MiRNome variations in milk fractions during feed restrictions of different intensities in dairy cows.

BACKGROUND: In dairy cows, diet is one factor that can affect their milk production and composition. However, the effect of feed restriction on milk miRNome has not yet been described. Indeed, milk is the body fluid with the highest RNA concentration, which includes numerous microRNA. Its presence in the four different milk fractions, whole milk, fat globules, mammary epithelial cells and extracellular vesicles, is still poorly documented. This study aimed to describe the effects of different feed restrictions on the miRNome composition of different milk fractions. RESULTS: Two feed restrictions were applied to lactating dairy cows, one of high intensity and one of moderate intensity. 2,896 mature microRNA were identified in the different milk fractions studied, including 1,493 that were already known in the bovine species. Among the 1,096 microRNA that were sufficiently abundant to be informative, the abundance of 1,027 of them varied between fractions: 36 of those were exclusive to one milk fraction. Feed restriction affected the abundance of 155 microRNA, with whole milk and milk extracellular vesicles being the most affected, whereas milk fat globules and exfoliated mammary epithelial cells were little or not affected at all. The high intensity feed restriction led to more microRNA variations in milk than moderate restriction. The target prediction of known microRNA that varied under feed restriction suggested the modification of some key pathways for lactation related to milk fat and protein metabolisms, cell cycle, and stress responses. CONCLUSIONS: This study highlighted that the miRNome of each milk fraction is specific, with mostly the same microRNA composition but with variations in abundance between fractions. These specific miRNomes were affected differently by feed restrictions, the intensity of which appeared to be a major factor modulating milk miRNomes. These findings offer opportunities for future research on the use of milk miRNA as biomarkers of energy status in dairy cows, which is affected by feed restrictions.

Milk metabolites as noninvasive indicators of nutritional status of mid-lactation Holstein and Montbéliarde cows.

The objective was to investigate the effects of feed restriction on concentrations of selected milk metabolites in mid-lactation Holstein and Montbéliarde cows and to explore their correlations with energy balance and classic plasma and milk indicators of nutritional status. Eight Holstein and 10 Montbéliarde cows (165 ± 21 d in milk) underwent 6 d of feed restriction during which feed allowance was reduced to meet 50% of their net energy for lactation (NEL) requirements. The experiment was divided in 4 periods: control (CON; d -3 to -1), restriction (RES; d 1 to 6), wk 1 (W1; d 7 to 13), and wk 2 (W2; d 14 to 18) after refeeding at ad libitum intake. Intake, milk production, energy balance and plasma metabolites were used to validate the feed restriction model. Concentrations of 7 milk metabolites: ß-hydroxybutyrate (BHB), glucose, glucose-6-phosphate, isocitrate, glutamate, uric acid, and free amino groups were measured in morning milk samples, and fatty acids were measured in pooled p.m. and a.m. samples. Feed restriction induced a negative energy balance (-42.5 ± 4.4 MJ/d), increased plasma nonesterified fatty acids and BHB, and decreased plasma glucose concentrations. Feed restriction increased milk glucose-6-phosphate and isocitrate (+38% and +39%, respectively) and decreased milk BHB, glucose, glutamate, uric acid and free amino group concentrations (-20%, -57%, -65%, -42%, and -14%, respectively), compared with pre- restriction. Milk concentrations of medium-chain fatty acids (e.g., sum of C10 to C15) decreased and those of long chain (e.g., 18:0, cis-9 18:1) increased during restriction. Breed differences were not detected for the majority of variables. All studied milk metabolites were significantly correlated with energy balance (Spearman correlation = 0.48, 0.63, -0.31, -0.45, and 0.61 for BHB, glucose, glucose-6-phosphate, isocitrate, and glutamate, respectively). Milk glucose and glutamate showed the strongest correlations with plasma metabolites and milk FA associated with lipomobilization. These results suggest that milk metabolites may be used as noninvasive indicators of negative energy balance and metabolic status of dairy cows.

Deep RNA-Seq reveals miRNome differences in mammary tissue of lactating Holstein and Montbéliarde cows.

