Dietary Macronutrients and Circulating Nonesterified Fatty Acids: A Secondary Analysis of the OMNI Heart Crossover Trial.

BACKGROUND: Nonesterified fatty acids (NEFAs) play key roles in the pathophysiology of diabetes and cardiovascular disease. OBJECTIVES: We sought to determine whether macronutrient content differences affect NEFA concentrations in a randomized crossover trial. METHODS: Total NEFAs were measured from postintervention specimens of participants in the OMNI Heart trial (Optimal Macronutrient Intake Trial to Prevent Heart Disease). OMNI Heart compared 3 healthful diets to evaluate their effect on systolic blood pressure and serum LDL cholesterol: carbohydrate-rich diet (58% carbohydrate); protein-rich diet (25% protein), about half from plant sources; and a diet rich in unsaturated fatty acids (21% unsaturated fat), predominantly monounsaturated. The trial included 164 participants who consumed the 3 diets, each for 6 wk. Data were analyzed from the 156 participants with unthawed serum available from the week 6 visit for all diet periods. We used ANCOVA and generalized estimating equations (GEEs) to compare serum NEFA concentrations across the 3 diet periods. RESULTS: The mean ± SD age of study participants was 52.9 ± 10.6 y and mean BMI was 30.3 ± 6.1 kg/m2. Fifty-five percent of participants were women and 55% were African American. Comparisons of adjusted mean serum NEFA concentrations after each diet intervention identified no statistically significant differences (58% carbohydrate: 0.144 ± 0.083 mEq/L; 25% protein: 0.143 ± 0.076 mEq/L; 21% unsaturated fat: 0.143 ± 0.084 mEq/L; ANCOVA, P = 0.99). Likewise, we observed no significant serum NEFA concentration difference by diet in adjusted GEE models. In adjusted models, serum NEFA concentrations were positively associated, as anticipated, with female sex and higher BMI. CONCLUSIONS: In this randomized crossover trial, we observed nearly identical serum NEFA concentrations after each of 3 healthful diets, regardless of macronutrient content.

AdipoRon alleviates fatty acid-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes by promoting autophagy.

During the transition period in dairy cows, high circulating concentrations of nonesterified fatty acids (NEFA) increase hepatic lipid deposits and are considered a major pathological factor for liver damage. We investigated whether AdipoRon, a synthetic small-molecule agonist of adiponectin receptors 1 and 2 shown to prevent liver lipid accumulation in nonruminants, could alleviate NEFA-induced lipid accumulation and mitochondrial dysfunction. Bovine hepatocytes were isolated from 5 healthy Holstein female newborn calves (1 d of age, 30-40 kg, fasting), and independently isolated hepatocytes from at least 3 different calves were used for each subsequent experiment. The composition and concentration of NEFA used in this study were selected according to hematological criteria of dairy cows with fatty liver or ketosis. First, hepatocytes were cultured with various concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 12 h. In a second experiment, hepatocytes were treated with AdipoRon at different concentrations (0, 5, 25, or 50 µM for 12 h) and times (25 µM for 0, 6, 12, or 24 h) with or without NEFA (1.2 mM) treatment. In the last experiment, hepatocytes were treated with AdipoRon (25 µM), NEFA (1.2 mM), or both for 12 h after treatment with or without the autophagy inhibitor chloroquine. Hepatocytes treated with NEFA had increased protein abundance of sterol regulatory element-binding protein 1c (SREBP-1c) and mRNA abundance of acetyl-CoA carboxylase 1 (ACACA), and decreased protein abundance of peroxisome proliferator-activated receptor α (PPARA), proliferator-activated receptor gamma coactivator-1 α (PGC-1α), mitofusin 2 (MFN2), cytochrome c oxidase subunit IV (COX IV), and mRNA abundance of carnitine palmitoyltransferase 1A (CPT1A), along with lower ATP concentrations. AdipoRon treatment reversed these effects, suggesting this compound had a positive effect on lipid metabolism and mitochondrial dysfunction during the NEFA challenge. In addition, upregulated expression of microtubule-associated protein 1 light chain 3-II (LC3-II, encoded by MAP1LC3) and downregulated expression of sequestosome-1 (SQSTM1, also called p62) indicated that AdipoRon enhanced autophagic activity in hepatocytes. The fact that chloroquine impeded the beneficial effects of AdipoRon on lipid accumulation and mitochondrial dysfunction suggested a direct role for autophagy during NEFA challenge. Our results suggest that autophagy is an important cellular mechanism to prevent NEFA-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes, which is consistent with other studies. Overall, AdipoRon may represent a promising therapeutic agent to maintain hepatic lipid homeostasis and mitochondrial function in dairy cows during the transition period.

Free fatty acid treatment of mouse preimplantation embryos demonstrates contrasting effects of palmitic acid and oleic acid on autophagy.

