Fat burning capacity in a mixed macronutrient meal protocol does not reflect metabolic flexibility in women who are overweight or obese.

Introduction: Metabolic flexibility, the ability to switch from glucose to fat as a fuel source, is considered a marker of metabolic health. Higher fat oxidation is often associated with greater flexibility and insulin sensitivity, while lower fat oxidation is linked to metabolic inflexibility and insulin resistance. However, our study challenges the universal validity of this relationship, uncovering a more nuanced understanding of the complex interplay between fuel source switching and fat oxidation, especially in the presence of insulin resistance. Methods: In an 8-week controlled feeding intervention, overweight to obese women with insulin resistance (as defined by McAuley's index) were randomized to consume either a diet based on the Dietary Guidelines for Americans 2010 (DGA) or a 'Typical' American Diet (TAD), n = 22 each. Participants were given a high-fat mixed macronutrient challenge test (MMCT) (60% fat, 28% carbohydrates, and 12% protein) at weeks 0, 2, and 8. Plasma lipids, metabolome, and lipidome were measured at 0, 0.5, 3, and 6h postprandial (PP); substrate oxidation measures were also recorded at 0,1 3, and 6h PP. Metabolic flexibility was evaluated as the change in fat oxidation from fasting to PP. Mixed model and multivariate analyses were used to evaluate the effect of diet on these outcomes, and to identify variables of interest to metabolic flexibility. Results: Intervention diets (DGA and TAD) did not differentially affect substrate oxidation or metabolic flexibility, and equivalence tests indicated that groups could be combined for subsequent analyses. Participants were classified into three groups based on the % of consumed MMCT fat was oxidized in the 6h post meal period at weeks 0, 2 and 8. Low fat burners (LB, n = 6, burned <30% of fat in MMCT) and high fat burners (HB, n = 7, burned > 40% of fat in MMCT) at all weeks. Compared to LB, HB group had higher fat mass, total mass, lean mass, BMI, lower HDLc and lower RER (p < 0.05), but not different % body fat or % lean mass. During week 0, at 1h PP, LB had an increase in % fat oxidation change from 0h compared to HB (p<0.05), suggesting higher metabolic flexibility. This difference disappeared later in the PP phase, and we did not detect this beyond week 0. Partial least squares discriminant analysis (PLSDA (regular and repeated measures (sPLSDA)) models identified that LB group, in the late PP phase, was associated with higher rates of disappearance of acylcarnitines (AC) and lysophosphatidylcholines (LPC) from plasma (Q2: 0.20, R 2 X: 0.177, R 2 Y: 0.716). Conclusion: In women with insulin resistance, a high fat burning capacity does not imply high metabolic flexibility, and not all women with insulin resistance are metabolically inflexible. LPCs and ACs are promising biomarkers of metabolic flexibility.

Illuminating the function of the orphan transporter, SLC22A10, in humans and other primates.

SLC22A10 is an orphan transporter with unknown substrates and function. The goal of this study is to elucidate its substrate specificity and functional characteristics. In contrast to orthologs from great apes, human SLC22A10, tagged with green fluorescent protein, is not expressed on the plasma membrane. Cells expressing great ape SLC22A10 orthologs exhibit significant accumulation of estradiol-17ß-glucuronide, unlike those expressing human SLC22A10. Sequence alignments reveal a proline at position 220 in humans, which is a leucine in great apes. Replacing proline with leucine in SLC22A10-P220L restores plasma membrane localization and uptake function. Neanderthal and Denisovan genomes show proline at position 220, akin to modern humans, indicating functional loss during hominin evolution. Human SLC22A10 is a unitary pseudogene due to a fixed missense mutation, P220, while in great apes, its orthologs transport sex steroid conjugates. Characterizing SLC22A10 across species sheds light on its biological role, influencing organism development and steroid homeostasis.

Illuminating the Function of the Orphan Transporter, SLC22A10 in Humans and Other Primates.

