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T Otonkoski

Publications and source records attributed to T Otonkoski.

At least 55 records · Page 3Linked to original sources
PubMed1996

Ontogeny and tissue distribution of human GAD expression.

One of the major beta-cell autoantigens associated with IDDM is GAD. Although GAD expression has been detected in adult islets, transcriptional expression of the GAD genes has not been reported during human pancreatic ontogeny. We therefore analyzed patterns of GAD gene transcription by quantitating the mRNAs encoding both the 65- and 67-kDa isoforms (GAD65 and GAD67, respectively) in human fetal, postnatal, and adult pancreases, as well as in isolated adult islets, and examined their tissue-specific expression. Significant levels of pancreatic GAD65 transcripts were already detected at 13 weeks of gestation and were expressed at higher levels in the fetal and infantile pancreas than in the adult pancreas. Isolated adult pancreatic islets were highly enriched in GAD65 mRNA. In contrast, GAD67 transcripts were not detectable in fetal and postnatal pancreases. In addition to the pancreas, marked GAD expression was detected in the brain, whereas other tissues examined contained either low or undetectable GAD transcripts. Triple immunofluorescent staining of fetal and adult pancreases revealed colocalization of GAD65 with alpha- and beta-cells. In the fetal pancreas, strong immunoreactivity for GAD65 was also evident in epithelial cells, which lacked expression of insulin or glucagon, some of which were present in the ductal epithelium, suggesting that GAD65 expression might correlate with endocrine determination. In summary, 1) this is the first demonstration of GAD65 expression in the human fetal pancreas, implicating a potential role during islet development, and 2) GAD65 may be a useful marker for the identification of primitive islet cells.

Adult↗
PubMed1996

Regulation of proliferation and differentiation of human fetal pancreatic islet cells by extracellular matrix, hepatocyte growth factor, and cell-cell contact.

Ex vivo expansion of human fetal pancreatic endocrine cells is important for biological studies and as a potential tissue source for transplantation in insulin-deficient states. In tissue culture experiments involving the use of hepatocyte growth factor/scatter factor and selected extracellular matrices, we obtained a 30-fold increase in cell number of human fetal pancreatic epithelial cells. This proliferation in monolayer culture was associated with marked downregulation of insulin and glucagon gene expression. However, gene expression increased when the cells were combined into three-dimensional aggregates, suggesting that cell-cell contact mediated mechanisms regulate the transcription of islet-specific genes, a process enhanced by nicotinamide (NIC). After transplantation into nude mice, either as cell suspensions or aggregates, only the cell aggregates treated with NIC developed into mature functional islet-like structures. These are the first experiments to describe the interactions of specific matrices and growth factors in the ex vivo expansion of human fetal pancreatic cells, and they also show the importance of cell aggregates in the context of cellular and molecular events that might positively influence islet cell transplantation.

Abortion, Induced↗
PubMed1995

Constitution of a biphasic insulin response to glucose in human fetal pancreatic beta-cells with glucagon-like peptide 1.

Insulin release from fetal beta-cells responds only minimally to acute glucose stimulation. Glucagon-like peptide-1 (GLP-1) is able to correct glucose sensitivity in some models of glucose-resistant beta-cells. We have now tested whether GLP-1 can induce glucose-responsive insulin release in human fetal islet-like cell clusters (ICCs). For this purpose we used perifusion and static incubation of ICCs and isolated adult human islets. In perifusion, the fetal ICCs responded with only minimal insulin release to 16.7 mmol/L glucose or 10 nmol/L GLP-1 combined with 1.67 mmol/L glucose. However, in the presence of GLP-1, the insulin response to glucose was markedly potentiated and appeared in a biphasic manner (10-fold first phase and 3-fold second phase). The first phase response was equal to that of adult human islets in similar experiments, whereas the second phase of the fetal cells was significantly lower. In static incubations, the relative insulin release responses to 16.7 mmol/L glucose plus 10 nmol/L GLP-1 were equal in the fetal and adult cells. The cAMP content of the cells was increased by glucose only in the presence of GLP-1. Our studies indicate that the glucoincretin hormone GLP-1 is able to constitute biphasic insulin release in the immature beta-cell, possibly as the result of cAMP-mediated priming of the glucose sensor mechanism.

