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Offspring of streptozotocin diabetic rats: size changes in Langerhans islets with time after birth1
Diabetes Research and Clinical Practice 41 (1998) 95 – 100
Offspring of streptozotocin diabetic rats: size changes in Langerhans islets with time after birth1 R.R. Rodrı´guez a,*, A. Renauld a, D. Celener b, R.L. Pe´rez a, M.C. Susemihl a a
Departments of Physiology, Histology and Embryology, Medical School, Buenos Aires Uni6ersity, Paraguay 2155, 7 ° piso, 1121 Buenos Aires, Argentina b Institute of Gastroenterology, ‘Pe´rez Companc Foundation’, Buenos Aires, Argentina Accepted 26 May 1998
Abstract It has been demonstrated that, in the diabetic rat, pregnancy and lactation are severely altered: in this study, we have measured the size of Langerhans islets of rat pups, the offspring of experimental diabetic mothers and nondiabetic controls. Diabetes was induced through streptozotocin administration (dose, 60 mg/kg body wt.). This drug was injected in every animal; their blood sugar was measured 1 week later (Haemo-Glukotest, Boehringer Mannheim), and they were then separated into three groups according to their fasting blood sugar levels: (a) severe diabetics (above 16.5 mM/l); (b) mild diabetics (6.5–16.5 mM/l); and (c) nondiabetic normals. They received insulin therapy (2–4 I.U./day) as the mild diabetics exhibited a slightly higher than normal fasting blood sugar, and the diabetic ones, above 15 mM/l. The areas of Langerhans islets of pups were measured 1 and 5 days after parturition; pancreas sections were dyed (haematoxylin–eosin) and morphometry was then performed using a digitalized magnetic tabloid connected to a Zeiss Morphomat 30 (Kontron). On the first day after parturition, the pancreas section areas in pups from mildly and severely diabetic mothers were smaller than those in neonates from nondiabetic controls (PB0.001). The areas in neonates from severely diabetic mothers showed a more intense decrease than those from mildly diabetic animals (P B0.01). On day 5 after delivery, the areas of Langerhans islets in offspring from normal mothers decreased and those in pups from diabetic mothers tended to normalize (P B 0.01), particularly those from the severely sick group (PB 0.01). We conclude that after parturition the offspring is no longer exposed to the high blood sugar levels found in both diabetic groups of mothers, thereby no hyperinsulinemia is needed; as time elapses, then, the area of their Langerhans islets tends to normalization. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Rat neonates; Experimental diabetes; Morphometry of Langerhans islets
* Corresponding author. Fax: +54 1 9636287. 1 This paper was partly presented at the Meetings of the XXXII Congress of the International Union of Physiological Sciences, Glasgow, 1 – 6 August 1993. 0168-8227/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0168-8227(98)00073-4
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R.R. Rodrı´guez et al. / Diabetes Research and Clinical Practice 41 (1998) 95–100
1. Introduction It has been described that in rat, during pregnancy, glucose crosses placenta freely and affects fetus metabolism via an unclearly understood mechanism [1]. In turn, in pregnant women, glucose, alanine and ketone bodies are transported across placenta and, as pregnancy advances, some insulin antagonistic hormones cross it as well [2]. During oral glucose tolerance test, glucose, triglycerides and insulin responses progressively increase with human pregnancy, whereas glucagon response decreases; triglycerides, then, serve to spare glucose as a maternal fluid and glucose uptake by fetus becomes very high [2]. On the other hand, administration of diabetogenic drugs and surgical pancreatectomy evoke experimental diabetes in rat. It has been observed that even prediabetes causes multiple defects in rat B-cells [3]; insulin sensitivity in diabetic rats is normal [4], but glucokinase activity — a glucose sensor in B-cell for insulin secretion — is intensely decreased in these animals [4]. Studies in rat and rabbit have demonstrated that offspring from experimental diabetic mothers show hyperfunction of endocrine pancreas [5]. Although it was early demonstrated [6] that pancreatic islets from prenatal and postnatal offspring from diabetic rats show beta-degranulation and glycogen infiltration, recent studies have demonstrated that chronic hyperglycemia is associated with a fasting-induced paradoxical increase in glucose-potentiated insulin secretion; this finding suggests the causative role for a lowered B-cell insulin content in experimental diabetic rats [7]. As expected, in rat, the diabetes of mothers during pregnancy implies an abnormal intrauterine medium for the development of the fetuses; since the availability of fuels for the fetus is primarily dependent upon the maternal metabolic state, diabetes-induced alternations in nutrient metabolism are then believed to result in perinatal complication in pups, including 40% death at birth; the abundant fuel supply from diabetic mothers to fetuses induces adaptation in the fetal metabolism and structural and functional changes in the fetal endocrine pancreas as well [8]. In the fetuses of rats with mild diabetes induced by
