A^-Nitro-L-Arginine Methyl Ester Reduces theIncidence of IDDM in BB/E RatsR. Mark Lindsay, William Smith, Sharon P. Rossiter, Margaret A. Mclntyre, Brent C. Williams,and Joyce D. Baird

Evidence that nitric oxide (NO) is involved in cytokine-mediated islet (J-cell dysfunction and destruction in vitrohas led to the hypothesis that increased production of NOmay contribute to the pathogenesis of insulin-dependentdiabetes mellitus (IDDM). This study demonstrates thatoral administration of A^-nitro-L-arginine methyl ester(an inhibitor of NO synthase) from 30 to 150 days of agesignificantly reduced (P < 0.05) the incidence of IDDM indiabetes-prone BB/E rats. This supports the idea that NOplays a significant role in the pathogenesis of IDDM inthis animal model. Diabetes 44:365-368, 1995

Current evidence suggests that the release of highly

toxic free radicals (either directly by infiltrating

macrophages or secondary to secretion of cyto-

kines, such as interleukin-1, from activated mac-

rophages) may be a critical event in the inflammatory

destruction of islet (3-cells that results in insulin-dependent

diabetes mellitus (IDDM) (1), and nitric oxide (NO) has been

proposed as the specific free-radical species that acts as the

primary effector molecule in cytokine-mediated p-cell dys-

function (decreased insulin secretion) and destruction in

vitro (2,3). A role for elevated NO production in the diabe-

togenic effect of streptozotocin (STZ) is indicated by the

partial prevention of low-dose STZ-induced diabetes in miceby in vivo administration of NO synthase inhibitors (4,5).Although development of diabetes in this animal model

involves an autoimmune component, the existence of other

mechanisms of diabetogenesis, such as direct release of NOfrom the nitroso moiety of STZ (6), complicate interpretationof these results. The aim of this study was to determine the

effect of oral administration of A^-nitro-L-arginine methyl

ester (L-NAME), an inhibitor of NO synthase, on the inci-

dence of spontaneous autoimmune IDDM in the Edinburgh,U.K., colony of BB rats (BB/E) (7,8).

RESEARCH DESIGN AND METHODSAnimals. A breeding colony of BB rats was established in Edinburgh in1982 from a small nucleus of animals (3 males and 4 females) donated by

From The Metabolic Unit, University of Edinburgh Department of Medicine, andthe Department of Pathology (M.A.M.), Western General Hospital, Edinburgh,U.K.

Address correspondence and reprint requests to Dr. R. Mark Lindsay, TheMetabolic Unit, University of Edinburgh Department of Medicine, Western GeneralHospital, Edinburgh EH4 2XU, U.K.

Received for publication 6 September 1994 and accepted in revised form 23November 1994.

BB/E, Biobreeding Edinburgh; DP, diabetes-prone; DR, diabetes-resistant;IDDM, insulin-dependent diabetes mellitus; L-NAME, A -̂nitro-L-arginine methylester; MAP, mean arterial pressure; NO, nitric oxide; STZ, streptozotocin.

Dr. P. Thibert from the original BB colony in Ottawa. The BB/E colonyconsists of two lines created by selectively breeding for and againstdiabetes, and these two lines have now been through 24 generations ofstrict brother-sister mating on site and have had their inbred statusconfirmed by skin grafting experiments. All animals are weighed twiceweekly from 40 days of age. If they fail to gain or lose weight, they aretested for glycosuria (Multistix SG reagent strips, Bayer Diagnostics,Basingstoke, U.K.). If glycosuria is detected, the blood glucose concen-tration is measured (Exactech blood glucose meter, Medisense Britain,Birmingham, U.K.) on a blood sample obtained by tail tipping withoutanesthesia. In the BB/E rat colony, a blood glucose concentration >18mmol/1 is invariably associated with ketonuria, weight loss, and the needfor daily injection of insulin (Bovine Ultratard U40, Novo Nordisk,Bagsvaerd, Denmark) to survive, and these parameters constitute ourcriterion for classifying an animal as having IDDM. In the high-incidencediabetes-prone (DP) main line, the incidence of IDDM is 50-60% and themean ± SD age at onset of diabetes is 96 ± 18 days. In the diabetes-resistant (DR) subline, the incidence of diabetes is <1%. All animals aremaintained at 20°C on 12-h light/dark cycles and fed Special DietServices (Witham, U.K.) rat and mouse Number 1 Expanded Feed.Experimental protocol. DP BB/E rats were randomized to an L-NAME-treated (n = 37) or an untreated (n = 36) group. These twogroups did not differ in mean ± SE of age (30 ± 0.5 vs. 30 ± 0.4 days,respectively), body weight (65 ± 2 vs. 63 ± 2 g), and sex (43 vs. 44%male). L-NAME (Sigma, Poole, U.K.) was dissolved daily in drinkingwater, and treated rats received a daily oral dose of 27 ± 1 mg/kg bodywt from 30 to 153 days of age. The bioavailability of L-NAME adminis-tered in this way was confirmed by measuring the mean arterial pressure(MAP) in all surviving animals at 153 days of age, that is, at the end ofthe experiment. MAP was measured directly in conscious animals via anindwelling carotid arterial cannula inserted 3 days previously underhalothane anesthesia. Immediately after determination of MAP, theserats were anesthetized with pentobarbital sodium (60 mg/kg i.p.), bloodsamples were collected from the inferior vena cava for measurement ofglycated hemoglobin (Glycotest II, Pierce and Warriner, Chester, U.K.)and plasma glucose concentration (Beckman Synchron CX3 multichan-nel analyzer, Beckman, High Wycombe, U.K.), and the pancreas andother organs were removed and fixed in 10% buffered formalin beforeroutine histological processing. Sections were stained with hematoxylinand eosin.

