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Endocrinology Vol. 141, No. 8 2752-2757
Copyright © 2000 by The Endocrine Society

ARTICLES

Islet Blood Flow in Multiple Low Dose Streptozotocin-Treated Wild-Type and Inducible Nitric Oxide Synthase-Deficient Mice1

Per-Ola Carlsson, Malin Flodström and Stellan Sandler

Department of Medical Cell Biology, Uppsala University (P.-O.C., M.F., S.S.), SE-751 23 Uppsala, Sweden; and Department of Immunology, The Scripps Research Institute (M.F.), La Jolla, California 92037

Address all correspondence and requests for reprints to: Per-Ola Carlsson, M.D., Ph.D., Department of Medical Cell Biology, Biomedical Center, Box 571, SE-751 23 Uppsala, Sweden. E-mail: per-ola.carlsson{at}medcellbiol.uu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study tested the hypothesis that changes in islet blood perfusion occur during the development of diabetes in the multiple low dose streptozotocin-treated mouse. Streptozotocin (40 mg/kg) or citrate buffer was given ip once daily for 5 consecutive days to wild-type and inducible nitric oxide synthase (iNOS)-deficient C57BL/6 x 129 SvEv hybrid mice. The blood flows were then determined by a microsphere technique. The islet blood perfusion was almost 2-fold higher in wild-type mice treated with streptozotocin than in those given vehicle. Whole pancreatic blood flow was also increased in the streptozotocin-treated wild-type mice. In iNOS-deficient mice, neither islet blood flow nor whole pancreatic blood flow was affected by repeated streptozotocin treatment. These combined findings suggest an increased islet blood perfusion in the prediabetic stage mediated by an iNOS-dependent mechanism. In combination with increased vasopermeability and expression of adhesion molecules on the islet endothelium, as previously described, this increased islet blood flow may be of crucial importance for the recruitment of inflammatory cells into the islets during the development of diabetes in this animal model. Indeed, an increased degree of insulitis was observed in wild-type mice compared with mice deficient in iNOS as well as a more rapid decrease in islet volume and an earlier debut of manifest diabetes. We also describe altered islet blood perfusion in the iNOS-deficient mice during basal conditions due to a compensatory increase in constitutive NOS activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 1 DIABETES is characterized by a selective destruction of the insulin-producing pancreatic ß-cells via an immune-mediated process (1, 2). An infiltration of inflammatory cells into the islets, i.e. insulitis, is generally recognized to precede the ß-cell destruction in both human type 1 diabetes subjects (3, 4, 5) and animal models of autoimmune diabetes, e.g. the nonobese diabetic (NOD) mouse (6, 7) and the multiple low dose streptozotocin (MLDS)-treated mouse (8, 9, 10). The exact mechanism for the subsequent ß-cell destruction remains controversial; it has been suggested that the infiltrating cells, either directly or through the release of inflammatory mediators such as cytokines and free radicals, are responsible for ß-cell damage and death (11, 12).

For inflammatory reactions, e.g. insulitis, to occur, the vascular endothelium is of crucial importance (13, 14). Surface receptors on endothelium and leukocytes are involved in the homing of mononuclear inflammatory cells to the islets (15, 16, 17, 18, 19). Moreover, changes in vascular permeability in islet blood vessels during the development of autoimmune diabetes have been described in several rodent models (20, 21, 22, 23, 24). An islet blood hyperperfusion may also augment homing to the pancreatic islets of inflammatory cells and soluble factors involved in ß-cell destruction during the development of autoimmune diabetes (13). We recently described a markedly increased islet blood perfusion in the prediabetic phase in the female NOD mouse (25). This islet blood flow increase could be identified as being due to enhanced production of the free radical nitric oxide (NO) by an inducible nitric oxide synthase (iNOS)-dependent mechanism (25).

Another frequently used animal model of autoimmune diabetes is the MLDS-treated mouse. An advantage of this model compared with diabetes-prone female NOD mice is that the study population is likely to be more homogenous, because the time of the initial ß-cell assault is given. Moreover, the recent introduction of a mouse deficient in iNOS (26, 27, 28) has provided an opportunity to selectively evaluate the role of NO produced by iNOS for any islet blood flow changes seen in this model of diabetes.

