R. F. L. James

GLUCOSE STIMULATES TRANSLOCATION OF THE HOMEODOMAIN TRANSCRIPTION FACTOR PDX1 FROM THE CYTOPLASM TO THE NUCLEUS IN PANCREATIC BETA -CELLS

One of the mechanisms whereby glucose stimulates insulin gene transcription in pancreatic β-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 of32Pi 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 β-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.

The effect of capsule composition on the biocompatibility of alginate-poly-l-lysine capsules

The encapsulation of islets of Langerhans in alginate-poly-l-lysine has been proposed as a method for the immunoprotection of transplanted islets. Although several capsule compositions have been reported, there has been no published study concerning the effect of capsule composition on the severity of the foreign body reaction. Empty capsules were prepared from high mannuronic acid alginate and were coated with: (1) poly-l-lysine alone, (2) poly-l-lysine plus high guluronic acid alginate, or (3) poly-l-lysine plus high mannuronic acid alginate. The capsules were placed in the renal subcapsular space or the peritoneal cavity, and retrieved after three weeks of histological examination. The recipients were WAG/01a, nude (athymic), diabetic BB, and non-diabetes prone BB rats. The severity of reaction to the capsules was determined by measuring the thickness of the pericapsular cell infiltrate or by a scoring system. The severity of the reaction to the capsules was strain-dependent in both the renal and peritoneal sites, with the BB and nude rats displaying the most severe responses. The degree of response was not affected by capsule composition in the renal subcapsular space, but in the peritoneum, the high mannuronic acid alginate capsules provoked the weakest response, and this type of capsule will be used for future transplantation work. The infiltrating cells were characterised by immunohistochemistry and electron microscopy and found to be mostly fibroblasts and macrophages.

Regulation of rat glutathione S-transferase A5 by cancer chemopreventive agents: Mechanisms of inducible resistance to aflatoxin B1

The rat can be protected against aflatoxin B1 (AFB1) hepatocarcinogenesis by being fed on a diet containing the synthetic antioxidant ethoxyquin. Evidence suggests that chemoprotection against AFB1 is due to increased detoxification of the mycotoxin by one or more inducible drug-metabolising enzymes. The glutathione S-transferase (GST) isoenzymes in rat liver that contribute to ethoxyquin-induced chemoprotection against AFB1 have been identified by protein purification. This approach resulted in the isolation of several heterodimeric class alpha GST, all of which contained the A5 subunit and possessed at least 50-fold greater activity towards AFB1-8,9-epoxide than previously studied transferases. Molecular cloning and heterologous expression of rat GSTA5-5 has led to the demonstration that it exhibits substantially greater activity for AFB1-8,9-epoxide than other rat transferases. The A5 homodimer can also catalyse the conjugation of glutathione with other epoxides, such as trans-stilbene oxide and 1,2-epoxy-3-(4-nitrophenoxy)propane, and possesses high catalytic activity for the reactive aldehyde 4-hydroxynonenal. Western blotting has shown that the A5 subunit is not only induced by ethoxyquin but that it is also induced by other cancer chemopreventive agents, such as butylated hydroxyanisole, oltipraz, benzyl isothiocyanate, indole-3-carbinol and coumarin. In addition to GSTA5, we have identified a novel aflatoxin-aldehyde reductase (AFAR) that is similarly induced by ethoxyquin. However, immunoblotting has shown that GSTA5 and AFAR are not always co-ordinately regulated by chemoprotectors. In order to gain a better understanding of the mechanisms responsible for the induction of GSTA5 protein, the GSTA5 gene has been cloned. It was isolated on two overlapping bacteriophage lambda clones and found to be approximately 12 kb in length. The transcriptional start site of GSTA5 has been identified 228 bp upstream from the ATG translational initiation codon. Computer-assisted analysis of the upstream sequence has indicated the presence of a putative antioxidant responsive element (located between -421 and -429 bp) which may be responsible for the induction of GSTA5 by chemopreventive agents.