SLC22A10 is classified as an orphan transporter with unknown substrates and function. Here we describe the discovery of the substrate specificity and functional characteristics of SLC22A10. The human SLC22A10 tagged with green fluorescent protein was found to be absent from the plasma membrane, in contrast to the SLC22A10 orthologs found in great apes. Estradiol-17ß-glucuronide accumulated in cells expressing great ape SLC22A10 orthologs (over 4-fold, p<0.001). In contrast, human SLC22A10 displayed no uptake function. Sequence alignments revealed two amino acid differences including a proline at position 220 of the human SLC22A10 and a leucine at the same position of great ape orthologs. Site-directed mutagenesis yielding the human SLC22A10-P220L produced a protein with excellent plasma membrane localization and associated uptake function. Neanderthal and Denisovan genomes show human-like sequences at proline 220 position, corroborating that SLC22A10 were rendered nonfunctional during hominin evolution after the divergence from the pan lineage (chimpanzees and bonobos). These findings demonstrate that human SLC22A10 is a unitary pseudogene and was inactivated by a missense mutation that is fixed in humans, whereas orthologs in great apes transport sex steroid conjugates.

Illuminating the Function of the Orphan Transporter, SLC22A10 in Humans and Other Primates.

SLC22A10 is classified as an orphan transporter with unknown substrates and function. Here we describe the discovery of the substrate specificity and functional characteristics of SLC22A10. The human SLC22A10 tagged with green fluorescent protein was found to be absent from the plasma membrane, in contrast to the SLC22A10 orthologs found in great apes. Estradiol-17ß-glucuronide accumulated in cells expressing great ape SLC22A10 orthologs (over 4-fold, p<0.001). In contrast, human SLC22A10 displayed no uptake function. Sequence alignments revealed two amino acid differences including a proline at position 220 of the human SLC22A10 and a leucine at the same position of great ape orthologs. Site-directed mutagenesis yielding the human SLC22A10-P220L produced a protein with excellent plasma membrane localization and associated uptake function. Neanderthal and Denisovan genomes show human-like sequences at proline 220 position, corroborating that SLC22A10 were rendered nonfunctional during hominin evolution after the divergence from the pan lineage (chimpanzees and bonobos). These findings demonstrate that human SLC22A10 is a unitary pseudogene and was inactivated by a missense mutation that is fixed in humans, whereas orthologs in great apes transport sex steroid conjugates.

Non-invasive metabolomics biomarkers of production efficiency and beef carcass quality traits.

The inter-cattle growth variations stem from the interaction of many metabolic processes making animal selection difficult. We hypothesized that growth could be predicted using metabolomics. Urinary biomarkers of cattle feed efficiency were explored using mass spectrometry-based untargeted and targeted metabolomics. Feed intake and weight-gain was measured in steers (n = 75) on forage-based growing rations (stage-1, 84 days) followed by high-concentrate finishing rations (stage-2, 84 days). Urine from days 0, 21, 42, 63, and 83 in each stage were analyzed from steers with the greater (n = 14) and least (n = 14) average-daily-gain (ADG) and comparable dry-matter-intake (DMI; within 0.32 SD of the mean). Steers were slaughtered after stage-2. Adjusted fat-thickness and carcass-yield-grade increased in greater-ADG-cattle selected in stage-1, but carcass traits did not differ between ADG-selected in stage-2. Overall 85 untargeted metabolites segregated greater- and least-ADG animals, with overlap across diets (both stages) and breed type, despite sampling time effects. Total 18-bile acids (BAs) and 5-steroids were quantified and associated with performance and carcass quality across ADG-classification depending on the stage. Stepwise logistic regression of urinary BA and steroids had > 90% accuracy identifying efficient-ADG-steers. Urine metabolomics provides new insight into the physiological mechanisms and potential biomarkers for feed efficiency.

Healthy eating index patterns in adults by sex and age predict cardiometabolic risk factors in a cross-sectional study.

