Human Islets Of Langerhans Research Articles

Acute effects of fatty acids on insulin secretion from rat and human islets of Langerhans

Fatty acids have both stimulatory and inhibitory effects on insulin secretion. Long-term exposure to fatty acids results in impaired insulin secretion whilst acute exposure has generally been found to enhance insulin release. However, there are conflicting data in the literature as to the relative efficacy of various fatty acids and on the glucose dependency of the stimulatory effect. Moreover, there is little information on the responses of human islets in vitro to fatty acids. We have therefore studied the acute effects of a range of fatty acids on insulin secretion from rat and human islets of Langerhans at different glucose concentrations. Fatty acids (0.5 mM) acutely stimulated insulin release from rat islets of Langerhans in static incubations in a glucose-dependent manner. The greatest effect was seen at high glucose concentration (16.7 mM) and little or no response was elicited at 3.3 or 8.7 mM glucose. Long-chain fatty acids (palmitate and stearate) were more effective than medium-chain (octanoate). Saturated fatty acids (palmitate, stearate) were more effective than unsaturated (palmitoleate, linoleate, elaidate). Stimulation of insulin secretion by fatty acids was also studied in perifused rat islets. No effects were observed at 3.3 mM glucose but fatty acids markedly potentiated the effect of 16.7 mM glucose. The combination of fatty acid plus glucose was less effective when islets had been first challenged with glucose alone. The insulin secretory responses to fatty acids of human islets in static incubations were similar to those of rat islets. In order to examine whether the responses to glucose and to fatty acids could be varied independently we used an animal model in which lactating rats are fed a low-protein diet during early lactation. Islets from rats whose mothers had been malnourished during lactation were still able to respond effectively to fatty acids despite a lowered secretory response to glucose. These data emphasise the complex interrelationships between nutrients in the control of insulin release and support the view that fatty acids play an important role in glucose homeostasis during undernutrition.

Metabotropic glutamate and GABA(B) receptors contribute to the modulation of glucose-stimulated insulin secretion in pancreatic beta cells.

The neurotransmitters glutamate and gamma-aminobutyric acid (GABA) could participate in the regulation of the endocrine functions of islets of Langerhans. We investigated the role of the metabotropic glutamate (mGluRs) and GABA(B) (GABA(B)Rs) receptors in this process. We studied the expression of mGluRs and GABA(B)Rs in rat and human islets of Langerhans and in pancreatic alpha-cell and beta-cell lines using RT-PCR and immunoblot analysis. Effects of mGluR and GABA(B) R agonists on insulin secretion were determined by radioimmunoassays and enzyme-linked immunoadsorbent assays (ELISAs). We detected mGluR3 and mGluR5 (but not mGluR1, 6 and 7) mRNAs in all of the samples examined. Trace amount of mGluR2 was found in MIN6 beta cells; mGluR4 was identified in rat islets; and mGluR8 expression was detected in rat islets, RINm5F and MIN6 cells. GABA(B)R1 a/b and 2 mRNAs were identified in islets of Langerhans and MIN6 cells. The expression of mGluR3, mGluR5, GABA(B)R1 a/b and GABA(B)R2 proteins was confirmed using specific antibodies. Group I (mGluR1/5) and group II (mGluR2/3) specific mGluR agonists increased the release of insulin in the presence of 3 to 10 mmol/l or 3 to 25 mmol/l glucose, respectively, whereas a group III (mGluR4/6-8) specific agonist inhibited insulin release at high (10-25 mmol/l) glucose concentrations. Baclofen, a GABA(B)R agonist, also inhibited the release of insulin but only in the presence of 25 mmol/l glucose. These data suggest that mGluRs and GABA(B)Rs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca(2+)-channels, cAMP generation and G-protein-mediated modulation of K(ATP) channels.

Differential effects of genistein on apoptosis induced by fluoride and pertussis toxin in human and rat pancreatic islets and RINm5F cells.

