Enhanced alloxan-induced β-cell damage and delayed recovery from hyperglycemia in mice lacking extracellular-superoxide dismutase

Free Radical Biology & Medicine, Vol. 27, Nos. 7/8, pp. 790 –796, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter

PII S0891-5849(99)00127-6

Original Contribution ENHANCED ALLOXAN-INDUCED ␤-CELL DAMAGE AND DELAYED RECOVERY FROM HYPERGLYCEMIA IN MICE LACKING EXTRACELLULAR-SUPEROXIDE DISMUTASE MARIE-LOUISE SENTMAN,* LENA M. JONSSON,†,‡

and

STEFAN L. MARKLUND*

*Departments of Clinical Chemistry and †Microbiology, Umeå University, Umeå and ‡Eurona Medical AB, Uppsala, Sweden (Received 23 February 1999; Accepted 28 April 1999)

Abstract—Alloxan is a diabetogenic agent which apparently acts through formation of superoxide radicals formed by redox cycling. Superoxide radicals are also formed by a variety of mechanisms in hyperglycemia. We exposed extracellular-superoxide dismutase (EC-SOD) null mutant and wild-type mice to alloxan, and followed up both the initial diabetes induction and the long-term course of the hyperglycemia. The null mutant mice responded with a modestly enhanced hyperglycemia compared to the wild type controls. In the long-term follow-up all mice eventually regained glycemic control, although it took longer for individuals with higher initial hyperglycemia. This delaying effect of the hyperglycemia was much more pronounced in the null mutant mice. These data suggest that the difference in initial diabetes induction between the groups is due to interception by EC-SOD of extracellular superoxide radicals produced by alloxan. The delayed recovery in the null mutant mice suggests that superoxide radicals released as a result of hyperglycemia impair ␤-cell regeneration and that EC-SOD provides some protection. Mouse islets were found to contain little EC-SOD, whereas the content of the cytosolic Cu- and Zn-containing SOD was very high. This low EC-SOD activity may contribute to the high alloxan susceptibility of ␤-cells, and may also cause a high susceptibility to superoxide radicals produced by activated inflammatory leukocytes and in hyperglycemia. © 1999 Elsevier Science Inc. Keywords—Alloxan, Superoxide dismutase, Superoxide radicals, Hyperglycemia, Extracellular space, Free radicals

INTRODUCTION

activated by the autoimmune reaction of type 1 diabetes. It has however also been shown that transgenic overexpression of the cytosolic CuZn-SOD reduces susceptibility of mice to alloxan [7,8], which suggests that the reactive superoxide radical might also be formed in the cytosol or pass through the plasma membrane. There are three mammalian SOD isoenzymes. CuZnSOD exists in the cytosol [9] and nucleus [10], Mn-SOD in the mitochondrial matrix [11], and the secreted extracellular-SOD (EC-SOD) [12] in the extracellular space. The major part of EC-SOD is anchored to heparan sulfate proteoglycans on cell surfaces [13,14] and in the tissue interstitial matrix [15], but the enzyme is also found in extracellular fluids. Because the substrate, the superoxide anion radical, poorly penetrates cell membranes [16], the SOD isoenzymes primarily exert their protective functions in their respective compartments. We have generated mice lacking EC-SOD by a targeted disruption of the gene encoding the enzyme [17]. To test the importance of extracellular superoxide radicals to

Alloxan has been widely used as a diabetogenic agent since the initial report of this action by Dunn et al, in 1943 [1]. It has been suggested that the selective destruction of pancreatic ␤-cells is mediated by oxygen free radicals formed by redox cycling [2– 4]. We have previously shown that superoxide dismutase (SOD) can protect against alloxan-induced ␤-cell damage, both when added to culture medium containing isolated pancreatic islets [5], and when injected intravenously into mice [6]. This suggests that superoxide radicals formed by alloxan redox-cycling on the exterior of ␤-cells contribute to their destruction. In that respect the alloxan model might mimic the toxicity potentially caused by superoxide radicals secreted by phagocytic leukocytes, attracted and Address correspondence to: Stefan L Marklund, MD, PhD, Department of Clinical Chemistry, Umeå University, SE-901 85 Umeå, Sweden; Tel: ⫹46 (90) 785-1239; Fax: ⫹46 (90) 777-296; E-Mail: [email protected]. 790

