Training alters the distribution of perilipin proteins in muscle following acute free fatty acid exposure.

KEY POINTS: The lipid droplet (LD)-associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, although whether the abundance and association of the PLIN proteins with LDs is related to the diverse lipid storage in muscle between trained and sedentary individuals is unknown. We show that lipid infusion augments IMTG content in type I fibres of both trained and sedentary individuals. Most importantly, despite there being no change in PLIN protein content, lipid infusion did increase the number of LDs connected with PLIN proteins in trained individuals only. We conclude that trained individuals are able to redistribute the pre-existing pool of PLIN proteins to an expanded LD pool during lipid infusion and, via this adaptation, may support the storage of fatty acids in IMTG. ABSTRACT: Because the lipid droplet (LD)-associated perilipin (PLIN) proteins promote intramuscular triglyceride (IMTG) storage, we investigated the hypothesis that differential protein content of PLINs and their distribution with LDs may be linked to the diverse lipid storage in muscle between trained and sedentary individuals. Trained (n = 11) and sedentary (n = 10) subjects, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic-euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed using confocal immunofluorescence microscopy for fibre type-specific IMTG content and PLIN associations with LDs. In both groups, lipid infusion increased IMTG content in type I fibres (trained: +62%, sedentary: +79%; P < 0.05) but did not affect PLIN protein content. At baseline, PLIN2 (+65%), PLIN3 (+105%) and PLIN5 (+53%; all P < 0.05) protein content was higher in trained compared to sedentary individuals. In trained individuals, lipid infusion increased the number of LDs associated with PLIN2 (+27%), PLIN3 (+73%) and PLIN5 (+40%; all P < 0.05) in type I fibres. By contrast, in sedentary individuals, lipid infusion only increased the number of LDs not associated with PLIN proteins. Acute free fatty acid elevation therefore induces a redistribution of PLIN proteins to an expanded LD pool in trained individuals only and this may be part of the mechanism that enables fatty acids to be stored in IMTG.

Effects of central administration of distinct fatty acids on hypothalamic neuropeptide expression and energy metabolism.

OBJECTIVE: To investigate the differential effects of acute central administration of distinct fatty acids (FA) on food intake, body weight and energy metabolism. DESIGN: Male Sprague-Dawley rats were treated with bolus intracerebroventricular injections of control hydroxypropyl-ß-cyclodextrin (HPB) or HPB complexed with 30 nmol of saturated palmitic acid (PA), monounsaturated oleic acid (OA) or polyunsaturated ω-3 docosahexaenoic acid (DHA). Food intake, body weight, neuropeptide expression and various serum parameters were assessed. RESULTS: When compared with controls, rats injected with either OA or DHA had significantly reduced food intake and body weight for 48 h following injections. No significant changes in food intake or body weight were observed in the PA group. In conjunction with reduced food intake, hypothalamic anorexigenic pro-opiomelanocortin (POMC) gene expression was significantly augmented in the OA and DHA groups, with essentially no changes observed in the PA group. Changes in serum measures of energy metabolism also changed coinciding with the observed differences in food intake. Moreover, central administration of SHU9119, a melanocortin-4-receptor (MC4R) antagonist, completely abolished the anorexigenic actions of OA, suggesting a role for OA-induced augmentation of hypothalamic POMC expression in mediating its central inhibition of food intake. CONCLUSIONS: The hypothalamus differentially senses FA and, specifically, that OA and DHA, but not PA, reduce food intake and body weight, which may be mediated through POMC/MC4R signaling.

Reducing dry period length to simplify feeding transition cows: milk production, energy balance, and metabolic profiles.

Sixty-five Holstein cows were used to evaluate management schemes involving altered dry period (DP) lengths on subsequent milk production, energy balance (EB), and metabolic variables. Cows were assigned to one of 3 treatments: traditional 56-d DP (fed a low-energy diet from -56 to -29 d and a moderate energy diet from -28 d to parturition; T), 28-d DP (continuously fed a high energy diet; S), and no planned DP (continuously fed a high energy diet; N). Prepartum DM intake (DMI), measured from 56 d prepartum through parturition, was lower for cows on the T treatment than for cows on the S treatment and was higher for cows on the N treatment than for cows on the S treatment. There were no differences in prepartum plasma glucose, and beta-hydroxybutryric acid; there was a treatment by time interaction for prepartum plasma nonesterified fatty acid (NEFA). There was no difference in prepartum liver triglyceride (TG); postpartum liver TG was decreased for cows on the N treatment compared with cows on the S treatment, but was similar for cows on the T and S treatments. Postpartum NEFA was similar between cows on the T and S treatments, but was greater for cows on the S treatment than for cows on the N treatment. Postpartum glucose was greater for cows on the N treatment compared with cows on the S treatment and tended to be greater for cows on the S treatment than for cows on the T treatment. There was no difference in postpartum solids-corrected milk (SCM) production or DMI by cows on the T vs. S treatment. However, there was a tendency toward lower postpartum SCM production by cows on the N vs. S treatment and a tendency for greater postpartum DMI by cows on the N vs. S treatment. Postpartum EB was greater for cows on the S vs. T treatment and the N vs. S treatment. In general, T and S management schemes had similar effects on DMI, SCM, and metabolic variables in the first 70 d of the subsequent lactation. Eliminating the DP improved energy and metabolic status.

