Role of FGF1, FGF2 and FGF7 in the Development of the Pancreas of Diabetic Hamsters

Home , FGF7

Institute of Experimental Morphology and Anthropology with Museum Bulgarian Anatomical Society Acta morphologica et anthropologica, 12 Sofia • 2007

M. Inchovska, V. Ogneva, Y. Martinova

Institute of Experimental Morphology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia

It has been established that in the early postnatal development after treatemenl with streptozotocin (STZ) the pancreas has the ability to regenerate B-cells. Little is known about the role of fibroblast growth factors (FGFs) in the development of the pancreas. That is why we have investigated the effects of FGF1,2 and 7, in vitro, on proliferation and differentiation of the diabetic hamster pancreatic epithelial cells in the early postnatal development. The following methods have been used: cultivation. H.E. staining, P.A.F. staining and histoautoradiography. Our results have shown that in diabetic hamster pancreases at the age pnd 5 FGF2 acts as a strong stimulator of ductal epithelial cell proliferation and new islet formation. Key words: fibroblast growth factors, diabetes, pancreatic cells, proliferative activity.

Introduction

Fibroblast growth factor receptor (FGFR) signalling has been implicated in processes of proliferation and differentiation in many organs, including the pancreas [12]. Numerous studies show that impairment of FGFR signalling in pancreatic p cells leads to diabetes, indicating that FGFR signalling plays a crucial role in controlling glucose homeostasis [1, 7,8,12]. It is well known that the formation of new p cells in the pancreas is a result of two processes: differentiation of ductal precursor cells that further assemble into islets, a process also called noogenesis, and proliferation of preexisting differentiated p cells [3, 10, 18]. It has been established that the injection of STZ at birth destroys 80% of the p cells thereby inducing a depletion of pancreatic insulin stores and a severe hyperglycemic state [2, 4, 14]. Various studies demonstrate that P cell regeneration that follows p cell destruction after STZ treatment involves both p cell proliferation and neogenesis [5, 9, 19]. It has not yet been established whether the new P cell masses display insulin secretion ability. Studies in the field of recovery of pancreatic islet B cells after STZ treatment in the early postnatal development give us grounds to investigate the effect of FGF1, 2 and 7 on the development of diabetic hamster pancreas in this period.

79 Materials and Methods

We induced experimental diabetes in female hamsters during the first 24 hours after mat ing of female and male hamsters by intraperitoneal STZ administration at a dose of 65 mg/ kg. For the study we used only these animals from the litter that had a blood glucose level above 11 mmol/1. We have studied three groups of diabetic hamsters: 20 hamsters at age pnd 1,20 hamsters at age pnd 5, and 20 hamsters at age pnd 10. Pups were decapitated, their pancreases were dissected and cut into small segments. One or two segments from the isolated pancreases were cultivated in each well of 96 — well plates. For the preparation of organ cultures from pancreases we used Iscove‘s medium, supplemented with fetal bovine serum (FBS) and 100 U/ml penicillin and 100pg/ml streptomycin. In the three groups of hamsters incubation medium was additionally supplemented with 4, 10 and 100 ng/1 FGF1, FGF2 and FGF7. Wc had also control wells in the present experiment in which pancreas segments were not treated with the fibroblast growth factors and STZ. The pancreatic pieces were incubated at 37 °C with 5% C 02 for 48 hours. After 48 hours, the medium was supplemented with 3H-thymidine in concentration 2 /rCi/ml for 8 hours. Then the pancreatic segments were Fixed in Bouin's solution for 24 hours, embedded in paraffin, sectioned and stained with H.E. and PA.F according to routine procedures. After 14 days of exposure the autoradiographs were stained with haematoxilin — Bomer. The proliferative activity of pancreatic cells was determined by the labelling index (LI) accord ing to the formula: LI (%) = number of labelled nuclei / number of counted nuclei x 100. From every slide of the experimental groups we estimated 200 cells. Statistical analysis was made by Student's t-test. The results are expressed as mean percentage ± S.E.