Treatment of mouse preimplantation embryos with elevated palmitic acid (PA) reduces blastocyst development, whereas cotreatment with PA and oleic acid (OA) together rescues blastocyst development to control frequencies. To understand the mechanistic effects of PA and OA treatment on early mouse embryos, we investigated the effects of PA and OA, alone and in combination, on autophagy during preimplantation development in vitro. We hypothesized that PA would alter autophagic processes and that OA cotreatment would restore control levels of autophagy. Two-cell stage mouse embryos were placed into culture medium supplemented with 100 µM PA, 250 µM OA, 100 µM PA and 250 µM OA, or potassium simplex optimization media with amino acid (KSOMaa) medium alone (control) for 18-48 h. The results demonstrated that OA cotreatment slowed developmental progression after 30 h of cotreatment but restored control blastocyst frequencies by 48 h. PA treatment elevated light chain 3 (LC3)-II puncta and p62 levels per cell whereas OA cotreatment returned to control levels of autophagy by 48 h. Autophagic mechanisms are altered by nonesterified fatty acid (NEFA) treatments during mouse preimplantation development in vitro, where PA elevates autophagosome formation and reduces autophagosome degradation levels, whereas cotreatment with OA reversed these PA effects. Autophagosome-lysosome colocalization only differed between PA and OA alone treatment groups. These findings advance our understanding of the effects of free fatty acid exposure on preimplantation development, and they uncover principles that may underlie the associations between elevated fatty acid levels and overall declines in reproductive fertility.

Lactation alters the relationship between liver lipid synthesis and hepatic fat stores in the postpartum period.

In mothers who are nursing their infants, increased clearance of plasma metabolites into the mammary gland may reduce ectopic lipid in the liver. No study to date has investigated the role of lactation on liver lipid synthesis in humans, and we hypothesized that lactation would modify fatty acid and glucose handling to support liver metabolism in a manner synchronized with the demands of milk production. Lactating (n = 18) and formula-feeding women (n = 10) underwent metabolic testing at 6-week postpartum to determine whether lactation modified intrahepatic triacylglycerols (IHTGs), measured by proton magnetic resonance spectroscopy. Subjects ingested oral deuterated water to measure fractional de novo lipogenesis (DNL) in VLDL-TG during fasting and during an isotope-labeled clamp at an insulin infusion rate of 10 mU/m2/min. Compared with formula-feeding women, we found that lactating women exhibited lower plasma VLDL-TG concentrations, similar IHTG content and similar contribution of DNL to total VLDL-TG production. These findings suggest that lactation lowers plasma VLDL-TG concentrations for reasons that are unrelated to IHTG and DNL. Surprisingly, we determined that the rate of appearance of nonesterified fatty acids was not related to IHTG in either group, and the expected positive association between DNL and IHTG was only significant in formula-feeding women. Further, in lactating women only, the higher the prolactin concentration, the lower the IHTG, while greater DNL strongly associated with elevations in VLDL-TG. In conclusion, we suggest that future studies should investigate the role of lactation and prolactin in liver lipid secretion and metabolism.

Fasting and Postload Nonesterified Fatty Acids and Glucose Dysregulation in Older Adults.

To evaluate the association of nonesterified fatty acids (NEFA) with dysglycemia in older adults, NEFA levels were measured among participants in the Cardiovascular Health Study (United States; enrolled 1989-1993). Associations with insulin sensitivity and pancreatic ß-cell function, and with incident type 2 diabetes mellitus (DM), were examined. The sample comprised 2,144 participants (aged 77.9 (standard deviation, 4.5) years). Participant data from the Cardiovascular Health Study visit in 1996-1997 was used with prospective follow-up through 2010. Fasting and postload NEFA showed significant associations with lower insulin sensitivity and pancreatic ß-cell function, individually and on concurrent adjustment. Over median follow-up of 9.7 years, 236 cases of DM occurred. Postload NEFA were associated with risk of DM (per standard deviation, hazard ratio = 1.18, 95% confidence interval: 1.08, 1.29), but fasting NEFA were not (hazard ratio = 1.12, 95% confidence interval: 0.97, 1.29). The association for postload NEFA persisted after adjustment for putative intermediates, and after adjustment for fasting NEFA. Sex and body mass index modified these associations, which were stronger for fasting NEFA with DM in men but were accentuated for postload NEFA in women and among leaner individuals. Fasting and postload NEFA were related to lower insulin sensitivity and pancreatic ß-cell function, but only postload NEFA were associated with increased DM. Additional study into NEFA metabolism could uncover novel potential targets for diabetes prevention in elders.

Effect of pegbovigrastim on fertility and culling in grazing dairy cows and its association with prepartum nonesterified fatty acids.