BACKGROUND: Associations between diet and cardiometabolic disease (CMD) risk may vary in men and women owing to sex differences in eating habits and physiology. The current secondary analysis sought to determine the ability of sex differences in dietary patterns to discriminate groups with or without CMD risk factors (CMDrf) in the adult population and if this was influenced by age. METHODS: Diet patterns and quality were evaluated using 24 h recall-based Healthy Eating Index (HEI-2015) in free-living apparently healthy men (n = 184) and women (n = 209) 18-65 y of age with BMIs of 18-44 kg/m2. Participants were stratified into low- and high-CMDrf groups based on the presence/absence of at least one CMDrf: BMI > 25 kg/m2; fasting triglycerides > 150 mg/dL; HDL cholesterol < 50 mg/dL-women or < 40 mg/dL-men; HOMA > 2; HbA1c > 5.7. Sex by age dietary patterns were stratified by multivariate analyses, with metabolic variable associations established by stepwise discriminant analysis. RESULTS: Diet quality increased with age in both sexes (P < 0.01), while women showed higher fruit, vegetable and saturated fat intake as a percentage of total energy (P < 0.05). The total-HEI score (i.e. diet quality) was lower in the high-CMDrf group (P = 0.01), however, diet quality parameters predicted CMDrf presence more accurately when separated by sex. Lower 'total vegetable' intake in the high-CMDrf group in both sexes, while high-CMDrf men also had lower 'total vegetables', 'greens and beans' intake, and high-CMDrf women had lower 'total fruits', 'whole-fruits', 'total vegetables', 'seafood and plant-proteins', 'fatty acids', and 'saturated fats' intakes (P < 0.05). Moreover, 'dairy' intake was higher in high-CMDrf women but not in men (sex by 'dairy' interaction P = 0.01). Sex by age diet pattern models predicted CMDrf with a 93 and 89% sensitivity and 84 and 92% specificity in women and men, respectively. CONCLUSIONS: Sex and age differences in dietary patterns classified participants with and without accepted CMDrfs, supporting an association between specific diet components and CMD risk that differs by sex. Including sex specific dietary patterns into health assessments may provide targeted nutritional guidance to reduce the burden of cardiovascular disease. TRIAL REGISTRATION: ClinicalTrials.gov : NCT02367287 . ClinicalTrials.gov : NCT02298725 .

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 .

Metabolomics Profile and Targeted Lipidomics in Multiple Tissues Associated with Feed Efficiency in Beef Steers.

A study of multiple tissues was conducted to identify potential metabolic differences in cattle differing in feed efficiency. Individual feed intake and body weight was measured on 144 steers during 105 days on a high-concentrate ration. Steers were selected according to differences in average daily gain (ADG) with those with the greatest ADG (n = 8; 1.96 ± 0.02 kg/day) and least ADG (n = 8; 1.57 ± 0.02 kg/day), whose dry matter intake was within 0.32 SD of the mean intake (10.10 ± 0.05 kg/day). Duodenum, liver, adipose, and longissimus-dorsi were collected at slaughter, and metabolomics profiles were performed by ultra performance liquid chromatography quadrupole-time of-flight mass spectrometry. Principal components analyses, t-tests, and fold changes in tissues profile were used to identify differential metabolites between ADG groups. These were primarily involved in α-linolenic metabolism, which was downregulated in the greatest ADG as compared to least-ADG group in duodenum, adipose, and longissimus-dorsi. However, taurine and glycerophospholipids metabolisms were both upregulated in the greatest ADG compared with least-ADG group in the liver. The phospholipids and cholesterol were quantified in the tissues. Lipid transport and oxidation were the main common metabolic mechanisms associated with cattle feed efficiency. Combining analyses of multiple tissues may offer a powerful approach for defining the molecular basis of differences in performance among cattle for key production attributes.

Quantification of Human Milk Phospholipids: the Effect of Gestational and Lactational Age on Phospholipid Composition.

Human milk (HM) provides infants with macro- and micronutrients needed for growth and development. Milk phospholipids are important sources of bioactive components, such as long-chain polyunsaturated fatty acids (LC-PUFA) and choline, crucial for neural and visual development. Milk from mothers who have delivered prematurely (<37 weeks) might not meet the nutritional requirements for optimal development and growth. Using liquid chromatography tandem-mass spectrometry, 31 phospholipid (PL) species were quantified for colostrum (<5 days postpartum), transitional (≥5 days and ≤2 weeks) and mature milk (>2 weeks and ≤15 weeks) samples from mothers who had delivered preterm (n = 57) and term infants (n = 22), respectively. Both gestational age and age postpartum affected the PL composition of HM. Significantly higher concentrations (p < 0.05) of phosphatidylcholine (PC), sphingomyelin (SM) and total PL were found in preterm milk throughout lactation, as well as significantly higher concentrations (p < 0.002) of several phosphatidylethanolamine (PE), PC and SM species. Multivariate analysis revealed that PLs containing LC-PUFA contributed highly to the differences in the PL composition of preterm and term colostrum. Differences related to gestation decreased as the milk matured. Thus, gestational age may impact the PL content of colostrum, however this effect of gestation might subside in mature milk.