streptozotocin, the increased glucose supply induces islet hyperplasia [9] and an increased B-cell activity in vitro and in vivo leading to B-cell degranulation and hyperinsulinemia [10]. In the fetuses of several diabetic rats, pancreases contain many well-structured islets of Langerhans, thereby capable of regulating the fetal glycemia automatically [11]; however, in these fetuses, the enormous blood glucose levels overstimulate the B-cells and most of them are finally exhausted, and degranulated [9]. Although B-cell degranulation was still found in neonates from streptozotocin-injected rats [12], 3–10 h after birth, withdrawal of the intense maternal glucose stimulus restores some of the B-cell granulation and Langerhans islets in the offspring do not further increase; it was reported that at the time of weaning, youngsters from diabetic mothers have reduced mass of endocrine tissue in their pancreases [13]. Morphometric studies showed reduced B-cell mass and low pancreatic insulin content in offsprings of experimental diabetic rats [14]; it is apparent that mechanisms other than mitotic, i.e. islets cell hyperplasia and/or neoformation of islet cells from precursors within. The islets, may be also involved in the rapid increase of B-cell mass [15,16]. Results obtained in women mimick these observations, because pancreatic islet hyperplasia and hyperinsulinemia are frequent features in fetuses and neonates during human diabetic pregnancy [17]. The findings summarized above suggest that (1) the size of Langerhans islets in young pups from normal and streptozotocin-treated diabetic rat mothers might be differently sized; and (2) this size might change with time elapsed after parturition on pups from these diabetic animals, namely at a time when the offspring is not submitted to the high maternal blood sugar levels any longer, rather to their own normal blood glucose concentration. Therefore, we performed some morphometric studies of Langerhans islets in pups from normal and experimental diabetic rats on days 1 and 5 after birth; diabetes was induced through streptozotocin administration before pregnancy start.
R.R. Rodrı´guez et al. / Diabetes Research and Clinical Practice 41 (1998) 95–100
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Table 1 Pregnancy in streptozotocin diabetic rats (6 months) No.
Non-injected controls Moderately diabetic Severely diabetic plus insulin Severely diabetic without insulin
(40) (30) (39) (31)
Pregnancy (No.) Deliveries
80 49 48 12
Newborns observed (No.)
No.
%
80 40 29 5
100 433 89 188 60 87 41 36
2. Materials and methods Female Wistar rats, weighing 150 – 200 g, 2 months of age, were used in this study. They were hosted in cages containing the same number of animals (eight). They were injected with streptozotocin intraperitoneally (dose 60 mg/kg body wt.); 1 week later they were separated into three groups according to their fasting blood sugar level: (A) severely diabetic (blood sugar above 16.5 mM/l); (B) mildly diabetic (blood sugar between 6.5 and 16.5 mM/l); and (C) streptozotocin uninjected nondiabetic controls. All these animals drank tap water and ate rat chow pellets ad libitum, were mated with uninjected normal males of the same strain and were located in individual cages. Some of the injected animals showing fasting blood sugar levels above 10 mM/l received one daily injection of 2 – 4 I.U. of lent pork insulin, depending upon the degree of hyperglycemia, which stayed hardly above than normal in the mildly diabetic rats and kept above 15 mM/l in those severely diabetic. As parturition occurred, only pups from rats delivering eight to 12 offspring (because those diabetic sometimes gave birth to less) were selected, 50% out of them were killed by beheading during the first hours, and the remaining 50% were killed 5 days later, which appeared a correct waiting time. Their pancreases (total number, 133) were immediately excised using small curve-tipped forceps, fixed in Bouin solution, sequentially cut and dyed with hematoxylin – eosin. Islet section areas were then measured using a digitalized magnetic tablet connected to a Zeiss Morphomat 30 (Kontron).