Engineering a glucose-responsive human insulin-secreting cell line from islets of Langerhans isolated from a patient with persistent hyperinsulinemic hypoglycemia of infancy

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a neonatal disease characterized by dysregulation of insulin secretion accompanied by profound hypoglycemia. We have discovered that islet cells, isolated from the pancreas of a PHHI patient, proliferate in culture while maintaining a beta cell-like phenotype. The PHHI-derived cell line (NES2Y) exhibits insulin secretory characteristics typical of islet cells derived from these patients, i.e. they have no KATP channel activity and as a consequence secrete insulin at constitutively high levels in the absence of glucose. In addition, they exhibit impaired expression of the homeodomain transcription factor PDX1, which is a key component of the signaling pathway linking nutrient metabolism to the regulation of insulin gene expression. To repair these defects NES2Y cells were triple-transfected with cDNAs encoding the two components of the KATP channel (SUR1 and Kir6.2) and PDX1. One selected clonal cell line (NISK9) had normal KATPchannel activity, and as a result of changes in intracellular Ca2+ homeostasis ([Ca2+] i ) secreted insulin within the physiological range of glucose concentrations. This approach to engineering PHHI-derived islet cells may be of use in gene therapy for PHHI and in cell engineering techniques for administering insulin for the treatment of diabetes mellitus.

The optimization of large-scale density gradient isolation of human islets.

The use of the COBE 2991 cell processor (COBE Laboratories, Colorado) for large-scale islet purification using discontinuous density gradients has been widely adopted. It minimizes many of the problems such as wall effects, normally encountered during centrifugation, and avoids the vortexing at interfaces that occurs during acceleration and deceleration by allowing the gradient to be formed and the islet-containing interface to be collected while continuing to spin. We have produced cross-sectional profiles of the 2991 bag during spinning which allow the area of interfaces in such step gradients to be calculated. This allows the volumes of the gradient media layers loaded on the machine to be adjusted in order to mazimize the area of the gradient interfaces. However, even using the maximal areas possible (144.5 cm2), clogging of tissue at such interfaces limits the volume of digest which can be separated on one gradient to 15 ml. We have shown that a linear continuous density gradient can be produced within the 2991 bag, that allows as much as 40 ml of digest to be successfully purified. Such a system combines the intrinsic advantages of the 2991 with those of continuous density gradients and provides the optimal method for density-dependent islet purification.

Impaired expression of transcription factor IUF1 in a pancreatic β-cell line derived from a patient with persistent hyperinsulinaemic hypoglycaemia of infancy (nesidioblastosis)

Persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI), or nesidioblastosis, is a rare disorder which may be familial or sporadic, and which is characterized by unregulated secretion of insulin and profound hypoglycaemia in the neonate. The defect has been linked in some patients to mutations in the sulphonyl urea receptor gene (SUR). The present study investigated potential defects in the regulation of the insulin gene by glucose in a β‐cell line (NES 2Y) derived from a patient with PHHI. The results show that the insulin promoter is unresponsive to glucose in PHHI, and that this defect can be attributed to impaired expression of the transcription factor IUF1. Because IUF1 is involved not only in linking glucose metabolism to the control of the insulin, but is also a major regulator of β‐cell differentiation during embryogenesis, we propose that impaired expression of IUF1 contributes to β‐cell dysfunction in PHHI by leading to abnormal β‐cell differentiation.

Cytotoxic effect of IFN-γ plus TNF-α on human islet cells

Abstract We have previously reported that the combination of IFN-γ plus TNF-α is able to induce the de novo expression of HLA class II on human β cells. In the present study, we have investigated the effect of these cytokines, alone or in combination, on the function and viability of human islet cells in vitro. Three hour insulin release was markedly reduced in human islet monolayer cultures after 4 days exposure to 1000 U/ml of the combination TNF-α plus IFN-γ (36.7 ± 7.7, % of the control ± SEM) or to TNF-α alone (49.5 ± 7% of the control) while IFN-γ had little effect. On direct inspection cell damage was clearly detected only in the cultures treated with TNF-α plus IFN-γ in which staining by indirect immunofluorescence (IFL) for insulin revealed that the number of β cells was also significantly reduced, thus suggesting a real cytotoxic effect of this cytokine combination. This effect was not β cell specific since glucagon release and the number of α cells were also reduced in the cultures exposed to IFN-γ plus TNF-α. 51Cr release experiments supported the cytoxicity of these cytokines to normal islet cells. There was a time course relationship between class II induction (2 days) and the cytotoxic effect of IFN-γ plus TNF-α (4 days) on the same islet cells. In conclusion, these results indicate that the combination of IFN-γ and TNF-α exerts a cytotoxic effect on human islet cells in vitro.

