L-Arginine has been reported to enhance brown adipose tissue developments in fetal lambs of obese ewes, but the underlying mechanism is unknown. The present study tested the hypothesis that L-arginine stimulates growth and development of brown adipocyte precursor cells (BAPCs) through activation of mammalian target of rapamycin cell signaling. BAPCs isolated from fetal lambs at day 90 of gestation were incubated   for 6 h in arginine-free DMEM, and then cultured in DMEM with concentrations of 50, 100, 200, 500 or 1000 μmol L-arginine/L for 24-96 h. Cell proliferation, protein turnover, the mammalian target of rapamycin (mTOR) signaling pathway and pre-adipocyte differentiation markers were determined. L-arginine treatment enhanced (P < 0.05) BAPC growth and protein synthesis, while inhibiting proteolysis in a dose-dependent manner. Compared with 50 and 100 μmol/L (the concentrations of arginine in the maternal plasma of obese ewes), 200 μmol L-arginine/L (the concentrations of arginine in the maternal plasma of obese ewes receiving arginine supplementation) increased (P < 0.05) the abundances of phosphorylated mTOR, P70 and 4EBP1, as well as the abundances of PGC1α, UCP1, BMP7 and PRDM16. These novel findings indicate that increasing extra-cellular arginine concentration from 50 to 200 µmol/L activates mTOR cell signaling in BAPCs and enhances their growth and development in a dose-dependent manner. Our results provide a mechanism for arginine supplementation to enhance the development of brown adipose tissue in fetal lambs.

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Abstract This study determined whether extracellular citrulline is degraded by ruminal bacteria of sheep. In the first experiment, whole rumen fluid (3 mL) from six adult Suffolk sheep was incubated at 37 °C with 5 mM l-glutamine (Gln), l-glutamate (Glu), l-arginine (Arg), or l-citrulline (Cit) for 0, 0.5, 1, and 2 h or with 0, 0.5, 2, or 5 mM Gln, Glu, Arg, or Cit for 2 h. An aliquot (50 µL) of the incubation solution was collected at the predetermined time points for amino acids (AA) analyses. Results showed extensive hydrolysis of Gln into Glu and ammonia, of Arg into l-ornithine and l-proline, but little or no degradation of extracellular Cit or Glu by ruminal microbes. In the second experiment, six adult Suffolk sheep were individually fed each of three separate supplements (8 g Gln , Cit, or urea) on three separate days along with regular feed (800 g/animal). Blood (2 mL) was sampled from the jugular vein prior to feeding (time 0) and at 0.5, 1, 2, and 4 h after consuming the supplement. Plasma was analyzed for AA, glucose, ammonia, and urea. The concentrations of Cit in the plasma of sheep consuming this AA increased (P &lt; 0.001) by 117% at 4 h and those of Arg increased by 23% at 4 h, compared with the baseline values. Urea or Gln feeding did not affect (P &gt; 0.05) the concentrations of Cit or Arg in plasma. These results indicate that Cit is not metabolized by ruminal microbes of sheep and is, therefore, absorbed as such by the small intestine and used for the synthesis of Arg by extrahepatic tissues.

In vitro and in vivo experiments were conducted to determine the metabolism of rumen-protected or unprotected l-citrulline (Cit) plus l-glutamine (Gln) by ruminal microbes. In the in vitro experiment, whole ruminal fluid (3 mL, containing microorganisms) from steers was incubated at 37 ºC with 5 mM Cit plus 6 mM Gln (in a rumen-protected or unprotected form) for 0, 0.5, 2, or 4 h after which times 50 µL samples were collected for AA and ammonia analyses. In the in vivo experiment, at 0.5 h before and 0, 0.5, 1, 2, 4, and 6 h after cannulated adult steers consumed 0.56 kg dried-distillers’ grain mixed with 70 g Cit plus 70 g Gln (in a rumen-protected or unprotected form), samples of ruminal fluid and jugular venous blood were obtained for AA analyses. Results from both in vitro and in vivo experiments demonstrated extensive hydrolysis of rumen-unprotected Gln into glutamate, but little degradation of the rumen-protected Gln or rumen-protected and unprotected Cit by ruminal microbes. Concentrations of Cit and arginine in the plasma of steers consuming rumen-protected or unprotected AA increased at 1 and 2 h after the meal, respectively, when compared with values at 0 h. Collectively, these novel findings indicate that ruminal microbes of adult steers do not degrade extracellular Cit in a rumen-protected or unprotected form. Our results refute the view that all dietary AAs are extensively catabolized by ruminal microorganisms and also have important implications for dietary supplementation with Cit to ruminants to enhance the concentration of arginine in their plasma and their productivity.