Mean body weight at 22 days (g)
33.0 24.5 18.5 18.5
The results obtained by these measurements showed a definite heteroscedasticity, whereby they 3 had to be submitted to 1/100 and
transformations to homogenize variance. They were then analyzed for variance for two factor experiments: (1) pups (1 and 5 days of age); and (2) diabetic state of their mothers (normal, mildly diabetic, severely diabetic), with repeated measures on one factor. Comparisons between pairs of these groups were performed (Student t-test) using the resulting error mean square as measure of dispersion.
3. Results Table 1 shows that in 40 normal rats, and during the observation period of 6 months, 80 normal pregnancies were studied, with 100% deliveries; after 22 days of lactation the average body weight of the pups was 33 g. On the other hand, in diabetic animals, the number of pregnancies and deliveries were reduced, more markedly in the severe diabetic rats and, in this group, those that were not treated with insulin. The mean pup body weight was markedly decreased due to insufficient lactation. After 1, 1.5 and 3 months of age the fasting blood sugar level showed that offspring from diabetic mothers had a transitory hyperglycemia (Table 2). Fig. 1 shows areas of the Langerhans islets observed in neonates from normal, mildly diabetic and severely diabetic rat mothers on days 1 and 5 after birth. In comparison to pups from
R.R. Rodrı´guez et al. / Diabetes Research and Clinical Practice 41 (1998) 95–100
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Table 2 Offspring from diabetics mothers showing fasting hyperglycemia Age (months)
1 1.5 3
Males
Females
No.
%
No.
%
26/65 29/65 0/65
40 45 0
22/72 16/72 0/72
30 22 0
nondiabetic controls, these areas decreased in all pups from diabetic mothers on day 1 (P B 0.001) and day 5 (PB0.01); on the 5th day the decrease and its significance as compared to the day 1 group increased. The areas observed in pups from severely diabetic mothers at day 5 from respective deliveries are above those observed in the same group at day 1; as in nondiabetic controls, in neonates from mildly diabetic rats these areas continued falling when days 1 and 5 after delivery are compared.
4. Discussion Our results reported above demonstrated that the blood sugar levels in rat offsprings at day 5 after delivery are decreased in respect those found in day 1 of parturition. This finding appears normal because these pups were fed by lactation, and normal rat milk is known to contain a low glucose concentration (8%); therefore fat and proteins predominate as metabolic fuels in these young animals, their serum IRI concentration remains low, gluconeogenesis and lipolysis become important in metabolic processes, the activity of their enzymes metabolizing glucose is poor, and glycogen storage in their liver and striated muscle is decreased [18]. It has been demonstrated that, in the diabetic rat, pregnancy and lactation are altered. Thus, its sex cycles (normally lasting 4 days) are more prolonged or disappear for quite long periods of time, fetus implant (which usually occurs between 7th and 9th day of pregnancy) frequently fails to happen and, at parturition, mothers sometimes
Fig. 1. Langerhans islets areas in nondiabetic, moderately and severely diabetic rats on days 1 and 5 after delivery. Significance of comparisons of results vs respective nondiabetics, vs moderately diabetics and day 5 vs 1 controls.
