Departments of 1 Pharmacological Research I, 2 Assay and Cell Technology, and 3 Histology, Novo Nordisk, DK-2880 Bagsvaerd, Denmark; and 4 Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, DK-1870 Copenhagen, Denmark
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ABSTRACT |
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Nonrodent models of diabetes are needed
for practical and physiological reasons. Induction of mild
insulin-deficient diabetes was investigated in male Göttingen
minipigs by use of streptozotocin (STZ) alone (75, 100, and 125 mg/kg)
or 125 mg/kg combined with pretreatment with nicotinamide (NIA; 0, 20, 67, 100, 150, and 230 mg/kg). Use of NIA resulted in a less steep slope
of the regression line between fasting plasma glucose and changing
doses compared with STZ [7.0 ± 1.4 vs. 29.7 ± 7.0 mM · mg
1 · kg
1,
P < 0.0001]. Intermediate NIA doses induced moderate
changes of glucose tolerance [glucose area under the curve increased
from 940 ± 175 to 1,598 ± 462 mM · min,
P < 0.001 (100 mg/kg) and from 890 ± 109 to
1,669 ± 691 mM · min, P = 0.003 (67 mg/kg)] with reduced insulin secretion [1,248 ± 602 pM · min after 16 days and 1,566 ± 190 pM · min
after 60 days vs. 3,251 ± 804 pM · min in normal animals
(P < 0.001)] and
-cell mass [5.5 ± 1.4 mg/kg after 27 days and 7.9 ± 4.1 mg/kg after 60 days vs.
17.7 ± 4.7 mg/kg in normal animals (P = 0.009)].
The combination of NIA and STZ provided a model characterized by
fasting and especially postprandial hyperglycemia and reduced, but
maintained, insulin secretion and
-cell mass. This model holds
promise as an important tool for studying the pathophysiology of
diabetes and development of new pharmacological agents for treatment of
the disease.
in vivo pharmacology; large-animal model; glucose tolerance; -cell reduction; glucose-stimulated insulin secretion.
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INTRODUCTION |
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THE STUDY OF THE PATHOPHYSIOLOGY and treatment of diabetes requires well characterized animal models that resemble aspects of the disease in humans. Various forms of diabetes occur spontaneously or can be induced in several species of animals. Most of the available models are based on rodents; however, nonrodent models of diabetes are urgently needed as a valuable supplement to rodents for both practical and physiological reasons.
The pig is useful as a model for human physiology and pathophysiology, because many organ systems resemble those of the human. Of special interest for the study of diabetes is the similarities to humans found in the clinical chemistry (7, 10, 12, 14, 24, 26, 55), nutrition and gastrointestinal tract (4, 8, 11, 20, 35, 40, 51), pancreas development and morphology (21, 36, 37, 44, 49, 54), and metabolism (3, 35). These characteristics make swine an interesting species for studies of metabolic abnormalities in diabetes. The Göttingen minipig is especially suitable for long-term studies because of its small size and ease of handling, even at full maturity (6).
Pancreatectomy has been investigated as a method of inducing diabetes
in pigs (33, 34, 50, 55). However, high rates of mortality
have been observed postoperatively (50, 55), meaning that
this technique should be used with great caution, and alternatives
should be considered because of welfare considerations. Chemical
induction of diabetes offers the advantage of preservation of both
exocrine and endocrine cell populations other than -cells, thus
resembling the situation in human diabetes (55). Several stable models have been established for overt type 1 diabetes in the
pig by the use of pharmacological induction of
-cell damage with
streptozotocin (STZ), either as single or repeated injections (2,
15, 16, 27-29, 46, 55). Substantially increased fasting
plasma glucose (FPG) levels and decreased insulin secretion in response
to glucose stimuli have been obtained as well as increases in plasma
triglycerides and total cholesterol (27, 29). Late complications typical of diabetes, such as capillary basement membrane
thickening and cataracts, have also been shown in diabetic minipigs
(28, 41).
