Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900 São Paulo, Brasil
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ABSTRACT |
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The activity of superoxide dismutase (SOD), catalase, and
glutathione peroxidase (GSP) in isolated rat pancreatic
islets exposed to high glucose concentration for a short period of time
(60 min) was determined. High glucose concentration (16.7 mM) did not
significantly alter catalase activity. GSP activity was
increased by glucose at 5.6 mM, remaining elevated at
higher concentrations up to 16.7 mM. However, the activity of SOD
increased with glucose concentration, and this increment was closely
correlated with the rate of insulin secretion
(r = 0.96). High potassium (30 mM) did
not increase SOD activity, suggesting that the increase in
intracellular ionic calcium concentration does not stimulate this
enzyme activity. -Ketoisocaproic acid and pyruvate, which are
metabolized through the TCA cycle, did not increase SOD activity,
indicating that the stimulation of SOD activity might be triggered by a
factor produced through glycolysis or the pentose phosphate pathway.
antioxidant enzymes; glycemia; pancreatic islets; insulin secretion
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INTRODUCTION |
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INSULIN SECRETION by pancreatic -cells is mainly
induced by circulating glucose (16). The glucose metabolism of the
-cells causes several cellular alterations that result in an
increase in intracellular ionic calcium concentration following insulin granule extrusion. These alterations are
1) closure of the ATP-sensitive potassium channels, resulting in
-cell membrane depolarization (17),
and the opening of the voltage-sensitive calcium channels (22);
2) activation of phospholipase C,
phospholipase A2, and adenyl
cyclase, which leads to the formation of substances that also increase
the intracellular ionic calcium concentration (e.g., inositol
trisphosphate) and/or potentiate the response of the exocytotic
apparatus to ionic calcium (e.g., protein kinase C, protein kinase A,
arachidonic acid, cAMP) (for reviews see Refs. 1 and 11).
In several tissues, under physiological conditions, glucose oxidation
results in the generation of hydrogen peroxide and oxygen free radicals
such as the hydroxyl radical (30). Reactive oxygen species are known to
interact with nucleic acids, proteins and the lipid bilayer of cell
membranes, leading to cell damage (13). Pancreatic islets
present low activity of antioxidant enzymes (14, 18), being highly
susceptible to cellular damage. Cytokines produced from macrophages and
lymphocytes infiltrating pancreatic islets in type 1 (insulin-dependent) diabetes may induce -cell damage by an increase
in the production of oxygen free radicals in the islet cells (5, 18,
25). The damage of islet
-cells induced by the production of oxygen
free radicals results from lipid peroxidation (21) and the consequent
production of aldehydes (24).
It has been demonstrated that a high glucose concentration causes overexpression of Cu/Zn superoxide dismutase (Cu/Zn-SOD), catalase, and glutathione peroxidase (GSP) in cultured human endothelial cells (7). However, the incubation of isolated rat pancreatic islets with high glucose concentration (30 mM) for 48 h (chronic exposure) does not show significant changes in activity and expression of SOD (the first cellular defense against free radicals), catalase, and GSP (26). These observations refer to a prolonged exposure to high glucose concentration, which does not usually occur under physiological conditions.
Pancreatic -cells are constantly subjected to transient increases in
glycemia, returning to normal levels after a short period of time.
Therefore, the purpose of this study was to investigate the activity of
the antioxidant enzymes (SOD, catalase, and GSP) in isolated rat
pancreatic islets exposed to high glucose concentrations for a short
period of time (60 min).
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MATERIALS AND METHODS |
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Animals. Female albino rats weighing 150-200 g (45-60 days old) were obtained from the Institute of Biomedical Sciences, USP, São Paulo, Brazil. The animals were kept in groups of five at 23°C in a room with a light-dark cycle of 12:12 h (lights on from 7:00 AM).
Chemicals and enzymes. Collagenase,
cytochrome c, glutathione (reduced
form), -ketoisocaproic acid,
-NADPH, purine,
t-butyl-hydroperoxide, and xanthine
oxidase were purchased from Sigma Chemical (St. Louis, MO); glutathione
reductase and pyruvate were from Boehringer Mannheim; and sodium azide
was from BDH Chemicals (Poole, England, UK).
Islets isolation and incubation. Rat
pancreatic islets were isolated as described by Lacy and Kostianovsky
(15). This method was slightly modified from our previous publications
(6, 10). Batches of 150 islets were incubated in 0.5 ml of
Krebs-Henseleit buffer (in mM: 139 Na+, 5 K+, 1 Ca
2+, 1 Mg2+, 124 Cl, and 24 HCO
3) at 37°C for 60 min in the
absence (control) and in the presence of 5.6, 8.3, 11.1, 14, or 16.7 mM glucose. A similar experiment was performed in the presence of pyruvate
(1 mM),
-ketoisocaproic acid (10 mM), and high potassium concentration (30 mM). After incubation, the medium was removed and the
islets were washed three times with phosphate buffer (10 mM sodium
phosphate at pH 7.4). Preliminary experiments established that this
procedure does not cause detectable loss of SOD activity. The islets
were then suspended in 150 µl phosphate buffer and homogenized by
sonication. The activity of total SOD, catalase, and GSP was then determined.