EC-SOD and alloxan hyperglycemia

cause toxicity towards ␤-cells by alloxan as well as by hyperglycemia, we exposed EC-SOD null mice to the toxin and followed up the development and course of the hyperglycemia. In this article we show that the mutant mice, compared with the wild-type mice, react with an enhanced hyperglycemia to alloxan and that the recovery of glycemic control is significantly delayed. MATERIALS AND METHODS

Mice Female mice about 12 weeks old, EC-SOD null-mutant and wild-type mice (background C57Bl/6 x 129/SV) were obtained from a breeding colony established at Umeå University [17]. A total of 77 wild type (also C57Bl/6 x 129/sv background) and 67 EC-SOD nullmutant mice entered the study which followed the “principles of laboratory animal care (National Institutes of Health publication No.86-23, revised 1985)” and was approved by the local animal ethics committee. Alloxan treatment Alloxan monohydrate (Sigma-Aldrich, St Louis, MO, USA), 10 mg/ml, was dissolved in sterile ice-cold water immediately before use. The spontaneous pH of the solution was low, 3.3, stabilizing the compound [18]; 35 mg/kg body weight alloxan was injected intravenously into overnight fasted EC-SOD null-mutant mice and wild-type mice. The animal genotypes were coded before the experiment and remained coded until after the 96-h blood glucose levels were analyzed. Nine sets of experiments were performed. After the alloxan injection the mice were given food ad libitum. Analysis of blood glucose Blood glucose levels were tested before the alloxan injection and at 24-, 48-, 72-, and 96-h after treatment, to monitor the immediate diabetogenesis. To follow up the course of and recovery from diabetes, blood glucose levels were monitored every third week thereafter. Animals showing blood glucose levels of ⬍ 10 mmol/l on two subsequent test occasions were considered nondiabetic and not analyzed further. Blood glucose levels were analyzed with a Glucometer Elite apparatus (Bayer Inc.) using 5 to 10 ␮l blood obtained from the tail vein. The method was continuously controlled with samples and controls analyzed in the routine clinical chemistry laboratory and showed a high precision and accuracy.

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x 129/SV). The pancreas was cut in 3 ⫻ 3 mm pieces and disintegrated at 37°C under vigorous shaking, with 2 pancreata per ml KRH buffer (0.13 mol/l NaCl, 4.7 mmol/l KCl, 1.2 mmol/l MgSO4, 1.2 mmol/l KH2PO4, 2.6 mmol/l CaCl2, 3 mmol/l glucose, 20 mmol/l N-[2hydroxyethyl]piperazine-N⬘-[2-ethanesulfonic acid]), containing 0.3% collagenase type V (Sigma-Aldrich). After 25 min, the disintegration was stopped by addition of 50-ml ice cold Hanks’ BSS (Gibco Ltd, Paisley, Scotland, UK), containing a tablet of Complete protease inhibitor cocktail (Boehringer Mannheim GmbH, Germany), the suspension was vortexed for 1 min and the islets collected by spinning up to 1200 rpm. The pellet was washed two additional times with ice-cold Hanks’ BSS and then suspended in ice-cold antiprotease containing KRH buffer. The islets were collected under dissection microscopes with micropipettes, centrifuged and stored as pellets at ⫺70° until analysis. Determination of SOD isoenzymes in islets and other tissues The combined pellet of islets from 54 mice was homogenized by sonication under cooling with ice in 0.7 ml, 50 mmol/l sodium phosphate (pH 7.4) with 0.3 mol/l KBr, containing the Complete protease inhibitor cocktail (one tablet per 25 ml). The other tissues were homogenized with an Ultraturrax in 10 volumes of the above buffer and sonicated. Thereafter, the homogenates were centrifuged at 20,000 ⫻ g for 15 min. The SOD activity of the supernatants was determined by the direct spectrophotometric method with KO2 [19] as modified [20]. To distinguish between the resistant Mn-SOD and the sensitive isoenzymes CuZn-SOD and EC-SOD, 3 mmol/l of cyanide was used. It should be noted that the KO2 assay method is ten times more sensitive for CuZn-SOD and EC-SOD activity than Mn-SOD activity. Because the glycoprotein EC-SOD binds to the lectin Concanavalin A it can be separated from the two other SOD isoenzymes using a ConA-Sepharose column [17,21]. The detection limit of the procedure is about 5 units of EC-SOD per gram wet weight tissue. When little material is available as in the islet extract, the detection limit becomes higher (Table 1). The CuZn-SOD activity is calculated as the total cyanide sensitive activity minus the EC-SOD activity. RESULTS

