A versatile delivery vehicle for cellular oxygen and fuels, or metabolic sensor? - A review and perspective on the functions of myoglobin.

A canonical view of the primary physiological function of myoglobin (Mb) is that it is an oxygen (O2) storage protein supporting mitochondrial oxidative phosphorylation, especially as the tissue O2 partial pressure (pO2) drops and Mb offloads O2. Besides O2 storage/transport, recent findings support functions for Mb in lipid trafficking and sequestration, interacting with cellular glycolytic metabolites such as lactate (LAC) and pyruvate (PYR) , and "ectopic" expression in some types of cancer cells and in brown adipose tissue (BAT). Data from Mb knockout (Mb-/-) mice and biochemical models suggest additional metabolic roles for Mb, especially regulation of nitric oxide (NO) pools, modulation of BAT bioenergetics, thermogenesis, and lipid storage phenotypes. From these and other findings in the literature over many decades, Mb's function is not confined to delivering O2 in support of oxidative phosphorylation, but also to serve as an O2-sensor that modulates intracellular pO2- and NO-responsive molecular signaling pathways. This paradigm reflects a fundamental change in how oxidative metabolism and cell regulation are viewed in Mb-expressing cells such as skeletal muscle, heart, brown adipocytes, and select cancer cells. Herein, we review historic and emerging views related to the physiological roles for Mb, and present working models illustrating the possible importance of interactions between Mb, gases, and small molecule metabolites in regulation of cell signaling and bioenergetics.

Effects of a diet based on the Dietary Guidelines on vascular health and TMAO in women with cardiometabolic risk factors.

BACKGROUND AND AIMS: Recent evidence links trimethylamine oxide (TMAO) to endothelial dysfunction, an early indicator of cardiovascular disease. We aimed to determine whether short-term consumption of a diet patterned after the 2010 Dietary Guidelines for Americans (DGA) would affect endothelial function, plasma TMAO concentrations, and cardiovascular disease risk, differently than a typical American Diet (TAD). METHODS AND RESULTS: An 8-wk controlled feeding trial was conducted in overweight/obese women pre-screened for insulin resistance and/or dyslipidemia. Women were randomized to a DGA or TAD group (n = 22/group). At wk0 (pre-intervention) and wk8 (post-intervention) vascular age was calculated; endothelial function (reactive hyperemia index (RHI)) and augmentation index (AI@75) were measured using EndoPAT, and plasma TMAO was measured by LC-MS/MS. Vascular age was reduced in DGA at wk8 compared to wk0 but TAD wk8 was not different from wk0 (DGA wk0: 54.2 ± 4.0 vs. wk8: 50.5 ± 3.1 (p = 0.05), vs. TAD wk8: 47.7 ± 2.3). Plasma TMAO concentrations, RHI, and AI@75 were not different between groups or weeks. CONCLUSION: Consumption of a diet based on the 2010 Dietary Guidelines for Americans for 8 weeks did not improve endothelial function or reduce plasma TMAO. CLINICALTRIALS.GOV: NCT02298725.

Myoglobin Interaction with Lactate Rapidly Releases Oxygen: Studies on Binding Thermodynamics, Spectroscopy, and Oxygen Kinetics.

Myoglobin (Mb)-mediated oxygen (O2) delivery and dissolved O2 in the cytosol are two major sources that support oxidative phosphorylation. During intense exercise, lactate (LAC) production is elevated in skeletal muscles as a consequence of insufficient intracellular O2 supply. The latter results in diminished mitochondrial oxidative metabolism and an increased reliance on nonoxidative pathways to generate ATP. Whether or not metabolites from these pathways impact Mb-O2 associations remains to be established. In the present study, we employed isothermal titration calorimetry, O2 kinetic studies, and UV-Vis spectroscopy to evaluate the LAC affinity toward Mb (oxy- and deoxy-Mb) and the effect of LAC on O2 release from oxy-Mb in varying pH conditions (pH 6.0-7.0). Our results show that LAC avidly binds to both oxy- and deoxy-Mb (only at acidic pH for the latter). Similarly, in the presence of LAC, increased release of O2 from oxy-Mb was detected. This suggests that with LAC binding to Mb, the structural conformation of the protein (near the heme center) might be altered, which concomitantly triggers the release of O2. Taken together, these novel findings support a mechanism where LAC acts as a regulator of O2 management in Mb-rich tissues and/or influences the putative signaling roles for oxy- and deoxy-Mb, especially under conditions of LAC accumulation and lactic acidosis.

Myoglobin-Pyruvate Interactions: Binding Thermodynamics, Structure-Function Relationships, and Impact on Oxygen Release Kinetics.

