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Quantifying sphingomyelin (SM) species by infusion-based mass spectrometry (MS) is complicated by the presence of isobaric phosphatidylcholine (PC) species, which generate a common

Placental tissue intracellular calcium (Ca

Bovine milk is a complex mixture of lipids, proteins, carbohydrates, and other factors of which lipids comprise 3-5% of the total mass. Rapid analysis and characterization of the triacylglycerols (TAG) that comprise about 95% of the total lipid is daunting given the numerous TAG species. In the attached methods paper, we demonstrate an improved method for identifying and quantifying TAG species by infusion-based "shotgun" lipidomics. Because of the broad range of TAG species in milk, a single internal standard was insufficient for the analysis and required sectioning the spectrum into three portions based upon mass range to provide accurate quantitation of TAG species. Isobaric phospholipid interferences were removed using a simple dispersive solid-phase extraction step. Using this method, > 100 TAG species were quantitated by acyl carbon number and desaturation level in a sample of commercially purchased bovine milk.

Both clinical and laboratory studies have shown that monocyte chemotactic protein-1 (MCP-1) is involved in cancer spread. To understand the role of MCP-1 in metabolism in the presence of metastasis, we conducted an untargeted metabolomic analysis of primary metabolism on plasma collected from a study showing that MCP-1 deficiency reduces spontaneous metastasis of Lewis lung carcinoma (LLC) to the lungs in mice fed a high-fat diet (HFD). In a 2 × 2 design, wild-type (WT) or Mcp-1 knockout ( Mcp-1-/-) mice maintained on the AIN93G standard diet or HFD were subcutaneously injected with LLC cells to induce lung metastasis. We identified 87 metabolites for metabolomic analysis from this study. Amino acid metabolism was altered considerably in the presence of LLC metastases with the aminoacyl-tRNA biosynthesis pathways as the leading pathway altered. The HFD modified lipid and energy metabolism, evidenced by lower contents of arachidonic acid, cholesterol, and long-chain saturated fatty acids and higher contents of glucose and pyruvic acid in mice fed the HFD. These findings were supported by network analysis showing alterations in fatty acid synthesis and glycolysis/gluconeogenesis pathways between the 2 diets. Furthermore, elevations of the citrate cycle intermediates (citric acid, fumaric acid, isocitric acid, and succinic acid) and glyceric acid in Mcp-1-/- mice, regardless of diet, suggest the involvement of MCP-1 in mitochondrial energy metabolism during LLC metastasis. The present study demonstrates that MCP-1 deficiency and the HFD altered plasma metabolome in mice bearing LLC metastases. These findings can be useful in understanding the impact of obesity on prevention and treatment of cancer metastasis.

BACKGROUND: Branched-chain fatty acids (BCFAs) are rumen-derived fatty acids comprising ∼2% of bovine-milk fatty acids. BCFAs possess anti-inflammatory properties and enriching the BCFA content of bovine milk may provide human health benefits. OBJECTIVE: We determined whether forage content impacts the BCFA content of milk from Holstein cows and identified fatty acid phenotypes in high vs. low BCFA–containing milks. METHODS: Holstein cows (n = 62), fed for 67 d in a crossover design, consumed a diet with high forage and low concentrate (HF:C) and a diet with low forage and high concentrate (LF:C). Milk samples were collected at the end of each treatment period and fatty acid content determined. Paired t-tests, 1-factor ANOVA, sparse partial least-squares discriminant analysis (sPLSDA), and Pearson's correlation analysis were used to analyze the data. RESULTS: The total milk fatty acid concentration for cows fed the HF:C diet was greater than that of cows fed the LF:C diet (4.2 ± 0.7 g/100 mL vs. 3.9 ± 0.9 g/100 mL). sPLSDA demonstrated separation of the dietary treatments, with BCFAs and odd-chain fatty acids as primary determinants. Total BCFA content in milk fat was elevated by HF:C intake compared with LF:C intake (1.80 vs. 1.68%). Quintile separation of high vs. low BCFA milks resulted in 4 groups: HF:C /low BCFAs, HF:C /high BCFAs; LF:C /low BCFAs, and LF:C /high BCFAs. Milks from the high BCFA quintiles had lower palmitic acid content (29.6% vs. 34.4%) but higher oleic acid content than milks from the low BCFA quintiles (19.7% vs. 17.0%). Some cows were identified as high BCFA producers or low BCFA producers regardless of diet. CONCLUSIONS: BCFA content of milk is diet-sensitive but variation in responses exists. The potential to produce milk with high BCFA content and lower SFA content needs further study.

Dietary malpractice is a risk factor for obesity. This study tested the hypothesis that consumption of a high-fat diet alters mammary metabolome in pubertal mice. We performed untargeted metabolomic analysis of primary metabolism on mammary glands from pubertal mice fed the AIN93G standard diet or a high-fat diet (HFD) for 3 weeks. We identified 97 metabolites for statistical comparisons. The HFD altered the amino acid metabolism considerably. This included elevated expression of branched-chain amino acids, non-essential amino acids (aspartic acid and glutamic acid), and methionine sulfoxide (oxidized methionine) and an alteration in the aminoacyl-tRNA biosynthesis pathway. Furthermore, elevations of fumaric acid and malic acid (both are citrate cycle intermediates) and glyceric acid (its phosphate derivatives are intermediates of glycolysis) in HFD-fed mice suggest an acceleration of both citrate cycle and glycolysis. Lower expression of glycerol, oleic acid, and palmitoleic acid, as well as decreased mammary expression of genes encoding lipid metabolism ( Acaca, Fads1, Fasn, Scd1, and Srebf1) in HFD-fed mice indicate an attenuated lipid metabolism in the presence of adequate dietary fat. In conclusion, consumption of the HFD for 3 weeks alters metabolic profile of pubertal mammary glands. This alteration may affect mammary development and growth in pubertal mice.

Adoption of an obesogenic diet such as a high-fat diet (HFD) results in obesity, bacterial dysbiosis, chronic inflammation, and cancer. Gut bacteria and their metabolites are recognized by interleukin-1 (IL-1R)/toll-like receptors (TLRs) which are essential to maintain intestinal homeostasis. Moreover, host extracellular microRNAs (miRNAs) can alter bacterial growth in the colon. Characterization of the underlying mechanisms may lead to identifying fecal oncogenic signatures reflecting colonic health. We hypothesize that an HFD accelerates the inflammatory process and modulates IL-1R/TLR pathways, gut microbiome, and disease-related miRNA in the colon. In this study, 4-week-old C57BL/6 mice were fed a modified AIN93G diet (AIN, 16% energy fat) or an HFD (45% energy fat) for 15 weeks. In addition to increased body weight and body fat composition, the concentrations of plasma interleukin 6 (IL-6), inflammatory cell infiltration, β-catenin, and cell proliferation marker (Ki67) in the colon were elevated68% in the HFD group compared to the AIN group. Using a PCR array analysis, we identified 14 out of 84 genes with a ≥ 24% decrease in mRNA content related to IL-1R and TLR pathways in colonic epithelial cells in mice fed an HFD compared to the AIN. Furthermore, the content of Alistipes bacteria, the Firmicutes/Bacteroidetes ratio, microRNA-29a, and deoxycholic and lithocholic acids (secondary bile acids with oncogenic potential) were 55% greater in the feces of the HFD group compared to the AIN group. Collectively, this composite, a multimodal profile may represent a unique HFD-induced fecal signature for colonic inflammation and cancer in C57BL/6 mice.

