The mTORC1/4EBP1/PPARγ Axis Mediates Insulin-Induced Lipogenesis by Regulating Lipogenic Gene Expression in Bovine Mammary Epithelial Cells

Guo, Zhixin; Cheng, Xiaoou; Feng, Xue; Zhao, Keyu; Zhang, Meng; Yao, Ruiyuan; Chen, Yuhao; Wang, Yanfeng; Huang, Hao; Wang, Zhigang

doi:10.1021/acs.jafc.9b01411

Cited by 19 publications

(12 citation statements)

References 57 publications

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“…Western blot was used to measure the indicated proteins and phosphorylated proteins as described ( 53 ). Briefly, BMECs were managed as four groups, i.e., control cells (uninfected cells), cells were infected by S. aureus 2, 4, and 8 h, respectively.…”

Section: Methodsmentioning

confidence: 99%

Intracellular Staphylococcus aureus Infection Decreases Milk Protein Synthesis by Preventing Amino Acid Uptake in Bovine Mammary Epithelial Cells

Chen

et al. 2021

Front. Vet. Sci.

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Staphylococcus aureus (S. aureus) is one of the main pathogens in cow mastitis, colonizing mammary tissues and being internalized into mammary epithelial cells, causing intracellular infection in the udder. Milk that is produced by cows that suffer from mastitis due to S. aureus is associated with decreased production and changes in protein composition. However, there is limited information on how mastitis-inducing bacteria affect raw milk, particularly with regard to protein content and protein composition. The main purpose of this work was to examine how S. aureus infection affects milk protein synthesis in bovine mammary epithelial cells (BMECs). BMECs were infected with S. aureus, and milk protein and amino acid levels were determined by ELISA after S. aureus invasion. The activity of mTORC1 signaling and the transcription factors NF-κB and STAT5 and the expression of the amino acid transporters SLC1A3 and SLC7A5 were measured by western blot or immunofluorescence and RT-qPCR. S. aureus was internalized by BMECs in vitro, and the internalized bacteria underwent intracellular proliferation. Eight hours after S. aureus invasion, milk proteins were downregulated, and the level of BMECs that absorbed Glu, Asp, and Leu from the culture medium and the exogenous amino acids induced β-casein synthesis declined. Further, the activity of mTORC1 signaling, NF-κB, and STAT5 was impaired, and SLC1A3 and SLC7A5 were downregulated. Eight hours of treatment with 100 nM rapamycin inhibited NF-κB and STAT5 activity, SLC1A3 and SLC7A5 expression, and milk protein synthesis in BMECs. Thus mTORC1 regulates the expression of SLC1A3 and SLC7A5 through NF-κB and STAT5. These findings constitute a model by which S. aureus infection suppresses milk protein synthesis by decreasing amino acids uptake in BMECs.

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Section: Methodsmentioning

confidence: 99%

Intracellular Staphylococcus aureus Infection Decreases Milk Protein Synthesis by Preventing Amino Acid Uptake in Bovine Mammary Epithelial Cells

Chen

et al. 2021

Front. Vet. Sci.

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“…Western blot was used to detect the expression levels of the indicated proteins and phosphorylated proteins, as previously described ( 35 ). Briefly, the supernatants were collected and boiled at 95°C with shaking for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Phosphoproteomics Analysis Reveals a Pivotal Mechanism Related to Amino Acid Signals in Goat Fetal Fibroblast

Huimin

Wang

et al. 2021

Front. Vet. Sci.

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In addition to serving as the building blocks for protein synthesis, amino acids serve as critical signaling molecules in cells. However, the mechanism through which amino acid signals are sensed in cells is not yet fully understood. This study examined differences in the phosphorylation levels of proteins in response to amino acid signals in Cashmere goat fetal fibroblasts (GFb). Amino acid deficiency was found to induce autophagy and attenuate mammalian/mechanistic target of rapamycin complex (mTORC1)/Unc-51-like autophagy activating kinase 1 (ULK1) signaling in GFb cells. A total of 144 phosphosites on 102 proteins positively associated with amino acid signaling were screened using phosphorylation-based proteomics analysis. The mitogen-activated protein kinase (MAPK) signaling pathway was found to play a potentially important role in the interaction network involved in the response to amino acid signals, according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and MAPK1/3 may serve as a central hub for the entire network. Motif analysis identified three master motifs, xxx_S_Pxx, xxx_S_xxE, and xxx_S_xDx, which were centered on those phosphosites at which phosphorylation was positively regulated by amino acid signaling. Additionally, the phosphorylation levels of three membrane proteins, the zinc transporter SLC39A7, the sodium-dependent neutral amino acid transporters SLC1A5 and SLC38A7, and three translation initiation factors, eukaryotic initiation factor (eIF)5B, eIF4G, and eIF3C, were positively regulated by amino acid signals. These pivotal proteins were added to currently known signaling pathways to generate a novel model of the network pathways associated with amino acid signals. Finally, the phosphorylation levels of threonine 203 and tyrosine 205 on MAPK3 in response to amino acid signals were examined by western blot analysis, and the results were consistent with the data from the phosphoproteomics analysis. The findings of this study provide new evidence and insights into the precise mechanism through which amino acid signals are sensed and conducted in Cashmere goat fetal fibroblasts.

