Time-Restricted Feeding Modifies the Fecal Lipidome and the Gut Microbiota.

Time-restricted feeding (TRF) has been identified as an approach to reduce the risk of obesity-related metabolic diseases. We hypothesize that TRF triggers a change in nutrient (e.g., dietary fat) absorption due to shortened feeding times, which subsequently alters the fecal microbiome and lipidome. In this report, three groups of C57BL/6 mice were fed either a control diet with ad libitum feeding (16% energy from fat) (CTRL-AL), a high-fat diet (48% energy from fat) with ad libitum feeding (HF-AL), or a high-fat diet with time-restricted feeding (HF-TRF) for 12 weeks. No changes in microbiota at the phylum level were detected, but eight taxonomic families were altered by either feeding timing or dietary fat content. The HF-AL diet doubled the total fecal fatty acid content of the CTRL-AL diet, while the HF-TRF doubled the total fecal fatty acid content of the HF-AL diet. Primary fecal bile acids were unaffected by diet. Total short-chain fatty acids were reduced by HF-AL, but this effect was diminished by HF-TRF. Each diet produced distinct relationships between the relative abundance of taxa and fecal lipids. The anti-obesogenic effects of TRF in HF diets are partly due to the increase in fat excretion in the feces. Furthermore, fat content and feeding timing differentially affect the fecal microbiota and the relationship between the microbiota and fecal lipids.

Associations among Milk Microbiota, Milk Fatty Acids, Milk Glycans, and Inflammation from Lactating Holstein Cows.

Milk oligosaccharides (MOs) can be prebiotic and antiadhesive, while fatty acids (MFAs) can be antimicrobial. Both have been associated with milk microbes or mammary gland inflammation in humans. Relationships between these milk components and milk microbes or inflammation have not been determined for cows and could help elucidate a novel approach for the dairy industry to promote desired milk microbial composition for improvement of milk quality and reduction of milk waste. We aimed to determine relationships among milk microbiota, MFAs, MOs, lactose, and somatic cell counts (SCC) from Holstein cows, using our previously published data. Raw milk samples were collected at three time points, ranging from early to late lactation. Data were analyzed using linear mixed-effects modeling and repeated-measures correlation. Unsaturated MFA and short-chain MFA had mostly negative relationships with potentially pathogenic genera, including Corynebacterium, Pseudomonas, and an unknown Enterobacteriaceae genus but numerous positive relationships with symbionts Bifidobacterium and Bacteroides. Conversely, many MOs were positively correlated with potentially pathogenic genera (e.g., Corynebacterium, Enterococcus, and Pseudomonas), and numerous MOs were negatively correlated with the symbiont Bifidobacterium. The neutral, nonfucosylated MO composed of eight hexoses had a positive relationship with SCC, while lactose had a negative relationship with SCC. One interpretation of these trends might be that in milk, MFAs disrupt primarily pathogenic bacterial cells, causing a relative increase in abundance of beneficial microbial taxa, while MOs respond to and act on pathogenic taxa primarily through antiadhesive methods. Further research is needed to confirm the potential mechanisms driving these correlations. IMPORTANCE Bovine milk can harbor microbes that cause mastitis, milk spoilage, and foodborne illness. Fatty acids found in milk can be antimicrobial and milk oligosaccharides can have antiadhesive, prebiotic, and immune-modulatory effects. Relationships among milk microbes, fatty acids, oligosaccharides, and inflammation have been reported for humans. To our knowledge, associations among the milk microbial composition, fatty acids, oligosaccharides, and lactose have not been reported for healthy lactating cows. Identifying these potential relationships in bovine milk will inform future efforts to characterize direct and indirect interactions of the milk components with the milk microbiota. Since many milk components are associated with herd management practices, determining if these milk components impact milk microbes may provide valuable information for dairy cow management and breeding practices aimed at minimizing harmful and spoilage-causing microbes in raw milk.

Changes in the Fecal Metabolome Accompany an Increase in Aberrant Crypt Foci in the Colon of C57BL/6 Mice Fed with a High-Fat Diet.

High-fat diet (HFD)-induced obesity is a risk factor for colon cancer. Our previous data show that compared to an AIN-93 diet (AIN), a HFD promotes azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation and microbial dysbiosis in C57BL/6 mice. To explore the underlying metabolic basis, we hypothesize that AOM treatment triggers a different fecal metabolomic profile in C57BL/6 mice fed the HFD or the AIN. We found that 65 of 196 identified metabolites were significantly different among the four groups of mice (AIN, AIN + AOM, HFD, and HFD + AOM). A sparse partial least squares discriminant analysis (sPLSDA) showed that concentrations of nine fecal lipid metabolites were increased in the HFD + AOM compared to the HFD, which played a key role in overall metabolome group separation. These nine fecal lipid metabolite concentrations were positively associated with the number of colonic ACF, the cell proliferation of Ki67 proteins, and the abundance of dysbiotic bacteria. These data suggest that the process of AOM-induced ACF formation may increase selective fecal lipid concentrations in mice fed with a HFD but not an AIN. Collectively, the accumulation of these critical fecal lipid species may alter the overall metabolome during tumorigenesis in the colon.

