Glutamine synthetase in muscle is required for glutamine production during fasting and extrahepatic ammonia detoxification

Abstract

The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a approximately 3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the approximately 4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was approximately 5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify approximately 2.5 micromol ammonia/g muscle.h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.

FIGURE 1.

Glutamine synthetase expression in MCK-Cre^tg/−/GS^fl/LacZ mice. A, MCK-Cre expression in muscle. Cryostat sections of gastrocnemius muscle of a 5-day-old neonatal and a 1-month-old MCK-Cre/R26R-lacZ transgenic mouse were stained for the presence of β-galactosidase activity. MCK-Cre-mediated recombination of the R26R locus, which causes LacZ expression, is hardly detectable at 5 days of age (panel A1) but is complete at 1 month of age (panel A2). B, GS protein expression in liver (L), kidney (K), cerebral cortex (C), and brown adipose tissue (B) is unaffected in GS^fl/LacZ mice with (−) or without (+) muscle-specific GS elimination. C and D, expression of GS and β-galactosidase in the muscle tissue. Serial sections of gastrocnemius muscle from Cre^tg/−/GS^+/+ (panels C1 and D1), Cre^tg/−/GS^+/LacZ (panels C2 and D2), and Cre^tg/−/GS^fl/LacZ (panels C3, C4, D3, and D4) were stained immunohistochemically for GS (C) and β-galactosidase (D). In Cre^tg/−/GS^+/LacZ mice, GS protein (panel C2) and β-galactosidase (panel D2) co-localize in muscle and adipose tissue. The expression pattern of β-galactosidase in Cre^tg/−/GS^+/LacZ and Cre^tg/−/GS^fl/LacZ mice is similar (panels D2–D4). In contrast, GS protein was absent in muscle tissue from Cre^tg/−/GS^fl/LacZ mice, whereas the neighboring adipose tissue maintained normal GS expression (panels C3 and C4 versus panels D3 and D4). M, muscle tissue; A (arrow), adipose tissue. Scale bars, 200 μm.

FIGURE 2.

Effect of genotype and gender on circulating concentrations and metabolism of glutamine, alanine, branched chain amino acids, and ammonia in the hindquarter of 4-hours fasted mice. A, arterial plasma concentration. B, net production (positive values) or consumption (negative values) across the hindquarter. All mice were transgenic for MCK-Cre. White bars, GS^+/+; gray bars, GS^+/LacZ; black bars, GS^fl/LacZ mice; bars without hatching, male mice; bars with hatching, female mice. *, p < 0.05; #, p < 0.01.

FIGURE 3.

The effect of fasting on circulating arterial concentrations and net production or consumption across the hindquarter of male control and GS-KO/M mice. A, glucose, lactate, and β-hydroxybutyrate (β-OH-butyrate). B, glutamine, alanine, and branched chain amino acids. C, glutamate, summed amino acids, and ammonia. The arterial levels of the respective compounds are shown in the left column, whereas production (positive values) or consumption (negative values) are shown in the right column. D, tissue concentration of glutamine, alanine, branched chain amino acids, glutamate, and summed amino acids in calf muscle. Filled diamonds represent control mice; open squares represent GS-KO/M mice. Significant effects of genotype or duration of fasting are mentioned in the text.

FIGURE 4.

Changes in GS mRNA and protein in calf muscle of fasting control and GS-KO/M mice. A, GS mRNA. B, GS protein. C, representative example of a Western blot used to quantify muscle GS protein. Black columns, control muscle; white columns, GS-KO/M muscle. AB, Western blot stained with Amido Black; WB, the same blot stained for the presence of GS. Note the difference in appearance of the GS band (42 kDa) in muscle and kidney (K) caused by the abundant presence of α1-actin (also 42 kDa) in muscle (see AB).

FIGURE 5.

The effect of infusion of incremental amounts of NH₄HCO₃ into the external jugular vein on circulating ammonia levels in control and GS-KO/M mice. Control (filled symbols) and GS-KO/M male mice (open symbols) were subjected to stepwise increments (every 80 min) in the rate of ammonia infusion. A, plasma concentration of ammonia. B, plasma glutamine and total amino acid concentration. C, plasma urea concentration. Plasma concentrations as a function of the infusion rate of ammonia were measured at the end of each 80-min infusion period. A shows individual observations of ammonia concentration at different infusion rates, whereas the concentrations of glutamine and total amino acids (B) and urea (C) are binned into groups with the indicated infusion rate. Black symbols, control mice; white symbols, GS-KO/M mice.