Male breast cancer, while uncommon, is a highly malignant disease. Monocyte chemotactic protein-1 (MCP-1) is an adipokine; its concentration in adipose tissue is elevated in obesity. This study tested the hypothesis that adipose-derived MCP-1 contributes to male breast cancer. In a 2x2 design, male MMTV-PyMT mice with or without adipose-specific Mcp-1 knockout [designated as Mcp-1-/- or wild-type (WT)] were fed the AIN93G standard diet or a high-fat diet (HFD) for 25 weeks. Mcp-1-/- mice had lower adipose Mcp-1 expression than WT mice. Adipose Mcp-1 deficiency reduced plasma concentrations of MCP-1 in mice fed the HFD compared to their WT counterparts. Mcp-1-/- mice had a longer tumor latency (25.2 weeks vs. 18.0 weeks) and lower tumor incidence (19% vs. 56%), tumor progression (2317% vs. 4792%), and tumor weight (0.23 g vs. 0.64 g) than WT mice. Plasma metabolomics analysis identified 56 metabolites that differed among the four dietary groups, including 22 differed between Mcp-1-/- and WT mice. Pathway and network analyses along with discriminant analysis showed that pathways of amino acid and carbohydrate metabolisms are the most disturbed in MMTV-PyMT mice. In conclusion, adipose-derived MCP-1 contributes to mammary tumorigenesis in male MMTV-PyMT. The potential involvement of adipose-derived MCP-1 in metabolomics warrants further investigation on its role in causal relationships between cancer metabolism and mammary tumorigenesis in this male MMTV-PyMT model.

Background Biological markers of vegetable and fruit (VF) intake are needed both for nutrition surveillance and for the evaluation of nutrition interventions. Optically assessed skin carotenoid status (SCS) has been proposed as a marker of intake but there are few published validity studies to date. Therefore, the objective of the study was to examine the concurrent validity of multiple methods of assessing VF intake cross-sectionally and seasonally over one year and to discuss the relative merits and limitations of each method. Methods Fifty-two 40–60 y old women completed a 1-year longitudinal study that included 1) SCS assessment using resonance Raman spectroscopy (RRS) and using pressure-mediated reflection spectroscopy (RS) at 12 timepoints, 2) thirty-six 24-h recalls using the Automated Self-Administered 24-Hour Dietary Assessment Tool (ASA24; total 1866 recalls), and 3) plasma carotenoid concentrations measured every 3 months. Pearson correlation coefficients and mixed linear models were used to estimate pairwise correlations between RRS, RS, ASA24, and plasma carotenoids. Results RS and RRS were strongly correlated at baseline and over the year (r = 0.86 and 0.76; respectively, P r = 0.70) and moderately across the year (r = 0.65), as was RRS (r = 0.77 and 0.69, respectively, all P r = 0.33; P = 0.016), but not with RS or plasma carotenoids. Across the year, self-reported VF intake was weakly correlated with both RS (r = 0.37; P = 0.008), RRS (r = 0.37; P = 0.007), and with plasma carotenoids (r = 0.36; P Conclusions SCS as measured by RS and RRS is moderately to strongly correlated with plasma carotenoid concentrations both cross-sectionally and longitudinally, indicating that it can be a powerful tool to assess carotenoid-rich VF intake in populations. Clinical trial registry This trial was registered at ClinicalTrials.gov as NCT01674296.

Saturated fatty acid (SFA) intake is associated with obesity, insulin resistance, and hepatic steatosis, but scant work examines the impact of SFA type upon these outcomes. We tested the hypothesis that an obesogenic diet prepared with medium chain SFA (MCSFA), mostly as lauric acid-derived from coconut oil, reduces obesity-induced outcomes compared to obesogenic diets prepared with increasing amounts long chain SFA (LCSFA), primarily palmitic acid. Mice were fed (16 weeks) a control, low fat diet or obesogenic diets prepared with differing content of MCSFA or LCSFA in which polyunsaturated and monounsaturated fatty acids (PUFA; MUFA) were kept constant. Inclusion of MCSFA in an obesogenic diet prevented hepatic lipid accumulation and lowered indices of insulin resistance. Obesogenic diets reduced hepatic levels of de novo lipogenesis proteins (SCD1 and FASN) but elevated the adipose levels of mRNA for the pro-inflammatory markers Mcp-1 and Tnfα. Lipidomic analysis of plasma indicated that MCSFA intake resulted in a different lipidomic signature than LCSFA intake, prevented elevation of pro-inflammatory ceramides, but elevated concentrations of some lipids associated with elevated cardiovascular disease risk. Intake of the obesogenic diets in an SFA-type dependent manner elevated plasma concentrations of several phosphatidylcholine (PC) lipids having the long chain PUFA (LCPUFA) arachidonic acid (ARA) and docosahexaenoic acid (DHA), altered phospholipid ethers, and changed the triacylglyceryl environments of these LCPUFA. Our data indicate that (1) MCSFA reduce the severity of some obesogenic co-morbidities, (2) SFA-type modulates lipidomic signatures associated with cardiovascular disease and diabetes, and (3) dietary SFA type impacts LCPUFA metabolism.

Metastasis is a devastating aspect of cancer. This study tested the hypothesis that metabolome of metastases differs from that of host organs by using the spontaneous metastasis model of Lewis lung carcinoma (LLC). In a 2 × 2 design, male C57BL/6 mice with or without a subcutaneous LLC inoculation were fed the standard AIN93G diet or a high-fat diet (HFD) for 12 weeks. Lung metastases from injected mice and the lungs from non-injected mice were harvested at the end of study for untargeted metabolomics of primary metabolism by using gas chromatography time-of-flight mass spectrometry. We identified 91 metabolites for metabolomic analysis. The analysis demonstrated that amino acid and energy metabolism were altered the most in LLC metastases compared to the lungs. A 60% decrease in glutamine and a 25-fold elevation in sorbitol were observed in metastases. Cholesterol and its metabolite dihydrocholesterol were 50% lower in metastases than in the lungs. The HFD elevated arachidonic acid and its precursor linoleic acid in the lungs from noncancer-bearing mice, reflecting the dietary fatty acid composition of the HFD. This elevation did not occur in metastases from HFD-fed LLC-bearing mice, suggesting alterations in lipid metabolism during LLC metastatic progression. Understanding the differences in metabolome between pulmonary LLC metastases and the normal healthy lungs can be useful in designing targeted studies for prevention and treatment of cancer spread using this LLC spontaneous metastasis model.

Consumption of a high-fat diet (HFD) links obesity to colon cancer in humans. Our data show that a HFD (45% energy fat versus 16% energy fat in an AIN-93 diet (AIN)) promotes azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation in a mouse cancer model. However, the underlying metabolic basis remains to be determined. In the present study, we hypothesize that AOM treatment results in different plasma metabolomic responses in diet-induced obese mice. An untargeted metabolomic analysis was performed on the plasma samples by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). We found that 53 of 144 identified metabolites were different between the 4 groups of mice (AIN, AIN + AOM, HFD, HFD + AOM), and sparse partial least-squares discriminant analysis showed a separation between the HFD and HFD + AOM groups but not the AIN and AIN + AOM groups. Moreover, the concentrations of dihydrocholesterol and cholesterol were inversely associated with AOM-induced colonic ACF formation. Functional pathway analyses indicated that diets and AOM-induced colonic ACF modulated five metabolic pathways. Collectively, in addition to differential plasma metabolomic responses, AOM treatment decreases dihydrocholesterol and cholesterol levels and alters the composition of plasma metabolome to a greater extent in mice fed a HFD compared to the AIN.