Adult↗
PubMed1994

Developmental gene expression in the human fetal pancreas.

Differential developmental regulation of pancreas-specific genes has not been reported for the human fetal pancreas. We have therefore undertaken a systematic, quantitative analysis of the transcriptional levels of various genes in the human pancreas at different stages of fetal and postnatal development. Using sensitive ribonuclease protection assays, in situ hybridization, and the polymerase chain reaction, our results indicate the following: 1) Transcriptional levels of insulin and amylin remain lower in the fetal than in the adult pancreas, whereas glucagon and somatostatin mRNA levels are consistently greater after 14 wk gestation than postnatally. These results are in agreement with previous immunohistochemical studies of these gene products. 2) The reg gene exhibits a 20-fold increase in mRNA levels after 16 wk gestation. The gene is expressed exclusively in the acinar cells and does not colocalize with insulin. This restricted exocrine expression does not indicate a direct role for the reg gene in islet development. 3) Glucose transporter 2 and glucokinase mRNA are detectable as early as 13 wk gestation and remain low throughout development. Glucose transporter 1 reaches adult transcriptional levels by 18 wk gestation. The early detection of glucose transporter 2 and glucokinase implies that lack of expression of these "glucose sensor" genes does not account for the known insensitivity of the fetal beta-cells to glucose.

Adult↗
PubMed1994

Acid beta-galactosidase: a developmentally regulated marker of endocrine cell precursors in the human fetal pancreas.

Isolation of endocrine cell precursors from the human fetal pancreas will be important to the study of islet cytodifferentiation and eventually for islet transplantation in insulin-dependent diabetes. These precursor cells, from which all four islet endocrine cell types arise, are present within fetal pancreatic ductal epithelium. After enzymatic digestion and culture of the fetal pancreas, we obtained cell clusters resembling islets, but with a high content of undifferentiated cells. Histochemical staining revealed very high acid beta-galactosidase activity in over 70% of cells within the clusters. After transplantation into athymic nude mice, the islet-like cell clusters gave rise to tissue rich in differentiated endocrine cells, but low in beta-galactosidase activity. The histochemical finding of high acid beta-galactosidase activity in endocrine precursor cells was confirmed by direct measurement of lysosomal enzyme activities. In addition, we found that the expression of acid beta-galactosidase was developmentally regulated, peaking at 18-24 weeks gestation and declining to low levels in adult islets. Using a fluorogenic beta-galactosidase substrate, we were able to isolate a subpopulation of cells high in acid beta-galactosidase activity using fluorescence-activated flow cytometry. Evidence identifying these cells as potential islet cell precursors includes, besides the transplantation experiments, the colocalization in vitro of tyrosine hydroxylase, a marker of embryonic islet cells. Thus, our results indicate that high acid beta-galactosidase activity serves as a marker for a population of fetal pancreatic cells with the potential to differentiate and grow into mature pancreatic endocrine cells.

Biomarkers↗
PubMed1994

Hepatocyte growth factor/scatter factor has insulinotropic activity in human fetal pancreatic cells.

Fetal mesenchyme-derived factors are likely to play an important role in pancreatic islet development and growth. We have used primary cultures of human fetal pancreatic tissue to identify growth factors that have morphogenic, mitogenic, and insulinotropic activity. The formation of islet-like cell clusters (ICCs) during a 6-day culture was stimulated two- to threefold by hepatocyte growth factor/scatter factor (HGF/SF) basic fibroblast growth factor (FGF)-2, and to a lesser extent by keratinocyte growth factor (FGF-7) and insulin-like growth factor-II (IGF-II). In contrast, transforming growth factor-beta (TGF-beta) had a strong inhibitory effect. The ICCs formed during HGF/SF stimulation consisted mainly of epithelial cells, whereas FGF-2-induced ICCs were predominantly nonepithelial. Furthermore, although both FGF-2 and HGF/SF increased the total insulin content of the cultures, only HGF/SF increased the insulin content per DNA. Quantitatively, HGF/SF stimulated a 2.3-fold increase in the proportion of insulin-positive cells and a 3-fold higher number of replicating beta-cells. Blocking of the IGF-I receptor inhibited ICC formation but did not affect their insulin content. Immunoneutralizing TGF-beta resulted in increased cell growth and insulin content, indicating the presence of an endogenous inhibitory TGF-beta activity in the model system. Our results suggest that HGF/SF may be an important component of the fetal mesenchyme-derived factors responsible for pancreatic islet development. HGF/SF also may prove valuable for supporting the in vitro growth of islet cells.

