L-leucine stimulates glutamate dehydrogenase activity and glutamate synthesis by regulating mTORC1/SIRT4 pathway in pig liver

The liver is the most essential organ for the metabolism of ammonia, in where most of ammonia is removed by urea and glutamine synthesis. Regulated by leucine, glutamate dehydrogenase(GDH) catalyzes the reversible inter-conversion of glutamate to ammonia. To determine the mechanism of leucine regulating GDH, pigs weighing 20 ± 1 kg were infused for 80 min with ammonium chloride or alanine in the presence or absence of leucine. Primary pig hepatocytes were incubated with or without leucine. In the in vivo experiments with either ammonium or alanine as the nitrogen source, addition of leucine significantly inhibited ureagenesis and promoted the production of glutamate and glutamine in the perfused pig liver(P < 0.05). Similarly, leucine stimulated GDH activity and inhibited sirtuin4(SIRT4)gene expression(P < 0.01). Leucine could also activate mammalian target of rapamycin complex 1(m TORC1) signaling(P < 0.05), as evidenced by the increased phosphorylation levels of ribosomal protein S6 kinase 1(S6 K1) and ribosomal protein S6(S6). Interestingly, the leucine-induced m TORC1 pathway activation suitably correlated with increased GDH activity and decreased expression of SIRT4.Similar results were observed in primary cultured hepatocytes. Notably, leucine exerted no significant change in GDH activity in SIRT4-deficient hepatocytes(P > 0.05), while m TORC1 signaling was activated.Leucine exerted no significant changes in both GDH activity and SIRT4 gene expression in rapamycin treated hepatocytes(P > 0.05). In conclusion, L-leucine increases GDH activity and stimulates glutamate synthesis from different nitrogen sources by regulating m TORC1/SIRT4 pathway in the liver of pigs.

MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-β and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes

Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.Results: By porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4(SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6(TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.Conclusions: MiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.