This randomized controlled trial on 4 commercial grazing dairy farms investigated whether treatment with pegbovigrastim (PEG) affected fertility and culling as measured during the full lactation. We also explored the effect of potential interactions of PEG treatment with parity, prepartum body condition score, prepartum nonesterified fatty acid concentration (pre-NEFA), and early-lactation clinical disease on these outcomes. Holstein cows were randomly assigned to 1 of 2 trial arms: a first PEG dose approximately 7 d before the expected calving date and a second dose within 24 h after calving (PEG: primiparous = 342; multiparous = 697) compared with untreated controls (control: primiparous = 391; multiparous = 723). Cox's proportional hazards regression models were used to analyze rate of first insemination, rate of pregnancy [within 150 and 305 d in milk (DIM)], and hazard of culling. Additional analyses were performed on data that were stratified by parity group and pre-NEFA class (low ≤0.3; high >0.3 mM). In high pre-NEFA cows, PEG treatment increased the rate of first insemination [hazard ratio (HR) = 1.15]. Early-lactation clinical mastitis (CM) and uterine disease (UD: retained placenta, metritis, or both) were associated with a reduced rate of pregnancy within 150 DIM (HR = 0.49 and 0.78, respectively). Pegbovigrastim treatment in high pre-NEFA cows with CM and UD increased the rate of pregnancy within 150 DIM (HR = 1.75 and 1.46, respectively). In high pre-NEFA cows, PEG treatment resulted in a lower hazard of culling (HR = 0.79). No treatment effect was detected in low pre-NEFA cows. This study shows that the effect of PEG treatment on fertility and culling interacts with pre-NEFA. In high pre-NEFA cows, PEG treatment increased the rate of first insemination, counteracted the negative association of early-lactation CM and UD with the rate of pregnancy, and decreased the hazard of culling.

Metabolic and antioxidant status during transition is associated with changes in the granulosa cell transcriptome in the preovulatory follicle in high-producing dairy cows at the time of breeding.

In this study, we hypothesized that early postpartum (pp) metabolic and oxidative stress conditions in dairy cows (particularly those with severe negative energy balance, NEB) are associated with long-term changes in granulosa cell (GC) functions in the preovulatory follicle at the time of breeding. Blood samples were collected at wk 2 and wk 8 pp from 47 healthy multiparous cows. Follicular fluid (FF) and GC were collected from the preovulatory follicle after estrous synchronization at wk 8. Several metabolic and antioxidant parameters were measured in blood and FF, and their correlations were studied. Subsequently, 27 representative GC samples were selected for RNA sequencing analysis. The GC gene expression data of LH-responsive genes and the estradiol:progesterone ratio in FF were used to identify pre- and post-LH surge cohorts. We compared the transcriptomic profile of subgroups of cows within the highest and lowest quartiles (Q4 vs. Q1) of each parameter, focusing on the pre-LH surge cohort (n = 16, at least 3 in each subgroup). Differentially expressed genes (DEG: adjusted P-value < 0.05, 5% false discovery rate) were determined using DESeq2 analysis and were functionally annotated. Blood and FF ß-carotene and vitamin E concentrations at wk 2, but not at wk 8, were associated with the most pronounced transcriptomic differences in the GC, with up to 341 DEG indicative for lower catabolism, increased oxidoreductase activity and signaling cascades that are known to enhance oocyte developmental competence, increased responsiveness to LH, and a higher steroidogenic activity. In contrast, elevated blood NEFA concentrations at wk 2 (and not at wk 8) were associated with a long-term carryover effect detectable in the GC transcriptome at wk 8 (64 DEG). These genes are related to response to lipids and ketones, oxidative stress, and immune responses, which suggests persistent cellular stress and oxidative damage. This effect was more pronounced in cows with antioxidant deficiencies at wk 8 (up to 148 DEG), with more genes involved in oxidative stress-dependent responses, apoptosis, autophagy and catabolic processes, and mitochondrial damage. Interestingly, within the severe NEB cows (high blood NEFA at wk 2), blood antioxidant concentrations (high vs. low) at wk 8 were associated with up to 194 DEG involved in activation of meiosis and other signaling pathways, indicating a better oocyte supportive capacity. This suggests that the cow antioxidant profile at the time of breeding might alleviate, at least in part, the effect of NEB on GC functions. In conclusion, these results provide further evidence that the metabolic and oxidative stress in dairy cows early postpartum can have long-term effects on GC functions in preovulatory follicles at the time of breeding. The interplay between the effects of antioxidants and NEFA illustrated here might be useful to develop intervention strategies to minimize the effect of severe NEB on fertility.

Increased postprandial nonesterified fatty acid efflux from adipose tissue in prediabetes is offset by enhanced dietary fatty acid adipose trapping.

The mechanism of increased postprandial nonesterified fatty acid (NEFA) appearance in the circulation in impaired glucose tolerance (IGT) is due to increased adipose tissue lipolysis but could also be contributed to by reduced adipose tissue (AT) dietary fatty acid (DFA) trapping and increased "spillover" into the circulation. Thirty-one subjects with IGT (14 women, 17 men) and 29 with normal glucose tolerance (NGT, 15 women, 14 men) underwent a meal test with oral and intravenous palmitate tracers and the oral [18F]-fluoro-thia-heptadecanoic acid positron emission tomography method. Postprandial palmitate appearance (Rapalmitate) was higher in IGT versus NGT (P < 0.001), driven exclusively by Rapalmitate from obesity-associated increase in intracellular lipolysis (P = 0.01), as Rapalmitate from DFA spillover was not different between the groups (P = 0.19) and visceral AT DFA trapping was even higher in IGT versus NGT (P = 0.02). Plasma glycerol appearance was lower in IGT (P = 0.01), driven down by insulin resistance and increased insulin secretion. Thus, we found higher AT DFA trapping, limiting spillover to lean organs and in part offsetting the increase in Rapalmitate from intracellular lipolysis. Whether similar findings occur in frank diabetes, a condition also characterized by insulin resistance but relative insulin deficiency, requires further investigation (Clinicaltrials.gov: NCT04088344, NCT02808182).NEW & NOTEWORTHY We found higher adipose tissue dietary fatty acid trapping, limiting spillover to lean organs, that in part offsets the increase in appearance rate of palmitate from intracellular lipolysis in prediabetes. These results point to the adaptive nature of adipose tissue trapping and dietary fatty acid spillover as a protective mechanism against excess obesity-related palmitate appearance rate from intracellular adipose tissue lipolysis.

