J. W. McFadden

Plasma ceramides are elevated in overweight Holstein dairy cows experiencing greater lipolysis and insulin resistance during the transition from late pregnancy to early lactation

Insulin resistance is a homeorhetic adaptation to parturition in dairy cows transitioning from late pregnancy to early lactation. An increase in prepartum adiposity can predispose periparturient cows to greater lipolysis and insulin resistance, thus increasing the risk for metabolic disease. Mechanisms mediating the development of insulin resistance in overweight peripartal dairy cows may depend on ceramide metabolism. The sphingolipid ceramide accumulates in plasma and tissues of overweight monogastric animals, and facilitates saturated fatty acid-induced insulin resistance. Considering this evidence, we hypothesized that plasma ceramides would be elevated in periparturient dairy cattle and that these sphingolipids would correlate with the magnitude of lipolysis and insulin resistance. To test our central hypothesis, multiparous Holstein cows were allocated into 2 groups according to their body condition score (BCS) at d -30 prepartum: lean (BCS <3.0; n=10) or overweight (BCS >4.0; n=11). Blood samples were collected at d -45, -30, -15, and -7, relative to expected parturition, and at d 4 postpartum. Plasma glucose, insulin, nonesterified fatty acids (NEFA), and β-hydroxybutyrate (BHBA) concentrations were measured, and insulin sensitivity was estimated. The concentrations of individual plasma ceramide and glycosylated ceramide were determined using liquid chromatography-based mass spectrometry. Results demonstrated that greater adiposity was associated with a greater loss in body condition during late pregnancy. Overweight cows had greater circulating concentrations of glucose, insulin, and NEFA, and lower insulin sensitivity relative to lean cows. We detected 30 different sphingolipids across 6 lipid classes with acyl chains ranging from 16 to 26 carbons. The most abundant plasma sphingolipids detected were C24:0-ceramide, C24:0-monohexosylceramide, and C16:0-lactosylceramide. Plasma concentrations of total ceramide and monohexosylceramide increased as lactation approached, and saturated ceramide and monohexosylceramide were elevated in cows with greater adiposity relative to those with a lean phenotype. Plasma ceramides (e.g., C24:0-ceramide) were positively correlated with plasma NEFA and inversely correlated with insulin sensitivity. Our data demonstrate a remodeled plasma sphingolipidome in dairy cows transitioning from late pregnancy to lactation characterized by a concomitant increase in plasma ceramides with the development of peripartal insulin resistance.

Increasing Fatty Acid Oxidation Remodels the Hypothalamic Neurometabolome to Mitigate Stress and Inflammation

Modification of hypothalamic fatty acid (FA) metabolism can improve energy homeostasis and prevent hyperphagia and excessive weight gain in diet-induced obesity (DIO) from a diet high in saturated fatty acids. We have shown previously that C75, a stimulator of carnitine palmitoyl transferase-1 (CPT-1) and fatty acid oxidation (FAOx), exerts at least some of its hypophagic effects via neuronal mechanisms in the hypothalamus. In the present work, we characterized the effects of C75 and another anorexigenic compound, the glycerol-3-phosphate acyltransferase (GPAT) inhibitor FSG67, on FA metabolism, metabolomics profiles, and metabolic stress responses in cultured hypothalamic neurons and hypothalamic neuronal cell lines during lipid excess with palmitate. Both compounds enhanced palmitate oxidation, increased ATP, and inactivated AMP-activated protein kinase (AMPK) in hypothalamic neurons in vitro. Lipidomics and untargeted metabolomics revealed that enhanced catabolism of FA decreased palmitate availability and prevented the production of fatty acylglycerols, ceramides, and cholesterol esters, lipids that are associated with lipotoxicity-provoked metabolic stress. This improved metabolic signature was accompanied by increased levels of reactive oxygen species (ROS), and yet favorable changes in oxidative stress, overt ER stress, and inflammation. We propose that enhancing FAOx in hypothalamic neurons exposed to excess lipids promotes metabolic remodeling that reduces local inflammatory and cell stress responses. This shift would restore mitochondrial function such that increased FAOx can produce hypothalamic neuronal ATP and lead to decreased food intake and body weight to improve systemic metabolism.