Myoglobin (Mb), besides its roles as an oxygen (O2) carrier/storage protein and nitric oxide NO scavenger/producer, may participate in lipid trafficking and metabolite binding. Our recent findings have shown that O2 is released from oxy-Mb upon interaction with lactate (LAC, anerobic glycolysis end-product). Since pyruvate (PYR) is structurally similar and metabolically related to LAC, we investigated the effects of PYR (aerobic glycolysis end-product) on Mb using isothermal titration calorimetry, circular dichroism, and O2-kinetic studies to evaluate PYR affinity toward Mb and to compare the effects of PYR and LAC on O2 release kinetics of oxy-Mb. Similar to LAC, PYR interacts with both oxy- and deoxy-Mb with a 1:1 stoichiometry. Time-resolved circular dichroism spectra revealed that there are no major conformational changes in the secondary structures of oxy- or deoxy-Mb during interactions with PYR or LAC. However, we found contrasting results with respect to binding affinities and substrate preference, where PYR has higher affinity toward deoxy-Mb when compared with LAC (which prefers oxy-Mb). Furthermore, PYR interaction with oxy-Mb releases a significantly lower amount of O2 than LAC. Taken together, our findings support the hypothesis that glycolytic end-products play a distinctive role in the Mb-rich tissues by serving as novel regulators of O2 availability, and/or by impacting other activities related to oxy-/deoxy-Mb toggling in resting vs. exercised or metabolically activated conditions.

On the potential role of globins in brown adipose tissue: a novel conceptual model and studies in myoglobin knockout mice.

Myoglobin (Mb) regulates O2 bioavailability in muscle and heart as the partial pressure of O2 (Po2) drops with increased tissue workload. Globin proteins also modulate cellular NO pools, "scavenging" NO at higher Po2 and converting NO2- to NO as Po2 falls. Myoglobin binding of fatty acids may also signal a role in fat metabolism. Interestingly, Mb is expressed in brown adipose tissue (BAT), but its function is unknown. Herein, we present a new conceptual model that proposes links between BAT thermogenic activation, concurrently reduced Po2, and NO pools regulated by deoxy/oxy-globin toggling and xanthine oxidoreductase (XOR). We describe the effect of Mb knockout (Mb-/-) on BAT phenotype [lipid droplets, mitochondrial markers uncoupling protein 1 (UCP1) and cytochrome C oxidase 4 (Cox4), transcriptomics] in male and female mice fed a high-fat diet (HFD, 45% of energy, ∼13 wk), and examine Mb expression during brown adipocyte differentiation. Interscapular BAT weights did not differ by genotype, but there was a higher prevalence of mid-large sized droplets in Mb-/-. COX4 protein expression was significantly reduced in Mb-/- BAT, and a suite of metabolic/NO/stress/hypoxia transcripts were lower. All of these Mb-/--associated differences were most apparent in females. The new conceptual model, and results derived from Mb-/- mice, suggest a role for Mb in BAT metabolic regulation, in part through sexually dimorphic systems and NO signaling. This possibility requires further validation in light of significant mouse-to-mouse variability of BAT Mb mRNA and protein abundances in wild-type mice and lower expression relative to muscle and heart.NEW & NOTEWORTHY Myoglobin confers the distinct red color to muscle and heart, serving as an oxygen-binding protein in oxidative fibers. Less attention has been paid to brown fat, a thermogenic tissue that also expresses myoglobin. In a mouse knockout model lacking myoglobin, brown fat had larger fat droplets and lower markers of mitochondrial oxidative metabolism, especially in females. Gene expression patterns suggest a role for myoglobin as an oxygen/nitric oxide-sensor that regulates cellular metabolic and signaling pathways.

Metabolic physiology and skeletal muscle phenotypes in male and female myoglobin knockout mice.

