Matthew J. Picklo

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.

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. 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. 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. 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. 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.

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.

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.

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.

The Agricultural Research Service (ARS) Beef Grand Challenge is a cooperative, multidisciplinary effort evaluating differences in performance of genetic lines across production environments representative of different geographical regions. Weaned spring-born calves (n = 120 per location), representing natural service matings to Angus, Hereford, Simmental, Charolais, or indicus-composite (Beefmaster or Brangus) bulls from the U.S. Meat Animal Research Center Germplasm Evaluation program in south central Nebraska, are sent to wheat pasture (central Oklahoma) and winter range (eastern Montana), and weaned fall-born calves (n=40 per location) are sent to summer grazing on shortgrass prairie (northeastern Colorado) and southern mixed-grass rangeland (western Oklahoma). All cattle are fed a finishing ration representative of the region that approximately matches energy content across locations. Each calving season has a matching counterpart of calves that remain in Nebraska on a calf-fed drylot program (receiving ration followed by longer finishing ration). Breeds and sires are represented equally, to the extent possible, at each location. To detect differences in breed effects at each location and average over yearly variation, the study is being replicated for 4 years. Weights, stress measures, carcass composition (marbling, yield grade, quality grade, etc.), steak tenderness and steak fatty acid composition are collected from each location. Additionally, rumen metagenomic composition, metagenomic samples, preharvest food safety samples, and feed intake measures are collected at some locations. Grazing impacts and supplemental range feeding are also being evaluated. One year of sampling has been completed, with numeric differences observed for marbling and tenderness as well as growth performance among locations. Statistical differences will be evaluated when replicate years are collected. The USDA is an equal opportunity provider and employer.

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.

Beef, while a prominent source of high-quality protein in the American diet, is a source of saturated fat. Beef production practices, such as breed selection and nutrition regimens, can influence the nutritional value of meat by reducing the saturated fatty acids (SFA) content. Thus, we sought to determine the extent to which implementing these practices reduces population-level SFA intake to meet the Dietary Guideline for Americans (DGA) recommendation of ≤10% en. Using reported intakes from the National Health and Nutrition Examination Survey 2001–2016, we determined the contribution of food types to total SFA intake. From these data, nutritional modeling methods were used in which the current fatty acid profile of beef was replaced with that from two different breeds (Angus, the predominant breed in the U.S., and Wagyu, a breed with lower SFA content) and three nutrition regimens (pasture-raised, 15% flaxseed supplementation, and 35% wet distiller's grain (WDG) supplementation) to estimate the contribution of each beef type to total SFA intake in the U.S. adult population. Four levels of replacement were compared - 10%, 25%, 50%, 100%. Daily per capita SFA intake was 10.9% en (10.8–11.0% en, 95% CI). Beef was the third greatest contributor (15%) to per capita SFA intake, behind grain (29%) and dairy (19%) dishes. In general, men consumed more beef fat than women (12.0 g vs. 6.6 g, respectively). SFA intake, as a percentage of total energy intake (% en), from beef was 2.1% en (2.1–2.2% en, 95% CI) in men and 1.6% en (1.6–1.7% en, 95% CI) in women. For each modeled beef, SFA intake decreased with each increase in replacement level. At 100% replacement, SFA intake was reduced by 0.5% (Angus), 2.8% (Wagyu), 1.9% (pasture-raised), 4.1% (15% flaxseed), and 2.6% (35% WDG). Data from this modeling study indicate that decreasing the SFA content of beef through breed selection and nutrition regimens can result in a per capita reduction in SFA intake in the American population. However, modeled SFA consumption remained greater (c. 10.5–10.7% en) than DGA recommended intakes in all age and gender groups. These results demonstrate the importance for Americans to reduce SFA intake from all sources, including beef, to meet daily intake recommendations. United States Department of Agriculture, Agricultural Research Service.