Effects of intravenous triacylglycerol emulsions on hepatic metabolism and blood metabolites in fasted dairy cows.

The objective was to determine the effects of intravenous infusion of triacylglycerol (TAG) emulsions derived from different lipid sources on energy metabolism during a 4-d fast. Six nonpregnant, nonlactating multiparous Holstein cows were randomly assigned to treatments in a replicated 3 x 3 Latin Square design. Treatments included intravenous infusion of tallow, linseed oil, or fish oil emulsions at a rate of 0.54 g of TAG/kg of body weight per day; infusions were concurrent with a 4-d fast. The emulsions were administered for 20 to 30 min every 4 h throughout the 4-d fast. Cows were fed ad libitum for 24 d between the fast/infusion periods. Infusion of tallow, linseed oil, or fish oil emulsions increased plasma concentrations of palmitic acid, linolenic acid, and eicosapentaenoic and docosahexaenoic acids, respectively. Infusion of linseed oil emulsion decreased plasma TAG concentrations compared with tallow and fish oil treatments, which were similar. Infusion of the tallow emulsion resulted in the highest concentrations of plasma nonesterified fatty acid (NEFA), insulin, and glucose, whereas the infusion derived from linseed oil had the lowest NEFA and beta-hydroxybutyric acid concentrations. The different TAG emulsions had no effect on total or peroxisomal oxidation of [1-(14C)]oleic acid in liver homogenates. Liver TAG content increased 12.0, 7.8, and 14.1 microg/microg of DNA during the fast for tallow, linseed oil, and fish oil treatments, respectively; linseed oil was different from fish oil and tended to be different from tallow.

Effects of conjugated linoleic acid isomers on lipid metabolism and gluconeogenesis in monolayer cultures of bovine hepatocytes.

The objective was to determine the effects of linoleic acid and different isomers of conjugated linoleic acid (CLA) at different concentrations on hepatic lipid and glucose metabolism in the bovine. Monolayer cultures of hepatocytes obtained from 7- to 10-d-old Holstein bull calves were exposed to treatments from 16 to 64 h after plating. The treatments included 1.0 mM palmitic acid plus either 0.1 or 1.0 mM of cis-9, cis-12 linoleic acid, cis-9, trans-11 CLA, or trans-10, cis-12 CLA. Metabolism of palmitic acid to cellular triacylglycerol (TAG) was decreased when media contained cis-9, trans-11 compared with trans-10, cis-12 CLA. Total cellular TAG content was increased for the CLA isomers compared to cis-9, cis-12 linoleic acid. Both CLA isomers increased palmitic acid incorporation into phospholipids, cholesterol, and media triacylglycerol compared with cis-9, cis-12 linoleic acid at a concentration of 1.0 mM. Increasing the concentration of treatment fatty acids from 0.1 to 1.0 mM decreased oxidation of palmitic acid to acid-soluble products, but no effects of fatty acids were observed. There was no treatment effect on rates of gluconeogenesis from propionic acid. Overall, CLA isomers elicited changes in palmitic acid metabolism to cellular and media triacylglycerol, and cellular phospholipids and cholesterol, but had little or no effect on other measured pathways of lipid metabolism or gluconeogenesis in bovine hepatocytes.

Effects of long chain fatty acids on lipid and glucose metabolism in monolayer cultures of bovine hepatocytes.

The objectives were to determine the long-term (48 h) effects of specific long chain fatty acids on hepatic lipid and glucose metabolism in monolayer cultures of bovine hepatocytes. From 16 to 64 h after plating, hepatocytes from three 7- to 10-d-old calves were exposed to one of the following treatments: 1 mM palmitic acid (1 mM C16:0), 2 mM palmitic acid (2 mM C16:0), or 1 mM palmitic acid plus 1 mM of either stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3), eicosapentaenoic (C20:5), or docosahexaenoic (C22:6) acid, or 0.5 mM each of eicosapentaenoic and docosahexaenoic acid (C20:5 + C22:6). The two treatments containing 2 mM of saturated fatty acids, 2 mM C16:0 and 1 mM C16:0 plus 1 mM C18:0, increased beta-hydroxybutyrate concentrations in the medium and [1-(14)C]palmitic acid oxidation to acid-soluble products compared with all other treatments. The treatment containing C22:6 increased total cellular triglyceride content and incorporation of [1-(14)C]palmitic acid into cellular triglycerides. The treatments containing C22:6 or C20:5 + C22:6 increased [1-(14)C]palmitic acid metabolism to phospholipids and cholesterol. The presence of C22:6 in the medium decreased metabolism of [2-(14)C]propionic acid either to glucose in the medium or to cellular glycogen. Overall, fatty acids differed in their effects on lipid and glucose metabolism in monolayer cultures of bovine hepatocytes with C22:6 eliciting the most profound changes.

