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Departments of Pathobiochemistry (I.M., N.I., S.T.) and Organic Manufacturing (M.S., Y.H.), Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan
Address all correspondence and requests for reprints to: Ichitomo Miwa, Ph.D., Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya 468-8503, Japan. E-mail: miwaichi{at}meijo-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-ketoisocaproate-induced insulin secretion, whereas leucine-induced
insulin secretion was stimulated. Insulin secretion induced by 10
mM glyceraldehyde was slightly decreased by HNE. On the
other hand, HNE severely decreased insulin secretion induced by 10
mM glyceraldehyde and 2.8 mM glucose. Glucose
utilization and glucose oxidation were significantly lowered in islets
treated with HNE. The amounts of fructose 1,6-bisphosphate and
dihydroxyacetone phosphate in islets were decreased by treatment with
HNE, whereas the amount of fructose 6-phosphate was increased. Our
study indicates that HNE and other lipid peroxidation products impair
insulin secretion induced by glucose probably through affecting both
the glycolytic pathway and the citric acid cycle. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß unsaturated double bond, spontaneously forms
Michael adducts with sulfhydryl groups of glutathione and proteins. HNE
also reacts with histidine and lysine residues of proteins to form
Michael adducts (4). The alkenal thus alters the catalytic and
structural properties of proteins (5, 6) and induces adverse biological
effects, such as toxicity toward mammalian cells, blockade of cell
proliferation, and inhibition of the synthesis of DNA, RNA, and protein
(3, 7). Recently, it was reported that the gene expression of various antioxidant enzymes, i.e. superoxide dismutase, catalase, and glutathione peroxidase, was substantially lower in mouse pancreatic islets than in various other tissues (8). This fact suggests that pancreatic islets would be more vulnerable to oxidative stress than other tissues. Another paper reported that cytokines might damage islet ß-cells by inducing oxygen free radical generation, lipid peroxidation, and consequently the formation of aldehydes such as HNE, hexanal, and malondialdehyde in the islet cells (9). These findings taken together with the above considerations raise the possibility that HNE may affect insulin secretion from pancreatic islets. In this study we examined the effect of HNE and other aldehydes on insulin secretion induced by glucose and other secretagogues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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= 13,750 liters/mol·cm),
and the solution was then brought to concentrations of 25–100
µM by the addition of Krebs-Ringer bicarbonate (KRB)
buffer (pH 7.4). KRB buffer contained 118 mM NaCl, 4.7
mM KC1, 2.5 mM CaCl2, 1.2
mM KH2PO4, 1.2
mM MgSO4, and 24.2 mM
NaHCO3 and was equilibrated with 5%
CO2/95% O2. KRB buffer containing 2 mg/ml BSA was used as a medium for the incubation of pancreatic islets with HNE. We found that the incubation markedly decreased the HNE concentration because of the covalent-bond formation between HNE and BSA. Therefore, we prepared HNE-treated BSA. HNE (20 µmol) and BSA (0.4 g) were dissolved in 10 ml KRB buffer containing 2.8 mM glucose and incubated for 2 h at 37 C under an atmosphere of 5% CO2/95% O2. The solution was thoroughly dialyzed against the same buffer and then centrifuged for 10 min at 10,000 x g at 4 C to eliminate sediments. The supernatant was used as a solution of HNE-treated BSA. When 100 µM HNE was incubated in KRB buffer containing 2 mg/ml untreated BSA for 1 h at 37 C, the HNE concentration decreased to 50 µM. On the other hand, the use of HNE-treated BSA instead of untreated BSA clearly reduced the loss of HNE; the HNE concentration after incubation was 85 µM.
Malondialdehyde was prepared by hydrolysis of malonaldehyde
bis(dimethylacetal) in 1 M HC1 for 10 min at 30 C. The
concentration of malondialdehyde was estimated at 245 nm ( =
13,700 liters/mol·cm). Just before use, the acidic solution was
neutralized with 2 M NaHCO3.
2-Nonenal, 2-octenal, 2-hexenal, and D-glyceraldehyde were
obtained from Aldrich Japan (Tokyo, Japan).
D-[5-3H]glucose,
D-[U-14C]glucose,
[3H]water, and
[14C]sodium bicarbonate were purchased from
NEN Life Science Products (Boston, MA). Hexanal, calf
thymus DNA, and sodium -ketoisocaproate came from Sigma
(St. Louis, MO). Malonaldehyde bis(dimethylacetal) and 2-butenal were
purchased from Tokyo Kasei (Tokyo, Japan). H33258 was obtained from
Calbiochem (La Jolla, CA). Hyamine came from Packard
(Meriden, CT).