In other studies, alloxan has been used for induction of diabetes in
pigs (11, 25, 41). This compound, which has -cell-toxic properties similar to those of STZ, in a dose of 200 mg/kg in Yucatan
minipigs induced severe diabetes with high mortality due to
hypoglycemia following acute hyperinsulinemia as a consequence of
massive
-cell damage (41). Doses of 80 mg/kg have been
reported to induce mild diabetes with moderate hyperglycemia and
partial loss of
-cell mass with impaired insulin secretion rates but normal fasting insulin levels in Göttingen minipigs
(25). Due to its greater selectivity toward
-cells, its
wider range between doses causing mild and severe changes in glucose
tolerance compared with alloxan (22, 23), and the more
extensive background literature on the effect of STZ in pigs, this
compound was chosen for reduction of
-cell mass in the present study.
Despite the widespread use of STZ, its use results in a wide variability in the extent of diabetes depending on species, strain, age, and laboratory, thus limiting the predictability of its effects. Furthermore, the efficacy of STZ varies even in an apparently uniform group of animals receiving the same dose of the compound (13, 47).
In the present study, it was therefore investigated whether the use of
nicotinamide (NIA) would have protective effects against the
diabetogenic action of STZ in the Göttingen minipig, as
previously reported in rats (31). The protective effect of
NIA against the effect of STZ has been shown in vivo to be both dose
and pretreatment time dependent, but even the most effective protective
dose of NIA did not completely prevent the diabetogenic effects of STZ (23, 30). Thus a combination of these two compounds might be useful in the establishment of a nonrodent model of mild
insulin-deficient diabetes, and in the present study, the dose-response
relations on glucose metabolism using two different approaches of
pharmacological induction of abnormalities in glucose tolerance are
investigated in adult male Göttingen minipigs. The first approach
was administration of a dose range of STZ (75, 100, and 125 mg/kg)
alone, the second being administration of different doses of NIA
(0-230 mg/kg) as a pretreatment in combination with STZ at a fixed
high dose (125 mg/kg). The aim of the study was to obtain a reliable
method of induction of impaired glucose tolerance and/or mild
insulin-deficient diabetes in the adult Göttingen minipig,
characterized by reduced -cell mass and disturbed residual insulin
secretion leading to a decreased ability to dispose of glucose and a
following, modest hyperglycemia during an oral glucose tolerance test (OGTT).
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MATERIALS AND METHODS |
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Animals
Adult male Göttingen minipigs 11-14 mo of age were obtained from the barrier unit at Ellegaard Göttingen Minipigs ApS, Dalmose, Denmark. Animals were housed in single pens under controlled conditions (temperature was kept between 18 and 22°C, relative air humidity was 30-70% with 4 air changes/h) with a 12:12-h light-dark cycle and fed twice daily: 140 g of SDS minipig diet (SDS, Essex, UK) and 240 g of a commercial swine fodder ("Svinefoder 22," Slangerup, Denmark) and allowed free access to water. The pigs were studied at least 2 wk after surgery and were trained carefully for all experimental procedures before the start of experiments.For the dosing studies with STZ alone, 14 animals weighing 18 ± 3 kg (range 14-23 kg) were used. For the dosing studies using NIA and STZ in combination, 38 animals weighing 22.5 ± 3.25 kg (range 16.9 to 28.9 kg) were used. These animals served as their own control and were studied both before and after induction of diabetes.
Principles of laboratory animal care were followed, and the type of study was approved by the Animal Experiments Inspectorate, Ministry of Justice, Denmark.
