Serum Metabolomic Markers of Dairy Consumption: Results from the Atherosclerosis Risk in Communities Study and the Bogalusa Heart Study.

BACKGROUND: Dairy consumption is related to chronic disease risk; however, the measurement of dairy consumption has largely relied upon self-report. Untargeted metabolomics allows for the identification of objective markers of dietary intake. OBJECTIVES: We aimed to identify associations between dietary dairy intake (total dairy, low-fat dairy, and high-fat dairy) and serum metabolites in 2 independent study populations of United States adults. METHODS: Dietary intake was assessed with food frequency questionnaires. Multivariable linear regression models were used to estimate cross-sectional associations between dietary intake of dairy and 360 serum metabolites analyzed in 2 subgroups of the Atherosclerosis Risk in Communities study (ARIC; n = 3776). Results from the 2 subgroups were meta-analyzed using fixed effects meta-analysis. Significant meta-analyzed associations in the ARIC study were then tested in the Bogalusa Heart Study (BHS; n = 785). RESULTS: In the ARIC study and BHS, the mean age was 54 and 48 years, 61% and 29% were Black, and the mean dairy intake was 1.7 and 1.3 servings/day, respectively. Twenty-nine significant associations between dietary intake of dairy and serum metabolites were identified in the ARIC study (total dairy, n = 14; low-fat dairy, n = 10; high-fat dairy, n = 5). Three associations were also significant in BHS: myristate (14:0) was associated with high-fat dairy, and pantothenate was associated with total dairy and low-fat dairy, but 23 of the 27 associations significant in the ARIC study and tested in BHS were not associated with dairy in BHS. CONCLUSIONS: We identified metabolomic associations with dietary intake of dairy, including 3 associations found in 2 independent cohort studies. These results suggest that myristate (14:0) and pantothenate (vitamin B5) are candidate biomarkers of dairy consumption.

The Combination of a Human Biomimetic Liver Microphysiology System with BIOLOGXsym, a Quantitative Systems Toxicology (QST) Modeling Platform for Macromolecules, Provides Mechanistic Understanding of Tocilizumab- and GGF2-Induced Liver Injury.

Biologics address a range of unmet clinical needs, but the occurrence of biologics-induced liver injury remains a major challenge. Development of cimaglermin alfa (GGF2) was terminated due to transient elevations in serum aminotransferases and total bilirubin. Tocilizumab has been reported to induce transient aminotransferase elevations, requiring frequent monitoring. To evaluate the clinical risk of biologics-induced liver injury, a novel quantitative systems toxicology modeling platform, BIOLOGXsym™, representing relevant liver biochemistry and the mechanistic effects of biologics on liver pathophysiology, was developed in conjunction with clinically relevant data from a human biomimetic liver microphysiology system. Phenotypic and mechanistic toxicity data and metabolomics analysis from the Liver Acinus Microphysiology System showed that tocilizumab and GGF2 increased high mobility group box 1, indicating hepatic injury and stress. Tocilizumab exposure was associated with increased oxidative stress and extracellular/tissue remodeling, and GGF2 decreased bile acid secretion. BIOLOGXsym simulations, leveraging the in vivo exposure predicted by physiologically-based pharmacokinetic modeling and mechanistic toxicity data from the Liver Acinus Microphysiology System, reproduced the clinically observed liver signals of tocilizumab and GGF2, demonstrating that mechanistic toxicity data from microphysiology systems can be successfully integrated into a quantitative systems toxicology model to identify liabilities of biologics-induced liver injury and provide mechanistic insights into observed liver safety signals.

Acetyl-CoA Carboxylase Inhibition Reverses NAFLD and Hepatic Insulin Resistance but Promotes Hypertriglyceridemia in Rodents.