R.R. Rodrı´guez et al. / Diabetes Research and Clinical Practice 41 (1998) 95–100
die or occasionally their offspring exhibit a high pre- or post-partum mortality. Furthermore, lactation is deficient in diabetic rat mothers, which is revealed in their offspring by a poor enhancement with time in the body weights of the pups. Moreover, these pups show also some temporary metabolic alterations. Diabetes mellitus and pregnancy complicate each other. However, the definite risk of diabetic complications during pregnancy decrease through normalization of blood glucose levels by insulin administration [19– 22]. In line with these findings, we have observed here that, in rats, the morphological intrauterine development of Langerhans islets tends to normalize and perinatal mortality falls when the mothers experimental condition is under exogenous insulin control, and just moderately higher than normal blood sugar levels are allowed. Furthermore, islet transplantation in diabetic pregnant rats normalizes glucose homeostasis in their offspring [21]. Wako and Kimura [1] have suggested that hyperglycemia of rat mothers causes hypertrophy of the pancreatic islets and increases insulin secretion in diabetes mellitus. It is therefore not surprising that the infants of diabetic mothers show morphological and functional features of hyperinsulinemia [22]. The basal hyperglycemia of diabetic rat mothers and of their offspring pre- and postpartum increases the need of a more intense insulin secretion in order to keep a rather normal blood sugar homeostasis. Therefore, it is expected for the pancreases of their young pups to become depleted, degranulated, and also smaller than those from rats with severe experimental disease, as found in this study 1 and 5 days after parturition. Some investigators have reported that the pancreases of youngsters from mild and severely diabetic rat mothers appear normal in size and composition; their basal insulinemias are at normal levels, as are their glycemias. However, continuous glucose infusion for 3 h in diabetic pregnant rats evidences marked differences with nondiabetic pregnant controls [23]; pups of mildly diabetic mothers exhibit abnormally high glycemic levels together with a decreased insulin response, while in the neonates of severely diabetic rat mothers, the blood sugar levels remain
99
within the normal range and this is associated with definitely elevated serum insulin levels [23], in line with our morphometric observations reported here. In summary then, in line with our previous findings [24], our results reported here let us conclude that the size of Langerhans islets in the pancreases of offsprings from diabetic rats is reduced due to the induction of experimental diabetes by streptozotocin. When the mother’s diabetic state is mild and insulin administration permits their proper blood glucose control, the intrauterine morphological development of Langerhans islets is less decreased than in severely diabetic rats, as shown by pancreases from pups of 1 and 5 days of age, respectively. When the severe diabetes was studied, the pups showed somewhat increased areas of pancreas sections in inverse relation to time elapsed from their recent delivery.
Acknowledgements The authors want to thank Dr Juan Anderson for generous donation of pork insulin from Beta Laboratories and Dr Claudio Gonzalez (Boehringer Mannheim, Argentina) for the supply of Haemo-Glukotest for blood glucose determinations. The skilled technical assistance of Irene Villarreal and Marisa M. Moriondo is fully acknowledged.
References [1] Y. Wako, S. Kimura, Muscle response to insulin in fetuses of diabetic pregnant rats, Agric. Biol. Chem. 48 (1984) 2669 – 2674. [2] R.G. Cutfield, D.H. Mintz, Clinical Guide to Diabetes Mellitus, Alan R. Liss, New York, NY, 1987, pp. 193 – 207. [3] Y. Tokuyama, J. Sturis, A.M. De Paoli, J. Takeda, M. Stoffel, J. Tang, X. Sun, K. Polonsky, G.I. Bell, Evolution of beta cell disfunction in the male Zucker diabetic fatty rat, Diabetes 44 (1995) 1447 – 1457. [4] H. Hosokawa, Y.A. Hosokawa, J.L. Leahy, Unregulated hexokinase activity in isolated islets from diabetic 90% pancreatectomized rats, Diabetes 44 (1995) 1328 – 1333.
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[5] E.K. Golab, Effects of streptozotocin-induced diabetes on pancreatic insulin content in the foetus, Diabetes 19 (1970) 610 – 613. [6] R.M. Pitkin, D.E. Van Orden, Fetal effects of maternal streptozotocin-diabetes, Endocrinology 94 (1974) 1247– 1253. [7] Y.A. Hosokawa, H. Hosokawa, C. Chen, J.L. Leahy, Mechanism of impaired glucose-potentiated insulin secretion in diabetic 90% pancreatectomy rats, J. Clin. Invest. 97 (1996) 180 – 186. [8] L.A. Aerts, K. Holemans, F.A. Van Assche, Maternal diabetes during pregnancy and the long-term consequences of the offspring, IDF Bull. 40 (1995) 24–28. [9] L. Aerts, F.A. Van Assche, Rat fetal endocrine pancreas in experimental diabetes, J. Endocrinol. (London) 73 (1977) 339 – 346. [10] A. Kevran, M. Guillaume, A. Jost, The endocrine pancreas of the fetus of diabetic pregnant rat, Diabetologia 15 (1978) 387 – 393. [11] A. Kevran, J. Randon, J.A. Girard, Dynamics of glucoseinduced plasma insulin increase in the rat fetus at different stages of gestation, Biol. Neonate 35 (1979) 242–248. [12] P.M. Pitkin, O.A. Van Orden, Foetal effects of maternal streptozotocin diabetes, Endocrinology 94 (1974) 1247– 1253. [13] L. Aerts, F.A. Van Assche, Endocrine pancreas in the offspring of rats with experimentally induced diabetes, J. Endocrinol. 88 (1981) 81–88. [14] U. Eriksson, A. Andersson, S. Effendic, C. Hellerstro¨m, Diabetes in pregnancy: effects on the normal and newborn rat with particular regard to body weight, serum insulin concentration and pancreatic contents of insulin, glucagon and somatostatin, Acta Endocrinol. (Copenhagen) 94 (1980) 354–364.