Surgical Implantation of Central Venous Catheters
Two central venous catheters (Certo 455, B. Braun Melsungen, Melsungen, Germany) were surgically inserted under general anesthesia induced with a combination of 0.83 mg/kg zolazepam, 0.83 mg/kg tiletamine (Zoletil 50 vet., Boehringer Ingelheim, Copenhagen, Denmark), 0.90 mg/kg xylazine [Rompun vet. (20 mg/ml), Bayer, Lyngby, Denmark], 0.83 mg/kg ketamine [Ketaminol vet. (100 mg/ml), Rosco, Taastrup, Denmark], and 0.20 mg/kg methadone [Metadon "DAK" (10 mg/ml), Nycomed, Roskilde, Denmark] and maintained with isoflurane (1-3%) (Forene, Abbot, Gentofte, Denmark) in 100% oxygen. Postsurgical analgesia was maintained by intramuscular injection of 0.03 mg/kg buprenorfine [Anorfin (0.3 mg/ml), GEA, Frederiksberg, Denmark] and 4 mg/kg carprofen [Rimadyl vet. (50 mg/ml), Pfizer, Ballerup, Denmark] before the end of anesthesia and for 3 days after surgery by intramuscular injection of 4 mg/kg carporfen once daily. Postsurgical infection was prevented by injection of dihydrostreptomycin sulfate (25 mg/kg) and benzylpenicillinprocain (20,000 IU/kg) [Streptocillin. vet. (250 mg + 200,000 IU benzylpenicillinprocain/ml), Boehringer Ingelheim] immediately after surgery and once daily for the following 2 days. All animals were allowed 2-3 wk of recovery after the surgical procedure and had normal behavior and eating patterns at the start of the study period.Protocol 1: Mixed-Meal OGTT
The mixed-meal OGTT was performed in all animals in the NIA and STZ combination study 1 wk before and 1 wk after exposure to NIA and STZ. The test was performed in nonrestrained, freely moving animals in their usual pens to reduce the amount of stress experienced by the animals during testing.After an 18-h overnight fast, animals were offered a mixed-meal OGTT of 25 g of SDS minipig fodder and 2 g/kg glucose (500 g/l, SAD, Copenhagen, Denmark). The meal was eaten from a bowl, rapidly and without stress, under supervision.
Blood samples were obtained from the jugular vein catheters at
t = 15,
5, 0, 15, 30, 45, 60, 90, 120, 150, and 180 min relative to the fodder and glucose load.
Protocol 2: Intravenous Glucose and Arginine Challenge
This test was performed 2 wk (16 ± 2 days; n = 6) or 2 mo (60 ± 0 days; n = 2) after dosing with NIA plus STZ in the 67 mg/kg NIA group and a control group of normal, age-matched, animals (n = 14) to evaluate insulin-secretory capacity. The test was performed in nonrestrained, freely moving animals to reduce the amount of stress experienced by the animals during testing.After an 18-h overnight fast, animals were dosed with an intravenous
bolus of glucose [500 g/l (0.3 mg/kg), SAD] at t = 0, and blood samples were obtained at t = 15,
10,
5,
1, 3, 5, 7, 10, 45, 50, 55, 61, 63, 65, 67, 70, 80, 85, 91, 93, 95, 97, and 100 min relative to the glucose load. At t = 60 min, another bolus of glucose [500 g/l (0.6 g/kg), SAD] was dosed
intravenously, and from t = 61 to t = 100 min, glucose was infused intravenously at 2 g · kg
1 · h
1 (200 g/l,
SAD). At t = 90 min, arginine (L-arginine,
Merck, art. 1542) (67 mg/kg) dissolved in sterile saline (0.9%, SAD)
was given intravenously. The insulin response to glucose and arginine
was calculated as the area under the curve (AUC; baseline subtracted) during 10 min immediately after dosing.
Protocol 3: Examination of STZ-Dosed Animals
The examination of animals dosed with STZ alone was limited to measurement of fasting plasma values of glucose, insulin, and glucagon.Handling and Analysis of Blood Samples
Blood samples (2 ml of whole blood) were immediately transferred to vials containing EDTA (1.6 mg/ml final concentration) and aprotinin [500 kallikrein inhibitor units (KIU)/ml full blood (Trasylol, 10,000 KIU/ml, Bayer)] and kept on ice until centrifugation. Samples were centrifuged (4°C, 10 min, 3,500 rpm), and plasma was separated and stored atInduction of Diabetes
Diabetes was induced by intravenous administration of STZ (Sigma S-0130) through the indwelling catheters over 2 min, either at variable doses alone [75 (n = 4), 100 (n = 3), or 125 (n = 7) mg/kg] or at a fixed dose of 125 mg/kg in combination with NIA to accomplish a partial protection of theVomiting was seen in all animals during the first hours after
administration of STZ, and this seemed to be unaffected by NIA pretreatment. Most animals were eating and behaving normally 24-48 h after dosing. Animals were offered SDS fodder 2 h after
administration of NIA and STZ and were observed frequently during the
first 2 days after administration of NIA and STZ. Blood glucose was
monitored regularly to avoid episodes of hypoglycemia due to sudden
hyperinsulinemia caused by necrosis of -cells.