Pharmacologic inhibition of acetyl-CoA carboxylase (ACC) enzymes, ACC1 and ACC2, offers an attractive therapeutic strategy for nonalcoholic fatty liver disease (NAFLD) through simultaneous inhibition of fatty acid synthesis and stimulation of fatty acid oxidation. However, the effects of ACC inhibition on hepatic mitochondrial oxidation, anaplerosis, and ketogenesis in vivo are unknown. Here, we evaluated the effect of a liver-directed allosteric inhibitor of ACC1 and ACC2 (Compound 1) on these parameters, as well as glucose and lipid metabolism, in control and diet-induced rodent models of NAFLD. Oral administration of Compound 1 preferentially inhibited ACC enzymatic activity in the liver, reduced hepatic malonyl-CoA levels, and enhanced hepatic ketogenesis by 50%. Furthermore, administration for 6 days to high-fructose-fed rats resulted in a 20% reduction in hepatic de novo lipogenesis. Importantly, long-term treatment (21 days) significantly reduced high-fat sucrose diet-induced hepatic steatosis, protein kinase C epsilon activation, and hepatic insulin resistance. ACCi treatment was associated with a significant increase in plasma triglycerides (approximately 30% to 130%, depending on the length of fasting). ACCi-mediated hypertriglyceridemia could be attributed to approximately a 15% increase in hepatic very low-density lipoprotein production and approximately a 20% reduction in triglyceride clearance by lipoprotein lipase (P ≤ 0.05). At the molecular level, these changes were associated with increases in liver X receptor/sterol response element-binding protein-1 and decreases in peroxisome proliferator-activated receptor-α target activation and could be reversed with fenofibrate co-treatment in a high-fat diet mouse model. Conclusion: Collectively, these studies warrant further investigation into the therapeutic utility of liver-directed ACC inhibition for the treatment of NAFLD and hepatic insulin resistance.

Combined Treatment with L-Carnitine and Nicotinamide Riboside Improves Hepatic Metabolism and Attenuates Obesity and Liver Steatosis.

Obesity characterized by adiposity and ectopic fat accumulation is associated with the development of non-alcoholic fatty liver disease (NAFLD). Treatments that stimulate lipid utilization may prevent the development of obesity and comorbidities. This study evaluated the potential anti-obesogenic hepatoprotective effects of combined treatment with L-carnitine and nicotinamide riboside, i.e., components that can enhance fatty acid transfer across the inner mitochondrial membrane and increase nicotinamide adenine nucleotide (NAD+) levels, which are necessary for ß-oxidation and the TCA cycle, respectively. Ldlr -/-.Leiden mice were treated with high-fat diet (HFD) supplemented with L-carnitine (LC; 0.4% w/w), nicotinamide riboside (NR; 0.3% w/w) or both (COMBI) for 21 weeks. L-carnitine plasma levels were reduced by HFD and normalized by LC. NR supplementation raised its plasma metabolite levels demonstrating effective delivery. Although food intake and ambulatory activity were comparable in all groups, COMBI treatment significantly attenuated HFD-induced body weight gain, fat mass gain (-17%) and hepatic steatosis (-22%). Also, NR and COMBI reduced hepatic 4-hydroxynonenal adducts. Upstream-regulator gene analysis demonstrated that COMBI reversed detrimental effects of HFD on liver metabolism pathways and associated regulators, e.g., ACOX, SCAP, SREBF, PPARGC1B, and INSR. Combination treatment with LC and NR exerts protective effects on metabolic pathways and constitutes a new approach to attenuate HFD-induced obesity and NAFLD.

Targeted metabolomics connects thioredoxin-interacting protein (TXNIP) to mitochondrial fuel selection and regulation of specific oxidoreductase enzymes in skeletal muscle.

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIP(SKM-/-)) Txnip deficiency. Compared with littermate controls, both TKO and TXNIP(SKM-/-) mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of ß-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability.

Increased Plasma Branched Short-Chain Fatty Acids and Improved Glucose Homeostasis: The Microbiome and Insulin Longitudinal Evaluation Study (MILES).

