Plasma leptin and mRNA expression of lipogenesis and lipolysis-related factors in bovine adipose tissue around parturition

The objective was to study changes in plasma leptin concentration parallel to changes in the gene expression of lipogenic- and lipolytic-related genes in adipose tissue of dairy cows around parturition. Subcutaneous fat biopsies were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. Blood samples were assayed for concentrations of leptin and non-esterified fatty acids (NEFA). Subcutaneous adipose tissue was analysed for mRNA abundance by real-time qRT-PCR encoding for leptin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), hormones-sensitive lipase (HSL), perilipin (PLIN), lipoprotein lipase (LPL), acyl-CoA synthase long-chain family member 1 (ACSL1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN) and glycerol-3-phosphate dehydrogenase 2 (GPD2). Body weight and body condition score of the cows were lower after parturition than before parturition. The calculated energy balance was negative in week 1 and 5 p.p., with higher negative energy balance in week 1 p.p. compared with that in week 5 p.p. On day 1 p.p., highest concentrations of NEFA (353.3 μmol/l) were detected compared with the other biopsy time-points (210.6 and 107.7 μmol/l, in week 8 a.p., and week 5 p.p. respectively). Reduced plasma concentrations of leptin during p.p. when compared with a.p. would favour increasing metabolic efficiency and energy conservation for mammary function and reconstitution of body reserves. Lower mRNA abundance of ACC and FASN expression on day 1 p.p. compared with other biopsy time-points suggests an attenuation of fatty acid synthesis in subcutaneous adipose tissue shortly after parturition. Gene expression of AdipoR1, AdipoR2, HSL, PLIN, LPL, ACSL1 and GPD2 was unchanged over time.     

Transition period-related changes in the abundance of the mRNAs of adiponectin and its receptors,of visfatin,and of fatty acid binding receptors in adipose tissue of high-yielding dairy cows

Adipose tissue expresses adipokines, which are involved in regulation of energy expenditure, lipid metabolism, and insulin sensitivity. To adapt for the transition from pregnancy to lactation, particularly in high-yielding dairy cows, adipokines, their receptors, and particular G-protein coupled receptors (GPRs) are of potential importance. Signaling by GPR 41 stimulates leptin release via activation by short-chain fatty acids; GPR 43/109A inhibits lipolysis, and GPR 109A thereby mediates the lipid-lowering effects of nicotinic acid and β–hydroxybutyrate. The aim of this study was to compare the mRNA expression of adiponectin and visfatin, adiponectin receptors 1 and 2 (AdipoR1/2), leptin receptor (obRb), insulin receptor as of the aforementioned GPRs during the transition period in high-yielding dairy cows. Biopsies from subcutaneous fat and blood samples were obtained from 10 dairy cows 1 week before and 3 weeks after calving. For AdipoR1 and AdipoR2 mRNA abundance as well as for leptin concentrations in plasma, a reduction (P ≤ .05) was observed postpartum; for visfatin and putative GPR 109A mRNA abundance in adipose tissue, there was a trend (P < .1) for analogous changes. In contrast, the mRNA content of obRb and GPR 41 in adipose tissue was higher (P ≤ .05) in samples from early lactation than in those from late gestation. Our results indicate decreasing adiponectin sensitivity in adipose tissue after calving, which might be involved in the reduced insulin sensitivity of adipose tissue during early lactation. In addition, visfatin, GPR 41, and GPR 109A may further modulate insulin sensitivity.     

Effects of short-term infusion with propionate on the mRNA expression of a putative G-protein coupled receptor 41 (GPR41) in adipose tissue of goats

Short chain fatty acids (SCFA) represent the main source for energy supply in ruminants. Propionate up-regulates leptin synthesis through the G protein-coupled receptor 41 (GPR41) in mice but the importance of the GPR41 in ruminants is not yet clarified. Here we characterise the short-term effects of intravenously infused propionate on a putative GPR41 mRNA in goat adipose tissue. Castrated male goats (Capra hircus) received propionate infusion or NaCl solution with equivalent sodium content for 260 min. A putative GPR41 mRNA was quantified in subcutaneous and perirenal adipose tissue by real-time RT-PCR. The mRNA concentration of the putative GPR41 mRNA increased (p = 0.029) in subcutaneous but not in perirenal adipose tissue (p = 0.756) of propionate-infused animals versus the NaCl group. We hypothesise that the differential response of the putative GPR41 mRNA in subcutaneous versus perirenal adipose tissue towards short-term propionate infusion could be involved in a differential nutrient sensing of SCFA in the two adipose depots of goats.     