Myoglobin (Mb) is a regulator of O2 bioavailability in type I muscle and heart, at least when tissue O2 levels drop. Mb also plays a role in regulating cellular nitric oxide (NO) pools. Robust binding of long-chain fatty acids and long-chain acylcarnitines to Mb, and enhanced glucose metabolism in hearts of Mb knockout (KO) mice, suggest additional roles in muscle intermediary metabolism and fuel selection. To evaluate this hypothesis, we measured energy expenditure (EE), respiratory exchange ratio (RER), body weight gain and adiposity, glucose tolerance, and insulin sensitivity in Mb knockout (Mb-/-) and wild-type (WT) mice challenged with a high-fat diet (HFD, 45% of calories). In males (n = 10/genotype) and females (n = 9/genotype) tested at 5-6, 11-12, and 17-18 wk, there were no genotype effects on RER, EE, or food intake. RER and EE during cold (10°C, 72 h), and glucose and insulin tolerance, were not different compared with within-sex WT controls. At ∼18 and ∼19 wk of age, female Mb-/- adiposity was ∼42%-48% higher versus WT females (P = 0.1). Transcriptomics analyses (whole gastrocnemius, soleus) revealed few consistent changes, with the notable exception of a 20% drop in soleus transferrin receptor (Tfrc) mRNA. Capillarity indices were significantly increased in Mb-/-, specifically in Mb-rich soleus and deep gastrocnemius. The results indicate that Mb loss does not have a major impact on whole body glucose homeostasis, EE, RER, or response to a cold challenge in mice. However, the greater adiposity in female Mb-/- mice indicates a sex-specific effect of Mb KO on fat storage and feed efficiency.NEW & NOTEWORTHY The roles of myoglobin remain to be elaborated. We address sexual dimorphism in terms of outcomes in response to the loss of myoglobin in knockout mice and perform, for the first time, a series of comprehensive metabolic studies under conditions in which fat is mobilized (high-fat diet, cold). The results highlight that myoglobin is not necessary and sufficient for maintaining oxidative metabolism and point to alternative roles for this protein in muscle and heart.

Exercise training and diet-induced weight loss increase markers of hepatic bile acid (BA) synthesis and reduce serum total BA concentrations in obese women.

Regular exercise has profound metabolic influence on the liver, but effects on bile acid (BA) metabolism are less well known. BAs are synthesized exclusively in the liver from cholesterol via the rate-limiting enzyme cholesterol 7 alpha-hydroxylase (CYP7A1). BAs contribute to the solubilization and absorption of lipids and serve as important signaling molecules, capable of systemic endocrine function. Circulating BAs increase with obesity and insulin resistance, but effects following exercise and diet-induced weight loss are unknown. To test if improvements in fitness and weight loss as a result of exercise training enhance BA metabolism, we measured serum concentrations of total BAs (conjugated and unconjugated primary and secondary BAs) in sedentary, obese, insulin-resistant women (N = 11) before (PRE) and after (POST) a ∼14-wk exercise and diet-induced weight loss intervention. BAs were measured in serum collected after an overnight fast and during an oral glucose tolerance test (OGTT). Serum fibroblast growth factor 19 (FGF19; a regulator of BA synthesis) and 7-alpha-hydroxy-cholesten-3-one (C4, a marker of CYP7A1 enzymatic activity) also were measured. Using linear mixed-model analyses and the change in V̇O2peak (mL/min/kg) as a covariate, we observed that exercise and weight loss intervention decreased total fasting serum BA by ∼30% (P = 0.001) and increased fasting serum C4 concentrations by 55% (P = 0.004). C4 was significantly correlated with serum total BAs only in the POST condition, whereas serum FGF19 was unchanged. These data indicate that a fitness and weight loss intervention modifies BA metabolism in obese women and suggest that improved metabolic health associates with higher postabsorptive (fasting) BA synthesis. Furthermore, pre- vs. postintervention patterns of serum C4 following an OGTT support the hypothesis that responsiveness of BA synthesis to postprandial inhibition is improved after exercise and weight loss.NEW & NOTEWORTHY Exercise and weight loss in previously sedentary, insulin-resistant women facilitates a significant improvement in insulin sensitivity and fitness that may be linked to changes in bile acid metabolism. Diet-induced weight loss plus exercise-induced increases in fitness promote greater postabsorptive bile acid synthesis while also sensitizing the bile acid metabolic system to feedback inhibition during a glucose challenge when glucose and insulin are elevated.

Weight Loss, but Not Dairy Composition of Diet, Moderately Affects Satiety and Postprandial Gut Hormone Patterns in Adults.

BACKGROUND: Inclusion of dairy in diet patterns has been shown to have mixed effects on weight loss. A prevailing hypothesis is that dairy improves weight loss by influencing endocrine systems associated with satiety and food intake regulation. OBJECTIVES: The objective of the current study was to evaluate the effect of weight loss with or without adequate dietary dairy on subjective and objective appetitive measures. METHODS: Men and women who were habitual low dairy consumers (n = 65, 20-50 y) participated in a 12-wk randomized controlled feeding weight loss trial. During the 12-wk intervention, a low-dairy (<1 serving dairy/d) was compared with an adequate-dairy (3-4 servings dairy/d) diet, both with a 500-kcal deficit/d. Test days, before and at the end of the intervention, began with 2 fasting blood draws and visual analog scale (VAS) measures, followed by a standard breakfast (25% of prescribed restricted calories), 5 postbreakfast blood draws and VASs, a standard lunch (40% of restricted energy amount), and 12 postlunch blood draws and VASs. Blood samples were used for satiety hormone measurements. On a separate day when matching standard meals were consumed, an ad libitum buffet meal was provided as dinner, at a self-selected time. Meal duration and intermeal interval were recorded. RESULTS: Weight loss (-6.1 kg), irrespective of dairy, resulted in reduced fasting insulin (-20%) and leptin (-25%), and increased fasting acylated ghrelin (+25%) and VAS desire to eat (+18%) (P < 0.05). There were no effects of dairy on objective or subjective satiety measures. Weight loss marginally reduced the intermeal interval (289 min compared with 276 min, P = 0.059) between lunch and the ad libitum buffet. CONCLUSIONS: These results do not support the hypothesis that inclusion of dairy in long-term dietary patterns influences appetite during weight loss. Weight loss per se has a modest impact on select systems that regulate hunger and satiety.This trial was registered at clinicaltrials.gov as NCT00858312.