BACKGROUND: Genetic polymorphisms are known to influence milk production and composition. However, the genomic mechanisms involved in the genetic regulation of milk component synthesis are not completely understood. MicroRNAs (miRNAs) regulate gene expression. Previous research suggests that the high developmental potential of the mammary gland may depend in part on a specific miRNA expression pattern. The objective of the present study was to compare the mammary gland miRNomes of two dairy cow breeds, Holstein and Montbéliarde, which have different mammogenic potentials that are related to differences in dairy performance. RESULTS: Milk, fat, protein, and lactose yields were lower in Montbéliarde cows than in Holstein cows. We detected 754 distinct miRNAs in the mammary glands of Holstein (n = 5) and Montbéliarde (n = 6) midlactating cows using RNA-Seq technology, among which 738 were known and 16 were predicted miRNAs. The 25 most abundant miRNAs accounted for 90.6% of the total reads. The comparison of their abundances in the mammary glands of Holstein versus Montbéliarde cows identified 22 differentially expressed miRNAs (Padj ≤ 0.05). Among them, 11 presented a fold change ≥2, and 2 (miR-100 and miR-146b) were highly expressed. Among the most abundant miRNAs, miR-186 is known to inhibit cell proliferation and epithelial-to-mesenchymal transition. Data mining showed that 17 differentially expressed miRNAs with more than 20 reads were involved in the regulation of mammary gland plasticity. Several of them may potentially target mRNAs involved in signaling pathways (such as mTOR) and lipid metabolism, thereby indicating that they could influence milk composition. CONCLUSION: We found differences in the mammary gland miRNomes of two dairy cattle breeds. These differences suggest a potential role for miRNAs in mammary gland plasticity and milk component synthesis, both of which are related to milk production and composition. Further research is warranted on the genetic regulation of miRNAs and their role in milk synthesis.

Extruded linseed alone or in combination with fish oil modifies mammary gene expression profiles in lactating goats.

Nutrition is a major factor that regulates ruminant milk components, particularly its fatty acid (FA) composition, which is an important determinant of milk nutritional quality. In the mammary gland, milk component biosynthesis involves a large number of genes under nutritional regulation that are not well understood. Thus, the objective of the present study was to evaluate the effects of extruded linseeds (EL) alone or in combination with fish oil (ELFO) on goat mammary gene expression. In total, 14 goats were fed one of the following three diets: a natural grassland hay basal diet (CTRL) alone, CTRL supplemented with 530 g/day of EL, or 340 g/day of EL plus 39 g/day of fish oil (ELFO). Mammary secretory tissues were collected after slaughter on day 28, to determine the expression of 14 lipogenic genes and five lipogenic enzyme activities and transcriptomic profiles. The mRNA abundance decreased for SCD1 (P<0.1) with ELFO v. CTRL, and for AZGP1 (P<0.1) and ACSBG1 (P<0.05) decreased with EL v. ELFO and the CTRL diets (only for ACSBG1), respectively. Transcriptomic analyses performed using a bovine microarray revealed 344 and 314 differentially expressed genes (DEG) in the EL and ELFO diets, respectively, compared with the CTRL diet, with 76 common DEGs. In total, 21 and 27 DEGs were involved in lipid metabolism and transport class in the EL and ELFO v. the CTRL diets, respectively, with eight common genes (ALDH3B1, ALDH18A1, DGKD1, ENPP1, IL7, NSMAF, PI4KA and SERINC5) down-regulated by these two treatments. In EL v. CTRL diets, a gene network related to lipid metabolism and transport was detected. Although this network was not detected in the ELFO v. CTRL analysis, five genes known to be involved in lipid metabolism and transport were up-regulated (SREBF1, PPARG and GPX4) or down-regulated (FABP1 and ENPP6) by ELFO. The protein metabolism and transport biological processes were largely altered by both EL and ELFO v. CTRL diets without changes in major milk protein secretion. Amino acid metabolism was highlighted as an enriched network by Ingenuity Pathway Analysis and was similar to cellular growth and proliferation function. Two regulation networks centered on the estrogen receptor (ESR1) and a transcriptional factor (SP1) were identified in EL and ELFO v. CTRL diets. In conclusion, these results show that these two supplemented diets, which largely changed milk FA composition, had more effects on mRNA linked to protein metabolism and transport pathways than to lipid metabolism, and could affect mammary remodeling.

Effects of body condition score at calving on indicators of fat and protein mobilization of periparturient Holstein-Friesian cows.

