Over-expression of human lipoprotein lipase in mouse mammary glands leads to reduction of milk triglyceride and delayed growth of suckling pups.
ABSTRACT: BACKGROUND: The mammary gland is a conserved site of lipoprotein lipase expression across species and lipoprotein lipase attachment to the luminal surface of mammary gland vascular endothelial cells has been implicated in the direction of circulating triglycerides into milk synthesis during lactation. PRINCIPAL FINDINGS: Here we report generation of transgenic mice harboring a human lipoprotein lipase gene driven by a mammary gland-specific promoter. Lipoprotein lipase levels in transgenic milk was raised to 0.16 mg/ml, corresponding to an activity of 8772.95 mU/ml. High lipoprotein lipase activity led to a significant reduction of triglyceride concentration in milk, but other components were largely unchanged. Normal pups fed with transgenic milk showed inferior growth performances compared to those fed with normal milk. CONCLUSION: Our study suggests a possibility to reduce the triglyceride content of cow milk using transgenic technology.
Project description:High concentrate diets are fed to early and mid-lactation stages dairy ruminants to meet the energy demands for high milk production in modern milk industry. The present study evaluated the effects of a high concentrate diet on milk fat and milk composition, especially, cis-9, trans-11 CLA content in milk and gene expression of lactating goats. Eight mid-lactating goats with rumen fistula were randomly assigned into a high concentrate diet (HCD) group and low concentrate diet (LCD) group. High concentrate diet feeding significantly increased lipopolysaccharides (LPS) in plasma and decreased milk fat content, vaccenic acid (VA) and cis-9, trans-11 CLA in milk of the lactating goats. The mRNA expression levels of sterol regulatory element binding protein B 1c (SREBP1c), lipoprotein lipase (LPL), fatty acid synthetase (FASN) and acetyl-CoA carboxylase ? (ACACA, ACC?) involving in lipid metabolism were analyzed, and ACACA and LPL all decreased in their expression level in the mammary glands of goats fed a high concentrate diet. DNA methylation rate of stearoyl-CoA desaturase (SCD) was elevated and decreased, and SCD mRNA and protein expression was reduced significantly in the mammary glands of goats fed a high concentrate diet. In conclusion, feeding a high concentrate diet to lactating goats decreases milk fat and reduced expression of SCD in the mammary gland, which finally induced cis-9, trans-11 CLA content in milk.
Project description:1. The mode of uptake of the precursors of milk fat by the mammary gland of the lactating goat has been examined by infusing radioactive fatty acids, glucerol or doubly labelled triglycerides into the mammary artery or jugular vein of animals surgically prepared to permit samples of arterial and venous blood to be withdrawn without disturbance to the animal. 2. Acetate was taken up by the mammary gland and incorporated into milk fat. The decrease in the specific radioactivity of blood acetate across the gland was evidence of acetate production, but there was no significant release of labelled lipid from the mammary gland. 3. When labelled long-chain fatty acids or glycerol were infused into the lactating goat, there was extensive transfer of radioactivity into milk in spite of the absence of net uptake of substrate by the mammary gland. The decrease in the specific radioactivity of each substrate across the mammary gland, however, showed that both fatty acids and glycerol were simultaneously taken up and released by mammary tissue. 4. The infusion of chylomicra and triglyceride emulsions labelled with (3)H and (14)C revealed that both glycerol and fatty acids were released during triglyceride uptake by mammary tissue. Changes in the (3)H/(14)C ratio during the transfer of triglyceride from blood into milk showed that at least 80% of the triglyceride was hydrolysed during uptake, but the potential re-utilization of both products of hydrolysis for triglyceride synthesis in mammary tissue implied that only a minimum value could be obtained from the change in the ratio. 5. The time-course of the transfer of (3)H and (14)C into milk and lymph were closely similar after the infusion of [2-(3)H]glycerol tri[1-(14)C]oleate or of a mixture of [2-(3)H]glycerol and [1-(14)C]oleate. 6. The results were consistent with the hypothesis that plasma triglycerides are extensively or completely hydrolysed during mammary uptake.