OBJECTIVES: Branched chain fatty acids (BCFA) are rumen-derived fatty acids comprising about 2% of bovine milk fatty acids. BCFA possess anti-inflammatory properties and enriching the BCFA content of bovine milk may provide human health benefits of milk consumption. In this work, we determined whether high vs low forage diets impact the BCFA content of milk from Holstein cows and identified fatty acid phenotypes in high vs low BCFA-containing milks. METHODS: In this study, 62 Holstein cows were fed for 70 days, in a cross-over design fashion, either a high forage: concentrate (HF: C) diet or a low forage: concentrate (LF: C) diet. At the end of the 70-day feeding period, cows were switched to the alternate feeding arm. Milk samples were collected prior to the start of the first feeding period and at the end of each treatment period. Milk fatty acid content was determined by fatty acid methyl ester analysis. Paired t-tests, one-way ANOVA, and sparse partial least squares discriminant analysis (sPLSDA) were used to analyze the data. RESULTS: The total milk fatty acid concentration at the end of HF: C diet period was greater than that of the LF: C diet (4.2 ± 0.9 g/100 mL vs 3.95 ± 0.9 g/100 mL). sPLSDA demonstrated clear separation of the dietary treatments, with BCFA and odd-chain fatty acids as primary determinants. Total BCFA content was elevated by HF: C intake (1.86 ± 0.13%) vs LF: C intake (1.77 ± 0.14%). Quintile separation of high vs low BCFA milks resulted in 4 groups (n = 12) HF: C/low BCFA, HF: C/high BCFA; LF: C/low BCFA, and LF: C/high BCFA. The total BCFA contents of the low BCFA milks were not different regardless of forage content (1.70 ± 0.06% and 1.61 ± 0.04%) and were lower than the high BFCA milks (2.04 ± 0.11% and 1.98 ± 0.11%). Milks from the high BCFA quintiles had lower saturated fatty acid (SFA) content but higher oleic acid content than milks from the low BCFA quintiles. Of the 62 cows, 5 cows were identified as high BCFA producers and 6 cows as low BCFA producers regardless of the LF: C or HF: C diets. CONCLUSIONS: The data demonstrate that the BCFA content of milk is diet-sensitive but variation in cow responses suggest factors such as genotype or rumen microbiome composition may play significant roles. The potential to produce milk with high BCFA content and lower SFA content needs further study. FUNDING SOURCES: USDA-ARS Projects 3062–53000-001–00D, 5090–31000-025–00D, 80–8040-05–01-0000–0000.

Background Supplementation with long chain n‐3 polyunsaturated fatty acids is used to reduce total circulating triacylglycerol (TAG) concentrations. However, in about 30% of people, supplementation with long chain n‐3 polyunsaturated fatty acids does not result in decreased plasma TAG. Lipidomic analysis may provide insight into this inter‐individual variability. Methods Lipidomic analyses using targeted, mass spectrometry were performed on plasma samples obtained from a clinical study in which participants were supplemented with 3 g/day of long chain n‐3 in the form of fish oil capsules over a 6‐week period. TAG species and cholesteryl esters (CE) were quantified for 130 participants pre‐ and post‐supplementation. Participants were segregated into 3 potential responder phenotypes: (1) positive responder (R pos ; TAG decrease), (2) non‐responder (R non ; lacking TAG change), and (3) negative responder (R neg ; TAG increase) representing 67%, 18%, and 15% of the study participants, respectively. Separation of the 3 phenotypes was attributed to differential responses in TAG with 50 to 54 carbons with 1 to 4 desaturations. Elevated TAG with higher carbon number and desaturation were common to all phenotypes following supplementation. Using the TAG responder phenotype for grouping, decreases in total CE and specific CE occurred in the R pos phenotype versus the R neg phenotype with intermediate responses in the R non phenotype. CE 20:5, containing eicosapentaenoic acid (20:5n‐3), was elevated in all phenotypes. A classifier combining lipidomic and genomic features was built to discriminate triacylglycerol response phenotypes and reached a high predictive performance with a balanced accuracy of 75%. Conclusions These data identify lipidomic signatures, TAG and CE, associated with long chain n‐3 response p henotypes and identify a novel phenotype based upon CE changes. Registration URL: https://www.ClinicalTrials.gov ; Unique Identifier: NCT01343342.

Metastasis, the spread of malignant cells from a primary tumor to distant organs, is the most devastating aspect of cancer. To understand the metabolism of metastases, we compared the metabolomes of pulmonary metastases of Lewis lung carcinoma (LLC) with that of normal lung tissues from mice fed an obesogenic high-fat diet. In a 2x2 design, male C57BL/6 mice, with or without a subcutaneous LLC inoculation, were fed the standard AIN93G diet or a high-fat diet (HFD), which provided 16% or 45% of energy from soybean oil, for 12 weeks. At the end of the study, lung metastases from injected mice and the lungs from non-injected mice were harvested for untargeted metabolomic analysis of primary metabolism by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). We identified 91 metabolites, 70 of which differed among the four groups. Pathway and network analyses along with the discriminant analysis showed that amino acid and carbohydrate metabolism were altered the most in metastases compared to the normal lung tissue (e.g., aminoacyl-tRNA biosynthesis pathways and the citric cycle). A 60% decrease in glutamine and a 25-fold elevation in sorbitol were observed in metastases. Cholesterol and its metabolite dihydrocholesterol were 50% lower in metastases than in the lungs. The HFD elevated arachidonic acid and its precursor linoleic acid in the lungs from non-LLC-bearing mice, which reflected dietary fatty acid composition of the HFD. However, such elevation did not occur in metastases from HFD-fed LLC-bearing mice, suggesting alterations in fatty acid metabolism during LLC metastatic progression. Differences in metabolomes between LLC pulmonary metastases and the normal healthy lungs identified in the present study can be useful in designing targeted studies for prevention and treatment of metastasis using this spontaneous metastasis model. Citation Format: Lin Yan, Sneha Sundaram, Bret M. Rust, Matthew J. Picklo, Michael R. Bukowski. Metabolomic differences between pulmonary metastasis of Lewis lung carcinoma and normal lungs from C57BL/6 mice fed an obesogenic high-fat diet [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 709.

OBJECTIVES: Obesogenic, high fat diets (HFD) increase hepatic long chain polyunsaturated fatty acid content (LCPUFA), but also lead to hepatic steatosis and insulin resistance. Fermentable fiber such as inulin (INU) may reduce these negative outcomes via a bile acid (BA)-liver signaling axis. This study tests the hypothesis that INU maintains elevated LCPUFA but reduces HFD-induced insulin resistance and fatty liver and investigates the potential relationship of bile acids. METHODS: Male mice (4 wk) were fed a low fat diet (LFD) (16% en fat) or HFD (48% en fat) containing 10% non-fermentable cellulose (CL) or 3% CL and 7% INU for 13 wks in a 2 × 2 factorial design. Tissue, plasma, and cecal contents were collected week 13. RESULTS: Intake of the HFD-CL increased hepatic LCPUFA content and steatosis and reduced insulin sensitivity vs LFD treatments. INU maintained HFD-induced LCPUFA elevation, improved insulin sensitivity, due to reductions in fasting glucose, but did not mitigate HFD-induced hepatic steatosis. When standardized to the hepatic triacylglycerol (TAG) content, LCPUFA (notably 22:6n-3) were increased by INU in the LFD only. The BAs with highest cecal concentrations were deoxycholic (DCA) and beta/omega murine cholic (B/OMCA) acids. INU increased cecal wt and decreased concentrations of DCA and B/OMCA. INU-induced B/OMCA reductions were enhanced by the HFD, but the DCA effects were independent of % en fat. The expression of multiple lipid metabolic genes was assessed. Of these, reductions in Scd1 expression occurred with both HFD and INU treatments compared to LFD-CL. SREBP1c, Fasn, Scd1, Elov5, Fads1, and Fads2 correlated with murine cholic acids and cholic acid in HFD-INU but only weakly in LFD-INU. These relationships were not evident with the CL-based diets. CONCLUSIONS: INU in an HFD provided mixed outcomes by maintaining elevated hepatic LCPUFA, reducing fasting plasma glucose concentrations but failing to reduce fasting insulin concentrations and hepatic steatosis. While cecal BAs and hepatic gene expression correlated with the INU treatments, these mechanistic changes did not mitigate HFD-induced hepatic steatosis. FUNDING SOURCES: USDA-ARS.

