Authors showed that almost complete elimination of zinc-insulin complex from cytoplasm of B-cells caused by 3 days prolonged administration of Glibenclamide to animals accompanied by complete disappearing of insulin and zinc-ions from B-cells. Next 6–7 days free of using of Glibenclamide result parallel complete recovery of amount of insulin and zinc in B-cells without any changes of histostructure of islets and function of B-cells.
Pancreatic B-cells of many sorts of animals and of human contained a large amount of zinc-ions which take part in process of forming deposited form of insulin in cells which concentrated in B-granules of cells. In pancreas tissue B-cells contain large amounts of zinc. The major role of zinc is the binding of insulin in hexamers [1]. Zinc ions and insulin create a hexameric, crystalline structure, comprising 2 zinc ions and 6 insulin molecules, which is stored in the secretory granules until secreted in response to metabolic de- mands [2]. Zinc in B-cell secretory granules is involved in the storage and stabilization of the insulin hexamere in B-cells [2, 3]. Zinc ions appear to play important significance in process of microcrystallization of the precipitated insulin granules. May be it is advantage in condensing the stored hormone [2].
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It is known that prescription of Sulphorea result elimination of complex insulin-zinc from B-cells [4]. It was showed that 1 week past of partial or almost complete elimination of zinc-insulin complex from B-cells, function of cells are restored. It is not investigated question: are eliminated from B-cells zinc and insulin, insu- lin only or insulin and part of zinc-ions. Previously it was reported that binding of zinc-ions in B-cells by diabetogenic or not diabetogenic chelat active chemicals result a complete binding of zinc-ions and dissociation of complex 1,5–2 h later: chemicals are removed from cells and zinc remains in the cytoplasm of B-cells [5].
Meanwhile now is not cleared what is insulin and zinc content in cytoplasm of B-cells more long time after elimination of insulin from B-cells.
Aim of work: to investigate insulin and zinc ions content as state of histostructure of pancreatic islets more long period later, as 30 days, after almost complete elimination of zinc-insulin complex from B-cells.
Materials and methods
26 rats Vistar 170–185 g were used. 2 % starch suspension of Glibenclamide (GB) used for peroral ad- ministration to animals in doses 10 and 25 mg/kg for 6 days 1 time daily. Blood Glucose control: 3 h, 24 h, 3 days 15 days and 30 days after past administration of GB). Histostructure of pancreas tissue and insulin content in B-cells were studied 30 days after administration of GB.
Histology. Pancreas tissue were fixed in Bouin. Sections 4–5 mcm were stained by hematoxylin and eosine; insulin staining by aldehyde fucshine [6], pseudoisocyanine [7–9] and zinc-ions — by 8-para(toluenesulphonylamino)quinolin (TSQ) [9]. Intensity of fluorescence of insulin and of zinc-ions was measured by fluorescent histofluorimetric complex constructed by G.G.Meyramov [10]. For transmission electron microscopy samples of pancreas tissue were fixed in 2,5 % Gluthar-aldehyde. Ultrafine sections of tissue contrasted by Reynolds [11] and were investigated on electron microscope JEM-7A.
Results
Blood Glucose concentration past administration of GB. Results showed that maximal decreasing of BG concentration was observed 3 h past first administration of 10 mg/kg (-approx. 20 %) and of 25 mg/kg (-28 %). 24 h past administration there are some not authentically prevalence of BG level in comparison be- fore administration of GB. Next period from 3rd days until 30th days BG concentrations was not changed.
Figure 1
Blood Glucose concentration past administration of GB
T a b l e 1
Insulin and Zinc content in pancreatic B-cells past administration of GB (parameter K)
T a b l e 2
Results of estimation of insulin and zinc content in B-cells showed evident decreasing amount as of in- sulin as of zinc-ions 3 hours after GB administration (Table 2; Fig. 1.1–1.6; Fig. 2.1, 2.2). Decreasing is more marked past administration of 25 mg/kg — for almost 30 % comparatively with approximately 20 % past ad- ministration of 10 mg/kg. Results showed that a coincidence of results between the contents in B-cells of insulin and zinc is available only for intact B-cells and after 24 h and 30 days past administration of drug. 3 hours after administration of GB results showed more intensive decreasing of insulin content com- paratively with content of zinc-ions (Table 2). Results obtained before action of GB were authentically pre- vailed in compared with zinc ions past administration of both doses of GB. 30 days past administration of both doses of GB insulin and zinc content in B-cells were restored completely. There are not any histological changes in pancreatic islets 30 days after action of GB (Fig. 1.2, 1.4).
We found discrepancy more marked differences between results measuring of insulin and zinc-ions content 3 h past action of GB 25 mg/kg (Table 2): approximately 30 % of insulin are eliminated from B-cells and 18–20 % of zinc-ions only eliminated contrary to parallelism of results 30 days past action of GB. It is possible to suppose that part of amount of zinc-ions after dissociation of complex is eliminated from B-cells and part remain in cells (Fig. 3).
- — Intact pancreatic islet. B-cells. Large number of B-granules contained zinc-insulin complex; ×3450;
- — Islet 3 h past administration to animal of GB 25 mg/kg. Marked decreasing of number of B-granules; ×2920
Figure 2. Transmission electron microscopy
Figure 3. Dissociation of complexes Zn+2-insulin and Zn+2-insulin-Dithizon in B-cells
Conclusions
- Elimination of insulin from B-cells by GB accompanied by partial decreasing of zinc-ions content 3 h after action and completely is restored 30 days Insulin content in B-cells reduced for approximately 30 % and of zinc-ions — for 18–20 % 3 hours after administration of 25 mg/kg of GB.
- There are not any histological changes of histostructure of pancreatic islets 30 days past elimination of zinc-insulin complex from B-cells. Amount of insulin and zinc-ions in B-cells restored completely 30 days
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