Multiple effects and stimulation of insulin secretion by the tyrosine kinase inhibitor genistein in normal mouse islets

Isolated perifused rat islets were stimulated with glucose, exogenous insulin, or carbachol. C-peptide and, where possible, insulin secretory rates were measured. Glucose (8 -10 mM) induced dose-dependent and kinetically similar patterns of C-peptide and insulin secretion. The addition of 100 nM bovine insulin had no effect on C-peptide release in response to 8 -10 mM glucose stimulation. The addition of 100 nM bovine insulin or 500 nM human insulin together with 3 mM glucose had no stimulatory effect on C-peptide secretion rates from perifused rat islets. Stimulation with carbachol plus 7 mM glucose enhanced both C-peptide and insulin secretion, and the further addition of 100 nM bovine insulin had no inhibitory effect on C-peptide secretory rates under this condition. Perifusion studies using pharmacologic inhibitors (genistein and wortmannin) of the kinases thought to be involved in insulin signaling potentiated 10 mM glucose-induced secretion. The results support the following conclusions. 1) C-peptide release rates accurately reflect insulin secretion rates from collagenase-isolated, perifused rat islets. 2) Exogenously added bovine insulin exerts no inhibitory effect on release to several agonists including glucose. 3) In the presence of 3 mM glucose, exogenously added bovine or human insulin do not stimulate endogenous insulin secretion.Insulin secretion from the pancreatic ␤-cell is tightly regulated by stimulatory signals generated during the intracellular metabolism of glucose and by neurohumoral agonists operative at the cell membrane (1-5). Most recently an additional layer of complexity has been added by reports suggesting that insulin exerts an autocrine stimulatory effect on insulin secretion from the ␤-cell (6, 7). This concept was based primarily on amperometric measurements using ␤-cells preincubated in 5-hydroxytryptamine (5HT).1 Because 5HT exposure exerts inhibitory effects on insulin release (8 -10), the precise physiologic significance of findings made with 5HT-preloaded ␤-cells is unclear. Moreover, previous studies exploring the potential role of insulin on stimulated secretion showed no effect (11) or supported the concept that insulin exerts a negative, not positive, feedback on its own secretion (12-15).There are at least three major issues that must be addressed in attempting to establish the impact of exogenously added insulin on endogenous insulin secretory rates. The first is technical; it is difficult to accurately measure endogenous insulin release rates in the presence of exogenous insulin. The second relates to concentration of insulin necessary to establish an effect of exogenously added hormone on the ␤-cell. Considering that the ␤-cell continually releases insulin into a small volume of interstitial fluid, the level of insulin bathing the ␤-cell may be quite high. For example, calculations based on islet cell volume, the insulin diffusion constant and insulin secretory rates, suggest that these levels may exceed 100 nM during glucose stimulation (16). Even at rest, levels o...

Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Effects of Glucose, Exogenous Insulin, and Carbachol on C-peptide and Insulin Secretion from Isolated Perifused Rat Islets

Zawalich

2002

“…Similar nonspecificity has been seen on other parameters when using a range of other tyrosine kinase inhibitors in islet cells (32). We therefore turned to the converse approach of using sodium orthovanadate, a tyrosine phosphatase inhibitor that augments tyrosine phosphorylation of several islet cell proteins (Ref.…”

Section: Resultsmentioning

confidence: 99%

Phospholipase C-γ Mediates the Hydrolysis of Phosphatidylinositol, but Not of Phosphatidylinositol 4,5-Bisphoshate, in Carbamylcholine-stimulated Islets of Langerhans

Mitchell

Kelly

Blewitt

et al. 2001

In pancreatic islets the activation of phospholipase C (PLC) by the muscarinic receptor agonist carbamyolcholine (carbachol) results in the hydrolysis of both phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) and phosphatidylinositol (PtdIns). Here we tested the hypothesis that PtdIns hydrolysis is mediated by PLC␥1, which is known to be regulated by activation of tyrosine kinases and PtdIns 3-kinase. PtdIns breakdown was more sensitive than that of PtdInsP 2 to the tyrosine kinase inhibitor, genistein. Conversely, the tyrosine phosphatase inhibitor, vanadate, alone promoted PtdIns hydrolysis and acted non-additively with carbachol. Vanadate did not stimulate PtdInsP 2 breakdown. Carbachol also stimulated a rapid (maximal at 1-2 min) tyrosine phosphorylation of several islet proteins, although not of PLC␥1 itself. Two structurally unrelated inhibitors of PtdIns 3-kinase, wortmannin and LY294002, more effectively attenuated the hyrolysis of PtdIns compared with PtdInsP 2 . Adenovirally mediated overexpression of PLC␥1 significantly increased carbachol-stimulated PtdIns hydrolysis without affecting that of PtdInsP 2 . Conversely overexpression of PLC␤1 up-regulated the PtdInsP 2 , but not PtdIns, response. These results indicate that the hydrolysis of PtdIns and PtdInsP 2 are independently regulated in pancreatic islets and that PLC␥1 selectively mediates the breakdown of PtdIns. The activation mechanism of PLC␥ involves tyrosine phosphorylation (but not of PLC␥ directly) and PtdIns 3-kinase. Our findings point to a novel bifurcation of signaling pathways downstream of muscarinic receptors and suggest that hydrolysis of PtdIns and PtdInsP 2 might serve different physiological ends.Many receptor tyrosine kinases and GPCRs 1 couple to PLC that catalyzes the cleavage of PtdInsP 2 and resultant generation of the two second messengers Ins(1,4,5)P 3 , which mobilizes Ca 2ϩ from intracellular stores, and DAG, which activates protein kinase C (1-4). These pathways are well described in the regulation of insulin secretion following occupation of m3 and m1 muscarinic receptors on the surface of pancreatic ␤Ϫcells (5-8). However we have shown that in addition to the classical pathway of PtdInsP 2 hydrolysis, exposure of pancreatic islets to the muscarinic receptor agonist carbachol also promotes the hydrolysis of PtdIns (9, 10). The latter predominates quantitatively over, and is a precursor of, PtdInsP 2 . Although hydrolysis of either phosphoinositide species results in generation of DAG, only PtdInsP 2 gives rise to a Ca 2ϩ signal via Ins(1,4,5)P 3 production. Therefore the two hydrolytic events may have different functional consequences. The inositol phosphate, corresponding to Ins(1,4,5)P 3 , that is derived from PtdIns, is Ins(1)P 1 . This has no biological function but can be used to quantify PtdIns hydrolysis, at least under the conditions verified in our previous studies (9).The PLC family is comprised of four major groups (PLCs ␤, ␥, ␦, and ⑀). Members of the PLC␤ family are activated by Gproteins in response to o...