After 2 days, insulin therapy (Insulatard, 100 IU/ml, Novo Nordisk, Bagsvaerd, Denmark) was initiated if necessary, on the basis of individual clinical examination, with the aim of keeping fasting plasma glucose (FPG) below 10 mM.
Histological Examination of Pancreas
Fixation and physical fractionation. Histological examination was performed 1 mo (27 ± 8 days; n = 7) or 2 mo (60 ± 0 days; n = 4) after dosing with NIA (67 mg/kg) and STZ and compared with data from normal animals (n = 5). Furthermore, an animal that received STZ alone was included for comparison. At the end of the study period, after euthanasia with pentobarbitone [20 ml/animal (200 mg/ml); Pharmacy of the Royal Veterinary and Agricultural University, Copenhagen, Denmark], the pancreas was immediately isolated in toto and fixed in paraformaldehyde (Bie & Berntsen) for 24 h. The following day, the pancreas was embedded in 3% agar solution (Meco-Benzon, cat. no. 303289, Copenhagen, Denmark) at 45°C. After cooling, the pancreas was cut into 3-mm slices (17), and every fifth tissue slice, starting at slice 1, 2, 3, 4, or 5, determined from a table of random numbers, was retained for sectioning into ~80 cubes of roughly equal size. Those cubes were arranged according to size, as practiced in the smooth fractionator method, with the largest cubes in the middle and the smallest cubes on the ends (5, 32). Every eighth cube, starting at cube 1, 2, 3 ... 8, determined from a table of random numbers, was retained and placed in cassettes for dehydration and paraffin infiltration in a tissue processor (Leica TP 1050, Copenhagen, Denmark). The 10-12 pancreas cubes were embedded in paraffin, and sections 3 µm thick were cut on a Leica RM 2165 microtome.
Immunohistochemistry. Sections were deparaffinized in xylen, brought to 99% ethanol, treated with 0.5% H2O2 for 20 min to block endogenous peroxidase activity, and rinsed with Tris-buffered saline (TBS). Sections were then immersed in 0.01 M citrate buffer, pH 6, preheated to 90°C, and submitted to antigen retrieval by microwave oven treatment for 3 × 5 min of heating at 80% (Polar Patent, Umeå, Sweden). The slides were subsequently cooled, still in the citrate buffer, by immersion of the jar in running tap water and rinsed in TBS + 0.01% Triton X-100 (TBS-T; Sigma, St. Louis, MO), and the tissue sections were "ringed" with a DAKO-pen (DAKO, Copenhagen, Denmark).
INSULIN STAINING. The following staining steps were carried out in an Autostainer (DAKO). All dilutions were carried out with TBS-T. Sections were blocked with 5% normal rabbit serum (X0902; DAKO) and then incubated for 60 min with primary antibody guinea pig anti-insulin (651041, ICN, Costa Mesa, CA) diluted 1:3,500 in 7% normal rabbit serum + 3% normal swine serum (X901; DAKO) in TBS-T. Sections were rinsed in TBS-T and incubated for 30 min in secondary antibody peroxidase-labeled rabbit anti-guinea pig IgG (P141; DAKO) diluted 1:300 in 7% rabbit + 3% swine serum in TBS-T. The sections were rinsed with TBS-T and then developed with 0.075% diaminobenzidine (DAB; DAKO) and 0.008% H2O2 in TBS-T for 3 min. After a rinse in TBS-T, the slides were washed for 5 min in running tap water, counterstained with Mayer's hematoxylin for 0.5 min, washed again in running tap water for 10 min, dehydrated, and mounted in Pertex (Histolab, Stockholm, Sweden). NON-Stereological estimation of - and non-
-cell mass.