The objective was to study the effects of body condition score (BCS) at calving on dairy performance, indicators of fat and protein mobilization, and metabolic and hormonal profiles during the periparturient period of Holstein-Friesian cows. Twenty-eight multiparous cows were classed according to their BCS (0 to 5 scale) before calving as low (BCS ≤ 2.5; n=9), medium (2.75 ≤ BCS ≤ 3.5; n=10), and high (BCS ≥ 3.75; n=9), corresponding to a mean of 2.33, 3.13, and 4.17 points of BCS, and preceding calving intervals of 362, 433, and 640 d, respectively. Cows received the same diets based on preserved grass to allow ad libitum feed intake throughout the study, and lactation diet contained 30% of concentrate (dry-matter basis). Measurements and sampling were performed between wk -4 and 7 relative to calving. No significant effects were observed of BCS group on dry matter intake (kg/d), milk yield, BCS loss, plasma glucose, and insulin concentrations. The high-BCS group had the lowest postpartum energy balance and the greatest plasma concentrations of leptin prepartum, nonesterified fatty acids and ß-hydroxybutyrate postpartum, insulin-like growth factor 1, and milk fat content. Milk fat yield was greater for the high- than the low-BCS group (1,681 vs. 1,417 g/d). Low-BCS cows had the greatest concentration of medium-chain fatty acids (e.g., sum of 10:0 to 15:0, and 16:0), and the lowest concentration and secretion of preformed fatty acids (e.g., cis-9 18:1) in milk fat. Milk protein secretion was lowest in the low-BCS group, averaging 924, 1,051, and 1,009 g/d for low-, medium-, and high-BCS groups, respectively. Plasma 3-methylhistidine was greater in wk 1 and 2 postpartum compared with other time points, indicating mobilization of muscle protein. Plasma creatinine tended to be lower and the 3-methylhistidine: creatinine ratio was greater in low- compared with medium- and high-BCS cows, suggesting less muscle mass but more intense mobilization of muscle protein in lean cows. High-BCS cows were metabolically challenged during early lactation due to intense mobilization of body fat. Conversely, limited availability of body fat in low-BCS cows was associated with increased plasma indicators of body protein mobilization during the first weeks of lactation, and lower milk protein secretion. These results should be confirmed using an experimental approach where calving BCS variation would be controlled by design.

The transcriptomic profiles of adipose tissues are modified by feed deprivation in lactating goats.

A major function of ruminant adipose tissue is to store lipids for use in productive functions. Body fat mobilization is required during periods of negative energy balance such as lactation or undernutrition. Until now, gene expression profiling of ruminant adipose tissue in response to nutritional restriction has not been performed. To gain a better understanding of the molecular mechanisms in adipose tissue in response to dietary factors, microarray analysis was used to compare the effects of two extreme nutritional conditions (control diet vs. 48-h feed deprivation) in the omental and perirenal adipose tissues of lactating goats (Capra hircus). We observed the altered expression of 456 and 199 genes in omental and perirenal adipose tissues, respectively. Similar biological processes were altered by feed deprivation in these two sites, although twice as many genes were differentially expressed in the omental than in the perirenal adipose tissue. Taken together, the transcriptional changes involved in lipid metabolism (decreased lipid synthesis and triglyceride storage capacity as well as increased fatty acid oxidation) were consistent with reduced energy deposition in goat adipose tissues in response to a 48-h fast. An inflammatory state of the adipose tissue was observed following the 48-h fast.

Glucose-6-phosphate dehydrogenase and leptin are related to marbling differences among Limousin and Angus or Japanese Black x Angus steers.

