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P. M. Jones, S. J. Persaud, and S. L. Howell

ABSTRACT

Protein kinase C (PKC) has been identified in islets of Langerhans and insulin-secreting tumour cells. Diacylglycerols (DAGs, the endogenous PKC activators) are generated in response to insulin secretagogues, although nutrient and non-nutrient secretagogues generate DAGs of different compositions and of different potencies as PKC activators. Exogenous activators of PKC stimulate insulin secretion from B cells, but attempts to define a physiological role for PKC by using inhibitors of this enzyme have produced ambiguous results. However, in studies using PKC-depleted B cells the loss of PKC activity does not inhibit glucose-induced insulin secretion, but markedly reduces responses to cholinergic agonists. These observations are supported by measurements of PKC activation which suggest that the enzyme is activated by cholinergic agonists, but not by nutrient secretagogues. Currently available experimental evidence therefore suggests that activation of PKC is not essential for nutrient-induced insulin secretion, but is required for the expression of a normal secretory response to cholinergic neurotransmitters.

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D. J. Slee, P. M. Jones, and S. L. Howell

ABSTRACT

Proinsulin conversion to insulin was studied using electrically permeabilized rat islets of Langerhans. Using high-performance liquid chromotography separation of radiolabelled islet proteins, we have demonstrated that conversion was dependent upon temperature, sensitive to pH and regulated by MgATP. Ammonium acetate was used to collapse the granular pH gradient, over a pH range of 3·5–7·4. Conversion was optimum at pH 4·5–5·5 and was reduced, but not abolished, at pH 7·4. Ca2+ (10 μm) and 4β-phorbol 12-myristate 13-acetate (500 nm), which are insulin secretagogues in permeabilized islets, caused no significant stimulation of conversion.

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A. M. Band, P. M. Jones, and S. L. Howell

ABSTRACT

There is growing evidence that arachidonic acid (AA) and/or its metabolites may be involved in the control of insulin secretion. We have now investigated the effect of AA on insulin secretion from rat islets, and the possible involvement of protein kinase C (PKC) in this process. Exogenous AA stimulated insulin secretion from intact islets at a substimulatory concentration of glucose (2 mm), but did not further enhance glucose-induced (20 mm) insulin secretion. AA-induced insulin secretion was temperature dependent. The secretory responses seen at 37°C were totally abolished by reducing the incubation temperature to ≤34°C. AA-induced insulin secretion was not dependent upon extracellular Ca2+ and was potentiated by omission of Ca2+ or bovine serum albumin from the media. PKC in rat islets can thus be stimulated by AA, but the stimulation of PKC is not required for AA-induced insulin secretion.

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T. Bjaaland, P. M. Jones, and S. L. Howell

ABSTRACT

The roles of calcium, cyclic AMP (cAMP), activation of protein kinase C (PKC) and the effect of ATP on glucagon secretion were investigated in intact and permeabilized rat islets of Langerhans, Ca2+ (10 nm-10 μm) stimulated glucagon secretion from electrically permeabilized islets in a dose-dependent manner. Forskolin and cAMP stimulated secretion from intact and permeabilized islets respectively, the latter at both sub-stimulatory (50 nm) and stimulatory (10 μm) Ca2+ concentrations. The tumour-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) increased secretion from both intact and permeabilized islets. In the latter, PMA increased glucagon release at both Ca2+ concentrations, the effect being enhanced at the stimulatory Ca2+ concentration, over and above that caused by Ca alone. Reduction of ATP content by incubation with the metabolic inhibitor 2,4-dinitrophenol resulted in an increased basal release of glucagon from intact islets, whilst arginine-induced glucagon secretion was abolished in both intact and permeabilized islets. Ca2+-induced glucagon secretion required MgATP in the permeabilized islets of Langerhans. These results suggest that Ca2+ acts as an initiator of glucagon secretion, whilst cAMP and activation of PKC may exert their effect as modulators of secretion. ATP is required for glucagon secretion in electrically permeabilized islets and is necessary for arginine-induced glucagon secretion in both intact and permeabilized islets.

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T. Bjaaland, C. S. T. Hii, P. M. Jones, and S. L. Howell

ABSTRACT

This study investigated the effect of pretreatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) on arginine-induced glucagon secretion. Isolated islets of Langerhans were pretreated by culturing for 18–24 h in the presence of 200 nm of the tumour-promoting phorbol ester PMA or 200 nm of the non-tumour-promoting phorbol ester 4-phorbol didecanoate (PDD). Islets pretreated with PMA did not secrete glucagon in response to 0·1 or 1 μm PMA on subsequent incubation, in contrast to PDD-pretreated islets which responded significantly on subsequent incubation with PMA. Pretreatment with PMA led to impairment of arginine-induced glucagon secretion. PMA-pretreated islets permeabilized by high-voltage discharge retained their normal secretory responses to calcium and cyclic AMP, but had an impaired secretory response to PMA. These results suggest (1) that protein kinase C (PKC) is likely to be present in the A cell, (2) that short-term culture in tumour-promoting phorbol ester leads to down-regulation of PKC, (3) that the PKC pathway is involved in arginine-induced glucagon secretion and (4) that pretreatment does not effect the A cell response to other intracellular mediators.

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K J Parker, P M Jones, C H Hunton, S J Persaud, C G Taylor, and S L Howell

ABSTRACT

The liberation of arachidonic acid (AA), by phospholipase A2 (PLA2), is the rate-limiting step in a number of cell signalling pathways. In the pancreatic β-cell, AA itself is thought to participate in the regulation of insulin secretion. Recently a Ca2+-sensitive, AA-selective cytosolic PLA2 (type IV cPLA2) has been isolated from the human monocyte U937 cell line. Although the DNA sequence of this enzyme implies a molecular weight of 85 kDa, the protein migrates with a molecular weight of 100-110 kDa on SDS-PAGE. In many cell types, cPLA2s which are reactive towards antibodies raised against the type IV cPLA2 have been shown to hydrolyse AA from membrane glycerophospholipids. Using a polyclonal antibody raised against a recombinant form of type IV cPLA2, we have detected an immunoreactive protein with a molecular weight of 93·5 kDa in rat islets of Langerhans. Furthermore, we have detected similar immunoreactive proteins in insulin-secreting β-cell lines and have shown co-expression of type IV cPLA2 immunoreactivity and insulin immunoreactivity in rat pancreatic β-cells. Under non-stimulatory conditions the 93·5 kDa immunoreactive protein detected in rat islets of Langerhans was located predominantly in the cytosolic fraction. We have shown that immunoprecipitation of the rat immunoreactive protein from rat islet homogenates significantly decreases the total dithiothreitol/β-mercaptoethanol-insensitive PLA2 activity by 56·4±7% This provides further evidence that the immunoreactive rat protein is a type IV cPLA2 and is responsible for a large component of the PLA2 activity in rat islets of Langerhans. It is possible that, in the rat β-cell, type IV cPLA2 couples the increase in intracellular Ca2+, brought about by insulin secretagogues, to the liberation of AA and the subsequent release of insulin.