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-Linolenic Acid–Restricted Diet1
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
2To whom correspondence should be addressed. E-mail: >okuyamah@phar." locator-type="email">locator-type="email">okuyamah@phar. nagoya-cu.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-linolenic acid (ALNA)-deficient diet. Rats fed a safflower oil diet
(ALNA-restricted) or a perilla oil diet (ALNA-sufficient) through two
generations were adapted to a 24-h cycle with light from 0700 to
1900 h. They were killed at 0500, 0900, 1300 and 1700 h to
determine the activities of four lysosomal enzymes in retina, including
ß-glucosidase, ß-glucuronidase, hexosaminidase and acid
phosphatase. The enzyme activities at 0500 h were the lowest and
then increased gradually until 1700 h, exhibiting similar
circadian rhythms in the two dietary groups. However, the activities at
each time point were significantly lower in the safflower group. In the
pineal gland, the activities were maximum at 1300 h, except for
ß-glucosidase, and were not different between groups. These diets had
qualitatively similar but quantitatively different effects on the fatty
acid compositions of the retina and the pineal gland. These results
indicate that decreased amplitudes in electroretinogram and altered
size distribution of phagosomes, as induced by a restricted intake of
ALNA, are associated with decreased lysosomal enzyme activities in the
retina but not in the pineal gland.
KEY WORDS: • -linolenic acid deficiency • docosahexaenoic acid • retina • lysosomal enzyme • circadian rhythm • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-linolenic [ALNA, 18:3(n-3)]
and linoleic acids [18:2(n-6)], respectively (Bazan 1990
, Green and Yavin 1993
, Scott and Bazan 1989
). A reduction in DHA and increases in AA and
22-carbon (n-6) PUFA levels caused by dietary deprivation of ALNA for
long periods (e.g., through two generations) have been shown to impair
retinal function (Bourre et al. 1989
, Watanabe et al. 1987
, Weisinger et al. 1996
, Wheeler et al. 1975
) and alter learning behavior (Bourre et al. 1989
, Nakashima et al. 1993
, Wainwright et al. 1994
, Yamamoto et al. 1987, 1988 and1991
)
in rodents. In rhesus monkeys, a restricted intake of (n-3) fatty acids
has been shown to decrease visual acuity (Neuringer et al. 1986
). Retarded retinal development has also been noted in
premature infants fed (n-3) fatty acid–restricted infant formulae
(Uauy et al. 1990
). These results suggest that DHA is
critical for the maintenance of retinal and neural functions, for which
(n-6) PUFA cannot compensate.
Among tissues, the retina contains the highest levels of DHA,
constituting about one half of the total fatty acids in the rod outer
segment (ROS) of photoreceptor cells (Anderson et al. 1994
, Bazan et al. 1982
). The ROS contains
rhodopsin and other signaling molecules that capture light signals and
transmit the information through the retinal synaptic circuitry to the
brain. Therefore, the ROS is susceptible to photoreactive radical
stress and requires daily renewal, during which its tip is shed and
then phagocytized by retinal pigment epithelial (RPE) cells (Bok 1985
, Young 1967
). Previously, we observed that
in RPE cells, the number of large phagosomes in the early morning after
light exposure was significantly lower in rats fed a diet low in ALNA
through two generations (Watanabe et al. 1993
),
suggesting altered phagocytotic processes. To elucidate biochemical
bases for the presumed impairments of phagocytotic processes,
activities of lysosomal enzymes and their circadian rhythms were
measured in the retina and pineal gland of rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4-Methylumbelliferyl-ß-D-glucopyranoside and 4-methylumbelliferyl-N-acetyl-ß-D-glucosaminide were purchased from Sigma Chemical (St. Louis, MO). 4-Methylumbelliferyl-ß-D-glucuronide, 4-methylumbelliferylphosphate and 4-methylumbelliferone were purchased from Wako Pure Chemical Industries (Osaka, Japan). Heptadecanoic acid was purchased from Funakoshi (Tokyo, Japan).