Quantifying Sphingomyelin in Dairy through Infusion-Based Shotgun Mass Spectrometry with Lithium-Ion-Induced Fragmentation.

Quantifying sphingomyelin (SM) species by infusion-based mass spectrometry (MS) is complicated by the presence of isobaric phosphatidylcholine (PC) species, which generate a common m/z 184 product ion in the presence of ammonium ions as a result of the phosphocholine headgroup. Lithium ion adducts of SM undergo a selective dehydration [Li + H2O + (CH3)3NC2H4PO4] with a corresponding neutral loss of -207 Da. This neutral loss was employed to create a SM-selective method for identifying target species, which were quantitated using multiple reaction monitoring (MRM). SM-selective fragments in MS3 were used to characterize the sphingosine base and acyl chain. These methods were used to identify 50 individual SM species in bovine milk ranging from SM 28:1 to SM 44:2, with d16:1, d17:1, d18:1, d19:1, and d20:1 bases, and acyl fatty acids ranging from 10 to 25 carbons and 0-1 desaturations. Spiked SM standards into milk had a recovery of 99.7%, and endogenous milk SM had <10% coefficient of variation for both intra- and interday variability, with limits of detection of 1.4-5.55 nM and limits of quantitation of 11.8-178.1 nM. This MS-MRM method was employed to accurately and precisely quantify SM species in dairy products, including bovine-derived whole milk, half and half, whipping cream, and goat milk.

Alteration in Plasma Metabolome in High-Fat Diet-Fed Monocyte Chemotactic Protein-1 Knockout Mice Bearing Pulmonary Metastases of Lewis Lung Carcinoma.

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.

Metabolomes of Lewis lung carcinoma metastases and normal lung tissue from mice fed different diets.

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.

Leptin Receptor Deficiency Results in Hyperphagia and Increased Fatty Acid Mobilization during Fasting in Rainbow Trout (Oncorhynchus mykiss).

Leptin is a pleiotropic hormone known for regulating appetite and metabolism. To characterize the role of leptin signaling in rainbow trout, we used CRISPR/Cas9 genome editing to disrupt the leptin receptor (LepR) genes, lepra1 and lepra2. We compared wildtype (WT) and mutant fish that were either fed to satiation or feed deprived for six weeks. The LepR mutants exhibited a hyperphagic phenotype, which led to heavier body weight, faster specific growth rate, increased viscero- and hepatosomatic indices, and greater condition factor. Muscle glycogen, plasma leptin, and leptin transcripts (lepa1) were also elevated in fed LepR mutant fish. Expression levels of several hypothalamic genes involved in feed regulation were analyzed (agrp, npy, orexin, cart-1, cart-2, pomc-a1, pomc-b). No differences were detected between fed WT and mutants except for pomc-b (proopiomelanocortin-b), where levels were 7.5-fold higher in LepR fed mutants, suggesting that pomc-b expression is regulated by leptin signaling. Fatty acid (FA) content did not statistically differ in muscle of fed mutant fish compared to WT. However, fasted mutants exhibited significantly lower muscle FA concentrations, suggesting that LepR mutants exhibit increased FA mobilization during fasting. These data demonstrate a key role for leptin signaling in lipid and energy mobilization in a teleost fish.

Identification of High and Low Branched-Chain Fatty Acid-Producing Phenotypes in Holstein Cows following High-Forage and Low-Forage Diets in a Crossover Designed Trial.

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.

Mammary Tumorigenesis and Metabolome in Male Adipose Specific Monocyte Chemotactic Protein-1 Deficient MMTV-PyMT Mice Fed a High-Fat Diet.

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.

Dietary Fat Chain Length, Saturation, and PUFA Source Acutely Affect Diet-Induced Thermogenesis but Not Satiety in Adults in a Randomized, Crossover Trial.

Structural differences in dietary fatty acids modify their rate of oxidation and effect on satiety, endpoints that may influence the development of obesity. This study tests the hypothesis that meals containing fat sources with elevated unsaturated fats will result in greater postprandial energy expenditure, fat oxidation, and satiety than meals containing fats with greater saturation. In a randomized, 5-way crossover design, healthy men and women (n = 23; age: 25.7 ± 6.6 years; BMI: 27.7 ± 3.8 kg/m2) consumed liquid meals containing 30 g of fat from heavy cream (HC), olive oil (OO), sunflower oil (SFO), flaxseed oil (FSO), and fish oil (FO). Energy expenditure and diet-induced thermogenesis (DIT) were determined by metabolic rate over a 240 min postprandial period. Serum concentrations of ghrelin, glucose, insulin, and triacylglycerol (TAG) were assessed. DIT induced by SFO was 5% lower than HC and FO (p = 0.04). Energy expenditure and substrate oxidation did not differ between fat sources. Postprandial TAG concentrations were significantly affected by fat source (p = 0.0001). Varying fat sources by the degree of saturation and PUFA type modified DIT but not satiety responses in normal to obese adult men and women.