Palmitic acid feeding increases ceramide supply in association with increased milk yield, circulating nonesterified fatty acids, and adipose tissue responsiveness to a glucose challenge

Reduced insulin action is a key adaptation that facilitates glucose partitioning to the mammary gland for milk synthesis and enhances adipose tissue lipolysis during early lactation. The progressive recovery of insulin sensitivity as cows advance toward late lactation is accompanied by reductions in circulating nonesterified fatty acids (NEFA) and milk yield. Because palmitic acid can promote insulin resistance in monogastrics through sphingolipid ceramide-dependent mechanisms, palmitic acid (C16:0) feeding may enhance milk production by restoring homeorhetic responses. We hypothesized that feeding C16:0 to mid-lactation cows would enhance ceramide supply and ceramide would be positively associated with milk yield. Twenty multiparous mid-lactation Holstein cows were enrolled in a study consisting of a 5-d covariate, 49-d treatment, and 14-d posttreatment period. All cows were randomly assigned to a sorghum silage-based diet containing no supplemental fat (control; n=10; 138±45 d in milk) or C16:0 at 4% of ration dry matter (PALM; 98% C16:0; n=10; 136±44 d in milk). Blood and milk were collected at routine intervals. Liver and skeletal muscle tissue were biopsied at d 47 of treatment. Intravenous glucose tolerance tests (300mg/kg of body weight) were performed at d -1, 24, and 49 relative to start of treatment. The plasma and tissue concentrations of ceramide and glycosylated ceramide were determined using liquid chromatography coupled with tandem mass spectrometry. Data were analyzed as repeated measures using a mixed model with fixed effects of treatment and time, and milk yield served as a covariate. The PALM treatment increased milk yield, energy-corrected milk, and milk fat yield. The most abundant plasma and tissue sphingolipids detected were C24:0-ceramide, C24:0-monohexosylceramide (GlcCer), and C16:0-lactosylceramide. Plasma concentrations of total ceramide and GlcCer decreased as lactation advanced, and ceramide and GlcCer were elevated in cows fed PALM. Palmitic acid feeding increased hepatic ceramide levels, a response not observed in skeletal muscle tissue. Plasma ceramides (e.g., C24:0-ceramide) were positively correlated with plasma NEFA and milk yield, and positively correlated with NEFA levels following a glucose challenge. Our data demonstrate a remodeled plasma and hepatic sphingolipidome in mid-lactation dairy cows fed PALM. The potential involvement in ceramide in homeorhetic nutrient partitioning to support lactation requires further consideration.

Temporal changes in sphingolipids and systemic insulin sensitivity during the transition from gestation to lactation

Reduced insulin action develops naturally during the peripartum to ensure maternal nutrient delivery to the fetus and neonate. However, increased insulin resistance can facilitate excessive lipolysis which in turn promotes metabolic disease in overweight dairy cattle. Increased fatty acid availability favors the accumulation of the sphingolipid ceramide and is implicated in the pathogenesis of insulin resistance, however, the relationship between sphingolipid metabolism and insulin resistance during the peripartum remains largely unknown. Our objectives were to characterize temporal responses in plasma and tissue sphingolipids in lean and overweight peripartal cows and to establish the relationships between sphingolipid supply and lipolysis, hepatic lipid deposition, and systemic insulin action. Twenty-one multiparous lean and overweight Holstein cows were enrolled in a longitudinal study spanning the transition from gestation to lactation (d -21 to 21, relative to parturition). Plasma, liver, and skeletal muscle samples were obtained, and sphingolipids were profiled using LC/MS/MS. Insulin sensitivity was assessed utilizing intravenous insulin and glucose challenges. Our results demonstrated the following: first, insulin resistance develops postpartum concurrently with increased lipolysis and hepatic lipid accumulation; second, ceramides and glycosylated ceramides accumulate during the transition from gestation to lactation and are further elevated in overweight cows; third, ceramide accrual is associated with lipolysis and liver lipid accumulation, and C16:0- and C24:0-ceramide are inversely associated with systemic insulin sensitivity postpartum; fourth, plasma sphingomyelin, a potential source of ceramides reaches a nadir at parturition and is closely associated with feed intake; fifth, select sphingomyelins are lower in the plasma of overweight cows during the peripartal period. Our results demonstrate that dynamic changes occur in peripartal sphingolipids that are influenced by adiposity, and are associated with the onset of peripartal insulin resistance. These observations are in agreement with a putative potential role for sphingolipids in facilitating the physiological adaptations of peripartum.

