Acylated Ghrelin Activates PI3K/mTOR Signaling Pathway by Promoting ThPOK Acetylation to Promote Milk Fat Synthesis in Bovine Mammary Epithelial Cells

Wang, Jiaxin; Cao, Yu; Long, Xiaoyu; Li, Feng; Jiang, Naiyuan; Sun, Mingyang; Xie, Yachun; Ge, Yusong; Guo, Wenjin; Liu, Juxiong; Fu, Shoupeng

doi:10.1021/acs.jafc.3c06977

Cited by 3 publications

(2 citation statements)

References 49 publications

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“…Cellular triglyceride (TAG) and TAG secreted into the culture medium by BMECs concentrations were measured using a TAG detection kit (E1013, E1003, Applygen) . Protein levels in the samples were determined using the BCA Protein Assay Kit (P0009, Beyotime Biotechnology).…”

Section: Methodsmentioning

confidence: 99%

Stigmasterol Activates the mTOR Signaling Pathway by Inhibiting ORP5 Ubiquitination to Promote Milk Synthesis in Bovine Mammary Epithelial Cells

Sun,

Cao,

Cheng

et al. 2024

J. Agric. Food Chem.

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Stigmasterol (ST), a phytosterol found in food, has various biological activities. However, the effect of ST on milk synthesis in dairy cows remains unclear. Therefore, bovine primary mammary epithelial cells (BMECs) were isolated, cultured, and treated with ST to determine the effect of ST on milk synthesis. The study revealed that 10 μM ST significantly increased milk synthesis in BMECs by activating the mammalian target of rapamycin (mTOR) signaling pathway. Further investigation revealed that this activation depends on the regulatory role of oxysterol binding protein 5 (ORP5). ST induces the translocation of ORP5 from the cytoplasm to the lysosome, interacts with the mTOR, recruits mTOR to target the lysosomal surface, and promotes the activation of the mTOR signaling pathway. Moreover, ST was found to increase ORP5 protein levels by inhibiting its degradation via the ubiquitin-proteasome pathway. Specifically, the E3 ubiquitin ligase membrane-associated cycle-CH-type finger 4 (MARCH4) promotes the ubiquitination and subsequent degradation of ORP5. ST mitigates the interaction between MARCH4 and ORP5, thereby enhancing the structural stability of ORP5 and reducing its ubiquitination. In summary, ST stabilizes ORP5 by inhibiting the interaction between MARCH4 and ORP5, thereby activating mTOR signaling pathway and enhancing milk synthesis.

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Section: Methodsmentioning

confidence: 99%

Stigmasterol Activates the mTOR Signaling Pathway by Inhibiting ORP5 Ubiquitination to Promote Milk Synthesis in Bovine Mammary Epithelial Cells

Sun,

Cao,

Cheng

et al. 2024

J. Agric. Food Chem.

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“…The culture induction medium for lactogenesis included 5 μg/mL insulin (Solarbio, China), 5 μg/mL hydrocortisone (Solarbio, China), and 20 ng/mL prolactin (ProSpec) in the basal medium . HEK-293T cells were seeded in high-glucose DMEM (Hyclone) containing 10% FBS (PAN-Biotech, Germany) and 100 μg/mL penicillin/streptomycin (Hyclone), and the culture environment was the same as those for BMECs …”

Section: Methodsmentioning

confidence: 99%

Bta-miR-200a Regulates Milk Fat Biosynthesis by Targeting IRS2 to Inhibit the PI3K/Akt Signal Pathway in Bovine Mammary Epithelial Cells