Effects of Turnout to Pasture and Dietary Fat Supplementation on Milk Fat Composition and Conjugated Linoleic Acid in Dairy Cows

Turnout to pasture involves considerable changes in the feed intake and metabolism of dairy cows. The aim of this study was to investigate how these changes affect milk production and milk fat composition, in cows selected for high (HFI) or low (LFI) milk fat content producing the same yield of 4% fat-corrected milk. Furthermore, two different concentrates, with high or low inclusion of soya oil, were studied. The experiment was conducted with 44 multiparous mid-lactating cows over a 4 week period. Samples of milk and blood were obtained before turnout to pasture, during transition to pasture and when cows were adapted to pasture. Milk yield decreased with 3.6 kg energy-corrected milk (ECM) on average during the first 5 days after turnout to pasture. This decrease, together with a drop in plasma insulin and increased plasma non-esterified fatty acids (NEFA), indicated nutritional insufficiency at turnout to pasture. Milk fat composition changed towards more long-chain fatty acids (LCFA) and fewer de novo synthesized fatty acids. This was probably caused by an increased supply of LCFA of feed and body origin to the udder. LFI cows showed higher levels of conjugated linoleic acid at pasture than HFI cows.     

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 β-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.     

日粮添加不同比例的中短链和长链脂肪酸对奶牛免疫功能的影响

为探讨不同比例的中短链和长链脂肪酸混合物对奶牛免疫功能的影响,试验选用72头产奶量、胎次及泌乳日龄相近的中国荷斯坦奶牛,按随机区组试验设计,分为对照组和5个处理组,对照组饲喂基础日粮,处理组在基础日粮上分别添加400 g/d 长链脂肪酸(LCFA,A组)、80 g/d中短链脂肪酸(SMCFA)+320 g/d LCFA(B组)、400 g/d 黄油(C组)、240 g/d SMCFA+160 g/d LCFA(D组)及400 g/d SMCFA(E组)。试验期为63 d,第1周为预试期,试验期最后1 d采集血液样品,用于测定血清中免疫球蛋白及细胞因子的含量。结果显示,日粮添加不同比例的中短链和长链脂肪酸对奶牛血清中免疫球蛋白M(IgM)及前列腺素(PGE₂)的含量没有显著影响(P＞0.05);A组免疫球蛋白A(IgA)的含量显著高于B、E组(P＜0.05),B、D组免疫球蛋白G(IgG)的含量显著高于其余4组(P＜0.05)。日粮中添加脂肪酸均能提高奶牛血清中IL-4及IL-10的含量,尤其是D组,增加效果极显著(P＜0.01)。因此,添加240 g/d SMCFA 和160 g/d LCFA更能提高泌乳中期奶牛的免疫性能。     

Leptin, GH, PRL, Insulin and Metabolic Parameters Throughout the Dry Period and Lactation in Dairy Cows

Leptin may play a role in the endocrine-metabolic processes that guarantee the physiological course of lactation in dairy cattle. This study was aimed at determining the changes in plasma concentrations of leptin and some of the main hormones and metabolites involved in the lactogenetic process in high-yielding dairy cows throughout lactation; we also wanted to assess whether leptin secretion is subjected to seasonal influences. Blood samples were collected from 23 Italian Friesian dairy cows from the end of a lactation to the ninth month of the subsequent one; in addition, blood was sampled from 47 dairy cows in different phases of lactation during February and July. Plasma concentrations of leptin, growth hormone (GH), insulin, prolactin (PRL), glucose, non-esterified fatty acids (NEFA) and urea were quantified by either validated radioimmunoassay (RIA) or enzymatic colorimetric methods. At the beginning of lactation, GH concentrations significantly increased, while a significant reduction occurred in leptin and insulin. This endocrine condition, such as the significant increase in NEFA plasma concentrations, is indicative of a marked lipid mobilization. In the more advanced stages of lactation, when both energy and protein balances become positive, leptin plasma concentrations increased, whereas GH and NEFA concentrations declined. During the summer months, a significant increase in leptin plasma concentrations, irrespective of the phase of lactation, was observed. Collectively, our findings suggest that, in dairy cows, leptin may represent a 'metabolic signal' of animal's status of fattening and nutritional level; in addition, leptin seems to be influenced by photoperiod and environmental temperature.     

