Citrulline stimulates locomotor activity in aged rats: Implication of the dopaminergic pathway

Accepted Manuscript Citrulline stimulates locomotor activity in aged rats: implication of the dopaminergic pathway C. Moinard, L. Tliba, J. Diaz, S. Le Plénier, L. Nay, N. Neveux, L. Cynober, A. Raynaud-Simon PII:

S0899-9007(17)30002-3

DOI:

10.1016/j.nut.2017.01.001

Reference:

NUT 9899

To appear in:

Nutrition

Received Date: 26 September 2016 Revised Date:

21 December 2016

Accepted Date: 1 January 2017

Please cite this article as: Moinard C, Tliba L, Diaz J, Le Plénier S, Nay L, Neveux N, Cynober L, Raynaud-Simon A, Citrulline stimulates locomotor activity in aged rats: implication of the dopaminergic pathway, Nutrition (2017), doi: 10.1016/j.nut.2017.01.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Citrulline stimulates locomotor activity in aged rats: implication of the dopaminergic

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pathway

3 C. Moinarda, L. Tlibaa, J. Diazb, S. Le Pléniera, L. Naya, N. Neveuxa, L. Cynobera,c, A.

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Raynaud-Simona,d *

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Biologiques de l'Université Paris Descartes, 4 avenue de l’Observatoire, 75270 Paris cedex

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06, France

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Laboratoire de Biologie de la Nutrition EA 4466, Faculté des Sciences Pharmaceutiques et

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b

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l'Université Paris Descartes,4 avenue de l’Observatoire, 75270 Paris Sorbonne Paris Cité,

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Cedex 06, France

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Jacques 75679 Paris cedex 14, France

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Paris cedex 18, France

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Service de Biochimie, Hôpitaux Hôtel-Dieu et Cochin, APHP, 27, rue du Faubourg Saint-

Département de Gériatrie, Hôpitaux Bichat et Beaujon APHP, 46, rue Henri Huchard, 75877

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Laboratoire de Physiologie, Faculté des Sciences Pharmaceutiques et Biologiques de

*Corresponding author: A. Raynaud-Simon, Hôpital Bichat. 46, rue Henri Huchard. 75877

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PARIS Cedex 18, France

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Phone: (+33) 1.40.25.73.69 Fax : (+33) 1.40.25.85.88 E-mail: [email protected]

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Abstract

Introduction: Citrulline (CIT)-enriched diet improves locomotor activity in aged rats,

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but the underlying mechanism is unknown. We hypothesized that CIT could act via the

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dopaminergic pathway. Methods: Sixty adult (3-mo–old) and aged (20-mo-old) rats

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were divided into four groups (n=15 each) stratified by age (adult vs old) and diet

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(Control vs CIT), i.e. Ad-Control, Ad-CIT, Old-Control, Old-CIT, and fed for 4 days on

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either a CIT-enriched diet (5 g/kg/day; Ad-CIT and Old-CIT) or an isonitrogeneous

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control diet (Ad-Control and Old-Control). Locomotor activity was evaluated in a Y-

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maze. On D5, animals were euthanized and brain (striatum) was removed to determine

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total and phosphorylated forms of tyrosine hydroxylase (TH) by immunohistochemistry.

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Results: CIT restored locomotor activity in aged rats (arm visits: Old-CIT 28±1 vs Old-

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Control 23±1, p<0.05), associated with an increase in total TH (Old-CIT 668±27 vs

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Old-Control 529±22, p<0.05) and phosphorylated forms of TH (Old-CIT 1012±39 vs

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Old-Control 589±69 p<0.05). Conclusion: In aged rats, CIT is able to stimulate

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locomotor activity via the dopaminergic pathway.

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Key words: locomotor activity, tyrosine hydroxylase, amino acids, aging

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1. Introduction

42 It is widely observed that physical activity declines as humans reach old age (1). Low

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physical activity is an important contributor to sarcopenia and frailty, and thus to falls,

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disability in activities of daily living, hospitalization, and death (2–5). Walking speed, a

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simple marker of frailty, is lower in elderly than younger persons (6).

