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Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6G 2P5 and * Division of Life Sciences, The University of Texas at San Antonio, San Antonio, TX 78249-0662
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • vitamin A • type I diabetes • retinol-binding protein • zinc • BB rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
When required, retinyl esters are rehydrolyzed to retinol, which is
then carried by retinol-binding protein
(RBP)4
into the circulation. In the blood, the retinol/RBP complex further
binds to transthyretin (TTR), at a ratio of 1:1:1 (Soprano and Blaner 1994
), and is then transported to the target cells.
Circulatory levels of retinol are regulated homeostatically over a wide
range of dietary intakes.
Impaired metabolic availability of vitamin A has been identified in
human subjects with type I diabetes, as evidenced by decreased levels
of plasma retinol and RBP, accompanied by increased urinary excretion
of the RBP (Basu et al. 1989
, Dubrey et al. 1997
). The metabolism of Zn, an important factor for the
synthesis of vitamin A carrier proteins, has also been shown to be
disturbed in the presence of diabetes as indicated by hyperzincuria
(Cunningham et al. 1994
, Heise et al. 1988
). Studies involving streptozotocin (STZ)-induced diabetic
rats have shown that circulatory levels of retinol as well as
11-cis-retinal concentrations in the retina of the eye, an
important component of rhodopsin, are reduced in diabetes, whereas the
hepatic storage of vitamin A is markedly elevated (Tuitoek et al. 1996c
). Insulin treatment of these rats normalized the
metabolic availability of vitamin A, whereas vitamin A supplementation
in the diet did not (Tuitoek et al. 1996a
and 1996c
),
indicating a linkage between vitamin A status and insulin secretion in
diabetes. Indeed, vitamin A has been reported to be required for normal
insulin secretion (Chertow et al. 1987
).
Most studies linking vitamin A metabolism and diabetes have been
carried out in STZ-induced diabetic rats. STZ is a nitrosourea
derivative that is toxic to the liver and kidney (Perloff et al. 1995
), the two major sites involved in vitamin A metabolism.
The possibility that vitamin A metabolic derangement is partially a
reflection of STZ toxicity rather than due solely to diabetes cannot be
excluded. Hence, to understand vitamin A metabolism in type I diabetes,
this study was undertaken to investigate the metabolic availability of
vitamin A in BB rats, which develop diabetes spontaneously and are
suitable animal models for human type I diabetes (Like et al. 1982
, Parfrey et al. 1989
). Feeding the NIH-07
diet, which consists of unrefined, natural ingredients and produces the
highest incidence of diabetes in BB rats (Scott 1994
),
we examined vitamin A status, including its carrier proteins at the
onset of diabetes. In addition, the zinc status, which is important in
the maintenance of vitamin A homeostasis, was also determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Diabetes-prone Biobreeding (BBdp) and normal (BBn) rat dams, originally from Health Canada (Animal Resources Division, Health Protection Branch, Ottawa, Canada), were obtained from the Department of Agricultural, Food and Nutritional Science, University of Alberta breeding colony. Animals were housed in a temperature- and humidity- controlled room with a 12-h light:dark cycle. All studies were reviewed and approved by the University of Alberta Animal Welfare Committee.
This study consisted of two experiments. In Experiment 1, the levels of vitamin A and its carrier proteins were determined at the onset of diabetes. Weaning (21-d-old) litter- and gender-matched BBdp and BBn rats (n = 12/group) were given free access to the NIH-07 diet (Zeigler Brothers, Gardmers, PA) and water up to 120 d of age. The NIH-07 diet contains (g/kg diet): protein 215; carbohydrate 514; fat 52; fiber 32; water 125. After diabetes was diagnosed (see below), the diabetic rats and their paired BBn rats were killed within 24 h by carbon dioxide asphyxiation.
In Experiment 2, the effects of vitamin A supplementation, either alone or in combination with zinc, on the metabolic availability of vitamin A were examined. BBn rats fed the NIH-07 diet were used as controls (group 1). Weanling (21-d-old) BBdp pups from each litter were evenly assigned to three treatment groups of 12 rats each. Group 2 received the NIH-07 diet only. Groups 3 and 4 received the basal diet supplemented with retinyl palmitate (18.5 µg/g diet) either alone or in combination with zinc (180 µg/g diet), respectively. All rats in BBdp groups were killed within 24 h after the diagnosis of diabetes. By 120 d of age, BBdp rats that had not developed diabetes were also killed.