100th anniversary of the discovery of insulin perspective: insulin and adipose tissue fatty acid metabolism.

Insulin inhibits systemic nonesterified fatty acid (NEFA) flux to a greater degree than glucose or any other metabolite. This remarkable effect is mainly due to insulin-mediated inhibition of intracellular triglyceride (TG) lipolysis in adipose tissues and is essential to prevent diabetic ketoacidosis, but also to limit the potential lipotoxic effects of NEFA in lean tissues that contribute to the development of diabetes complications. Insulin also regulates adipose tissue fatty acid esterification, glycerol and TG synthesis, lipogenesis, and possibly oxidation, contributing to the trapping of dietary fatty acids in the postprandial state. Excess NEFA flux at a given insulin level has been used to define in vivo adipose tissue insulin resistance. Adipose tissue insulin resistance defined in this fashion has been associated with several dysmetabolic features and complications of diabetes, but the mechanistic significance of this concept is not fully understood. This review focusses on the in vivo regulation of adipose tissue fatty acid metabolism by insulin and the mechanistic significance of the current definition of adipose tissue insulin resistance. One hundred years after the discovery of insulin and despite decades of investigations, much is still to be understood about the multifaceted in vivo actions of this hormone on adipose tissue fatty acid metabolism.

Nonesterified Fatty Acids and Kidney Function Decline in Older Adults: Findings From the Cardiovascular Health Study.

RATIONALE & OBJECTIVE: Circulating nonesterified fatty acids (NEFAs) make up a small portion of circulating lipids but are a metabolically important energy source. Excessive circulating NEFAs may contribute to lipotoxicity in many tissues, including the kidneys. We investigated the relationship between total circulating NEFA concentration and kidney outcomes in older, community-dwelling adults. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 4,698 participants≥65 years of age in the Cardiovascular Health Study who underwent total fasting serum NEFA concentration measurements in 1992-1993. EXPOSURE: Fasting serum NEFA concentration at one time point. OUTCOME: Three primary outcomes: estimated glomerular filtration rate (eGFR) decline of≥30%, the composite of eGFR decline≥30% or kidney failure with replacement therapy, and change in eGFR. These outcomes were assessed over 4- and 13-year periods. ANALYTICAL APPROACH: Logistic regression for the dichotomous outcomes and mixed effects models for the continuous outcome, with sequential adjustment for baseline covariates. Inverse probability of attrition weighting was implemented to account for informative attrition during the follow-up periods. RESULTS: Serum NEFA concentrations were not independently associated with kidney outcomes. In unadjusted and partially adjusted analyses, the highest quartile of serum NEFA concentration (compared with lowest) was associated with a higher risk of≥30% eGFR decline at 4 years and faster rate of decline of eGFR. No associations were evident after adjustment for comorbidities, lipid levels, insulin sensitivity, medications, and vital signs: the odds ratio for the eGFR decline outcome was 1.33 (95% CI, 0.83-2.13), and the difference in eGFR slope in the highest versus lowest quartile of serum NEFA concentration was-0.15 (95% CI, -0.36 to 0.06) mL/min/1.73m2 per year. LIMITATIONS: Single NEFA measurements, no measurements of post-glucose load NEFA concentrations or individual NEFA species, no measurement of baseline urine albumin. CONCLUSIONS: A single fasting serum NEFA concentration was not independently associated with long-term adverse kidney outcomes in a cohort of older community-living adults.

Effect of hyperketonemia on the diurnal patterns of energy-related blood metabolites in early-lactation dairy cows.

Most dairy cows experience a period of energy deficit in early lactation, resulting in increased plasma concentrations of nonesterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB). Our objectives were to determine (1) the diurnal variation in plasma BHB and NEFA, (2) the correlation between plasma NEFA and BHB when accounting for diurnal changes, and (3) the effect of hyperketonemia (HYK) on the diurnal pattern of blood metabolites. Jugular catheters were placed in 28 multiparous Holstein cows between 3 and 9 days in milk, and blood samples were collected every 2 h for 96 h. Cows were retrospectively classified as HYK positive (HYK; n = 13) if they had plasma BHB concentrations ≥1.2 mmol/L for ≥3 study days, or HYK negative (non-HYK; n = 15) if they had plasma BHB concentrations ≥1.2 mmol/L for ≤2 study days. Generalized linear mixed models were used to analyze concentrations of analytes over time and differences in metabolites between HYK groups. The correlation between total plasma NEFA and BHB was analyzed by calculating the area under the curve for plasma NEFA and BHB for all cows. Plasma NEFA reached a peak approximately 2 h before morning feed delivery, falling to a nadir in the late evening. Plasma BHB was at a nadir at the time of morning feed delivery, peaking 4 h later. We observed a strong positive correlation between daily plasma NEFA and BHB. Additionally, HYK cows had greater concentrations of plasma NEFA and BHB than non-HYK cows. The HYK cows also experienced a greater magnitude of change in BHB throughout the day than the non-HYK cows. Our results suggest that the time relative to feeding should be considered when analyzing plasma metabolites, as classification of energy status may change throughout a day.