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Slower mobility in the elderly may be linked to multiple physiologic or pathologic causes, but

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a decline in dopaminergic activity in the aging brain is likely to be implicated. Clinical

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observations of bradykinesia, gait disturbance and rigidity are common in elderly people,

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even in subjects that do not present with Parkinson’s disease or related conditions (7). Tissue

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concentration of striatal dopamine and D2 dopamine receptor availability decrease

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physiologically with age (8–12). The tyrosine hydroxylase (TH) activity is the rate-limiting

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step in the synthesis of dopamine from tyrosine. Measurements on human and rat brain

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samples have consistently shown that tyrosine hydroxylase quantity and activity severely

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decreases with age in the nigrostriatal tract (13-16). The age-related locomotor impairment

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can be reversed by the dopamine receptor stimulant apomorphine and by the biosynthetic

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precursor of dopamine, L-dopa (17), or by transplantation of fetal cells from dopamine-rich

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neural tissue (18), demonstrating a causal relationship between low dopaminergic activity and

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age-related locomotor impairment. Thus, strategies designed to improve dopaminergic

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activity would likely improve mobility in elderly people.

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New concepts have emerged on amino acid properties. Beside their contribution as substrates

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for protein and energy metabolism, recent studies have highlighted roles of some amino acids

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as signaling molecules. In particular, citrulline demonstrates important regulatory properties

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on muscle protein synthesis (see (19) for a recent review). In refeeding of malnourished aged

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rats, a citrulline-enriched diet improved muscle mass, muscle strength and locomotor activity

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ACCEPTED MANUSCRIPT measured as number of arms visited in a Y-maze in 10 minutes (20). We concluded that

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improvement in locomotor activity could partly be attributed to improved muscle function by

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citrulline. However, an effect of citrulline via the central neurological system cannot be ruled

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out. Indeed, we previously observed that oral administration of citrulline to aged rats

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increases tissue CIT concentrations and improves specific age-related raft changes in the

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hippocampus (21).

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Based on these observations, we hypothesized that citrulline could also have an effect on

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locomotor activity via the central neurological system and, given that locomotor activity is

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under control of the dopaminergic system, via the dopaminergic pathway. The aim of the

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present study was to determine the effect of citrulline administration on locomotor activity

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and dopamine activity in healthy aged rats. Dopamine pathway was assessed

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immunohistochemically by total tyrosine hydroxylase (TH) and by the activated

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(phosphorylated) form of TH (pSer40-TH), as tyrosine is metabolized directly to dopamine by

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this enzyme.

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2. Materials and methods

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This program was approved by the regional animal ethics committee under number

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P2CM05508. Citrulline was a gift from Kyowa Hakko (Tokyo, Japan).

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2.1. Study design

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This study used 60 Sprague-Dawley rats (30 three-month-old adults and 30 twenty-month-old

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aged rats) divided into four groups of 15 rats each stratified by age (adult or old) and diet

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(Control or CIT), i.e. Ad-Control, Ad-CIT, Old-Control, Old-CIT. All four groups were fed

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ad libitum (standard regimen; UAR A04, Safe, France) for a 1-week equilibration period (D-7

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ACCEPTED MANUSCRIPT to D0) in which rats and food intakes were recorded. Mean food intake was 24 grams/day.

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Then, from day 1 to day 4 (D1to D4), the rats were fed at 90% of their spontaneous food

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intake recorded the previous week (i.e. 21.6 g of food per day). The 10% dietary restriction

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was performed: (i) to ensure that the rats ate all the given food, (ii) and that the rats received

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equal amounts of CIT or isonitrogenous control throughout the experiment). The chow diet

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was supplemented with either CIT (5 g/kg/day; Ad-CIT and Old-CIT) or an isonitrogenous

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amino acids mixture (alanine:glycine:serine:histidine, 1:1:1:1 molar ratio; Ad-Control and

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Old-control). We showed in previous studies that neither this amino acid mixture

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supplementation nor a 10%-food restriction affect motoricity per se (22).

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2.2. Locomotor activity and spontaneous alternation behavior

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The rats were tested for spontaneous alternation and motor activity behavior in a Y-maze on

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D4. The apparatus consisted of a symmetrical Y-maze with 40-cm-long, 15-cm-wide and 35-

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cm-high arms, as described previously (20,22). The apparatus was illuminated from above (35

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lux) and the floor was covered with a small amount of sawdust. Behavior was observed by a

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trained experimenter. The rats were put in one arm of the maze, and then sequence and

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number of arm entries were recorded over a 10-minute period. An arm visit was recorded

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when a rat moved all four paws into the arm. Alternations were scored by evaluation of

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response sequences in which entering the arm least-recently visited was considered an

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alternation response. Proportion of alternations was computed by dividing number of

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alternations by total number of arm visits. To obtain an accurate evaluation of the qualitative

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aspects of behavior, we set a minimum threshold of 9 visits was to calculate percentage of

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spontaneous alternation behaviors. This test made it possible to determine two behavioral

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parameters, as of the alternation score is a good indicator of attention and/or related functions,

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and total arm visits is a good indicator of locomotor activity.