Background/aim Time restricted feeding (TRF) mitigates the high-fat diet-enhanced mammary tumorigenesis in a MMTV-PyMT breast cancer model. Materials and methods We performed untargeted metabolomic and targeted transcriptomic analyses on mammary tumors from MMTV-PyMT mice fed a standard AIN93G diet, a high-fat diet (HFD), or HFD with TRF (12 h, dark phase) and mammary glands from wild-type mice fed the AIN93G diet. Results The metabolic profile of mammary tumors differed from that of mammary glands; there was no impact of TRF upon tumor metabolome. TRF did reduce elevated expression of Hmgcr, Srebp1, Fads2, and Ppard in mammary tumors, indicating a down-regulation of lipid metabolism. Conclusion The null effect of TRF on the metabolomic profile does not rule out changes in more refined intracellular signaling pathways. It suggests that the protection of TRF against mammary tumorigenesis may rely upon its action on the host rather than a direct effect on tumor metabolism.

Time-restricted feeding (TRF) can reduce adiposity and lessen the co-morbidities of obesity. Mice consuming obesogenic high-fat (HF) diets develop insulin resistance and hepatic steatosis, but have elevated indices of long-chain polyunsaturated fatty acids (LCPUFA) that may be beneficial. While TRF impacts lipid metabolism, scant data exist regarding the impact of TRF upon lipidomic composition of tissues. We (1) tested the hypothesis that TRF of a HF diet elevates LCPUFA indices while preventing insulin resistance and hepatic steatosis and (2) determined the impact of TRF upon the lipidome in plasma, liver, and adipose tissue. For 12 weeks, male, adult mice were fed a control diet ad libitum, a HF diet ad libitum (HF-AL), or a HF diet with TRF, 12 hours during the dark phase (HF-TRF). HF-TRF prevented insulin resistance and hepatic steatosis resulting from by HF-AL treatment. TRF-blocked plasma increases in LCPUFA induced by HF-AL treatment but elevated concentrations of triacylglycerols and non-esterified saturated fatty acids. Analysis of the hepatic lipidome demonstrated that TRF did not elevate LCPUFA while reducing steatosis. However, TRF created (1) a separate hepatic lipid signature for triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine species and (2) modified gene and protein expression consistent with reduced fatty acid synthesis and restoration of diurnal gene signaling. TRF increased the saturated fatty acid content in visceral adipose tissue. In summary, TRF of a HF diet alters the lipidomic profile of plasma, liver, and adipose tissue, creating a third distinct lipid metabolic state indicative of positive metabolic adaptations following HF intake.

Machine learning and classical statistics were used to unravel the complex data set generated by the analysis of 77 fatty acids in milk from 76 cows fed 3 forages. Multivariate analysis of variance – principal components analysis (mANOVA-PCA) was used to deconvolute the data and determine the variance contributed by the main experimental factors: forage, the arbitrary cow groups, cow-to-cow variability, and milking day. All four factors contributed significantly to the data variance at the 99% confidence level as did the cross-factor variances. Cow-to-cow variance constituted 51% of the total variance of the data set and had to be removed before the significance of the other factors could be determined. The 3 forages were then found to generate milk fatty acid profiles that were significantly different. The PCA loadings showed that the major contributors to separate clustering of the forages were the less concentrated branched chain fatty acids. Fatty acids with even numbers of carbons were more concentrated, contributed high signal levels, and high noise, but were not significant in discriminating between forages. Classical paired analysis also removed the cow-to-cow variance and allowed discrimination between forages. The results showed that forages can significantly impact the fatty acid composition of milk.

Plasma levels of n-3 long chain polyunsaturated fatty acids (LCPUFA) are associated with a reduction in risk of cardiovascular disease and other chronic, age-related diseases like Alzheimer's disease. In this work, we tested the hypothesis that n-3 LCPUFA fatty acids in human plasma are incorporated into selective lipid species following intake of n-3 LCPUFA rich marine fish. To test this hypothesis, we performed lipidomic analysis on plasma samples from a clinical trial in which participants consumed increasing amounts of farmed Atlantic salmon (Salmo salar). Under basal conditions, n-3 and n-6 LCPUFA were selectively incorporated into plasma phosphatidylcholine (PC) species containing saturated fatty acids (SFA) versus unsaturated fatty acids as the complementary fatty acids. LCPUFA were incorporated into selective triacylglycerol (TAG) species with complementary diacylglyceryl environments of 34:1 or 34:2 (for 20:5 and 22:5) and 36:2>36:3>36:4 and 36:1 (for 20:4 and 22:6). High n-3 LCPUFA marine fish intake resulted in selective increases of PC SFA_n-3 LCPUFA species and LCPUFA-containing TAG species. Changes in cholesteryl esters and phosphatidylethanolamines also occurred following fish intake. Our results highlight the importance of discriminating phospholipid and TAG species and dietary background when evaluating lipidomic outcomes and disease associations.

OBJECTIVES: Metastasis is a hallmark of cancer. Diet-induced obesity enhances lung metastasis from the primary tumor of Lewis lung carcinoma (LLC) in mice and the lipidomic profile of LLC primary tumor reflects the fatty acid composition of the diet. The objective of this study was to evaluate the impact of feeding an obesogenic, high-fat diet (HFD) on the lipidomic composition of resulting LLC metastases in mice. METHODS: Lung metastases were obtained from LLC primary tumor-bearing mice fed the AIN93G diet or a HFD providing 16% or 45% of energy from soybean oil. Triacylglycerols (TAG) in the metastases were analyzed by using a mass spectrometry method wherein the ion source was calibrated with an equimolar TAG standard. Neutral mass losses were used to determine the relative content of selected fatty acids across Brutto structures. Lipidomic data were analyzed by using MetaboAnalyst software (Version 4.0) with a false discovery rate of 0.05. RESULTS: Analysis of TAG composition in metastases identified 51 separate TAGs by Brutto structure. Six TAGs were significantly different between the two diet groups. Concentrations of TAGs containing 48 and 50 acyl carbons were lower and those containing 54 acyl carbons were higher in the HFD-fed mice. In mice fed the HFD, TAG 54:6 was higher whereas TAGs 48:1 and 50:1 were lower in the metastases than in mice-fed the AIN93G diet. The volcano plot analysis of TAGs showed that differences in 3 TAGs between the two groups were at least two-fold. In neutral loss analysis, the acyl chain C and saturation values of 36:4 and 36:5 corresponding to 18:2_18:2 and 18:2_18:3, respectively, were significantly higher in the HFD group than in the AIN93G group. Increases in these linoleic and linolenic acid-containing species reflected the higher concentrations of these fatty acids in the HFD. CONCLUSIONS: The lipidomic profile of LLC metastases reflects the fatty acid composition of the diet. It suggests that lipidomic alteration of the metastases by an obesogenic diet may contribute to the aggressiveness of metastatic growth in the lungs. FUNDING SOURCES: USDA ARS 3062-51000-050-00D.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries, and the gut-liver axis is implicated in liver disease pathogenesis. We hypothesize that advanced liver steatosis accompanies an increase in hepatic inflammation, colonic secondary bile acids (BAs) and secondary BA-producing bacteria in mice fed a high-fat (HF) diet model of obesity. Four-week old male C57BL/6 mice were fed an HF (45% energy) or a low-fat (LF) (10% energy) diet for 21 weeks. At the end of the study, body weight and body fat percentage in the HF group were 0.23- and 0.41-fold greater than those in the LF group, respectively. Similarly, the HF group exhibited an increase in hepatic lipid droplets, inflammatory cell infiltration, inducible nitric oxide synthase, and hepatocellular ballooning (but without hepatic Mallory bodies) which are key histological features of advanced hepatic steatosis. Furthermore, RNA sequencing, qPCR and immunohistological methods found that nicotinamide n-methyltransferase and selenoprotein P, two inflammation-related hepatic genes, were upregulated in the HF group. Consistent with the hepatic inflammation, the levels of proinflammatory plasma-cytokines (TNF-α and IL6), colonic secondary BAs (LCA, DCA) and secondary BA producing bacteria (e.g., lactobacillaceae/Lachnospiraceae) were at least 0.5-fold greater in the HF group compared with the LF group. Taken together, the data demonstrate that advanced liver-steatosis is concurrent with an elevated level of hepatic inflammation, colonic secondary bile acids and their associated bacteria in mice fed an HF diet. These data suggest a potential gut-liver crosstalk at the stage of advanced liver-steatosis.