Provision of shelter during the prepartum period: Effects on behavior, blood analytes, and health status in dairy cows in winter.

This study aimed to assess the effect of shelter provision during the prepartum period on lying, ruminating, and feeding behavior in outdoor-housed dairy cows exposed to winter weather conditions in a temperate climate. We also aimed to determine whether shelter provision during the prepartum period influenced blood analytes related to energy metabolism, body cleanliness, and health status. In this study, 2 cohorts of 12 multiparous Holstein prepartum cows were tested in winter. Twenty-five days before their expected calving date, the cows in each cohort were paired and randomly assigned to an open paddock without shelter or to one with access to an artificial shelter until calving. Shelter use, lying time, number of lying bouts, duration of lying bouts, rumination time, and feeding time were continuously recorded during the 3 wk before calving. Cows were assessed weekly for body cleanliness throughout the 3-wk prepartum period. A blood sample was taken from the coccygeal vein of each cow at wk -3, -2, -1, 1, 2, and 3 relative to calving date, and were assayed for nonesterified fatty acids (NEFA), ß-hydroxybutyrate, and haptoglobin concentrations. Cases of clinical health disorders after calving were also recorded. The cows spent 60% of their daily time in the shelters, and for 75% of that time they were lying down. Cows with access to shelter during the prepartum period lay down around 3.2 h/d more than cows without shelter on wk -3 and -2 relative to calving. The cows with shelter access spent less time feeding during the morning (wk -3: 29.7 min; wk -2: 12.1 min; wk -1: 17.3 min) and afternoon (wk -3: 18.2 min; wk -2: 21.9 min) than cows without shelter access. As well, cows with shelter access showed a higher body cleanliness score (∼92 vs. ∼48%) and lower NEFA concentrations (wk -2: 0.27 vs. 0.44 mmol/L; wk -1: 0.46 vs. 0.64 mmol/L) in the precalving period, and lower haptoglobin concentrations in the first week postpartum (0.34 vs. 0.79 mg/mL) than cows without shelter access. We observed no treatment differences in daily rumination time, ß-hydroxybutyrate concentrations, or postpartum health disorders. Despite the small number of dairy cows used in this study, these findings suggest that having access to a shelter during the prepartum period increases lying time, improves body cleanliness, and reduces adipose mobilization. Therefore, it is important to provide a protected area for the welfare of prepartum dairy cows exposed to winter climate conditions.

Effect of diet and nonesterified fatty acid levels on global transcriptomic profiles in circulating peripheral blood mononuclear cells in early lactation dairy cows.

After calving, lipid mobilization caused by increased nutrient demands for lactation leads to elevated circulating concentrations of nonesterified fatty acids (NEFA). Excessive NEFA levels have previously been identified as a major risk factor for postpartum immunosuppression. The aim of this study was to investigate changes in global transcriptomic gene expression of peripheral blood mononuclear cells (PBMC) in dairy cows offered different early lactation diets (high concentrate, n = 7; medium, n = 8; or low, n = 9) and with differing circulating levels of NEFA. Cows were classified as having NEFA concentrations of either <500 µM (low, n = 6), 500 to 750 µM (medium, n = 8) or >750 µM (high, n = 10) at 14 d in milk. Plasma urea concentrations were greater for cows on the high concentrate diet but ß-hydroxybutyrate and glucose concentrations did not differ significantly between either dietary treatments or NEFA groups. Cows with high NEFA weighed more at drying off and suffered greater body condition score loss after calving. The PBMC were isolated at 14 d in milk, and RNA was extracted for RNA sequencing. Differential gene expression was analyzed with DESeq2 with q-value for false discovery rate control followed by Gene Ontology Enrichment. Although there were no differentially expressed genes associated with lactation diet, 304 differentially expressed genes were identified between cows with high and low circulating NEFA, with 118 upregulated and 186 downregulated. Gene Ontology enrichment analysis demonstrated that biological adhesion and immune system process were foremost among various PBMC functions which were altered relating to body defenses and immunity. High NEFA concentrations were associated with inhibited cellular adhesion function by downregulating 20 out of 26 genes (by up to 17-fold) related to this process. Medium NEFA concentrations altered a similar set of functions as high NEFA, but with smaller enrichment scores. Localization and immune system process were most significant, with biological adhesion ranking only eleventh. Our results demonstrated that increased circulating NEFA concentrations, but not diet, were associated with immune system processes in PBMC in early lactation cows. Leukocyte cell-to-cell adhesion was inhibited when the NEFA concentration exceeded 750 µM, which would reduce the efficiency of diapedesis and so contribute to decreased body defense mechanisms and predispose animals to infection.