This work investigated the metabolic basis for the variability of carcass and i.m. adiposity in cattle. Our hypothesis was that the comparison of extreme breeds for adiposity might allow for the identification of some metabolic pathways determinant for carcass and i.m. adiposity. Thus, 23- to 28-mo-old steers of 3 breeds, 2 with high [Angus or Japanese Black x Angus (J. Black cross)] and 1 with low (Limousin) i.m. and carcass adiposity, were used to measure activities or mRNA levels, or both, of enzymes involved in de novo lipogenesis [acetyl-coA carboxylase, fatty acid synthase (FAS), glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme], circulating triacylglycerol (TAG) uptake (lipoprotein lipase), and fatty acid esterification (glycerol-3-phosphate dehydrogenase), as well as the mRNA level of leptin, an adiposity-related factor. In a first study, enzyme activities were assayed in the s.c. adipose tissue (AT), the oxidative rectus abdominis, and the glycolytic semitendinosus muscles from steers finished for 6 mo. Compared with Angus or J. Black cross, Limousin steers had a 27% less (P = 0.003) rib fat thickness, and 23 and 29% less (P < or = 0.02) FAS and G6PDH activities in s.c. AT. In rectus abdominis and semitendinosus, the 75% less (P < 0.001) TAG content was concomitant with 50% less (P < 0.001) G6PDH activity. In a second study, enzyme activities plus mRNA levels were assayed in an oxido-glycolytic muscle, the longissimus thoracis (LT), in the i.m. AT dissected from LT, and in s.c. AT from the same Limousin steers and from Angus steers finished for 10 mo. Compared with Angus, the 50% less (P < 0.001) rib fat thickness in Limousin contrasted with the 1.1- to 5.8-fold greater (P < or = 0.02) mRNA levels or activities, or both, of acetyl-coA carboxylase, G6PDH, lipoprotein lipase, and glycerol-3-phosphate dehydrogenase in s.c. AT. Conversely, the 90% less (P < 0.001) TAG content in Limousin LT was concomitant to the 79 and 83% less (P < or = 0.002) G6PDH activity and leptin mRNA level. Such differences could arise from a greater number of adipocytes in LT from Angus steers because no difference was found between Limousin and Angus for G6PDH activity and leptin mRNA in i.m. AT. We conclude that FAS and G6PDH in s.c. AT could be involved in differences in carcass adiposity, but this relationship disappeared when the fatness increased strongly. Leptin and G6PDH are related to the expression of marbling whatever the body condition and thus could be relevant indicators of marbling in beef cattle.

Butters rich either in trans-10-C18:1 or in trans-11-C18:1 plus cis-9, trans-11 CLA differentially affect plasma lipids and aortic fatty streak in experimental atherosclerosis in rabbits.

Dairy fat contains high amounts of saturated fatty acids (FA), which are associated with cardiovascular disease (CVD) risk. Manipulation of dairy cows nutrition allows to decrease the saturated FA content of milk fat, and is associated with increases either in conjugated linoleic acid (CLA) and trans-11-C18:1 contents, or in trans-10-C18:1 content. CLA putatively exhibits beneficial properties on CVD risk, whereas trans FA are suspected to be detrimental. The present study compared the effects of a trans-10-C18:1-rich butter (T10 butter), a trans-11-C18:1+CLA-rich butter (T11-CLA butter) and a standard butter (S butter) on lipid parameters linked to the CVD risk and fatty streaks. Thirty-six White New Zealand rabbits were fed one of the three butters (12% of the diet, plus 0.2% cholesterol) for 6 (experiment 1) or 12 (experiment 2) weeks. Liver lipids, plasma lipids and lipoprotein concentrations (experiments 1 and 2) and aortic lipid deposition (experiment 2) were determined. The T10 butter increased VLDL-cholesterol compared with the two others, and total and LDL-cholesterol compared with the T11-CLA butter ( P < 0.05). The T10 butter also increased non-HDL/HDL ratio and aortic lipid deposition compared with the T11-CLA butter ( P < 0.05). The T11-CLA butter non-significantly reduced aortic lipid deposition compared with the S butter, and decreased HDL-cholesterol and increased liver triacyglycerols compared with the two other butters ( P < 0.05). These results suggest that, compared with the S butter, the T10 butter had detrimental effects on plasma lipid and lipoprotein metabolism in rabbits, whereas the T11-CLA butter was neutral or tended to reduce the aortic lipid deposition.

Influence of the diet and grazing on adipose tissue lipogenic activities and plasma leptin in steers.

The objectives of the two experiments were to determine the respective effects and interactions of diet type (grass v. maize diets) and physical activity (grazing v. zero grazing) on lipogenic enzyme activities and adipose cell size in subcutaneous, perirenal and intermuscular adipose tissues and on plasma metabolites and hormones in Charolais steers. After weaning, the steers were assigned to two (Experiment 1, n = 24) or three (Experiment 2, n = 24) groups, with steers in Experiment 1 grazed grass or indoors maize-silage-fed and steers in Experiment 2 grazed grass, indoors cut grass- or indoors maize-silage-fed. Both experiments lasted for 23 months. All grass-fed animals were fed grass silage during the two winter seasons. During the two summer seasons, steers fed on grass were rotationally grazed on a perennial rye-grass pasture while steers fed on cut grass were fed indoors on freshly cut grass alone. Steers fed on maize silage were fed maize silage indoors during the entire experiment. All animals were reared for similar body weight and growth rates and slaughtered at the same age (31 to 32 months). Activities of lipogenic enzymes were significantly lower in the three adipose tissue sites of steers fed cut grass compared with maize silage, although there were less-marked effects in intermuscular adipose tissue. Plasma insulin and glucose concentrations were also lower in steers fed cut grass whereas plasma leptin concentration was similar. As body fat content was not affected by nutritional treatment, it is suggested that the decrease in potential lipogenic activity was associated with the nature of the diet and not to differences in available net energy. In other respects, grazed grass compared with eating cut grass did not affect lipogenic enzyme activities but decreased plasma leptin concentrations in the older steers and increased plasma non-esterified fatty acids and glucose concentrations without affecting adipose tissue weight and adipose cell size.