Ceramides (CER) are biologically active sphingolipid precursors that are mechanistically linked to several pathogenic states including cancer, insulin resistance, and neurodegeneration. CER are commonly quantified through mass spectrometry-based methods founded upon a product ion scan (PIS) in positive mode to produce a characteristic m/z 264 ion. The ionization efficiency (IE) of CER species decreases with an increase in CER mass, thus quantitation of CER typically involves application of mass-dependent response factors (RF) for each CER species. In this work, we observed that the RF were systematically dependent on the number of fatty acid acyl carbons and the collision energy (CE) used to generate the m/z 264 ion. Using these complimentary trends, we determined an "isosbestic" CE where the RF for all CER species were equivalent, allowing for CER quantitation without postcollection correction factors. A comparison of this common CE/common RF method to the multiple RF method demonstrated good agreement between the two methods. Use of the common CE/common RF method will reduce data processing and reduce the use of multiple CER species standards.

Protein-glutathione mixed disulphides (PSSG) are an important redox-sensitive posttranslational modification. Quantitation of protein-glutathione mixed disulphides (PSSG) is achieved by the reduction of the disulphide bond to liberate glutathione (GSH); however, this method leaves the assay susceptible to contamination by cytosolic GSH and glutathione disulphide (GSSG) captured during protein precipitation. The method herein describes a workflow in which protein from mouse liver is precipitated and adventitious GSH contamination is removed by reaction with N-ethylmaleimide. The sample is divided into two equal aliquots, a control aliquot that allows for direct quantitation of adventitious GSSG and a chemically reduced aliquot that contains GSH from both the GSSG and PSSG disulphides. Determining the concentration of adventitious GSSG allows for correction of the latter value to provide an accurate assay of PSSG. This assay also provides quantitation of cytosolic GSH and GSSG.

Breast cancer is a rare but aggressive disease in men. Approximately, 90% of all breast cancer in men are invasive carcinomas, 25% of which have distant metastasis at the time of clinical presentation. Obesity is a risk factor for breast cancer. Monocyte chemotactic protein-1 (MCP-1) is an adipose-derived cytokine whose concentrations are elevated by obesity. This study tested the hypothesis that adipose-derived MCP-1 contributes to male breast cancer. In a 2x2 design, male MMTV-PyMT mice with or without adipose specific Mcp-1 knockdown [designated as Mcp-1-/- or wild-type (WT)] were fed the standard AIN93G diet or an obesogenic, high-fat diet (HFD) for 25 weeks. The AIN93G diet and HFD contained 16% and 45% of energy from soybean oil, respectively. Mcp-1-/- mice had lower adipose Mcp-1 expression than WT mice. Adipose Mcp-1 deficiency reduced plasma concentrations of MCP-1 in mice fed the HFD. Mice fed the HFD had a greater tumor progression rate than mice fed the AIN93G diet (4679% vs 2430%), regardless of genotype. Mcp-1-/- mice, compared to WT mice, had a longer tumor latency (25.2 weeks vs 18.0 weeks) and lower tumor incidence (19% vs 56%), tumor progression rate (2317% vs 4792%), and tumor weights (0.23 g vs 0.64 g). Metabolomic analysis of plasma identified 87 metabolites, 56 of which differed among the four groups, including 24 that differed between the diets and 22 that differed between the genotypes. Sparse partial least squares-discriminant analysis identified 10 major metabolites that were altered the most among the four groups. These included amino acids (alanine, isoleucine, leucine, phenylalanine, threonine, and valine) and carbohydrate metabolites (fumaric acid, glucuronic acid, hexuronic acid, and malic acid). The integrated pathway analysis of the 87 identified metabolites showed that protein biosynthesis and carbohydrate metabolism pathways are the most disturbed in MMTV-PyMT mice. In conclusion, adipose-derived MCP-1 contributes to mammary tumorigenesis and alters metabolism in male MMTV-PyMT mice. Citation Format: Lin Yan, Sneha Sundaram, Bret M. Rust, Matthew J. Picklo, Michael R. Bukowski. Tumorigenic and metabolomic impacts of adipose-derived monocyte chemotactic protein-1 in a model of obesity-enhanced, male breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2561.

OBJECTIVES: Consumption of a high fat diet (HFD) links obesity to colon cancer in humans. Consistent with this observation, our data show that a HFD (45% energy fat versus 16% energy fat in AIN-93 diet, AIN) promotes azoxymethane (AOM) induced colonic aberrant crypt foci (ACF) formation in a mouse cancer model. However, the underlying metabolic basis remains to be determined. We hypothesize that AOM treatment results in different plasma metabolomic responses in AOM mouse colon cancer model fed with a HFD compared to the AIN. METHODS: Four-week-old male C57BL/6 mice were fed the AIN or HFD in the context of with or without AOM treatment for 16 weeks. At the end of this study, plasma samples were extracted and derivatized by silylation and methyloximation then analyzed by time-of-flight mass spectrometry using an untargeted metabolomic technique. RESULTS: Untargeted metabolomic profiling showed that 53 of 144 identified metabolites were different between the 4 groups of mice (AIN only, AIN + AOM; HFD only, HFD + AOM). Sparse partial least squares discriminant analysis (two-dimensional plots) showed a separation between the HFD and HFD + AOM groups but not AIN and AIN + AOM groups. Major metabolites responsible for this separation were beta-sitosterol, dihydrocholesterol, alpha-tocopherol, methylphosphate, citric acid, cholesterol, isocitric acid, myristic acid, arachidonic acid, and nicotinamide. Importantly, the levels of dihydrocholesterol and cholesterol were inversely associated with AOM-induced colonic ACF formation. Functional pathway and enrichment analyses indicated that diets and AOM-induced colonic ACF modulated 5 metabolic pathways: (a) citrate cycle (TCA cycle), (b) arginine biosynthesis, (c) aminoacyl-tRNA biosynthesis, (d) alanine, aspartate and glutamate metabolism, (e) glyoxylate and dicarboxylate metabolism. CONCLUSIONS: Collectively, we demonstrate that AOM decreases dihydrocholesterol and cholesterol levels, and alters the composition of plasma metabolome to a greater extent in mice fed a HFD compared to the AIN. FUNDING SOURCES: This work was supported by U.S. Department of Agriculture, Agricultural Research Service, research project 3062-51000-056-00D.

Adoption of an obesogenic diet low in calcium and vitamin D (CaD) leads to increased obesity, colonic inflammation, and cancer. However, the underlying mechanisms remain to be elucidated. We tested the hypothesis that CaD supplementation (from inadequacy to adequacy) may reduce colonic inflammation, oncogenic signaling, and dysbiosis in the colon of C57BL/6 mice fed a Western diet. Male C57/BL6 mice (4-weeks old) were assigned to 3 dietary groups for 36 weeks: (1) AIN76A as a control diet (AIN); (2) a defined rodent “new Western diet” (NWD); or (3) NWD with CaD supplementation (NWD/CaD). Compared to the AIN, mice receiving the NWD or NWD/CaD exhibited more than 0.2-fold increase in the levels of plasma leptin, tumor necrosis factor α (TNF-α) and body weight. The levels of plasma interleukin 6 (IL-6), inflammatory cell infiltration, and β-catenin/Ki67 protein (oncogenic signaling) were increased more than 0.8-fold in the NWD (but not NWD/CaD) group compared to the AIN group. Consistent with the inflammatory phenotype, colonic secondary bile acid (inflammatory bacterial metabolite) levels increased more than 0.4-fold in the NWD group compared to the NWD/CaD and AIN groups. Furthermore, the abundance of colonic Proteobacteria (e.g., Parasutterela), considered signatures of dysbiosis, was increased more than four-fold; and the α diversity of colonic bacterial species, indicative of health, was decreased by 30% in the NWD group compared to the AIN and NWD/CaD groups. Collectively, CaD adequacy reduces colonic inflammation, β-catenin oncogenic signaling, secondary bile acids, and bacterial dysbiosis in mice fed with a Western diet.