Diet affects glycosylation of serum proteins in women at risk for cardiometabolic disease.

BACKGROUND: Glycoproteomics deals with glycoproteins that are formed by post-translational modification when sugars (like fucose and sialic acid) are attached to protein. Glycosylation of proteins influences function, but whether glycosylation is altered by diet is unknown. OBJECTIVE: To evaluate the effect of consuming a diet based on the Dietary Guidelines for Americans on circulating glycoproteins that have previously been associated with cardiometabolic diseases. DESIGN: Forty-four women, with one or more metabolic syndrome characteristics, completed an 8-week randomized controlled feeding intervention (n = 22) consuming a diet based on the Dietary Guidelines for Americans (DGA 2010); the remaining consumed a 'typical American diet' (TAD, n = 22). Fasting serum samples were obtained at week0 (baseline) and week8 (post-intervention); 17 serum proteins were chosen for targeted analyses. Protein standards and serum samples were analyzed in a UHPLC-MS protocol to determine peptide concentration and their glycan (fucosylation or sialylation) profiles. Data at baseline were used in correlational analyses; change in proteins and glycans following intervention were used in non-parametric analyses. RESULTS: At baseline, women with more metabolic syndrome characteristics had more fucosylation (total di-fucosylated proteins: p = 0.045) compared to women with a lesser number of metabolic syndrome characteristics. Dietary refined grain intake was associated with increased total fucosylation (ρ = - 0.530, p < 0.001) and reduced total sialylation (ρ = 0.311, p = 0.042). After the 8-week intervention, there was higher sialylation following the DGA diet (Total di-sialylated protein p = 0.018, poly-sialylated orosomucoid p = 0.012) compared to the TAD diet. CONCLUSIONS: Based on this study, glycosylation of proteins is likely affected by dietary patterns; higher sialylation was associated with a healthier diet pattern. Altered glycosylation is associated with several diseases, particularly cancer and type 2 diabetes, and this study raises the possibility that diet may influence disease state by altering glycosylation. CLINICAL TRIAL REGISTRATION: NCT02298725 at clinicaltrials.gov; https://clinicaltrials.gov/ct2/show/NCT02298725 .

Carnitine palmitoyltransferase 2 knockout potentiates palmitate-induced insulin resistance in C2C12 myotubes.

Saturated fatty acids (SFAs) are implicated in muscle inflammation/cell stress and insulin resistance, but the catalog of factors involved is incomplete. SFA derivatives that accumulate with mismatched FA availability and FA oxidation (FAO) are likely involved, and evidence has emerged that select acylcarnitines should be considered. To understand if excessive long-chain acylcarnitine accumulation and limited FAO associate with lipotoxicity, carnitine palmitoyltransferase 2 knockout C2C12 cells were generated (CPT2 KO). CPT2 KO was confirmed by Western blot, increased palmitoylcarnitine accumulation, and loss of FAO capacity. There was no effect of CPT2 KO on palmitic acid (PA) concentration-dependent increases in media IL-6 or adenylate kinase. PA at 200 and 500 µM did not trigger cell stress responses (phospho-Erk, -JNK, or -p38) above that of vehicle in WT or CPT2 KO cells. In contrast, loss of CPT2 exacerbated PA-induced insulin resistance (acute phospho-Akt; 10 or 100 nM insulin) by as much as ~50-96% compared with WT. Growing cells in carnitine-free media abolished differences between WT and CPT2 KO, but this did not fully rescue PA-induced insulin resistance. The results suggest that PA-induced insulin resistance stems in part from palmitoylcarnitine accumulation, further supporting the hypothesis that select acylcarnitines participate in cell signaling and, when in excess, can compromise cell function. Since carnitine-free conditions could not fully rescue insulin signaling, and CPT2 KO did not alter cell stress responses, the majority of PA-induced "lipotoxicity" in C2C12 myotubes cannot be attributed to palmitoylcarnitine alone.

Coupling of energy intake and energy expenditure across a temperature spectrum: impact of diet-induced obesity in mice.