Short-chain fatty acids (SCFAs) have been extensively studied for potential beneficial roles in glucose homeostasis and risk of diabetes; however, most of this research has focused on butyrate, acetate, and propionate. The effect on metabolism of branched SCFAs (BSCFAs; isobutyrate, isovalerate, and methylbutyrate) is largely unknown. In a cohort of 219 non-Hispanic White participants and 126 African American participants, we examined the association of BSCFA with dysglycemia (prediabetes and diabetes) and oral glucose tolerance test-based measures of glucose and insulin homeostasis, as well as with demographic, anthropometric, lifestyle, and lipid traits, and other SCFAs. We observed a bimodal distribution of BSCFAs, with 25 individuals having high levels (H-BSCFA group) and 320 individuals having lower levels (L-BSCFA group). The prevalence of dysglycemia was lower in the H-BSCFA group compared with the L-BSCFA group (16% vs. 49%; P = 0.0014). This association remained significant after adjustment for age, sex, race, BMI, and levels of other SCFAs. Consistent with the lower rate of dysglycemia, fasting and postprandial glucose levels were lower and the disposition index was higher in the H-BSCFA group. Additional findings in H-BSCFA versus L-BSCFA included lower fasting and postprandial C-peptide levels and lower insulin clearance without differences in insulin levels, insulin sensitivity, insulin secretion, or other variables examined, including diet and physical activity. As one of the first human studies associating higher BSCFA levels with lower odds of dysglycemia and improved glucose homeostasis, this study sets the stage for further investigation of BSCFA as a novel target for prevention or treatment of diabetes.

Serum Metabolomic Markers of Protein-Rich Foods and Incident CKD: Results From the Atherosclerosis Risk in Communities Study.

Rationale & Objective: While urine excretion of nitrogen estimates the total protein intake, biomarkers of specific dietary protein sources have been sparsely studied. Using untargeted metabolomics, this study aimed to identify serum metabolomic markers of 6 protein-rich foods and to examine whether dietary protein-related metabolites are associated with incident chronic kidney disease (CKD). Study Design: Prospective cohort study. Setting & Participants: A total of 3,726 participants from the Atherosclerosis Risk in Communities study without CKD at baseline. Exposures: Dietary intake of 6 protein-rich foods (fish, nuts, legumes, red and processed meat, eggs, and poultry), serum metabolites. Outcomes: Incident CKD (estimated glomerular filtration rate < 60 mL/min/1.73 m2 with ≥25% estimated glomerular filtration rate decline relative to visit 1, hospitalization or death related to CKD, or end-stage kidney disease). Analytical Approach: Multivariable linear regression models estimated cross-sectional associations between protein-rich foods and serum metabolites. C statistics assessed the ability of the metabolites to improve the discrimination of highest versus lower 3 quartiles of intake of protein-rich foods beyond covariates (demographics, clinical factors, health behaviors, and the intake of nonprotein food groups). Cox regression models identified prospective associations between protein-related metabolites and incident CKD. Results: Thirty significant associations were identified between protein-rich foods and serum metabolites (fish, n = 8; nuts, n = 5; legumes, n = 0; red and processed meat, n = 5; eggs, n = 3; and poultry, n = 9). Metabolites collectively and significantly improved the discrimination of high intake of protein-rich foods compared with covariates alone (difference in C statistics = 0.033, 0.051, 0.003, 0.024, and 0.025 for fish, nuts, red and processed meat, eggs, and poultry-related metabolites, respectively; P < 1.00 × 10-16 for all). Dietary intake of fish was positively associated with 1-docosahexaenoylglycerophosphocholine (22:6n3), which was inversely associated with incident CKD (HR, 0.82; 95% CI, 0.75-0.89; P = 7.81 × 10-6). Limitations: Residual confounding and sample-storage duration. Conclusions: We identified candidate biomarkers of fish, nuts, red and processed meat, eggs, and poultry. A fish-related metabolite, 1-docosahexaenoylglycerophosphocholine (22:6n3), was associated with a lower risk of CKD.

In this study, we aimed to identify associations between protein-rich foods (fish, nuts, legumes, red and processed meat, eggs, and poultry) and serum metabolites, which are small biological molecules involved in metabolism. Metabolites significantly associated with a protein-rich food individually and collectively improved the discrimination of the respective protein-rich food, suggesting that these metabolites should be prioritized in future diet biomarker research. We also studied associations between significant diet-related metabolites and incident kidney disease. One fish-related metabolite was associated with a lower kidney disease risk. This finding supports the recent nutritional guidelines recommending a Mediterranean diet, which includes fish as the main dietary protein source.

Metabolomic Association and Risk Prediction With Heart Failure in Older Adults.

