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Ajinomoto Company, Central Research Laboratories, 210-8681 Kawasaki, Japan
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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KEY WORDS: • amino acid deficiency • L-lysine • hypothalamus • norepinephrine • circadian rhythm • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). In experimental animals, Lys deficiency can be induced
by feeding animals a Lys-deficient diet. Recognition of and
subsequent anorectic response to such a diet were shown to be brain
mediated (Gietzen 1998
, Torii 1998
). In
fact, whole-brain Lys levels declined significantly in
Lys-deficient rats (Mori et al. 1991
).
Specifically, hypothalamic neuronal populations were reported as
integration centers during Lys deficiency (Gietzen 1993
,
Hawkins et al. 1995
, Mori et al. 1991
,
Torii et al. 1996
). Magnetic resonance imaging revealed
activation of the hypothalamus during the deficiency (Torii 1998
). In parallel, norepinephrine
(NE)2
levels were increased in homogenized tissue of the ventromedial
hypothalamus (VMH) of amino acid–deficient rats (Gietzen et al. 1989
). Additionally, microdialysis revealed a specific decrease
in the VMH NE, but not lateral hypothalamus (LH) NE, release in rats
fed a Lys-deficient diet for 1 wk (Smriga et al. 2000
). In spite of these results, the neurochemical components
of the hypothalamic response to an amino acid (e.g., Lys) deficiency
are largely unknown. Here we studied the early circadian pattern of VMH
NE release by microdialysis measurement of interstitial NE during the
first 26 h of Lys deficiency. We hypothesized that NE might
provide the initial signal of nutritional Lys deficiency.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. Rats were implanted with guide cannulae for the microdialysis probes
(Eicom, Kyoto, Japan). The cannulae were placed just above the left
VMH, as described (Hawkins et al. 1995
), and rats were
allowed to recover for 1 wk. Rats were treated in compliance with the
U.S. Public Health Service Policy on Human Care and Use of Laboratory
Animals and the NIH guidelines (NRC 1985
).
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Microdialysis recording started at 1200 h (4 h after the probe
insertion). Data were averaged over 1-h periods. The results from the
rats drinking distilled water were not significantly different from
those of rats drinking Gly solution. Thus, although they were included
in the statistical analysis, they were not shown in Figure 1
for the sake of clarity. The dialysis probe was perfused at a rate of
1.0 µL/min with a modified Ringer’s solution (147 mmol/L
NaCl, 10 mmol/L KCl, 1.1 mmol/L
CaCl2·2H2O, 1.1 mmol/L
MgCl2·6H2O, pH 6.0). The
outflow was connected by a teflon tube to a HPLC auto-injector
system (Eicom).
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At the end of the experiments, the rats were anesthetized with ether and their brains fixed with formalin solution. Brains were kept at 4°C in several changes of 150 g/L sucrose/0.1 mol/L phosphate buffer for a few days. Brain sections were cut on a cryotome and stained with toluidine blue. All rats were found to have a probe placed successfully into the VMH.
Time and group differences were analyzed at the end of experiments using one-way ANOVA followed by unpaired t test and two-way ANOVA with post-hoc Duncan’s multiple range test, as appropriate. Differences were considered significant at P < 0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The results of the present study (Fig. 1)
show circadian oscillations
in the VMH NE release in free-moving, unstressed normally fed rats.
VMH NE release decreased during the afternoon period of the light phase
(Fig. 1)
. The NE released at the onset of the dark period (1900 h) was
significantly lower than that at 1200–1400 h. The NE release increased
gradually during the dark period. The NE released at 1900 h was
significantly lower than on the following day at 0300 h and
0500–1300 h. These data are in good agreement with the recent report
of Choi et al. (1998)
, which suggested that the VMH is a
diet-entrained oscillator. Our results did not differentiate
between the light- and the diet-entrained oscillations. However,
the above-mentioned study and the involvement of the VMH in general
food intake regulation (Choi et al. 1998
) indicates that
the oscillations observed (Fig. 1)
could be considered to be
diet-entrained. Indeed, >85% of the rats’ food and fluid intakes
occurred during the dark phase (1900–0700 h), especially during the
initial 5–6 h (data not shown). Because the results were averaged over
1-h periods (3 values obtained every 20 min), it is possible that
information about shorter magnitude NE oscillations (<1h) was not
obtained.