Project description:We examined the effects of reproductive stage and fasting on lipoprotein lipase (LPL) activity and mRNA in the mouse mammary gland. Heparin-releasable and cell-associated LPL activity rose immediately after birth, followed 1-2 days later by an increase in LPL mRNA. Fasting decreased LPL activity in the mammary gland at all reproductive stages. During lactation, both milk and heparin-releasable LPL were substantially decreased by an overnight fast, whereas cell-associated LPL was less affected and LPL mRNA did not change. These studies indicate that the extracellular, heparin-releasable, fraction of mammary LPL activity responds most rapidly to alterations in physiological state, usually accompanied by smaller changes in cellular enzyme activity. Changes in the level of LPL mRNA were seen only during the transition from pregnancy to lactation, and these tended to follow, rather than precede, changes in enzyme activity. We conclude that in the mammary gland as in adipose tissue, LPL is regulated primarily at the translational and post-translational level.
Project description:1. Measurements were made of milk yield, mammary blood flow and arteriovenous differences of each plasma lipid fraction, and their specific radioactivities, during the infusion of [U-(14)C]stearate, [U-(14)C]oleate, [U-(14)C]palmitate and [1-(14)C]acetate into fed lactating goats. 2. Entry rates of fatty acids into the circulation were 4.2mg./min./kg. body wt. for acetate, and 0.18, 0.28 and 0.42mg./min./kg. for stearate, oleate and palmitate respectively. Acetate accounted for 23% of the total carbon dioxide produced by the whole animal, and contributed to the oxidative metabolism of the mammary gland to about the same extent. Corresponding values for each of the long-chain acids were less than 1%. 3. There were no significant arteriovenous differences of phospholipids, sterols or sterol esters, and their fatty acid composition showed no net changes during passage through the mammary gland. 4. There were large arteriovenous differences of plasma triglycerides, and their fatty acid composition showed marked changes across the gland. The proportions of palmitate and stearate fell, and that of oleate increased. 5. Arteriovenous differences of plasma free fatty acids (FFA) were small and variable, but a large fall in the specific radioactivity of each of the long-chain acids examined indicated substantial uptake of plasma FFA, accompanied by roughly equivalent FFA release from mammary tissue. The uptake of FFA was confirmed by the extensive transfer of radioactivity into milk. The FFA of milk were similar in composition and radioactivity to the milk triglyceride fatty acids, and quite unlike plasma FFA. 6. The formation of large amounts of oleic acid (18-21 mg./min.) from stearic acid was demonstrated. 7. During the terminal stages of the [(14)C]acetate infusion, milk triglyceride fatty acids of chain length C(4)-C(14) showed specific radioactivities that were 75-90% of that of blood acetate, and that of palmitate was roughly one-quarter of this value. Oleate and stearate were unlabelled. 8. The results confirmed that milk fatty acids of chain length C(4)-C(14) arise largely from blood acetate, and palmitate is derived partly from acetate and partly from plasma triglyceride, the latter fraction being almost the sole precursor of oleate and stearate.