Diets and animals.
A semipurified diet (Central Laboratory for Experimental Animals, Clea
Japan, Tokyo, Japan) contained (g/100 g) 48.0 cornstarch, 25.1 milk
casein, 8.2 cellulose, 5.1 sucrose, 2.0 okanol (a mixture of
carbohydrates, mainly -starch), 6.1 mineral
mixture,4
1.4 vitamin
mixture,5
0.4 DL-methionine, 0.6 choline chloride and 3 of the
indicated oil. Safflower oil consisted mainly of 16:0 (6.7 g/100 g
total fatty acids), 18:0 (2.5), 18:1(n-9) (14.7), 18:2(n-6) (75) and
ALNA (<0.6); perilla oil consisted of 16:0 (5.5), 18:0 (1.6),
18:1(n-9) (14.1), 18:2(n-6) (14.0) and ALNA (64.5). All rats were
maintained under controlled lighting (24-h cycle with light from 0700
to 1900 h) at 23 ± 2°C and 50 ± 10% humidity.
Female Donryu rats (F0) (SLC,
Shizuoka, Japan) at 4 wk of age were fed the safflower oil
(ALNA-restricted) or perilla oil (ALNA-sufficient) diet for 7 wk. The
F0 rats were mated at 11 wk of age and
the litters (F1) were weaned at 3 wk.
The male F1 pups were fed the same
diets for 6 wk and used for the experiments at 9 wk of age. The rats
(n = 9/group) were decapitated at 0500, 0900, 1300
and 1700 h in a room illuminated with a dim red light. The retina
and pineal gland were excised quickly, frozen and maintained at
-80°C until assayed.
Lysosomal enzyme assays.
The 200-µL assay mixture consisted of the following:
for ß-glucosidase assay, 5.0 mmol/L
4-methylumbelliferyl-ß-D-glucopyranoside in 0.2 mol/L
sodium acetate buffer at pH 5.0 containing 6 g/L sodium taurocholate;
for ß-glucuronidase assay, 1.5 mmol/L
4-methylumbelliferyl-ß-D-glucuronide in 0.2 mol/L sodium
acetate buffer at pH 5.0; for hexosaminidase assay, 1.0 mmol/L
4-methylumbelliferyl-N-acetyl-ß-D-glucosaminide
in 0.12 mol/L citrate phosphate buffer at pH 5.0; and for acid
phosphatase assay, 5.6 mmol/L 4-methylumbelliferylphosphate in 0.2
mol/L sodium acetate buffer at pH 5.0. Assay mixtures were
incubated at 37°C for 15 min. To measure the enzyme activities in the
retina, the following amounts of homogenates (µg
protein/assay) were used: ß-glucosidase (80 µg);
ß-glucuronidase (40 µg); hexosaminidase (20
µg); and acid phosphatase (20 µg). To
measure the enzyme activities in the pineal gland, the following
amounts of homogenates (µg protein/assay) and incubation times were used: ß-glucosidase (20 µg, 60
min); ß-glucuronidase (10 µg, 30 min);
hexosaminidase (10 µg, 30 min); and acid phosphatase
(10 µg, 15 min). The reactions were terminated by
adding 3.0 mL of 0.1 mol/L ammonium hydroxide-glycine buffer (pH
10.5). The release of 4-methylumbelliferone from fluorogenic substrates
was measured fluorometrically as described by Vaughan et al. (1987)
. The specific activity was expressed as nanomoles
substrate hydrolyzed per minute per milligram of protein using
4-methylumbelliferone as the standard. The linearity of the assays was
assessed with respect to the incubation time and the amounts of protein
used.
Lipid analysis.