Transfection with SV40 gene of human pancreatic endocrine cells

At present, only islet cell lines of animal origin have been successfully generated (e.g. RIN, HIT). A fully differentiated human beta cell line would be advantageous for diabetes research. We now report the generation of a human endocrine pancreatic cell line obtained by transfection using a plasmid containing the early region of SV40 viral DNA. Viral integration and transcription was assessed by Southern and Northern blotting. This cell line has been growing continuously for more than 2 years and maintains several of the characteristics of the parental cells from which they were generated. The presence of Neuron Specific Enolase, Protein Gene Product 9.5, cytokeratin, microvilli, cytoplasmic electrodense granules and the secretion of insulin, glucagon and somatostatin supports the neuroendocrine origin of this cell line. However, hormone production progressively decreased and finally stopped at passage 8. Flow cytometric analysis showed that HLA expression in this cell line is readily induced by IFN-gamma and modulated by TNF-alpha. The establishment of this human endocrine cell line indicates the feasibility of immortalizing human islets by transfection with viral oncogenes. To obtain a fully differentiated cell line it may be necessary to use other DNA constructs which immortalize the cells without fully transforming their phenotype.

The effect of insulin treatment and of islet transplantation on the resistance artery function in the STZ-induced diabetic rat

1 This study was designed to investigate the influence of insulin treatment and islet transplantation on the smooth muscle contractility and endothelium‐dependent and independent relaxation of resistance arteries in the chemically induced streptozotocin (STZ) diabetic rat after 6–8 weeks, and 12–14 weeks of diabetes, compared to non‐diabetic age‐matched controls. 2 The morphology, and contractile responses to high potassium physiological salt solution (KPSS), KPSS containing 10−5 m noradrenaline (NAK), and concentration‐response curves to noradrenaline (NA) of mesenteric resistance arteries were recorded, along with the endothelium‐dependent relaxation responses to acetylcholine (ACh) and bradykinin (BK), and endothelium‐independent relaxation to sodium nitroprusside (SNP). Concentration‐response curves were then repeated in the presence of a nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine (L‐NOARG). 3 Insulin‐treated diabetic rats in the 12 week study demonstrated enhanced vascular contractility to KPSS, NAK and NA, compared to age‐matched non‐diabetic controls. 4 Incubation with L‐NOARG resulted in both a significant increase in maximum contractile response, and sensitivity (pD2) to NA in the untreated diabetic group (6 weeks). A significant shift in sensitivity was also seen in the insulin‐treated diabetic group. In the 12 week study, incubation with L‐NOARG resulted in an increased maximum contractile response and sensitivity to NA in the insulin‐treated diabetics. An increase in sensitivity was also observed in the untreated diabetic group. 5 Endothelium‐dependent relaxation to ACh was significantly augmented in the untreated diabetics (6‐weeks), compared to the control group. In the 12‐week study, relaxation to both ACh and BK was not significantly different in any of the experimental groups when compared to the sham‐operated non‐diabetic controls. 6 Incubation with L‐NOARG resulted in a significant attenuation of the maximum relaxation response to ACh and BK in all of the experimental groups, in the 6‐ and the 12‐week study. 7 There was no significant difference in the maximum relaxation response or sensitivity to sodium nitroprusside between the diabetic groups and their age‐matched controls in either the 6‐week or the 12‐week study. 8 The results of this study suggest an enhanced release of nitric oxide in the early stages of diabetes, which is more evident in the untreated diabetic rats than the insulin treated, and appears to normalize as the duration of diabetes progresses. This study also shows that the alteration in vascular reactivity of the resistance arteries can be restored to within normal limits by the transplantation of islets of Langerhans, and that islet transplantation is an effective strategy in the correction of the metabolic abnormalities associated with insulin‐dependent diabetes.

Optimisation of gene transfer into vascular endothelial cells using electroporation

OBJECTIVESnWe have examined the conditions required to obtain optimum transfection efficiencies for human umbilical vein endothelial cells by transduction with a plasmid conferring neomycin resistance.nnnMATERIALS AND METHODSnPreliminary studies examined the effects of electric discharges using the Biorad Gene Pulser on endothelial cells. Post-electroporation, there was a significant decrease in cell survival with increasing voltages (100-400 volts; p = 0.03), capacitances [125-960 microFarads (microF); p = 0.02], number of electric pulses (1-2; p = 0.03) and decreasing cell concentrations (p = 0.01). The optimal cell concentration was 3 x 10(6) cells/ml. Transfection studies utilised the neomycin resistance expressing plasmid, pTCF; transfectants were selected with the neomycin analogue G-148.nnnRESULTSnElectro-transfection was optimised with increasing voltages (p = 0.02) and capacitances (p = 0.01) using a single pulse. Optimal transfection was obtained using 400 volts with a capacitance of 960 microF using a single pulse; the median transfection efficiency was 10%. Transduced endothelial cells stably expressed the plasmid for 12 days and at least two cell passages.nnnCONCLUSIONSnThe results indicate that endothelial cells can be efficiently transduced by electroporation to stably express an introduced gene. This may have important implications in vascular surgery.