Post-Transcriptional Processing of the Small Intestinal Alkaline Phosphatase in the Postnatal Developing Pig

猪的全身能量和营养物质利用效率在出生和哺乳期间较高，在断奶过渡期间急剧下降，在断奶后生长期间下降。断奶相关肠道疾病和生长迟缓是当前全球生猪生产面临的主要挑战。目前使用饲料抗生素来抑制断奶猪营养中的发病率和死亡率，导致与抗生素耐药性相关的全球公共卫生威胁不断上升。我的NSERC Discovery项目的长期目标是了解猪小肠水解酶和营养转运蛋白在隐窝绒毛轴和肠道发育过程中影响肠道功能的表达和调控因子。断奶标志着哺乳动物从哺乳到以固体食物为食的主要肠道适应。肠道碱性磷酸酶（IAP）是一种丰富的根尖膜蛋白，其主要生理功能是解毒细菌内毒素脂多糖。因此，IAP的表达对于保护肠道免受炎症和感染以及维持全身健康状态至关重要。早期断奶通过降低IAP基因在转录和转录后水平的表达，抑制了IAP最大酶活性，显著降低了仔猪顶端IAP的丰度和亲和力。肠内氨基酸利用率和膳食益生元可调节IAP的表达和功能。我推测，低蛋白饲粮可以通过对IAP生物合成和IAP糖基化的转录后调控，显著提高断奶仔猪的IAP表达、功能和肠内健康状况，因为饲粮中增加了营养AA的供应，即晶体谷氨酸、谷氨酰胺、亮氨酸和可发酵的非粘性可溶性纤维作为益生元。该研究的具体目标是研究提高营养膳食AA利用率和改变所选非粘性可溶性纤维组成在调节空肠IAP分数合成速率和IAP功能中的作用，并支持两名研究生进行拟议的研究。主要研究方法包括显色和纯化生理底物测定IAP动力学，同位素标记法测定体内IAP分数合成率，免疫沉淀法测定IAP糖基化和化学谱法测定IAP糖基化，免疫印迹法测定肠道蛋白翻译途径起始和延伸因子丰度。本研究将有助于进一步了解饲粮因素对断奶仔猪肠道内源性碱性磷酸酶表达和功能上调的影响，并制定改善肠道健康的替代策略。

Efficiency of whole body energy and nutrient utilization is high at birth and during suckling, then drops dramatically during the weanling transition and becomes low during the post-weaning growth in pigs. Weaning-associated enteric diseases and growth retardation are major challenges facing the current global swine production. Current use of feed antibiotics for curbing morbidity and mortality in weanling swine nutrition contributes to the rising global public health threat associated with antibiotic resistance. Long-term goals of my NSERC Discovery program aim to understand the expression and regulatory factors of the small intestinal hydrolases and nutrient transporters in affecting gut functions along the crypt-villus axis and during the gut development in pigs. Weaning marks a major intestinal adaptation from suckling to feeding on solid diets in mammals. Intestinal alkaline phosphatase (IAP) is an abundant apical membrane protein with a primary physiological function of detoxification of bacterial endotoxin lipopolysaccharides. Thus, expression of IAP is pivotal in protecting the gut from inflammation and infection and in maintaining whole body health status. Early weaning dampens the IAP maximal enzyme activity by reducing the IAP gene expression at the transcriptional and post-transcriptional levels with considerable decreases in the apical IAP abundance and affinity in piglets. Enteral amino acid (AA) availability and dietary prebiotics could modulate IAP expression and functionality. I hypothesize that IAP expression, functionality and enteral health status in weanling piglets fed a low-protein diet could be substantially enhanced by post-transcriptional regulation of IAP biosynthesis and IAP glycosylation because of increases in dietary supply of trophic AA, i.e., crystalline glutamate, glutamine, leucine and selected fermentable non-viscous soluble fibres serving as prebiotics. Specific objectives for the proposed research are to investigate roles of increasing these trophic dietary AA availability and changing the selected non-viscous soluble fibre composition in regulating the jejunal IAP fractional synthesis rate and IAP functionality, and in supporting two graduate students to conduct the proposed research. Major methodology for the proposed research includes IAP kinetics measured with chromogenic and purified physiological substrates, in vivo IAP fractional synthesis rate by isotopic labeling, IAP glycosylation by immune-precipitation and chemical profiling, and intestinal protein translational pathway initiation and elongation factor abundances by Western blotting. The proposed research will contribute to improved understanding of dietary factors in up-regulating intestinal endogenous alkaline phosphatase expression and functionality and the development of alternative strategies to improve gut health in weanling pigs.