Tan,

Yang,

Qiu

et al. 2024

J. Agric. Food Chem.

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Milk fat synthesis has garnered significant attention due to its influence on the quality of milk. Recently, an increasing amount of proofs have elucidated that microRNAs (miRNAs) are important post-transcriptional factor involved in regulating gene expression and play a significant role in milk fat synthesis. MiR-200a was differentially expressed in the mammary gland tissue of dairy cows during different lactation periods, which indicated that miR-200a was a candidate miRNA involved in regulating milk fat synthesis. In our research, we investigated the potential function of miR-200a in regulating milk fat biosynthesis in bovine mammary epithelial cells (BMECs). We discovered that miR-200a inhibited cellular triacylglycerol (TAG) synthesis and suppressed lipid droplet formation; at the same time, miR-200a overexpression suppressed the mRNA and protein expression of milk fat metabolismrelated genes, such as fatty acid synthase (FASN), peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding protein 1 (SREBP1), CCAAT enhancer binding protein alpha (CEBPα), etc. However, knocking down miR-200a displayed the opposite results. We uncovered that insulin receptor substrate 2 (IRS2) was a candidate target gene of miR-200a through the bioinformatics online program TargetScan. Subsequently, it was confirmed that miR-200a directly targeted the 3′untranslated region (3′-UTR) of IRS2 via real-time fluorescence quantitative PCR (RT-qPCR), western blot analysis, and dualluciferase reporter gene assay. Additionally, IRS2 knockdown in BMECs has similar effects to miR-200a overexpression. Our research set up the mechanism by which miR-200a interacted with IRS2 and discovered that miR-200a targeted IRS2 and modulated the activity of the PI3K/Akt signaling pathway, thereby taking part in regulating milk fat synthesis in BMECs. Our research results provided valuable information on the molecular mechanisms for enhancing milk quality from the view of miRNA-mRNA regulatory networks.

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Integrated metagenomics and metabolomics analyses revealed biomarkers in β-casein A2A2-type cows

Zhao,

Wang,

et al. 2024

Front. Vet. Sci.

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In Holstein cows, β-casein, one of the most critical proteins in milk, exists in two main genotypes, A1 and A2. Herein, 45 Holstein cows [categorized into three groups based on β-casein A1A1, A1A2, and A2A2 genotypes (N = 15)] with the same feeding management and litter size were enrolled to explore differences in rumen microflora and metabolites across various β-casein genotypes. Rumen fluids were collected for metagenomics and metabolomics analyses. Metabolomics and weighted gene co-expression network analysis (WGCNA) revealed that arachidonic acid (AA), adrenic acid (AdA), glycocholic acid (GCA), and taurocholic acid (TCA) were significantly and positively correlated with milk fat % in dairy cows (p < 0.05). Furthermore, macro-genomics and Spearman’s correlation analysis revealed significant positive correlations (p < 0.05) between the characteristic flora (g_Acetobacter, g_Pseudoxanthomonas, g_Streptococcus, and g_Pediococcus) and the five characteristic metabolites in the rumen of A2A2 dairy cows. Moreover, functional enrichment analysis revealed more genes enriched to the TRP channel’s inflammatory mediator-regulated pathway and the mTOR signaling pathway in A2A2 genotyped cows. Additionally, the regulatory effects of AA on bovine mammary epithelial cells (BMECs) were examined using CCK-8, EdU, and qRT-PCR assays, revealing that AA promoted triglyceride (TG) synthesis and upregulated the milk fat marker genes including SREBF1, ACSS2, AGPAT6, and FASN. Overall, we identified characteristic microorganisms and metabolites in A2A2 Holstein cows and established that AA could be a biomarker for higher milk fat %.

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Acylated Ghrelin Activates PI3K/mTOR Signaling Pathway by Promoting ThPOK Acetylation to Promote Milk Fat Synthesis in Bovine Mammary Epithelial Cells

Cited by 3 publications

References 49 publications

Stigmasterol Activates the mTOR Signaling Pathway by Inhibiting ORP5 Ubiquitination to Promote Milk Synthesis in Bovine Mammary Epithelial Cells

Stigmasterol Activates the mTOR Signaling Pathway by Inhibiting ORP5 Ubiquitination to Promote Milk Synthesis in Bovine Mammary Epithelial Cells

Bta-miR-200a Regulates Milk Fat Biosynthesis by Targeting IRS2 to Inhibit the PI3K/Akt Signal Pathway in Bovine Mammary Epithelial Cells

Integrated metagenomics and metabolomics analyses revealed biomarkers in β-casein A2A2-type cows

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