Induction of hyperglycemia by alloxan Preparation of islets The pancreatic gland was rapidly excised from 54 about 12-weeks-old mice of the wild type strain (C57Bl6

The diabetogenic effects of several different doses of alloxan were tested, and 35 mg/kg was chosen. This dose resulted in a light to intermediate degree (medians 15–20

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M.-L. SENTMAN et al. Table 1. Content of SOD Isoenzymes in Pancreatic Islets and Other Tissues of the Mouse EC-SOD

Islets (pool of 54) Pancreas (n ⫽ 5) Braina (n ⫽ 10) Hearta (n ⫽ 10) Intestine (n ⫽ 5) large small Kidneya (n ⫽ 10) Livera (n ⫽ 10) Lunga (n ⫽ 10)

CuZn-SOD

Mn-SOD

U/g Wet weight

U/mg Protein

U/g Wet weight

U/mg Protein

U/g Wet weight

U/mg Protein

58 ⫾ 9 7⫾8 65 ⫾ 11

⬍ 0.49 0.56 ⫾ 0.10 0.16 ⫾ 0.20 0.59 ⫾ 0.14

13,500 ⫾ 740 10,400 ⫾ 3500 14,300 ⫾ 12,000

330 130 ⫾ 10 260 ⫾ 91 130 ⫾ 27

200 ⫾ 13 416 ⫾ 82 1950 ⫾ 280

2.2 1.93 ⫾ 0.08 10 ⫾ 1.9 18 ⫾ 2.2

230 ⫾ 100 68 ⫾ 29 620 ⫾ 82 42 ⫾ 22 3300 ⫾ 490

2.7 ⫾ 1.3 0.93 ⫾ 0.39 4.9 ⫾ 1.3 0.35 ⫾ 0.17 34 ⫾ 9

13,500 ⫾ 5600 19,400 ⫾ 8000 37,500 ⫾ 4500 92,800 ⫾ 3200 21,300 ⫾ 1200

160 ⫾ 68 270 ⫾ 110 290 ⫾ 34 780 ⫾ 54 220 ⫾ 51

272 ⫾ 120 230 ⫾ 97 2790 ⫾ 620 1320 ⫾ 120 308 ⫾ 56

3.1 ⫾ 1.3 3.2 ⫾ 1.4 22 ⫾ 1 11 ⫾ 1 3.1 ⫾ 0.9

The tissues were extracted and analyzed as described under Materials and Methods. The results are presented as means ⫾ SD. The SOD levels in islets were determined from a pool of islets from 54 mice. The wet weight of this tissue material was not possible to determine. The EC-SOD content was below the detection limit for the amount of material available. Note that the SOD assay is ten times less sensitive for Mn-SOD activity than for EC-SOD and CuZn-SOD activity, see Materials and Methods. a Part of the data for these organs are from a previous study [17].

mmol/l) of hyperglycemia after 3 to 4 d in the present mouse strains (Fig. 1). Although there were no differences in the fasting blood glucose levels before the alloxan injections (wild type mice 3.4 ⫾ 0.7 mmol/l [SD], EC-SOD null mice 3.6 ⫾ 0.5 mmol/l [SD]), the EC-SOD null mice responded with a slightly stronger hyperglycemia during the initial 4-d period (Fig. 1).

mouse organs. Many organs including islets, whole pancreas, brain, heart, and liver contained very little ECSOD. The CuZn-SOD content of islets was high, second only to the liver. The Mn-SOD content was relatively low in islets.

