David B. Ebenstein

Substituting dietary monounsaturated fat for saturated fat is associated with increased daily physical activity and resting energy expenditure and with changes in mood

BACKGROUND The Western diet increases risk of metabolic disease. OBJECTIVE We determined whether lowering the ratio of saturated fatty acids to monounsaturated fatty acids in the Western diet would affect physical activity and energy expenditure. DESIGN With the use of a balanced design, 2 cohorts of 18 and 14 young adults were enrolled in separate randomized, double-masked, crossover trials that compared a 3-wk high-palmitic acid diet (HPA; similar to the Western diet fat composition) to a low-palmitic acid and high-oleic acid diet (HOA; similar to the Mediterranean diet fat composition). All foods were provided by the investigators, and the palmitic acid (PA):oleic acid (OA) ratio was manipulated by adding different oil blends to the same foods. In both cohorts, we assessed physical activity (monitored continuously by using accelerometry) and resting energy expenditure (REE). To gain insight into a possible mood disturbance that might explain changes in physical activity, the Profile of Mood States (POMS) was administered in cohort 2. RESULTS Physical activity was higher during the HOA than during the HPA in 15 of 17 subjects in cohort 1 (P = 0.008) (mean: 12% higher; P = 0.003) and in 12 of 12 subjects in the second, confirmatory cohort (P = 0.005) (mean: 15% higher; P = 0.003). When the HOA was compared with the HPA, REE measured during the fed state was 3% higher for cohort 1 (P < 0.01), and REE was 4.5% higher in the fasted state for cohort 2 (P = 0.04). POMS testing showed that the anger-hostility score was significantly higher during the HPA (P = 0.007). CONCLUSIONS The replacement of dietary PA with OA was associated with increased physical activity and REE and less anger. Besides presumed effects on mitochondrial function (increased REE), the dietary PA:OA ratio appears to affect behavior. The second cohort was derived from a study that was registered at clinicaltrials.gov as R01DK082803.

Determination of steady-state protein breakdown rate in vivo by the disappearance of protein-bound tracer-labeled amino acids: a method applicable in humans

A method to determine the rate of protein breakdown in individual proteins was developed and tested in rats and confirmed in humans, using administration of deuterium oxide and incorporation of the deuterium into alanine that was subsequently incorporated into body proteins. Measurement of the fractional breakdown rate of proteins was determined from the rate of disappearance of deuterated alanine from the proteins. The rate of disappearance of deuterated alanine from the proteins was calculated using an exponential decay, giving the fractional breakdown rate (FBR) of the proteins. The applicability of this protein-specific FBR approach is suitable for human in vivo experimentation. The labeling period of deuterium oxide administration is dependent on the turnover rate of the protein of interest.

Characterization of the bovine milk proteome in early-lactation Holstein and Jersey breeds of dairy cows

UNLABELLED Milk is a highly nutritious natural product that provides not only a rich source of amino acids to the consumer but also hundreds of bioactive peptides and proteins known to elicit health-benefitting activities. We investigated the milk protein profile produced by Holstein and Jersey dairy cows maintained under the same diet, management and environmental conditions using proteomic approaches that optimize protein extraction and characterization of the low abundance proteins within the skim milk fraction of bovine milk. In total, 935 low abundance proteins were identified. Gene ontology classified all proteins identified into various cellular localization and function categories. A total of 43 low abundance proteins were differentially expressed between the two dairy breeds. Bioactive proteins involved in host-defense, including lactotransferrin (P=0.0026) and complement C2 protein (P=0.0001), were differentially expressed by the two breeds, whereas others such as osteopontin (P=0.1788) and lactoperoxidase (P=0.2973) were not. This work is the first to outline the protein profile produced by two important breeds of dairy cattle maintained under the same diet, environment and management conditions in order to observe likely true breed differences. This research now allows us to better understand and contrast further research examining the bovine proteome that includes these different breeds. BIOLOGICAL SIGNIFICANCE Within the last decade, the amount of research characterizing the bovine milk proteome has increased due to growing interest in the bioactive proteins that are present in milk. Proteomic analysis of low abundance whey proteins has mainly focused on human breast milk; however, previous research has highlighted the presence of bioactive proteins in bovine milk. Recent publications outlining the cross-reactivity of bovine bioactive proteins on human biological function highlight the need for further investigation into the bovine milk proteome. The rationale behind this study is to characterize and compare the low abundance protein profile in the skim milk fraction produced from Holstein and Jersey breeds of dairy cattle, which are two major dairy cattle breeds in the USA. A combination of fractionation strategies was used to efficiently enrich the low abundance proteins from bovine skim milk for proteomic profiling. A total of 935 low abundance proteins were identified and compared between the two bovine breeds. The results from this study provide insight into breed differences and similarities in the milk proteome profile produced by two breeds of dairy cattle.

High-performance liquid chromatographic technique for non-derivatized leucine purification: evidence for carbon isotope fractionation.