Mammary lipid metabolism and milk fatty acid secretion in alpine goats fed vegetable lipids.

Fourteen Alpine goats at midlactation were fed a diet of hay and concentrate (55:45), without (control) or with formaldehyde-treated linseed (FLS) or oleic sunflower oil (OSO) at 11.2 or 3.5% of dry matter intake, respectively, in a 3 x 3 Latin Square design with three 3-wk periods. Milk yield was lower in goats fed FLS than control or OSO (2.13 vs. 2.32 kg/d). Milk fat content was higher with FLS or OSO than control (40.8 vs. 33.8 g/kg). Formaldehyde-treated linseed and OSO caused a significant decrease (23 and 18%, respectively) of C10 to C17 fatty acids secretion compared with control. The secretion of cis-9 C18:1 and cis-9, trans-11 C18:2 were increased 1.44- and 1.54-fold for FLS and 1.78- and 1.36-fold for OSO, compared with control. The C18:3 (n-3) secretion was increased 2.61-fold with FLS compared with control. Milk cis-9 C14:1/C14:0, cis-9 C16:1/C16:0, and cis-9 C18:1/C18:0 ratios decreased with the supplemented diets compared with control. Mammary stearoyl-CoA desaturase mRNA and activity were decreased by the lipid supplements, whereas no significant change was observed for acetyl-CoA carboxylase and fatty acid synthase. The activities of glucose-6-phosphate dehydrogenase, malic enzyme, and glycerol-3-phosphate dehydrogenase were not affected by the lipid supplements. Mammary lipoprotein lipase mRNA increased with OSO, whereas lipoprotein lipase activity tended to decrease with FLS compared with control. Milk lipoprotein lipase activity sharply decreased with lipid supplement (by 59 and 71%, for FLS and OSO, respectively). The changes in milk fatty acid profile due to FLS and OSO supplements were partly related to changes in the levels of mammary enzyme activities or mRNA.

Plasma leptin concentration in adult cattle: effects of breed, adiposity, feeding level, and meal intake.

An ovine-specific RIA, shown to be reliable for bovine leptin determination, was used to study the effects of breed, body fatness, feeding level, and meal intake on plasma leptin level in adult cattle. Eighteen fat Charolais, fat Holstein, and lean Holstein adult cows were either well-fed (130% of maintenance energy requirements [MER]) or underfed (60% of MER) for 3 wk. The breed tended to have a small effect on plasma leptin level, which was decreased by 70% (P < 0.05) in lean compared to fat Holstein cows. A strong curvilinear relationship was found between mean adipocyte volume and plasma leptin concentrations in well-fed (r = +0.95) and underfed (r = +0.91) cows. Underfeeding caused a significant decrease in plasma leptin levels from 8.0+/-3.1 to 6.1+/-2.3 ng/mL (P < 0.01). Nine adult Holstein cows initially fed at 130% of MER (control) were underfed to 21% of MER for 7 d, and five of them were refed to 237% of MER for 21 d. Plasma leptin measured 1 h before meal distribution was decreased from 5.9+/-0.4 to 3.8+/-0.2 ng/mL (P < 0.01) by underfeeding and increased to reach 8.8+/-1.0 ng/mL (P < 0.01) after refeeding. It was positively related to plasma glucose (r = +0.52, P < 0.01) and negatively related to plasma NEFA (r = -0.67, P < 0.001). Plasma leptin measured 4 h after meal distribution was positively related to feeding level and to plasma 3-OH-butyrate (r = +0.61, P < 0.005) and negatively related to plasma NEFA (r = -0.56, P < 0.01). Differences between pre- and postprandial leptin concentrations showed a decrease after meal intake in control and well-fed cows (-7 and -19%, P < 0.01, respectively) and an increase in underfed cows (+12%, P < 0.01). Leptin response to meal intake was positively related to glucose response (r = +0.66, P < 0.001) and negatively related to 3-OH-butyrate response (r = -0.78, P < 0.001). By using the "multispecies" commercial RIA, leptin concentrations were lower and we observed similar physiological responses, although less related to other hormones or metabolites. These data provide evidence, first, that a specific RIA for ruminant leptin determination is necessary to better understand leptin regulation, and second, that plasma leptin is strongly related to adipose cell size and positively related to feeding level in adult cattle, and that an effect of meal intake could be mediated by glucose and(or) ketone bodies.