Total lipids were extracted from the retina and pineal gland with
chloroform/methanol according to the method of Bligh and Dyer (1959)
. Fatty acids were converted to their methyl esters by
treatment with 50 g/L HCl in methanol and were quantified by capillary
column gas liquid chromatography (Shimazu,, Kyoto, Japan) using
heptadecanoic acid as the internal standard, as described previously
(Yamamoto et al. 1987
).
Statistic analysis.
The lysosomal enzyme activities were compared by two-way ANOVA, with time and diet as variables. Differences in the fatty acid compositions of the two dietary groups were analyzed using Student’s t test. A difference was considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Weight gain, litter size and protein contents in the retina and pineal
gland were not significantly different between the two dietary groups
(data not shown). Total fatty acid contents in the two tissues were
also not significantly different between the two groups (Table 1
). In the safflower oil group, the restricted ALNA intake resulted in
lower 22:6(n-3) levels in the retina and the pineal gland, <40 and
16% of that in the perilla oil group, respectively, which was
compensated for mainly by a greater 22:5(n-6) level, as described
previously in the brain (Yamamoto et al. 1987
). However,
the total C-22 PUFA content in the retina was slightly lower
(P < 0.05) in the safflower oil group. The 20:4(n-6)
level in the retina of the safflower oil group was 50% higher than
that in the ALNA-sufficient group. Furthermore, the decrease in the
pineal gland 22:6(n-3) in the safflower oil group was not sufficiently
compensated for by the increase in the 22-carbon (n-6) PUFA level; the
total C-22 PUFA content in the safflower oil group was <50% of that
of the perilla oil group. The 20:4(n-6) level in the pineal gland of
the safflower oil group was >100% higher; the total C-20 plus C-22
PUFA levels as well as the total fatty acid contents were not different
between the two dietary groups. A significant difference in the ALNA
content in the pineal gland of the two dietary groups was also noted.
|
In the retina, circadian rhythms in activity were observed for all four
enzymes (Fig. 1A
–D
, P < 0.01 in two-way ANOVA using time as a
variable). The activities at 0500 h during the dark period were
the lowest and then gradually increased after light exposure. The
safflower oil diet led to significantly lower activities of all
lysosomal enzymes in the retina compared with those in the perilla oil
group (P < 0.01 in two-way ANOVA). At 0900 h,
when the activities were first measured after light exposure, the
activities of ß-glucosidase, ß-glucuronidase, hexosaminidase and
acid phosphatase in the safflower oil group were lower by 20, 27, 27
and 25%, respectively. In the perilla oil group, the ß-glucuronidase
and hexosaminidase activities had reached a plateau at 0900 h,
whereas in the safflower oil, all of the enzyme activities increased
gradually until 1700 h.
|
–H
; P < 0.05 in two-way
ANOVA), no significant differences in the enzyme activities were
observed between the two dietary groups. The circadian patterns in the
pineal gland were different from those in the retina; the activities of
ß-glucuronidase, hexosaminidase and acid phosphatase were higher at
1300 h than at other time references, whereas ß-glucuronidase
activity increased gradually from 0500 to 1700 h in both groups. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Young 1967
). Previously, we noted that the number of small phagosomes
was greater but that of large phagosomes was lower in the safflower oil
group at 0930 h (Watanabe et al. 1993
), suggesting
altered phagocytotic processes. Retinal lysosomal enzyme activities had
been shown by Vaughan et al. (1987)
to exhibit circadian
rhythms associated with phagocytosis, which is activated by light
exposure. Consistent with these observations, lysosomal enzyme
activities in the retina increased relatively rapidly after light
exposure (from 0500 to 0900 h) in the perilla oil group compared
with those in the safflower oil group. The lysosomal enzyme activities
of the whole retina reflect not only those in RPE cells but also those
in the other retinal cells. However, their circadian rhythms
corresponded roughly to those of the size-distribution of
phagosomes in RPE layers (Watanabe et al. 1993
). Here,
we first demonstrated that a restricted intake of ALNA decreases
retinal lysosomal enzyme activities, suggesting that a reduction in DHA
level lowers the turnover rates of the ROS.