A high-performance liquid chromatographic (HPLC) method, utilizing an ion-pairing agent with a reversed-phase column, isocratic elution and ultraviolet detection, was developed for the separation of non-derivatized leucine from a mixture of amino acids. Fractionation of leucine isotopes during HPLC separation was observed, creating potential problems for collection of leucine plasma or tissue protein hydrolyzates for subsequent isotopic determinations. It was demonstrated that [13C]leucine and [12C]leucine have different retention times (p less than 0.01). It is concluded from this study that partial collection of leucine eluted from an HPLC column will result in erroneous estimation of the isotopic enrichment of leucine.

Lipidomic evidence that lowering the typical dietary palmitate to oleate ratio in humans decreases the leukocyte production of proinflammatory cytokines and muscle expression of redox-sensitive genes ☆

We recently reported that lowering the high, habitual palmitic acid (PA) intake in ovulating women improved insulin sensitivity and both inflammatory and oxidative stress. In vitro studies indicate that PA can activate both cell membrane toll-like receptor-4 and the intracellular nucleotide oligomerization domain-like receptor protein (NLRP3). To gain further insight into the relevance to human metabolic disease of dietary PA, we studied healthy, lean and obese adults enrolled in a randomized, crossover trial comparing 3-week, high-PA (HPA) and low-PA/high-oleic-acid (HOA) diets. After each diet, both hepatic and peripheral insulin sensitivities were measured, and we assessed cytokine concentrations in plasma and in supernatants derived from lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMCs) as well as proinflammatory gene expression in skeletal muscle. Insulin sensitivity was unaffected by diet. Plasma concentration of tumor necrosis factor-α was higher during the HPA diet. Lowering the habitually high PA intake by feeding the HOA diet resulted in lower secretion of interleukin (IL)-1β, IL-18, IL-10, and tumor necrosis factor-α by PBMCs, as well as lower relative mRNA expression of cJun and NLRP3 in muscle. Principal components analysis of 156 total variables coupled to analysis of covariance indicated that the mechanistic pathway for the differential dietary effects on PBMCs involved changes in the PA/OA ratio of tissue lipids. Our results indicate that lowering the dietary and tissue lipid PA/OA ratio resulted in lower leukocyte production of proinflammatory cytokines and muscle expression of redox-sensitive genes, but the relevance to diabetes risk is uncertain.

Dietary saturated fat and monounsaturated fat have reversible effects on brain function and the secretion of pro-inflammatory cytokines in young women

BACKGROUND Previous literature suggests that a higher ratio of palmitic acid (PA)/oleic acid (OA) in the diet induces inflammation, which may result in deficient brain insulin signaling, and, secondarily, impaired physical activity, sleep efficiency, and cognitive functioning. OBJECTIVE We hypothesized that lowering the typical dietary PA/OA would affect the activation of relevant brain networks during a working memory task and would also lower secretion of pro-inflammatory cytokines. DESIGN In 12 female subjects participating in a randomized, cross-over trial comparing 3-week high PA diet (HPA) and low PA and a high OA diet (HOA), we evaluated functional magnetic resonance imaging (fMRI) using an N-back test of working memory, cytokine secretion by lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC), and plasma cytokine concentrations. RESULTS Brain activation during the HPA diet compared to the HOA diet was increased in regions of the basal ganglia including the caudate and putamen (p<0.005). In addition, compared to the HOA diet, during the HPA diet, the plasma concentrations of IL-6 (p=0.04) and IL-1β (p=0.05) were higher, and there was a higher secretion of IL-18 (p=0.015) and a trend for higher IL-1β secretion (p=0.066) from LPS-stimulated PBMCs. CONCLUSIONS The HPA diet resulted in increased brain activation in the basal ganglia compared to the HOA diet as well as increased secretion of pro-inflammatory cytokines. These data provide evidence that short-term (2week) diet interventions impact brain network activation during a working memory task and that these effects are reversible since the order of the study diets was randomized. These data are consistent with the hypothesis that lowering the dietary PA content via substitution with OA also could affect cognition.

Increased palmitate intake: higher acylcarnitine concentrations without impaired progression of β-oxidation.

Palmitic acid (PA) is associated with higher blood concentrations of medium-chain acylcarnitines (MCACs), and we hypothesized that PA may inhibit progression of FA β-oxidation. Using a cross-over design, 17 adults were fed high PA (HPA) and low PA/high oleic acid (HOA) diets, each for 3 weeks. The [1-13C]PA and [13-13C]PA tracers were administered with food in random order with each diet, and we assessed PA oxidation (PA OX) and serum AC concentration to determine whether a higher PA intake promoted incomplete PA OX. Dietary PA was completely oxidized during the HOA diet, but only about 40% was oxidized during the HPA diet. The [13-13C]PA/[1-13C]PA ratio of PA OX had an approximate value of 1.0 for either diet, but the ratio of the serum concentrations of MCACs to long-chain ACs (LCACs) was significantly higher during the HPA diet. Thus, direct measurement of PA OX did not confirm that the HPA diet caused incomplete PA OX, despite the modest, but statistically significant, increase in the ratio of MCACs to LCACs in blood.

