Background： The objective of this study was to study how changing the ratio of Lys to Thr, Lys to His, and Lys to Val affects the expression of lipogenic genes and microRNA （miRNA） in bovine mammary epithelial cell...Background： The objective of this study was to study how changing the ratio of Lys to Thr, Lys to His, and Lys to Val affects the expression of lipogenic genes and microRNA （miRNA） in bovine mammary epithelial cells. Results： Triplicate cultures with the respective ＂optimal＂ amino acid （AA） ratio （OPAA = Lys：Met 2.9：1; Thr：Phe 1.05：1; Lys：Thr 1.8：1; Lys：His 2.38：1; Lys：Val 1.23：1） plus rapamycin （OPAARMC; positive control）, OPAA, Lys：Thr 2.1：1 （LT2.1）, Lys：Thr 1.3：1 （LT1.3）, Lys：His 3.05：1 （LH3.0）, or Lys：Val 1.62：1 （LV1.6） were incubated in lactogenic medium for 12 h. The expression of 15 lipogenic genes and 7 miRNA were evaluated. Responses to LT2.1, LT1.3, LH3.0, and LV1.6 relative to the control （OPAARMC） included up-regulated expression ofACSS2, FABP3, ACACA, FASN, SCD, LPIN1, INSIG1, SREBF1, PPARD, and NR1H3 （commonly known as LXR-a）. Furthermore, LV1.6 up-regulated expression of ACSL1, DGAT1, and RXRA and down-regulated PPARG expression. Although no effect of OPAA on expression of PPARG was observed, compared with the control, OPAA up-regulated expression of the PPAR targets ACSS2, FABP3, ACACA, FASN, SCD, LPIN1, INSIG1, and SREBF1. Compared with the control, the expression of the anti-lipogenic MIR27AB was down-regulated by OPAA, LT2.1, LT1.3 and LH3.0. In contrast, compared with the control, the expression of the pro-lipogenic MIR21 was up-regulated by LT2.1, LT1.3, LH3.0, and LV1.6. Conclusions： The observed up-regulation of lipogenic gene networks and the changes in expression of key miRNA involved in the control of lipogenic balance are indicative of a potentially important role of EAA ratios and mTOR signaling in the regulation of milk fat synthesis.展开更多
Background: Milk protein is crucial for milk quality in sows and health of newborn piglets. Plasma amino acids(AA)in sows are important precursors for milk protein synthesis in the mammary gland. In order to study the...Background: Milk protein is crucial for milk quality in sows and health of newborn piglets. Plasma amino acids(AA)in sows are important precursors for milk protein synthesis in the mammary gland. In order to study the regulation of AA transported in sow mammary glands and possible underlying mechanisms, we measured the expression of genes coding for milk proteins, AA transporter expressions, and plasma AA concentrations in sows at three different physiological stages(D-17, D1 and D17 of lactation), and then further investigated the regulation of AA transport across the cell membrane by adaptive mechanisms using pig mammary epithelial cells(PMEC) as an in vitro model.PMEC were cultured in DMEM:F12 with 4 amino acid concentrations(0 × AA complex, 1 × AA complex, 5 × AA complex,and 25 × AA complex). Classes of AA complexes evaluated in this study included neutral AAs(L-Ala + L-Ser + L-Cys), acidic AAs(L-Asp, L-Glu) and neutral + basic AAs(L-Ala + L-Ser + L-Cys + L-Lys).Results: Our results indicated that m RNA expression of genes coding for milk protein(αs1-casein, αs2-casein,β-casein and κ-casein) increased significantly with the advance of physiological stage(P < 0.05), and plasma concentrations of most AAs including threonine, serine, glutamate, alanine, valine, cysteine, methionine, isoleucine and tyrosine were greater at D1 of lactation compared with D-17 and D17 of lactation(P < 0.05). Additionally, protein and gene expressions of AA transporters including excitatory AA transporter 3(EAAT3), alanine/serine/cysteine/threonine transporter(ASCT1) and sodium-coupled neutral AA transporter 1(SNAT2) were greater in lactating sow mammary glands compared with sow mammary glands in late pregnancy(P < 0.05). The m RNA expressions of SLC38 A2, SLC1 A1,SLC6 A14 increased significantly in the cell mediums supplemented with 5 × and 25 × of AA complexes compared with those cells cultured in DMEM/F12 cell medium(P < 0.05). The m RNA expressions of SLC38 A, SLC1 A4, and SLC6 A14 also increased in EBSS cell medium compared to DMEM/F12. However, only m RNA expression of SLC38 A decreased when AA complex was added into EBSS(P < 0.05).Conclusion: AA transportation was positively regulated in sow mammary glands with the advance of physiological stage from late pregnancy to peak of lactation and AA transporters in PMECs were adaptively regulated by changed AA concentrations.展开更多