Short communication: Net uptake of nonesterified long chain fatty acids by the perfused caudate lobe of the caprine liver.

The objective was to determine whether net uptake of various nonesterified long chain fatty acids differs in the caprine liver. Caudate lobes were isolated from four mature goats and perfused (1 ml/min x g wet tissue) with buffer containing 0.3 mM of each palmitic, stearic, oleic, linoleic, linolenic, eicosapentaenoic, and docosahexaenoic acids. The amount of fatty acid in the perfusate decreased over time for all fatty acids with the exception of stearic acid. There was no net uptake of stearic acid, which was significantly different from all other fatty acids examined, with the exception of oleic acid. Net hepatic uptake of oleic acid was numerically, but not significantly lower than palmitic, linoleic, linolenic, eicosapentaenoic, and docosahexaenoic acids. It was concluded that net uptake of fatty acids was similar for all fatty acids tested with the exception of stearic acid.

Metabolic fate of long-chain unsaturated fatty acids and their effects on palmitic acid metabolism and gluconeogenesis in bovine hepatocytes.

The objectives were to determine the metabolic fate of different long-chain fatty acids, and their effects on palmitic acid metabolism and gluconeogenesis in bovine hepatocytes. Hepatocytes were isolated from four ruminating calves and exposed in suspension for 3 h to one of the following treatments: 1 mM palmitic acid (1C16), 2 mM palmitic acid (2C16), or 1 mM palmitic acid plus either 1 mM oleic (C18:1), linoleic (C18:2), linolenic (C18:3), eicosapentaenoic (C20:5), or docosahexaenoic acid (C22:6). Oxidation of [1-(14)C]palmitic acid or one of the [1-(14)C]-labeled treatment fatty acids to CO2 or incorporation into cellular triglycerides (TG), phospholipids, cholesterol, and cholesterol esters were measured. Rates of oxidation to CO2 were 3- to 4-fold higher for C22:6 than for other fatty acids, with the exception of C20:5, which had intermediate rates of oxidation to CO2. In general, treatments 2C16 and C18:1 yielded the highest rates of incorporation into most cellular lipids, whereas the polyunsaturated fatty acids were poor substrates for incorporation into cellular lipids. The most pronounced change was a large reduction of polyunsaturated fatty acid incorporation into cellular TG compared to 1C16, 2C16, and C18:1. The unsaturated fatty acids also influenced palmitic acid metabolism. The addition of C20:5 yielded the highest rates of palmitic acid oxidation to CO2 followed by addition of C18:1 and C22:6. Treatments containing polyunsaturated fatty acids decreased palmitic acid metabolism to TG and total cellular lipids compared with treatments 2C16 and C18:1. Rates of gluconeogenesis from propionate were significantly higher for the treatment containing C18:1. Long-chain fatty acids vary in their routes of metabolism and influence palmitic acid metabolism and gluconeogenesis in bovine hepatocytes.

Effects of hyperinsulinaemia under euglycaemic condition on liver fat metabolism in dairy cows in early and mid-lactation.

The objective was to examine the effects of insulin under euglycaemic conditions on liver long chain fatty acids (LCFA) metabolism with special focus on the aetiology of hepatic lipidosis in early lactation. A 4-day hyperinsulinaemic-euglycaemic clamp (clamp) was conducted on four dairy cows starting in weeks 4 and 17 postpartum. Insulin was infused continuously (1 microg/kg BW per h) and a 50% glucose solution was infused to maintain euglycaemia. Liver biopsies were taken 6 days before, the last day of, and 5 days after the clamp, and blood samples were taken in the same period. In the liver tissue, the relative triglyceride content decreased (P < 0.01) and the glycogen content increased (P < 0.0001) in response to the clamp. Hepatic in vitro palmitate oxidation capacity was lowest during the clamp period and could be explained by a significant decrease in incomplete oxidation (ketogenesis) (P < 0.04) and a tendency to a decreased complete oxidation of palmitate (P < 0.10). Plasma non-esterified fatty acids concentration was decreased during the clamp in early lactation (P < 0.05) but there was no effect on the mid-lactation clamp. The present study shows that increased insulin under euglycaemic conditions seems to depress hepatic LCFA oxidation capacity. However, in terms of preventing hepatic lipidosis, the anti-lipolytic effect of insulin on adipose tissue, which results in decreased mobilization of and hence hepatic load with LCFA, appears more important.