In the present study we aimed to determine whether similar changes in islet blood perfusion occur in the prediabetic stage of MLDS-treated mice as in diabetes-prone NOD mice. For this purpose, control and iNOS-deficient mice were subjected to MLDS treatment. Interestingly, differences in islet blood flow between vehicle-treated iNOS-deficient mice and controls seemed to exist. In separate experiments, we therefore also determined islet blood flow in nonpretreated animals and evaluated the effects of pharmacological NO inhibition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Wild-type (+/+) and iNOS-deficient (-/-) male C57BL/6 x 129SvEv hybrid mice, aged 9–15 weeks, were used in all experiments. Breeding pairs of mice deficient in iNOS were provided by J. S. Mudgett (Merck Research Laboratories, Rahway, NJ) and J. D. MacMicking and C. Nathan (Cornell University Medical College, New York, NY). The mice were generated by gene targeting in embryonic stem cells as previously described (26). Homozygous iNOS mutants were maintained by interbreeding the F2 generation. The colony has subsequently been barrier-bred under pathogen-free conditions in the Animal Department, Biomedical Center (Uppsala Sweden). Barrier-bred male C57BL/6 x 129SvEv wild-type mice were purchased from Taconic Farms, Inc. (Germantown, NY). All animals had free access to autoclaved tap water and pelleted food throughout the course of the study. The experiments were approved by the local animal ethics committee at Uppsala University and the Scripps Research Institute.

MLDS
Mice deficient in iNOS and wild-type mice were treated with ip injections of streptozotocin (40 mg/kg BW, dissolved in citrate buffer, pH 4.5) or vehicle (citrate buffer, pH 4.5) during 5 consecutive days (8). Day 1 was defined as the day of the first injection of streptozotocin. Blood glucose concentrations were measured in blood obtained from the tail vein on days 1, 3, and 5 using blood glucose reagent strips (Medisense, Baxter Travenol, Deerfield, IL). On day 5, some of the animals were allocated to blood flow measurements. Other animals were followed with repeated blood glucose measurements for up to 42 days after initiation of treatment.

Blood flow measurements and assessment of islet volume
The blood flow to the duodenum, ileum, colon, and pancreas and its islets was determined with a microsphere technique, as previously described and evaluated (29). Briefly, iNOS-deficient and wild-type mice were anesthetized with an ip injection of 0.02 ml/g BW Avertin [a 2.5% (vol/vol) solution of 10 g 97% (vol/vol) 2.2.2-tribromoethanol (Sigma, St. Louis, MO) in 10 ml 2-methyl-2-butanol (Kemila AB, Stockholm, Sweden)], heparinized, and placed on an operating table maintained at body temperature (38 C). Polyethylene catheters were inserted into the ascending aorta via the right carotid artery and into the right femoral artery. The former catheter was connected to a pressure transducer (PDCR 75, Druck, Groby, UK) to allow continuous monitoring of the mean arterial blood pressure, which was allowed to stabilize for 10–15 min. Nonpretreated animals (see above) were then injected iv with 0.25 ml saline or Nw-nitro-L-arginine (NNA; 25 mg/kg) dissolved in saline. Approximately 9 x 104 nonradioactive microspheres (NEN Life Science Products-DuPont Pharmaceuticals, Wilmington, DE), with a diameter of 11 µm, were injected 15 min later during 10 sec via the aortic catheter. Starting 5 sec before the microsphere injection and continuing for a total of 60 sec, an arterial blood reference sample was collected by free flow from the femoral catheter at a rate of approximately 0.10 ml/min. The exact withdrawal rate in each experiment was confirmed by weighing the sample.

Arterial blood was then collected from the femoral catheter for determination of blood glucose concentrations with test reagent strips (Medisense) and for serum insulin determinations with an enzyme-linked immunosorbent assay (Insulin ELISA Kit, Mercodia, Uppsala, Sweden), using a rat insulin standard (Novo Research Institute, Bagsvaerd, Denmark).