.
[15] R. Pictet, W.J. Rutter, Development of the embryonic endocrine pancreas. In: D.F. Steiner, N. Freinkel (Eds.), Handbook of Physiology, Section 7, vol. 1, Am. Physiol. Soc., Baltimore, MD, 1972, pp. 25 – 66. [16] R.M. Melamed, C.J. Benı´tez, S.J. Hollt, Intermediate cells of the pancreas. I. Ultrastructural characterization, J. Cell Sci. 11 (1972) 449 – 475. [17] K.F. Wellman, B.W. Volk, The islets of diabetic mothers. In: B.W. Volk, K.E. Wellman (Eds). The Diabetic Pancreas, Plenum Press, New York, NY, 1977, pp. 365 – 380. [18] E. Blazquez, E. Montoya, C. Lo´pez Quijada, Relationship between insulin concentrations in plasma and pancreas of foetal and weanling rats, J. Endocrinol. 48 (1970) 553 – 561. [19] K. Healy, L. Jovanovic-Peterson, J.M. Peterson, Pancreatic disorders of pregnancy. Pregestational diabetes, Endocrinol. Metab. Clin. North Am. 24 (1995) 73 – 101. [20] E.A. Reeci, C.J. Homko, Assessment and management of pregnancies complicated by pregestational and gestational diabetes mellitus, J. Assoc. Acad. Minor Phys. 5 (1994) 87 – 97. [21] L. Aerts, E.A. Van Assche, Islet transplantation in diabetic pregnant rats normalizes homeostasis in their offspring, J. Dev. Physiol. 17 (1992) 283 – 287. [22] F.A. Van Assche, W. Gepts, The cytological composition of the foetal endocrine pancreas in normal and pathological conditions, Diabetologia 7 (1971) 434 – 441. [23] L. Aerts, F. Sodoyez-Goffaux, J.C. Sodoyez, W. Malaisse, F.A. Van Assche, The diabetic intrauterine milien has a long-lasting effect on insulin secretion by beta-cells and an insulin uptake by target tissues, Am. J. Obstet. Gynecol. 5 (1988) 1237 – 1292. [24] R.R. Rodrı´guez, D. Celener, A. Renauld, M.C. Susemihl, Fetal morphology of the pancreas in offspring from diabetic rats. XXXI Cong. Int. Union Physiol. Sci., Glasgow, 1 – 6 August 1993.
Offspring of streptozotocin diabetic rats: size changes in Langerhans islets with time after birth1 R.R. Rodrı´guez a,*, A. Renauld a, D. Celener b, R.L. Pe´rez a, M.C. Susemihl a a
Departments of Physiology, Histology and Embryology, Medical School, Buenos Aires Uni6ersity, Paraguay 2155, 7 ° piso, 1121 Buenos Aires, Argentina b Institute of Gastroenterology, ‘Pe´rez Companc Foundation’, Buenos Aires, Argentina Accepted 26 May 1998
Abstract It has been demonstrated that, in the diabetic rat, pregnancy and lactation are severely altered: in this study, we have measured the size of Langerhans islets of rat pups, the offspring of experimental diabetic mothers and nondiabetic controls. Diabetes was induced through streptozotocin administration (dose, 60 mg/kg body wt.). This drug was injected in every animal; their blood sugar was measured 1 week later (Haemo-Glukotest, Boehringer Mannheim), and they were then separated into three groups according to their fasting blood sugar levels: (a) severe diabetics (above 16.5 mM/l); (b) mild diabetics (6.5–16.5 mM/l); and (c) nondiabetic normals. They received insulin therapy (2–4 I.U./day) as the mild diabetics exhibited a slightly higher than normal fasting blood sugar, and the diabetic ones, above 15 mM/l. The areas of Langerhans islets of pups were measured 1 and 5 days after parturition; pancreas sections were dyed (haematoxylin–eosin) and morphometry was then performed using a digitalized magnetic tabloid connected to a Zeiss Morphomat 30 (Kontron). On the first day after parturition, the pancreas section areas in pups from mildly and severely diabetic mothers were smaller than those in neonates from nondiabetic controls (PB0.001). The areas in neonates from severely diabetic mothers showed a more intense decrease than those from mildly diabetic animals (P B0.01). On day 5 after delivery, the areas of Langerhans islets in offspring from normal mothers decreased and those in pups from diabetic mothers tended to normalize (P B 0.01), particularly those from the severely sick group (PB 0.01). We conclude that after parturition the offspring is no longer exposed to the high blood sugar levels found in both diabetic groups of mothers, thereby no hyperinsulinemia is needed; as time elapses, then, the area of their Langerhans islets tends to normalization. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Rat neonates; Experimental diabetes; Morphometry of Langerhans islets
* Corresponding author. Fax: +54 1 9636287. 1 This paper was partly presented at the Meetings of the XXXII Congress of the International Union of Physiological Sciences, Glasgow, 1 – 6 August 1993. 0168-8227/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0168-8227(98)00073-4
