Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes

Abstract

SummaryThe cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. Culture of Hep G2 cells without glutamine resulted in very high levels of protein, again with no change in mRNA abundance. Insulin was without effect in both C2C12 and Hep G2 cells. In 3T3 L1 adipocytes glucocorticoids increased the abundance of both glutamine synthetase mRNA and protein, insulin added alone had no effect but in the presence of glucocorticoids resulted in lower mRNA levels than seen with glucocorticoids alone, although protein levels remained high under such conditions. In contrast to the other cell lines glutamine synthetase protein levels were relatively unchanged by culture in the absence of glutamine. The results support the hypothesis that in myocytes, and hepatomas, but not in adipocytes, glutamine acts to moderate glutamine synthetase induction by glucocorticoids. Keywords glutamine; glutamine synthetase; skeletal muscle; liver; adipocyte; insulin; glucocorticoids Glutamine is the most abundant free α amino acid in most mammalian species. The plasma glutamine pool is turning over very rapidly since it plays important roles in the interorgan transport of carbon, nitrogen and energy [1]. Although present in the diet, most ingested glutamine is metabolized by the small intestinal mucosa and thus the large body pool of glutamine is synthesized de novo [2]. The only enzyme capable of glutamine synthesis is glutamine synthetase (L-glutamate:ammonia ligase (ADP) EC 6.3.1.2) which is found in relatively high activity in skeletal muscle, adipose tissue, liver, lungs, brain and small intestine, but its regulation is poorly understood [3][4][5][6][7][8][9]. The enzyme is not known to be subject to regulation by allosteric or covalent modification mechanisms and those agents that show stimulatory or inhibitory actions in vitro are unlikely to play any physiological role [5,7]. The enzyme is however, subject to long-term regulation via changes in the amount of enzyme protein. Diabetes increases expression of the enzyme in skeletal muscle and adipose tissue and

FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3′-UTR: possible contribution to ammonia detoxification

“…On the other hand, insulin, an anabolic hormone, suppresses glucocorticoid-induced GS mRNA level in cells such as adipocytes (2,34). Meanwhile, exogenous glutamine addition does not affect the GS mRNA level, but it decreases the GS protein level in cells (15,34), which was also observed in the present study. FOXO1 is a catabolic transcriptional regulator that is known to have an opposite effect to insulin (24).…”

“…For example, glucocorticoid, a catabolic hormone, upregulates GS mRNA expression in various cells (2,9,34). On the other hand, insulin, an anabolic hormone, suppresses glucocorticoid-induced GS mRNA level in cells such as adipocytes (2,34). Meanwhile, exogenous glutamine addition does not affect the GS mRNA level, but it decreases the GS protein level in cells (15,34), which was also observed in the present study.…”

“…Tamoxifen treatment increased the FOXO1-ER fusion protein level in this experiment. Glutamine suppressed the GS level (0.5-fold), which is consistent with previous studies (15,34). In the presence of tamoxifen, GS protein levels were increased twofold both in glutamine-free and glutamine-containing medium (Fig.…”

“…For example, glucocorticoid, a catabolic hormone, upregulates GS mRNA expression in various cells (2,9,34). On the other hand, insulin, an anabolic hormone, suppresses glucocorticoid-induced GS mRNA level in cells such as adipocytes (2,34).…”

“…Then, we analyzed the GS protein level in the cells. The GS protein level is known to be downregulated by glutamine in the medium (15,34). Thus, we performed experiments in both the presence and absence of glutamine in the medium.…”