Simple, Rapid Lipidomic Analysis of Triacylglycerols in Bovine Milk by Infusion-Electrospray Mass Spectrometry.

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.

Hepatic Fatty Acid and Transcriptome Profiles during the Transition from Vegetable- to Fish Oil-Based Diets in Rainbow Trout (Oncorhynchus mykiss).

A finishing diet strategy is effective at increasing fillet long-chain n-3 fatty acid content in fish consuming sustainable plant oil-based diets. This study investigates the outcomes of a fish oil finishing diet upon the hepatic fatty acid and transcriptome profile in rainbow trout (Oncorhynchus mykiss). Fish were placed on one of three feeding treatments: (1) FO: a fish oil (FO) diet for 20 weeks, (2) VO/FO: a vegetable oil (VO) diet during weeks 1-12 then the FO diet for 8 weeks, or (3) VO/fd/FO: the VO diet between weeks 1-12, 2 weeks of feed deprivation, then the FO diet for 6 weeks. Hepatic fatty acid and transcriptome profiles were analyzed at week 12, 14, and 20. Hepatic fatty acid profiles at week 12 were similar to dietary profiles; transcriptomic analyses indicated 131 differentially regulated genes (DEG) between VO- and FO-fed fish, characterized by VO-induced up-regulation of cholesterol and long-chain fatty acyl-CoA synthesis and oxidation-reduction processes. At week 14, the hepatic fatty acid profile was similar between VO/FO and FO, although concentrations of 18:3n-3 remained higher in the VO/FO group. Thirty-three DEG were detected at week 14 with enrichment of genes associated with extracellular matrix assembly, supporting liver remodeling during the early finishing diet period. Only five DEG were detected at week 20 between VO/FO and FO. Collectively, these findings suggest that it takes several weeks for liver to reach a homeostatic state, even after the hepatic fatty acid equilibration following a finishing diet.

Time-restricted feeding mice a high-fat diet induces a unique lipidomic profile.

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.

Impact of beef consumption on saturated fat intake in the United States adult population: Insights from modeling the influences of bovine genetics and nutrition.

We sought to determine the impact of breed and finishing ration that reduces the saturated fat (SFA) content of beef on SFA intake (%E) in adults. Using National Health and Nutrition Examination Surveys (2001-2016), we replaced the current fatty acid profile of beef with that from two breeds (Angus, Wagyu) and three finishing rations (pasture, 15% flaxseed supplementation, 35% wet distiller's grain (WDG) supplementation). Dietary replacement levels in the model were 10%, 25%, 50%, 100%. Overall, men consumed more beef fat than women (12.0 g (11.6-12.4 g, 95%CI) and 6.6 g (6.4-6.9 g, 95%CI), respectively). The contribution of beef fat to SFA intake was 2.1%E (2.1-2.2%E, 95%CI) in men and 1.6%E (1.6-1.7%E, 95%CI) in women. SFA intake decreased with each increased replacement level for all beef types. At 100% replacement, SFA intake decreased 0.5% (Angus), 2.8% (Wagyu), 1.9% (pasture), 4.1% (flaxseed), 2.6% (WDG). Our findings demonstrate that breed and finishing ration that reduces the SFA content of beef can decrease population-level SFA intake.

Metabolome of Mammary Tumors Differs from Normal Mammary Glands But Is Not Altered by Time-restricted Feeding Under Obesogenic Conditions.

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.

Plasma Metabolomic Changes in Mice With Time-restricted Feeding-attenuated Spontaneous Metastasis of Lewis Lung Carcinoma.

BACKGROUND/AIM: Time-restricted feeding (TRF) during the dark phase of the day restores metabolic homeostasis in mice. MATERIALS AND METHODS: We performed untargeted metabolomic analysis on plasma from mice subjected to TRF that attenuates high-fat diet-enhanced spontaneous metastasis of Lewis lung carcinoma (LLC). RESULTS: Twenty-four of 152 identified metabolites differed among the four dietary groups (non-LLC-bearing mice fed the AIN93G diet and LLC-bearing mice fed the AIN93G, the high-fat diet (HFD), or TRF of the HFD). Component 1 of sparse partial least squares-discriminant analysis showed a clear separation between non-LLC-bearing and LLC-bearing mice. Major metabolites responsible for the changes were elevations in α-tocopherol, docosahexaenoic acid, cholesterol, dihydrocholestrol, isoleucine, leucine, and phenylalanine and decreases in lactic acid and pyruvic acid in LLC-bearing mice particularly those fed the HFD. Time-restricted feeding shifted the metabolic profile of LLC-bearing mice towards that of non-LLC-bearing controls. CONCLUSION: Time-restricted feeding improves metabolic profile of LLC-bearing mice.