Early pregnancy alters the metabolic responses to restricted nutrition in sheep

This study investigated whether a 27-day period of nutrition at half-maintenance during early pregnancy (up to Day 14) could alter maternal endocrine responses. Forty-six ewes were fed all or half of their maintenance requirements and slaughtered on Day 14 of the oestrous cycle or pregnancy. We used real time RT-PCR to study gene expression of growth hormone receptor (GHR) and leptin in adipose tissue and GHR, GHR1A and of the insulin-like growth factor I (IGF-I) in the liver. Blood profiles of metabolites and metabolic hormones were also determined. Throughout the experiment, underfed animals presented lower body weight and body condition, greater plasma concentrations of non-esterified fatty acids (NEFA), and lower plasma concentrations of leptin, compared to adequately fed animals. Undernutrition affected the patterns of gene expression in adipose and hepatic tissues, and the responses differed between pregnant and non-pregnant ewes. In adequately fed ewes, pregnancy up-regulated leptin mRNA expression in adipose tissue, a response that was impaired in underfed ewes. The hepatic expression of IGF-I mRNA was increased by pregnancy in underfed animals while no effect was observed in adequately fed ewes. It remains to be determined whether the changes in the endocrine milieu are paralleled by modifications in uterine gene expression that could alter the environment of the embryo during early pregnancy.     

Decrease in stearic acid proportions in adipose tissues and liver lipids in fatty liver of dairy cows

Samples of liver and perirenal, mesenteric and subcutaneous fat were collected from 16 sick necropsied dairy cows to evaluate the fatty acid profiles in the hepatic and adipose tissues associated with advanced fatty liver or hepatic lipidosis. Hepatic triglyceride and eight fatty acids were measured in the hepatic and adipose tissues. Six cows had more than 3% triglyceride on fresh weight in their livers and were classified as having fatty liver. Stearic and linoleic acid proportions in the liver decreased markedly with increased hepatic triglyceride levels, while the proportion of palmitic and oleic acids increased. The most striking fluctuations in hepatic lipidosis were manifested as decreased stearic acid in the adipose tissues including subcutaneous fat with the trend of decreasing stearic acid. Palmitic acid was elevated in hepatic and perirenal fat in fatty liver cows. In instances of advanced hepatic lipidosis, palmitoleic acid increased in only subcutaneous fat and not in perirenal or mesenteric fat. In addition to the proportions of hepatic fatty acids in fatty liver, this study also clarified the fluctuations observed in the profiles of fatty acids of the adipose tissues in cows with advanced hepatic lipidosis, particularly the decline in the proportions of stearic acid.     

Effect of Prepartum Energetic Supplementation on Productive and Reproductive Characteristics, and Metabolic and Hormonal Profiles in Dairy Cows under Grazing Conditions

The effect of cracked corn grain supplementation (3.5 kg/day) during 3 weeks before the expected calving date on milk production and composition, body condition score (BCS), metabolic and hormonal profiles and length of postpartum anoestrus was evaluated in multiparous Holstein dairy cows under grazing conditions (Energy supplemented group, n = 10; Control group, n = 10). Body condition score was weekly recorded during the peripartum period, from days −21 to +35 (parturition = day 0). Non-esterified fatty acids, β-hydroxybutyrate, cholesterol, urea, insulin, insulin-like growth factor I (IGF-I), leptin, thyroxine (T₄) and 3,3'5-triiodothyroinine (T₃) were weekly determined in plasma from days −21 to +35. The reinitiation of ovarian cyclicity was twice weekly determined by ovarian ultrasonography and confirmed by plasma progesterone concentrations. Cows fed energy concentrate prepartum had higher BCS during the prepartum and postpartum and produced more milk. Non-esterified fatty acids plasma concentrations were significantly higher in the energy group, while cholesterol was higher in the control group. Treated cows had higher levels of plasma insulin, IGF-I and leptin pre-calving. IGF-I, leptin and T₄ were diminished during the early postpartum period in both groups. Insulin levels were also diminished in the control group, but levels remained high in the energy-supplemented group. Treated cows ovulated sooner after parturition than controls. We conclude that Energetic supplementation prepartum in cows under grazing conditions increased milk production and reduced the reinitiation of ovarian activity, consistent with a better EB (BCS), higher prepartum levels of IGF-I, leptin and insulin, and higher insulin levels during early postpartum.     

