Insulin secretion by a transplantable rat islet cell tumour

Abstract

Summary. Investigation of the subcellular and molecular components of insulin secretion has been made difficult by the small quantities of material available. The recent development of a transplantable rat islet cell tumour of high insulin content and state of differentiation suggested a system more amenable to analysis. To validate the tumour as a model of secretion we have studied its release of insulin. In acute experiments in vitro immunoreactive insulin release was increased by leucine, glucagon, theophylline and dibutyryl cyclic AMP, though not by glucose. Leucine (20 mmol/1) plus theophylline (5 mmol/1) caused an abrupt, sustained and rapidly reversible stimulation of two-to fivefold. The response was inhibited by antagonists of cellular oxidative phosphorylation (cyanide, 2,4--dinitrophenol, antimycin A), calcium flux (EGTA, verapamil, Mg2 § calmodulin (trifluoperazine), microtubules (vinblastine, colchicine) and by adrenaline and somatostatin. These findings suggest that the tumour secretes insulin by an exocytotic mechanism similar to that of normal islet tissue. Key words: Islet cell tumour, B cell, insulin secretionThe pancreatic B cell has proved difficult to investigate at the subcellular and molecular levels. The classical strategy of subcellular fractionation, purification and reassembly has been greatly hindered by the shortage of starting material [3,4]. In this context the description recently of a transplantable rat islet cell tumour is of considerable interest. The tumour contains large amounts of insulin and consists predominantly of well-granulated cells the morphology of which closely resembles that of rat B cells [1,2].That this islet cell tumour might offer a largescale model of insulin secretion is an attractive possibility. Realisation of this potential, however, depends upon establishing that the tumour is indeed capable of the exocytosis of insulin. We here report a series of experiments in which the characteristics of insulin release by the turnout were defined. The effect of putative secretagogues, the dynamics of insulin release, and the influence of potentiators and inhibitors of secretion are described. Materials and MethodsThe islet cell tumour [1, 2] was maintained by serial subcutaneous transplantation within a colony of inbred albino NEDH strain rats [2]. BuffersTissue was handled in a Krebs Ringer solution [5] containing NaC1 115mmol/1, KC1 5.0mmol/1, NaI-ICO 3 24mmol/1, MgC12 1.0 mmol/1 and CaC12 2.5 mmol/1, freshly equilibrated with 5% CO 2 95% 02. Except where specified glucose 2.8 mmol/l, bovine plasma albumin (Armour Pharmaceuticals, Eastbourne, UK) 1.0 mg/ml, and 5.0 mmol/1 each of sodium pyruvate, monosodium glutamate and disodium fumarate were included (using 95 mmol/l NaC1 to maintain the final sodium ion concentration at 139 mmol/ 1). When individual experiments required further additions the osmolality of control buffers was balanced using sucrose.Chemicals were purchased from Fisons Scientific Apparatus, Loughborough, UK and British Drug House Chemicals, Poole,...

Five new insulin-producing cell lines with differing secretory properties

“…The The response of the cell lines to leucine plus theo¬ phylline and the lack of response to glucose resemble the responses of the parent tumour line (Sopwith et al 1981). The greater response to potassium, however, suggests that the relatively poor response to leucine plus theophylline is not simply due to a lack of stored insulin available for exocytosis.…”

Defective regulation of glucokinase in rat pancreatic islet cell tumours

“…This may explain the sluggish and decreased insulin secretory response of the larger streptozotocin tumours to glucose stimulation (Lenzen et al 1985a). In radiation-induced transplantable pancreatic islet cell tumours the nearly 90% reduction in glucokinase activity, with a 150% increase in hexokinase activity may explain the insulin secre¬ tory unresponsiveness to glucose stimulation (Chick et al 1977;Sopwith et al 1981;Flau et al 1986a), and the high, ultimately fatal, basal cir¬ culating insulin of the B-cell tumour bearing rats (Flatt et al 1986a). In these tumours, glucokinase activity contributed only 15% to the total glucose phosphorylation capacity (Fig.…”

“…These tumours contain more than 90% pancreatic B-cells and exhibit a wide range of growth rates and degrees of dif¬ ferentiation, accompanied by variable changes in insulin secretory responsiveness and enzymatic activities (Rakieten et al 1971;Creutzfeldt et al 1980;Lenzen et al 1985a). In contrast, the radia¬ tion-induced transplantable NEDH rat pancreatic B-cell tumours display a rapid growth rate and high degree of dedifferentiation with a lack of an insulin secretory response to glucose stimulation (Chick et al 1977;Sopwith et al 1981;Flau et al 1986a). …”

In vivo activity and in vitro specificity of CD4+ Th1 and Th2 cells derived from the spleens of diabetic NOD mice.

“…Rat insulinoma tissue (22) was propagated by subcutaneous implantation in NEDH rats (23). The tissue was homogenized in isotonic sucrose medium (24) centrifuged at 1,700 g for 10 min at 40C to yield a postnuclear supernatant, which was then centrifuged at 45,000 g for 30 min to produce a crude membrane fraction (CMF) used in the majority of these studies.…”