Enzyme assays. The activities of SOD,
catalase, and GSP were measured by spectrophotometric
assays. The extraction medium for the measurement of the enzyme
activities was 10 mM sodium phosphate buffer at pH 7.4. Total SOD
activity was measured by inhibition of the cytochrome
c reduction rate induced by superoxide anions, monitored at 550 nm at 25°C, utilizing the
xanthine/xanthine oxidase system as the source of
O2. SOD competes for superoxide and
decreases the reduction rate of cytochrome c (12). One unit of SOD is defined as
that amount of enzyme that inhibits by 50% the rate of cytochrome
c reduction, under specified
conditions. The activity of Mn-SOD was determined after adding cyanide
to the assay medium. The procedure used for catalase and GSP assays was
similar to those reported by Beutler (2) and Wendel (29), respectively.
Catalase activity was determined by measuring the consumption of
hydrogen peroxide at 230 nm and 30°C. The activity of GSP was
measured by following the rate of oxidation of the reduced form of
glutathione. The formation of oxidized glutathione was monitored by a
decrease in the concentration of NADPH, measured at 340 nm and
37°C, caused by the addition of glutathione reductase to the
medium. The enzyme assays were performed in a Gilford (Response model)
spectrophotometer. A similar procedure has been previously used by
Pereira et al. (20) and Pithon-Curi et al. (20a).
Expression of the results. The activities of the enzymes are expressed as micromoles per minute per milligram of protein. The rates of insulin release are presented as microunits per 60 min per islet.
Protein determination. Protein from the islet homogenates was measured by the method of Bradford (4), using BSA as standard.
Statistical analysis. Results are presented as means ± SE from 8 pools of 150 islets each. ANOVA was employed to indicate significant effects of glucose. The level of significance was set for P < 0.05.
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RESULTS AND DISCUSSION |
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A short time exposure of the pancreatic islets to high glucose
concentration (16.7 mM) did not significantly alter catalase activity
(data not shown). In contrast, GSP activity (in
µmol · min1 · mg
protein
1) was increased
(2.7-fold) by glucose even at 5.6 mM concentration (Fig.
1) and was 1.64 ± 0.08 in the absence
and 4.37 ± 0.17 in the presence of 5.6 mM glucose (means ± SE
of 8 determinations). This effect remained unchanged in the presence of
8.3 mM (4.66 ± 0.17) and 16.7 mM (4.75 ± 0.21) glucose. These
findings support the proposition that the activity of hydrogen
peroxide-consuming enzymes is not efficiently influenced by glucose
concentrations. In fact, inactivation of hydrogen peroxide has been
proposed to be of critical importance for the removal of reactive
oxygen species in insulin-producing cells (26).
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High glucose concentrations have been reported to produce cellular
stress in vascular endothelial cells and to increase SOD expression
(7). However, exposure of pancreatic islets to 30 mM glucose for 48 h
does not show significant alteration in Cu/Zn-SOD and Mn-SOD
activities, abundance of the mRNA of the enzymes, and the corresponding
protein contents (26). In the present study, SOD activity in
1-h-incubated pancreatic islets increased with the increase in glucose
concentration (Fig. 2). The increment of
this enzyme activity was also closely correlated with the rate of
insulin secretion (r = 0.96; Fig. 2).
To elucidate whether glucose stimulates Cu/Zn-SOD or Mn-SOD activity,
the measurement of total SOD activity was performed in the presence of
cyanide (2 mM) (20). The addition of cyanide (an inhibitor of Cu/Zn-SOD activity) altered the increase in total SOD activity caused by high
glucose concentrations (data not shown). Therefore, glucose does not
acutely affect Mn-SOD activity, as has been reported to occur in
pancreatic islets incubated for 90 min in the presence of
interleukin-1 (3).
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The observations above led us to postulate that the activity of
Cu/Zn-SOD is acutely stimulated by glucose. This may function as an
important mechanism to prevent the toxic effects of high glucose
concentrations. However, when a high glucose concentration is
maintained for a prolonged period of time (such as 48 h), this regulation is somehow lost (26), which might favor the occurrence of
glucocytotoxicity. In fact, it has been postulated that SOD counteracts
islet vascular alterations in low-dose streptozotocin-treated mice (19)
and plays an important role in longevity and degenerative diseases
(28). Whether the findings presented herein are linked to the
observations of Vinci et al. (27) remains to be established. These
authors reported that Cu (II) is able to prevent the interleukin-1 inhibition of glucose-induced insulin release and glucose oxidation in
isolated pancreatic islets.