BACKGROUND: Older adults have markedly increased risks of heart failure (HF), specifically HF with preserved ejection fraction (HFpEF). Identifying novel biomarkers can help in understanding HF pathogenesis and improve at-risk population identification. This study aimed to identify metabolites associated with incident HF, HFpEF, and HF with reduced ejection fraction and examine risk prediction in older adults. METHODS: Untargeted metabolomic profiling was performed in Black and White adults from the ARIC study (Atherosclerosis Risk in Communities) visit 5 (n=3719; mean age, 75 years). We applied Cox regressions to identify metabolites associated with incident HF and its subtypes. The metabolite risk score (MRS) was constructed and examined for associations with HF, echocardiographic measures, and HF risk prediction. Independent samples from visit 3 (n=1929; mean age, 58 years) were used for replication. RESULTS: Sixty metabolites (hazard ratios range, 0.79-1.49; false discovery rate, <0.05) were associated with incident HF after adjusting for clinical risk factors, eGFR, and NT-proBNP (N-terminal pro-B-type natriuretic peptide). Mannonate, a hydroxy acid, was replicated (hazard ratio, 1.36 [95% CI, 1.19-1.56]) with full adjustments. MRS was associated with an 80% increased risk of HF per SD increment, and the highest MRS quartile had 8.7× the risk of developing HFpEF than the lowest quartile. High MRS was also associated with unfavorable values of cardiac structure and function. Adding MRS over clinical risk factors and NT-proBNP improved 5-year HF risk prediction C statistics from 0.817 to 0.850 (∆C, 0.033 [95% CI, 0.017-0.047]). The association between MRS and incident HF was replicated after accounting for clinical risk factors (P<0.05). CONCLUSIONS: Novel metabolites associated with HF risk were identified, elucidating disease pathways, specifically HFpEF. An MRS was associated with HF risk and improved 5-year risk prediction in older adults, which may assist at at-risk population identification.

Metabolomic Markers of Ultra-Processed Food and Incident CKD.

BACKGROUND: High ultra-processed food consumption is associated with higher risk of CKD. However, there is no biomarker for ultra-processed food, and the mechanism through which ultra-processed food is associated with CKD is not clear. Metabolomics can provide objective biomarkers of ultra-processed food and provide important insights into the mechanisms by which ultra-processed food is associated with risk of incident CKD. Our objective was to identify serum metabolites associated with ultra-processed food consumption and investigate whether ultra-processed food-associated metabolites are prospectively associated with incident CKD. METHODS: We used data from 3751 Black and White men and women (aged 45-64 years) in the Atherosclerosis Risk in Communities study. Dietary intake was assessed using a semiquantitative 66-item food frequency questionnaire, and ultra-processed food was classified using the NOVA classification system. Multivariable linear regression models were used to identify the association between 359 metabolites and ultra-processed food consumption. Cox proportional hazards models were used to investigate the prospective association of ultra-processed food-associated metabolites with incident CKD. RESULTS: Twelve metabolites (saccharine, homostachydrine, stachydrine, N2, N2-dimethylguanosine, catechol sulfate, caffeine, 3-methyl-2-oxovalerate, theobromine, docosahexaenoate, glucose, mannose, and bradykinin) were significantly associated with ultra-processed food consumption after controlling for false discovery rate <0.05 and adjusting for sociodemographic factors, health behaviors, eGFR, and total energy intake. The 12 ultra-processed food-related metabolites significantly improved the prediction of ultra-processed food consumption (difference in C statistics: 0.069, P <1×10 -16 ). Higher levels of mannose, glucose, and N2, N2-dimethylguanosine were associated with higher risk of incident CKD after a median follow-up of 23 years. CONCLUSIONS: We identified 12 serum metabolites associated with ultra-processed food consumption and three of them were positively associated with incident CKD. Mannose and N2, N2-dimethylguanosine are novel markers of CKD that may explain observed associations between ultra-processed food and CKD. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/CJASN/2023_03_08_CJN08480722.mp3.

Metabolomic Profiles Associated With Blood Pressure Reduction in Response to the DASH and DASH-Sodium Dietary Interventions.

