Abstract
Intramuscular fat (IMF) deposition is one of the most important factors to affect meat quality in livestock and induce insulin resistance and adverse metabolic phenotypes for humans. However, the key regulators involved in this process remain largely unknown. Although liver kinase B1 (LKB1) was reported to participate in the development of skeletal muscles and classical adipose tissues. Due to the specific autonomic location of intramuscular adipocytes, deposited between or within muscle bundles, the exact roles of LKB1 in IMF deposition need further verified. Here, we cloned the goat LKB1 coding sequence with 1,317 bp, encoding a 438 amino acid peptide. LKB1 was extensively expressed in detected tissues and displayed a trend from decline to rise during intramuscular adipogenesis. Functionally, knockdown of LKB1 by two individual siRNAs enhanced the intramuscular preadipocytes differentiation, accompanied by promoting lipid accumulation and inducing adipogenic transcriptional factors and triglyceride synthesis-related genes expression. Conversely, overexpression of LKB1 restrained these biological signatures. To further explore the mechanisms, the RNA-seq technique was performed to compare the difference between siLKB1 and the control group. There were 1,043 differential expression genes (DEGs) were screened, i.e., 425 upregulated genes and 618 downregulated genes in the siLKB1 group. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis predicted that the DEGs were mainly enriched in the focal adhesion pathway and its classical downstream signal, the PI3K-Akt signaling pathway. Specifically, knockdown of LKB1 increased the mRNA level of focal adhesion kinase (FAK) and vice versa in LKB1-overexpressed cells, a key component of the activated focal adhesion pathway. Convincingly, blocking this pathway by a specific FAK inhibitor (PF573228) rescued the observed phenotypes in LKB1 knockdown adipocytes. In conclusion, LKB1 inhibited goat intramuscular adipogenesis through the focal adhesion pathway. This work expanded the genetic regulator networks of IMF deposition and provided theoretical support for improving human health and meat quality from the aspect of IMF deposition.
Highlights
Intramuscular fat (IMF) was regarded as the most intramuscular adipocytes deposited between primary and secondary muscle bundles of perimysium, and a small population was founded within muscle bundles (Li et al, 2020)
The data showed that the full length of the liver kinase B1 (LKB1) gene was 1,380 bp and was cloned by PCR using cDNA of longissimus dorsi as a template, including 1,317 bp complete open reading frame (ORF) region sequence, encoding a 438 amino acid peptide, and 63 bp 3′ untranslated region (3′UTR) sequence (Figure 1A)
The phylogenetic trees of LKB1 proteins, constructed by clustalx1.83 and mega5.0, showed that goat LKB1 protein had the closest relationship with those of cattle and pig, and the farthest relationship with that of Danio rerio and Drosophila (Figure 1D). These results suggest that the function of the LKB1 gene was relative conservation among species
Summary
Intramuscular fat (IMF) was regarded as the most intramuscular adipocytes deposited between primary and secondary muscle bundles of perimysium, and a small population was founded within muscle bundles (Li et al, 2020). IMF was referred to as marbling fat, which was highly desirable for improving the tenderness and flavor of meat quality (Hocquette et al, 2010; Hunt et al, 2016; Sun et al, 2019), while increasing IMF content in humans was associated with the occurrence of insulin resistance and adverse metabolic phenotypes, such as type 2 diabetes (Lim, 2014). There are intensive efforts for elucidating the molecular mechanism of IMF deposition to enhance meat quality or defend against metabolic syndrome because of ectopic fat accumulation in skeletal muscle. MyoD-Cre-derived Lkb deletion of muscle stem cells (satellite cells) and their descendent mature muscles increased lipid accumulation in proliferating myoblasts and myofibers (Shan et al, 2015). Intramuscular adipocyte was located within perimysium or muscle bundles, whose hyperplasia and hypertrophy were associated with reduced contractile function, insulin resistance, and type 2 diabetes (Li et al, 2020). The specific autonomic location and absence of maker genes of intramuscular adipocyte limited researchers to explore the molecular mechanism of its deposition in vivo
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