Rats were housed in a room maintained at 23 C with a fixed 12-h light, 12-h dark cycle and were given free access to rat chow and water. Experimental protocols were approved by the animal use committee of the Faculty of Pharmacy, Meijo University.
Incubation of islets and measurement of insulin secretion
Pancreatic islets were isolated from female Wistar rats
(weighing 300–350 g; Clea Japan, Tokyo, Japan) according to the method
described previously (11). Batches of 5 islets were treated with
aldehyde (e.g. HNE and 2-hexanal) for 1 h at 37 C in 1
ml KRB buffer supplemented with 5 mM glucose and
2 mg/ml HNE-treated BSA. The islets were washed once with KRB buffer
containing 2.8 mM glucose and 2 mg/ml BSA at room
temperature, and then incubated for 1 h at 37 C in 1 ml KRB buffer
supplemented with 2 mg/ml BSA together with 20 mM
glucose, 10 mM
D-glyceraldehyde, 10 mM
D-glyceraldehyde plus 2.8
mM glucose, 15 mM
-ketoisocaproate, or 15 mM
L-leucine. All incubations were performed in an
atmosphere of 5% CO2-95%
O2. In control experiments, the first incubation
of islets was performed in the absence of aldehyde. Of the aldehydes
used, 2-nonenal and 2-octenal were dissolved in ethanol and diluted to
yield a final solvent concentration of 0.1%. Insulin in the medium
obtained after the second incubation was assayed as described
previously (12). The basal insulin secretion induced in the presence of
2.8 mM glucose was 19 ± 1 µU/h·islet
(mean ± SEM of 25 determinations). The
insulin secretion values were expressed by subtraction of basal
secretion. In this article 1 determination refers to 1 incubation well
containing 5 islets.
Measurement of glucose utilization
Glucose utilization by islets was determined by measuring the
formation of 3H2O from
D-[5-3H]glucose (13). Briefly,
batches of 10 islets were incubated for 1 h at 37 C in the
presence or absence of 100 µM HNE, washed with KRB
buffer containing 2.8 mM glucose and 2 mg/ml BSA, and again
incubated for 1 h in 400 µl KRB buffer containing glucose (2.8
or 20 mM) and 2 mg/ml BSA as well as 1.6 µCi
D-[5-3H]glucose. After
the second incubation, 20 µl 3 M HCl and 50 µl ethanol
were added to stop metabolism, and then a piece of filter paper was put
into each microtube. The microtubes were placed in 20-ml glass
scintillation vials that contained 1 ml purified water. The vials were
stoppered and kept at 30 C for 24 h.
3H2O standard was treated
in the same way to correct for incomplete equilibration during the
diffusion step. Radioactivity in the purified water was determined.
Measurement of glucose oxidation
Glucose oxidation by islets was determined by measuring the
formation of 14CO2 from
D-[U-14C]glucose (14). Briefly,
batches of 8 islets were incubated for 1 h at 37 C in the presence
or absence of 100 µM HNE, washed once, and again
incubated for 1 h in 300 µl KRB buffer containing glucose (2.8
or 20 mM) and 2 mg/ml BSA as well as 0.6 µCi
D-[U-14C]glucose. After the second
incubation, 15 µl 3 M HCl were added to each microtube to
stop metabolism and to liberate
14CO2 from the medium. The
microtubes were placed in 20-ml glass scintillation vials that
contained 0.6 ml hyamine, and then the vials were stoppered and kept at
room temperature for 2 h to permit absorption of
14CO2.
NaH14CO3 standard was
treated in the same way to correct for incomplete
14CO2 absorption.
Radioactivity in the hyamine was determined.
Measurement of glycolytic intermediates
Batches of 40–50 islets were incubated for 1 h at 37 C in
the presence or absence of 100 µM HNE, washed once, and
again incubated for 1 h in 5 ml KRB buffer containing glucose (2.8
or 20 mM) and 2 mg/ml BSA. After removal of the medium, the
islets were quickly washed with ice-cold KRB buffer and then sonicated
in 70 µl 0.4 M HClO4. The sonicate
was centrifuged, and the supernatant was neutralized with 200
mM HEPES-NaOH buffer (pH 7.0) and 1 SC
K2CO3. After centrifugation
of the neutralized solution, the supernatant obtained was analyzed for
glycolytic intermediates as described previously (15).