Obesity and its metabolic sequelae are implicated in dysfunction of the somatosensory, sympathetic, and hypothalamic systems. Because these systems contribute to integrative regulation of energy expenditure (EE) and energy intake (EI) in response to ambient temperature (Ta) changes, we hypothesized that diet-induced obesity (DIO) disrupts Ta-associated EE-EI coupling. C57BL/6N male mice were fed a high-fat diet (HFD; 45% kcal) or low-fat diet (LFD; 10% kcal) for ∼9.5 wk; HFD mice were then split into body weight (BWT) quartiles (n = 8 each) to study DIO-low gainers (Q1) versus -high gainers (Q4). EI and indirect calorimetry (IC) were measured over 3 days each at 10°C, 20°C, and 30°C. Responses did not differ between LFD, Q1, and Q4; EI and BWT-adjusted EE increased rapidly when transitioning toward 20°C and 10°C. In all groups, EI at 30°C was not reduced despite lower EE, resulting in positive energy balance and respiratory exchange ratios consistent with increased de novo lipogenesis, energy storage, and relative hyperphagia. We conclude that 1) systems controlling Ta-dependent acute EI/EE coupling remained intact in obese mice and 2) rapid coupling of EI/EE at cooler temperatures is an important adaptation to maintain energy stores and defend body temperature, but less critical at thermoneutrality. A post hoc analysis using digestible EI plus IC-calculated EE suggests that standard IC assumptions for EE calculation require further validation in the setting of DIO. The experimental paradigm provides a platform to query the hypothalamic, somatosensory, and sympathetic mechanisms that drive Ta-associated EI/EE coupling.

Identification and regulation of the xenometabolite derivatives cis- and trans-3,4-methylene-heptanoylcarnitine in plasma and skeletal muscle of exercising humans.

Little is known about xenometabolites in human metabolism, particularly under exercising conditions. Previously, an exercise-modifiable, likely xenometabolite derivative, cis-3,4-methylene-heptanoylcarnitine, was reported in human plasma. Here, we identified trans-3,4-methylene-heptanoylcarnitine, and its cis-isomer, in plasma and skeletal muscle by liquid chromatography-mass spectrometry. We analyzed the regulation by exercise and the arterial-to-venous differences of these cyclopropane ring-containing carnitine esters over the hepatosplanchnic bed and the exercising leg in plasma samples obtained in three separate studies from young, lean and healthy males. Compared with other medium-chain acylcarnitines, the plasma concentrations of the 3,4-methylene-heptanoylcarnitine isomers only marginally increased with exercise. Both isomers showed a more than twofold increase in the skeletal muscle tissue of the exercising leg; this may have been due to the net effect of fatty acid oxidation in the exercising muscle and uptake from blood. The latter idea is supported by a more than twofold increased net uptake in the exercising leg only. Both isomers showed a constant release from the hepatosplanchnic bed, with an increased release of the trans-isomer after exercise. The isomers differ in their plasma concentration, with a four times higher concentration of the cis-isomer regardless of the exercise state. This is the first approach studying kinetics and fluxes of xenolipid isomers from tissues under exercise conditions, supporting the hypothesis that hepatic metabolism of cyclopropane ring-containing fatty acids is one source of these acylcarnitines in plasma. The data also provide clear evidence for an exercise-dependent regulation of xenometabolites, opening perspectives for future studies about the physiological role of this largely unknown class of metabolites.

Net release and uptake of xenometabolites across intestinal, hepatic, muscle, and renal tissue beds in healthy conscious pigs.

Xenometabolites from microbial and plant sources are thought to confer beneficial as well as deleterious effects on host physiology. Studies determining absorption and tissue uptake of xenometabolites are limited. We utilized a conscious catheterized pig model to evaluate interorgan flux of annotated known and suspected xenometabolites, derivatives, and bile acids. Female pigs (n = 12, 2-3 mo old, 25.6 ± 2.2 kg) had surgically implanted catheters across portal-drained viscera (PDV), splanchnic compartment (SPL), liver, kidney, and hindquarter muscle. Overnight-fasted arterial and venous plasma was collected simultaneously in a conscious state and stored at -80°C. Thawed samples were analyzed by liquid chromatography-mass spectrometry. Plasma flow was determined with para-aminohippuric acid dilution technology and used to calculate net organ balance for each metabolite. Significant organ uptake or release was determined if net balance differed from zero. A total of 48 metabolites were identified in plasma, and 31 of these had at least one tissue with a significant net release or uptake. All bile acids, indole-3-acetic acid, indole-3-arylic acid, and hydrocinnamic acid were released from the intestine and taken up by the liver. Indole-3-carboxaldehyde, p-cresol glucuronide, 4-hydroxyphenyllactic acid, dodecanendioic acid, and phenylacetylglycine were also released from the intestines. Liver or kidney uptake was noted for indole-3-acetylglycine, p-cresol glucuronide, atrolactic acid, and dodecanedioic acid. Indole-3-carboxaldehyde, atrolactic acid, and dodecanedioic acids showed net release from skeletal muscle. The results confirm gastrointestinal origins for several known xenometabolites in an in vivo overnight-fasted conscious pig model, whereas nongut net release of other putative xenometabolites suggests a more complex metabolism.NEW & NOTEWORTHY Xenometabolites from microbe origins influence host health and disease, but absorption and tissue uptake of these metabolites remain speculative. Results herein are the first to demonstrate in vivo organ uptake and release of these metabolites. We used a conscious catheterized pig model to confirm gastrointestinal origins for several xenometabolites (e.g., indolic compounds, 4-hydroxyphenyllactic acid, dodecanendioic acid, and phenylacetylgycine). Liver and kidney were major sites for xenometabolite uptake, likely highlighting liver conjugation metabolism and renal excretion.