Thirty minutes of moderate-intensity downhill or level running has no effect on postprandial lipemia: A randomized controlled trial.

Elevated postprandial triglyceride (TG) concentrations are linked to a relatively high risk of cardiovascular disease. Eccentric endurance exercise, such as downhill walking and running, can provide metabolic benefits similar to concentric exercise. However, whether eccentric exercise affects postprandial lipemia remains unknown. Nine healthy young men performed level running (trial) or downhill running (DR trial, -15% slope) at 60% [INSIDE:1]O2max or rest (CON trial) for 30 min in a randomized crossover design. The participants were fed a high-fat meal the next day. Blood and expired gas samples were collected before and 0.5, 1, 2, 3, 4, 5, and 6 h after the meal. Muscle soreness was measured using a visual analog scale. The DR trial induced mild muscle damage. During the 6-h postprandial period, serum TG concentrations and area under the curve (AUC) were similar across the three trials. The DR trial had a significantly higher AUC of nonesterified fatty acid concentrations and a significantly lower AUC of glucose concentrations than the CON trial. The results suggested that neither moderate-intensity DR nor running a level surface had a significant effect on lipemia after a high-fat meal. However, DR improved the postprandial glycemic response.

Evaluating acute inflammation's effects on hepatic triglyceride content in experimentally induced hyperlipidemic dairy cows in late lactation.

Inflammation appears to be a predisposing factor and key component of hepatic steatosis in a variety of species. Objectives were to evaluate effects of inflammation [induced via intravenous lipopolysaccharide (LPS) infusion] on metabolism and liver lipid content in experimentally induced hyperlipidemic lactating cows. Cows (765 ± 32 kg of body weight; 273 ± 35 d in milk) were enrolled in 2 experimental periods (P); during P1 (5 d), baseline data were obtained. At the start of P2 (2 d), cows were assigned to 1 of 2 treatments: (1) intralipid plus control (IL-CON; 3 mL of saline; n = 5) or (2) intralipid plus LPS (IL-LPS; 0.375 µg of LPS/kg of body weight; n = 5). Directly following intravenous bolus (saline or LPS) administration, intralipid (20% fat emulsion) was intravenously infused continuously (200 mL/h) for 16 h to induce hyperlipidemia during which feed was removed. Blood samples were collected at -0.5, 0, 4, 8, 12, 16, 24, and 48 h relative to bolus administration, and liver biopsies were obtained on d 1 of P1 and at 16 and 48 h after the bolus. By experimental design (feed was removed during the first 16 h of d 1), dry matter intake decreased in both treatments on d 1 of P2, but the magnitude of reduction was greater in LPS cows. Dry matter intake of IL-LPS remained decreased on d 2 of P2, whereas IL-CON cows returned to baseline. Milk yield decreased in both treatments during P2, but the extent and duration was longer in LPS-infused cows. Administering LPS increased circulating LPS-binding protein (2-fold) at 8 h after bolus, after which it markedly decreased (84%) below baseline for the remainder of P2. Serum amyloid A concentrations progressively increased throughout P2 in IL-LPS cows (3-fold, relative to controls). Lipid infusion gradually increased nonesterified fatty acids and triglycerides in both treatments relative to baseline (3- and 2.5-fold, respectively). Interestingly, LPS infusion blunted the peak in nonesterified fatty acids, such that concentrations peaked (43%) higher in IL-CON compared with IL-LPS cows and heightened the increase in serum triglycerides (1.5-fold greater relative to controls). Liver fat content remained similar in IL-LPS relative to P1 at 16 h; however, hyperlipidemia alone (IL-CON) increased liver fat (36% relative to P1). No treatment differences in liver fat were observed at 48 h. In IL-LPS cows, circulating insulin increased markedly at 4 h after bolus (2-fold relative to IL-CON), and then gradually decreased during the 16 h of lipid infusion. Inducing inflammation with simultaneous hyperlipidemia altered the characteristic patterns of insulin and LPS-binding protein but did not cause fatty liver.

Association of prepartum lying time with nonesterified fatty acids and stillbirth in prepartum dairy heifers and cows.