Project description:Human hepatic lipase (hHL) is mainly localized on the hepatocyte cell surface where it hydrolyzes lipids from remnant lipoproteins and high density lipoproteins and promotes their hepatic selective uptake. Furthermore, hepatic lipase (HL) is closely associated with obesity in multiple studies. Therefore, HL may play a key role on lipid homeostasis in liver and white adipose tissue (WAT). In the present study, we aimed to evaluate the effects of hHL expression on hepatic and white adipose triglyceride metabolism in vivo. Experiments were carried out in hHL transgenic and wild-type mice fed a Western-type diet. Triglyceride metabolism studies included ?-oxidation and de novo lipogenesis in liver and WAT, hepatic triglyceride secretion, and adipose lipoprotein lipase (LPL)-mediated free fatty acid (FFA) lipolysis and influx. The expression of hHL promoted hepatic triglyceride accumulation and de novo lipogenesis without affecting triglyceride secretion, and this was associated with an upregulation of Srebf1 as well as the main genes controlling the synthesis of fatty acids. Transgenic mice also exhibited more adiposity and an increased LPL-mediated FFA influx into the WAT without affecting glucose tolerance. Our results demonstrate that hHL promoted hepatic steatosis in mice mainly by upregulating de novo lipogenesis. HL also upregulated WAT LPL and promoted triglyceride-rich lipoprotein hydrolysis and adipose FFA uptake. These data support the important role of hHL in regulating hepatic lipid homeostasis and confirm the broad cardiometabolic role of HL.
Project description:Bile salt-stimulated lipase (BSSL) is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL) can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of transgenic cloned cows would therefore be a mean to meet a medical need. In the present study, a vector pBAC-hLF-hBSSL was constructed, which efficiently expressed active rhBSSL in milk of transgenic cloned cows to a concentration of 9.8 mg/ml. The rhBSSL purified from cow milk had the same enzymatic activity, N-terminal amino acid sequence, amino acid composition and isoelectric point and similar physicochemical characteristics as human native BSSL. Our study supports the use of transgenic cattle for the cost-competitive, large-scale production of therapeutic rhBSSL.
Project description:Pten is a tumor suppressor gene regulating many cellular processes, including growth, adhesion, and apoptosis. In the aim of investigating the role of Pten during mammary gland development and lactation of dairy cows, we analyzed Pten expression levels in the mammary glands of dairy cows by using western blotting, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Dairy cow mammary epithelial cells (DCMECs) were used to study the function of Pten in vitro. We determined concentrations of ?-casein, triglyceride, and lactose in the culture medium following Pten overexpression and siRNA inhibition. To determine whether Pten affected DCMEC viability and proliferation, cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected. Pten expression was also assessed by adding prolactin and glucose to cell cultures. When Pten was overexpressed, proliferation of DCMECs and concentrations for ?-casein, triglyceride, and lactose were significantly decreased. Overexpression of Pten down-regulated expression of MAPK, CYCLIN D1, AKT, MTOR, S6K1, STAT5, SREBP1, PPAR?, PRLR, and GLUT1, but up-regulated 4EBP1 in DCMECs. The Pten siRNA inhibition experiments revealed results that opposed those from the gene overexpression experiments. Introduction of prolactin (PRL) increased secretion of ?-casein, triglyceride, and lactose, but decreased Pten expression levels. Introduction of glucose also increased ?-casein and triglyceride concentrations, but did not significantly alter Pten expression levels. The Pten mRNA and protein expression levels were decreased 0.3- and 0.4-fold in mammary glands of lactating cows producing high quality milk (milk protein >3.0%, milk fat >3.5%), compared with those cows producing low quality milk (milk protein <3.0%, milk fat <3.5%). In conclusion, Pten functions as an inhibitor during mammary gland development and lactation in dairy cows. It can down-regulate DCMECs secretion of ?-casein, triglyceride, and lactose, and plays a critical role in lactation related signaling pathways.