Xenometabolite signatures in the UC Davis type 2 diabetes mellitus rat model revealed using a metabolomics platform enriched with microbe-derived metabolites.

The gut microbiome has the potential to create or modify xenometabolites (i.e., nonhost-derived metabolites) through de novo synthesis or modification of exogenous and endogenous compounds. While there are isolated examples of xenometabolites influencing host health and disease, wide-scale characterization of these metabolites remains limited. We developed a metabolomics platform ("XenoScan") using liquid chromatography-mass spectrometry to characterize a range of known and suspected xenometabolites and their derivatives. This assay currently applies authentic standards for 190 molecules, enriched for metabolites of microbial origin. As a proof-of-principle, we characterized the cecal content xenometabolomics profile in adult male lean Sprague-Dawley (LSD) and University of California, Davis type 2 diabetes mellitus (UCD-T2DM) rats at different stages of diabetes. These results were correlated to specific bacterial species generated via shotgun metagenomic sequencing. UCD-T2DM rats had a unique xenometabolite profile compared with LSD rats, regardless of diabetes status, suggesting that at least some of the variation is associated with host genetics. Furthermore, modeling approaches revealed that several xenometabolites discriminated UCD-T2DM rats at early stages of diabetes versus those at 3 mo postdiabetes onset. Several xenometabolite hubs correlated with specific bacterial species in both LSD and UCD-T2DM rats. For example, indole-3-propionic acid negatively correlated with species within the Oscillibacter genus in UCD-T2DM rats considered to be prediabetic or recently diagnosed diabetic, in contrast to gluconic acid and trimethylamine, which were positively correlated with Oscillibacter species. The application of a xenometabolite-enriched metabolomics assay in relevant milieus will enable rapid identification of a wide variety of gut-derived metabolites, their derivatives, and their potential biochemical origins of xenometabolites in relationship to host gastrointestinal microbial ecology.NEW & NOTEWORTHY We debut a liquid chromatography-mass spectrometry (LC/MS) platform called the XenoScan, which is a metabolomics platform for xenometabolites (nonself-originating metabolites). This assay has 190 in-house standards with the majority enriched for microbe-derived metabolites. As a proof-of-principle, we used the XenoScan to discriminate genetic differences from cecal samples associated with different rat lineages, in addition to characterizing diabetes progression in rat model of type 2 diabetes. Complementing microbial sequencing data with xenometabolites uncovered novel microbial metabolism in targeted organisms.

Skeletal muscle interstitial fluid metabolomics at rest and associated with an exercise bout: application in rats and humans.

Blood or biopsies are often used to characterize metabolites that are modulated by exercising muscle. However, blood has inputs derived from multiple tissues, biopsies cannot discriminate between secreted and intracellular metabolites, and their invasive nature is challenging for frequent collections in sensitive populations (e.g., children and pregnant women). Thus, minimally invasive approaches to interstitial fluid (IF) metabolomics would be valuable. A catheter was designed to collect IF from the gastrocnemius muscle of acutely anesthetized adult male rats at rest or immediately following 20 min of exercise (~60% of maximal O2 uptake). Nontargeted, gas chromatography-time-of-flight mass spectrometry analysis was used to detect 299 metabolites, including nonannotated metabolites, sugars, fatty acids, amino acids, and purine metabolites and derivatives. Just 43% of all detected metabolites were common to IF and blood plasma, and only 20% of exercise-modified metabolites were shared in both pools, highlighting that the blood does not fully reflect the metabolic outcomes in muscle. Notable exercise patterns included increased IF amino acids (except leucine and isoleucine), increased α-ketoglutarate and citrate (which may reflect tricarboxylic acid cataplerosis or shifts in nonmitochondrial pathways), and higher concentration of the signaling lipid oleamide. A preliminary study of human muscle IF was conducted using a 20-kDa microdialysis catheter placed in the vastus lateralis of five healthy adults at rest and during exercise (65% of estimated maximal heart rate). Approximately 70% of commonly detected metabolites discriminating rest vs. exercise in rats were also changed in exercising humans. Interstitium metabolomics may aid in the identification of molecules that signal muscle work (e.g., exertion and fatigue) and muscle health.