The diets modified the (n-6) and (n-3) fatty acids in the pineal gland
more than in the retina (Table 1)
. The results were in agreement with
those of Sarda et al. (1991),
indicating that the pineal
gland is highly sensitive to the restriction of (n-3) fatty acid intake
compared with the brain. Nevertheless, no significant difference was
observed in the activities of pineal gland lysosomal enzymes or in
their circadian rhythms in the two dietary groups. These results
indicate that basic circadian rhythms and lysosomal hydrolysis are
retained in the pineal gland under these dietary conditions. The
mammalian pineal gland is considered to be a neuroendocrine organ,
functioning almost exclusively in the synthesis and secretion of
melatonin (Reiter 1991
). Melatonin synthesis in the
pineal gland of rats is higher at night and melatonin is degraded
mainly during the daytime (Reiter 1991
). Restricted
intake of (n-3) fatty acids decreased the adenosine-dependent
melatonin release in cultured rat pineal gland in ex vivo experiments
(Gazzah et al. 1993
) and the decreased release was
normalized by administering DHA-rich phospholipids
(Zaouali-Ajina et al. 1999
). Thus, the (n-3) fatty acid
status affects the pineal gland melatonin release, although restricted
ALNA intake did not affect the activities of the pineal lysosomal
enzymes (Fig. 1E
–H
).
Early response genes such as zif-268, c-fos and
tis-1 are rapidly and transiently expressed in cultured rat
RPE cells during the phagocytosis of the ROS isolated from the bovine
retina (Ershov et al. 1996
). These inductions are
considered to modulate the expression of the gene cascade needed for
intracellular responses after ROS phagocytosis. However, these genes
are not activated in the RPE cells during the phagocytosis of
nonspecific compounds such as latex beads. These observations suggest
that the phagocytized substances regulate the biochemical events in RPE
cells after phagocytosis. Phagocytosis may be receptor dependent, with
a mannose receptor (Boyle et al. 1991
) and a scavenger
receptor CD36 (Ryeom et al. 1996
) considered putative
ROS receptors. The expressions of these molecules may affect the
phagocytosis and subsequent degradation. It remains to be elucidated
which process of ROS turnover is mainly affected by restricted ALNA
intake. However, an unequivocal biochemical variable was presented here
as a possible basis for the altered retinal functions and size
distribution of phagosomes resulting from restricted ALNA intake by
rats.
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FOOTNOTES |
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3 Abbreviations used: AA, arachidonic acid; ALNA,
-linolenic acid; DHA, docosahexaenoic acid; PUFA, polyunsaturated
fatty acid; ROS, rod outer segment; RPE, retinal pigment epithelium.
4 The mineral mixtures contained (g/kg) 13.554
CaCO3, 17.3 KH2PO4, 15.0
CaHPO4 · 2H2O, 8.0
MgSO4 · 7H2O, 6.0 NaCl, 1.9
Fe(C6H5O7) · 5H2O,
0.06 5ZnO · 2CO2 · 4H2O, 0.0126
CuSO4 · 5H2O, 0.004
CoCl2 · 6H2O, 0.0154
Ca(IO3)2, 0.154
MnSO4 · 4H2O and 15.5 cornstarch.
5 The vitamin mixtures contained (g/kg) 0.02
retinyl acetate, 0.004 cholecalciferol, 0.2 -tocopherol, 0.003
menadione, 0.015 thiamin, 0.0156 riboflavin, 0.0102
pyridoxine · HCl, 0.05 cyanocobalamin, 0.005 biotin, 0.04 calcium
pantothenate, 0.1015 p-aminobenzoic acid, 0.1015 niacin,
0.15 inositol, 0.002 folic acid, 3.0 choline chloride and 13.7822
cornstarch.
Manuscript received May 5, 2000. Revision accepted September 4, 2000.
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