BACKGROUND: The DASH (Dietary Approaches to Stop Hypertension) diets reduced blood pressure (BP) in the DASH and DASH-Sodium trials, but the underlying mechanisms are unclear. We identified metabolites associated with systolic BP or diastolic BP (DBP) changes induced by dietary interventions (DASH versus control arms) in 2 randomized controlled feeding studies-the DASH and DASH-Sodium trials. METHODS: Metabolomic profiling was conducted in serum and urine samples collected at the end of diet interventions: DASH (n=219) and DASH-Sodium (n=395). Using multivariable linear regression models, associations were examined between metabolites and change in systolic BP and DBP. Tested for interactions between diet interventions and metabolites were the following comparisons: (1) DASH versus control diets in the DASH trial (serum), (2) DASH high-sodium versus control high-sodium diets in the DASH-Sodium trial (urine), and (3) DASH low-sodium versus control high-sodium diets in the DASH-Sodium trial (urine). RESULTS: Sixty-five significant interactions were identified (DASH trial [serum], 12; DASH high sodium [urine], 35; DASH low sodium [urine], 18) between metabolites and systolic BP or DBP. In the DASH trial, serum tryptophan betaine was associated with reductions in DBP in participants consuming the DASH diets but not control diets (P interaction, 0.023). In the DASH-Sodium trial, urine levels of N-methylglutamate and proline derivatives (eg, stachydrine, 3-hydroxystachydrine, N-methylproline, and N-methylhydroxyproline) were associated with reductions in systolic BP or DBP in participants consuming the DASH diets but not control diets (P interaction, <0.05 for all tests). CONCLUSIONS: We identified metabolites that were associated with BP lowering in response to dietary interventions. REGISTRATION: URL: https://www. CLINICALTRIALS: gov/ct2/show/NCT03403166; Unique identifier: NCT03403166 (DASH trial). URL: https://www. CLINICALTRIALS: gov/ct2/show/NCT00000608; Unique identifier: NCT00000608 (DASH-Sodium trial).

Metabolomics of Dietary Intake of Total, Animal, and Plant Protein: Results from the Atherosclerosis Risk in Communities (ARIC) Study.

Background: Dietary consumption has traditionally been studied through food intake questionnaires. Metabolomics can be used to identify blood markers of dietary protein that may complement existing dietary assessment tools. Objectives: We aimed to identify associations between 3 dietary protein sources (total protein, animal protein, and plant protein) and serum metabolites using data from the Atherosclerosis Risk in Communities Study. Methods: Participants' dietary protein intake was derived from a food frequency questionnaire administered by an interviewer, and fasting serum samples were collected at study visit 1 (1987-1989). Untargeted metabolomic profiling was performed in 2 subgroups (subgroup 1: n = 1842; subgroup 2: n = 2072). Multivariable linear regression models were used to assess associations between 3 dietary protein sources and 360 metabolites, adjusting for demographic factors and other participant characteristics. Analyses were performed separately within each subgroup and meta-analyzed with fixed-effects models. Results: In this study of 3914 middle-aged adults, the mean (SD) age was 54 (6) y, 60% were women, and 61% were Black. We identified 41 metabolites significantly associated with dietary protein intake. Twenty-six metabolite associations overlapped between total protein and animal protein, such as pyroglutamine, creatine, 3-methylhistidine, and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid. Plant protein was uniquely associated with 11 metabolites, such as tryptophan betaine, 4-vinylphenol sulfate, N-δ-acetylornithine, and pipecolate. Conclusions: The results of 17 of the 41 metabolites (41%) were consistent with those of previous nutritional metabolomic studies and specific protein-rich food items. We discovered 24 metabolites that had not been previously associated with dietary protein intake. These results enhance the validity of candidate markers of dietary protein intake and introduce novel metabolomic markers of dietary protein intake.

Whole-Genome Sequencing Analysis of Human Metabolome in Multi-Ethnic Populations.

Circulating metabolite levels may reflect the state of the human organism in health and disease, however, the genetic architecture of metabolites is not fully understood. We have performed a whole-genome sequencing association analysis of both common and rare variants in up to 11,840 multi-ethnic participants from five studies with up to 1666 circulating metabolites. We have discovered 1985 novel variant-metabolite associations, and validated 761 locus-metabolite associations reported previously. Seventy-nine novel variant-metabolite associations have been replicated, including three genetic loci located on the X chromosome that have demonstrated its involvement in metabolic regulation. Gene-based analysis have provided further support for seven metabolite-replicated loci pairs and their biologically plausible genes. Among those novel replicated variant-metabolite pairs, follow-up analyses have revealed that 26 metabolites have colocalized with 21 tissues, seven metabolite-disease outcome associations have been putatively causal, and 7 metabolites might be regulated by plasma protein levels. Our results have depicted the genetic contribution to circulating metabolite levels, providing additional insights into understanding human disease.