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On D5, the rats were sacrificed by decapitation, and the brain was rapidly dissected and

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frozen in dry-ice-cooled (-40°C) isopentane. All samples were stored at -80°C until analysis.

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The samples were excised from striatal brain until to be used for immunohistochemistry and

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amino acid analysis.

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Tissues were ground and deproteinized with 10% trichloroacetic acid solution containing 0.5

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mM EDTA. Supernatants were stored at -80°C until analysis. Amino acids were measured by

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ion exchange chromatography using a JEOL (Tokyo, Japan) amino acid autoanalyzer. Results

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are expressed in µmol/L plasma and nmol/g tissue.

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2.4. Immunohistochemistry of total and phosphorylated tyrosine hydroxylase

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All rat brains were cut in the coronal plane through the striatum using a Leica JUNG-CM3000

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cryostat (at -20°C). Brain sections (10 µm-thick) were then mounted on SuperFrost-Plus

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slides (Menzel-Glasser) and stored at -75°C until use for immunohistochemistry. To perform

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tyrosine hydroxylase (TH) or pSer40-TH immunohistochemistry, cryo-sections from different

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animal groups were simultaneously processed as described (23).

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Density levels of TH and pSer40-TH immunoreactivity in the dorsolateral region of the rat

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striatum were analyzed under the ×40 lens of a Leica microscope. The immunoperoxidase-

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stained sections from different animal groups were processed with identical illumination

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intensity and settings to get digitized microscopic images (converted into 8-bit grayscale. To

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evaluate overall staining density in each image, the background measured in the corpus

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callosum bordering the dorsal striatum was subtracted from dorsolateral-sector measurements.

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Staining density was analyzed using Image J software (Image J, National Institute of Health,

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Bethesda, MD).

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Data are expressed as means ± SEM. Comparisons between sets of data were performed using

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one-way analysis of variance (ANOVA) followed by the Fisher’s PLSD test on Statview

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software. Differences at p<0.05 were considered significant.

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3. Results

147 3.1 Locomotor activity

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In the control groups, aged rats visited significantly fewer Y-maze arms than adult rats (Ad-

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Control 28±1 vs Old-Control 23±1, p<0.05). Citrulline supplementation did not affect

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locomotor activity in adults but totally restored locomotor activity in aged rats (Old-Control

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23±1 vs Old-CIT 28±1, p<0.05). Spontaneous alternation in visits in the different Y-maze

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arms ranged from 60% to 70% and was not affected by diet (data not shown).

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3.2. Amino acid concentrations in plasma and striatum (Table 1)

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CIT supplementation was associated with increased plasma CIT, arginine and ornithine and

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decreased plasma glutamine in adult and old rats (CIT vs control, p<0.05). Interestingly,

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plasma concentration of tyrosine was only increased in adult rats (Ad-CIT vs Ad-Control,

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p<0.05). Other amino acids were unaffected by CIT supplementation (data not shown).

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In adult rats, CIT supplementation significantly increased striatal CIT, arginine, ornithine and

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tyrosine concentrations (Ad-CIT vs Ad-control, p<0.05). In aged rats, CIT supplementation

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led to a similar pattern but only arginine was significantly increased (Old-Control vs Old-CIT,

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p<0.05) as the increases in CIT, tyrosine and ornithine failed to reach significance (p=0.1,

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0.18 and 0.06, respectively).

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3.3. Total and phosphorylated forms of tyrosine hydroxylase in the striatum

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This parameter was only determined in aged rats since CIT did not affect locomotor activity

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in adult rats. CIT supplementation significantly increased both total and phosphorylated forms

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of TH (Old-Control vs Old-CIT, p<0.05, Figure 1).

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ACCEPTED MANUSCRIPT 4. Discussion

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Although a number of exploratory studies have focused on amino acid metabolism in the

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aging brain, this is, to our knowledge, the first report to show that oral administration of an

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amino acid, i.e. CIT, corrects age-induced locomotor impairment in animals. We show that

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CIT supplementation increases locomotor activity in old rats, associated with increased

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plasma and striatal CIT concentrations and striatal immunoreactivity of TH and pSer40-TH,

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the key enzyme for the dopamine synthesis.