The experiment was designed to determine the effect of protected lysine (Lys) and methionine (Met) supply on milk protein profile in grazing dairy cows specifically in the caseins (CNs) and α-lactalbumin fractions. T...The experiment was designed to determine the effect of protected lysine (Lys) and methionine (Met) supply on milk protein profile in grazing dairy cows specifically in the caseins (CNs) and α-lactalbumin fractions. Twelve multiparous mid lactation Holstein cows producing 24 (±4.76) kg of milk were assigned to one of two treatments (six cows per treatment) during an experimental period of 21 days. In the control (C) group, cows grazed a Pennisetum clandestinum pasture and were supplemented with a commercial concentrate according to milk production. In the Met-Lys treatment, cows received the same ration supplemented with protected Lys and Met. Milk yield and composition and milk protein profile were measured at the start and the end (21st day) of the experimental period. The Tricine-SDS-PAGE and the Gel-Quant Express Analysis (Invitrogen) software were used to determine milk protein composition. Statistical analysis was performed using the SAS’s PROC MIXED procedure through a mixed model that included the animal as a random effect and the treatments as a fixed effect adjusted by covariables. Milk production averaged 23.7 (±2.0) kg cow-1 day-1 without differences between treatments (P P -1 day-1) compared to C (24.2 kg cow-1 day-1). Milk protein content (g/kg) did not differ (C = 30.4;Met-Lys = 31.1) and lactose content tended (P P β-CN also increased (P < 0.05) after protected aminoacid supply (C = 9.58;Met-Lys = 10.35). It can be concluded that milk protein composition was improved by protected Lys-Met supply without altering other compositional parameters of milk composition. Milk nutritional quality and its potential yield for cheese-making were positively enhanced.展开更多
A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis （MCE） coupled with laser-induced fluorescence （LIF） detection was developed. Seven kinds of standard amino a...A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis （MCE） coupled with laser-induced fluorescence （LIF） detection was developed. Seven kinds of standard amino ac- ids were derivated with sulfoindocyanine succinimidyl ester （Cy5） and then perfectly measured by MCE-LIF within 150 s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions： 100 mmolo L^-1 sodium borate solution （pH 10.0） as running buffer solution, 0.8 kV as injection voltage, 2.2 kV as separation voltage etc. The linear range of the detection of amino acids was from 0.01 μmol·L^-1 to 1.0 μmol·L^-1 and the detection limit was as low as about 1.0 μmol·L^-1. This MCE-LIF method was applied to the measurements of free amino acids in actual milk samples and satisfactory experimental results were achieved.展开更多
基金J.X.Liu’s laboratory is recipient of funds from China Mo ST(Grant No.2011CB100801)supported by Hatch funds allocated to University of Illinois(ILLU-538-914J.J.Loor)
文摘Background： The objective of this study was to study how changing the ratio of Lys to Thr, Lys to His, and Lys to Val affects the expression of lipogenic genes and microRNA （miRNA） in bovine mammary epithelial cells. Results： Triplicate cultures with the respective ＂optimal＂ amino acid （AA） ratio （OPAA = Lys：Met 2.9：1; Thr：Phe 1.05：1; Lys：Thr 1.8：1; Lys：His 2.38：1; Lys：Val 1.23：1） plus rapamycin （OPAARMC; positive control）, OPAA, Lys：Thr 2.1：1 （LT2.1）, Lys：Thr 1.3：1 （LT1.3）, Lys：His 3.05：1 （LH3.0）, or Lys：Val 1.62：1 （LV1.6） were incubated in lactogenic medium for 12 h. The expression of 15 lipogenic genes and 7 miRNA were evaluated. Responses to LT2.1, LT1.3, LH3.0, and LV1.6 relative to the control （OPAARMC） included up-regulated expression ofACSS2, FABP3, ACACA, FASN, SCD, LPIN1, INSIG1, SREBF1, PPARD, and NR1H3 （commonly known as LXR-a）. Furthermore, LV1.6 up-regulated expression of ACSL1, DGAT1, and RXRA and down-regulated PPARG expression. Although no effect of OPAA on expression of PPARG was observed, compared with the control, OPAA up-regulated expression of the PPAR targets ACSS2, FABP3, ACACA, FASN, SCD, LPIN1, INSIG1, and SREBF1. Compared with the control, the expression of the anti-lipogenic MIR27AB was down-regulated by OPAA, LT2.1, LT1.3 and LH3.0. In contrast, compared with the control, the expression of the pro-lipogenic MIR21 was up-regulated by LT2.1, LT1.3, LH3.0, and LV1.6. Conclusions： The observed up-regulation of lipogenic gene networks and the changes in expression of key miRNA involved in the control of lipogenic balance are indicative of a potentially important role of EAA ratios and mTOR signaling in the regulation of milk fat synthesis.
基金financially supported by the National Natural Science Foundation of China(No.31402082)
文摘Background: Milk protein is crucial for milk quality in sows and health of newborn piglets. Plasma amino acids(AA)in sows are important precursors for milk protein synthesis in the mammary gland. In order to study the regulation of AA transported in sow mammary glands and possible underlying mechanisms, we measured the expression of genes coding for milk proteins, AA transporter expressions, and plasma AA concentrations in sows at three different physiological stages(D-17, D1 and D17 of lactation), and then further investigated the regulation of AA transport across the cell membrane by adaptive mechanisms using pig mammary epithelial cells(PMEC) as an in vitro model.PMEC were cultured in DMEM:F12 with 4 amino acid concentrations(0 × AA complex, 1 × AA complex, 5 × AA complex,and 25 × AA complex). Classes of AA complexes evaluated in this study included neutral AAs(L-Ala + L-Ser + L-Cys), acidic AAs(L-Asp, L-Glu) and neutral + basic AAs(L-Ala + L-Ser + L-Cys + L-Lys).Results: Our results indicated that m RNA expression of genes coding for milk protein(αs1-casein, αs2-casein,β-casein and κ-casein) increased significantly with the advance of physiological stage(P < 0.05), and plasma concentrations of most AAs including threonine, serine, glutamate, alanine, valine, cysteine, methionine, isoleucine and tyrosine were greater at D1 of lactation compared with D-17 and D17 of lactation(P < 0.05). Additionally, protein and gene expressions of AA transporters including excitatory AA transporter 3(EAAT3), alanine/serine/cysteine/threonine transporter(ASCT1) and sodium-coupled neutral AA transporter 1(SNAT2) were greater in lactating sow mammary glands compared with sow mammary glands in late pregnancy(P < 0.05). The m RNA expressions of SLC38 A2, SLC1 A1,SLC6 A14 increased significantly in the cell mediums supplemented with 5 × and 25 × of AA complexes compared with those cells cultured in DMEM/F12 cell medium(P < 0.05). The m RNA expressions of SLC38 A, SLC1 A4, and SLC6 A14 also increased in EBSS cell medium compared to DMEM/F12. However, only m RNA expression of SLC38 A decreased when AA complex was added into EBSS(P < 0.05).Conclusion: AA transportation was positively regulated in sow mammary glands with the advance of physiological stage from late pregnancy to peak of lactation and AA transporters in PMECs were adaptively regulated by changed AA concentrations.
文摘The experiment was designed to determine the effect of protected lysine (Lys) and methionine (Met) supply on milk protein profile in grazing dairy cows specifically in the caseins (CNs) and α-lactalbumin fractions. Twelve multiparous mid lactation Holstein cows producing 24 (±4.76) kg of milk were assigned to one of two treatments (six cows per treatment) during an experimental period of 21 days. In the control (C) group, cows grazed a Pennisetum clandestinum pasture and were supplemented with a commercial concentrate according to milk production. In the Met-Lys treatment, cows received the same ration supplemented with protected Lys and Met. Milk yield and composition and milk protein profile were measured at the start and the end (21st day) of the experimental period. The Tricine-SDS-PAGE and the Gel-Quant Express Analysis (Invitrogen) software were used to determine milk protein composition. Statistical analysis was performed using the SAS’s PROC MIXED procedure through a mixed model that included the animal as a random effect and the treatments as a fixed effect adjusted by covariables. Milk production averaged 23.7 (±2.0) kg cow-1 day-1 without differences between treatments (P P -1 day-1) compared to C (24.2 kg cow-1 day-1). Milk protein content (g/kg) did not differ (C = 30.4;Met-Lys = 31.1) and lactose content tended (P P β-CN also increased (P < 0.05) after protected aminoacid supply (C = 9.58;Met-Lys = 10.35). It can be concluded that milk protein composition was improved by protected Lys-Met supply without altering other compositional parameters of milk composition. Milk nutritional quality and its potential yield for cheese-making were positively enhanced.
文摘A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis （MCE） coupled with laser-induced fluorescence （LIF） detection was developed. Seven kinds of standard amino ac- ids were derivated with sulfoindocyanine succinimidyl ester （Cy5） and then perfectly measured by MCE-LIF within 150 s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions： 100 mmolo L^-1 sodium borate solution （pH 10.0） as running buffer solution, 0.8 kV as injection voltage, 2.2 kV as separation voltage etc. The linear range of the detection of amino acids was from 0.01 μmol·L^-1 to 1.0 μmol·L^-1 and the detection limit was as low as about 1.0 μmol·L^-1. This MCE-LIF method was applied to the measurements of free amino acids in actual milk samples and satisfactory experimental results were achieved.