An important question remains: What is the mechanism for the effect of
glucose on SOD activity? Induction of insulin secretion by high
potassium concentration (30 mM) instead of glucose did not increase SOD
activity (221.0 ± 7.9 µmol · min1 · mg
protein
1 in islets exposed
to high potassium concentration vs. 240.0 ± 7.0 for the control
group; means ± SE of 4 experiments). This observation supports the proposition that neither the increase in
intracellular ionic calcium concentration nor the events involved in
the process of exocytosis of the insulin granules seem to stimulate this enzyme activity.
It is well known that Cu/Zn-SOD activity is found in the cytosol,
whereas Mn-SOD activity is restricted to mitochondria (20). Therefore,
the authors believe that the stimulation of SOD activity might occur by
a cytosolic factor produced through glycolysis or the pentose phosphate
pathway. In fact, -ketoisocaproic acid (10 mM) and pyruvate (1 mM),
which are metabolized through the TCA cycle in the mitochondria leading
to insulin release (23), did not increase the SOD activity compared
with control (incubation in the absence of substrates; 216.0 ± 6.0 µmol · min
1 · mg
protein
1 for
-ketoisocaproate, 140.0 ± 1.0 for pyruvate, and 240.0 ± 7.0 for the control groups; means ± SE of 4 experiments). In contrast to glucose, pyruvate caused a significant
reduction (41.7%) of the SOD activity. As also reported by others (9),
pyruvate is able to react with hydrogen peroxide and may exert
protective effects against cellular damages caused by the oxygen
reactive species. Therefore, further studies are needed to elucidate
this important point.
In conclusion, the findings presented support the proposition that glucose acutely stimulates Cu/Zn-SOD activity in pancreatic islets. This may make up an important physiological mechanism to protect the islets against oxygen toxicity due to the periodic exposure to high glucose concentration that occurs after feeding episodes.
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ACKNOWLEDGEMENTS |
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We are grateful for the expert technical assistance of Marlene S. Rocha and José C. B. Gonçalves.
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FOOTNOTES |
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This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundaçao de Amparo a Pesquisa do Estado de São Paulo, and Programa de Apoio a Núcleos de Exelência (168/97).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: A. R. Carpinelli, Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, Ed. Bio I, Av. Prof. Lineu Prestes 1524, 05508-900 São Paulo, Brasil.
Received 2 October 1998; accepted in final form 25 November 1998.
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REFERENCES |
---|
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---|
1.
Ashcroft, F. M.,
and
S. J. H. Ashcroft.
Insulin: Molecular Biology to Pathology. Oxford, UK: IRL, 1992, p. 103-130.
2.
Beutler, E.
Red Cell Metabolism. A Manual of Biochemical Methods. New York: Grune & Stratton, 1975, p. 89-90.
3.
Borg, L. A. H.,
E. Gagliero,
S. Sandler,
N. Welsh,
and
D. L. Eizirik.
Interleukin-1 increases the activity of superoxide dismutase in rat pancreatic islets.
Endocrinology
130:
2851-2857,
1992[Abstract].
4.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
5.
Burkart, V.,
T. Koike,
H. H. Brenner,
and
H. Kolb.
Oxygen radicals generated by the enzyme xanthine oxidase lyse rat pancreatic islet cells in vitro.
Diabetologia
35:
1028-1034,
1992[Medline].
6.
Carpinelli, A. R.,
R. Curi,
and
W. J. Malaisse.
Long-term regulation of pancreatic B-cell responsiveness to D-glucose by food availability, feeding schedule, and diet composition.
Physiol. Behav.
52:
1193-1196,
1992[Medline].
7.
Ceriello, A.,
P. Russo,
P. Amstad,
and
P. Cerretti.
High glucose induces antioxidant enzymes in human endothelial cells in culture.
Diabetes
45:
471-477,
1996[Abstract].
9.
De Groot, M. J.
The influence of lactate, pyruvate and glucose as exogenous substrates on free radical defense mechanisms in isolated rat hearts during ischaemia and reperfusion.
Mol. Cell. Biochem.
146:
147-155,
1995[Medline].
10.
El Razi Neto, S.,
T. M. T. Zorn,
R. Curi,
and
A. R. Carpinelli.
Impairment of insulin secretion in pancreatic islets isolated from Walker 256 tumor-bearing rats.
Am. J. Physiol.
271 (Cell Physiol. 40):
C804-C809,
1996
11.