Results

The H.E. stained slides have shown well developed ductal epithelium on the periphery of the slides as well as acinar cells in the central part of slides obtained from pancreases of diabetic hamsters at age pnd 1 and treated with FGF1 at a concentration of 4 ng/1 (Fig. 1). The histoautoradiographic study showed an increased proliferative activity of pancreatic epithelial cells in the three groups of diabetic hamsters — pnd 1, pnd 5 and pnd 10, and in all concentrations of the investigated FGFs. We have observed a large number of prolifer ating ductal cells as well as proliferating cells around the ducts on slides obtained from pancreases of the diabetic hamsters at age pnd 5 and treated with FGF2 at a concentration of 10 ng/1 (Fig. 2). We have also observed proliferating exocrine cells on slides obtained from pancreases of the diabetic hamsters at age pnd 5 and after treatment with FGF1 at a concentration of 4 ng/1. Slides stained with PAF provided additional knowledge about the role of FGFs in the early postnatal development of the pancreas of diabetic hamsters. We have observed insu lin - synthesizing cells in the ductal epithelium on slides obtained from pancreases of the diabetic hamsters at age pnd 5 treated with FGF1 at a concentration of 4 ng/1 as well as on slides obtained from pancreases of the diabetic hamsters at age pnd 10 and treated with FGF1 at a concentration of 10 ng/1 (Fig. 3). Insulin-synthesizing cells scattered throughout the acinar cells have also been detected on slides obtained from pancreases of the diabetic hamsters at age pnd 5 and treated with FGF2 at a concentration of 4 ng/1 (Fig. 4). We could also see insulin - positive islets of Langerhans on slides obtained from pancreases of the diabetic hamsters at age pnd 5 and treated with FGF1 at a concentration of 100 ng/1» (Fig. 5).

80 Fig. 1. Ductal epithelial cells in pancreas of diabetic hamsters atagepnd 1 after treatement with FGF1 at a concentration of 4 ng/1 (x 25)

Fig. 2. Autoradiograph of diabetic hamster pancreas at age pnd 5 after treatement with FGF2 at a concentration of 10 ng/1 (x 5)

6 Acta morphologica et anthropologica, 12 81 Fig. 3. Insulin - synthesizing cells in the ductal epithelium of diabetic hamster pancreas at age pnd 5 treated with FGF1 at a concentration of 4 ng/1 (x 25)

Fig. 4. Insulin - synthesizing cells scattered throughout the acinar cells in diabetic hamster pancreas at age pnd 5 after trealement with FGF2 at a concentration of 4 ng/1 (x 25)

82 Fig. 5. Insulin - positive islets of Langerhans in diabetic hamster pancreas at age pnd 5 after treatement with FGF1 at a concentration of 100 ng/1 (x 100)

We have determined the proliferative activity of pancreatic cells by the labelling index (LI). The control for experiments for pnd 1 had labelling index 4,2 ± 0,4%, control for experiments for pnd 5 had labelling index 6 %, control for experiments for pnd 10 had labelling index 6+ 0,4%. FGF1 has shown the strongest effect (18,7 ± 0,6%,p< 0,05 in comparison with the control) at age pnd 5 and at a concentration of 10 ng/1 and the lowest mitogenic effect - (7 ± 1,6%) at pnd 1 and at a concentration of 100 ng/1. FGF2 has displayed the strongest mitogenic effect (26,2 ± 0,4%, p< 0,01 in comparison with the control) at a concentration of 10 ng/1 and at age pnd 5 and the lowest (13 ± 0,4%, p< 0,001 in comparison with the control) — at a concentration of 4 ng/1 and at age pnd 1. FGF7 has shown the strongest mitogenic effect (16,2 ± 0,6%, p< 0,05 in comparison with the control) at a concentration of 10 ng/1 and at age pnd 5 and the lowest effect (5,5 ± 1,6%) — at a concentration of 100 ng/1 and at age pnd 1. The most prominent stimulatory effect (26, 2 ± 0,6 %,p< 0,01 in comparison with the control) was registered at a concentration of 10 ng/1 of FGF2 and at age pnd 5 and the lowest mitogenic effect — (5,5 ± 1,6%) was registered at a concentration of 100 ng/1 FGF7 at age pnd 1.

Discussion

It is generally admitted that the process of neogenesis mostly takes place during natal and early postnatal development. S t e i n e r et al., consider that B cells have a poor regenerative activity and that treatment with STZ leads to B cell destruction and irreversible diabetes [16]. Conversely, in rats treated with STZ at birth or at age pnd 2,3 a destruction of the B cell mass causes diabetes followed by a rapid reappearance of B cell masses [2,9,14].

83 While data related to the development the pancreas accumulate, little is known about the factors that control the proliferation and differentiation of the B cells in normal and diabetic animals. It is established that FGF1, FGF2 and FGF7 are among the growth factors that are expressed in B cells. That is why the purpose of our study was to establish the effect of these FGFs in the early postnatal development of diabetic hamsters and to get a further insight into the process of regeneration of beta cells after treatment with STZ. It is accepted that all endocrine cells of the Langerhans islets arise from the ductal epithelial stem cells through sequential differentiation [11, 15, 17]. Y a m a m o t o et al. reported that most of the cells that had BrdU — positive nuclei after in vivo treatment with STZ were ductal cells, and only a few pancreatic islet cells showed positive reaction to BrdU. Therefore, division of ductal epithelial cells is promoted after loss of B - cells [20]. In our study most of the cells that had 3H - thymidine marked nuclei after in vivo treat ment with STZ were epithelial cells of ducts. Our results have shown that in diabetic hamsters pancreases at age pnd 5 FGF2 acts as a strong stimulator of ductal epithelial cell proliferation and new islet formation.

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