The animals were killed, and the whole pancreas, the adrenal glands, and parts of the duodenum (proximal part), ileum (distal part), and colon (descending part) were dissected free from fat and lymph nodes, blotted, weighed, and placed between object slides. Before placement between object slides, each pancreas was cut into 20–24 pieces. The islets were visualized by a freeze-thawing technique (30), and the islet volume percentage was determined by a point-counting method (29, 31). For this purpose, the number of intersections overlapping islets was counted at a magnification of x400 in a stereo microscope equipped with both dark- and brightfield illuminations (Wild M3Z, Wild Heerbrugg Ltd., Heerbrugg, Switzerland). Approximately 20–24 different fields were counted in each mouse pancreas (corresponding to 2400–2900 points).

The total number of microspheres in the exocrine and endocrine parts of the pancreas, intestines, and adrenal glands was then estimated with the aid of a stereo microscope (29). The number of microspheres in the arterial reference sample was determined by transferring the blood to glass microfiber filters with a pore size of less than 0.2 µm (Whatman, London, UK), and counting the microspheres in a microscope equipped with transmitted light. All microsphere counting and evaluations of islet volume percentage were performed by an observer unaware of the origin of the samples.

The blood flow values were calculated according to the formula Qorg = Qref x Norg/Nref, where Qorg is organ blood flow (milliliters per min), Qref is the withdrawal rate of the reference sample (milliliters per min), Norg is number of microspheres present in the organ, and Nref is the number of microspheres present in the reference sample. The microsphere contents of the adrenal glands were used to confirm that the microspheres had been adequately mixed in the arterial circulation. A less than 10% difference in numbers of microspheres between the right and left adrenal glands was taken to indicate sufficient mixing. When the islet blood flow was expressed per islet weight, the latter was estimated by multiplying pancreatic weight with the islet volume fraction of the whole pancreas in each animal (25).

Estimation of degree of insulitis
Separate iNOS-deficient and wild-type animals treated with multiple injections of streptozotocin (see above) or vehicle were used for histological evaluation of the degree of insulitis. The pancreatic glands were removed, fixed in a 10% formalin solution, and embedded in paraffin. Sections, 5 µm thick, were cut and stained with hematoxylin and eosin. Pancreatic islet histology was ranked according to an arbitrary scale, as illustrated previously (32). Rank A denotes normal islet structure; rank B denotes mononuclear cell infiltration in the periinsular area; rank C denotes heavy mononuclear cell infiltration into a majority of islets; rank D denotes the presence of only a few residual islets, showing an altered cellular architecture and pyknotic cell nuclei. The pancreatic sections were evaluated by an observer unaware of the origin of the sections.

Statistical analysis
Values are expressed as the mean ± SEM. Multiple comparisons between data were performed using ANOVA (StatView, Abacus Concepts, Berkeley, CA), with correction of P values using the Bonferroni method (33). When only two groups were compared, probabilities of differences were calculated using Student’s unpaired t test. For all comparisons, P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MLDS regimen
Wild-type mice treated with MLDS gradually developed hyperglycemia (Fig. 1Go). On day 21, eight of nine (89%) had blood glucose levels above 11.1 mmol/liter. By comparison, only two of eight (25%) MLDS-treated iNOS-deficient mice were diabetic (blood glucose, >11.1 mmol/liter) at this time (Fig. 1Go). Also on days 28, 35, and 42, mice deficient in iNOS had markedly lower blood glucose than wild-type mice after MLDS treatment (Fig. 1Go). Vehicle-treated iNOS-deficient and wild-type mice showed no increase in blood glucose throughout the course of the study (Fig. 1Go).



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  		Figure 1. Blood glucose concentrations in wild-type (filled symbols) and iNOS-deficient (open symbols) male 129SvEv x C57BL/6 hybrid mice given streptozotocin (STZ; 40 mg/kg BW; circles) or vehicle (citrate buffer; boxes) ip for 5 days. Blood glucose levels were monitored during a 42-day period after the first injection. All values are given as the mean ± SEM for five to nine animals. *, P < 0.05 compared with the corresponding iNOS-deficient mice. All comparisons were made using ANOVA, with corrections of P values using the Bonferroni method.

