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

ABSTRACT: Glutamate dehydrogenase is an important enzyme in the hepatic regulation of nitrogen and energy metabolism. It catalyzes one of the most relevant anaplerotic reactions. Although its relevance in liver homeostasis has been widely described, its daily pattern and responsiveness to restricted feeding protocols has not been studied. We explored the daily variations of liver glutamate dehydrogenase transcription, protein, activity, and histochemical and subcellular location in a protocol of daytime food synchronization in rats. Restricted feeding involved food access for 2?h each day for three weeks. Control groups included food ad libitum as well as acute fasting (21?h fasting) and refeeding (22?h fasting followed by 2?h of food access). Glutamate dehydrogenase mRNA, protein, activity, and histological location were measured every 3?h by qPCR, Western blot, spectrophotometry, and immunohistochemistry, respectively, to generate 24-h profiles. Restricted feeding promoted higher levels of mitochondrial glutamate dehydrogenase protein and activity, as well as a loss of 24-h rhythmicity, in comparison to ad libitum conditions. The rhythmicity of glutamate dehydrogenase activity detected in serum was changed. The data demonstrated that daytime restricted feeding enhanced glutamate dehydrogenase protein and activity levels in liver mitochondria, changed the rhythmicity of its mRNA and serum activity, but without effect in its expression in hepatocytes surrounding central and portal veins. These results could be related to the adaptation in nitrogen and energy metabolism that occurs in the liver during restricted feeding and the concomitant expression of the food entrainable oscillator. Impact statement For the first time, we are reporting the changes in daily rhythmicity of glutamate dehydrogenase (GDH) mRNA, protein and activity that occur in the liver during the expression of the food entrained oscillator (FEO). These results are part of the metabolic adaptations that modulate the hepatic timing system when the protocol of daytime restricted feeding is applied. As highlight, it was demonstrated higher GDH protein and activity in the mitochondrial fraction. These results contribute to a better understanding of the influence of the FEO in the energy and nitrogen handling in the liver. They could also be significant in the pathophysiology of hepatic diseases related with circadian abnormalities.

SUBMITTER: Vazquez-Martinez O

PROVIDER: S-EPMC5407590 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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

Vázquez-Martínez Olivia O Méndez Isabel I Turrubiate Isaías I Valente-Godínez Héctor H Pérez-Mendoza Moisés M García-Tejada Paola P Díaz-Muñoz Mauricio M

Experimental biology and medicine (Maywood, N.J.) 20170316 9

Glutamate dehydrogenase is an important enzyme in the hepatic regulation of nitrogen and energy metabolism. It catalyzes one of the most relevant anaplerotic reactions. Although its relevance in liver homeostasis has been widely described, its daily pattern and responsiveness to restricted feeding protocols has not been studied. We explored the daily variations of liver glutamate dehydrogenase transcription, protein, activity, and histochemical and subcellular location in a protocol of daytime f ...[more]

PMID: 28440738

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Project description:The cerebellum harbors a circadian clock that can be shifted by scheduled mealtime and participates in behavioral anticipation of food access. Large-scale two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry was used to identify day-night variations in the cerebellar proteome of mice fed either during daytime or nighttime. Experimental conditions led to modified expression of 89 cerebellar proteins contained in 63 protein spots. Five and 33 spots were changed respectively by time-of-day or feeding conditions. Strikingly, several proteins of the heat-shock protein family (i.e., Hsp90aa1, 90ab1, 90b1, and Hspa2, 4, 5, 8, 9) were down-regulated in the cerebellum of daytime food-restricted mice. This was also the case for brain fatty acid protein (Fabp7) and enzymes involved in oxidative phosphorylation (Ndufs1) or folate metabolism (Aldh1l1). In contrast, aldolase C (Aldoc or zebrin II) and pyruvate carboxylase (Pc), two enzymes involved in carbohydrate metabolism, and vesicle-fusing ATPase (Nsf) were up-regulated during daytime restricted feeding, possibly reflecting increased neuronal activity. Significant feeding × time-of-day interactions were found for changes in the intensity of 20 spots. Guanine nucleotide-binding protein G(o) subunit alpha (Gnao1) was more expressed in the cerebellum before food access. Neuronal calcium-sensor proteins [i.e., parvalbumin (Pvalb) and visinin-like protein 1 (Vsnl1)] were inversely regulated in daytime food-restricted mice, compared to control mice fed at night. Furthermore, expression of three enzymes modulating the circadian clockwork, namely heterogeneous nuclear ribonucleoprotein K (Hnrnpk), serine/threonine-protein phosphatases 1 (Ppp1cc and Ppp1cb subunits) and 5 (Ppp5), was differentially altered by daytime restricted feeding. Besides cerebellar proteins affected only by feeding conditions or daily cues, specific changes in in protein abundance before food access may be related to behavioral anticipation of food access and/or feeding-induced shift of the cerebellar clockwork.

Project description:1. When rat liver slices were incubated aerobically with [U-(14)C]glutamate the concentration of (14)C within the slices remained lower (about 50%) than in the medium. The maximal concentration of (14)C in the liver was reached within minutes. In rat kidney-cortex slices by contrast, (14)C reached concentrations more than six times those of the medium. 2. In both liver and kidney (14)C appeared in the respiratory CO(2), indicating penetration of glutamate carbon into the mitochondria. In kidney slices the rate of glutamate oxidation per unit weight was about five times that in liver slices. 3. Taking into account the conversion of glutamate into glucose that occurs in the kidney but not in the liver, the flux rates of glutamate through the kidney were calculated to be about 15 times those through the liver when the external glutamate concentration was 5mm. 4. Anaerobically the glutamate concentrations in medium and tissue rapidly became equal in both liver and kidney. Thus the maintenance of concentration gradients depended on the expenditure of energy. 5. [U-(14)C]Succinate behaved similarly to glutamate. [U-(14)C]Serine was taken up more rapidly by the kidney than by the liver slices, but the concentrations reached in the liver did not remain below those of the medium. [(14)C]Urea was distributed evenly between medium and tissue water. 6. Incubation of liver slices with [(3)H]inulin indicated an extracellular space of liver slices of 26%. 7. When glutamate was generated within liver slices or the perfused liver on addition of oxaloacetate, pyruvate and a source of nitrogen, the concentration of glutamate in the tissue after 1hr. was 70-97 times that in the medium. Thus the exit of glutamate from the liver cell, like its entry, is restricted. This is borne out by measurements of the specific activity of extra- and intra-cellular glutamate on addition of [U-(14)C]glutamate medium. 8. Liver homogenates removed added glutamate and dicarboxylic acids 20-30 times as fast as did the perfused liver. 9. It is concluded that a major permeability barrier restricts the entry and exit through the outer liver cell membrane.

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

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

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