Increasing palmitic acid intake enhances milk production and prevents glucose-stimulated fatty acid disappearance without modifying systemic glucose tolerance in mid-lactation dairy cows

Feeding saturated fatty acids may enhance milk yield in part by decreasing insulin sensitivity and shifting glucose utilization toward the mammary gland. Our objective was to evaluate the effects of palmitic acid (C16:0) on milk production and insulin sensitivity in cows. Twenty multiparous mid-lactation Holstein cows were enrolled in a study consisting of a 5-d covariate, 49-d treatment, and 14-d posttreatment period. All cows received a common sorghum silage-based diet and were randomly assigned to a diet containing no supplemental fat (control; n=10; 138±45d in milk) or C16:0 at 4% of ration DM (PALM; 98% C16:0; n=10; 136±44d in milk). Blood and milk were collected at routine intervals. Intravenous glucose tolerance tests (300mg/kg of body weight) were performed at d -1, 24, and 49 relative to start of treatment. Data were analyzed as repeated measures using a mixed model with fixed effects of treatment and time, and milk yield served as a covariate. The PALM treatment increased milk yield by wk 7. Furthermore, PALM increased milk fat yield and energy-corrected milk at wk 3 and 7. Changes in milk production occurred in parallel with enhanced energy intake. Increased milk fat yield during PALM treatment was due to increased C16:0 and C16:1 incorporation; PALM had no effect on concentration of milk components, BW, or body condition score. Two weeks posttreatment, energy-corrected milk and milk fat yield remained elevated in PALM-fed cows whereas yields of milk were similar between treatments. Increased milk fat yield after PALM treatment was due to increased de novo lipogenesis and uptake of preformed fatty acids. The basal concentration of nonesterified fatty acids (NEFA) in plasma increased by d 4, 6, and 8 of PALM treatment, a response not observed thereafter. Although PALM supplementation did not modify insulin, glucose, or triacylglycerol levels in plasma, total cholesterol in plasma was elevated by wk 3. Estimated insulin sensitivity was lower during the first week of PALM treatment; however, glucose disposal following glucose tolerance tests was not modified. In contrast, C16:0 feeding reduced glucose-stimulated NEFA disappearance by wk 7. Results demonstrate that increasing dietary energy from C16:0 for 7wk improves milk yield and milk composition without modifying systemic glucose tolerance. Reduced glucose-stimulated NEFA disappearance with C16:0 feeding and elevated circulating NEFA may reflect changes in adipose tissue insulin sensitivity.

Jugular-infused methionine, lysine and branched-chain amino acids does not improve milk production in Holstein cows experiencing heat stress

Poor utilization of amino acids contributes to losses of milk protein yield in dairy cows exposed to heat stress (HS). Our objective was to test the effect of essential amino acids on milk production in lactating dairy cows exposed to short-term HS conditions. To achieve this objective, 12 multiparous, lactating Holstein cows were assigned to two environments (thermoneutral (THN) or HS) from days 1 to 14 in a split-plot type cross-over design. All cows received 0 g/day of essential amino acids from days 1 to 7 (negative control (NC)) followed by an intravenous infusion of l-methionine (12 g/day), l-lysine (21 g/day), l-leucine (35 g/day), l-isoleucine (15 g/day) and l-valine (15 g/day, methionine, lysine and branched-chain amino acids (ML+BCAA)) from days 8 to 14. The basal diet was composed of ryegrass silage and hay, and a concentrate mix. This diet supplied 44 g of methionine, 125 g of lysine, 167 g of leucine, 98 g of isoleucine and 109 g of valine per day to the small intestine of THN cows. Temperature-humidity index was maintained below 66 for the THN environment, whereas the index was maintained above 68, peaking at 76, for 14 continuous h/day for the HS environment. Heat stress conditioning increased the udder temperature from 37.0°C to 39.6°C. Cows that received the ML+BCAA treatment had greater p.m. rectal and vaginal temperatures (0.50°C and 0.40°C, respectively), and respiration rate (8 breaths/min) compared with those on the NC treatment and exposed to a HS environment. However, neither NC nor ML+BCAA affected rectal or vaginal temperatures and respiration rates in the THN environment. Compared with THN, the HS environment reduced dry matter intake (1.48 kg/day), milk yield (2.82 kg/day) and milk protein yield (0.11 kg/day). However, compared with NC, the ML+BCAA treatment increased milk protein percent by 0.07 points. For the THN environment, the ML+BCAA treatment increased concentrations of milk urea nitrogen. For the HS environment, the ML+BCAA treatment decreased plasma concentrations of arginine, ornithine and citrulline; however, differences were not observed for the THN environment. In summary, HS elicited expected changes in production; however, infusions of ML+BCAA failed to increase milk protein yield. Lower dry matter intake and greater heat load in response to ML+BCAA contributed to the lack of response in milk production in HS cows. The ML+BCAA treatment may have reduced the breakdown of muscle protein in heat-stressed cows.