“…3, lanes 1, 7, and 10, top and bottom panels; Fig. 4, lanes 1, 3, 5, 7, and 9 of the left panel and lanes 1,5,7, and 9 of the right panel).…”

Section: Resultsmentioning

confidence: 99%

“…The top and bottom panels each show a separate experiment. Samples in lanes 13 and 14 contained methanol equal to that in samples applied to lanes [5][6][7][8][9][10][11][12]. The concentration of Q 1 and Q 2 was 1 mM except in mixtures shown in lanes 9, 11, and 12 of the bottom panel, where the concentration of Q 2 was 0.5 mM.…”

Section: Figmentioning

confidence: 99%

Immunochemical Identification of Coenzyme Q0-Dihydrolipoamide Adducts in the E2 Components of the α-Ketoglutarate and Pyruvate Dehydrogenase Complexes Partially Explains the Cellular Toxicity of Coenzyme Q0

MacDonald

Husain

Hoffmann-Benning

et al. 2004

Coenzyme Q 0 (Q 0 ), a strong electrophile, is toxic to insulin-producing cells. Q 0 was incubated with rat and human pancreatic islets and INS-1 insulinoma cells, and its attachment to cellular proteins was studied with Western analysis using antiserum raised against the benzoquinone ring structure of ubiquinone (anti-Q). Q 0 covalently bonded to two proteins, one of 50 kDa and another of 70 kDa. Both proteins were found to be mitochondrial in human and rat islet cells and in many rat organs. Mitochondria were incubated with Q 0 , and affinity-purified anti-Q was used to immunoprecipitate the 50-kDa protein. Amino acid sequencing identified it as dihydrolipoamide succinyltransferase, the E2 component of the ␣-ketoglutarate dehydrogenase complex (KDC). Western analysis also showed that Q 0 bonds to the E2 components of the purified KDC and the pyruvate dehydrogenase complex (PDC). Dihydrolipoamide acetyltransferase, the E2 of the PDC, has a molecular mass of 70 kDa, and the 70-kDa protein was inferred to be this enzyme. Q 0 was found to bond only to proteins containing dihydrolipoate, and in preparations of mitochondria, thiol reducing agents facilitated the attachment of Q 0 , but oxidizing agents prevented it, suggesting that Q 0 bonds to thiols of dihydrolipoamide. Incubation of human or pig PDC with Q 0 followed by matrix-assisted laser desorption ionization time-of-flight and liquid chromatography/electrospray ionization mass spectrometry analyses of chymotrypsin-digested peptides of PDC E2 confirmed that Q 0 bonds to the dihydrolipoamide in these proteins. In mitochondria, coenzymes Q 1 and Q 2 did not bond to the 50-kDa protein but competed with the bonding of Q 0 to this protein. The prevention by Q 1 of the bonding of Q 0 to KDC E2, as well as other characteristics of the Q 0 effect, are reminiscent of the action of Q 0 on the mitochondrial permeability transition pore described previously (Fontaine, E., Ichas, F., and Bernardi, P. (1998) J. Biol. Chem. 273, 25734 -25740).Ubiquinone analogs (1, 2) and other quinones (3-6) stimulate insulin release from pancreatic islets, but they also kill the insulin-producing cell. The insulin release is not simply due to insulin leaked from dying cells because numerous inhibitors of cellular respiration that kill the cell, such as rotenone, antimycin A, cyanide, and dinitrophenol, inhibit insulin release rather than stimulate it (7, 8). The injurious effects of these compounds may be due, in part, to redox cycling that collapses the proton gradient that maintains the electrical potential of the inner mitochondrial membrane. However, many quinones, because of their electrophilicity, can induce various patterns of oxidative damage to cells. Also, some ubiquinone analogs influence the mitochondrial permeability transition pore (PTP) 1 (9 -11). To learn more about the roles of quinones in these processes, we raised antisera to the ring structure of ubiquinone (anti-Q antibodies) and used them in Western analysis and for immunoprecipitation. This enabled us to identify se...