Clonal pancreatic beta-cell lines have been used widely for the study of the factors involved in the regulation of apoptosis but it has not been firmly established that the response of normal islets mirrors that found in transformed beta-cells. In the present work, the role of pertussis toxin (Ptx)-sensitive G-proteins in the control of beta-cell apoptosis was studied in isolated rat and human islets of Langerhans and compared with the clonal beta-cell line, RINm5F. Annexin-V and deoxycarboxyfluoroscein diacetate staining was used to identify viable, apoptotic and necrotic cells directly, under fluorescence illumination. Treatment of human and rat islet cells with the G-protein activator fluoride (NaF; 5 mM) caused a marked increase in apoptosis that was further potentiated in islets pretreated with Ptx. The tyrosine kinase inhibitor genistein (100 microM) also increased islet cell apoptosis and the combination of 100 microM genistein and 5 mM NaF did not lead to any diminution of the apoptotic response. This latter effect was quite different from that seen in RINm5F cells where the combination of 100 microM genistein and 5 mM NaF resulted in much less apoptosis than was observed with either agent alone. In islets treated with a lower concentration of genistein (25 microM; that did not, itself, increase cell death), the drug attenuated NaF-induced apoptosis and also blocked the enhancement mediated by Ptx. These results revealed that human (and rat) islets are equipped with a Ptx-sensitive pathway that may be regulated by tyrosine phosphorylation and is anti-apoptotic. However, they also define conditions under which marked differences in response between RINm5F cells and normal islets were observed and they suggest that care should be taken when extrapolating data obtained with clonal cell lines to the situation in normal islet cells.

Insulin receptor activation inhibits insulin secretion from human islets of Langerhans

There is no consensus on the role of insulin secreted from pancreatic β-cells in regulating its own secretion, either in rodent islets or in human islets. We have now investigated whether there is an autocrine signalling role for insulin in human islets by determining insulin receptor expression and assessing the effects of insulin receptor activation using a non-peptidyl insulin mimetic termed L-783,281. Human insulin receptor mRNA was detected by PCR amplification of human islet cDNA, and translation of the message in human islets was confirmed by Western blotting. Perifusion experiments revealed that both glucose-stimulated and basal insulin secretion were significantly inhibited following human islet insulin receptor activation with L-783,281, and that signalling through phosphatidylinositol 3-kinase (PI 3-kinase) was responsible, at least in part, for this inhibitory effect. These studies indicate that human islets express insulin receptors and that they are functionally coupled to a PI 3-kinase-dependent inhibition of insulin secretion.

Glucose Regulates Islet Amyloid Polypeptide Gene Transcription in a PDX1- and Calcium-dependent Manner

Islet amyloid polypeptide (IAPP) and insulin are expressed in the beta-cells of the islets of Langerhans. They are co-secreted in response to changes in glucose concentration, and their mRNA levels are also regulated by glucose. The promoters of both genes share similar cis-acting sequence elements, and both bind the homeodomain transcription factor PDX1, which plays an important role in the regulation of the insulin promoter and insulin mRNA levels by glucose. Here we examine the role of PDX1 in the regulation of the human IAPP promoter by glucose. The experiments were facilitated by the availability of a human beta-cell line (NES2Y) that lacks PDX1. NES2Y cells also lack operational K(ATP) channels, resulting in a loss of control of calcium signaling. We have previously used these cells to show that glucose regulation of the insulin gene is dependent on PDX1, but not calcium. In the mouse beta-cell line Min6, glucose (16 mm) stimulated a 3.5-4-fold increase in the activity of a -222 to +450 IAPP promoter construct compared with values observed in 0.5 mm glucose. In NES2Y cells, glucose failed to stimulate transcriptional activation of the IAPP promoter. Overexpression of PDX1 in NES2Y cells failed to reinstate glucose-responsive control of the IAPP promoter. Glucose effects on the IAPP promoter were observed only in the presence of PDX1 when normal calcium signaling was restored by overexpression of the two K(ATP) channel subunits SUR1 and Kir6.2. The importance of calcium was further emphasized by an experiment in which glucose-stimulated IAPP promoter activity was inhibited by the calcium channel blocker verapamil (50 microm). Verapamil was further shown to inhibit the stimulatory effect of glucose on IAPP mRNA levels. These results demonstrate that like the insulin promoter, glucose regulation of the IAPP promoter is dependent on the activity of PDX1, but unlike the insulin promoter, it additionally requires the activity of another, as yet uncharacterized factor(s), the activity of which is calcium-dependent.

