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Differential gene expression in ATZ-globule-free and ATZ-globule-containing hepatocytes in PIZ mouse liver

ABSTRACT: Alpha-1-antitrypsin deficiency is a disease of the liver and lung where accumulation of misfolded ATZ protein forms large protein aggregates or globules that result in significant cellular stess. From Birth to 2 months of age, virtually every hepatocyte has numerous globules of the mutant ATZ protien. At 2 months of age small colonies of globule free hepatocytes arise and over time the liver becomes largely globule free. this analysis aims to identify differential gene expression profiles in the globule free vs the globule containing cells to idenitfy key factors and pathways regulating clearance of the mutant ATZ protien. We used Laser Capture Microdisection to isolate RNA from ATZ-globule-free hepatocytes and ATZ-globule-containing hepatocytes and subjected them to microarray analysis to detail the differential global gene expression profiles.

ORGANISM(S): Mus musculus

PROVIDER: GSE84763 | GEO | 2017/08/25

SECONDARY ACCESSION(S): PRJNA331089

REPOSITORIES: GEO

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Project description:During lactation, mammary epithelial secretory cells secrete huge amounts of milk from their apical side. The major milk proteins, the caseins, are secreted by exocytosis, while milk fat globules are released by budding, enwrapped by the plasma membrane. Due to the number and large size of milk fat globules, the membrane surface needed for their secretion might exceed that of the apical plasma membrane. In order to identify the cellular compartments that may provide membrane during the budding of milk fat globules, a large-scale proteomics analysis of both cytoplasmic lipid droplets and secreted milk fat globules membranes was performed in mouse cells. The differential analysis of the protein profiles of these two organelles strongly suggest that, in addition to the apical plasma membrane, at least the endoplasmic reticulum, the secretory vesicles containing caseins, and potentially the mitochondria contribute to the formation of the milk fat globule membrane. Moreover, the specific analysis of the membrane-associated as well as the raft-associated proteins reinforces this possibility and points to a role for lipid rafts in milk product secretion. The subcellular localization of several major proteins of the cytoplasmic lipid droplets or of the milk fat globule membrane was investigated by immunofluorescence in lactating mammary epithelial cells. Furthermore, the localization of GM1 ganglioside, a known marker of lipid rafts and of free cholesterol, showed a clear association with both cytoplasmic lipid droplets and secreted milk fat globules. Additionally, the presence of some SNARE proteins that may be involved in casein exocytosis and of two Rab GTPases on both cytoplasmic lipid droplets and milk fat globules was demonstrated by immunofluorescence. Altogether, our results provide evidence for a pivotal role of the endoplasmic reticulum as membrane contributor to milk fat globule budding, and suggest that some SNARE proteins may spatio-temporally coordinate the secretion of milk products.

Project description:Intake of high-fat foods raises postprandial plasma triglycerides and inflammatory markers, which may depend on the type of fat ingested. Dairy products are commonly consumed, but not much is known about the impact of milk fat and the milk fat globule membrane on postprandial inflammation. Here, we aimed to study the effect of milk fat with and without milk fat globule membrane and vegetable blend fat on post-prandial inflammation, with a focus on blood monocyte gene expression. We performed a randomized, double-blind cross-over trial in middle-aged healthy male and female volunteers (BMI 22–27 kg/m2). The participants consumed a meal shake containing 95.5 g of fat consisting of either a vegetable fat blend (VEGE), anhydrous milk fat (AMF, without milk fat globule membrane), or cream (CREAM, containing milk fat globule membrane). Blood monocytes were collected at 0 and 6 hours postprandially and used for bulk RNA sequencing and ex vivo stimulation with LPS. Consumption of all three shakes significantly decreased the percentage of classical monocytes and increased the percentages of intermediate monocytes and non-classical monocytes. No differences in these measures were observed between shakes. Using a threshold of p < 0.01, 787 genes were differentially regulated postprandially between the three shakes. 89 genes were differentially regulated postprandially between AMF and VEGE, 373 genes between AMF and CREAM, and 667 genes between VEGE and CREAM, indicating that the effect of CREAM on monocyte gene expression was distinct from AMF and VEGE. Pathway analyses showed that VEGE significantly increased the expression of genes involved in inflammatory pathways, whereas this was less pronounced after AMF and not observed after CREAM. In addition, CREAM significantly down-regulated the expression of genes involved in energy metabolism-related pathways, such as glycolysis, TCA cycle, and oxidative phosphorylation, as well as HIF1 signaling. Compared to the consumption of an anhydrous milk fat without milk fat globule membrane and a vegetable fat blend, the consumption of cream with milk fat globule membrane downregulated inflammatory pathways in blood monocytes, thus suggesting a potential inflammation inhibitory effect of milk fat globule membrane.

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Differential gene expression in ATZ-globule-free and ATZ-globule-containing hepatocytes in PIZ mouse liver

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