A comparative study of mitochondrial respiration in circulating blood cells and skeletal muscle fibers in women.

Skeletal muscle mitochondrial respiration is thought to be altered in obesity, insulin resistance, and type 2 diabetes; however, the invasive nature of tissue biopsies is an important limiting factor for studying mitochondrial function. Recent findings suggest that bioenergetics profiling of circulating cells may inform on mitochondrial function in other tissues in lieu of biopsies. Thus, we sought to determine whether mitochondrial respiration in circulating cells [peripheral blood mononuclear cells (PBMCs) and platelets] reflects that of skeletal muscle fibers derived from the same subjects. PBMCs, platelets, and skeletal muscle (vastus lateralis) samples were obtained from 32 young (25-35 yr) women of varying body mass indexes. With the use of extracellular flux analysis and high-resolution respirometry, mitochondrial respiration was measured in intact blood cells as well as in permeabilized cells and permeabilized muscle fibers. Respiratory parameters were not correlated between permeabilized muscle fibers and intact PBMCs or platelets. In a subset of samples (n = 12-13) with permeabilized blood cells available, raw measures of substrate (pyruvate, malate, glutamate, and succinate)-driven respiration did not correlate between permeabilized muscle (per mg tissue) and permeabilized PBMCs (per 106 cells); however, complex I leak and oxidative phosphorylation coupling efficiency correlated between permeabilized platelets and muscle (Spearman's ρ = 0.64, P = 0.030; Spearman's ρ = 0.72, P = 0.010, respectively). Our data indicate that bioenergetics phenotypes in circulating cells cannot recapitulate muscle mitochondrial function. Select circulating cell bioenergetics phenotypes may possibly inform on overall metabolic health, but this postulate awaits validation in cohorts spanning a larger range of insulin resistance and type 2 diabetes status.

Exercise plasma metabolomics and xenometabolomics in obese, sedentary, insulin-resistant women: impact of a fitness and weight loss intervention.

Insulin resistance has wide-ranging effects on metabolism, but there are knowledge gaps regarding the tissue origins of systemic metabolite patterns and how patterns are altered by fitness and metabolic health. To address these questions, plasma metabolite patterns were determined every 5 min during exercise (30 min, ∼45% of V̇o2peak, ∼63 W) and recovery in overnight-fasted sedentary, obese, insulin-resistant women under controlled conditions of diet and physical activity. We hypothesized that improved fitness and insulin sensitivity following a ∼14-wk training and weight loss intervention would lead to fixed workload plasma metabolomics signatures reflective of metabolic health and muscle metabolism. Pattern analysis over the first 15 min of exercise, regardless of pre- versus postintervention status, highlighted anticipated increases in fatty acid tissue uptake and oxidation (e.g., reduced long-chain fatty acids), diminution of nonoxidative fates of glucose [e.g., lowered sorbitol-pathway metabolites and glycerol-3-galactoside (possible glycerolipid synthesis metabolite)], and enhanced tissue amino acid use (e.g., drops in amino acids; modest increase in urea). A novel observation was that exercise significantly increased several xenometabolites ("non-self" molecules, from microbes or foods), including benzoic acid-salicylic acid-salicylaldehyde, hexadecanol-octadecanol-dodecanol, and chlorogenic acid. In addition, many nonannotated metabolites changed with exercise. Although exercise itself strongly impacted the global metabolome, there were surprisingly few intervention-associated differences despite marked improvements in insulin sensitivity, fitness, and adiposity. These results and previously reported plasma acylcarnitine profiles support the principle that most metabolic changes during submaximal aerobic exercise are closely tethered to absolute ATP turnover rate (workload), regardless of fitness or metabolic health status.

Is palmitate truly proinflammatory? Experimental confounders and context-specificity.