Pro Islet Amyloid Polypeptide (ProIAPP) Immunoreactivity in the Islets of Langerhans

Islet amyloid is typically found in type 2 diabetes mellitus and is believed to participate in the beta cell deterioration. The islet amyloid fibril consists of the 37-amino-acid islet amyloid polypeptide (IAPP) but its pathogenesis is only partly understood. We developed several different rabbit antisera against the flanking peptides of the IAPP precursor (proIAPP) and the proIAPP processing sites in order to study the possible occurrence of unprocessed proIAPP or parts thereof in islet amyloid. We applied these antisera in an immunohistochemical study on islet amyloid deposits present in a newly generated mouse strain that over-expresses human IAPP but is devoid of mouse IAPP. Male mice of this strain develop severe islet amyloidosis when given a high fat diet. Generally, the antisera showed no immunoreactivity with the amyloid. However, in scattered single beta cells, where amyloid could be seen intracellularly, immunoreactivity with one or more of the antisera co-localized with the amyloid. Although virtually all amyloid in human islets of Langerhans is found extracellularly, we propose that the initial amyloid formation occurs intracellularly, perhaps by not fully processed or folded (pro)IAPP. This amyloid, which may develop rapidly under certain circumstances, probably leads to cell death. If not degraded these amyloid spots may then act as nidus for further amyloid formation from fully processed IAPP, secreted from surrounding beta cells.

Dissociation between Fas expression and induction of apoptosis in human islets of Langerhans.

There is increasing evidence that inappropriate induction of apoptosis in pancreatic beta-cells may precede the development of type 1 diabetes in animal models and in man. One mechanism by which this has been proposed to occur involves up-regulation of the death receptor Fas on beta-cells, resulting in apoptosis of the Fas-bearing beta-cells upon ligation of the receptor. We have examined this hypothesis in isolated human islets of Langerhans and show that--in contrast to data obtained with rodent beta-cells--expression of Fas per se is not sufficient to allow induction of apoptosis upon addition of agonistic anti-Fas serum.

Effects of prolonged exposure to pancreatic glucagon on the function, antigenicity and survival of isolated human islets.

Certain clinical conditions are associated with inappropriately high levels of circulating glucagon. To date, little information is available about the direct effects of prolonged exposure of human islet cells to pancreatic glucagon. In the present study we evaluated the function, antigenicity and survival of human islets exposed for 24 h to human pancreatic glucagon. We prepared human islets of Langerhans by collagenase digestion and density-gradient purification, incubated them for 24 h with 44 or 430 pmol/l pancreatic glucagon at physiological (5.5 mmol/l) glucose level, and evaluated their insulin release function, which was then compared with that obtained from islets kept at high (11.1 mmol/l) glucose concentration. In addition, aliquots of the islets were evaluated to assess their chemotactic properties towards human monocyte-macrophage cells, and their potency to induce cytokine release from human lymphocytes. Finally, survival of the islet cells cultured under varying conditions was evaluated, and an assessment was performed of mRNA expression of Bcl-2 and Bax proteins. The insulin secretion results demonstrated that, compared to the control islets, the islets previously exposed to either 44 or 430 pmol/l glucagon exhibited changes in insulin release in response to glucose, consisting of augmented secretion at low glucose challenge, and no further significant increase at high glucose stimulation, similar to the effects observed with islets pre-cultured with high glucose. These effects were reversible, as documented by the recovery of normal islet sensitivity to glucose after an additional 24-h culture in medium lacking glucagon. Compared to control islets, the culture medium from islets pre-cultured with high glucagon or high glucose showed an increased chemotactic potency towards human monocyte-macrophage cells. In addition, human lymphocytes released a greater amount of tumour necrosis factor alpha when co-cultured with the islets pre-exposed to high glucagon or high glucose, whereas no significant difference was observed (in comparison with control islets) as regards the release of gamma-interferon, interleukin-2 and interleukin-10. The TUNEL technique and RT-PCR showed, respectively, no major difference in cell survival and expression of mRNA encoding for Bcl-2 and Bax protein between control islets and islets kept for 24 h in the presence of high glucagon or high glucose. Our results show that in vitro exposure of human islets to pancreatic glucagon for 24 h causes changes in the function and antigenicity of isolated human islets that are similar to the changes observed after pre-culture with increased glucose levels. Under our experimental conditions, these changes were not accompanied by any evidence of cytotoxicity.