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“…We identified three distinct subtypes of adipose stem and progenitor cells (ASPC1-3, Figure 3A; Supplementary Table S6). ASPC1 showed greater expression of PPARG (Figures 3B,C), a master regulator of lipid biosynthesis and adipocyte differentiation (Schadinger et al, 2005;Guo et al, 2019), as well as SLC1A3, LIPE, GPAM and LMO4, suggesting that these cells are committed adipocyte precursors (Hepler et al, 2018). In contrast, ASPC2 and ASPC3 showed increased expression of PDGFRA (Figures 3B,C), a known marker of fibroadipogenic progenitor cells (FAP), which have the capability to differentiate into adipocytes or activated fibroblasts (Contreras et al, 2021;Dohmen et al, 2022).…”

Section: Adipose Stem and Progenitor Cell Subpopulations Have Adipoge...mentioning

confidence: 98%

Single-nuclei analysis reveals depot-specific transcriptional heterogeneity and depot-specific cell types in adipose tissue of dairy cows

Michelotti

Kisby

Flores

et al. 2022

Front. Cell Dev. Biol.

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Adipose tissue (AT) is an endocrine organ with a central role on whole-body energy metabolism and development of metabolic diseases. Single-cell and single-nuclei RNA sequencing (scRNA-seq and snRNA-seq, respectively) analyses in mice and human AT have revealed vast cell heterogeneity and functionally distinct subtypes that are potential therapeutic targets to metabolic disease. In periparturient dairy cows, AT goes through intensive remodeling and its dysfunction is associated with metabolic disease pathogenesis and decreased productive performance. The contributions of depot-specific cells and subtypes to the development of diseases in dairy cows remain to be studied. Our objective was to elucidate differences in cellular diversity of visceral (VAT) and subcutaneous (SAT) AT in dairy cows at the single-nuclei level. We collected matched SAT and VAT samples from three dairy cows and performed snRNA-seq analysis. We identified distinct cell types including four major mature adipocytes (AD) and three stem and progenitor cells (ASPC) subtypes, along with endothelial cells (EC), mesothelial cells (ME), immune cells, and pericytes and smooth muscle cells. All major cell types were present in both SAT and VAT, although a strong VAT-specificity was observed for ME, which were basically absent in SAT. One ASPC subtype was defined as adipogenic (PPARG+) while the other two had a fibro-adipogenic profile (PDGFRA+). We identified vascular and lymphatic EC subtypes, and different immune cell types and subtypes in both SAT and VAT, i.e., macrophages, monocytes, T cells, and natural killer cells. Not only did VAT show a greater proportion of immune cells, but these visceral immune cells had greater activation of pathways related to immune and inflammatory response, and complement cascade in comparison with SAT. There was a substantial contrast between depots for gene expression of complement cascade, which were greatly expressed by VAT cell subtypes compared to SAT, indicating a pro-inflammatory profile in VAT. Unprecedently, our study demonstrated cell-type and depot-specific heterogeneity in VAT and SAT of dairy cows. A better understanding of depot-specific molecular and cellular features of SAT and VAT will aid in the development of AT-targeted strategies to prevent and treat metabolic disease in dairy cows, especially during the periparturient period.

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The mTORC1/4EBP1/PPARγ Axis Mediates Insulin-Induced Lipogenesis by Regulating Lipogenic Gene Expression in Bovine Mammary Epithelial Cells

Cited by 19 publications

References 57 publications

Intracellular Staphylococcus aureus Infection Decreases Milk Protein Synthesis by Preventing Amino Acid Uptake in Bovine Mammary Epithelial Cells

Intracellular Staphylococcus aureus Infection Decreases Milk Protein Synthesis by Preventing Amino Acid Uptake in Bovine Mammary Epithelial Cells

Phosphoproteomics Analysis Reveals a Pivotal Mechanism Related to Amino Acid Signals in Goat Fetal Fibroblast

Single-nuclei analysis reveals depot-specific transcriptional heterogeneity and depot-specific cell types in adipose tissue of dairy cows

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