 
Histological examination of pancreas from MLDS-treated animals 10 days after treatment initiation showed essentially normal islet morphology, although inflammatory changes were seen in some of the wild-type mice (Table 1Go). Fourteen and 21 days after treatment initiation, islet inflammatory changes were observed preferentially in the wild-type MLDS-treated mice and to a lesser extent in the corresponding iNOS-deficient mice (Table 1Go). All of the citrate buffer-treated animals (wild-type or iNOS-deficient) examined 10, 14, or 21 days after initiation of treatment had normal islet morphology (data not shown).


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  		Table 1. Pancreatic islet morphology of iNOS-deficient (-/-) and control (+/+) male 129 SvEv x C57BL/6 hybrid mice 10, 14, or 21 days after initiation of multiple low dose streptozotocin (MLDS) treatment (40 mg/kg BW) for 5 days

 
Blood flow measurements were performed on day 5 after initiation of MLDS treatment. At this time, both mice deficient in iNOS and wild-type mice remained normoglycemic (data not shown). A slight decrease in islet volume was seen in the wild-type mice, but not in the iNOS-deficient mice, subjected to MLDS treatment compared with that in citrate buffer-treated mice (Table 2Go). Likewise, a tendency for a decreased serum insulin concentration was seen in wild-type mice subjected to MLDS-treatment compared with citrate buffer-treated mice [0.32 ± 0.03 (MLDS; n = 10) vs. 0.65 ± 0.15 ng/ml (citrate; n = 11); P = 0.057]. In iNOS-deficient mice there were no differences in serum insulin concentration between the two groups [0.81 ± 0.22 (MLDS; n = 9) vs. 0.57 ± 0.17 ng/ml (citrate; n = 10); P = 0.374]. Mean arterial blood pressure was approximately 90 mm Hg in all of the animals subjected to blood flow measurements (Table 2Go).


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  		Table 2. Mean arterial blood pressure, whole pancreatic blood flow (PBF), islet blood flow (IBF), and islet volume in iNOS-deficient (-/-) and control (+/+) male 129 SvEv x C57BL/6 hybrid mice after administration of streptozotocin (STZ; 40 mg/kg BW) or vehicle (citrate buffer) ip for 5 days

 
MLDS treatment increased both whole pancreatic blood flow and islet blood flow (when expressed per islet weight) in wild-type mice (Table 2Go). However, due to the decreased islet volume in MLDS-treated animals, the blood flow to the whole islet organ (per g pancreas) remained unaffected (Table 2Go). In iNOS-deficient mice, MLDS treatment had no effect on either whole pancreatic or islet blood flow (Table 2Go). Duodenal and ileal blood flows were not affected by MLDS treatment in either wild-type or iNOS-deficient mice (Table 3Go). Colonic blood flow was increased by MLDS treatment in wild-type, but not in iNOS-deficient, mice (Table 3Go).


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  		Table 3. Duodenal, ileal, and colonic blood flow in iNOS-deficient (-/-) and control (+/+) male 129 SvEv x C57BL/6 hybrid mice after administration of streptozotocin (STZ; 40 mg/kg BW) or vehicle (citrate buffer) ip for 5 days

 
Pharmacological NOS inhibition
Blood glucose and serum insulin concentrations were similar in both iNOS-deficient and control mice and were not affected by iv administration of the NOS inhibitor NNA (data not shown). Moreover, mean arterial blood pressure did not differ between mice deficient in INOS and wild-type mice (Table 4Go). Fifteen minutes after the administration of NNA, mean arterial blood pressure had increased by 30–40% in all animals compared with pretreatment levels (Table 4Go).


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  		Table 4. Mean arterial blood pressure, whole pancreatic blood flow (PBF), islet blood flow (IBF), and islet volume in iNOS-deficient (-/-) and control (+/+) male 129 SvEv x C57BL/6 hybrid mice 15 min after iv administration of saline or N-{varpi}-nitro-L-arginine (NNA; 25 mg/kg)

 
During basal conditions, mice deficient in iNOS had a selective increase in islet blood flow compared with control mice (Table 4Go). By comparison, no differences were seen in whole pancreatic (Table 4Go), duodenal, ileal, or colonic (Table 5Go) blood flow. Administration of NNA markedly decreased islet blood flow in both iNOS-deficient and wild-type mice (Table 4Go). After NNA treatment, there were no differences in islet blood perfusion between iNOS-deficient and control mice (Table 4Go). NNA also decreased whole pancreatic blood flow in the iNOS-deficient mice (Table 4Go). However, this decline was relatively less than the decrease in islet blood flow, as reflected in a decreased fraction of islet blood flow of the whole pancreatic blood flow (Table 4Go). Duodenal, ileal, and colonic blood flows were not affected by NNA treatment in either strain (Table 5Go).


