Glutamine synthetase and alanine transaminase expression are decreased in livers of aged vs. young beef cows and GS can be upregulated by 17β-estradiol implants1

Abstract

Aged beef cows (≥ 8 yr of age) produce calves with lower birth and weaning weights. In mammals, aging is associated with reduced hepatic expression of glutamine synthetase (GS) and alanine transaminase (ALT), thus impaired hepatic Gln-Glu cycle function. To determine if the relative protein content of GS, ALT, aspartate transaminase (AST), glutamate transporters (EAAC1, GLT-1), and their regulating protein (GTRAP3-18) differed in biopsied liver tissue of (a) aged vs. young (3 to 4 yr old) nonlactating, nongestating Angus cows (Exp. 1 and 2) and (b) aged mixed-breed cows with and without COMPUDOSE (17β-estradiol) ear implants (Exp. 3), Western blot analyses were performed. In Exp. 1, 12 young (3.62 ± 0.01 yr) and 13 aged (10.08 ± 0.42 yr) cows grazed the same mixed forage for 42 d (August-October). In Exp. 2, 12 young (3.36 ± 0.01 yr) and 12 aged (10.38 ± 0.47 yr) cows were individually fed (1.03% of BW) a corn-silage-based diet to maintain BW for 20 d. For both Exp. 1 and 2, the effect of cow age was assessed by ANOVA using the MIXED procedure of SAS. Cow BW did not change ( ≥ 0.17). Hepatic ALT (78% and 61%) and GS (52% and 71%) protein content (Exp. 1 and 2, respectively) was decreased ( ≤ 0.01), whereas GTRAP3-18 (an inhibitor of EAAC1 activity) increased ( ≤ 0.01; 170% and 136%) and AST, GLT-1, and EAAC1 contents did not differ ( ≥ 0.17) in aged vs. young cows. In Exp. 2, free concentrations (nmol/g) of Glu, Ala, Gln, Arg, and Orn in liver homogenates were determined. Aged cows tended to have less ( = 0.10) free Gln (15.0%) than young cows, whereas other AA concentrations did not differ ( 0.26). In Exp. 3, 14 aged (> 10 yr) cows were randomly allotted ( = 7) to sham or COMPUDOSE (25.7 mg of 17β-estradiol) implant treatment (TRT), and had ad libitum access to alfalfa hay for 28 d. Blood and liver biopsies were collected 14 and 28 d after implant treatment. Treatment, time after implant (DAY), and TRT × DAY effects were assessed by ANOVA using the MIXED procedure of SAS. Cow BW was not affected ( ≥ 0.96). Implant increased ( ≤ 0.02) total plasma estradiol by 220% (5.07 vs. 1.58 pg/mL) and GS protein by 300%, whereas the relative content of other proteins was not altered ( ≥ 0.16). We conclude that hepatic expression of ALT and GS are reduced in aged vs. young cows, and administration of 17β-estradiol to aged cows increases plasma estradiol and hepatic GS, but not that of other proteins that support hepatic Glu metabolism.

Investigation of Liver Injury of Polygonum multiflorum Thunb. in Rats by Metabolomics and Traditional Approaches

“…Part of Amino acid was transformed into ionic ammonia by the kidney and excreted with the urine. Glutamine was produced by amino acid and glutamic acid which was originated from α-ketoglutaric acid by ALT and AST enzyme catalyzation in tricarboxylic acid cycle (Sözen et al, 2011 ; Miles et al, 2015 ). The long term administration of Raw PM resulted in liver damage with the decreased function of transforming blood ammonia to urea.…”

Hepatic glutamate transport and glutamine synthesis capacities are decreased in finished vs. growing beef steers, concomitant with increased GTRAP3-18 content

“…In general, Western blot analysis of liver tissue and LPM vesicles was performed as previously described by us (Howell et al 2001 ; Miles et al 2015 ). For liver homogenates, 1 g of liver tissue was homogenized on ice for 30 s (setting 11, POLYTRON, Model PT10/35; Kinematic, Inc., Neuchâtel) in 7.5 mL of 4 °C sample extraction buffer solution (0.25 mM sucrose, 10 mM HEPES–KOH pH 7.5, 1 mM EDTA, and 50 µL of protease inhibitor (Sigma, St. Louis, MO).…”

“…For liver homogenates (30 μg/lane) and LPM (15 μg/lane, protein quantified during preparation), proteins were separated by 12% SDS-PAGE and electrotransferred to a 0.45 µm nitrocellulose membrane (Bio-Rad, Hercules, CA). Blots were stained with Fast-Green (Fisher, Pittsburgh, PA), and the relative amount of stained protein per lane/sample determined by densitometric analyses and recorded as arbitrary units (Miles et al 2015 ; Howell et al 2001 ).…”

“…Two high-affinity (μM), concentrative, Glu transporters (GLT-1 and EAAC1) that demonstrate system activity (Na + -dependent l -Glu and l -aspartate uptake that is inhibited by d -aspartate) are expressed by the intestinal epithelia, liver, and kidney of sheep (Howell et al 2001 , 2003 ; Xue et al 2010 ) and cattle (Howell et al 2001 ; Miles et al 2015 ). In mouse neuronal cells, the function of EAAC1 (solute carrier 1A1/SLC1A1) and GLT-1 (SLC1A2) is inhibited by endoplasmic reticulum-localized GTRAP3-18 (glutamate transporter-associated protein 3–18; a.k.a.…”

“…However, ADP-ribosylation factor-like 6 interacting protein 1 (ARL6IP1) binds and inhibits GTRAP3-18, and indirectly promotes EAAC1 activity (Aoyama and Nakaki 2012 , 2013 ; Akiduki and Ikemoto 2008 ). The ARL6IP1/GTRAP3-18/EAAC1 triad is highly expressed in rat liver (Akiduki and Ikemoto 2008 ), whereas cow liver expresses at least EAAC1 and GTRAP3-18 (Miles et al 2015 ). The objective of this study was to determine whether the hepatic activities and protein content of system transporters (EAAC1, GLT-1), system regulatory proteins (GTRAP3-18, ARL6IP1), and glutamine synthetase (GS) and glutathione (GSH) contents changed as beef steers developed from predominantly lean (growing) to predominantly lipid (finished) deposition growth phases.…”