Non-
- and
-endocrine cell mass was evaluated stereologically in
two to three sections 250 µm apart in an Olympus BX-50 microscope (Olympus, Copenhagen, Denmark) with video camera and monitor at a total
on-screen magnification of ×960. The sections were analyzed by point
counting of frames after systematic uniform random sampling using a
PC-controlled motorized stage and the CAST-GRID software (Olympus).
Statistics
Calculations of fasting values and AUC [baseline = 0 for glucose and insulin during OGTT (protocol 1), baseline subtracted for glucagon during OGTT (protocol 1) and for insulin during glucose and arginine stimulation test (protocol 2)], and statistical evaluation of results was performed using paired two-tailed Student's t-test, the Kruskal-Wallis test, and linear regression using Excel (2000) and GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). Comparison of slopes of regression lines was performed using the method described by Zar (56). P values of ![]() |
RESULTS |
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Normal Plasma Profiles During OGTT (Protocol 1)
On the basis of the OGTT performed in all 38 animals before administration of NIA and STZ, curves showing the normal plasma concentrations of glucose, insulin, and glucagon were obtained. Normal FPG and fasting plasma insulin (FPI) are summarized in Table 1. The glucose area ander the curve (AUCglucose) was 980 ± 200 mM · min, and the 2-h plasma glucose (2-hPG) was 4.7 ± 1.2 mM. Similarly, normal AUCinsulin was 42,087 ± 21,637 pM · min. Normal fasting plasma glucagon (FPGa) was 86 ± 22 ng/l, and AUCglucagon was
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Fasting Plasma Values After Administration of STZ (Protocol 3)
Changes in FPG, FPI, and FPGa were evaluated by comparison with the normal values obtained in the 38 animals (Table 1). FPG was significantly increased in the 100 and 125 mg/kg STZ groups but not in the 75 mg/kg STZ group, and there was a significant correlation between log STZ dose and FPG (r2 = 0.5924, P = 0.001) (Fig. 1). FPI was significantly decreased in the 100 and 125 mg/kg STZ groups but not in the 75 mg/kg STZ group, there being a significant correlation between log dose STZ and FPI (r2 = 0.6157, P = 0.002). No significant change was detected in FPGa (data not shown).
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Plasma Profiles During OGTT (Protocol 1) After Administration Of NIA and STZ
A significant increase in FPG was found in the 100, 67, 20, and 0 mg/kg NIA groups but not in the 230 and 150 mg/kg NIA groups (Table 2), the correlation being significant between log dose of NIA and FPG (r2 = 0.4339, P < 0.001) (Fig. 1). A significant decrease in FPI was found in the 67 mg/kg NIA group, but this most probably is due to high values of FPI before NIA + STZ in this group, since changes in all other groups were nonsignificant. There was no significant correlation between log dose of NIA and FPI (r2 = 0.0052, P = 0.687).
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No significant changes in FPGa were detected in any of the dosing groups.
A significant increase in AUCglucose was found in all
dosing groups except the 230 mg/kg NIA group (Table
3). Changes in glucose levels are
illustrated in Fig. 2, and there was a
significant correlation between the log NIA dose and
AUCglucose (r2 = 0.2970, P < 0.001) (data not shown). Furthermore, there was a
significant correlation between AUCglucose and FPG
(r2 = 0.880, P < 0.0001)
(Fig. 3). A significant decrease in
AUCinsulin was found in the 150, 67, and 0 mg/kg NIA
groups. In the other groups, a nonsignificant trend toward lowering of
AUCinsulin was seen. When AUCinsulin after 20 mg/kg NIA + STZ were compared with the AUCinsulin (in
pM · min) from the 38 normal OGTT profiles, there was a
significant decrease (from 42,087 ± 21,637 to 7,970 ± 2,702, P = 0.03). There was no significant
correlation between log NIA dose and AUCinsulin
(r2 = 0.00875, P = 0.599). No significant changes in AUCglucagon were found in
any of the groups.
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Significant increases in 2h-PG were found in the 100, 20, and 0 mg/kg NIA groups, and nonsignificant increases were seen in the 150 and 67 mg/kg NIA groups. A nonsignificant decrease was seen in the 230 mg/kg NIA group, and there was a significant correlation between log NIA dose and 2-hPG (r2 = 0.2989, P < 0.001) (data not shown).