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  		Table 5. Duodenal, ileal, and colonic blood flow in iNOS-deficient (-/-) and control (+/+) male 129 SvEv x C57BL/6 hybrid mice 15 min after iv administration of saline or Nw-nitro-L-arginine (NNA; 25 mg/kg)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we used MLDS treatment as a model of type 1 diabetes. This animal model is characterized by an initial ß-cell assault, triggering an infiltration of mononuclear cells and subsequent ß-cell destruction (9, 10). The autoimmune reaction appears similar to what is seen in human subjects during the development of autoimmune diabetes (3, 4, 5). The protocol used for low dose streptozotocin treatment was previously evaluated in the same strain of mice as that used in the present study and was found to cause ß-cell destruction and diabetes in more than 80% of treated mice within 14 days of treatment initiation (34). This was also confirmed in the present study. In this study the majority of animals were, however, allocated to blood flow measurements on day 5 after treatment initiation. An increase in islet blood perfusion, as previously seen in diabetes-prone NOD mice, was presently observed in MLDS-treated mice. It should be noted that all animals remained normoglycemic; thus, they were studied in the prediabetic phase. Experiments performed in mice after the development of manifest diabetes could not be performed due to the marked decrease in islet mass present in these latter animals. In contrast to the study in NOD mice (25), a concomitant increase in whole pancreatic blood flow was seen in the MLDS-treated animals. To investigate whether the increased whole pancreatic and islet blood perfusion was due to a mechanism similar to that seen in the diabetic-prone NOD mice, i.e. increased NO production by iNOS, experiments were conducted in mice deficient in iNOS. Interestingly, in the iNOS-deficient mice, no changes in blood perfusion of either the whole pancreas or the islets were recorded. It seems likely therefore that increased NO production by iNOS is involved in the increased islet blood flow in the MLDS model of type 1 diabetes.

It should be noted that the present findings are in contrast to several previous morphological studies of the islet vasculature, which have suggested that islet blood perfusion may be decreased due to vasoconstriction or destruction of islet capillaries during the development of autoimmune diabetes (35, 36, 37, 38, 39). This discrepancy may be explained by the finding that the number of blood microvessels within an organ does not necessarily reflect the blood perfusion of that organ, as the flow in single capillaries varies considerably. Thus, even if a reduction in the capillary area occurs, this may be compensated for by an increased flow in the remaining capillaries.

The significance of islet blood hyperperfusion for the development of autoimmune diabetes is unknown. However, acute vasodilation is known to decrease shear stress and cause margination of leukocytes, which may enhance the accumulation of inflammatory cells (13, 40). Moreover, the acute islet vasodilation may contribute together with signals from inflammatory cells to an increased expression of endothelial surface molecules, e.g. integrins, which are necessary for the adhesion of leukocytes to the endothelium and the subsequent transmigration into tissues (13, 14). Unfortunately, the degree of islet infiltration, i.e. insulitis, in the freeze-thawed pancreas preparations from wild-type and iNOS-deficient mice was impossible to determine with accuracy. However, in paraffin-embedded pancreas preparations from MLDS-treated normal and iNOS-deficient mice, a markedly decreased insulitis were previously observed on day 21 after initiation of MLDS treatment (34) in iNOS-deficient animals. This was accompanied by a reduced sensitivity of the iNOS-deficient mice to develop manifest diabetes (34). In the present study in which histological examination was performed at 10, 14, as well as 21 days after initiation of MLDS treatment, islet inflammatory changes also seemed more pronounced in wild-type MLDS-treated animals than in the corresponding iNOS-deficient mice. Moreover, on day 5 after initiation of MLDS treatment, a decrease in islet volume was seen in wild-type, but not in iNOS-deficient, mice compared with that in citrate buffer-treated controls.