Food intake and body weight were monitored throughout the studies. BBdp rats >50 d old were tested for glycosuria by Testape (Eli Lilly, Indianapolis, IN) three times each week. Glucose levels were determined in blood samples taken from the tail vein by Glucometer II (Ames Miles, Toronto, Canada). Diabetes was diagnosed on the basis of glycosuria >2+ and subsequently hyperglycemia (blood glucose >11 mmol/L). Rats were killed after overnight food deprivation. Blood was collected in heparinized tubes. To avoid light-induced oxidation of vitamin A, separated plasma was protected from light and stored at -20°C. Livers were removed, cleaned, immersed in liquid nitrogen and stored at -72°C for later analysis.
Determination of vitamin A.
Plasma and liver vitamin A were assayed by HPLC as described
(Tuitoek et al. 1996c
). Chromatography was performed on
a LC-18 (15.0 cm x 4.6 mm) (Supelco, Mississauga, ON)
reverse-phase column with 3-µm packing, with a
mobile phase consisting of methanol/water (95:5, v/v). Detection was
carried out by UV absorption at 325 nm. Quantification was obtained by
the injection of known amounts of retinol; calculations with the use of
a Shimadzu Class-VP Chromatography data system (Schimadzu,
Columbia, MD). The standard added to plasma, taken through the
extraction procedure and injected into the HPLC, resulted in the
recovery of 82–88%.
Radioimmunoassay.
Plasma and liver RBP and TTR were assayed by RIA (Tuitoek et al. 1996b
). Liver samples were homogenized in 19 volumes of 250
mmol/L sucrose with a Polytron homogenizer (Brinkmann, Westbury, NY) at
speed 5 for 15 s and then diluted with an equal volume of an
aqueous Triton X-100 solution (20 g/L) to release the proteins trapped
in subcellular organelles. The plasma samples and Triton X-100–treated
liver homogenates were diluted appropriately with assay buffer (50
mmol/L Tris-HCl containing 10 g bovine serum albumin/L, pH
8.6). After the addition of 125I-labeled proteins, assay
buffer and anti-rat antiserum for RBP or TTR, all samples were
incubated in the dark at 4°C for 3 d. After being mixed with
goat anti-rabbit 
-globulin antiserum for RBP and polyethylene
glycol for TTR, all samples were incubated at 4°C to precipitate the
antibody-bound 125I-protein; this was collected by
centrifugation at 2800 x g for 15 min at 4°C. The
precipitates were washed with barbital albumin buffer, and the wash
supernatants were combined. The precipitates representing
antibody-bound RBP-125I or TTR-125I and the
wash supernatants representing free protein were assayed for
125I separately in a Packard 500C AutoGamma Counter
(Packard Instrument, Meriden, CT). After calculation of the bound/free
ratios of RBP-125I or TTR-125I, the amounts of
the proteins were determined from the standard curve. To determine the
molar concentrations of RBP and TTR, molecular weights of 21422 and
54892, based on the amino acid sequence of rat RBP and TTR,
respectively, were used.
RNA isolation and Northern blot analysis.
Total RNA was isolated from the liver samples using TriZol (Gibco BRL,
Burlington, Canada). RNA quantification and quality determination were
carried out by UV spectrophotometry at 260 and 280 nm. Total RNA (15
µg) was analyzed by Northern blotting after
electrophoresis in 10 g/L agarose gel and transferred to a MSI
Nitropure nitrocellulose membrane (MSI Laboratories, Westborough, MA)
as described by Reimer et al. (1997)
. The 28S and 18S
ribosomal RNA bands were used to check the integrity of RNA and
compensate for any loading discrepancies. The radioautograms were
quantified using GS-670 imaging laser densitometry (BioRad
Laboratories, Mississuage, Canada); values were normalized to the 28S
and 18S ribosomal RNA bands. Using Random Primers DNA Labeling System
(Life Technologies, Burlington, Canada) the cDNA probe for rat RBP was
labeled with [32P] dATP (3000 Ci/mmol, Amersham Canada,
Oakville, Canada).
