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.
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J Chen, L Wang, YS Boeg, B Xia, and J Wang
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.
R Wang, J Li, and N Yashpal
The limitation of available islets for transplantation is a major obstacle for the treatment of diabetes through islet therapy. However, islet monolayers expanded ex vivo may provide a source of progenitor cells and a model to help understand islet development from precursor cell types. The existence of progenitor cells within the islets is highly likely, yet, to date, no fully defined or characterized postnatal stem cell has been isolated, expanded or marked. Our study evaluates the expression of progenitor markers, including the haematopoietic stem cell marker c-Kit, in epithelial monolayers derived from postnatal rat islets through immunofluorescence and RT-PCR, and the ability of precursor-rich monolayers to reform islet-like structures. Islets formed confluent monolayers when cultured on a type I collagen gel which lacked endocrine phenotypes but were positive for cytokeratin 20 and contained an increased proportion of proliferating c-Kit-expressing cells, with the proportion reaching a maximum of 45+/-6% at 8 weeks of culture. Evaluation of transcription factors at the mRNA level revealed constant PDX-1, ngn3 and Pax4 expression, while undifferentiated cell markers, such as Oct4 and alpha-fetoprotein, were also detected frequently after 4 weeks of culture. Changing the extracellular matrix protein to laminin-rich Matrigel, the monolayers re-formed islet-like clusters that secreted insulin in a glucose-responsive fashion. Our data show that islets can be expanded ex vivo to form epithelial monolayers with rich undifferentiating cell populations that are characterized by cells expressing the progenitor markers. These monolayers are capable of extensive proliferation and retain plasticity to form new islet cells, and c-Kit-expressing cells may play an important role in new islet cluster formation.
Jia Fang Wang and David J Hill
Pancreatic islets and acinar tissue develop from duct epithelium and share expression of several transcription factors and other molecular markers also involved with the development of neural tissues. We examined rat pancreatic tissue from fetal life until adulthood for the expression of N-myc downstream regulated gene 4 (Ndrg4), a gene shown to be expressed during neuronal cell differentiation. Isolated pancreatic ducts from neonatal rats were maintained in culture and gave rise to clusters of cells expressing nestin (NES) and PDX-1, which subsequently contained immunoreactive glucagon. Using reverse transcription PCR (RT-PCR), we identified mRNA expression and immunoreactive protein presence for NDRG4 in cultured duct-derived cells, and brain of neonatal rats. By PCR cloning of the ductal cell-derived DNA the molecular form of NDRG4 expressed in pancreatic ducts and ARIP rat pancreatic cells was identified as NDRG4A2, and its presence in intact pancreas of fetal and neonatal rats was demonstrated by immunohistochemistry. Incubation of ARIP cells with glucagon-like polypeptide-1 (GLP-1), increased the expression of NDRG4A2 and PDX-1, while decreasing DNA synthesis and promoting the appearance of glucagon-positive cells. This inhibitory effect of GLP-1 on DNA synthesis and the stimulatory effect on endocrine differentiation were reversed when the translation of NDRG4A2 was prevented using siRNA. These findings indicate that NDRG4A2 is expressed in pancreatic duct cells under GLP-1 control and may be related to a reduction in proliferation and the onset of the pancreas cell differentiation.
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
YL Wang-Fisher, J Han, and W Guo
Acipimox is a nicotinic acid-derived antilipolytic drug devoid of major side effects, and has been used in a number of human trials. This work reports the effects of Acipimox on leptin production from isolated rat adipocytes, in comparison with nicotinic acid and insulin. For cells isolated from normal animals, all these three reagents stimulated leptin release to a similar extent. Acipimox and nicotinic acid were more potent than insulin in stimulating leptin release from cells isolated from diabetic animals, probably because of impaired insulin sensitivity in cells from these diseased animals. Co-incubation of Acipimox with norepinephrine or dibutyryl cAMP diminished its stimulatory effects on leptin release, in parallel with increased lipolysis, suggesting that intracellular free fatty acids play an important role in mediating leptin production in adipocytes.
R Wang, J Li, N Yashpal, and N Gao
There has over the last several years been renewed interest in developing a system for generating new islets and a search for a self-renewing population in the pancreas. In particular, the neural stem cell marker nestin has been implicated as an islet precursor marker and its immunoreactivity has been localized in the islets of Langerhans. This study examines islet-derived epithelial monolayers expanded ex vivo to provide a source of nestin-expressing progenitor cells – a model that will help us understand the role of nestin-expressing cells in islet cell development. When cultured on a type I collagen gel, islets formed confluent monolayers which lacked endocrine phenotypes but were positive for cytokeratin 20 and contained an increased proportion of proliferating nestin-expressing cells, reaching a maximum of 54±10%. Co-expression studies demonstrated that the nestin-positive cells are heterogeneous, with some nestin-expressing cells co-localizing with the transcription factor PDX-1 and glucose transporter type 2 or lack of co-expression with vimentin. When clonal populations of nestin-positive cells were expanded and subjected to a differentiation protocol, only a population that expressed the transcription factor PDX-1 at the mRNA level was capable of re-expressing insulin at the mRNA and protein level. In conclusion, these studies demonstrate that expanded nestin-expressing cells in vitro from islet-derived epithelial monolayers are heterogeneous; clonal analysis of nestin-positive cells reveals that a distinct subpopulation of nestin/PDX-1-expressing cells is capable of forming insulin-producing cells.