Technical note: Characterization of ceramide in bovine lipoproteins

The hepatic synthesis and export of ceramide is enhanced in diabetic monogastrics. Moreover, ceramide in lipoproteins can mediate the development of insulin resistance. We have previously demonstrated that circulating ceramide increases during the progression of insulin resistance in postpartum dairy cows. Considering that the origins of circulating ceramide required investigation, our objective was to develop a method to characterize the ceramide profile of lipoprotein fractions collected from dairy cows. Serum was collected from 4 nonpregnant and nonlactating Holstein dairy cows. Serum lipoproteins were isolated using size exclusion chromatography by fast protein liquid chromatography (SEC-FPLC). Measurement of triacylglycerol (TAG), phospholipid, total cholesterol, and protein was performed using standard colorimetry practices. Following lipid extraction, fractions were analyzed using electrospray ionization tandem mass spectrometry. Data were analyzed as repeated measures using a mixed model. Lipoprotein isolation using SEC-FPLC and subsequent colorimetric analyses confirmed the presence of 4 distinct fractions: TAG-rich, low density (LDL), and large (buoyant) and small (dense) high density lipoprotein (HDL) subclasses. As expected, the fraction representing mixed very low density lipoproteins and chylomicrons primarily contained TAG. Low density lipoprotein fractions were equally enriched with cholesterol and phospholipid. Buoyant HDL contained elevated levels of cholesterol, phospholipid, and protein. In contrast, the fraction containing dense HDL primarily contained protein. Our method revealed that LDL are enriched with ceramides. Ceramides were also compartmentalized to a lesser extent within both HDL subclasses and TAG-rich lipoproteins. Comparable to whole serum, C16:0-ceramide was the predominant ceramide quantified in all lipoprotein subclasses. Interestingly, the proportion of C24:0-ceramide to total ceramide was elevated in TAG-rich lipoproteins, relative to all other lipoprotein subclasses. We conclude that SEC-FPLC coupled with mass spectrometry is a means to quantify ceramides in lipoprotein fractions. Moreover, ceramides are enriched within bovine LDL, and lipoprotein ceramide profiles reflect levels observed in whole serum. Future investigation will need to determine the biological importance of lipoprotein ceramides with distinct C-chains at amide residues.

Hot topic: Ceramide inhibits insulin sensitivity in primary bovine adipocytes

In nonruminants, the sphingolipid ceramide inhibits insulin sensitivity by inactivating protein kinase B (AKT) within the insulin-signaling pathway. We have established that ceramide accrual develops with impaired systemic insulin action in ruminants during the transition from gestation to lactation, dietary palmitic acid supplementation, or controlled nutrient restriction. We hypothesized that ceramide promotes AKT inactivation and antagonizes insulin sensitivity in primary bovine adipocytes. Stromal-vascular cells were grown from bovine adipose tissue explants and cultured in differentiation media. To modify ceramide supply, we treated differentiated adipocytes with (1) myriocin, an inhibitor of de novo ceramide synthesis, or (2) cell-permeable C2:0-ceramide. Insulin-stimulated AKT activation (i.e., phosphorylation) and 2-deoxy-D-[3H]-glucose (2DOG) uptake were measured. Treatment of adipocytes with myriocin consistently decreased concentrations of ceramide, monohexosylceramide, and lactosylceramide. The insulin-stimulated ratio of phosphorylated AKT to total AKT was increased with myriocin but decreased with C2:0-ceramide. Moreover, adipocyte insulin-stimulated 2DOG uptake was decreased with C2:0-ceramide and increased with myriocin. We conclude that ceramide inhibits insulin-stimulated glucose uptake by downregulating AKT activation in primary bovine adipocytes.