The objective of the present study was to assess the association of prepartum lying time (LT) and the coefficient of variation (CV) of LT within 7 d before calving with prepartum serum nonesterified fatty acid (NEFA) concentration at 7 ± 3 d prepartum (dpp) and stillbirth. Prepartum pregnant Holstein heifers and cows from 3 dairy herds were used (n = 1,044). Animals were housed in freestall barns using a prepartum pen 21 d before the expected calving date and were moved into a contiguous maternity pen at parturition. Monthly, cohorts of 20 to 36 animals (heifers and cows combined) were enrolled at each farm and electronic data loggers (IceQube, IceRobotics, Edinburgh, UK) were fitted to the hind leg of individual animals to assess their behavioral activity. Stillbirth was defined as a calf born dead or died during the first 24 h after parturition in dams with normal gestation length. The LT was recorded for the last 7 dpp to assess differences among dams with stillbirth versus those with a calf born alive. Mean LT within 7 d before blood NEFA collection was assessed to determine the association with prepartum serum NEFA at 7 ± 3 dpp. Blood samples for the assessment of serum NEFA concentration were collected from prepartum animals at 14 ± 3 and at 7 ± 3 dpp. Blood samples for total serum calcium concentration were collected from postpartum cows within 48 h after parturition to assess differences among cows with stillbirth versus those with a calf born alive. Data were analyzed using CORR, MIXED, or GLIMMIX procedures of SAS (SAS Institute Inc., Cary, NC). Dams experiencing dystocic births had a greater proportion of stillbirth, but herd, parity, and season did not have an effect. Dams with a stillborn calf had reduced LT and increased CV of LT within the last 7 dpp compared with dams with a calf born alive, regardless of parity. Multiparous cows with a stillborn calf had higher prepartum serum NEFA concentration compared with multiparous cows with a calf born alive, but this did not differ for first-calf heifers. Regardless of parity, the proportion of postpartum cows with hypocalcemia was higher for dams with a stillborn calf compared with those with a calf born alive. Regardless of parity, LT of prepartum dams was strongly correlated with the CV of LT (as LT increased, the CV decreased), and prepartum dams with a mean LT between 11 and 15 h/d had the lowest serum NEFA concentration compared with dams with LT of 8 to 10 or >16 h/d. Serum NEFA concentrations at 7 ± 3 dpp was positively correlated with the CV of LT within 7 d before blood sample. These results show that the dam's prepartum LT and its consistency over time are associated with prepartum serum NEFA and calf survival at calving.

Sirtuin 3 improves fatty acid metabolism in response to high nonesterified fatty acids in calf hepatocytes by modulating gene expression.

Sirtuin 3 (SIRT3), a mitochondrial deacetylase, is a key regulator of energy metabolism in the liver. In nonruminants, the hepatic abundance of SIRT3 is decreased in individuals with nonalcoholic fatty liver diseases, and recovery of SIRT3 alleviates hepatic triacylglycerol (TG) deposition. However, the level of SIRT3 expression and its effects on lipid metabolism in dairy cows have not been characterized. Here we studied the hepatic expression of SIRT3 in cows with fatty liver and the role of SIRT3 in fatty acid metabolism in bovine hepatocytes. This in vivo study involved 10 healthy cows and 10 cows with fatty liver, from which we collected samples of liver tissue and blood. Primary hepatocytes were isolated from Holstein calves and treated with 0, 0.5, or 1.0 mM nonesterified fatty acids (NEFA) for 24 h or transinfected with SIRT3 overexpression adenovirus (Ad-SIRT3)/SIRT3-short interfering (si)RNA for 48 h. Cows with fatty liver displayed lower serum glucose concentrations but higher serum NEFA and ß-hydroxybutyrate concentrations relative to healthy cows. Cows with fatty liver also had significant lower mRNA and protein abundance of hepatic SIRT3. Incubation of primary hepatocytes with NEFA reduced SIRT3 abundance in primary hepatocytes in a dose-dependent manner. Fatty acid (1 mM) treatment also markedly increased the abundance of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) but significantly decreased the abundance of carnitine palmitoyltransferase I (CPT1A), carnitine palmitoyltransferase II (CPT2), and acyl-CoA oxidase (ACO). Knockdown of SIRT3 by SIRT3-siRNA downregulated the mRNA abundance of CPT1A, CPT2, and ACO. In contrast, overexpression of SIRT3 by Ad-SIRT3 upregulated the mRNA abundance of CPT1A, CPT2, and ACO; downregulated the mRNA abundance of ACC1 and FAS; and consequently, decreased intracellular TG concentrations. Overexpression of SIRT3 ameliorated exogenous NEFA-induced TG accumulation by downregulating the abundance of ACC1 and FAS and upregulating the abundance of CPT1A, CPT2, and ACO in calf hepatocytes. Our data demonstrated that cows with fatty liver had lower hepatic SIRT3 contents, and an increase in SIRT3 abundance by overexpression mitigated TG deposition by modulating the expression of lipid metabolism genes in bovine hepatocytes. These data suggest a possible role of SIRT3 as a therapeutic target for fatty liver disease prevention in periparturient dairy cattle.

Diagnostic performance of cytology for assessment of hepatic lipid content in dairy cattle.