Targeted expression of human vitamin D receptor in adipocytes decreases energy expenditure and induces obesity in mice.

Our previous studies demonstrated a high fat diet-resistant lean phenotype of vitamin D receptor (VDR)-null mutant mice mainly due to increased energy expenditure, suggesting an involvement of the VDR in energy metabolism. Here, we took a transgenic approach to further define the role of VDR in adipocyte biology. We used the aP2 gene promoter to target the expression of the human (h) VDR in adipocytes in mice. In contrast to the VDR-null mice, the aP2-hVDR Tg mice developed obesity compared with the wild-type counterparts without changes in food intake. The increase in fat mass was mainly due to markedly reduced energy expenditure, which was correlated with decreased locomotive activity and reduced fatty acid ß-oxidation and lipolysis in the adipose tissue in the transgenic mice. Consistently, the expression of genes involved in the regulation of fatty acid transport, thermogenesis, and lipolysis were suppressed in the transgenic mice. Taken together, these data confirm an important role of the VDR in the regulation of energy metabolism.

Inactivation of the vitamin D receptor in APC(min/+) mice reveals a critical role for the vitamin D receptor in intestinal tumor growth.

Emerging evidence supports an inhibitory role for vitamin D in colorectal carcinogenesis; however, the mechanism remains unclear. The adenomatous polyposis coli (APC)/ß-catenin pathway plays a critical role in colorectal carcinogenesis. The purpose of our study is to explore the interactions of vitamin D and APC/ß-catenin pathways in intestinal tumor development. APC(min/+) mice with genetic inactivation of the vitamin D receptor (VDR) were generated through breeding. Intestinal tumorigenesis was compared between APC(min/+) and APC(min/+) VDR(-/-) mice at different ages. No differences were seen in the number of small intestinal and colonic tumors between APC(min/+) and APC(min/+) VDR(-/-) mice aged 3, 4, 6 and 7 months. The size of the tumors, however, was significantly increased in APC(min/+) VDR(-/-) mice in all age groups. Immunostaining showed significant increases in ß-catenin, cyclin D1, phosphorylated Stat-3 and MSH-2 levels and decreases in Stat-1 in APC(min/+) VDR(-/-) tumors compared to APC(min/+) tumors. These observations suggest that VDR signaling inhibits tumor growth rather than tumor initiation in the intestine. Thus, the increased tumor burden in APC(min/+) VDR(-/-) mice is likely due to the loss of the growth-inhibiting effect of VDR. This study provides strong evidence for the in vivo relevance of the interaction demonstrated in vitro between the vitamin D and ß-catenin signaling pathways in intestinal tumorigenesis.

Plasma Metabolites Associated with a Protein-Rich Dietary Pattern: Results from the OmniHeart Trial.

SCOPE: Lack of biomarkers is a challenge for the accurate assessment of protein intake and interpretation of observational study data. The study aims to identify biomarkers of a protein-rich dietary pattern. METHODS AND RESULTS: The Optimal Macronutrient Intake Trial to Prevent Heart Disease (OmniHeart) trial is a randomized cross-over feeding study which tested three dietary patterns with varied macronutrient content (carbohydrate-rich; protein-rich with about half from plant sources; and unsaturated fat-rich). In 156 adults, differences in log-transformed plasma metabolite levels at the end of the protein- and carbohydrate-rich diet periods using paired t-tests is examined. Partial least-squares discriminant analysis is used to identify a set of metabolites which are influential in discriminating between the protein-rich versus carbohydrate-rich dietary patterns. Of 839 known metabolites, 102 metabolites differ significantly between the protein-rich and the carbohydrate-rich dietary patterns after Bonferroni correction, the majority of which are lipids (n = 35), amino acids (n = 27), and xenobiotics (n = 24). Metabolites which are the most influential in discriminating between the protein-rich and the carbohydrate-rich dietary patterns represent plant protein intake, food or beverage intake, and preparation methods. CONCLUSIONS: The study identifies many plasma metabolites associated with the protein-rich dietary pattern. If replicated, these metabolites may be used to assess level of adherence to a similar dietary pattern.

