Islet transplantation is associated with a high rate of early graft failure, a problem that remains poorly understood. It is probable that the destruction of the islet microenvironment and loss of tropic support that occur during isolation lead to compromised survival. The purpose of this study was to determine the role of matrix-integrin interactions on beta-cell survival and function following islet isolation. Canine islets were obtained by conventional methods. Immediately after isolation, the peri-insular basement membrane (BM) was absent. The ability of islets maintained in suspension culture to attach to a collagen matrix declined progressively over 6 days. Attachment could be blocked by an arginine-glycine-aspartate (RGD) motif-presenting synthetic peptide, thereby implicating an integrin-mediated process. Characterization of cell surface integrins by immunocytochemistry (ICC) demonstrated that the expression of integrins alpha3, alpha5 and alphaV diminished during the culture period. This change was coincident with both a decrease in beta-cell function (proinsulin gene expression, islet insulin content and stimulated insulin release) and a rise in beta-cell death from apoptosis, as determined by in situ cell death detection (TUNEL) assay. These adverse events were prevented or delayed by exposure of islets to matrix proteins. In conclusion, routine islet isolation disrupts the cell-matrix relationship leading to a variety of structural and functional abnormalities, including apoptotic cell death. These alterations can be diminished by restoration of a culture microenvironment that includes matrix proteins.
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R Wang, J Li, and L Rosenberg
We have previously shown that isolated islets embedded in type 1 collagen gel in the presence of a defined medium undergo transdifferentiation within 96 h to duct epithelial structures. The aim of this study was to identify the factors implicated in this process. Freshly isolated canine islets were embedded in type 1 collagen gel, Matrigel or agarose for up to 120 h and cultured in (i) Dulbecco's modified Eagle's medium (DMEM)/F12 plus cholera toxin (CT), (ii) medium CMRL1066 plus CT, (iii) CMRL1066 plus forskolin and (iv) CMRL1066 alone. At 16 h, intracellular levels of cAMP (fmol/10(3) islets) were increased in groups i-iii (642+/-17, 338+/-48, 1128+/-221) compared with group iv (106+/-19, P<0.01). Epithelial differentiation correlated with the total amount of intracellular cAMP measured over 120 h. Islet-epithelial transformation during the initial 36 h was associated with a wave of apoptosis which was followed by a wave of cell proliferation. During epithelial differentiation there was a progressive loss of all islet hormones and the concomitant expression of cytoskeletal proteins characteristic of duct epithelial cells. Islets in collagen and Matrigel demonstrated high rates of epithelial differentiation (63+/-2% and 71+/-4% respectively) compared with those in agarose gel (0+/-0%, P<0.001). Islets suspended in DMEM/F12 plus CT supplemented with soluble laminin or fibronectin did not undergo transformation. Prior incubation of freshly isolated islets with an integrin-binding arginine-glycine-aspartate motif-presenting synthetic peptide also reduced islet transformation. These studies confirm the biological potential of islets of Langerhans to differentiate to duct epithelial structures. cAMP-mediated signal transduction and an appropriate integrin-matrix interaction are necessary for this process to proceed.
J Chen, L Wang, YS Boeg, B Xia, and J Wang
Secreted by white adipose tissue as a hormone, resistin was identified as a possible link between obesity and insulin resistance. High circulating resistin levels were observed to correlate with obesity. Administration of resistin lowered the glucose tolerance threshold and impaired insulin activity; whereas anti-resistin antibodies had the opposite effects. However, contradictory data were subsequently reported in regard to the correlation between resistin expression level and obesity or type 2 diabetes. Two additional proteins that share a highly homologous C-terminus with resistin have been identified in mouse, and one in human, forming a resistin-related protein family. Resistin was shown to dimerize through a disulfide bond formed by the N-terminal-most cysteine (Cys26). Here we demonstrate that while Cys26 is both necessary and sufficient for homodimer formation, all three resistin family members can also interact with one another regardless of the presence of Cys26 through non-covalent interactions. Furthermore, protein crosslinking analysis indicated that resistin and resistin beta, but not resistin alpha, exist as multimers, probably with a dimer as the subunit. The multiple protein complex formation is obviously at a level higher than the Cys26 disulfide bonding. These results suggest the potential importance of considering intermolecular interactions among resistin family members in studying their functions.