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A decrease in CIT concentrations has been described in various regions of the aging brain

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(24–26) together with an alteration in arginase and NO synthase (25,26) that was associated

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with a decrease in exploratory behavior (26). Thus, CIT administration inducing an increase

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in brain CIT concentration is capable of correcting an aged-related process. We did not

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perform the comparison between adult and old rats TH levels, which is a limitation in our

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study, but the CIT-induced increase in TH activity does suggest it was lower in old rats than

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in adult rats, as previously described (13-16).

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Interestingly, it has been shown that CIT administration is also able to modulate hippocampic

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eNOS expression, reduce neuronal death and capillary loss, and limit memory decline in a

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murine model of ischemic stroke (28). Thus, CIT may also affect the vascular system in the

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brain, improving oxygenation of the tissue. Alternatively, arginine (derived from CIT) could

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increase cAMP, activate protein kinase A and mediate TH phosphorylation. Such hypothesis

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is in line with in vitro study showing that arginine, in neurons, is able to increase cAMP

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concentration (29). However, it remains to be confirm by an in vitro approach.

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We previously found that CIT also induced an increase in muscle protein synthesis, muscle

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mass and strength that probably contribute to the increase in locomotor activity (21).

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However, this effect on muscle cannot explain the co-increase of both CIT concentrations and

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TH activity in the striatum. Furthermore, the CIT-mediated effect on locomotor activity in old

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tyrosine concentrations did not increase significantly in response to CIT treatment.

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The mechanism by which CIT increases dopaminergic activity in the striatum remains to be

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elucidated. However, given the clinical importance of improving mobility in the elderly along

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with the excellent tolerance to CIT in humans of all ages (30), CIT administration could be a

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candidate for integration into multimodal care strategies for frail elderly subjects—and

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ultimately even as an adjuvant treatment in Parkinson’s disease.

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Conflict of interest S. Le Plénier, L. Cynober and C. Moinard are shareholders of Citrage Company.

Grants

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This work was supported by a grant from the French Ministry of Research (contrat

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quadriennal EA 4466).

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Figure legends

302 Figure 1: Determination of total forms (left) and phosphorylated forms (right) of tyrosine

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hydroxylase in healthy aged rats fed at 90% of spontaneous food intake with either CIT (5

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g/kg/d) (Old-CIT) or an isonitrogenous control diet (Old-control) for 5 days. Results are

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expressed in arbitrary units (A.U.).

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ANOVA + Fischer’s PLSD test:

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Values in a row with no common superscript letter are statistically different at p<0.05.

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Figure 2: Illustration of total forms (up) and phosphorylated forms (bottom) of tyrosine

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hydroxylase in healthy aged rats fed at 90% of spontaneous food intake with either CIT (5

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g/kg/d) (Old-CIT) or an isonitrogenous control diet (Old-control) for 5 days.

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Table 1: Effects of the diets on amino acid concentrations in plasma and striatum.

Ad-Control

Ad-CIT

Old-CIT317

Old-Control

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Plasma (µmol/L) 3217 ± 279*

98 ± 16

ARG

145 ± 7

1091 ± 55*

123 ± 21

ORN

47±2

230 ± 12*

45 ± 8

TYR

58 ± 3

76 ± 6*

79 ± 19

Striatum (nmol/g)

319 1820 ± 695*

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79 ± 4

320 825 ± 77*

321 268 ± 102 75 ± 8 322

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CIT

324 242 ± 107

CIT

ND

1524 ± 102*

ARG

69 ± 6

1637 ± 86*

134 ± 13

325 868 ± 213*

ORN

ND

30 ± 9*

23 ± 3

44 ± 4

TYR

65 ± 6

265 ± 21*

95 ± 7

327 129 ± 12 328

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Healthy rats were fed at 90% of spontaneous food intake with either CIT (5 g/kg/d; Ad-CIT

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and Old-CIT) or an isonitrogenous control diet (Ad-Control and Old-Control) for 4 days.

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ND: non-detectable.

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2-way ANOVA + Fischer’s PLSD test; * p<0.05 vs control

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ACCEPTED MANUSCRIPT Highlight :

Oral administration of citrulline corrects age-induced locomotor impairment

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Citrulline is able to increase total and phosphorylated for of tyrosine hydroxylase in striatum

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In aged rats, Citrulline is able to stimulate locomotor activity via the dopaminergic pathway

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