Serum metabolomic signatures of plant-based diets and incident chronic kidney disease.

BACKGROUND: Greater adherence to plant-based diets is associated with a lower risk of incident chronic kidney disease (CKD). Metabolomics can help identify blood biomarkers of plant-based diets and enhance understanding of underlying mechanisms. OBJECTIVES: Using untargeted metabolomics, we aimed to identify metabolites associated with 4 plant-based diet indices (PDIs) (overall PDI, provegetarian diet, healthful PDI, and unhealthful PDI) and incident CKD in 2 subgroups within the Atherosclerosis Risk in Communities study. METHODS: We calculated 4 PDIs based on participants' responses on an FFQ. We used multivariable linear regression to examine the association between 4 PDIs and 374 individual metabolites, adjusting for confounders. We used Cox proportional hazards regression to evaluate associations between PDI-related metabolites and incident CKD. Estimates were meta-analyzed across 2 subgroups (n1 = 1762; n2 = 1960). We calculated C-statistics to assess whether metabolites improved the prediction of those in the highest quintile compared to the lower 4 quintiles of PDIs, and whether PDI- and CKD-related metabolites predicted incident CKD beyond the CKD prediction model. RESULTS: We identified 82 significant PDI-metabolite associations (overall PDI = 27; provegetarian = 17; healthful PDI = 20; unhealthful PDI = 18); 11 metabolites overlapped across the overall PDI, provegetarian diet, and healthful PDI. The addition of metabolites improved prediction of those in the highest quintile as opposed to the lower 4 quintiles of PDIs compared with participant characteristics alone (range of differences in C-statistics = 0.026-0.104; P value ≤ 0.001 for all tests). Six PDI-related metabolites (glycerate, 1,5-anhydroglucitol, γ-glutamylalanine, γ-glutamylglutamate, γ-glutamylleucine, γ-glutamylvaline), involved in glycolysis, gluconeogenesis, pyruvate metabolism, and γ-glutamyl peptide metabolism, were significantly associated with incident CKD and improved prediction of incident CKD beyond the CKD prediction model (difference in C-statistics for 6 metabolites = 0.005; P value = 0.006). CONCLUSIONS: In a community-based study of US adults, we identified metabolites that were related to plant-based diets and predicted incident CKD. These metabolites highlight pathways through which plant-based diets are associated with incident CKD.

Butyrate-Producing Bacteria and Insulin Homeostasis: The Microbiome and Insulin Longitudinal Evaluation Study (MILES).

Gut microbiome studies have documented depletion of butyrate-producing taxa in type 2 diabetes. We analyzed associations between butyrate-producing taxa and detailed measures of insulin homeostasis, whose dysfunction underlies diabetes in 224 non-Hispanic Whites and 129 African Americans, all of whom completed an oral glucose tolerance test. Stool microbiome was assessed by whole-metagenome shotgun sequencing with taxonomic profiling. We examined associations among 36 butyrate-producing taxa (n = 7 genera and 29 species) and insulin sensitivity, insulin secretion, disposition index, insulin clearance, and prevalence of dysglycemia (prediabetes plus diabetes, 46% of cohort), adjusting for age, sex, BMI, and race. The genus Coprococcus was associated with higher insulin sensitivity (ß = 0.14; P = 0.002) and disposition index (ß = 0.12; P = 0.012) and a lower rate of dysglycemia (odds ratio [OR] 0.91; 95% CI 0.85-0.97; P = 0.0025). In contrast, Flavonifractor was associated with lower insulin sensitivity (ß = -0.13; P = 0.004) and disposition index (ß = -0.11; P = 0.04) and higher prevalence of dysglycemia (OR 1.22; 95% CI 1.08-1.38; P = 0.0013). Species-level analyses found 10 bacteria associated with beneficial directions of effects and two bacteria with adverse associations on insulin homeostasis and dysglycemia. Although most butyrate producers analyzed appear to be metabolically beneficial, this is not the case for all such bacteria, suggesting that microbiome-directed therapeutic measures to prevent or treat diabetes should be targeted to specific butyrate-producing taxa rather than all butyrate producers.