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.     

重组脂联素对皖南花猪脂肪细胞脂联素及其受体mRNA表达的影响

脂联素（Adp）是主要由脂肪组织分泌的细胞因子,有重要的生理作用。本试验旨在研究重组脂联素（rAdp）对皖南花猪脂肪细胞脂联素及其受体2,AMP激活蛋白激酶（AMPK）、过氧化物增殖剂活化受体α（PPARα）mR-NA表达的影响。选择10d皖南花猪皮下脂肪组织分离前体脂肪细胞,增殖培养至80%融合后换分化培养基培养,细胞分化后用0、2和10mg/L rAdp分别处理12和48h。油红O染色法鉴定脂肪细胞,MTT方法检测细胞活力;酶法测定培养液中甘油释放量,荧光定量RT-PCR方法检测脂肪细胞脂联素（Adp）、脂联素受体1（AdpR1）、脂联素受体2（AdpR2）、PPARα和AMPK mRNA表达。结果显示,rAdp处理后,脂肪细胞活力总体有降低趋势,10mg/L处理48h达到显著水平（P〈0.05）;rAdp处理对甘油释放的抑制作用未达到差异水平。rAdp处理12h后,脂肪细胞AdpR1和AdpR2mRNA表达显著升高（P〈0.01）,但无剂量依赖性;rAdp处理48h后,脂肪细胞AdpmRNA表达显著下降（P〈0.05）。rAdp处理12h后,脂肪细胞PPARαmRNA表达显著升高（P〈0.01）,且有剂量效应性;而AMP AMPK mRNA表达均无显著性变化。结果提示,重组脂联素处理猪原代脂肪细胞有降低细胞活力和抑制脂肪细胞甘油释放量的趋势,能显著上调AdpR1、AdpR2和PPARα基因的表达,从而刺激脂肪酸氧化和甘油三酯的水解作用。     

Differential effects of propionate or β-hydroxybutyrate on genes related to energy balance and insulin sensitivity in bovine white adipose tissue explants from a subcutaneous and a visceral depot

Ruminants rely on short-chain fatty acids (SCFA) as principal energy source. Herein, we compared the effects of propionate, β-hydroxybutyrate (BHB) and insulin on mRNA abundance of energy balance-related genes by short-term incubation (4 h) in bovine subcutaneous (SC) and retroperitoneal (RP) adipose tissue (AT) explants in vitro. Propionate either significantly (p < 0.05), or as a trend (p ≤ 0.1) affected mRNA abundance of genes such as adiponectin system in both depots in treated samples versus controls. Propionate increased adiponectin receptor 1 (AdipoR1) and AdipoR2 mRNA only in SC AT. β-hydroxybutyrate decreased mRNA abundance of adiponectin and AdipoR1 in SC AT as a trend. The mRNA abundance of free fatty acid receptor 2/3 (FFAR2/3) and other genes of interest (GOI) was increased during differentiation in bovine preadipocyte culture. The mRNA abundance of all the GOI remained unchanged after short-term insulin stimulation. In total, propionate, BHB or insulin during short-term treatment exert divergent effects on the mRNA abundance of GOI in both depots in vitro. Our results indicate that the bovine adiponectin system might be more sensitive to propionate than to BHB. We demonstrated the presence of FFAR2/3 mRNA not only in both AT depots but also in differentiating preadipocytes isolated from bovine SC AT. Therefore, we established that SCFA are able to exert insulin-independent effects on bovine adipose tissue, which might be independent from propionate uptake-related events.     

Effect of energy density in the diet and milking frequency on hepatic long chain fatty acid oxidation in early lactating dairy cows

The objective of this study was to examine the effects of diet energy density (high versus low) and increased milk yield, induced by increased milking frequency (two versus three times daily), on the hepatic status of triacylglycerol (TAG) and glycogen content and hepatic long chain fatty acid (LCFA) oxidation capacity in early lactation in a 2 x 2 factorial design. Forty multiparous Danish-Holstein dairy cows were used from 8 weeks before to 8 weeks after calving. Liver biopsies and blood samples were taken in weeks -2, 2, and 7 from calving. The cows fed the high energy density diet, compared with the cows fed the low energy density diet, had an 18 and 28% higher milk production and net energy intake, respectively. Milk yield was increased by 10% when the cows were milked three times compared with twice daily. Complete (CO2 production) and incomplete (ketone body production) LCFA oxidation capacity in the liver were 35 and 32% higher, respectively, and liver TAG content was 48% lower for the cows fed the high energy density diet compared with the low energy density diet. Overall there was no effect of milking frequency on liver parameters. However, a significant interaction between diet and milking frequency showed that the cows milked three times daily and fed the low energy density diet had the lowest liver LCFA oxidation (CO2 and ketone body) capacity. Furthermore, these cows had the numerically highest liver TAG content. The results for liver LCFA metabolism are discussed in relation to the plasma concentration of metabolites and insulin. In conclusion, cows in early lactation given a high energy density diet will, in general, have a lower risk of high TAG infiltration in the liver.     