Measurement of enzyme activities
Pancreatic islets were incubated in the presence or absence of
100 µM HNE for 1 h at 37 C in 1 ml KRB buffer
containing 5 mM glucose and 2 mg/ml HNE-treated BSA. The
islets were washed with the medium used for their homogenization. The
preparation of islet extracts and the assay of glucokinase and
hexokinase activities were performed as described previously (16). The
6-phosphofructokinase (PFK) and glyceraldehyde-3-phosphate
dehydrogenase activities in the islet extracts were assayed as
described by Trus et al. (17) and Laychock (18),
respectively.
Assay of DNA
The content of DNA in homogenates of pancreatic islets was
assayed by fluorometry according to the method of Labarca and Paigen
(19).
Statistical analysis
The statistical analyses were performed by the method of Dunnett
with the level of significance at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In
a treatment-time study, glucose-induced insulin secretion was
significantly decreased when islets were treated with 100
µM HNE for more than 15 min (Fig. 2).
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).
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). In contrast,
-ketoisocaproate-induced insulin secretion was significantly
decreased by treatment with the aldehyde.
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). Insulin secretion induced by
glyceraldehyde alone was significantly decreased by 50 and 100
µM HNE, but the decrease was only about 15% at either
concentration (Fig. 4). On the contrary, the secretion induced by a
mixture of glucose and glyceraldehyde was markedly decreased by
treatment of islets with 50 or 100 µM HNE. In this case,
the dependency of the decrease on the HNE concentration was quite
similar to that (Fig. 1
) seen for glucose-induced insulin
secretion.
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) and glucose oxidation (Fig. 6) measured in the presence of 20
mM glucose. HNE treatment decreased the amounts of fructose
1,6-bisphosphate and dihydroxyacetone phosphate in islets incubated
with 20 mM glucose, on the one hand, and increased the
amount of fructose 6-phosphate, on the other hand (Fig. 7).
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, none of the
enzymes tested was affected by treatment of islets with 100
µM HNE for 1 h.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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100
µM) used in this study are likely to occur in
pancreatic islets at least under diabetic conditions, which strongly
suggests that the HNE inhibition of glucose-induced insulin secretion
takes place in vivo.
All of the aldehydes listed in Table 1 are known to be produced as
lipid peroxidation products (24, 29, 30). Table 1 illustrates that some
alkenals among lipid peroxidation products, in addition to HNE, act to
impair glucose-induced insulin secretion. It should be noted that
shorter chain alkenals, 2-hexenal and 2-butenal, were more potent than
longer chain ones, 2-nonenal and 2-octenal. The concentration
dependency of the inhibitory potency of 2-hexenal and 2-butenal was
quite similar to that of HNE (data not shown).
The widely accepted mechanism for glucose-induced insulin secretion is
that the metabolism of glucose via the glycolytic pathway and the
citric acid cycle leads to an increase in the ATP/ADP ratio, resulting
in ß-cell depolarization induced by inhibition of ATP-sensitive
K+ channels and in subsequent activation of
voltage-dependent Ca2+ channels (31, 32). The
rise in the intracellular Ca2+ concentration is
thought to play a role in insulin release through activation of protein
kinases that interact with components of the secretory machinery (33).
-Ketoisocaproate is oxidized via the citric acid cycle to produce
ATP after its conversion to acetyl-coenzyme A and is thought to
stimulate insulin secretion by the mechanism common to islet fuels
including glucose (34). Leucine, in contrast to its deamination product
-ketoisocaproate, is believed to stimulate insulin secretion by two
intramitochondrial mechanisms: firstly, the catabolism of leucine
itself (35, 36), and secondly, the allosteric activation of glutamate
dehydrogenase resulting in an increase in the formation of
-ketoglutarate, one of the intermediates of the citric acid cycle,
from glutamate (37, 38). We recently found that glyceraldehyde not only
stimulates insulin secretion via metabolism through the glycolytic
pathway and the citric acid cycle after its phosphorylation, but also
induces the activation of phospholipase C by glyceraldehyde
3-phosphate, a phosphorylation product of the triose, resulting in the
stimulation of insulin secretion without the closure of ATP-sensitive
K+ channels (15). The mechanism for potentiation
of glyceraldehyde-induced insulin secretion by glucose is not yet
known, although glucose metabolism has been shown to be responsible for
the potentiation (21).