Abortion, Legal↗
PubMed1994

Opposite effects of beta-cell differentiation and growth on reg expression in human fetal pancreatic cells.

Reg is a gene associated with regeneration of pancreatic islets. We have previously shown that nicotinamide induces differentiation of human fetal beta-cells in tissue culture and that hepatocyte growth factor/scatter factor (HGF/SF) is mitogenic for the fetal beta-cells. We now tested whether these conditions, supporting either differentiation or growth, are associated with changes in reg gene expression in human fetal pancreatic cells. Culture for 7 days with 10 mM nicotinamide led to a fourfold decrease in reg mRNA levels (23 +/- 12% of control, n = 5, P < 0.001). In contrast, HGF/SF increased reg expression threefold (302 +/- 68% of control, n = 4, P < 0.05). Nicotinamide, which does not alter the differentiation level of adult beta-cells, did not significantly affect reg expression in adult human islets (84 +/- 4% of control, n = 2, NS). Thus, a higher level of endocrine differentiation is associated with a lower level of reg expression, and a higher rate of beta-cell replication results in increased reg transcription. These results provide the first evidence of a molecular marker, the reg gene, to distinguish between proliferation and differentiation of human beta-cells.

Amino Acid Isomerases↗
PubMed1993

Maturation and function of human fetal pancreatic cells after cryopreservation.

If transplantation of endocrine tissue is to become a therapy for a significant number of insulin-dependent diabetic individuals, tissue sources other than adult human islets will be required. Because human fetal pancreatic cells may offer that alternative, we have tried to find the optimal combination of tissue culture and cryopreservation methods for use in transplantation. Islet-like cell clusters (ICCs), cryopreserved according to reported methods for adult human islets, survived poorly after thawing. In contrast, the yield of ICCs was comparable after collagenase digestion of fresh and cryopreserved pancreatic fragments. However, the ICCs derived from cryopreserved tissue contained a higher proportion of nonepithelial cells, and the recovery of insulin was only 28%, as compared with freshly cultured cells. These ICCs had a lower DNA content and a higher rate of DNA synthesis. Moreover, cryopreserved cells released a higher fraction of their insulin content in basal conditions, and their response to theophylline stimulation was slightly lower. ICCs generated from cryopreserved fragments were able to mature morphologically and functionally in vivo after transplantation into athymic nude mice. However, the level and magnitude of the C-peptide response did not equal that of grafted freshly cultured ICCs. Our results indicate that it is possible to generate ICCs from cryopreserved human fetal pancreas with the capacity, after transplantation, to release insulin appropriately in response to glucose. However, possibly because undifferentiated pancreatic cells may be particularly vulnerable to cryopreservation, current methods may need to be improved for optimal tissue retrieval.

Animals↗
PubMed1993

Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells.

The effects of nicotinamide (NIC) on human fetal and adult endocrine pancreatic cells were studied in tissue culture. Treatment of the fetal cells with 10 mM NIC resulted in a twofold increase in DNA content and a threefold increase in insulin content. This was associated with the development of beta cell outgrowths from undifferentiated epithelial cell clusters and an increase in the expression of the insulin, glucagon, and somatostatin genes. DNA synthesis was stimulated only in the undifferentiated cells. Half-maximal doses for the insulinotropic and mitogenic effects of NIC were 5-10 and 1-2 mM, respectively. Islet-like cell clusters cultured with NIC responded to glucose stimulation with a biphasic increase in insulin release (fourfold peak), whereas control cells were unresponsive to glucose. Both control and NIC-treated cells developed into functional islet tissue after transplantation into athymic nude mice. As compared with adult islets, the insulinotropic action of NIC could only be demonstrated in the fetal cells. Our results indicate that NIC induces differentiation and maturation of human fetal pancreatic islet cells. This model should be useful for the study of molecular mechanisms involved in beta cell development.

Adult↗
PubMed1993

Somatostatin regulation of beta-cell function in the normal human fetuses and in neonates with persistent hyperinsulinemic hypoglycemia.