Effects of photoperiod and feeding level on adipose tissue and muscle lipoprotein lipase activity and mRNA level in dry non-pregnant sheep.

The aim of the present study was to investigate the effects of photoperiod and feeding level on lipid metabolism in ovine perirenal and subcutaneous adipose tissues (AT) and in skeletal and cardiac muscles. Twenty dry non-pregnant ovariectomised ewes were divided into two groups and subjected to either 8 h or 16 h light/d, and underfed at 22 % energy requirements for 7 d. Half of the ewes in each group were slaughtered and the remaining ewes were refed at 190 % energy requirements for 14 d, until slaughtering. Refeeding increased (2.6-4.3-fold) malic enzyme (ME), fatty acid synthase (FAS), glucose-6-phosphate dehydrogenase (G6PDH) and glycerol-3-phosphate dehydrogenase (G3PDH) activities in subcutaneous AT as well as lipoprotein lipase (LPL) activity in perirenal (3.5-fold) and subcutaneous (10-fold) AT and to a lesser extent (1.4-fold) in the skeletal longissimus thoracis and cardiac muscles. Moreover, variations of LPL mRNA level followed variations of LPL activity: refeeding increased perirenal AT- and cardiac muscle-mRNA levels (7.4- and 2-fold respectively). The main finding of this study is that, for a given level of food intake, long days (compared with short days) increased the LPL activity in the longissimus thoracis muscle and, in refed ewes, the activities of LPL and ME in subcutaneous AT. Furthermore, long days increased LPL mRNA level in cardiac muscle and perirenal AT. Thus, our results show that there are direct effects of photoperiod on sheep AT lipogenic potential, as well as on muscle LPL activity, which are not caused by changes in nutrient availability.

Leptin in ruminants. Gene expression in adipose tissue and mammary gland, and regulation of plasma concentration.

This paper reviews data on leptin gene expression in adipose tissue (AT) and mammary gland of adult ruminants, as well as on plasma leptin variations, according to genetic, physiological, nutritional and environmental factors. AT leptin mRNA level was higher in sheep and goat subcutaneous than visceral tissues, and the opposite was observed in cattle; it was higher in fat than in lean selection line in sheep; it was decreased by undernutrition and increased by refeeding in cattle and sheep, and not changed by adding soybeans to the diet of lactating goats; it was increased by injection of NPY to sheep, and by GH treatment of growing sheep and cattle. Insulin and glucocorticoids in vitro increased AT leptin mRNA in cattle, and leptin production in sheep. Long daylength increased AT lipogenic activities and leptin mRNA, as well as plasma leptin in sheep. Mammary tissue leptin mRNA level was high during early pregnancy and was lower but still expressed during late pregnancy and lactation in sheep. Leptin was present in sheep mammary adipocytes, epithelial and myoepithelial cells during early pregnancy, late pregnancy and lactation, respectively. Plasma leptin in cattle and sheep was first studied thanks to a commercial "multi-species" kit. It was positively related to body fatness and energy balance or feeding level, and decreased by beta-agonist injection. The recent development of specific RIA for ruminant leptin enabled more quantitative study of changes in plasma leptin concentration, which were explained for 35--50% by body fatness and for 15--20% by feeding level. The response of plasma leptin to meal intake was related positively to glycemia, and negatively to plasma 3-hydroxybutyrate. The putative physiological roles of changes in leptin gene expression are discussed in relation with published data on leptin receptors in several body tissues, and on in vivo or in vitro effects of leptin treatment.

Adipose tissue metabolism and its role in adaptations to undernutrition in ruminants.

