Coordinated alteration of mRNA-microRNA transcriptomes associated with exosomes and fatty acid metabolism in adipose tissue and skeletal muscle in grazing cattle

Abstract

Objective: On the hypothesis that grazing of cattle prompts organs to secrete or internalize circulating microRNAs (c-miRNAs) in parallel with changes in energy metabolism, we aimed to clarify biological events in adipose, skeletal muscle, and liver tissues in grazing Japanese Shorthorn (JSH) steers by a transcriptomic approach.

Methods: The subcutaneous fat (SCF), biceps femoris muscle (BFM), and liver in JSH steers after three months of grazing or housing were analyzed using microarray and quantitative polymerase chain reaction (qPCR), followed by gene ontology (GO) and functional annotation analyses.

Results: The results of transcriptomics indicated that SCF was highly responsive to grazing compared to BFM and liver tissues. The 'Exosome', 'Carbohydrate metabolism' and 'Lipid metabolism' were extracted as the relevant GO terms in SCF and BFM, and/or liver from the >1.5-fold-altered mRNAs in grazing steers. The qPCR analyses showed a trend of upregulated gene expression related to exosome secretion and internalization (charged multivesicular body protein 4A, vacuolar protein sorting-associated protein 4B, vesicle associated membrane protein 7, caveolin 1) in the BFM and SCF, as well as upregulation of lipolysisassociated mRNAs (carnitine palmitoyltransferase 1A, hormone-sensitive lipase, perilipin 1, adipose triglyceride lipase, fatty acid binding protein 4) and most of the microRNAs (miRNAs) in SCF. Moreover, gene expression related to fatty acid uptake and inter-organ signaling (solute carrier family 27 member 4 and angiopoietin-like 4) was upregulated in BFM, suggesting activation of SCF-BFM organ crosstalk for energy metabolism. Meanwhile, expression of plasma exosomal miR-16a, miR-19b, miR-21-5p, and miR-142-5p was reduced. According to bioinformatic analyses, the c-miRNA target genes are associated with the terms 'Endosome', 'Caveola', 'Endocytosis', 'Carbohydrate metabolism', and with pathways related to environmental information processing and the endocrine system.

Conclusion: Exosome and fatty acid metabolism-related gene expression was altered in SCF of grazing cattle, which could be regulated by miRNA such as miR-142-5p. These changes occurred coordinately in both the SCF and BFM, suggesting involvement of exosome in the SCF-BFM organ crosstalk to modulate energy metabolism.

Keywords: Exosome; Grazing Cattle; Lipid Metabolism; Organ Crosstalk; Subcutaneous Fat; microRNA.

Figure 1

Gene expression related to exosome biogenesis, secretion, and internalization in SCF, BFM, and liver grazing cattle. The ratios of the normalized gene expression of grazing cattle to housed cattle are shown in columns (level of housed cattle is indicated with dashed line). RPL7 was used as the internal control. SCF and BFM indicate subcutaneous fat and biceps femoris muscle, respectively. RPL7, ribosomal protein L7. Error bars indicate standard error. * and + indicate differences between grazing and housed at p<0.05 and p<0.10, respectively.

Figure 2

MicroRNA expression in plasma exosomes of grazing cattle. The ratios of the normalized gene expression of grazing cattle to housed cattle are shown in columns (level of housed cattle is indicated with dashed line). miR-15a was used as the internal control. Error bars indicate standard error. * and + indicate differences between grazing and housed cattle at p<0.05 and p<0.10, respectively.

Figure 3

Changes in non-esterified fatty acid (NEFA) concentration during grazing period. Closed circles and open squares indicate grazing and housed in cattle, respectively. + indicates a difference between grazing and housed at p<0.10.

Figure 4

Gene expression related to adipogenesis and lipid metabolism in subcutaneous fat of grazing cattle. The ratios of the normalized gene expression of grazing cattle to housed cattle are shown in columns (level of housed cattle is indicated with dashed line). Ribosomal protein L7 (RPL7) was used as the internal control. Error bars indicate standard error. * and + indicate differences between grazing and housed cattle at p<0.05 and p<0.10, respectively.

Figure 5

MicroRNA expression in subcutaneous fat of grazing cattle. The ratios of the normalized gene expression of grazing cattle to housed cattle are shown in columns (level of housed cattle is indicated with dashed line). RNA U6A small nuclear (RNU6) was used as the internal control. Error bars indicate standard error. * and + indicate differences between grazing and housed at p<0.05 and p<0.10, respectively.

Figure 6

mRNA expression in the biceps femoris muscle of grazing cattle. The ratios of the normalized gene expression of grazing cattle to housed cattle are shown as columns (level of housed cattle is indicated with dashed line). β-Actin was used as the internal control. Error bars indicate standard error. ** and + indicate differences between grazing and housed at p<0.01 and p<0.10, respectively.

Figure 7

Hypothesized adipose-skeletal muscle organ crosstalk for energy homeostasis in grazing cattle. Lipolysis in subcutaneous fat (SCF) induced by grazing stimulation could generate non-esterified fatty acid (NEFA) and contribute to increases in circulating NEFA, which could then result in NEFA uptake by the biceps femoris muscle (BFM), with NEFA serving as an energy substrate in cattle. Exosome traffic might be involved in SCF-BFM organ crosstalk regarding energy homeostasis.