Molecular and biochemical modifications of liver glutamine synthetase elicited by daytime restricted feeding

Abstract

Restricted feeding schedule induced circadian rhythmicity inmRNA levels of GS and the loss of the rhythmic pattern in mitochondrial GS activity. GS activity in liver homogenates displayed a robust rhythmic pattern in AL that was not modified by RFS. The presence of GS and its zonal distribution did not show rhythmic pattern in both groups. However, acute Fa and Fa–Re diminished GS protein and activity in liver homogenates. Hepatic glutamine concentrations showed a 24-h rhythmic pattern in both groups, in an antiphasic pattern. In conclusion, daytime RFS influences the liver GS system at different levels, that could be part of rheostatic adaptations associated to the FEO, and highlight the plasticity of this system.

Restricted feeding modulates the daily variations of liver glutamate dehydrogenase activity, expression, and histological location

“…The relative mRNA expression of Glud1 in the liver was analyzed by quantitative real time polymerase chain reaction (RT-qPCR) using a protocol previously reported. 17 Primer sequences were synthesized by Sigma-Aldrich Co. (St. Louis, MO, USA), and the corresponding sequences were: for Glud1 , forward 5′-ACAGCAGAGTTCCAGGACAG -3′, reverse 5′-GTCTATGTGAAGGTCACGCC-3′ (GenBank number NM_012570.2) and for ribosomal protein S18 (rps18), forward 5′-TTCAGCACATCCTGCGAGTA-3′, reverse 5′-TTGGTGAGGTCAATGTCTGC-3′ (GenBank number BC126072.1). Amplification for Glud1 and housekeeping rps18 was performed in SYBR Green Master Mix, according to the following protocol: DNA denaturation at 95℃ for 10 min, followed by 40 amplification cycles consisting of 10 s at 95℃, 30 s at 63℃ for Glud1 or 60℃ for rps18 and 30 s at 72℃.…”

“…23 Quantitative real time polymerase chain reaction The relative mRNA expression of Glud1 in the liver was analyzed by quantitative real time polymerase chain reaction (RT-qPCR) using a protocol previously reported. 17 Primer sequences were synthesized by Sigma-Aldrich Co. (St. Louis, MO, USA), and the corresponding sequences were: for Glud1, forward 5 0 -ACAGCAGAGTTCCAGG ACAG -3 0 , reverse 5 0 -GTCTATGTGAAGGTCACGCC-3 0 (GenBank number NM_012570.2) and for ribosomal protein S18 (rps18), forward 5 0 -TTCAGCACATCCTGCGAGTA-3 0 , reverse 5 0 -TTGGTGAGGTCAATGTCTGC-3 0 (GenBank number BC126072.1). Amplification for Glud1 and housekeeping rps18 was performed in SYBR Green Master Mix, according to the following protocol: DNA denaturation at 95 C for 10 min, followed by 40 amplification cycles consisting of 10 s at 95 C, 30 s at 63 C for Glud1 or 60 C for rps18 and 30 s at 72 C. 17 Western-blot Tissue sampling and subcellular fractionation were done according to the report of Aguilar-Delfín et al 24 Liver homogenates and mitochondrial fractions were subjected to denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.…”

“…17 Primer sequences were synthesized by Sigma-Aldrich Co. (St. Louis, MO, USA), and the corresponding sequences were: for Glud1, forward 5 0 -ACAGCAGAGTTCCAGG ACAG -3 0 , reverse 5 0 -GTCTATGTGAAGGTCACGCC-3 0 (GenBank number NM_012570.2) and for ribosomal protein S18 (rps18), forward 5 0 -TTCAGCACATCCTGCGAGTA-3 0 , reverse 5 0 -TTGGTGAGGTCAATGTCTGC-3 0 (GenBank number BC126072.1). Amplification for Glud1 and housekeeping rps18 was performed in SYBR Green Master Mix, according to the following protocol: DNA denaturation at 95 C for 10 min, followed by 40 amplification cycles consisting of 10 s at 95 C, 30 s at 63 C for Glud1 or 60 C for rps18 and 30 s at 72 C. 17 Western-blot Tissue sampling and subcellular fractionation were done according to the report of Aguilar-Delfín et al 24 Liver homogenates and mitochondrial fractions were subjected to denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Equal amounts of proteins (40 mg of homogenates, 80 mg of cytoplasmic fraction and 25 mg of mitochondrial fraction, as measured by Bradford method) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Darmstadt, Germany).…”

“…In all these cases, no clear correlation was observed among the 24-h profiles of the mRNA, the protein, and the activity of the enzyme. 17,18,26 The RF protocol promoted a distinctive result: a specific enhancement of liver GDH protein and activity in the mitochondrial fraction (Figure 2, panel B, and Figure 3, panel A). The increased GDH within mitochondria in RF rats was not directly associated with an elevation of its transcriptional activity (Figure 1).…”

“…[10][11][12] Using a food restriction protocol (2 h of daytime food access for three weeks), we have reported a set of biochemical adaptations in liver mitochondrial activity and metabolic nitrogen-handling responses that could influence GDH functional properties: (1) enhanced mitochondrial synthesis of ATP, 13 (2) reduced mitochondrial lipid peroxidative activity, 14 (3) increased gluconeogenesis and ureagenesis, 15,16 and (4) modified activity and expression of glutamine synthetase and GABA transaminase. 17,18 This protocol has also been used to study an alternative circadian clock, different from the suprachiasmatic nucleus, which is entrained by food access (known as the food entrainable oscillator, FEO). 19 FEO expression promoted by repeated cycles of fasting and re-feeding over at least three weeks elicits a chronostatic/rheostatic adaptation in the liver, including a behavioral anticipatory activity without a detrimental impact on metabolic networks and cellular energy status, 20 which is not present after a single cycle of fasting and re-feeding.…”