Phosphatidylcholine (PC) species in human plasma are used as biomarkers of disease. PC biomarkers are often limited by the inability to separate isobaric PCs. In this work, we developed a targeted shotgun approach for analysis of isobaric and isomeric PCs. This approach is comprised of two MS methods: a precursor ion scanning (PIS) of mass m/z 184 in positive mode (PIS m/z +184) and MS3 fragmentation in negative mode, both performed on the same instrument, a hybrid triple quadrupole ion-trap mass spectrometer. The MS3 experiment identified the FA composition and the relative abundance of isobaric and sn-1, sn-2 positional isomeric PC species, which were subsequently combined with absolute quantitative data obtained by PIS m/z +184 scan. This approach was applied to the analysis of a National Institute of Standards and Technology human blood plasma standard reference material (SRM 1950). We quantified more than 70 PCs and confirmed that a majority are present in isobaric and isomeric mixtures. The FA content determined by this method was comparable to that obtained using GC with flame ionization detection, supporting the quantitative nature of this MS method. This methodology will provide more in-depth biomarker information for clinical and mechanistic studies.

OBJECTIVES: Assess the differences in triacylglycerol (TAG) and cholesterol ester (CE) species in the plasma of individuals displaying heterogeneous lipid responses following long chain n-3 (LCn-3) polyunsaturated fatty acid supplementation. METHODS: We performed a targeted, mass spectrometry (MS), infusion-based lipidomic analysis on plasma samples obtained from a clinical study in which participants were supplemented with 3 g/day of LCn-3 in the form of fish oil capsules over a 6-week period. Triacylglycerol (TAG) species and cholesteryl esters (CE) were quantified for 130 participants pre- and post-supplementation. RESULTS: Based on the change of total TAG concentrations following supplementation, participants were segregated into three response phenotypes: (1) positive responders (R+; TAG decrease 10%) representing 87/130 (67%), 24/130 (18%), and 19/130 (15%) of the study samples, respectively. There were no phenotypic differences for age, sex, body-mass index, glycemia, or ApoB concentrations. Sparse partial least squares discriminant analysis separated the three phenotypes with component 1 attributed to changes in TAG 50–53: X with 0–3 desaturations with R + having reductions in these TAG. Separation along component 2 identified lower mass TAG 46–48: X with 1–3 desaturations likely containing 14:0. This latter effect impacted mostly NR and R- phenotypes. Analysis of individual TAG species per response phenotype revealed TAG species that did not align with the overall TAG response phenotype. Using the TAG response phenotype for grouping, we performed SPLDA analysis for CE responses. We observed that distinction of the TAG response phenotypes qualitatively applies to CE in which separation along component 1 (65% of variance) was due to differences in CE 18:0, 18:1, and 14:0. CE 20:5 was elevated equally (>300%) between all phenotypes indicating LCn-3 intake. However, CE 22:6 was elevated R− (86%) to a greater extent vs. Res+ (55%) and NR (49%) phenotypes. CONCLUSIONS: Our data identify lipidomic signatures (TAG and CE) associated with LCn-3 response phenotypes in humans and provide insight into the variability of lipid metabolism in humans. FUNDING SOURCES: USDA-NIFA, USDA-ARS and CIHR MOP-229,488.

OBJECTIVES: Adoption of a Western diet, low in calcium and vitamin D (CaD), is a global problem leading to increased obesity, colonic inflammation and neoplasia. However, the mechanisms underlying this relationship remains to be elucidated. We tested the hypothesis that CaD supplementation may reduce colonic inflammation, oncogenic signaling and dysbiosis in the colon of C57BL/6 mice fed a Western diet. METHODS: Male C57/BL6 mice (4-week old) were assigned to 3 dietary groups (n = 20/group) for 36 weeks: (1) AIN76A as a control diet (AIN); (2) a defined rodent “new Western diet” (NWD); or (3) NWD with CaD supplementation (NWD/CaD). We used biochemical, mass spectrometry, histological and multi-omics approaches to determine the interplay between CaD supplementation, colonic inflammation and microbiome composition. RESULTS: Mice receiving the NWD or NWD/CaD exhibited >0.2- fold increase in the concentrations of plasma leptin, tumor necrosis factor α (TNF-α) and body weight when compared with those on the AIN diet. However, the concentrations of plasma interleukin 6 (IL6), inflammatory cell infiltration and b-catenin/Ki67 protein (oncogenic signaling) were increased >0.8 - fold in the NWD (but not NWD/CaD) group when compared with the AIN group. Consistent with the inflammatory phenotype: (1) colonic secondary bile acid (an inflammatory bacterial metabolite) concentrations increased >0.4 - fold in the NWD group when compared with the NWD/CaD and AIN groups; (2) in contrast, α diversity of colonic bacterial species, a healthy gut microbiome marker, was decreased by 30% in the NWD group when compared with the AIN and NWD/CaD groups. CONCLUSIONS: This study demonstrates that, in a mouse model, CaD supplementation reduces colonic inflammation, oncogenic signaling and bacterial dysbiosis in the context of Western diet consumption. FUNDING SOURCES: This work was supported by U.S. Department of Agriculture, Agricultural Research Service, research project 3062–51,000-056–00D.

Protein-glutathione mixed disulphides (PSSG) are an important redox-sensitive posttranslational modification. Quantitation of protein-glutathione mixed disulphides (PSSG) is achieved by the reduction of the disulphide bond to liberate glutathione (GSH); however, this method leaves the assay susceptible to contamination by cytosolic GSH and glutathione disulphide (GSSG) captured during protein precipitation. The method herein describes a workflow in which protein from mouse liver is precipitated and adventitious GSH contamination is removed by reaction with N-ethylmaleimide. The sample is divided into two equal aliquots, a control aliquot that allows for direct quantitation of adventitious GSSG and a chemically reduced aliquot that contains GSH from both the GSSG and PSSG disulphides. Determining the concentration of adventitious GSSG allows for correction of the latter value to provide an accurate assay of PSSG. This assay also provides quantitation of cytosolic GSH and GSSG.

Metabolic reprogramming of lipid metabolism is a hallmark of cancer. Consumption of a high-fat obesogenic diet enhances spontaneous metastasis using a Lewis lung carcinoma (LLC) model. In order to gain further insights into the mechanisms by which dietary fats impact cancer progression, we conducted a lipidomic analysis of primary tumors originated from LLC from mice fed with a standard AIN93G diet or a soybean oil-based high-fat diet (HFD). Hierarchical clustering heatmap analysis of phosphatidylcholine (PC) lipids and phosphatidylethanolamine (PE) lipids demonstrated an increase in polyunsaturated fatty acids (PUFA)-containing phospholipids and a decrease in monounsaturated fatty acids (MUFA)-containing lipids in tumors from mice fed the HFD. The quantities of 51 PC and 24 PE lipids differed in primary tumors of LLC from mice fed the control diet and the HFD. Analysis of triacylglycerol (TAG) lipids identified differences in 32 TAG (by brutto structure) between the two groups; TAG analysis by neutral loss identified 46 PUFA-containing TAG species that were higher in mice fed with the HFD than in the controls. Intake of the HFD did not alter the expression of the de novo lipogenesis enzymes (fatty acid synthase, acetyl-CoA carboxylase-1, and stearoyl-CoA desaturase-1). Our results demonstrate that the dietary fatty acid composition of the HFD is reflected in the higher order lipidomic composition of primary tumors. Subsequent studies are needed to investigate how these lipidomic changes may be used for targeted dietary intervention to reduce tumor growth and malignant progression.