Based primarily on cell culture results, saturated fatty acids (SFAs) are proposed to promote inflammation and contribute to metabolic dysfunction through Toll-like receptor activation. Studies are often complicated by a requirement for carriers (e.g., BSA) or solvents (e.g., ethanol) to increase SFA solubility. To ascertain whether these factors influence interpretations of SFA-associated inflammation activity, we measured responses of RAW264.7 monocyte/macrophages and C2C12 myotubes to various BSA, ethanol, and cyclodextrin (alternative FA carrier) conditions. Fatty acid-free, low-endotoxin BSA preparations (0.33% to 2% wt/vol) activated whereas 0.5-1.0% ethanol inhibited RAW264.7 TNFα release. Ethanol modestly increased IL-6 secretion in C2C12 myotubes. Cyclodextrins (0.3-6.0 mM) were tested as alternative carriers of palmitate, but their usefulness was limited due to toxicity and solubility issues. Using a lower-inflammation BSA source and no ethanol, ∼24-h sodium palmitate treatment (≤600 µM) failed to trigger RAW264.7 TNFα release and, in fact, significantly dampened BSA-induced inflammation by >50%. In C2C12 myotubes, only high palmitate concentrations (500-600 µM) elicited IL-6 secretion (>2.5-fold increase). Acute palmitate (200 or 500 µM) treatment did not activate MAP kinase pathways above that of fresh BSA-containing media alone in either cell type. These results highlight the importance of experimental conditions in studies exploring SFA inflammation effects. The limited (or even anti-inflammatory) effects of palmitate that we observed indicate that immunomodulatory effects of SFAs are context-specific. Thus, caution is needed when interpreting the literature related to putative proinflammatory effects of SFA.

The serum metabolomics signature of type 2 diabetes is obscured in Alzheimer's disease.

There is evidence for systemic metabolic impairment in Alzheimer's disease (AD), and type 2 diabetes (T2D) increases AD risk. Although studies analyzing blood metabolomics signatures have shown differences between cognitively healthy (CH) and AD subjects, these signatures have not been compared with individuals with T2D. We utilized untargeted analysis platforms (primary metabolism and complex lipids) to characterize the serum metabolome of 126 overnight-fasted elderly subjects classified into four groups based upon AD status (CH or AD) and T2D status [nondiabetic (ND) or T2D]. Cognitive diagnosis groups were a priori weighted equally with T2D subjects. We hypothesized that AD subjects would display a metabolic profile similar to cognitively normal elderly individuals with T2D. However, partial least squares-discriminant analysis (PLS-DA) modeling resulted in poor classification across the four groups (<50% classification accuracy of test subjects). Binary classification of AD vs. CH was poor, but binary classification of T2D vs. ND was good, providing >79.5% and >76.9% classification accuracy for held-out samples using primary metabolism and complex lipids, respectively. When modeling was limited to CH subjects, T2D discrimination improved for the primary metabolism platform (>89.5%) and remained accurate for complex lipids (>73% accuracy). Greater abundances of glucose, fatty acids (C20:2), and phosphatidylcholines and lower abundances of glycine, maleimide, octanol, and tryptophan, cholesterol esters, phosphatidylcholines, and sphingomyelins were identified in CH subjects with T2D relative to those without T2D. In contrast, T2D was not accurately discriminated within AD subjects. Results herein suggest that AD may obscure the typical metabolic phenotype of T2D.

Diabetes-associated alterations in the cecal microbiome and metabolome are independent of diet or environment in the UC Davis Type 2 Diabetes Mellitus Rat model.

The composition of the gut microbiome is altered in obesity and type 2 diabetes; however, it is not known whether these alterations are mediated by dietary factors or related to declines in metabolic health. To address this, cecal contents were collected from age-matched, chow-fed male University of California, Davis Type 2 Diabetes Mellitus (UCD-T2DM) rats before the onset of diabetes (prediabetic PD; n = 15), 2 wk recently diabetic (RD; n = 10), 3 mo (D3M; n = 11), and 6 mo (D6M; n = 8) postonset of diabetes. Bacterial species and functional gene counts were assessed by shotgun metagenomic sequencing of bacterial DNA in cecal contents, while metabolites were identified by gas chromatography-quadrupole time-off-flight-mass spectrometry. Metagenomic analysis showed a shift from Firmicutes species in early stages of diabetes (PD + RD) toward an enrichment of Bacteroidetes species in later stages of diabetes (D3M + D6M). In total, 45 bacterial species discriminated early and late stages of diabetes with 25 of these belonging to either Bacteroides or Prevotella genera. Furthermore, 61 bacterial gene clusters discriminated early and later stages of diabetes with elevations of enzymes related to stress response (e.g., glutathione and glutaredoxin) and amino acid, carbohydrate, and bacterial cell wall metabolism. Twenty-five cecal metabolites discriminated early vs. late stages of diabetes, with the largest differences observed in abundances of dehydroabietic acid and phosphate. Alterations in the gut microbiota and cecal metabolome track diabetes progression in UCD-T2DM rats when controlling for diet, age, and housing environment. Results suggest that diabetes-specific host signals impact the ecology and end product metabolites of the gut microbiome when diet is held constant.