Duration Of Reduction Of -Cell Function and Mass
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During the histological examination of pancreata from animals dosed
with NIA + STZ, no signs of development of tumors from surviving
-cells were found.
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DISCUSSION |
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Attempts to genetically select a strain of diabetic pigs have been
made (38, 39, 40) but have, so far, not been confirmed to
be successful (19). The use of pancreatectomized animals as a model of diabetes (15, 33, 34, 50) has the
disadvantage of also removing the exocrine function of the pancreas and
the non--cell endocrine cells of the islets of Langerhans, and
although performance of partial pancreatectomy might be useful for
induction of mild diabetes, this method clearly is more invasive
compared with the administration of STZ.
Pigs are more resistant to the diabetogenic effects of STZ than rats (2, 13, 15, 16, 23, 27-29, 31, 46, 55) and nonhuman primates (42), and a wide individual variability in the response to STZ was seen in the present study, as has also been shown in nonhuman primates (42). In the present study, only male animals were included to reduce variability in glucose tolerance due to estrous cycling, and probably the diversity in genetic background is the best explanation for the wide variability seen, because diet and nutritional status were standardized in the present experiment.
Previous observations showing that 35-40 mg/kg STZ did not influence glucose metabolism in pigs (15, 29) are consistent with data from the present study. In the present study, all of the animals in the 100 mg/kg STZ group and five of seven animals in the 125 mg/kg STZ group were classified as severely diabetic, whereas the remaining two animals from the 125 mg/kg STZ group had impaired fasting glucose (IFG). Thus these data demonstrate that adult male Göttingen minipigs can be made severely diabetic by using a dose of STZ of 100-125 mg/kg or above. This is in accord with previous observations in minipigs and domestic pigs, with 100-150 mg/kg STZ inducing overt diabetes (9, 15, 16, 55). The response to 100 or 125 mg/kg STZ showed some individual variation, indicating individual grades of sensitivity to STZ alone, in accord with previous observations (13, 47). The effects of STZ alone, expressed as increased FPG and decreased FPI and FPC, are significantly correlated with log dose of STZ and show a clear dose-response relationship, and despite the individual variation, this dosing regimen seems to be a reliable method for pharmacological induction of overt diabetes. However, on the basis of the present results, the dosing window in which induction of mild, insulin-deficient diabetes can be accomplished seems very narrow due to the steepness of the dose-response curve to STZ alone.
The present results show that NIA is indeed capable of partially
protecting -cells from the damaging effects of STZ in the Göttingen minipig, as has also been shown previously in rats (23, 30, 31).
The use of NIA seems feasible for induction of mild type 1 insulin-deficient diabetes, since the changes in FPG in response to
changing doses of NIA are significantly smaller compared with changing
doses of STZ. This can be seen when the regression lines for log dose
NIA or STZ and FPG (Fig. 2) are compared, with the regression line
based on changing STZ dose having a much steeper slope (30 ± 7)
compared with that for the changing NIA dose (7 ± 2)
(P < 0.001). NIA in a dose of 67 mg/kg in combination
with STZ seems to be a useful regimen for induction of mild
insulin-deficient diabetes.
However, the protective effect of NIA is not complete, since even the
highest dose of NIA could not fully prevent the effect on the
-cells, as has also been shown in rats (23, 30).
The ratio of insulin to glucose at 30 min during the OGTT was decreased
even in the animals that had normal glucose tolerance (NGT)
post-NIA+STZ dosing compared with pre-NIA+STZ values (25.8 ± 11.5 vs. 61.2 ± 28.0, P < 0.001). Furthermore, there
was a gradual decrease in the ratio post-NIA+STZ dosing from the NGT
animals, through the glucose-intolerant (17.7 ± 11.1) and mildly
diabetic animals (9.2 ± 8.8), to the overtly diabetic animals
(2.6 ± 3.2, P < 0.001; Fig.
6), which is similar to what has been
observed in humans (52). The fact that this ratio
is also decreased in the animals with NGT post-NIA+STZ dosing compared
with pre-NIA+STZ values indicates some deterioration of
insulin-secretory response to glucose even in these animals.