Project description:1. Starvation caused a marked decrease in the activity of ornithine decarboxylase in mammary gland, together with a lesser decrease in the activity of S-adenosylmethionine decarboxylase and a marked fall in milk production. Liver ornithine decarboxylase and S-adenosylmethionine decarboxylase activities were unaffected. 2. Refeeding for 2.5 h was without effect on ornithine decarboxylase in mammary gland, but it returned the S-adenosylmethionine decarboxylase activity in mammary gland to control values and elevated both ornithine decarboxylase and S-adenosylmethionine decarboxylase in liver. 3. Refeeding for 5 h returned the activity of ornithine decarboxylase in mammary gland to fed-state values and resulted in further increases in S-adenosylmethionine decarboxylase in mammary gland and liver and in ornithine decarboxylase in liver. 4. Prolactin deficiency in fed rats resulted in decreased milk production and decreased activity of ornithine decarboxylase in mammary gland. The increase in ornithine decarboxylase activity normally seen after refeeding starved rats for 5 h was completely blocked by prolactin deficiency. 5. In fed rats, injection of streptozotocin 2.5 h before death caused a decrease in the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase in mammary gland, which could be reversed by simultaneous injection of insulin. Insulin deficiency also prevented the increase in S-adenosylmethionine decarboxylase in liver and mammary gland normally observed after refeeding starved rats for 2.5 h.
Project description:Lipoprotein lipase (LPL) serves as a central factor in hydrolysis of triacylglycerol and uptake of free fatty acids from the plasma. However, there are limited data concerning the action of LPL on the regulation of milk fat synthesis in goat mammary gland. In this investigation, we describe the cloning and sequencing of the LPL gene from Xinong Saanen dairy goat mammary gland, along with a study of its phylogenetic relationships. Sequence analysis showed that goat LPL shares similarities with other species including sheep, bovine, human and mouse. LPL mRNA expression in various tissues determined by RT-qPCR revealed the highest expression in white adipose tissue, with lower expression in heart, lung, spleen, rumen, small intestine, mammary gland, and kidney. Expression was almost undetectable in liver and muscle. The expression profiles of LPL gene in mammary gland at early, peak, mid, late lactation, and the dry period were also measured. Compared with the dry period, LPL mRNA expression was markedly greater at early lactation. However, compared with early lactation, the expression was lower at peak lactation and mid lactation. Despite those differences, LPL mRNA expression was still greater at peak, mid, and late lactation compared with the dry period. Using goat mammary epithelial cells (GMEC), the in vitro knockdown of LPL via shRNA or with Orlistat resulted in a similar degree of down-regulation of LPL (respectively). Furthermore, knockdown of LPL was associated with reduced mRNA expression of SREBF1, FASN, LIPE and PPARG but greater expression of FFAR3. There was no effect on ACACA expression. Orlistat decreased expression of LIPE, FASN, ACACA, and PPARG, and increased FFAR3 and SREBF1 expression. The pattern of LPL expression was similar to the changes in milk fat percentage in lactating goats. Taken together, results suggest that LPL may play a crucial role in fatty acid synthesis.
Project description:Milk casein and triglyceride content are important production traits in goats. Studies on mechanisms in milk casein secretion and mammary gland development is essential for milk goat breeding. miRNAs play an important role in goat lactation. While novel-miR-3880 is highly expressed at goat peak lactation stage, its molecular mechanism has not been studied. The purpose of the present study was to explore the relationship between novel-miR-3880 and lactation, as well as to construct a network among novel-miR-3880, ciRNA13761, and E74 like ETS transcription factor 2 (ELF2), thus further exploring their potential roles in milk components and mammary gland development. ELF2 was previously proven to be important in cell survival and proliferation, and 3'-UTR of ELF2 was predicted to have binding sites of novel-miR-3880. Our study found that the overexpression of novel-miR-3880 exerted anti-apoptotic and proliferative roles in GMEC, induced a boost in triglyceride synthesis, and caused a decrease in ? s1-, ? s2-, and ?-casein, but an increase in ?-casein secretion. Furthermore, treatment in mice indicated that novel-miR-3880 could promote mammary gland development and extend the lactation period, while novel-miR-3880 expression was found to be suppressed by ciRNA13761 as a miRNA sponge. The present study explores a mechanism of triglyceride synthesis and casein secretion, and reveals a crosstalk between ciRNA13761/novel-miR-3880/ELF2 axis and PI3K/AKT/mTOR/S6K1 pathway, to gain a better understanding of lactation traits in dairy goats.