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R.R. Rodrı´guez et al. / Diabetes Research and Clinical Practice 41 (1998) 95–100
1. Introduction It has been described that in rat, during pregnancy, glucose crosses placenta freely and affects fetus metabolism via an unclearly understood mechanism [1]. In turn, in pregnant women, glucose, alanine and ketone bodies are transported across placenta and, as pregnancy advances, some insulin antagonistic hormones cross it as well [2]. During oral glucose tolerance test, glucose, triglycerides and insulin responses progressively increase with human pregnancy, whereas glucagon response decreases; triglycerides, then, serve to spare glucose as a maternal fluid and glucose uptake by fetus becomes very high [2]. On the other hand, administration of diabetogenic drugs and surgical pancreatectomy evoke experimental diabetes in rat. It has been observed that even prediabetes causes multiple defects in rat B-cells [3]; insulin sensitivity in diabetic rats is normal [4], but glucokinase activity — a glucose sensor in B-cell for insulin secretion — is intensely decreased in these animals [4]. Studies in rat and rabbit have demonstrated that offspring from experimental diabetic mothers show hyperfunction of endocrine pancreas [5]. Although it was early demonstrated [6] that pancreatic islets from prenatal and postnatal offspring from diabetic rats show beta-degranulation and glycogen infiltration, recent studies have demonstrated that chronic hyperglycemia is associated with a fasting-induced paradoxical increase in glucose-potentiated insulin secretion; this finding suggests the causative role for a lowered B-cell insulin content in experimental diabetic rats [7]. As expected, in rat, the diabetes of mothers during pregnancy implies an abnormal intrauterine medium for the development of the fetuses; since the availability of fuels for the fetus is primarily dependent upon the maternal metabolic state, diabetes-induced alternations in nutrient metabolism are then believed to result in perinatal complication in pups, including 40% death at birth; the abundant fuel supply from diabetic mothers to fetuses induces adaptation in the fetal metabolism and structural and functional changes in the fetal endocrine pancreas as well [8]. In the fetuses of rats with mild diabetes induced by
streptozotocin, the increased glucose supply induces islet hyperplasia [9] and an increased B-cell activity in vitro and in vivo leading to B-cell degranulation and hyperinsulinemia [10]. In the fetuses of several diabetic rats, pancreases contain many well-structured islets of Langerhans, thereby capable of regulating the fetal glycemia automatically [11]; however, in these fetuses, the enormous blood glucose levels overstimulate the B-cells and most of them are finally exhausted, and degranulated [9]. Although B-cell degranulation was still found in neonates from streptozotocin-injected rats [12], 3–10 h after birth, withdrawal of the intense maternal glucose stimulus restores some of the B-cell granulation and Langerhans islets in the offspring do not further increase; it was reported that at the time of weaning, youngsters from diabetic mothers have reduced mass of endocrine tissue in their pancreases [13]. Morphometric studies showed reduced B-cell mass and low pancreatic insulin content in offsprings of experimental diabetic rats [14]; it is apparent that mechanisms other than mitotic, i.e. islets cell hyperplasia and/or neoformation of islet cells from precursors within. The islets, may be also involved in the rapid increase of B-cell mass [15,16]. Results obtained in women mimick these observations, because pancreatic islet hyperplasia and hyperinsulinemia are frequent features in fetuses and neonates during human diabetic pregnancy [17]. The findings summarized above suggest that (1) the size of Langerhans islets in young pups from normal and streptozotocin-treated diabetic rat mothers might be differently sized; and (2) this size might change with time elapsed after parturition on pups from these diabetic animals, namely at a time when the offspring is not submitted to the high maternal blood sugar levels any longer, rather to their own normal blood glucose concentration. Therefore, we performed some morphometric studies of Langerhans islets in pups from normal and experimental diabetic rats on days 1 and 5 after birth; diabetes was induced through streptozotocin administration before pregnancy start.