Determination of zinc.
Zinc levels in the plasma, liver and urine were determined using flame
atomic absorption spectrophotometry (Perkin Elmer 4000, Norwalk, CT)
with a zinc standard (Fisher Scientific, Edmonton, AB, Canada) as
described by Butrimovitz (1977)
, Luterotti (1992)
and Kiilerich (1980)
, respectively. All
glassware was rinsed with a 20% (v/v) nitric acid solution followed by
deionized water to avoid any contamination.
Determination of plasma total cholesterol and triglyceride.
The plasma samples were analyzed for total cholesterol and triglyceride using enzymatic kits obtained from Sigma Biochemicals (St. Louis, MO; Catolog # 7921 and 336, respectively).
Statistical analysis.
Statistical analyses were performed by SAS computer program (Version 6.12, SAS Institute, Cary, NC). The level of significance was set at P < 0.05. A paired Student’s t test was used to determine the differences between age-paired diabetic biobreeding (BBd) and BBn rats for Experiment 1. For Experiment 2, data were analyzed by two-way ANOVA that included the effect of gender. If no effect of gender was found, groups classified according to different treatments were compared using one-way ANOVA. Significant differences between groups were compared using Student Newman Keuls test procedure.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Experiment 1, BBd rats were characterized by hyperlipidemia
(Fig. 1
) in addition to hyperglycemia. Plasma triglyceride and total
cholesterol concentrations of BBd rats were significantly greater than
those of BBn rats. The onset age of diabetes among BBdp rats was 89
± 4.4 d. The BBd rats had lower concentrations of plasma
retinol in parallel with its carrier proteins, RBP and TTR, in both
plasma and the liver compared with those of BBn rats (Table 1
). The hepatic concentrations of free retinol were also lower in BBd
rats, whereas the total vitamin A levels were not significantly
different from controls. Abundance of the hepatic RBP mRNA was also
lower in BBd rats (Fig. 2
). In addition, the BBd rats exhibited biochemical evidence of zinc
deficiency as indicated by a lower plasma concentration accompanied by
a higher urinary excretion of this trace element (Fig. 3
). Liver zinc concentrations, however, were not affected in BBd rats.
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After vitamin A supplementation for up to 3 mo, the liver total vitamin
A concentrations were markedly greater in BBd rats, whereas the plasma
concentrations of vitamin A and its carrier proteins, including RBP and
TTR, did not differ from BBd rats fed the basal NIH-07 diet
(Table 2
). Although supplemental vitamin A plus zinc intake did not affect
plasma vitamin A, this treatment increased the abundance of RBP mRNA in
the liver of the BBd rats (Fig. 4
), whereas liver RBP concentrations were unaffected (Table 2)
.
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. Urinary zinc concentrations, however, were markedly
greater in rats supplemented with zinc and vitamin A than in the group
given vitamin A alone. Urinary zinc in BBd rats fed the basal diet did
not differ from either supplemented group. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
and STZ-induced diabetic rats (Tuitoek et al. 1996b
and 1996c
), in which the plasma levels of retinol, RBP and TTR were
all reduced markedly. The plasma and hepatic vitamin A concentrations
in those BBdp rats without diabetes are similar to those of control
rats, suggesting that vitamin A deficiency in BBd rats is due primarily
to hyperglycemia. To determine whether the vitamin A deficiency in BBd
rats could be normalized, they were fed a diet supplemented with
vitamin A, either alone or in combination with zinc. Neither of these
treatments, however, altered the reduced plasma levels of vitamin A.
Although the total vitamin A concentrations in the liver were
unaffected, the hepatic levels of free retinol were markedly reduced in
BBd rats. The transformation of retinyl ester to free retinol in the
liver is regulated by the bile salt–dependent BSDREH) and bile
salt–independent (BSIREH) hepatic retinyl ester hydrolases
(Blaner and Olson 1994
). BSIREH is thought to be
involved in the initial hydrolysis of dietary retinyl esters delivered
to the liver from the gut. Because of its high specific activity in
vitamin A storage site hepatic stellate cells, BSDREH is believed to be
more important for the later retinyl ester hydrolysis and retinol
release in the liver than BSIREH (Harrison 1993
).