Changes in the amount and metabolism of adipose tissue (AT) occur in underfed ruminants, and are amplified during lactation, or in fat animals. The fat depot of the tail of some ovine breeds seems to play a particular role in adaptation to undernutrition; this role could be linked to its smaller adipocytes and high sensitivity to the lipolytic effect of catecholamines. Glucocorticoids and growth hormone probably interact to induce teleophoretic changes in the AT responses to adenosine and catecholamines during lactation. Fat mobilization in dry ewes is related both to body fatness and to energy balance. The in vivo beta-adrenergic lipolytic potential is primarily related to energy balance, whereas basal postprandial plasma non-esterified fatty acids (NEFA) are related to body fatness, and preprandial plasma NEFA is the best predictor of the actual body lipid loss. Several mechanisms seem to be aimed at avoiding excessive fat mobilization and/or insuring a return to the body fatness homeostatic set point. As well as providing the underfed animal with fatty acids as oxidative fuels, AT acts as an endocrine gland. The yield of leptin by ruminant AT is positively related to body fatness, decreased by underfeeding, beta-adrenergic stimulation and short day length, and increased by insulin and glucocorticoids. This finding suggests that the leptin chronic (or acute) decrease in lean (or underfed respectively) ruminants is, as in rodents, a signal for endocrine, metabolic and behavioural adaptations aimed at restoring homeostasis.

Lipoprotein lipase activity and mRNA are up-regulated by refeeding in adipose tissue and cardiac muscle of sheep.

Previous studies in rodents have shown that the lipoprotein lipase (LPL) regulation is complex and often opposite in adipose tissue (AT) and muscle in response to the same nutritional treatment. However, neither LPL responses nor the molecular mechanisms involved in the nutritional regulation have been studied in both AT and muscle of ruminant species. To explore this, we measured the LPL activity and mRNA levels in perirenal AT and cardiac muscle (CM) of control, 7-d-underfed or 14-d-refed ewes. Underfeeding decreased (P < 0.01) LPL activity both in AT (-59%) and CM (-31%), and these activities were restored (P < 0.01) by refeeding (AT, +248%; CM, +34%). Variations of LPL mRNA level measured by real-time reverse transcription-polymerase chain reaction or by Northern blot followed variations of LPL activity: underfeeding decreased AT- and CM-LPL mRNA levels (-58 and -53%, respectively), and refeeding restored (P < 0.01) them in CM (+117%) and increased them over the baseline in AT (+640%). Quantification of either 3.4- or 3.8-kb LPL mRNA levels revealed a predominant (P < 0.001) expression of the 3.4-kb mRNA in AT (60%) and of the 3.8-kb mRNA in CM (56%), without any preferential regulation of one of these mRNA species by the nutritional status. This work reveals a tissue-specific expression pattern of the ovine LPL gene and a pretranslational nutritional regulation of its expression, which is achieved in the same direction in perirenal AT and CM. The different regulation of CM-LPL between ewes and rats probably arises from peculiarities of ruminant species for nutrient digestion and absorption and liver lipogenesis.

Messenger RNAs encoding lipoprotein lipase, fatty acid synthase and hormone-sensitive lipase in the adipose tissue of underfed-refed ewes and cows.

The mechanisms involved in the nutritional regulation of genes encoding lipogenic (lipoprotein lipase (LPL) and fatty acid synthase (FAS)) and lipolytic (hormone-sensitive lipase (HSL)) enzymes were investigated by comparing the levels of the corresponding mRNAs in the adipose tissue (AT) of underfed or underfed-refed ewes and cows. Refeeding sharply increased LPL and FAS activities (19-25- and 6-8-fold, respectively) and moderately increased (2-4 fold) the activities of glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME) and glycerol-3-phosphate dehydrogenase (G3PDH). Northern blot analysis revealed three LPL transcripts and a single FAS transcript in cow and ewe AT. A single HSL mRNA was detected in cow AT and two transcripts in ewe AT. Refeeding sharply increased LPL and FAS mRNA levels, while restriction slightly increased (cows) or had no effect (ewes) on the HSL mRNA levels. This suggests that nutritional factors regulate sharply the expression of LPL and FAS genes by pretranslational mechanisms, but less clearly that of HSL gene.

Effects of photoperiod and feeding level on perirenal adipose tissue metabolic activity and leptin synthesis in the ovariectomized ewe.