J F Wang, G P Becks, and D J Hill
In this study, we have found that IGF-binding protein-3 (IGFBP-3) in calf serum added to tissue culture medium is degraded by cultured FRTL-5 cells and a major 31 kDa fragment of IGFBP-3 is produced. When FRTL-5 rat thyroid cells were cultured in 6H medium (modified F-12M medium containing TSH, insulin, hydrocortisone, somatostatin, transferrin, and glycyl-histidyl-lysine) containing 5% calf serum, both 44–46 and 31 kDa IGFBPs were found in conditioned medium by ligand blot analysis using 125I-labelled IGF-II. However, predominantly the 44–46 kDa IGFBP was detected in unconditioned 6H medium containing 5% calf serum. When calf serum in the media was replaced by human serum similar results were obtained, and the 44–46 kDa and 31 kDa IGFBPs were recognized using a human IGFBP-3 antibody following Western blot analysis. FRTL-5 cells secreted only small amounts of an endogenous 29 kDa IGFBP, thought to be IGFBP-5.
To separate the 31 kDa fragment of IGFBP-3 from the endogenous IGFBP-5, culture media were fractionated by concanavalin-A–Sepharose chromatography and aliquots of both flow-through and eluate from the column were analyzed by ligand blotting. A 31 kDa IGFBP was found in the eluate fractions from concanavalin-A–Sepharose chromatography following the separation of conditioned 6H medium supplemented with calf serum, suggesting that this species was an N-linked glycoprotein and could be derived from the degradation of serum IGFBP-3 by FRTL-5 cells.
Using a modified zymographic assay, we examined whether the degradation of IGFBP-3 could depend on the cell membrane. Confluent FRTL-5 cells were washed with PBS and overlaid with liquid agarose solution. After the agarose had solidified, unconditioned 6H medium containing 5% calf serum was incubated with the cells at 37 °C for 16 h. Both 44–46 and 31 kDa IGFBP species were found in the overlying, conditioned medium by ligand blot. However, the 31 kDa IGFBP was not found in medium in the absence of FRTL-5 cells, and no IGFBP could be found in serum-free conditioned medium from agarose-covered FRTL-5 cells. This suggests that the 44–46 kDa IGFBP-3 in serum was degraded to yield a 31 kDa fragment, while any endogenous IGFBP-5 could not pass out of the agarose. The degradation of 44–46 kDa IGFBP-3 in the modified zymographic assay was inhibited by phenylmethylsulfonyl fluoride, EDTA, and aprotinin, but not by leupeptin. In summary, these results indicated that IGFBP-3 in calf serum added to culture medium could be degraded by FRTL-5 cells and that this may involve calcium-dependent serine proteases.
Journal of Endocrinology (1997) 152, 265–274
J F Wang, D J Hill, and G P Becks
Isolated sheep thyroid follicles release insulin-like growth factors (IGF)-I and -II together with IGF-binding proteins (IGFBPs). We previously showed that TSH suppresses the biosynthesis and release of IGFBPs in vitro which may increase the tissue availability of IGFs, allowing a synergy with TSH which potentiates both thyroid growth and function. Many of the actions of TSH on thyroid cell function are dependent upon activation of adenylate cyclase, although increased synthesis of inositol trisphosphate and activation of protein kinase C (PKC) have also been implicated. We have now examined whether probable changes in intracellular cyclic adenosine monophosphate (cAMP) or PKC are involved in TSH-mediated suppression of IGFBP release. Confluent primary cultures of ovine thyroid cells were maintained in serum-free Ham's modified F-12M medium containing transferrin, somatostatin and glycyl-histidyl-lysine (designated 3H), and further supplemented with sodium iodide (10−8–10−3 mol/l), dibutyryl cAMP (0·25–1 mmol/l), forskolin (5–20 μmol/l) or 12-0-tetradecanoylphorbol-13-acetate (TPA; 10−11–10−6 mol/l), with or without exposure to TSH (200 μU/ml). The uptake and organification of Na [125I] by cells was examined after test incubations of up to 48 h, and IGFBPs in conditioned media were analysed by ligand blot using 125I-labelled IGF-II. The PKC activity in the cytosol and plasma membrane fractions of cells was measured by phosphorylation of histone using [γ-32P]ATP, and PKC immunoreactivity was visualized by Western immunoblot analysis. While dibutyryl cAMP or forskolin largely reproduced the stimulatory effect of TSH on iodine organification, they did not mimic the inhibitory effect of TSH on the secretion of IGFBPs of 43, 34, 28 and 19 kDa. Incubation with physiological or pharmacological concentrations of iodide (10−6–10−3 mol/l) for up to 48 h significantly decreased TSH action on iodide uptake and organification but did not alter the inhibitory action of TSH on IGFBP release. Incubation of cells with 10−11–10−6 mol TPA/l for 24 h inhibited the subsequent ability of TSH both to potentiate iodine organification and to suppress IGFBP release. In 3H medium, PKC activity was predominantly recovered from the membrane fraction but, following incubation for 48 h with TSH, the enzyme was no longer translocated to the membrane and was recovered predominantly from the cytosol. An 80 kDa species of immunoreactive PKC was recovered from the membranes of cells cultured in 3H medium, but its presence in membrane was decreased following incubation with TSH. The actions of TSH on intracellular PKC distribution were reversed by prior incubation with TPA, which itself stimulated the appearance of membrane PKC immunoreactivity. These results suggest that the ability of TSH to suppress IGFBP release does not depend primarily on cAMP stimulation, but may involve changes in the activation of PKC, possibly inhibition or down-regulation.
Journal of Endocrinology (1994) 141, 231–242
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