Short communication: Circulating fatty acylcarnitines are elevated in overweight periparturient dairy cows in association with sphingolipid biomarkers of insulin resistance

Defects in mitochondrial fatty acid processing are associated with the development of fatty liver disease, inflammation, and insulin resistance in overweight nonruminants. Surplus fatty acids (FA) and defects in FA oxidation favor the accumulation of fatty acylcarnitines (FAC) and the sphingolipid ceramide. Moreover, elevated circulating FAC and ceramide concentrations are inversely related to insulin action. Because we have previously demonstrated that plasma ceramide levels increase during the transition from gestation to lactation, our aim was to determine whether changes in plasma medium- and long-chain FAC levels are related to circulating FA and sphingolipids in peripartal dairy cows. We hypothesized that plasma FAC levels would be higher in overweight cows experiencing increased lipolysis, and that FAC levels would be positively associated with elevations in plasma ceramides. Twenty-one multiparous Holstein cows were grouped according to body condition score (BCS) at d -30 prepartum as lean (BCS <3.0; n = 10) or overweight (BCS >4.0; n = 11). Blood was collected at d -30, -15, -7, and 4, relative to parturition. Circulating FAC and ceramide levels were determined using liquid chromatography and tandem mass spectrometry. To investigate the potential contributions of sphingomyelin (SM) hydrolysis to ceramide accrual, we also determined plasma SM levels during the peripartum period. Data were analyzed under a mixed model with the fixed effects of adiposity and time, and the random effect of cow. Relative to lean cows, overweight cows had elevated FAC during the transition from gestation to lactation. Circulating FAC levels were positively associated with FA, ceramide, and dihydro-SM levels. Although circulating FAC levels increased in all cows during the peripartum, enhanced prepartum adiposity contributed to a greater rise in plasma FA and FAC. Our results support on-going efforts to determine whether altered mitochondrial FA processing promotes the accumulation of the insulin resistance biomarker ceramide in blood and liver.

Efficacy of nutritional interventions to lower circulating ceramides in young adults: FRUVEDomic pilot study

The 2010 USDA Dietary Guidelines for Americans (DGA) recommends a diet largely composed of fruit and vegetables. Consuming a diet high in fruit and vegetables and low in refined carbohydrates and saturated fat may reduce an individuals risk for type 2 diabetes, nonalcoholic fatty liver disease, low‐grade chronic inflammation, and metabolic syndrome (MetS). Several recent studies have implicated the bioactive sphingolipid ceramide as an associative and causative biomarker for the development of these conditions. Considering that the intake of fruit and vegetables is frequently inadequate in young adults, we performed a pilot investigation to assess the efficacy of a free‐living fruit and vegetable intervention on overall metabolic health, circulating ceramide supply, and inflammatory status in young adults. We discovered that adoption of the recommended DGA for fruit and vegetable intake for 8 weeks decreased waist circumference, systolic blood pressure, and circulating cholesterol. Lipidomics analysis revealed that nutritional intervention can lower circulating ceramides, including C24:0 ceramide, a known inhibitor of insulin signaling. Unexpectedly, we observed an increase in C16:0 ceramide, suggesting that this form of ceramide in circulation is not associated with metabolic disease in humans. We also observed an improved inflammatory status with enhanced fruit and vegetable intake that was correlated with ceramide concentrations. These data suggest that adopting the recommended DGA is associated with a reduction of many, but not all, ceramide species and may help to prevent or mitigate MetS. Future research needs to assess whether the ceramide‐lowering ability of nutritional intervention is associated with reduced risk of developing metabolic disease.