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97
Table 1 Pregnancy in streptozotocin diabetic rats (6 months) No.
Non-injected controls Moderately diabetic Severely diabetic plus insulin Severely diabetic without insulin
(40) (30) (39) (31)
Pregnancy (No.) Deliveries
80 49 48 12
Newborns observed (No.)
No.
%
80 40 29 5
100 433 89 188 60 87 41 36
2. Materials and methods Female Wistar rats, weighing 150 – 200 g, 2 months of age, were used in this study. They were hosted in cages containing the same number of animals (eight). They were injected with streptozotocin intraperitoneally (dose 60 mg/kg body wt.); 1 week later they were separated into three groups according to their fasting blood sugar level: (A) severely diabetic (blood sugar above 16.5 mM/l); (B) mildly diabetic (blood sugar between 6.5 and 16.5 mM/l); and (C) streptozotocin uninjected nondiabetic controls. All these animals drank tap water and ate rat chow pellets ad libitum, were mated with uninjected normal males of the same strain and were located in individual cages. Some of the injected animals showing fasting blood sugar levels above 10 mM/l received one daily injection of 2 – 4 I.U. of lent pork insulin, depending upon the degree of hyperglycemia, which stayed hardly above than normal in the mildly diabetic rats and kept above 15 mM/l in those severely diabetic. As parturition occurred, only pups from rats delivering eight to 12 offspring (because those diabetic sometimes gave birth to less) were selected, 50% out of them were killed by beheading during the first hours, and the remaining 50% were killed 5 days later, which appeared a correct waiting time. Their pancreases (total number, 133) were immediately excised using small curve-tipped forceps, fixed in Bouin solution, sequentially cut and dyed with hematoxylin – eosin. Islet section areas were then measured using a digitalized magnetic tablet connected to a Zeiss Morphomat 30 (Kontron).
Mean body weight at 22 days (g)
33.0 24.5 18.5 18.5
The results obtained by these measurements showed a definite heteroscedasticity, whereby they 3 had to be submitted to 1/100 and
transformations to homogenize variance. They were then analyzed for variance for two factor experiments: (1) pups (1 and 5 days of age); and (2) diabetic state of their mothers (normal, mildly diabetic, severely diabetic), with repeated measures on one factor. Comparisons between pairs of these groups were performed (Student t-test) using the resulting error mean square as measure of dispersion.
3. Results Table 1 shows that in 40 normal rats, and during the observation period of 6 months, 80 normal pregnancies were studied, with 100% deliveries; after 22 days of lactation the average body weight of the pups was 33 g. On the other hand, in diabetic animals, the number of pregnancies and deliveries were reduced, more markedly in the severe diabetic rats and, in this group, those that were not treated with insulin. The mean pup body weight was markedly decreased due to insufficient lactation. After 1, 1.5 and 3 months of age the fasting blood sugar level showed that offspring from diabetic mothers had a transitory hyperglycemia (Table 2). Fig. 1 shows areas of the Langerhans islets observed in neonates from normal, mildly diabetic and severely diabetic rat mothers on days 1 and 5 after birth. In comparison to pups from
R.R. Rodrı´guez et al. / Diabetes Research and Clinical Practice 41 (1998) 95–100
98
Table 2 Offspring from diabetics mothers showing fasting hyperglycemia Age (months)
1 1.5 3
Males
Females
No.
%
No.