To investigate the insulin-inhibitory effects of somatostatin during fetal development, and the defective regulation of insulin release in persistent hyperinsulinemic hypoglycemia of infancy (PHHI), we made perifusion experiments with islet-like cell clusters obtained from 14 human fetal pancreases (HFP) before 20 weeks of gestation and 6 neonates pancreatectomized because of PHHI (5 cases of diffuse and 1 adenomatous islet cell hyperplasia). Somatostatin-14 (600 nmol/L) suppressed insulin release only marginally (20% and 25% suppression in 2 and 20 mmol/L glucose, respectively) in HFP, whereas supplementation of the perifusate with 14 nmol/L glucagon potentiated the suppression to 57%. Somatostatin-28 (600 nmol/L) caused a 35% inhibition in insulin release from the HFP. In static incubation of HFP, somatostatin-14 caused a weak dose-dependent inhibition of insulin release, but had no effect on (pro)insulin biosynthesis. In the PHHI infants, somatostatin infusion suppressed hyperinsulinemia effectively before pancreatectomy. In vitro, 600 nmol/L somatostatin-14 suppressed insulin release clearly better than in the HFP experiments (35-61% inhibition). Glucose (20 mmol/L) induced a 2- to 3-fold increase in insulin release in two clinically mild cases of PHHI, but no response at all in the four others. We conclude that human fetal beta-cells are relatively insensitive to somatostatin, possibly because of low cellular cAMP levels. Furthermore, insulin release from PHHI cells is poorly stimulated by glucose but effectively blocked by somatostatin, supporting the concept of somatostatin deficiency in PHHI.

Abortion, Induced↗
PubMed1993

Insulin-like growth factor-II and transforming growth factor-alpha in developing human fetal pancreatic islets.

To understand the development of the human pancreas better, we studied the expression and regulation of insulin, insulin-like growth factor-II (IGF-II) and transforming growth factor-alpha (TGF-alpha) genes in the human fetal pancreas and islet-like cell clusters (ICC) from the second trimester human fetuses. Northern blot analysis revealed an abundant expression of IGF-II, insulin and TGF-alpha mRNAs in the intact pancreas and the cultured ICCs. Furthermore, transcripts for insulin receptor, type-1 and -2 IGF receptors, and GH receptor could be amplified by polymerase chain reaction analysis from the pancreas and the ICCs. With in-situ hybridization, IGF-II mRNA was found in abundance in both the exocrine and endocrine pancreas, exceeding the amount of insulin mRNA. In ICCs, insulin mRNA-containing cells were present as small clusters in the periphery and in the centre of the clusters corresponding to the immunolocation of insulin. The ICCs also contained many epidermal growth factor-, insulin- and type-1 IGF receptor- and TGF-alpha-positive cells. When the ICCs were cultured in the presence of various secretagogues, only dibutyryl cyclic AMP was found to up-regulate insulin mRNA (39%; P < 0.05). IGF-II mRNA was also under cyclic AMP-dependent regulation (threefold increase; P = 0.025). Furthermore, blocking the type-1 IGF receptor with a monoclonal receptor antibody drastically reduced insulin expression (87%; P = 0.005) and additionally down-regulated IGF-II mRNA (49%; P = 0.005). IGF-1, IGF-II, TGF-alpha or epidermal growth factor-receptor antibody had no significant effect on either insulin or IGF-II mRNA. Exogenous TGF-alpha inhibited the release of insulin by the ICCs. It was concluded that IGF-II and TGF-alpha may be involved in the regulation of islet growth and differentiation.

Base Sequence↗
PubMed1991

Lack of glucose-induced functional maturation during long-term culture of human fetal islet cells.

To investigate the long-term effects of glucose on the function of human fetal islets we cultured islet-like cell clusters (ICC) obtained from 12 human fetuses with a mean age of 16.1 weeks in media containing 2.8, 11.1 or 16.7 mM glucose. On the 8th day of culture, the ICC that had been maintained in 16.7 mM glucose contained 60% less insulin than the ICC cultured in 2.8 mM glucose. However, insulin release was similar in both groups, and was not affected by a 24-h incubation in high vs. low glucose. Also (pro) insulin biosynthesis was not significantly affected. During a 24-day culture period, the total release of insulin and glucagon was similar in all glucose concentrations. The ICC released about 75% of their insulin content but only 15% of their glucagon content during the last 48 h of the 24-day culture period, again regardless of glucose concentration in media. Insulin release was insensitive to acute glucose and leucine challenges in perifusion experiments after culture for 1, 5, 8 or 16 days in 11.1 mM glucose, whereas glucagon was always a potent stimulus. In conclusion, the function of cultured young human fetal islet cells is remarkably independent of glucose, even during prolonged exposure. Moreover, the primary role of glucagon in fetal life may be that of a paracrine stimulator of beta-cell function.