Calculation of diabetes incidence. The cumulative incidence ofdiabetes in the L-NAME-treated and untreated groups was calculated asthe percentage of diabetic rats among surviving animals at each timepoint.Statistical analysis. Statistical significance of differences betweenL-NAME-treated and untreated rats in the number of diabetic rats andthe incidence of diabetes was assessed using the Mantel-Haenszel(log-rank) test (9). Results in Table 2 are presented as means ± SE, anddifferences were assessed by unpaired Student's t test (populationvariances not assumed to be equal).

RESULTS AND DISCUSSIONL-NAME treatment significantly reduced (P < 0.05) both thenumber of DP BB/E rats developing IDDM (Fig. LA) and theincidence of diabetes (Fig. IB) (Mantel-Haenszel test statis-tic t = 4.513 and 4.275, respectively), but the mean age atonset of diabetes was not affected (102 ± 5 vs. 99 ± 7 days).Table 1 shows that the number of experimental deaths(namely, deaths occurring during or after either cannulation

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NITRIC OXIDE SYNTHESIS IN DIABETOGENESIS

Start of L-NAMEtreatment

End of L-NAMEtreatment

FIG. 1. Effect of treatment with L-NAME (daily oral dose = 27 ± 1mg/kg body wt) on the cumulative number (A ) and incidence (B ) ofIDDM in DP BB/E rats. The difference between untreated (A) andL-NAME-treated (D) rats is significant (P < 0.05) for both the numberof untreated rats and the incidence of IDDM using the Mantel-Haenszel(log-rank) test. Death of a nondiabetic L-NAME-treated rat is indicatedb y « .

or anesthesia between 150 and 153 days of age) wereidentical in the L-NAME-treated and untreated groups. Diffi-culty in regulating the diabetes also accounted for a similarnumber of deaths in diabetic animals in both groups. How-ever, the number of unexplained excess deaths in nondia-betic L-NAME-treated animals was striking. Figure IB showsthat these deaths occurred between 117 and 149 days of age(mean ± SE = 135 ± 3) and account for the different shapeof the curves from 130 days of age onward depicting thecumulative number (Fig. LA) and incidence (Fig. IB) of ratsdeveloping IDDM. Using our database for the age at onset ofIDDM in the Edinburgh colony, we calculated that fewerthan one (0.49 animal) of the seven nondiabetic L-NAME-treated rats who died after 130 days would be expected todevelop IDDM at this late age. Postmortem examination wasperformed blind in six of the nine unexplained deaths innondiabetic L-NAME-treated animals and six diabetic L-NAME-treated rats killed at the end of the experiment(M.A.McL). All animals in the former group showed severehypertensive vasculitis with fibrinoid necrosis of the arterialwall (Fig. 2), while none of the latter group showed any signof vasculitis. Severe hypertensive vasculitis was observed inall tissues examined (including small intestine, kidney,esophagus, and pancreas) from nondiabetic L-NAME-treatedrats and is consistent with the fact that these animals weresignificantly more hypertensive than diabetic L-NAME-treated rats (Table 2). Although sustained hypertension (dueto inhibition of the constitutive isoform of NO synthase)associated with chronic in vivo administration of L-NAME tonondiabetic rats has previously been reported (10), this is thefirst report to document histological evidence of severe

TABLE 1Animal numbers

Experimental deathsOther causes of deathAnimals surviving at

153 days of age

Untreated rats

Nondiabetic IDDM

3

12

15

15

L-NAME-Treated rats

Nondiabetic IDDM

39

12

14

8

At the beginning of the experiment, n = 36 (untreated) and 37(treated).

hypertensive vasculitis following long-term treatment (3-4months) with an inhibitor of NO synthase.

The attenuated hypertension observed in diabetic BB/Erats treated with L-NAME confirms the reported effects of NOsynthase inhibition in STZ-induced diabetic rats (11-13) andpatients with IDDM (14). These studies concluded that NOsynthesis or effects are impaired in established diabeticsubjects and may contribute to the pathogenesis of diabeticvascular complications. However, evidence for a role ofelevated NO production in diabetic vascular dysfunction inSTZ-induced diabetic rats (15,16), possibly linked to thepolyol pathway (13), is also available. It is possible that thelonger exposure to more severe hypertension of L-NAME-treated rats who did not develop diabetes (mean age at death= 135 ± 3 days) compared with treated rats developingIDDM at a mean age of 102 days also contributed to theirmore severe pathology and consequent death.