The marked inhibition of insulin secretion induced by glucose and by glyceraldehyde plus glucose implies that some step(s) responsible for glucose-induced insulin secretion is susceptible to the action of HNE. The data that HNE only slightly decreased glyceraldehyde-induced insulin secretion and increased leucine-induced insulin secretion suggest that the inhibitory effect of the aldehyde on distal secretion steps, including ß-cell depolarization, Ca2+ influx, and subsequent events, is marginal, if any. The same data are also likely to exclude the possibility that the inhibitory effect of HNE on glucose-induced insulin secretion was due to a nonspecific toxic effect on pancreatic islets.
The attenuation by HNE of islet glucose utilization in the presence of
20 mM glucose is indicative that the glycolytic pathway is
affected by the aldehyde. The citric acid cycle would also be one of
the possible targets of HNE, because the compound severely impaired
-ketoisocaproate-induced insulin secretion. An appreciable decrease
in islet glucose oxidation by HNE would be a demonstration that the
glycolytic pathway, the citric acid cycle, or both are the action sites
of the aldehyde. We do not have any proper explanation for the
mechanism(s) of the potentiation of leucine-induced insulin secretion
by HNE. It also remains to be answered whether the HNE potentiation of
leucine-induced insulin secretion is biologically meaningful. Less
marked inhibition of glyceraldehyde-induced insulin secretion may
indicate that the machinery for stimulation of insulin secretion
through the activation of phospholipase C by glyceraldehyde 3-phosphate
is kept intact in islets treated with HNE.
The decrease in fructose 1,6-bisphosphate and dihydroxyacetone
phosphate contents in concord with the increase in fructose 6-phosphate
content by treatment of islets with HNE strongly suggests that the
aldehyde affects the PFK step in the glycolytic pathway, but not the
glucose phosphorylation step. Contrary to these data, islet PFK
activity was not decreased by HNE treatment. The PFK activity shown in
Table 1, however, was assayed in the presence of 2 mM AMP
(an allosteric activator of the enzyme). It is possible that the
HNE-induced change in intracellular levels of allosteric effectors,
e.g. citric acid and AMP, may decrease the activity of
PFK.
It has been suggested that whatever defect, e.g. insulin resistance or impaired insulin secretion, initiates the disturbance in glucose metabolism, it will be followed by worsening of hyperglycemia (39). It seems worthwhile to investigate whether the impairment of insulin secretion by HNE and other lipid peroxidation products is involved in such a vicious cycle responsible for the development of type 2 diabetes, because hyperglycemia causes an increase in oxidative stress and the subsequent acceleration of lipid peroxidation.
During the course of the present study, Ihara et al. reported the presence of HNE-protein adducts in pancreatic islets (40). They stated that quantitative immunohistochemical analyses with specific antibodies revealed higher levels of HNE- modified proteins as well as 8-hydroxy-2'-deoxyguanosine (a marker for oxidative stress) in the pancreatic ß-cells of GK rats, a model of type 2 diabetes, than in the control Wistar rats, with the levels increasing proportionally with age and fibrosis of the pancreatic islets. This observation is compatible with the view that HNE and other lipid peroxidation products are implicated in the impairment of insulin secretion in diabetic animals and subjects.
In summary, we have demonstrated for the first time that lipid peroxidation products, HNE and other aldehydes, at concentrations likely to occur in tissues impair insulin secretion induced by glucose. The present study suggests the possibility that lipid peroxidation products formed by oxidative stress due to hyperglycemia in diabetes are involved in the worsening of glucose-induced insulin secretion in type 2 diabetes.
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Footnotes |
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Received June 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and
ß-anomeric preference of glucose-induced insulin secretion at
physiological and higher glucose concentrations, respectively. Biochem
Biophys Res Commun 180:709–715[CrossRef][Medline]
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) or absence (
) of 100 µM HNE for 1
h at 37 C and then assayed for insulin secretion induced by 15
mM leucine (Leu) or 15 mM
). Values are the
mean ± SEM of 14–16 determinations. The control
values for insulin secretion induced by glyceraldehyde and by
glyceraldehyde plus glucose were 96 ± 4 and 210 ± 16
µU/h·islet, respectively. +, P < 0.05; *,
P < 0.01; **, P < 0.001
(compared with the control).