Leptin is secreted by adipose tissue and plays a pivotal role in regulating both body energy homeostasis and reproductive function in rodents. Among livestock, sheep is a seasonal breeder whose reproductive period is initiated by short daylength. We show that plasma leptin and leptin gene expression in perirenal adipose tissue were decreased when ovariectomized Lacaune ewes were exposed to short days (8 versus 16 h light/d). This effect of the photoperiod occurred despite the nutritional status, with leptin levels lower in underfed than in refed ewes, and without significant changes in perirenal fat mass and adipocyte size. Plasma prolactin and leptin followed a similar pattern suggesting a relationship between the two hormones. These findings indicate, for the first time, that adipose tissue leptin is modulated by daylength independently of food intake, body fatness and gonadal activity. Furthermore, plasma non-esterified fatty acids of underfed ewes subjected to short days were more elevated than for underfed ewes on long days. On the other hand, refed ewes placed under long daylength tended to have a higher adipose tissue lipogenic activity than refed ewes on short days. We propose that these adaptations of leptin production and lipogenic activity with long photoperiod are of physiological significance for body fat deposition, which naturally occurs during long days when food is abundant. Conversely, a low leptin level during short days may enhance the sensitivity to food deprivation during the natural reproductive season, where any food shortage would decrease plasma leptin under a threshold critical for reproduction. Furthermore, in this situation, the observed enhanced ability to mobilize body fat may be related to the necessity to cope with energy shortage.

Insulin and/or dexamethasone regulation of lactate production and its relationship to glucose utilization by ovine and bovine adipose tissue explants incubated for 7 days.

This study investigated the hormonal regulation of lactate production and the lactate production/glucose utilization ratio in sheep and cow adipose tissue (AT) explants. The effects of insulin (2 mU/mL) and/or dexamethasone (DEX, 100 nM) were measured in perirenal AT of adult non-lactating non-pregnant cows (n = 5) or ewes (n = 5) given a restricted diet followed by overfeeding. The AT explants were incubated for 7 days. Under basal conditions, the lactate production was two times greater in sheep than in cow AT. Insulin increased lactate production in the two species, whereas DEX decreased it. DEX, in the presence of insulin, increased lactate production from day 3 to day 7. The lactate production/glucose utilization ratio was about two times greater in sheep than in cow AT. The presence of insulin increased, and that of DEX decreased this ratio in sheep AT, whereas it was not changed in cow AT. DEX, in the presence of insulin, increased this ratio in the two species after 4 or 5 days.

Lipoprotein lipase and glucose-6-phosphate dehydrogenase activities in bovine and ovine adipose tissue incubated for 7 days: effects of insulin and/or dexamethasone.

The in vitro regulation of lipoprotein lipase (LPL) and glucose-6-phosphate dehydrogenase (G6PDH) activity in bovine and ovine adipose tissue was investigated. Adult non-lactating non-pregnant cows (n = 5) or ewes (n = 5) were given limited amounts of feed for 8 or 10 days and then overfed for 10 (ewes) or 21 (cows) days. Perirenal adipose tissue explants were incubated for 2, 4 or 7 days. Regardless of the experimental conditions, the activity of LPL and G6PDH after 2 days of incubation was lower than in fresh tissue. Insulin significantly increased LPL activity in bovine but not in ovine adipose tissue, and it had no effect on G6PDH activity in the two species. Dexamethasone addition to the insulin-supplemented medium significantly increased LPL activity in ovine adipose tissue, whereas it was decreased in bovine adipose tissue on days 4 and 7. Moreover, dexamethasone addition to the insulin-supplemented medium did not change G6PDH activity in the two species on day 2, whereas it was increased in bovine and ovine adipose tissue on days 4 and 7. Therefore, the effects of insulin and/or dexamethasone on LDL and G6PDH differed with ruminant species and incubation time.

Insulin and(or) dexamethasone regulation of glucose and acetate utilization in ovine and bovine adipose tissue explants incubated for seven days.

An in vitro system was used to study the regulation of glucose and acetate utilization by bovine and ovine adipose tissue (AT). Adult nonlactating, nonpregnant cows (n = 5) and ewes (n = 5) were given limited amounts of feed for 8 to 10 d, then overfed for 10 (ewes) or 21 (cows) d. Perirenal AT explants were incubated for 7 d. The effect of 2 mU/mL of insulin on glucose utilization was significant in bovine but not in ovine AT during the 1st d in vitro, whereas the relationship was reversed from the 4th d of incubation. The effect of insulin on acetate utilization was significant in bovine but not in ovine AT. The addition of dexamethasone (DEX, 100 nM) to the insulin-supplemented medium increased (P < .05) glucose and acetate utilization by ovine AT. However, DEX did not affect substrate utilization by bovine AT in the presence of insulin. The DEX alone decreased glucose and acetate utilization by bovine and ovine AT during the first 4 d of incubation. So, the effects of insulin and(or) DEX on substrate utilization differed with species and incubation time.