Although the mean plasma glucose concentrations ofnondiabetic rats were higher (Table 2) than we have previ-ously observed in conscious animals (17), the values aresimilar to those reported for anesthetized nondiabetic rats(18). We are confident that this represents transient anes-thetic-induced hyperglycemia because the values for gly-cated hemoglobin are similar to those we see in consciousDR BB/E rats (unpublished data).

Histological analysis of pancreatic biopsies obtained fromrats with IDDM in both L-NAME-treated and untreatedgroups showed a picture similar to that we have describedpreviously (19), in which virtually all the islets throughoutthe pancreas are of an end-stage type (that is, islets in whichthe central core of (3-cells has disappeared, leaving onlyclumps or rings of glucagon-containing cells, and only occa-sional inflammatory cells are seen). Pancreatic biopsies fromrats that did not develop IDDM showed a variable degree ofinflammatory infiltration in 25% of the animals in both theL-NAME-treated and untreated groups with no obvious dif-ference between them. However, it is possible that thecellular and/or cytokine profile of the infiltrate differs intreated and untreated animals, and further investigation isnecessary. Previous studies with STZ-induced diabetic micehave reported that insulitis was reduced (4) or unchanged(5) by NO synthase inhibition. The nature of the differencebetween treated animals that do or do not subsequentlydevelop IDDM is also unknown but, again, may be related todifferences in the components of the infiltrate at differentstages in the natural history of the disease process (19).

Inhibitors of NO synthase have previously been shown toprotect against both cytokine-induced inhibition of insulinsecretion (20-22) and the cytotoxicity of cytokines andactivated macrophages on pancreatic islet (3-cells in vitro

366 DIABETES, VOL. 44, MARCH 1995

R.M. LINDSAY AND ASSOCIATES

£ -«r'tfft .,5*.-.

FIG. 2. Section of small intestine from a nondiabetic L-NAME-treated rat stained with hematoxylin and eosin (magnification X80) showing athrombosed submucosal artery with fibrinoid necrosis and circumferential chronic inflammatory cell infiltrate.

(23,24). In vivo administration of LrNAME (4) or L-Mr-mono-methyl-arginine (5) reduces hyperglycemia in low-dose-STZ-induced diabetic mice. This is the first study to investigatethe effect of NO synthase inhibition on the development ofspontaneous autoimmune IDDM in an appropriate animalmodel. The significant decrease in both the number ofdiabetic rats and overall incidence of IDDM in DP BB/E ratstreated with L-NAME supports the hypothesis that elevatedNO production has a significant role in the development ofIDDM. Although the L-NAME treatment regimen used in thecurrent study clearly inhibits the constitutive isoform of NOsynthase, the extent of inhibition of inducible NO synthase isunknown. However, the recent demonstration of enhancedexpression of inducible NO synthase in the pancreas ofprediabetic BB rats (25) and the observation that NO syn-thase inhibition delayed development of diabetes in the

nonobese diabetic (NOD) mouse following adoptive transfer(26) suggest that elevated NO production has a significantrole in the development of diabetes in these autoimmuneanimal models of IDDM. Failure to completely suppress thedevelopment of IDDM in the BB/E rat by L-NAME treatmentmay be due to incomplete inhibition of inducible NO syn-thase or may indicate that development of diabetes involvesboth NO-sensitive and NO-insensitive phases as proposed forthe NOD mouse (26).

In conclusion, chronic administration of L-NAME duringthe prediabetic period significantly reduces the incidence ofIDDM in BB/E rats. To clarify the role of systemic hyperten-sion associated with L-NAME treatment, we are investigatingthe use of selective inhibitors of inducible NO synthase (theisoform most likely to produce elevated NO levels in pancre-atic p-cells) that do not cause systemic hypertension (15,16).

TABLE 2Characteristics of surviving nondiabetic and diabetic BB/E rats at the end of the experiment (i.e., 153 days of age)

Untreated rats L-NAME-Treated rats

MAP (mmHg)Plasma glucose concentration (mmol/1)Glycated hemoglobin (%)Body weight (g)Daily insulin dose (U)n

Nondiabetic

113 ± 210.1 ± 0.63.7 ± 0.1318 ± 24

—12

IDDM

119 ± 215.3 ± 1.5*6.1 ± 0.3t311 ± 142.7 ± 0.1

15

Nondiabetic

171 ± 6$10.2 ± 0.43.8 ± 0.2308 ± 15

—12

IDDM

140 ± 4tt15.2 ± 1.9*6.0 ± 0.4t305 ± 132.8 ± 0.2

8

Data are means ± SE. *P < 0.05, tP < 0.001, significantly different from nondiabetic rats. $P < 0.001, significantly different from untreatedgroup.

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NITRIC OXIDE SYNTHESIS IN DIABETOGENESIS

These studies should enable further definition of the role ofelevated NO production in the development of IDDM.

ACKNOWLEDGMENTWe are extremely grateful to the Cunningham Trust forsupporting the BB/E rat colony for the past 4 years.

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