Weiye Wang, Lishan Wang, Akira Endoh, Geoffrey Hummelke, Christina L Hawks, and Peter J Hornsby
In order to establish whether there are differences in DNA-binding proteins between zona fasciculata (ZF) and zona reticularis (ZR) cells of the human adrenal cortex, we prepared nuclear extracts from separated ZF and ZR cells. The formation of DNA–protein complexes was studied using an element in the first intron of the type I and type II 3β-hydroxysteroid dehydrogenase genes (HSD3B1 and HSD3B2). Using the element in the HSD3B2 gene as a probe, a complex (C1) was formed with extracts from ZF cells but was formed only at a low level with ZR cell extracts. Another pair of complexes (C2/C3) was formed with both ZF and ZR cell extracts. The ZF-specific protein forming C1 was enriched by column chromatography on DEAE-Sepharose and carboxymethyl-Sepharose. Oligonucleotide competition analysis on the enriched fraction gave results consistent with those obtained on the unfractionated material. A further enrichment was brought about by passing the protein over an oligonucleotide affinity column based on the HSD3B2 element. The protein bound to the column was identified as α-enolase by mass spectrometry. Although α-enolase is a glycolytic enzyme, it binds to specific DNA sequences and has been found to be present in nuclei of various cell types. We performed immunohistochemistry on sections of adult human adrenal cortex and found α-enolase to be located in nuclei of ZF cells but to be predominantly cytoplasmic in ZR cells. Transfection of an α-enolase expression vector into NCI-H295R human adrenocortical cells increased HSD3B2 promoter activity, suggesting a possible functional role for this protein in regulation of HSD3B2 expression.
J.-F. Wang, L. J. Fraher, and D. J. Hill
We have characterized an insulin-like growth factor (IGF)-binding protein present in ovine amniotic fluid. Using an activated charcoal-binding assay, whole amniotic fluid specifically bound approximately 20–30% of 125I-labelled human (h) IGF-II added, while the binding of 125I-labelled hIGF-I was minimal. Radioimmunoassay for IGF-I or -II in ovine biological fluids showed that values in amniotic fluid were 9- to 13-fold less than in fetal plasma, while gel filtration of amniotic fluid on Sephadex G-50 eluted with 1 mol acetic acid/l revealed no additional binding activity which had been complexed to IGFs at neutral pH. Together, these observations suggest that the binding activity in amniotic fluid is largely unsaturated. Competition studies for the displacement of 125I-labelled IGF-II binding to amniotic fluid by increasing amounts of unlabelled IGF-I or -II, using the charcoal assay, showed that IGF-II was 30-fold more potent than IGF-I. Scatchard analysis revealed a single class of binding site for IGF-II, with a binding affinity of 0·68 ±0·18 litres/nmol (mean ± s.d., n = 3). Ligand blot analysis of amniotic fluid by separation on 8% SDS-PAGE, transfer to nitrocellulose membranes, incubation with 125I-labelled IGF-II and autoradiography revealed a single band of IGF-binding protein with approximate molecular size of 38 kDa. Additional IGF-binding species of 20, 28, 48 and > 180 kDa were present in ovine fetal plasma. Separation of amniotic fluid on Concanavalin A–Sepharose revealed that it had little carbohydrate content. These results show that ovine amniotic fluid contains an unsaturated, non-glycosylated IGF-binding protein with high affinity for IGF-II. These characteristics differ from those of the IGF-binding proteins purified from human amniotic fluid.