Ontogeny of Human Langerhans Islets. A Review of Some Light- and Electron-Microscopical, Immunohistochemical, and Functional Data on Fetal Development of the Endocrine Pancreas (Review)

The parenchymal cells of the islets of Langerhans belong to the extensive human neuroendocrine system. Its messenger substances are biogenic amines and neurohormonal peptides. Like other neuroendocrine cells, the islet cells might have originated from the neural crest. However, in the fetal life, their stem cells are located in the epithelium of the pancreatic ductuli. As early as at the 8th gestational week, these stem cells have been found to contain secretory granules of the neuroendocrine type. Evidences for production of insulin, somatostatin, glucagon, and PP (the pancreatic polypeptide) have been obtained immunohistochemically in the samples from the 10–12th gestational weeks. In the samples from the 14th week, cell clusters have been observed, which are outgrowing from the ductular epithelium and forming primitive Langerhans islets. The insulin cells predominate markedly and are shown to respond functionally to glucose stimulation. By the 16th week, the islets become vascularized, with the primary innervation. The completely formed endocrine pancreas, as it is observed at birth, is revealed at the 26th gestational week. Based on some light-microscopical, ultrastructural, and immunohistochemical characteristics of the islet parenchymal cells and their supply with blood vessels and nerves, three phases of the gland embryonal/fetal development are identified.

Improved method for the isolation and purification of human islets of Langerhans using Liberase™ enzyme blend

To determine the effects of procedural modifications, 23 human islet isolations were analyzed. Isolations were divided into two groups based on the enzyme used. The influence of Liberase™, with an improved method of mechanical disassociation of pancreas, was compared to an automated method using Sevac collagenase. Pancreases were processed within 10 h of cross clamping. Following ductal injection of the enzyme, tissue was placed in the digestion chamber for disassociation. Purification was accomplished using a COBE 2991 cell processor and continuous gradients of Hypaque EuroFicoll. Isolations in Group I (Sevac) had an average yield of 138,602 ± 128,364 islet equivalents (IE) (2083 ± 1679 IE/g) with a purity of 85 ± 11%. Group II (Liberase) showed an average yield of 389,586 ± 191,161 IE (5,958 ± 3,083 IE/g) with a purity of 90 ± 6.8%. Viability was confirmed by fluorescein diacetate and propidium iodide staining, static incubations, and perifusions. In conclusion, the combination of the enzyme blend, Liberase, and a more gentle system of disassociation has proven to be a more productive method of islet isolation with higher purity than the previously published methods.

GLUCAGON DECREASES CYTOKINE INDUCTION OF NITRIC OXIDE SYNTHASE AND ACTION ON INSULIN SECRETION IN RIN5F CELLS AND RAT AND HUMAN ISLETS OF LANGERHANS

Nitric oxide synthase, induced by cytokines in insulin-containing cells, produces nitric oxide which inhibits function and may promote cell killing. Since glucagon was shown to prevent inducible nitric oxide synthase (iNOS) expression in rat hepatocytes it was of interest to examine the action of glucagon (and cyclic AMP) on iNOS induction in insulin-producing cells. Cultured RIN5F cells and primary rat and human islets of Langerhans were treated with interleukin 1β (IL-1β) or a combination of cytokines, and were co-treated or pre-treated with glucagon. In RIN5F cells, the activity of iNOS induced by IL-1β (10pM, 24h), was significantly reduced by glucagon (1000nM), which raises cyclic AMP, and by forskolin (1–10μM), a non specific activator of adenylate cyclase. Glucagon and forskolin also decreased iNOS expression in RIN5F cells, and rat and human islets, as shown by Western blotting. The inhibitory action of IL-1β (100pM, 24h) on rat islet insulin secretion was partially reversed by 1-h pre-treatment with glucagon (10–1000nM), while the contrasting stimulatory effect of 48-h treatment with cytokines on insulin secretion from human islets was similarly prevented by glucagon (1000nM) pre-treatment. These results suggest that glucagon inhibits iNOS expression in insulin-containing cells and imply that glucagon could modulate the inhibitory effects of cytokines.