Decreasing the Ratio of Dietary Linoleic to α-Linolenic Acid from 10 to 4 by Changing Only the Former Does Not Prevent Adiposity or Bone Deterioration in Obese Mice.

BACKGROUND: Linoleic acid (LA; 18:2n-6) has been considered to promote low-grade chronic inflammation and adiposity. Studies show adiposity and inflammation are inversely associated with bone mass. OBJECTIVES: This study tested the hypothesis that decreasing the dietary ratio of LA to α-linolenic acid (ALA, 18:3n-3), while keeping ALA constant, mitigates high-fat diet (HF)-induced adiposity and bone loss. METHODS: Male C57BL/6 mice at 6 wk old were assigned to 4 treatment groups and fed 1 of the following diets ad libitum for 6 mo: a normal-fat diet (NF; 3.85 kcal/g and 10% energy as fat) with the ratio of the PUFAs LA to ALA at 6; or HFs (4.73 kcal/g and 45% energy as fat) with the ratio of LA to ALA at 10:1, 7:1, or 4:1, respectively. ALA content in the diets was kept the same for all groups at 1% energy. Bone structure, body composition, bone-related cytokines in serum, and gene expression in bone were measured. Data were analyzed using 1-factor ANOVA. RESULTS: Compared with those fed the NF, mice fed the HFs had 19.6% higher fat mass (P < 0.01) and 13.5% higher concentration of serum tartrate-resistant acid phosphatase (TRAP) (P < 0.05), a bone resorption cytokine. Mice fed the HFs had 19.5% and 12.2% lower tibial and second lumbar vertebral bone mass, respectively (P < 0.01). Decreasing the dietary ratio of LA to ALA from 10 to 4 did not affect body mass, fat mass, serum TRAP and TNF-α, or any bone structural parameters. CONCLUSIONS: These data indicate that decreasing the dietary ratio of LA to ALA from 10 to 4 by simply reducing LA intake does not prevent adiposity or improve bone structure in obese mice.

Increasing Dietary Fish Oil Reduces Adiposity and Mitigates Bone Deterioration in Growing C57BL/6 Mice Fed a High-Fat Diet.

BACKGROUND: Intake of total fat is linked to obesity and inversely associated with bone density in humans. Epidemiologic and animal studies show that long-chain n-3 (ω-3) PUFAs supplied as fish oil (FO) are beneficial to skeletal health. OBJECTIVE: This study tested the hypothesis that increasing dietary FO would decrease adiposity and improve bone-related outcomes in growing obese mice. METHODS: Male C57BL/6 mice at 6 wk old were assigned to 6 treatment groups and fed either a normal-fat diet (3.85 kcal/g and 10% energy as fat) or a high-fat diet (HF; 4.73 kcal/g and 45% energy as fat) containing either 0%, 3%, or 9% energy as FO (0FO, 3FO, and 9FO, respectively) ad libitum for 6 mo. Bone structure, body composition, and serum bone-related cytokines were measured. RESULTS: The HF diet increased the expression of the adipose tissue tumor necrosis factor α (Tnfa) and serum concentrations of leptin and tartrate-resistant acid phosphatase (TRAP), and decreased serum concentrations of osteocalcin and bone-specific alkaline phosphatase (P < 0.05). FO decreased fat mass (P < 0.05), serum TRAP (P < 0.05), and adipose tissue Tnfa expression (P < 0.01). Bone content of long-chain n-3 PUFAs was increased and n-6 PUFAs were decreased with the elevation in dietary FO content (P < 0.01). Compared with mice fed 9FO, animals fed 3FO had higher femoral bone volume/total volume (25%), trabecular number (23%), connectivity density (82%), and bone mass of second lumbar vertebrae (12%) and lower femoral trabecular separation (-19%). Mice fed the 3FO HF diet had 42% higher bone mass than those fed the 0FO HF diet. CONCLUSIONS: These data indicate increasing dietary FO ≤3% energy can decrease adiposity and mitigate HF diet-induced bone deterioration in growing C57BL/6 mice possibly by reducing inflammation and bone resorption. FO at 9% diet energy had no further beneficial effects on bone of obese mice.

Simplified Mass Spectrometric Analysis of Ceramides using a Common Collision Energy.

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.

Quantitation of Glutathione, Glutathione Disulphide, and Protein-Glutathione Mixed Disulphides by High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

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.