Arginine↗
PubMed1989

EGF- and TGF-alpha-like peptides in human fetal gut.

The presence of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) immunoreactivities in fetal human tissues was studied immunohistochemically at different gestational ages. EGF and TGF alpha immunoreactivities were detected from the 20th gestational wk. EGF immunoreactivity was limited to the small intestine, but TGF alpha immunoreactive cells were present in the colon also. According to radioreceptor assay, the intestine of a 19-wk-old human fetus contained 10 times more EGF receptor-binding substance than EGF, as measured by immunofluorometric assay. Chromatographic analysis suggests that TGF alpha-like peptides account for at least part of this activity, as so argues in favor of the presence of TGF alpha- and EGF-like peptides in the human fetal gut. Whether they are synthesized in the fetus is yet unknown.

Digestive System↗
PubMed1988

Insulin and glucagon secretory responses to arginine, glucagon, and theophylline during perifusion of human fetal islet-like cell clusters.

The secretory responsiveness of human fetal pancreatic endocrine cells was studied by perifusion of cultured islet-like cell clusters (ICC). ICC were obtained from 7 fetuses at 13-15 weeks gestation and 21 fetuses at 17-22 weeks gestation. The ICC were challenged with glucose (20 mmol/L), arginine (10 mmol/L), glucagon (1.4 mumol/L), and theophylline (10 mmol/L) combined with zero, low (2 mmol/L), or high (20 mmol/L) glucose. At 13-15 weeks, glucose and arginine enhanced insulin release in some experiments, whereas glucagon and theophylline were always potent stimuli (mean response, 4-fold regardless of the glucose concentration). At 17-22 weeks, both glucose alone (20 mmol/L) and arginine (10 mmol/L, with 2 mmol/L glucose) induced a small (1.4- to 1.5-fold) increase in insulin release. When arginine was combined with 20 mmol/L glucose, the response was potentiated to become a 2.3-fold increase. In contrast, glucagon was equally effective in 2 and 20 mmol/L glucose (2.9- and 2.6-fold responses, respectively) and produced a half-maximal response even in the absence of glucose. In this age range the most potent stimulus for insulin release was clearly theophylline. The effect of theophylline was also remarkably independent of the glucose concentration of the perifusate (5.6-, 8.1-, and 8.6-fold responses at 0, 2, and 20 mmol/L glucose, respectively). Glucagon release from the ICC of the 17- to 22-week-old fetuses was low (mean basal glucagon release, 2.9; insulin, 24.8 fmol/100 ICC/min). Glucagon release was not affected by 20 mmol/L glucose, but was stimulated by arginine and theophylline. These findings suggest that in the fetal pancreas, in contrast to the adult organ, insulin release results from elevation of intracellular cAMP concentrations (by glucagon or theophylline) relatively independent of the exogenous glucose concentration. Therefore, glucagon may have an important role in regulating insulin release during the early development of human fetal B-cells.

Arginine↗
PubMed1988

Morphology, yield and functional integrity of islet-like cell clusters in tissue culture of human fetal pancreata obtained after different means of abortion.

Morphology, yield and function were studied in cultured islet-like cell clusters (ICC) from 140 human fetal pancreata obtained after abortions of different types performed at 11-23 weeks of gestation (12 by hysterotomy, 75 by mechanical dilation and extraction, and 53 induced with prostaglandin). After collagenase digestion and culture in medium supplemented with 10% human serum, up to 2000 free-floating ICC were formed from a single pancreas. Randomly scattered insulin- and glucagon-immunoreactive cells were found in the medullary part of the ICC. More than 100 ICC developed in 100% of the hysterotomies and 87% of the mechanical abortions, but in only 53% of the prostaglandin-induced abortions. