Updates from the Metabolism Community

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://staging-morgridgeorgproduscentral.kinsta.cloud/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