Amino acids regulate energy utilization through mammalian target of rapamycin complex 1 and adenosine monophosphate activated protein kinase pathway in porcine enterocytes

As major fuels for the small intestinal mucosa, dietary amino acids (AA) are catabolized in the mitochondria and serve as sources of energy production. The present study was conducted to investigate AA metabolism that supply cell energy and the underlying signaling pathways in porcine enterocytes. Intestinal porcine epithelial cells (IPEC-J2) were treated with different concentrations of AA, inhibitor, or agonist of mammalian target of rapamycin complex 1 (mTORC1) and adenosine monophosphate activated protein kinase (AMPK), and mitochondrial respiration was monitored. The results showed that AA treatments resulted in enhanced mitochondrial respiration, increased intracellular content of pyruvic acid and lactic acid, and increased hormone-sensitive lipase mRNA expression. Meanwhile, decreased citrate synthase, isocitrate dehydrogenase alpha, and carnitine palmitoyltransferase 1 mRNA expression were also observed. We found that AA treatments increased the protein levels of phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated-p70 ribosomal protein S6 kinase, and phosphorylated-4E-binding protein 1. What is more, the protein levels of phosphorylated AMPK α (p-AMPKα) and nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase sirtuin-1 (SIRT1) were decreased by AA treatments in a time depending manner. Mitochondrial bioenergetics and the production of tricarboxylic acid cycle intermediates were decreased upon inhibition of mTORC1 or AMPK. Moreover, AMPK activation could up-regulate the mRNA expressions of inhibitor of nuclear factor kappa-B kinase subunit beta (Ikbkβ), integrin-linked protein kinase (ILK), unconventional myosin-Ic (Myo1c), ribosomal protein S6 kinase beta-2 (RPS6Kβ2), and vascular endothelial growth factor (VEGF)-β, which are downstream effectors of mammalian target of rapamycin (mTOR). The mRNA expressions of phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (PIK3CD) and 5′-AMP-activated protein kinase subunit gamma-1 (PRKAG1), which are upstream regulators of mTOR, were also up-regulated by AMPK activation. On the other hand, AMPK activation also down-regulated FK506-binding protein 1A (FKBP1A), serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform, phosphatase and tensin homolog (PTEN), and unc-51 like autophagy activating kinase 1 (Ulk1), which are up-stream regulators of mTORC1. Taken together, these data indicated that AA regulated cellular energy metabolism through mTOR and AMPK pathway in porcine enterocytes. These results demonstrated interactions of AMPK and mTORC1 pathways in AA catabolism and energy metabolism in intestinal mucosa cells of piglets, and also provided reference for using AA to remedy human intestinal diseases.     

茉莉酸甲酯对匍枝筋骨草细胞生长和β-蜕皮甾酮积累的影响

匍枝筋骨草悬浮细胞中含有较高β-蜕皮甾酮,为了进一步提高其β-蜕皮甾酮含量,通过添加茉莉酸甲酯(MeJA)进行一系列试验研究。以4~10代筋骨草悬浮细胞为试验材料,测定不同处理浓度和处理时间的MeJA对细胞生长、β-蜕皮甾酮积累的影响,细胞生长量采用称量计数,β-蜕皮甾酮含量则使用高效液相进行检测。结果表明:匍枝筋骨草悬浮细胞生长曲线及β-蜕皮甾酮积累曲线符合Logistics模型。在细胞快速生长的初始期(第4天)或中期(第7天)添加不同浓度的MeJA,对细胞生长影响相对较小,生长曲线均有小高峰,分别出现在处理后第5天和第3天,干物质积累量分别达到0.60和0.62 g。而在细胞快速生长的高峰期添加MeJA,细胞生长曲线呈下降趋势,细胞鲜重和干重显著低于CK(P<0.05)。在细胞快速生长的初始期或中期添加MeJA后细胞鲜重与β-蜕皮甾酮积累量呈显著相关。在细胞快速生长的初始期或中期添加10~50 μmol·L^-1 MeJA后,细胞鲜重与CK对比表现为显著增加,其中添加50 μmol·L^-1 MeJA后细胞鲜重最佳,最高可达到35.90 g,显著高于其他处理(P<0.05)。而同样条件下β-蜕皮甾酮表现为积累量小幅增加,最高量为0.5095 mg·g^-1。添加100~200 μmol·L^-1 MeJA均会抑制细胞的生长,其中添加200 μmol·L^-1 MeJA细胞鲜重与CK相比显著下降,抑制率最高可达到38.88%。而添加100~200 μmol·L^-1 MeJA后β-蜕皮甾酮积累量表现为大幅提升,最高可达3.5315 mg·g^-1,为同期CK的14.44倍(P<0.01)。在细胞快速生长的高峰期添加10~200 μmol·L^-1 MeJA后,细胞鲜重与CK相比都表现为下降,说明在此时添加这些浓度MeJA可抑制细胞的生长,最高抑制率可达31.01%。而在细胞快速生长的高峰期添加10~50 μmol·L^-1 MeJA后,β-蜕皮甾酮积累量可在短时间内大量激增,β-蜕皮甾酮积累量最高达到1.4136 mg·g^-1,是CK的5.06倍(P<0.01)。添加100~200 μmol·L^-1 MeJA则积累量较少。在细胞快速生长的中期添加100 μmol·L^-1 MeJA条件下对细胞的刺激较小,β-蜕皮甾酮积累量最高,达到3.5315 mg·g^-1。     

Effect of feeding rice whole crop silage on growth rate,levels of vitamin A, β‐carotene,vitamin E and IGF‐1 in plasma and skeletal muscle protein degradation in Japanese black calves

This study evaluated the effects of rice whole crop silage (RWCS) on growth, plasma levels of vitamin A, β‐carotene, vitamin E and IGF‐1, and expression of genes involved in muscle protein degradation and synthesis in Japanese Black calves. Eleven calves were divided into RWCS (fed RWCS ad libitum and concentrate, n = 5) and control groups (fed hay ad libitum and concentrate, n = 6). Final body weight and dairy gain were significantly larger in the RWCS group compared with the control group. Plasma β‐carotene and vitamin E concentrations were significantly higher in the RWCS group compared with control group. Although plasma vitamin E concentration in the RWCS group significantly increased from 4 to 9 months of age, it did not increase in the control group. At 6 months of age in the RWCS group, ubiquitin B (p < 0.05) and calpain 1 (p = 0.097) mRNA expression were lower than control group, but they were not different between groups at 9 months of age. These results indicate that RWCS increases plasma β‐carotene level and promotes muscle growth because of a decrease in the rate of protein degradation, but the effect is lost with the increase in plasma vitamin E level.     

A possible solution to model nonlinearity in elimination and distributional clearances with α2‐adrenergic receptor agonists: Example of the intravenous detomidine and methadone combination in sedated horses

The alpha(α)₂‐agonist detomidine is used for equine sedation with opioids such as methadone. We retrieved the data from two randomized, crossover studies where detomidine and methadone were given intravenously alone or combined as boli (STUDY 1) (Gozalo‐Marcilla et al., 2017, Veterinary Anaesthesia and Analgesia, 2017, 44 , 1116) or as 2‐hr constant rate infusions (STUDY 2) (Gozalo‐Marcilla et al., 2019, Equine Veterinary Journal, 51 , 530). Plasma drug concentrations were measured with a validated tandem Mass Spectrometry assay. We used nonlinear mixed effect modelling and took pharmacokinetic (PK) data from both studies to fit simultaneously both drugs and explore their nonlinear kinetics. Two significant improvements over the classical mammillary two‐compartment model were identified. First, the inclusion of an effect of detomidine plasma concentration on the elimination clearances (Cls) of both drugs improved the fit of detomidine (Objective Function Value [OFV]: ?160) and methadone (OFV: ?132) submodels. Second, a detomidine concentration‐dependent reduction of distributional Cls of each drug further improved detomidine (OFV: ?60) and methadone (OFV: ?52) submodel fits. Using the PK data from both studies (a) helped exploring hypotheses on the nonlinearity of the elimination and distributional Cls and (b) allowed inclusion of dynamic effects of detomidine plasma concentration in the model which are compatible with the pharmacology of detomidine (vasoconstriction and reduction in cardiac output).