Urine Metabolites Associated with the Dietary Approaches to Stop Hypertension (DASH) Diet: Results from the DASH-Sodium Trial.

SCOPE: Serum metabolomic markers of the Dietary Approaches to Stop Hypertension (DASH) diet are previously reported. In an independent study, the similarity of urine metabolomic markers are investigated. METHODS AND RESULTS: In the DASH-Sodium trial, participants are randomly assigned to the DASH diet or control diet, and received three sodium interventions (high, intermediate, low) within each randomized diet group in random order for 30 days each. Urine samples are collected at the end of each intervention period and analyzed for 938 metabolites. Two comparisons are conducted: 1) DASH-high sodium (n = 199) versus control-high sodium (n = 193), and 2) DASH-low sodium (n = 196) versus control-high sodium. Significant metabolites identified using multivariable linear regression are compared and the top 10 influential metabolites identified using partial least-squares discriminant analysis to the results from the DASH trial. Nine out of 10 predictive metabolites of the DASH-high sodium and DASH-low sodium diets are identical. Most candidate biomarkers from the DASH trial replicated. N-methylproline, chiro-inositol, stachydrine, and theobromine replicated as influential metabolites of DASH diets. CONCLUSIONS: Candidate biomarkers of the DASH diet identified in serum replicated in urine. Replicated influential metabolites are likely to be objective biomarkers of the DASH diet.

Combined vitamin D analog and AT1 receptor antagonist synergistically block the development of kidney disease in a model of type 2 diabetes.

We recently showed that losartan and paricalcitol are synergistic in the treatment of diabetic nephropathy in a model of type 1 diabetes. To test this strategy in a model of type 2 diabetes, we treated 2-month-old diabetic Lprdb/db mice with losartan, paricalcitol, or a combination of losartan and paricalcitol for 3 months. Vehicle-treated diabetic mice developed progressive albuminuria and glomerular abnormalities with mesangial expansion and glomerulosclerosis compared to their non-diabetic littermate control mice. Accompanying damage of the glomerular filtration barrier was a marked reduction in podocyte number as well as reduced expression of slit diaphragm proteins. Further, there was increased glomerular expression of extracellular matrix proteins, monocyte chemoattractant protein-1 and transforming growth factor-beta. Losartan or paricalcitol each alone moderately ameliorated albuminuria and glomerular damage. However, their combined use showed a dramatic therapeutic synergism, manifested by prevention of progressive albuminuria, restoration of the glomerular filtration barrier, reversal of the decline in slit diaphragm proteins, reduced synthesis of extracellular matrix proteins, and reduction of glomerulosclerosis. These effects were accompanied by blockade of the compensatory increase of renin production and angiotensin I/II accumulation in the kidney. Thus, our study further shows that vitamin D analogs can increase the efficacy of AT1 receptor blockade, leading to a more effective prevention of kidney disease in type 2 diabetes.

Loss of vitamin D receptor produces polyuria by increasing thirst.

Vitamin D receptor (VDR)-null mice develop polyuria, but the underlying mechanism remains unknown. In this study, we investigated the relationship between vitamin D and homeostasis of water and electrolytes. VDR-null mice had polyuria, but the urine osmolarity was normal as a result of high salt excretion. The urinary responses to water restriction and to vasopressin were similar between wild-type and VDR-null mice, suggesting intact fluid-handling capacity in VDR-null mice. Compared with wild-type mice, however, renin and angiotensin II were dramatically upregulated in the kidney and brain of VDR-null mice, leading to a marked increase in water intake and salt appetite. Angiotensin II-mediated upregulation of intestinal NHE3 expression partially explained the increased salt absorption and excretion in VDR-null mice. In the brain of VDR-null mice, expression of c-Fos, which is known to associate with increased water intake, was increased in the hypothalamic paraventricular nucleus and the subfornical organ. Treatment with an angiotensin II type 1 receptor antagonist normalized water intake, urinary volume, and c-Fos expression in VDR-null mice. Furthermore, despite a salt-deficient diet to reduce intestinal salt absorption, VDR-null mice still maintained the increased water intake and urinary output. Together, these data indicate that the polyuria observed in VDR-null mice is not caused by impaired renal fluid handling or increased intestinal salt absorption but rather is the result of increased water intake induced by the increase in systemic and brain angiotensin II.