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The protection by NIA seems to be negligible when doses of 20 mg/kg or below are used. In the dosing range 150-67 mg/kg NIA, mild diabetes could be induced, even though some variability existed within the dosing groups, with the diabetic animals having moderately increased FPG and higher residual insulin-secretory capacity compared with diabetic animals from the 20 and 0 mg/kg NIA groups.
Administration of NIA and STZ in combination has been known to induce
insulin-producing tumors in the pancreas of rats (45). In
the present study, no tumors were found during histological examination
of pancreatic tissues from animals for up to 2 mo after dosing with
NIA + STZ. Diabetes induced by STZ has previously been shown to be
stable in some experiments (9, 55), whereas other studies
have shown a gradual improvement in glucose tolerance (27). In the present study, the elevated FPG after
NIA + STZ was stable in the seven tested animals for a period of
from 2 wk to 2 mo after dosing. Furthermore, there was a very
significant reduction in insulin-secretory response to both glucose and
arginine after the same period of time. Finally, the in vivo measures
of reduced insulin-secretory capacity were confirmed histologically by
reduced -cell mass in the model. The stability of the IGT/diabetes in this model beyond 2 mo after dosing has yet to be investigated in detail.
In conclusion, these studies have shown that varying degrees of glucose
intolerance and diabetes can be induced in male Göttingen minipigs with STZ and that NIA has protective effects in -cells in
these animals, as has also been shown in rats. There seems to be a
rational reason for including NIA together with STZ for induction of
diabetes in the minipig, because NIA produces a less steep
dose-response curve with respect to effects of changing doses on FPG
compared with STZ. Even though the protection of 67 mg/kg NIA was too
high to induce frank diabetes in some of the animals, this dose still
results in a significant change in glucose tolerance, making it a
suitable model for studies of new pharmacological agents for the
treatment of diabetes. The model has proven to be stable for up to 2 mo, both functionally and histologically. The characteristics of the
model include a reduced
-cell mass and disturbed residual
insulin-secretory capacity, leading to a decreased ability to dispose
of glucose, seen as both fasting, and especially postprandial
hyperglycemia. The pathogenesis of latent autoimmune diabetes in adults
(LADA) is closely related to a primary reduction in
-cell function,
whereas obesity and the metabolic syndrome are not thought to be of
primary importance (43, 48, 53). Because the primary
defect in the present model is reduction of
-cell mass and function,
whereas obesity, insulin resistance, and other characteristics of the
metabolic syndrome are not involved as a primary defect, this model of
insulin-deficient diabetes is of special interest for the study of
LADA. Induction of insulin resistance and obesity, possibly by high-fat
feeding, would further improve the usefulness of this model as a tool
in diabetes research, thereby including major characteristics of type 2 diabetes, and studies are ongoing to investigate this possibility. Furthermore, the good condition of the animals after induction of
diabetes with NIA+STZ makes this a valuable alternative to pancreatectomy for induction of diabetes with respect to animal welfare
measures. Thus a model of IGT/mild insulin-deficient diabetes has been
developed that can be very useful in short- and long-term studies of
pathophysiology and treatment of human diabetes/IGT due to reduced
insulin secretion capacity.
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ACKNOWLEDGEMENTS |
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We thank Helle Nygaard, Line Mürer, Margit Nelboe Jeppesen, Lene Sejersen Winther, Lotte Gotlieb Sørensen, Anne Grethe Juul, Nanna Kasmira Nowa Hansen, Annemette Petersen, Jannie Neuman, Susanne Primdal, Steen Kryger, Karsten Larsen, Ejnar Eriksen, and Hans Rasmussen for excellent technical assistance.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. O. Larsen, Dept. of Pharmacological Research I, Pharmacology Research and Development, Novo Allé, 6A1.117, DK-2880 Bagsvaerd, Denmark (E-mail: mmla{at}novonordisk.com).
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. Section 1734 solely to indicate this fact.
First published February 19, 2002;10.1152/ajpendo.00564.2001
Received 21 December 2001; accepted in final form 12 February 2002.
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