%
26/65 29/65 0/65
40 45 0
22/72 16/72 0/72
30 22 0
nondiabetic controls, these areas decreased in all pups from diabetic mothers on day 1 (P B 0.001) and day 5 (PB0.01); on the 5th day the decrease and its significance as compared to the day 1 group increased. The areas observed in pups from severely diabetic mothers at day 5 from respective deliveries are above those observed in the same group at day 1; as in nondiabetic controls, in neonates from mildly diabetic rats these areas continued falling when days 1 and 5 after delivery are compared.
4. Discussion Our results reported above demonstrated that the blood sugar levels in rat offsprings at day 5 after delivery are decreased in respect those found in day 1 of parturition. This finding appears normal because these pups were fed by lactation, and normal rat milk is known to contain a low glucose concentration (8%); therefore fat and proteins predominate as metabolic fuels in these young animals, their serum IRI concentration remains low, gluconeogenesis and lipolysis become important in metabolic processes, the activity of their enzymes metabolizing glucose is poor, and glycogen storage in their liver and striated muscle is decreased [18]. It has been demonstrated that, in the diabetic rat, pregnancy and lactation are altered. Thus, its sex cycles (normally lasting 4 days) are more prolonged or disappear for quite long periods of time, fetus implant (which usually occurs between 7th and 9th day of pregnancy) frequently fails to happen and, at parturition, mothers sometimes
Fig. 1. Langerhans islets areas in nondiabetic, moderately and severely diabetic rats on days 1 and 5 after delivery. Significance of comparisons of results vs respective nondiabetics, vs moderately diabetics and day 5 vs 1 controls.
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die or occasionally their offspring exhibit a high pre- or post-partum mortality. Furthermore, lactation is deficient in diabetic rat mothers, which is revealed in their offspring by a poor enhancement with time in the body weights of the pups. Moreover, these pups show also some temporary metabolic alterations. Diabetes mellitus and pregnancy complicate each other. However, the definite risk of diabetic complications during pregnancy decrease through normalization of blood glucose levels by insulin administration [19– 22]. In line with these findings, we have observed here that, in rats, the morphological intrauterine development of Langerhans islets tends to normalize and perinatal mortality falls when the mothers experimental condition is under exogenous insulin control, and just moderately higher than normal blood sugar levels are allowed. Furthermore, islet transplantation in diabetic pregnant rats normalizes glucose homeostasis in their offspring [21]. Wako and Kimura [1] have suggested that hyperglycemia of rat mothers causes hypertrophy of the pancreatic islets and increases insulin secretion in diabetes mellitus. It is therefore not surprising that the infants of diabetic mothers show morphological and functional features of hyperinsulinemia [22]. The basal hyperglycemia of diabetic rat mothers and of their offspring pre- and postpartum increases the need of a more intense insulin secretion in order to keep a rather normal blood sugar homeostasis. Therefore, it is expected for the pancreases of their young pups to become depleted, degranulated, and also smaller than those from rats with severe experimental disease, as found in this study 1 and 5 days after parturition. Some investigators have reported that the pancreases of youngsters from mild and severely diabetic rat mothers appear normal in size and composition; their basal insulinemias are at normal levels, as are their glycemias. However, continuous glucose infusion for 3 h in diabetic pregnant rats evidences marked differences with nondiabetic pregnant controls [23]; pups of mildly diabetic mothers exhibit abnormally high glycemic levels together with a decreased insulin response, while in the neonates of severely diabetic rat mothers, the blood sugar levels remain
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within the normal range and this is associated with definitely elevated serum insulin levels [23], in line with our morphometric observations reported here. In summary then, in line with our previous findings [24], our results reported here let us conclude that the size of Langerhans islets in the pancreases of offsprings from diabetic rats is reduced due to the induction of experimental diabetes by streptozotocin. When the mother’s diabetic state is mild and insulin administration permits their proper blood glucose control, the intrauterine morphological development of Langerhans islets is less decreased than in severely diabetic rats, as shown by pancreases from pups of 1 and 5 days of age, respectively. When the severe diabetes was studied, the pups showed somewhat increased areas of pancreas sections in inverse relation to time elapsed from their recent delivery.
Acknowledgements The authors want to thank Dr Juan Anderson for generous donation of pork insulin from Beta Laboratories and Dr Claudio Gonzalez (Boehringer Mannheim, Argentina) for the supply of Haemo-Glukotest for blood glucose determinations. The skilled technical assistance of Irene Villarreal and Marisa M. Moriondo is fully acknowledged.
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