Biliary structure and hepatic function are affected in diabetes
(Watkins et al. 1995
). Diabetic BB rats often have a
significantly altered bile flow and biliary secretions of bile acid,
cholesterol, phospholipid, sodium, potassium, chloride and bicarbonate
(Gonzalez et al. 1992
). It is thus possible that a
diabetes-induced modification of bile salt metabolism may account
for this impaired vitamin A hydrolysis activity in BBd rats.
The transport of free retinol from the liver to target cells is
accomplished by RBP, which circulates as a 1:1 complex with TTR. Due to
the high molecular weight of TTR, its binding to RBP may prevent the
glomerular filtration and renal catabolism of RBP. Significantly lower
concentrations of these retinol carrier proteins in the plasma and the
liver of BBd rats, in parallel with lower RBP mRNA abundance, were
observed. The underlying mechanism for these results is not clear. In
an early study, a zinc-deficient diet significantly reduced plasma
vitamin A, RBP and hepatic RBP concentrations, thus linking zinc status
to vitamin A metabolism (Brown et al. 1976
). Later
experiments demonstrated that hepatic RBP synthesis is dependent on
adequate dietary levels of zinc (Smith 1980
), and
another study has shown that cellular hepatic RBP (cRBP) is
dramatically lower in zinc-deficient rats compared with their
pair-fed controls with adequate zinc intake (Mobarhan et al. 1992
). These results suggest that zinc deficiency can impair
hepatic cellular transportation of vitamin A via cRBP and mobilization
of vitamin A from the liver via RBP. Type I diabetes is characterized
by hyperzincuria, which also was demonstrated in our study. The BBd
rats thus had significantly higher urinary and lower plasma
concentrations of zinc than those of their nondiabetic counterparts.
The hepatic levels of the trace element, however, were unaffected in
the presence of hyperglycemia. It should be pointed out that the
hepatic content of zinc may not be a reflection of total body zinc
because severely zinc-deficient rats have depressed circulatory
zinc levels, whereas hepatic concentrations are affected only minimally
compared with their pair-fed controls (McClain et al. 1980
). It is reasonable to suggest, therefore, that the lower
circulatory and hepatic levels of RBP in BBd rats are, at least in
part, a metabolic consequence of zinc deficiency. The evidence that
reduced abundance of RBP mRNA was improved after zinc plus vitamin A
supplementation, but not by vitamin A alone, supports this hypothesis.
In contrast, the plasma and hepatic levels of RBP remained depressed
after zinc supplementation despite the improved hepatic abundance of
RBP mRNA in BB rats. These results cannot be explained at this time.
Nevertheless, zinc deficiency has been reported to be associated with
decreased concentrations of RBP and TTR in rat plasma and liver
(Bates et al. 1981
, Smith et al. 1974
,
Smith 1980
).
In summary, the metabolic availability of vitamin A is impaired in BB
rats after the onset of hyperglycemia. The mechanism for this
abnormality is not understood. Possible factors may include alteration
in hepatic retinyl hydrolase activity, synthesis of retinol carrier
proteins and perturbed zinc metabolism. A recent study has suggested
that there is a need for vitamin A supplementation in type 1 diabetic
patients with marginal serum retinol levels (Granado et al. 1998
). There is little evidence, however, to confirm that such
therapy would be of any benefit (American Diabetes Association 1996
). In STZ-induced diabetic rats, a 12-fold increase in
vitamin A intake did not show any effect on the degree of hyperglycemia
and glycosuria (Seifer et al. 1981
). In fact, such
supplementation appears to enhance the hepatic load of the vitamin
(Tuitoek et al. 1996c
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the National Science of Engineering Research Council of Canada.
4 Abbreviations used: BBd, diabetic biobreeding; BBdp, biobreeding diabetes prone; BBn, biobreeding normal; BSIREH, bile salt-independent retinyl ester hydrolases; BSDREH, bile salt-dependent retinyl ester hydrolases; cRBP, cellular retinol-binding protein; RBP, retinol binding protein; STZ, streptozotocin; TTR, transthyretin.
Manuscript received October 8, 1999. Initial review completed November 9, 1999. Revision accepted February 21, 2000.
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5). Bars with different letters are significantly different,
P < 0.05.