The objective of our study was to characterize the diagnostic performance of cytology for assessing hepatic lipid content (HLC) in dairy cows by comparing microscopic evaluation of lipid vacuolation in touch imprint slide preparations of liver biopsies with quantitative measurement of triglyceride concentration ([TG]; mg/mg of wet weight) in paired biopsy samples. Our study also sought to compare the diagnostic performance of liver cytology, plasma nonesterified fatty acid concentration ([NEFA]), and plasma ß-hydroxybutyrate concentration ([BHB]) derived from a measurement performed on whole blood, for assessing HLC. Chemical extraction of TG from liver tissue remains the gold standard for quantifying HLC, largely because available blood tests, although useful for detecting some types of pathology, such as increased lipid mobilization, ketosis, or hepatocellular injury, are nonspecific as to etiology. Veterinary practitioners can sample bovine liver for cytological evaluation in a fast, minimally invasive, and inexpensive manner. Thus, if highly predictive of HLC, cytology would be a practical diagnostic tool for dairy veterinarians. In our study, liver biopsy samples from Holstein cows (219 samples from 105 cows: 52 from cows 2 to 20 d prepartum, 105 from cows 0 to 10 d in milk, 62 from cows 18 to 25 d in milk) were used to prepare cytology slides and to quantify [TG] using the Folch extraction method followed by the Hantzch condensation reaction and spectrophotometric measurement. An ordinal scale (0-4) based on amount of hepatocellular cytoplasm occupied by discrete clear vacuoles was used by 3 blinded, independent observers to rank HLC in Wright-Giemsa-stained slides. Interobserver agreement in cytology scoring was good. Corresponding plasma [NEFA] and [BHB] measurements were available for 187 and 195 of the 219 samples, respectively. Liver [TG] correlated more strongly with cytology score than with NEFA or BHB, and receiver operating characteristic curve analysis showed that cytology had better diagnostic performance than either NEFA or BHB for correctly categorizing [TG] at thresholds of 5, 10, and 15%. Hepatic lipidosis in high-producing dairy cows is of major clinical and economic importance, and this study demonstrates that cytology is an accurate means of assessing HLC. Additional work is indicated to evaluate the diagnostic utility of liver cytology.

Glucose-dependent insulinotropic polypeptide promotes lipid deposition in subcutaneous adipocytes in obese type 2 diabetes patients: a maladaptive response.

Glucose-dependent insulinotropic polypeptide (GIP) beyond its insulinotropic effects may regulate postprandial lipid metabolism. Whereas the insulinotropic action of GIP is known to be impaired in type 2 diabetes mellitus (T2DM), its adipogenic effect is unknown. We hypothesized that GIP is anabolic in human subcutaneous adipose tissue (SAT) promoting triacylglycerol (TAG) deposition through reesterification of nonesterified fatty acids (NEFA), and this effect may differ according to obesity status or glucose tolerance. Twenty-three subjects categorized into four groups, normoglycemic lean (n = 6), normoglycemic obese (n = 6), obese with impaired glucose regulation (IGR; n = 6), and obese T2DM (n = 5), participated in a double-blind, randomized, crossover study involving a hyperglycemic clamp with a 240-min GIP infusion (2 pmol·kg-1·min-1) or normal saline. Insulin, NEFA, SAT-TAG content, and gene expression of key lipogenic enzymes were determined before and immediately after GIP/saline infusions. GIP lowered NEFA concentrations in the obese T2DM group despite diminished insulinotropic activity (mean NEFA AUC0-4 h ± SE, 41,992 ± 9,843 µmol·l-1·min-1 vs. 71,468 ± 13,605 with placebo, P = 0.039, 95% CI: 0.31-0.95). Additionally, GIP increased SAT-TAG in obese T2DM (1.78 ± 0.4 vs 0.86 ± 0.1-fold with placebo, P = 0.043, 95% CI: 0.1-1.8). Such effect with GIP was not observed in other three groups despite greater insulinotropic activity. Reduction in NEFA concentration with GIP correlated with adipose tissue insulin resistance for all subjects (Pearson, r = 0.56, P = 0.005). There were no significant gene expression changes in key SAT lipid metabolism enzymes. In conclusion, GIP appears to promote fat accretion and thus may exacerbate obesity and insulin resistance in T2DM.

Degree of free fatty acid saturation influences chocolate rejection in human assessors.

In foods, free fatty acids (FFAs) traditionally have been viewed as contributing an odor, yet evidence has accumulated that FFAs also contribute a unique taste ("oleogustus"). However, minimal work has been conducted using actual foods to test the contribution of FFA to taste preferences. Here, we investigate flavor, taste, and aroma contributions of added FFA in chocolate, as some commercial manufacturers already use lipolysis of triglycerides to generate unique profiles. We hypothesized that small added concentrations of FFAs would increase preferences for chocolate, whereas higher added concentrations would decrease preferences. We also hypothesized a saturated fatty acid (stearic C18) would have a lesser effect than a monounsaturated (oleic C18:1), which would have a lesser effect than a polyunsaturated (linoleic C18:2) fatty acid. For each, paired preference tests were conducted for 10 concentrations (0.04% to 2.25%) of added FFAs compared with the control chocolate without added FFAs. Stearic acid was tested for flavor (tasting and nares open), whereas the unsaturated fatty acids were tested for both aroma (orthonasal only and no tasting) and taste (tasting with nares blocked to eliminate retronasal odor). We found no preference for any added FFA chocolate; however, rejection was observed independently for both taste and aroma of unsaturated fatty acids, with linoleic acid reaching rejection at lower concentrations than oleic acid. These data indicate that degree of unsaturation influences rejection of both FFA aroma and taste in chocolate. Thus, alterations of FFA profiles in foods should be approached cautiously to avoid shifting concentrations of unsaturated fatty acids to hedonically unacceptable levels.