Long-term microdialysis measurements are burdened by technical problems
associated mainly with the glial barrier build-up (Di Chiara et al. 1996
). Nevertheless, the level of NE in the samples did
not change between the starting phase of our experiments (1200–1300 h)
and the same phase on the next day. This agrees with the conclusions of
Di Chiara et al. (1996)
, who documented constant
dialysis probe sensitivity to monoamines within the first 24 h
after probe insertion.
The effects of Lys deficiency on the VMH NE circadian release.
Our findings showed for the first time that the circadian VMH NE
oscillations are depressed in rats fed a Lys-deficient diet, with
the changes appearing within the first night/day cycle. The depression
began during the later stages of the dark phase (0500 h), after a
natural feeding period during the same dark phase. These results are
consistent with the findings of Gietzen and colleagues (Gietzen et al. 1989
, Wang et al. 1998
), who proposed
that the VMH NE is a neurochemical regulator of early amino acid
deficiency. However, Wang et al. (1998)
reported a
decrease in VMH NE beginning 30 min after the introduction of an
L-threonine (Thr)-deficient diet, whereas we did not find a
significant effect until 10 h into the dark phase. The differences
could originate from the different amino acids tested (Lys vs. Thr) and
a different route of administration. In the above study, rats were
infused with a large dose (2 g) of liquid diet directly into their
stomachs. In our experiment, rats freely consumed powdered diet,
possibly resulting in smaller gastric concentrations at any single time
point and in different postingestive responses. Additionally, our
results (Fig. 1)
suggest depression of NE oscillations, rather than a
straightforward decrease of VMH NE release. Indeed, although NE release
in Lys-deficient rats was significantly decreased from 0500 to
1200 h, no differences were found at 1300 h. Because the
timing of the experiments was not described in the study of Wang et al. (1998)
, no direct comparisons are possible.
It is interesting to compare the effects of Lys and
L-tyrosine (Tyr) deficiency on hypothalamic NE release. Tyr
is a precursor of NE. A Tyr-deficient diet reduces brain Tyr levels
(Fernstrom and Fernstrom 1995
), but its effects on brain
NE are controversial, with most studies (e.g., McTavish et al. 1999
) showing no direct influence. Lys, on the other hand, is
not a precursor of NE and does not affect NE synthesis. Thus, we
contend that the data depicted in Figure 1
suggest a specific role for
NE in the integration of signals about the availability of dietary Lys.
There were no significant differences between the intakes of the normal
and Lys-deficient diets (P > 0.25). Therefore, the
depression of the VMH NE release during the later stages of the dark
phase was not attributable to a decrease in food intake. Because
previous reports (Smriga et al. 2000
, Torii 1998
) documented significant decreases in food and fluid
intakes in rats fed a Lys-deficient diet for 1 wk, we suggest that
the first 24 h of the deficiency, during which the volume of food
and fluid consumed is stable, might be classified as an early
deficiency.
There were no apparent changes in the fluid intake volumes among the
six experimental groups (data not shown). Nevertheless, comparable to
the results of Markison et al. (1998)
, rats fed the
Lys-deficient diet slightly (P > 0.7) increased
the number of licking trials, without regard to the fluid offered
(distilled water, Gly or Lys solution) (data not shown), suggesting an
increase in the rats’ motivation to seek a source of Lys. Taken
together, the intake data indicate that the recognition of early Lys
deficiency does not result from taste and/or olfactory cues (see also
Rogers and Leung 1977
).
Consequently, the significant depression of the VMH NE release (Fig. 1)
in Lys-deficient rats was likely triggered by metabolic changes.
Indeed, we have already reported an involvement of hepatoportal amino
acid sensors in the early recognition of an amino acid deficiency (for
review, see Torii 1998
). Postingestive signals also
reach the brain chemosensor, the anterior piriform cortex
(Gietzen et al. 1998
). Relays to the hypothalamus from
either source may trigger a rapid decline in VMH NE oscillations and
influence the hypophagia that characterizes prolonged Lys deficiency.