Effects of a four-day hyperinsulinemic-euglycemic clamp in early and mid-lactation dairy cows on plasma concentrations of metabolites, hormones, and binding proteins.

The effects of insulin, using a 4 d hyperinsulinemic-euglycemic clamp, on plasma concentrations of hormone, metabolites, and binding proteins were evaluated in four Holstein dairy cows during wk 4 and 17 of lactation. Insulin was infused at 1 microg/kg/hr for 96 hr during the clamp period. Compared with the pre-clamp period, plasma insulin concentrations increased 7-fold and 4-fold during the clamp periods in early and mid-lactation, respectively. The total amount of glucose infused was higher (P < 0.05) during the clamp in early lactation. The clamp decreased plasma concentrations of non-esterified fatty acids (P < 0.001) during early lactation while differences in mid-lactation were minor. The clamp also decreased plasma concentration of beta-hydroxybutyrate (P < 0.001), plasma urea nitrogen (P < 0.001), and true protein (P < 0.01) although the patterns of decline differed between early and mid-lactation. Growth hormone (GH) concentrations decreased (P < 0.001) and insulin-like growth factor-1 (IGF-1) increased (P < 0.01) during the clamp period suggesting a direct effect of insulin on the un-coupling of the GH/IGF-1 axis. Levels of IGF binding protein-2 (IGFBP-2) decreased (P < 0.01) during the clamp period. The relative proportion of IGFBP-2 decreased (P < 0.001) and that of IGFBP-3 increased (P < 0.001) during the clamp period. There were no interactions between the clamp period and stage of lactation on GH, IGF-1, or IGFBPs. Overall, most plasma variables measured were affected in the same way during the two clamps, but the pattern of change often varied with stage of lactation.

Peripartum responses of dairy cows fed energy-dense diets for 3 or 6 weeks prepartum.

Pregnant cows (n = 189) in two commercial dairy farms were assigned randomly to be fed energy-dense diets for either 3 or 6 wk before expected calving. Cows fed diets for less than or equal to 26 d were designated the short (S) treatment group, and those fed greater than 26 d were the long (L) treatment group. Cows in L tended to have improved energy status during the first 2 wk postpartum, as indicated by higher insulin concentrations and a tendency for lower nonesterified fatty acid concentrations. Treatment did not affect plasma beta-hydroxybutyrate concentrations. Cows in L tended to gain more body condition during the late dry period. Total body condition loss from parturition through 6 wk postpartum was not different between treatments, but the rate of change varied over this period. Cows in S lost more body condition during the first 3 wk postpartum than cows in L. In farm 1 only, cows in L lost more body condition from 3 to 6 wk postpartum and had a higher incidence of metritis and a longer interval to first service than cows in S. Cows in L had higher milk protein content through 60 d in milk compared with cows in S. Additionally, cows in L in farm 1 produced 4.4 kg/d less milk, tended to have lower milk fat content and yields, and higher somatic cell counts through 150 d in milk than cows in S. Overall, increasing the length of time cows were fed the energy-dense diet prepartum elicited significant changes in farm 1, but had little effect in farm 2. Based on these results, L treatment may improve energy status immediately postpartum, but long-term effects varied between farms, perhaps due to other unmeasured management differences.

Peripartum responses of dairy cows to partial substitution of corn silage with corn grain in diets fed during the late dry period.

We randomly assigned 189 cows in a commercial dairy farm to dietary treatments with supplemental corn grain (SC) or without supplemental corn grain (NC) approximately 3 wk before expected parturition. Diets formulated were similar except that dry ground corn replaced 21% of the corn silage in one diet. Cows fed SC had reduced plasma beta-hydroxybutyrate and tended to have increased plasma insulin concentrations prepartum compared with cows fed NC. Treatment did not affect nonesterified fatty acid concentrations prepartum, any blood variables postpartum, or incidences of health disorders. Effects of treatment on production responses were highly dependent on parity as indicated by parity x treatment x time interactions for milk and protein yields. Primiparous cows fed SC had lower milk protein yield, higher somatic cell count and days open compared with cows fed NC. The SC diet resulted in lower milk yields in early lactation and increased somatic cell count and days open for cows in second parity. However, cows in third parity or greater fed the SC diet yielded more milk and protein in early lactation, and had decreased somatic cell counts and days open. Increasing the corn grain concentration of the diet fed prepartum was advantageous to third and greater parity cows in this experiment, but showed no benefits during lactation for cows in first or second parities.