of Biomolecular Mass Spectrometry (LBMS)
• Custom galactose-1-phosphatse assay developed by LBMS

This publication reveals that disruption of the poorly characterized mitochondrial phosphatase, Ptc7p, perturbs mitochondrial phosphorylation of ~20 matrix proteins, partially inactivates citrate synthase, and decreases mitochondrial respiratory function.

Key techniques performed in Madison:

• LC-MS/MS-based phosphoproteomics
• Recombinant protein purification, including site-specific phospho-incorporation
• Citrate synthase assays
• Blue native PAGE
• Yeast liquid culture and plate-based assays
• Molecular modeling of point mutations

This publication shows slight alteration in the positioning and geometric configuration of double bonds in isomers of linoleic acid have substantial effects on the onset of arthritis. Tissue fatty acids and joint cytokines are also presented.

Key techniques performed in Madison:

• GC fatty acid analysis conducted in ME Cook lab
• Lumines Systems Cytokine array analysis conducted in the shared Pharmacokinetics, Pharmacodynamics, Pharmacogenetics Laboratory (3Plab)
• Arthritic mouse experiment conducted in Animal Sciences Vivarium