Human islets of Langerhans express the C isoform of calcium/calmodulin‐dependent protein kinase II

Background There is considerable evidence that calcium/calmodulin-dependent protein kinase II (CaM kinase II) plays a key role in insulin secretion and the enzyme provides a candidate gene for Type 2 diabetes. Since several isoforms of the enzyme exist, it is essential to define which are expressed by the β-cell. Methods A human islet cDNA library in λZAPII was screened with a probe for the 5′-end of human γ CaM kinase II. Since this region is very homologous between the different isoforms, it is expected that isoforms other than γ would be detected. From each of the six positive clones obtained, DNA was prepared and subjected to PCR using primers spanning the variable region in which the main variability of CaM kinase II isoforms resides. PCR products were purified and sequenced in both directions. The β-cell line MIN6 was screened for CaM kinase II δ by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blotting. Results The sequences of five of the human islet PCR products indicated that the clones corresponded to the γB isoform whose expression in human islets we have previously documented. The other PCR product, however, gave a sequence containing the variable domains II and VII characteristic of CaM kinase II δ. This sequence and the absence of other domains in this region identified the clone as CaM kinase II δC. The expression of CaM kinase II δ in MIN6 β-cells was confirmed by RT-PCR and by Western blotting. Conclusions Human islets of Langerhans express the δC isoform of CaM kinase II. Copyright © 1999 John Wiley & Sons, Ltd.

Characterization of monoamine oxidase isoforms in human islets of langerhans

In this paper, we describe the characterization of the expression of monoamine oxidase (MAO) in whole pancreas and in isolated islets of Langerhans from human. Classical monamine oxidase activity assays reveal that both isoforms A & B are present in human pancreas. Two complementary approaches indicated that both MAO A and B are expressed in isolated islet: RT-PCR using specific primers revealed amplification products with the expected size for MAO-A and MAO-B: two peptides corresponding to MAO A (~ 61 kDa) and B (~ 55 kDa) were detected using a polyclonal anti MAO-A/MAO-B antiserum. Western blotting and subsequent densitometric analysis indicate that whole and endocrine pancreas express the two isoforms with different relative proportions. Islets appear to express almost twice as much MAO protein as whole pancreas, in near equal proportions of the two isoforms, whereas whole pancreas expresses more MAO-A than the B isoform. The expression of MAO A and B in islets could be the first step toward the characterization of the functional properties of these enymes in the endocrine pancreas.

Glucagon-like peptide 1 has a physiological role in the control of postprandial glucose in humans: studies with the antagonist exendin 9-39.

Glucagon-like peptide 1(7-36) amide (GLP-1) is postulated to be the major physiological incretin in humans, but evidence is indirect. We report the first studies examining the physiological role of GLP-1 in the postprandial state in humans using the GLP-1 antagonist exendin 9-39. Exendin 9-39 completely blocked GLP-1-induced glucose-stimulated insulin release from perifused human islets of Langerhans. In healthy fasted volunteers, intravenous infusion of exendin 9-39 at 500 pmol x kg(-1) x min(-1) in the hyperglycemic state abolished the insulinotropic effect of a physiological dose of GLP-1 and fully reversed the glucose-lowering effect of GLP-1. Nine healthy subjects consumed a 150-g oral glucose tolerance test and were infused with 500 pmol x kg(-1) x min(-1) exendin 9-39 or saline. Exendin 9-39 increased the peak postprandial glucose level (exendin 9-39, 8.67 +/- 0.35 vs. saline, 7.67 +/- 0.35 mmol/l, P < or = 0.005) and increased postprandial plasma glucose incremental area under the curve by 35% (exendin 9-39, 152 +/- 19 vs. saline, 113 +/- 16 mmol x min x l(-1), P < or = 0.05). This could be explained as partly secondary to the blockade of glucose-induced suppression of glucagon and maybe also to an increased rate of gastric emptying. Thus, in humans exendin 9-39 acts as an antagonist of GLP-1 both in vitro and in vivo. When infused alone, exendin 9-39 causes a deterioration in postprandial glycemic control, suggesting that GLP-1 may be important for maintenance of normal postprandial glucose homeostasis in humans.