Depression of VMH NE release was observed in Lys-deficient rats
that had access to distilled water, as well as in those that had access
to Gly solution (400 mmol/L). However, consistent with our previous
behavioral experiments (Hawkins et al. 1995
), we found
that provision of Lys solution (400 mmol/L) to Lys-deficient rats
restored the VMH NE circadian pattern. These results show that the
postingestive benefits of Lys ingestion contributed to recovering the
VMH NE circadian pattern.
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FOOTNOTES |
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Manuscript received December 1, 1999. Revision accepted February 25, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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1.
Choi S., Wong L. S., Yamato C., Dallman M. F. Hypothalamic ventromedial nuclei amplify circadian rhythm. J. Neurosci. 1998;18:3843-3852
2. Di Chiara G., Tanda G., Carboni E. Estimation of in vivo neurotransmitter release by brain microdialysis: the issue of validity. Behav. Pharmacol. 1996;7:640-657[Medline]
3. Fernstrom M. H., Fernstrom J. D. Acute tyrosine depletion reduces tyrosine hydroxylation rate in rat central nervous system. Life Sci 1995;57:97-102
4. Gietzen D. W. Neural mechanisms in the responses to amino acid deficiency. J. Nutr. 1993;123:610-625
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Gietzen D. W., Erecius L. F., Rogers Q. R. Neurochemical changes after imbalanced diets suggest a brain circuit mediating anorectic responses to amino acid deficiency in rats. J. Nutr. 1998;128:771-781
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7. Hawkins R. L., Inoue M., Mori M., Torii K. Effects of inhibin, follistatin, or activin infusion into the lateral hypothalamus on operant behavior of rats fed lysine deficient diet. Brain Res 1995;704:1-9[Medline]
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Markison S., Gietzen D. W., Spector A. C. Essential amino acid deficiency enhances long-term intake but not short-term licking of the required nutrient. J. Nutr. 1998;129:1604-1612
9. McTavish S. F., Cowen P. J., Sharp T. Effect of tyrosine-free amino acid mixture on regional brain catecholamine synthesis and release. Psychopharmacology (Berlin) 1999;141:182-188[Medline]
10. Mori M., Kawada T., Ono T., Torii K. Taste preference, protein nutrition and L-amino acid homeostasis in male Sprague-Dawley rats. Physiol. Behav. 1991;49:987-996[Medline]
11. National Research Council Guide for the Care and Use of Laboratory Animals. Publication no. 85–23 (rev.) 1985 National Institutes of Health Bethesda, MD.
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Rand W. M., Young V. R. Statistical analysis of nitrogen balance with reference to the lysine requirement in adults. J. Nutr. 1999;129:1920-1926
13. Rogers Q. R., Leung P.M.B. The control of food intake: when and how are amino acids involved?. Kare M. R. Maller O. eds. The Chemical Senses and Nutrition 1977:213-249 Academic Press New York, NY.
14. Smriga M., Mori M., Torii K. Interstitial norepinephrine in the hypothalamus of rats fed L-lysine deficient diet. McCarty R. eds. Stress: Neural, Endocrine and Molecular Studies 2000 Gordon and Breach New York, NY. (in press)
15. Torii K. Central mechanisms of umami taste perception and effect of dietary protein on the preference for amino acids and sodium chloride in rats. Food. Rev. Int. 1998;2:273-308
16. Torii K., Yokawa T., Tabuchi E., Hawkins R. L., Mori M., Kondoh T., Ono T. Recognition of deficient nutrient intake in the brain of rats with the L-lysine deficiency monitored by functional magnetic resonance imaging, electrophysiologically and behaviorally. Amino Acids 1996;10:73-81
17. Wang C. X., Yang H., Perrot J. C., Gietzen D. W. Inhibition of norepinephrine release in the rat ventromedial hypothalamic nucleus in essential amino acid deficiency. Neurosci. Lett. 1998;259:53-55
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