Journal of Endocrinology (1990) 127, 325–333
H. S. Wang, J. Lim, J. English, L. Irvine, and T. Chard
Serum levels of insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein-1 (IGFBP-1) have been determined by radioimmunoassay in the maternal circulation (n = 91) and in the umbilical artery (n = 56) and vein (n = 90) of man. In both the umbilical artery and vein, the concentration of serum IGF-I showed an inverse correlation with birthweight (P < 0·005 and P < 0·001 respectively); the mean serum IGF-I levels in the small-for-gestational-age (SGA) group were significantly higher than those in average-for-gestational-age (AGA) neonates (P <0·01 and P < 0·001 respectively). However, maternal serum IGF-I showed no association with birthweight and there was no significant difference between the SGA and AGA groups. These observations imply that the production of IGF-I in the maternal and fetal compartments is independent and that there is unlikely to be transfer of IGF-I across the placenta. Serum IGFBP-1 levels in both maternal and umbilical cord blood (artery and vein) showed an inverse relation to birthweight (P <0·001, P<0·005 and P<0·001 respectively). Increased IGFBP-1 levels in the umbilical artery and vein were observed in the SGA group. These findings suggest that IGFBP-1 might inhibit the action of IGF-I in both the maternal and the fetal compartments and that the rise in IGFBP-1 could be a primary factor in retardation of fetal growth. Alternatively, circulating IGF-I and IGFBP-1 levels may only be a secondary reflection of local tissue events involved in fetal growth.
Journal of Endocrinology (1991) 129, 459–464
F. J. DARBY, D. Y. WANG, and A. L. GREENBAUM
(1) Changes in the content of lactose, DNAP, RNAP and acid-soluble ribonucleotides have been followed in the involuting mammary tissue of rats, and of rats treated with reserpine, for periods up to 6 days after removal of the pups.
(2) After a rise in the first 24 hr. after separation from the litter, the lactose content declines and is virtually absent after 5 days. Administration of 75 μg. reserpine a day retards the rate of this disappearance.
(3) The DNAP level falls progressively after the first 24 hr. in the normally involuting gland. Reserpine prevents this decline during the first 4 days. Seventy-two hours after weaning there is a sharp peak in the DNAP content of involuting glands from reserpine-treated rats. A similar pattern of change is found for the RNAP content.
(4) Reserpine decreases the rate of decline of acid-soluble ribonucleotides in the involuting gland, particularly of UMP, of UDP-N-acetyl-glucosamine and of those in the eluate which contains GDP and ADP-ribose.
(5) The findings are discussed in relation to the chemical changes found in involuting mammary tissue and to the histological picture presented by the gland after reserpine treatment.
H Wang, E Isaksson, B Von Schoultz, JM Cline, and L Sahlin
The effects of oestrogen are mediated by two specific intracellular receptors, oestrogen receptors (ER) alpha and beta, which function as ligand-activated transcriptional regulators. Ovariectomized macaques (Macaca fascicularis) were used to study the regulation of ERalpha and ERbeta in the endometrium by immunohistochemistry and in situ hybridization after long-term hormone treatment. Animals were treated continuously for 35 Months with either conjugated equine oestrogen (CEE), medroxyprogesterone acetate (MPA), combined CEE/MPA, or tamoxifen (TAM). Treatment with CEE/MPA down-regulated ERalpha in the superficial glands. In the superficial stroma the ERalpha level was lower in the CEE/MPA group than in the CEE and MPA groups. ERbeta immunostaining was faint with minor variation in response to treatment, but increased in the superficial stroma after MPA treatment. The ratio of ERbeta/ERalpha increased in superficial stroma and gland after CEE/MPA treatment, and also in stroma after MPA and TAM. Cystic endometrial hyperplasia was observed in TAM-treated animals, in combination with a high level of ERalpha protein expression. The present data show that long-term hormone treatment affects the ERalpha and ERbeta protein levels in the endometrium. The balance between ERalpha and ERbeta seems to be important for the proliferative response to oestrogen.