Characterization of key genes of the renin–angiotensin system in mature feline adipocytes and during in vitro adipogenesis

Obesity is a growing health problem in humans as well as companion animals. In the development and progression of obesity‐associated diseases, the members of the renin–angiotensin system (RAS) are proposed to be involved. Particularly, the prevalence of type 2 diabetes mellitus in cats has increased enormously which is often been linked to obesity as well as to RAS. So far, reports about the expression of a local RAS in cat adipocytes are missing. Therefore, we investigated the mRNA expression of various RAS genes as well as the adipocyte marker genes adiponectin, leptin and PPAR‐γ in feline adipocytes using quantitative PCR. To characterize the gene expression during adipogenesis, feline pre‐adipocytes were differentiated into adipocytes in a primary cell culture and the expression of RAS key genes measured. All major RAS components were expressed in feline cells, but obvious differences in the expression between pre‐adipocytes and the various differentiation stages were found. Interestingly, the two enzymes ACE and ACE2 showed an opposite expression course. In addition to the in vitro experiments, mature adipocytes were isolated from subcutaneous and visceral adipose tissue. Significant differences between both fat depots were found for ACE as well as AT1 receptor with greater expression in subcutaneous than in visceral adipocytes. Visceral adipocytes had significantly higher adiponectin and PPAR‐γ mRNA level compared to the subcutaneous fat cells. Concerning the nutritional status, a significant lower expression of ACE2 was measured in subcutaneous adipocytes of overweight cats. In summary, the results show the existence of a potentially functional local RAS in feline adipose tissue which is differentially regulated during adipogenesis and dependent on the fat tissue depot and nutritional status. These findings are relevant for understanding the development of obesity‐associated diseases in cats such as diabetes mellitus.     

Coordinated gene expression in adipose tissue and liver differs between cows with high or low NEFA concentrations in early lactation

Dairy cows with high and low plasma non-esterified fatty acid (NEFA) concentrations in early lactation were compared for plasma parameters and mRNA expression of genes in liver and subcutaneous adipose tissue. The study involved 16 multiparous dairy cows with a plasma NEFA concentration of >500 μmol/l [n = 8, high NEFA (HNEFA)] and <140 μmol/l [n = 8, low NEFA (LNEFA)] in the first week post-partum (pp). Blood samples, adipose and liver tissues were collected on day 1 (+1d) and at week 3 pp (+3wk). Blood plasma was assayed for concentrations of metabolites and hormones. Subcutaneous adipose and liver tissues were analysed for mRNA abundance by real-time qRT-PCR encoding parameters related to lipid metabolism. Results showed that mean daily milk yield and milk fat quantity were higher in HNEFA than in LNEFA cows (p < 0.01), and the NEB was more negative in HNEFA than in LNEFA in +3wk too (p < 0.05). HNEFA cows had slightly lower (p < 0.1) insulin concentrations than LNEFA cows across the study period, and the body condition score decreased more from +1d to +3wk in HNEFA than in LNEFA (p = 0.09). The mRNA abundance of genes in the liver related to fatty acid oxidation (carnitine palmitoyltransferase 2 and very long chain acyl-coenzyme A dehydrogenase) and ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2) were lower in HNEFA than in LNEFA cows. No differences between the two groups were observed for mRNA expression of genes in adipose tissue. The number of calculated significant correlation coefficients (moderately strong) between parameters in the liver and in adipose tissue was nearly similar on +1d, and higher for HNEFA compared with LNEFA cows in +3wk. In conclusion, dairy cows with high compared with low plasma NEFA concentrations in early lactation show differentially synchronized mRNA expression of genes in adipose tissue and liver in +3wk that suggests a different orchestrated homeorhetic regulation of lipid metabolism.