Nrf2 negatively regulates STING indicating a link between antiviral sensing and metabolic reprogramming.

The transcription factor Nrf2 is a critical regulator of inflammatory responses. If and how Nrf2 also affects cytosolic nucleic acid sensing is currently unknown. Here we identify Nrf2 as an important negative regulator of STING and suggest a link between metabolic reprogramming and antiviral cytosolic DNA sensing in human cells. Here, Nrf2 activation decreases STING expression and responsiveness to STING agonists while increasing susceptibility to infection with DNA viruses. Mechanistically, Nrf2 regulates STING expression by decreasing STING mRNA stability. Repression of STING by Nrf2 occurs in metabolically reprogrammed cells following TLR4/7 engagement, and is inducible by a cell-permeable derivative of the TCA-cycle-derived metabolite itaconate (4-octyl-itaconate, 4-OI). Additionally, engagement of this pathway by 4-OI or the Nrf2 inducer sulforaphane is sufficient to repress STING expression and type I IFN production in cells from patients with STING-dependent interferonopathies. We propose Nrf2 inducers as a future treatment option in STING-dependent inflammatory diseases.

Rumen Fluid Metabolomics Analysis Associated with Feed Efficiency on Crossbred Steers.

The rumen has a central role in the efficiency of digestion in ruminants. To identify potential differences in rumen function that lead to differences in average daily gain (ADG), rumen fluid metabolomic analysis by LC-MS and multivariate/univariate statistical analysis were used to identify differences in rumen metabolites. Individual feed intake and body-weight was measured on 144 steers during 105 d on a high concentrate ration. Eight steers with the greatest ADG and 8 steers with the least-ADG with dry matter intake near the population average were selected. Blood and rumen fluid was collected from the 16 steers 26 d before slaughter and at slaughter, respectively. As a result of the metabolomics analysis of rumen fluid, 33 metabolites differed between the ADG groups based on t-test, fold changes and partial least square discriminant analysis. These metabolites were primarily involved in linoleic and alpha-linolenic metabolism (impact-value 1.0 and 0.75, respectively; P < 0.05); both pathways were down-regulated in the greatest-ADG compared with least-ADG group. Ruminal biohydrogenation might be associated with the overall animal production. The fatty acids were quantified in rumen and plasma using targeted MS to validate and evaluate the simple combination of metabolites that effectively predict ADG.

Choline and choline metabolite patterns and associations in blood and milk during lactation in dairy cows.

Milk and dairy products are an important source of choline, a nutrient essential for human health. Infant formula derived from bovine milk contains a number of metabolic forms of choline, all contribute to the growth and development of the newborn. At present, little is known about the factors that influence the concentrations of choline metabolites in milk. The objectives of this study were to characterize and then evaluate associations for choline and its metabolites in blood and milk through the first 37 weeks of lactation in the dairy cow. Milk and blood samples from twelve Holstein cows were collected in early, mid and late lactation and analyzed for acetylcholine, free choline, betaine, glycerophosphocholine, lysophosphatidylcholine, phosphatidylcholine, phosphocholine and sphingomyelin using hydrophilic interaction liquid chromatography-tandem mass spectrometry, and quantified using stable isotope-labeled internal standards. Total choline concentration in plasma, which was almost entirely phosphatidylcholine, increased 10-times from early to late lactation (1305 to 13,535 µmol/L). In milk, phosphocholine was the main metabolite in early lactation (492 µmol/L), which is a similar concentration to that found in human milk, however, phosphocholine concentration decreased exponentially through lactation to 43 µmol/L in late lactation. In contrast, phosphatidylcholine was the main metabolite in mid and late lactation (188 µmol/L and 659 µmol/L, respectively), with the increase through lactation positively correlated with phosphatidylcholine in plasma (R2 = 0.78). Unlike previously reported with human milk we found no correlation between plasma free choline concentration and milk choline metabolites. The changes in pattern of phosphocholine and phosphatidylcholine in milk through lactation observed in the bovine suggests that it is possible to manufacture infant formula that more closely matches these metabolites profile in human milk.