Glucose Stimulates Translocation of the Homeodomain Transcription Factor PDX1 from the Cytoplasm to the Nucleus in Pancreatic β-Cells

One of the mechanisms whereby glucose stimulates insulin gene transcription in pancreatic beta-cells involves activation of the homeodomain transcription factor PDX1 (pancreatic/duodenal homeobox-1) via a stress-activated pathway involving stress-activated protein kinase 2 (SAPK2, also termed RK/p38, CSBP, and Mxi2). In the present study we show, by Western blotting and electrophoretic mobility shift assay, that in human islets of Langerhans incubated in low glucose (3 mM) PDX1 exists as an inactive 31-kDa protein localized exclusively in the cytoplasm. Transfer of the islets to high (16 mM) glucose results in rapid (within 10 min) conversion of PDX1 to an active 46-kDa form that was present predominantly in the nucleus. Activation of PDX1 appears to involve phosphorylation, as shown by incorporation of 32Pi into the 46-kDa form of the protein. These effects of glucose could be mimicked by chemical stress (sodium arsenite), or by overexpression of SAPK2 in the beta-cell line MIN6. Overexpression of SAPK2 also stimulated PDX1-dependent transcription of a -50 to -250 region of the human insulin gene promoter linked to a firefly luciferase reporter gene. The effects of glucose were inhibited by the SAPK2 inhibitor SB 203580, and by wortmannin and LY 294002, which inhibit phosphatidylinositol 3-kinase, although the effects of stress (arsenite) were inhibited only by SB 203580. These results demonstrate that glucose regulates the insulin gene promoter through activation and nuclear translocation of PDX1 via the SAPK2 pathway.

IL-1 produced and released endogenously within human islets inhibits beta cell function.

Resident macrophages have been suggested to participate in the initiation of beta cell damage during the development of autoimmune diabetes. The purpose of this study was to determine if the endogenous production and release of interleukin 1 (IL-1) in human islets of Langerhans by resident macrophages results in the inhibition of beta cell function. Treatment of human islets with a combination of tumor necrosis factor (TNF) + lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) stimulates inducible nitric oxide synthase (iNOS) expression, nitric oxide production, and inhibits glucose-stimulated insulin secretion. The IL-1 receptor antagonist protein (IRAP) prevents TNF + LPS + IFN-gamma-induced iNOS expression and nitrite production, and attenuates the inhibitory effects on glucose-stimulated insulin secretion by human islets. Inhibition of iNOS activity by aminoguanidine also attenuates TNF + LPS + IFN-gamma-induced inhibition of insulin secretion by human islets. These results indicate that the inhibitory effects of TNF + LPS + IFN-gamma are mediated by nitric oxide, produced by the actions of IL-1 released endogenously within human islets. Reverse transcriptase polymerase chain reaction was used to confirm that TNF + LPS + IFN-gamma stimulates the expression of both IL-1alpha and IL-1beta in human islets. Two forms of evidence indicate that resident macrophages are the human islet cellular source of IL-1: culture conditions that deplete islet lymphoid cells prevent TNF + LPS + IFN-gamma-induced iNOS expression, nitric oxide production, and IL-1 mRNA expression by human islets; and IL-1 and the macrophage surface marker CD69 colocalize in human islets treated with TNF + LPS + IFN-gamma as determined by immunohistochemical analysis. Lastly, nitric oxide production is not required for TNF + LPS + IFN-gamma-induced IL-1 release in human islets. However, cellular damage stimulates IL-1 release by islet macrophages. These findings support the hypothesis that activated islet macrophages may mediate beta cell damage during the development of insulin-dependent diabetes by releasing IL-1 in human islets followed by cytokine-induced iNOS expression by beta cells.

Human islets of Langerhans express Fas ligand and undergo apoptosis in response to interleukin-1beta and Fas ligation.