After streptozotocin treatment, increased colonic blood flow was also observed in wild-type mice. By comparison, in iNOS-deficient mice colonic blood flow was unaffected, which suggests this to be mediated by an iNOS-dependent mechanism. The significance of this finding is unclear.

When performing experiments on mice deficient in iNOS, the citrate buffer-treated controls had increased islet blood flow compared with citrate buffer-treated wild-type mice. We therefore performed separate experiments in nonpretreated wild-type and iNOS-deficient mice. Indeed, the mice deficient in iNOS had a markedly higher islet blood flow, whereas whole pancreatic, duodenal, ileal, and colonic blood flow did not differ from that in controls. Inhibition of NO production with NNA caused a marked decrease in islet blood flow in both mice deficient in iNOS and wild-type controls. This once again underlines the exquisite sensitivity of the islet blood perfusion to NO as previously described in rats (41) and mice (25, 42). After treatment with the nonselective NOS inhibitor NNA, no differences were seen in islet blood flow between the two groups of mice. Taken together, this suggests a selective compensatory increased cNOS activity in the islets of the iNOS-deficient mice. In contrast, as judged from the blood flow measurements, the exocrine parts of the pancreas as well as the duodenum, ileum, and colon seemed not to have a similarly increased cNOS activity. It may therefore be speculated that the production of NO by iNOS is important for normal islet function. A similar phenomenon of a compensatory increase in other biological systems to maintain an important physiological homeostasis has been seen repeatedly in different knockout mice (27, 43). This study, therefore, underlines the caution that must be taken when interpreting results obtained in knockout models.

In this context, it should be noted that a chronically increased tissue blood perfusion per se is not connected with an increased capacity for inflammatory cells to migrate into the tissue (13, 14). In contrast, in the absence of other inflammatory tissue reactions, e.g. increased vasopermeability and expression of endothelial adhesion molecules, a paradoxically increased shear stress and centripetal moving of blood cells occur (13, 40). Thus, the higher islet blood flow seen in the iNOS-deficient mice may actually protect from leukocyte infiltration into the islets. This given, that also the increased vasopermeability and/or the enhanced expression of endothelial adhesion molecules, previously described in the islets during the development of type 1 diabetes (15, 16, 17, 18, 19, 20, 21, 22, 23, 24), are affected by iNOS deficiency. Interestingly, in several other organs an enhanced microvascular leakage has repeatedly been described after induction of iNOS (44, 45, 46), whereas there are conflicting results concerning the influence of increased iNOS activation on integrin molecule levels; both an increased and a decreased expression of vascular adhesion molecules have been described after iNOS activation (47, 48). It remains to be determined whether the increased vasopermeability and/or expression of vascular integrins in the islets seen during development of autoimmune diabetes are iNOS dependent.

In conclusion, the present study describes an increased islet blood perfusion in the prediabetic phase of MLDS-treated mice. Similar results have previously been obtained in diabetes-prone NOD mice (25). In both animal models, there is strong evidence to suggest that this increase in islet blood flow is due to excessive production of NO caused by increased iNOS activity. This increased islet blood flow may augment homing to the pancreatic islets of inflammatory cells and thus accelerate the development of autoimmune diabetes. Furthermore, we also describe an altered islet blood perfusion in nontreated iNOS-deficient mice. This finding once again underlines the tendency for compensatory processes in knockout models.


   Acknowledgments
 
The technical assistance of Ms. Astrid Nordin, Eva Törnelius, and Dr. Björn Tyrberg is gratefully acknowledged.


   Footnotes
 
1 This work was supported by grants from the Swedish Medical Research Council (17X-109 and 17X-8273), the Swedish-American Diabetes Research Program funded by the Juvenile Diabetes Foundation and the Wallenberg Foundation, the Swedish Diabetes Association, the NOVO Nordic Fund, the Family Ernfors Fund, the American National Multiple Sclerosis Society, the Swedish Society of Medicine, Harald and Greta Jeanssons Stiftelse, and Svenska barndiabetesfonden. Back

Received December 27, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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