Recovery of glycemic control

We here show that EC-SOD null mice respond to alloxan with greater blood glucose increase than the wild type mice (Figs. 1 and 2). In the initial 4-d development period of hyperglycemia, the difference between the groups was significant, although modest (Fig. 1). The recovery of glycemic control was markedly delayed in the EC-SOD null mice and appeared to be negatively influenced by high glucose concentrations (Fig. 2A and 2B). The alloxan response, both in vivo and in vitro, is known to be influenced by a variety of metabolic factors [22]. We have previously analyzed CuZn-SOD, MnSOD, and other antioxidant enzymes in a variety of tissues as well as a series of metabolites in blood in the EC-SOD null mice and found no differences versus wild-type mice [17]. This suggests that the differences found here in alloxan response and in recovery of glycemic control are caused by the lack of EC-SOD and are not due to other differences between the mice. Alloxan is readily reduced to dialuric acid, and these two compounds form a redox cycle producing superoxide radical in the presence of reductants and molecular oxygen [2– 4]. The present results together with previous findings [5– 8] indicate that the superoxide radicals formed contribute to the ␤-cell toxicity of alloxan. From the effects of SOD added in vitro [5] and intravenously injected [6] we previously concluded that the ␤-cell toxicity was primarily due to superoxide radicals produced at the exterior of cells. The modest effect due to

After the diabetes induction, the blood glucose levels of all mice with 96-h blood glucose levels over 10 mmol/l were monitored at 3-week intervals. Seventy-one percent of the EC-SOD null mice and 60% of the controls entered this phase of the study. The glucose levels gradually declined (data not shown) and, in the end, all mice regained a degree of glycemic control, in the present study defined as blood glucose levels below 10 mmol/l on two subsequent test occasions. Overall there was a correlation between the 96-h blood glucose levels and time required to regain glycemic control (Fig. 2A). This dependence on the hyperglycemia was significantly more pronounced in the EC-SOD null group, p ⬍ .02 by analysis of covariance. When the blood glucose levels at 3 weeks were plotted against remaining time to glycemic control, again the data indicated that the recovery of the EC-SOD null mice was significantly more affected by the hyperglycemia, p ⬍ .001 (Fig. 2B). Content of SOD isoenzymes in pancreatic islets, and other organs of the mouse To further characterize this model system, the contents of SOD isoenzymes were determined in pancreatic islets and other organs of control mice (Table 1). The EC-SOD contents were highly variable between different

DISCUSSION

EC-SOD and alloxan hyperglycemia

the absence of EC-SOD (Fig. 1), together with findings of a more distinct protective effect of transgenic overexpression of the cytosolic CuZn-SOD [7,8], might seem to contradict that notion. Although some loss of EC-SOD might have occurred during isolation of the islets, they apparently contain little EC-SOD (Table 1). Of importance for the protection of ␤-cells may also be the ECSOD activity in plasma, about 300 U/ml [13]. Taken together the protection of islets by EC-SOD appears to be modest, and still loss of that activity resulted in a discernible increase in alloxan susceptibility. In comparison the plasma SOD activity was about 5000 U/ml in the mice protected against alloxan by intravenously injected SOD [6], and a SOD activity of 4000 U/ml in the culture medium was required to protect islets in vitro [5]. Mouse pancreatic islets show a very high CuZn-SOD activity (Table 1). Immunohistochemistry studies in dogs and humans have indicated that there is more or at least as much CuZn-SOD in ␤-cells as in other cells of the

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islets [23,24]. This indicates that the data for whole islets are also representative for ␤-cells. In the transgenic strains that showed resistance against alloxan there were apparently marked increases in ␤-cell CuZn-SOD activity [7,8], suggesting that the cells contained extremely large amounts of CuZn-SOD. Even a minor leakage of CuZn-SOD to the exterior or entrance of superoxide anion radicals through anion channels [25] and carriers into the cytosol might explain the protection seen in the transgenic mice. The permeability of the ␤-cell membrane for the anion chloride is high [26], suggesting that significant transport of superoxide anion radical into the cytosol might occur. We conclude that the notion of a primarily extracellular production of superoxide by alloxan is still viable, but not proven. The low EC-SOD content of islets may be one of the factors that contribute to the susceptibility of ␤-cells to alloxan. It may also be of relevance for the response of islets to the inflammatory reaction of type 1 diabetes.

