Metabolic transition of milk triacylglycerol synthesis in response to varying levels of palmitate in porcine mammary epithelial cells

Abstract

BackgroundMilk in mammals is a key source of lipids for offspring, providing both critical energy and essential fatty acids. For lactating sows, palmitic acid is one of the most abundant fatty acids in milk, providing 10~12% of the suckling pig total dietary energy supply. However, the effects of exogenous palmitic acid on milk fat synthesis in sow mammary glands are not well-known. In this study, we investigated the effects of palmitic acid on lipogenic genes in porcine mammary epithelial cells (pMECs) to explore the role of exogenous palmitic acid in mediating milk triacylglycerols (TAG) synthesis.MethodsPorcine mammary epithelial cells were cultured for 24 h in the presence of different concentrations of palmitate (0, 25, 50, 100, 200, 400, and 600 μM). The effect of palmitate on cell viability was tested via MTT assay. Intracellular lipid accumulation was measured through Oil Red O staining, and TAG levels were quantified by enzymatic colorimetric methods. Expression of genes and proteins involved in milk fat biosynthesis were assayed with quantitative real-time polymerase chain reaction (qPCR) and Western blotting, respectively.ResultsIncubation with palmitate promoted cellular lipid synthesis in a dose-dependent manner, as reflected by the increased TAG content and enhanced formation of cytosolic lipid droplets. The increased lipid synthesis by palmitate was probably attributable to the upregulated mRNA expression of genes associated with milk fat biosynthesis, including long-chain fatty acid uptake (LPL, CD36), intracellular activation and transport (ACSL3, FABP3), TAG synthesis (GPAM, AGPAT6, DGAT1), lipid droplet formation (PLIN2), and regulation of transcription (PPARγ). Western blot analysis of CD36 and DGAT1 proteins confirmed the increased lipid synthesis with increasing incubation of palmitate. However, the genes involved in fatty acid de novo synthesis (ACACA, FASN), fatty acid desaturation (SCD), and regulation of transcription (SREBP1, INSIG1) were inversely affected by incubation with increasing concentrations of palmitate. Western blot analysis of ACACA protein confirmed this decrease associated with increasing levels of palmitate.ConclusionsResults from this study suggest that palmitate stimulated the cytosolic TAG accumulation in pMECs, probably by promoting lipogenic genes and proteins that are involved in lipid synthesis. However, addition of palmitate decreased the fatty acid de novo synthesis in pMECs.

Aging-associated alterations in the mammary gland revealed by single-cell RNA sequencing

“…Of note, the decrease in expression of the redox regulators Gpx3 and Fabp4 in aged AV cells may reflect a change in their redox state. In addition, the enhanced expression of the master alveolar-lineage transcription factor Elf5 and milk-related genes Spp1 22-24 and Plin2 25,26 in aged AV cells may potentially reflect their shift toward a slightly more mature state in the alveolar lineage, consistent with previous report 27 as well as our observation (Supplementary figure 3) that aged mammary glands showed signs of activated alveolar cells and increased secretory material independent of pregnancy.…”

“…For example, within the mammary epithelium, the dramatic expansion of the AV population with age, from a low-abundance cell type in young mammary glands to the most dominant population in older glands, reflects a significant increase in proliferation of these cells relative to the other cell types. In addition to their increase in relative proportion, aged AV cells also exhibit higher expression levels of genes associated with mature alveolar cells, including the master alveolar transcription factor Elf5 37,38 , as well as milk-related genes Plin2 25,26 and Spp1 22-24 , suggesting a shift toward a more differentiated mature alveolar state with age. These changes are consistent with previous report 27 as well as our observation that aged nulliparous mice exhibit higher levels of budding alveoli and dilated ducts filled with secretory material without pregnancy, and are likely caused by the continuous stimulation by hormones in the estrous cycle 27 .…”

Metabolic Transition of Milk Triacylglycerol Synthesis in Response to Varying Levels of Three 18-Carbon Fatty Acids in Porcine Mammary Epithelial Cells

“…The pMECs were isolated, purified, and cultured from the mammary gland of a 17-day lactating Large White sow according to our previously described protocol [ 48 ]. Isolated cells were maintained in a basal Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) (Gibco, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), Insulin-Transferrin Selenium (ITS) (5 μg/mL; ScienCell, Carlsbad, CA, USA), epidermal growth factor (EGF) (10 ng/mL; Gibco, Grand Island, NY, USA), IGF-1 (10 ng/mL; Sigma-Aldrich, St. Louis, MO, USA), hydrocortisone (5 μg/mL; Sigma-Aldrich, St. Louis, MO, USA), penicillin (100 U/mL) and streptomycin (100 μg/mL; Gibco, Grand Island, NY, USA).…”

AQP3 Facilitates Proliferation and Adipogenic Differentiation of Porcine Intramuscular Adipocytes

“…The procedure of PCR reaction was pre-denaturation for 5 min, followed by denaturation for 10 s, annealing for 30 s, and extension of 30 s for 35 cycles. Primers targeting AQP3 [21], PPARγ [22] (peroxisome proliferator activated receptor γ), FABP4 [23] (adipocyte fatty-acid binding protein 4), mGPAT [24] (mitochondrial glycerol-3-phosphate acyltransferase), Perilipin 1 [25], cyclin B [26], and β-actin [27] were picked out in previous reports. Primers for SCD (stearoyl-CoA desaturase), CD36, C/EBPα (CCAAT/enhancer binding protein α), ELOVL6 (elongase of long-chain fatty acids family), FASN (fatty acid synthetase), ACACA (acetyl-CoA carboxylase), DGAT2 (diacylglycerol O-acyltransferase 2), cyclin E, and cyclin D were designed online (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Sangon Biotech (Shanghai, China).…”