IDDM results from a progressive loss of pancreatic beta-cells that, in humans, may be triggered by a combination of genetic and environmental factors. Recently, attention has been focused on the hypothesis that the loss of beta-cells is initiated by inappropriate induction of apoptosis. We now demonstrate that human islets of Langerhans undergo apoptosis upon exposure to interleukin-1beta. The cytokine also sharply increases the number of cells that enter apoptosis on treatment with a stimulatory anti-Fas antibody. Western blotting and immunocytochemistry clearly show for the first time that human pancreatic beta-cells normally express Fas ligand. The results suggest that human islet cells are primed to undergo apoptosis by interleukin-1beta and that this involves the close association between cell-surface Fas and its ligand.

Human Islets of Langerhans Express Multiple Isoforms of Calcium/Calmodulin-Dependent Protein Kinase II

Previous studies have provided evidence for the presence of calcium/calmodulin-dependent protein kinase II (CaM kinase II) in rodent islets of Langerhans, and β-cell CaM kinase II activity has been correlated with insulin secretion. In this study we provide the first conclusive evidence for the expression of CaM kinase II in human islets of Langerhans and show that multiple isoforms are expressed. Screening of a human islet cDNA library resulted in the isolation of a 999bp partial cDNA clone encoding CaM kinase II. The nucleotide sequence of the islet clone showed a high degree of homology (94.8%) to the two γ isoforms of CaM kinase II previously isolated from human T lymphocytes (γBand γC). In order to obtain full length sequence for the islet clone, rapid amplification of cDNA ends (RACE) was used to amplify the 3′ end of the islet clone from human islet poly A+RNA. Two distinct γ isoforms of CaM kinase II were amplified from the islet RNA. They were identified as γBand γE; the latter is distinguished from γBby a 114bp insertion within the association domain of the cDNA. Using reverse transcriptase polymerase chain reaction (RT-PCR) we also detected in human islets of Langerhans the novel β3isoform of CaM kinase II previously reported to be expressed in neonatal rat islets.

A truncated isoform of Ca 2+/calmodulin-dependent protein kinase II expressed in human islets of Langerhans may result from trans-splicing

Calcium/calmodulin-dependent protein kinase II (CaM kinase II) has been proposed to play a key role in glucose stimulated insulin secretion. Using the rapid amplification of cDNA ends technique we amplified the 3′ end of the CaM kinase II γ gene from human islet RNA. A novel cDNA was detected composed of 5′ sequence from the human CaM kinase II γ gene joined to the 3′ end of the human signal recognition particle 72 (SRP72) gene. We predict that this mRNA species will code for a truncated form of CaM kinase II, designated γ SRP, comprising the entire catalytic and regulatory domains of the protein and with a predicted molecular weight of 37 kDa. We mapped the human SRP72 gene to chromosome 18 and, as the CaM kinase II γ gene was previously mapped to human chromosome 10q22, we suggest this novel cDNA may have resulted from trans-splicing.

The effect of the putative endogenous imidazoline receptor ligand, clonidine-displacing substance, on insulin secretion from rat and human islets of Langerhans.

1. The effects of a rat brain extract containing clonidine-displacing substance (CDS), a putative endogenous imidazoline receptor ligand, on insulin release from rat and human isolated islets of Langerhans were investigated. 2. CDS was able to potentiate the insulin secretory response of rat islets incubated at 6 mM glucose, in a dose-dependent manner. The magnitude of this effect was similar to that in response to the well-characterized imidazoline secretagogue, efaroxan. 3. CDS, like other imidazoline secretagogues, was also able to reverse the inhibitory action of diazoxide on glucose-induced insulin release, in both rat and human islets. 4. These effects of CDS on secretion were reversed by the imidazoline secretagogue antagonists, RX801080 and the newly defined KU14R, providing the first evidence that imidazoline-mediated actions of CDS can be blocked by specific imidazoline antagonists. 5. The effects of CDS on insulin secretion were unaffected when the method of preparation involved centri-filtration through a 3,000 Da cut-off membrane or when the extract was treated with protease. These results confirm that the active principle is of low molecular weight and is not a peptide. 6. Overall, the data suggest that CDS behaves as a potent endogenous insulin secretagogue acting at the islet imidazoline receptor.