The aim of this study was to investigate the effect of various cooking techniques on the fatty acid and oxylipin content of farmed rainbow trout. Rainbow trout is an excellent source of long-chain omega-3 (n-3) polyunsaturated fatty acids (PUFA) which have beneficial health effects. Fillets of 2-year-old farmed rainbow trout were baked, broiled, microwaved, or pan-fried in corn (CO), canola (CaO), peanut (PO), or high oleic sunflower oil (HOSO). Fatty acids and oxidized lipids were extracted from these samples and their respective raw fillet samples. Fatty acid content was determined using gas chromatography and oxylipin content by mass spectroscopy. The values obtained from each cooking method were compared to those obtained from the respective raw fillets using paired t tests. PUFA content was not altered when samples were baked, broiled, microwaved, or pan-fried in CO or CaO. Pan-frying in PO reduced α-linolenic acid (18:3n-3), eicosadienoic acid (20:2n-6), and dihomo-γ-linolenic acid (20:3n-6), while pan-frying in HOSO reduced 18:3n-3, eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), docosahexaenoic acid (22:6n-3), linoleic acid (18:2n-6), 18:3n-6, 20:2n-6, 20:3n-6, docosatrienoic acid (22:2n-6), and adrenic acid (22:4n-6) compared to raw fish. Cooking decreased the omega-6 (n-6) PUFA-derived oxylipins, but caused no change in 20:5n-3 or 22:6n-3-derived oxylipins of the fillets. In conclusion, pan-frying was the only cooking method to alter the fatty acid content of the fillets, while observed changes in oxylipin content varied by cooking method. As the physiological impact of oxylipins is currently unknown, these results suggest that the cooking methods which optimize the consumption of n-3 PUFA from rainbow trout are baking, broiling, microwaving, or pan-frying in CO, CaO, or PO.

Protein S-glutathionylation is a posttranslational modification that links oxidative stimuli to reversible changes in cellular function. Protein–glutathione mixed disulfide (PSSG) is commonly quantified by reduction of the disulfide and detection of the resultant glutathione species. This methodology is susceptible to contamination by free unreacted cellular glutathione (GSH) species, which are present in 1000-fold greater concentration. A liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based method was developed for quantification of glutathione and glutathione disulfide (GSSG), which was used for the determination of PSSG in biological samples. Analysis of rat liver samples demonstrated that GSH and GSSG coprecipitated with proteins similar to the range for PSSG in the sample. The use of [13C2,5N]GSH and [13C4,5N2]GSSG validated these results and demonstrated that the release of GSH from PSSG did not occur during sample preparation and analysis. These data demonstrate that GSH and GSSG contamination must be accounted for when determining PSSG content in cellular/tissue preparations. A protocol for rinsing samples to remove the adventitious glutathione species is demonstrated. The fragmentation patterns for glutathione were determined by high-resolution mass spectrometry, and candidate ions for detection of PSSG on protein and protein fragments were identified.

The increasing worldwide incidence of colon cancer has been linked to obesity and consumption of a high-fat Western diet. To test the hypothesis that a high-fat diet (HFD) promotes colonic aberrant crypt (AC) formation in a manner associated with gut bacterial dysbiosis, we examined the susceptibility to azoxymethane (AOM)-induced colonic AC and microbiome composition in C57/BL6 mice fed a modified AIN93G diet (AIN, 16% fat, energy) or an HFD (45% fat, energy) for 14 weeks. Mice receiving the HFD exhibited increased plasma leptin, body weight, body fat composition and inflammatory cell infiltration in the ileum compared with those in the AIN group. Consistent with the gut inflammatory phenotype, we observed an increase in colonic AC, plasma interleukin-6, tumor necrosis factor-α, monocyte chemoattractant protein-1 and inducible nitric oxide synthase in the ileum of the HFD-AOM group compared with the AIN-AOM group. Although the HFD and AIN groups did not differ in bacterial species number, the HFD and AIN diets resulted in different bacterial community structures in the colon. The abundance of certain short-chain fatty acid (SCFA) producing bacteria (e.g., Barnesiella) and fecal SCFA (e.g., acetic acid) content were lower in the HFD-AOM group compared with the AIN and AIN-AOM groups. Furthermore, we identified a high abundance of Anaeroplasma bacteria, an opportunistic pathogen in the HFD-AOM group. Collectively, we demonstrate that an HFD promotes AC formation concurrent with an increase of opportunistic pathogenic bacteria in the colon of C57BL/6 mice.

OBJECTIVES: Obesity is associated with several diseases such as cardiovascular disease and non-alcoholic fatty liver disease. Time-restricted feeding (TRF) is effective in reducing adiposity and lessening the co-morbidities of obesity. We have demonstrated that mice consuming a high-fat (HF) diet (48%en fat), primarily composed of long chain saturated fatty acids display elevated content of hepatic triacylglycerol (TAG) and the long-chain polyunsaturated fatty acids (LCPUFAs) arachidonic acid (20:4n6, ARA) and docosahexaenoic acid (22:6n-3, DHA). We tested the hypothesis that TRF of the HF diet reduces adiposity and hepatic TAG content while maintaining elevated hepatic LCPUFA concentrations compared to mice eating the HF diet ad libitum. METHODS: Male mice (12 weeks old) were fed (12 weeks) a low-fat diet ad libitum (LF-AL, 16%en fat) or a HF diet (48%en fat) either ad libitum (HF-AL) or restricted to feeding for 12 hours per day during the dark phase (HF-TRF). Fasting blood glucose and plasma insulin were measured. Plasma and hepatic levels of TAG and cholesterol were analyzed. Hepatic gene expression of mediators of de novo lipogenesis (DNL) and the circadian rhythm were examined using qPCR. Fatty acid methyl ester (FAME) analysis determined fatty acid content within the livers. RESULTS: Blood glucose, plasma insulin, and plasma cholesterol were elevated in the HF-AL group, but were attenuated by TRF. Intake of the HF diet elevated hepatic ARA and DHA content over that of the LF diet. Hepatic TAG content, while elevated in the HF-AL mice, was not different between HF-TRF and LF-AL mice. HF-AL mice had a greater body mass and % body fat than the LF-AL and HF-TRF animals. Lipidomic analysis coupled with multivariate analysis of hepatic TAG species demonstrated TRF induced distinct differences between the TAG structures and their concentrations. Hepatic gene expression of several DNL mediators were reduced by HF-AL intake and were further reduced within the HF-TRF group. TRF restored or partially restored mRNA levels of several circadian genes altered by the HF-AL diet. CONCLUSIONS: These data suggest that limiting HF diet intake to certain hours of the day ameliorates comorbidities associated with obesity, modifies hepatic lipidomic composition, and increases the hepatic content of LCPUFA. FUNDING SOURCES: This work was supported by USDA-ARS project 3062-51000-053-00D.