Fig. 1. Development of blood glucose levels following alloxan injection. Alloxan was injected intravenously into EC-SOD null (E) and wild type mice (●), whereafter the blood glucose levels were monitored every 24 h for 4 d. The horizontal bars indicate the medians of the groups. The statistical significances for the differences between the groups were calculated by the nonparametric Mann-Whitney U-test, and are indicated in the figure.

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Fig. 2. Correlations between (A) 96-h and (B) 3-week blood glucose levels and remaining time to glycemic control. All mice showing 96-h blood glucose levels above 10 mmol/l were monitored with glucose determinations every third week. The animals were considered to have regained a degree of glycemic control if nonfasting blood glucose levels below 10 mmol/l were found on two subsequent occasions. In (B) only data for animals showing blood glucose levels ⬎10 mmol/l at 3 weeks are plotted. The hatched and full lines show the linear regressions for the (E) EC-SOD null and (●) wild type groups. The significances for the linear regressions for the null and wild type mice were ⬍ .0001 and ⬍ .02 respectively in (A) and ⬍ .0001 and ⬍ .0001 in (B). In each group three mice died before regaining glycemic control. Their 96-h blood glucose levels were 29.5, 40.5, 47.0 and 28.6, 36.6, 42.0 mmol/l in the null mutants and wild type controls, respectively.

EC-SOD and alloxan hyperglycemia

Production of nitric oxide by inflammatory leukocytes and ␤-cells has been implicated in the development of diabetes [27,28]. Nitric oxide reacts with diffusion-limited rate with superoxide radical to form the noxious peroxynitrite [29]. This reaction should be virtually unopposed by the low EC-SOD activity of the islet extracellular space, whereas better protection should be afforded the ␤-cell interior by CuZn-SOD. An increase in protein nitrotyrosine, a reaction product of peroxynitrite, has been seen in islets in diabetic NOD mice [30]. Alloxan is highly unstable and shows a half-life of only a few minutes under physiologic conditions [18]. The alloxan-induced hyperglycemia, however, develops over several days (Fig. 1). This is in accordance with histochemical studies that show degenerative changes in the ␤-cells developing over at least 40 h [31]. After diabetes induction all mice eventually regained a degree of glycemic control (Fig. 2). This reversibility of alloxan diabetes has previously been reported [32,33] and may be due to differentiation and proliferation of ␤-cell precursors or stem cells in pancreatic islets and ducts [32, 34,35]. There was a correlation between both the 96-h and 3-week glucose levels and remaining time to glycemic control (Fig. 2 A and 2B). Part of this effect should be caused by fewer remaining ␤-cells and possibly in parallel a larger alloxan-induced insult to cells with a proliferative potential in animals showing high glucose levels at those early times. However, there was a much larger glucose-related delay in EC-SOD null mice. If it is assumed that the glucose levels at 96 h and 3 weeks reflect the number and insulin-secreting capacity of ␤-cells of the mice and is independent of the prevalent concentration of superoxide radicals, the much larger glucose related delay in recovery in EC-SOD null mice suggests a negative effect of superoxide radicals in ␤-cell regeneration. High glucose concentrations are known to cause oxidant stress by a variety of mechanisms. Glucose itself [36] as well as the various primary and secondary adducts formed with protein amino groups [37] autooxidize under formation of superoxide radicals. Hyperglycemia causes increased tissue superoxide radical production [38]. The advanced glycation end products react with cell surface receptors resulting in oxidant stress including formation of superoxide radicals [39]. Overall, evidence for enhanced oxidant stress in hyperglycemia is abundant, and such stress is suggested to contribute to a variety of diabetic complications. The present findings suggest that enhanced glucose-induced production of superoxide radicals impairs ␤-cell regeneration, and that endogenous EC-SOD provides some protection. The finding may have relevance for maintenance and regeneration of ␤-cells in diabetes, further supporting the importance of glycemic control in the disease, and sug-

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gesting that therapy with EC-SOD or other antioxidants might be beneficial. Acknowledgements — The study was supported by the Swedish Medical Research Council, grant 12566. The skillful technical assistance of ¨ berg, E. Bern, and K Lejon is gratefully acknowledged. A. O

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SOD—superoxide dismutase EC-SOD— extracellular-SOD