A Ilieva, S Yuan, RN Wang, D Agapitos, DJ Hill, and L Rosenberg
The purpose of this study was to characterize the trophic effect of pancreatic duct cells on the islets of Langerhans. Ductal epithelium and islets were isolated from hamster pancreata. In addition, duct-conditioned medium (DCM) was prepared from primary duct cultures that had been passaged twice to remove other cellular elements. Three experimental groups were then established: Group 1, 100 islets alone; Group 2, 100 islets+80 duct fragments; and Group 3, 100 islets in 25% DCM. All tissues were embedded in rat tail collagen for up to 12 days and the influence of pancreatic ductal epithelium on islet cell survival was examined. By day 12, 20.6+/-3. 0% (S.E.M.) of the islets cultured alone developed central necrosis, compared with 6.7+/-2.0% of the islets co-cultured with ducts and 5.6+/-1.5% of the islets cultured in DCM (P<0.05). The presence of apoptotic cell death was determined by a TdT-mediated dUTP-biotin nick end labelling (TUNEL) assay and by a specific cell death ELISA. DNA fragmentation in islets cultured alone was significantly increased compared with islets cultured either in the presence of duct epithelium or in DCM (P<0.05). More than 80% of TUNEL-positive cells were situated in the inner 80% of the islet area, suggesting that most were beta-cells. DCM was analysed for known growth factors. The presence of a large amount of IGF-II (34 ng/ml) and a much smaller quantity of nerve growth factor (4 ng/ml) was identified. When the apoptosis studies were repeated to compare islets alone, islets+DCM and islets+IGF-II, the cell death ELISA indicated that IGF-II produced the same beneficial result as DCM when compared with islets cultured alone. We conclude that pancreatic ductal epithelium promotes islet cell survival. This effect appears to be mediated in a paracrine manner by the release of IGF-II from cells in the ductal epithelium.
D L Crombie, J S Hayes, R B Heap, and M-W Wang
Anti-progesterone treatment using specific anti-progesterone antibodies or a progesterone receptor (PR) antagonist during first pregnancy impairs postpartum maternal behaviour in mice. This effect is demonstrable only if the treatment is given during pregnancy but not immediately after parturition. The purpose of the present studies was to investigate if maternal behaviour is also impaired by anti-progesterone treatment in subsequent pregnancies.
Studies with a monoclonal antibody to progesterone (DB3; 4·5 nmol/mouse) showed that injection of females on day 17 of second pregnancy did not cause maternal rejection but the latency of pup retrieval was prolonged especially during the first 3 days of lactation. This phenomenon was not observed in animals that had previous experience of full length lactation. Experiments were carried out with mifepristone (RU486; 10 μg/mouse) injected at day 17 of first, second or third pregnancies. Pup rejection (22·5% vs 12·3%) and prolongation of the retrieval latency (62·3 ± 13·3 vs 19·7 ± 6·5 s; P<0·02) were observed following the first pregnancy. No abnormal behavioural effects were found in mothers treated in second or third pregnancy who had prior full length lactation experience. Control females subjected to only one pup retrieval test after first delivery rejected their pups if treated in their second pregnancy (27.3% vs 4·4%; P<0·001) and displayed a marginal prolongation of the retrieval latency period (20·9 ± 7·0 vs 7·4 ± 2·6 s). Anti-progesterone treatment had no negative influence when administered during third pregnancy.
To determine whether treatment with RU486 (50 μg/mouse, day 17) during first pregnancy has any residual effects, maternal response was monitored after completion of second pregnancy where no treatment was given. Females who exhibited both maternal rejection and prolonged retrieval latency following first pregnancy did not demonstrate any carryover effects during second lactation, indicating that there is no long-term consequence of RU486 treatment. These results suggest that: (i) anti-progesterone treatment of pregnant mice prevents maternal recognition and disrupts postpartum behaviour in females who had no, or very limited, nursing experience; (ii) there is a progesterone-dependent imprinting mechanism during the first pregnancy that is disrupted by anti-progesterone antibody or PR antagonist; and (iii) this imprinting mechanism and first lactation are important components of the consolidation of neural pathways that are associated with the establishment of normal maternal behaviour.
Journal of Endocrinology (1995) 147, 331–337