OBJECTIVES: The gut-liver axis is implicated in the pathogenesis of liver diseases; however, the mechanistic link between the gut and fatty liver disease severity remains to be characterized. We tested the hypothesis that advanced liver-steatosis accompanies an increase in the concentrations of colonic proinflammatory bile acids and hepatic inflammation in mice fed a high fat (HF) diet. METHODS: Four-week-old male C57BL/6 mice (n = 14/group) were fed a HF (45% energy fat) or a low-fat (LF) (10% energy fat) diet for 21 wks. We used biochemical, immunohistological, gas & liquid chromatography with mass spectrometry, RNA sequencing and real-time PCR methods to examine the level of colonic proinflammatory bile acids and hepatic inflammation. RESULTS: At the end of the study, body weight and body fat percentage in the HF group were 0.23- and 0.41- fold greater than the LF group, respectively. Compared with the LF group, the HF group exhibited an increase in hepatic lipid droplets, inflammatory cell infiltration, inducible nitric oxide synthase, and hepatocellular ballooning cells (without hepatic Mallory bodies) which are key histological features of advanced hepatic steatosis. Furthermore, nicotinamide n-methyltransferase and selenoprotein P, two hepatic proinflammation-related genes, were upregulated in the HF group. With unique colonic bile acid profiles, the levels of colonic hydrophobic (secondary) bile acids (lithocholic acids and deoxycholic acids) were at least 0.5- fold greater in the HF group compared with the LF group. It is known that these hydrophobic bile acids induce proinflammatory cytokine production (TNF-α and IL-6) in the colon, and exacerbate hepatic inflammation via portal venous circulation. This potential colon-liver axis is consistent with results that the plasma concentrations of TNF-α and IL-6 were 0.5-fold greater in the HF group compared the LF group. CONCLUSIONS: Our results demonstrate that advanced liver-steatosis accompanies an increase in colonic proinflammatory bile acid levels and hepatic inflammation, which suggests a cross-talk between colon and liver in mice fed a HF diet. FUNDING SOURCES: This work was funded by the US Department of Agriculture, Agricultural Research Service, CRIS project 3062-51000-050-00D.

OBJECTIVES: Maternal high-fat (HF) diet predisposes offspring to metabolic syndrome, in part, by inducing alterations during placental development resulting in abnormal fetal development and offspring birth weight. We previously reported in a rat model that a maternal malnutrition diet increases fetal inflammation, and placental vascularization, and decreases offspring birth weight followed by adult-onset obesity. In the current study, we investigated when during gestational development a maternal HF diet induces changes in placental weight, lipid content, and inflammation. We further investigated whether probiotic supplementation offsets HF-diet induced adverse outcomes. METHODS: Two-month old female C57BL/6 mice were fed diets of 16% fat (normal-fat, NF) or 45% fat (high-fat, HF) for 8 weeks followed by 4 weeks of probiotic (Bifidobacterium animalis subsp. lactis, BB-12) supplementation. Fetuses and placentae were examined mid- (D12) and late- (D19) gestation RESULTS: Placental length, width, and weight as well as fetal weight were decreased in the HF group at D12. Probiotic supplementation reversed the HF diet-induced reduction in placental weight at D12. These beneficial effects of probiotic supplementation were absent at D19 as fetal weights from all HF diet fed groups weighed less regardless of probiotic supplementation. Probiotic supplementation reduced placental TNF-alpha mRNA at D12 and IL-10 mRNA at D19. Triacylglyceride concentration was increased at D19 by HF diet, in part, by the preceding increases in lipoprotein lipase (LPL) and plasma membrane-associated fatty-acid binding protein (FABPpm) mRNA expression at D12. CONCLUSIONS: These findings suggest that maternal HF diet alters placental size and weight as well as fetal weight and that probiotic supplementation renders a protective effect against these changes in mid-gestation. Future studies are needed to determine whether the protective mechanism of probiotic supplementation in mid-gestation has beneficial effects on offspring developmental programming. FUNDING SOURCES: USDA Agricultural Research Service Project #3062-51000-052-00D.

A family of copolymer hydrogels containing different mass percentages of vinylimidazole, acrylamide and N,N′-methylenebisacrylamide were used to bind copper(II) ions. The resultant copper-loaded gels demonstrated spectroscopic features that indicated copper was bound in a distorted square planar geometry. The hydrolysis activity of these the most active of these systems towards bis(3-nitrophenyl)phosphate at pH 8 was 2.78 × 10−6 s−1, five orders of magnitude greater than the uncatalyzed reaction. While these systems obey Michaelis–Menten kinetics, they also subject both competitive and non-competitive inhibition from excess substrate and excess hydroxide due to constraints based in the coordination geometry of the copper(II) active sites.

In the absence of base, the reaction of [Fe{sup II}(TMCS)]PF{sub 6} (1, TMCS = 1-(2-mercaptoethyl)-4,8,11-trimethyl-1,4,8,11-tetraazacyclotetradecane) with peracid in methanol at -20 C did not yield the oxoiron(IV) complex (2, [Fe{sup IV}(O)(TMCS)]PF{sub 6}), as previously observed in the presence of strong base (KO{sup t}Bu). Instead, the addition of 1 equiv of peracid resulted in 50% consumption of 1. The addition of a second equivalent of peracid resulted in the complete consumption of 1 and the formation of a new species 3, as monitored by UV-vis, ESI-MS, and Moessbauer spectroscopies. ESI-MS showed 3 to be formulated as [Fe{sup II}(TMCS) + 2O]{sup +}, while EXAFS analysis suggested that 3 was an O-bound iron(II)-sulfinate complex (Fe-O = 1.95 {angstrom}, Fe-S = 3.26 {angstrom}). The addition of a third equivalent of peracid resulted in the formation of yet another compound, 4, which showed electronic absorption properties typical of an oxoiron(IV) species. Moessbauer spectroscopy confirmed 4 to be a novel iron(IV) compound, different from 2, and EXAFS (Fe{double_bond}O = 1.64 {angstrom}) and resonance Raman ({nu}{sub Fe{double_bond}O} = 831 cm{sup -1}) showed that indeed an oxoiron(IV) unit had been generated in 4. Furthermore, both infrared and Raman spectroscopy gave indications that 4 contains a metal-bound sulfinate moietymore » ({nu}{sub s}(SO{sub 2}) {approx} 1000 cm{sup -1}, {nu}{sub as}(SO{sub 2}) {approx} 1150 cm{sup -1}). Investigations into the reactivity of 1 and 2 toward H{sup +} and oxygen atom transfer reagents have led to a mechanism for sulfur oxidation in which 2 could form even in the absence of base but is rapidly protonated to yield an oxoiron(IV) species with an uncoordinated thiol moiety that acts as both oxidant and substrate in the conversion of 2 to 3.« less

Dysregulation of adipokines is a hallmark of obesity. Polyunsaturated fatty acids in fish oil may exert anti-inflammatory effects on adipose tissue mitigating the dysregulation of adipokines thereby preventing obesity. This study investigated the effects of high-fat diets containing different amounts of n3 polyunsaturated fatty acids (PUFA) on adiposity and adipokine production in mice. Mice were fed a low-fat or a high-fat diet with 16 or 45 % of energy from corn oil (low n3 PUFA) in comparison with a high-fat diet containing soybean or high-oleic sunflower oil (adequate n3 PUFA) or flaxseed or fish oil (high n3 PUFA) for 11 weeks. High-fat diets, regardless of types of oils, significantly increased body fat mass and body weights compared to the low-fat diet. Adipose fatty acid composition and contents reflected dietary fatty acid profiles. The high-fat fish oil diet significantly increased adiponectin and reduced leptin concentrations in both plasma and adipose tissue; it did not elevate plasma insulin concentration compared to the high-fat corn oil diet. All high-fat diets elevated concentrations of plasminogen activator inhibitor-1 (PAI-1) and monocyte chemoattractant protein-1 (MCP-1) but lowered resistin concentrations in both plasma and adipose tissue. In conclusion, fish oil may be beneficial in improving insulin sensitivity by upregulation of adiponectin and downregulation of leptin production; n3 and n6 PUFA do not play a role at the dietary levels tested in reducing adiposity and production of pro-inflammatory cytokines (leptin, PAI-1, MCP-1 and resistin) and anti-inflammatory cytokine adiponectin.