Flatt, P. R.
Nutrient Regulation of Insulin Secretion. London: Portland, 1992, p. 83-289.
12.
Flohé, L.,
and
F. Otting.
Superoxide dismutase assays.
Methods Enzymol.
105:
93-104,
1984[Medline].
13.
Halliwell, B.,
and
J. M. C. Gutteridge.
Free Radicals in Biology and Medicine. Oxford, UK: Clarendon, 1995.
14.
Harris, E. D.
Regulation of antioxidant enzymes.
FASEB J.
6:
2675-2683,
1992
15.
Lacy, P. E.,
and
Y. Kostianovsky.
Method for isolation of intact islets of Langerhans from the rat pancreas.
Diabetes
16:
35-39,
1967[Medline].
16.
Malaisse, W. J.,
A. Sener,
A. Herchuelz,
and
J. C. Hutton.
Insulin release: the fuel hypothesis.
Metabolism
28:
373-386,
1979[Medline].
17.
Meissner, H. P.,
and
H. Schmelz.
Membrane potential of -cells in pancreatic islets.
Pflügers Arch.
351:
195-206,
1974[Medline].
18.
Oberley, L. W.
Free radicals and diabetes.
Free Radic. Biol. Med.
5:
113-124,
1988[Medline].
19.
Papaccio, G.,
S. Frascatore,
and
F. A. Pisanti.
An increase in superoxide dismutase counteracts islet vascular alterations in low-dose streptozocin-treated mice.
Histochemistry
101:
215-221,
1994[Medline].
20.
Pereira, B.,
L. F. B. P. Costa Rosa,
D. A. Safi,
E. J. H. Bechara,
and
R. Curi.
Hormonal regulation of superoxide dismutase, catalase and glutathione peroxidase activities in rat macrophages.
Biochem. Pharmacol.
50:
2093-2098,
1995[Medline].
20a.
Pithon-Curi, T. C.,
M. Pires-de-Melo,
A. C. Palanch,
C. K. Miyasaka,
and
R. Curi.
Percentage of phagocytosis, production of O2, H2O2, and NO, and antioxidant enzyme activities of rat neutrophils in culture.
Cell Biochem. Funct.
16:
43-49,
1998[Medline].
21.
Rabinovitch, A.,
W. L. Suarez,
P. D. Thomas,
K. Strynadka,
and
I. Simpson.
Cytotoxic effects of cytokines on rat islets: evidence for involvement of free radicals and lipid peroxidation.
Diabetologia
35:
409-413,
1992[Medline].
22.
Satin, L. S.,
and
D. L. Cook.
Voltage-gated Ca2+ current in pancreatic islets.
Pflügers Arch.
404:
385-387,
1985[Medline].
23.
Sener, A.,
S. Kawazu,
J. C. Hutton,
A. C. Boschero,
D. Ghislain,
G. Somers,
A. Herchuelz,
and
W. J. Malaisse.
The stimulus-secretion coupling of glucose-induced insulin release. Effect of exogenous pyruvate on islet function.
Biochem. J.
176:
217-232,
1978[Medline].
24.
Suarez-Pinzon, W. L.,
K. Strynadka,
and
A. Rabinovitch.
Destruction of rat pancreatic islet -cells by cytokines involves the production of cytotoxic aldehydes.
Endocrinology
137:
5290-5295,
1996[Abstract].
25.
Sumoski, W.,
H. Baquerizo,
and
A. Rabinovitch.
Oxygen free radical scavengers protect rat islet cells from damage by cytokines.
Diabetologia
32:
792-796,
1989[Medline].
26.
Tiedge, M.,
S. Lortz,
J. Drinkgern,
and
S. Lenzen.
Relation between antioxidant enzyme gene expression and antioxidant defense status of insulin-producing cells.
Diabetes
46:
1733-1742,
1997[Abstract].
27.
Vinci, C.,
V. Caltabiano,
A. M. Santoro,
A. M. Rabuazzo,
M. Buscema,
R. Purrello,
E. Rizzarelli,
R. Vigneri,
and
F. Purrello.
Copper addition prevents the inhibitory effects of interleukin-1 on rat pancreatic islets.
Diabetologia
38:
39-45,
1995[Medline].
28.
Warner, H. R.
Superoxide dismutase, ageing and degenerative disease.
Free Radic. Biol. Med.
17:
249-258,
1994[Medline].
29.
Wendel, A.
Glutathione peroxidase.
Methods Enzymol.
3:
325-339,
1978.
30.
Wolff, S. P.,
and
R. T. Dean.
Glucose autoxidation and protein modification: the potential role of oxidative glycosylation in diabetes.
Biochem. J.
245:
243-250,
1987[Medline].