Ratio of dietary rumen degradable protein to rumen undegradable protein affects nitrogen partitioning but does not affect the bovine milk proteome produced by mid-lactation Holstein dairy cows

Little is known about the bovine milk proteome or whether it can be affected by diet. The objective of this study was to determine if the dietary rumen degradable protein (RDP):rumen undegradable protein (RUP) ra-tio could alter the bovine milk proteome. Six Holstein cows (parity: 2.5 ± 0.8) in mid lactation were blocked by days in milk (80 ± 43 d in milk) and milk yield (57.5 ± 6.0 kg) and randomly assigned to treatment groups. The experiment was conducted as a double-crossover design consisting of three 21-d periods. Within each period, treatment groups received diets with either (1) a high RDP:RUP ratio (RDP treatment: 62.4:37.6% of crude protein) or (2) a low RDP:RUP ratio (RUP treatment: 51.3:48.7% of crude protein). Both diets were isonitrogenous and isoenergetic (crude protein: 18.5%, net energy for lactation: 1.8 Mcal/kg of dry matter). To confirm N and energy status of cows, dry matter intake was determined daily, rumen fluid samples were collected for volatile fatty acid analysis, blood samples were collected for plasma glucose, β-hydroxybutyrate, urea nitrogen, and fatty acid analysis, and total 24-h urine and fecal samples were collected for N analysis. Milk samples were collected to determine the general milk composition and the protein profile. Milk samples collected for high-abundance protein analysis were subjected to HPLC analysis to determine the content of α-casein, β-casein, and κ-casein, as well as α-lactalbumin and β-lactoglobulin. Samples collected for low-abundance protein analysis were fractionated, enriched using ProteoMiner treatment, and separated using sodium dodecyl sulfate-PAGE. After excision and digestion, the peptides were analyzed using liquid chromatography (LC) tandem mass spectrometry (MS/MS). The LC-MS/MS data were analyzed using PROC GLIMMIX of SAS (version 9.4, SAS Institute Inc., Cary, NC) and adjusted using the MULTTEST procedure. All other parameters were analyzed using PROC MIXED of SAS. No treatment differences were observed in dry matter intake, milk yield, general milk composition, plasma parameters, or rumen volatile fatty acid concentrations, indicating no shift in total energy or protein available. Milk urea N and plasma urea N concentrations were higher in the RDP group, indicating some shift in N partitioning due to diet. A total of 595 milk proteins were identified, with 83% of these proteins known to be involved in cellular processes. Although none of the low-abundance proteins identified by LC-MS/MS were affected by diet, feeding a diet high in RUP decreased β-casein, κ-casein, and total milk casein concentration. Further investigations of the interactions between diet and the milk protein profile are needed to manipulate the milk proteome using diet.

Exploration of the bovine colostrum proteome and effects of heat treatment time on colostrum protein profile

Heat treatment of colostrum is performed on modern dairy farms to reduce pathogenic contamination before hand-feeding the colostrum to newborn calves; however, limited data are available concerning effects of heat treatment on biologically active proteins in colostrum. The objective of this exploratory study was to investigate effects of heat treatment and length of heat treatment on colostrum protein profile. Colostrum samples were collected from Holstein cows within 12 h after parturition and assigned to the following groups: heat treatment at 60°C for 0 (untreated control), 30, 60, or 90 min. Samples were fractionated using acid precipitation, followed by ultracentrifugation and ProteoMiner (Bio-Rad Laboratories, Hercules, CA) treatment, and tandem-mass tagging was used to comparatively assess the low abundance protein profile. A total of 162 proteins were identified with more than 2 peptides in the low abundance protein enriched fraction. Of these, 62 differed in abundance by more than 2-fold in heat treated samples compared with the unheated control. The majority of proteins affected by heat treatment were involved in immunity, enzyme function, and transport-related processes; affected proteins included lactadherin, chitinase-3-like protein 1, and complement component C9. These results provide a foundation for further research to determine optimum heat treatment practices to ensure newborn calves are fed colostrum-containing proteins with the highest nutritional and biological value.

Comparative proteomics dataset of skimmed milk samples from Holstein and Jersey dairy cattle.

Milk samples were collected from Holstein and Jersey breeds of dairy cattle maintained under the same management practices and environmental conditions over a seven-day period. Milk samples were collected twice daily from six cows of each breed as previously described (Tacoma et al., 2016) [1]. Samples were composited within individual cow over the experimental period and skimmed to remove the fat layer. Skimmed milk samples were fractionated using CaCl2 precipitation, ultracentrifugation and ProteoMiner treatment to remove the high abundance milk proteins. Separation of the low abundance proteins was achieved using SDS-PAGE. Differential protein abundances were analyzed by mass spectrometry-based proteomic approaches followed by statistical analyses of the peptide count data. The complete list of low-abundance proteins identified in both breeds is provided in the dataset as well as the total number of distinct sequenced peptides and gene ontology functions for each protein. The relative abundance of a select few proteins is depicted using the SIEVE software.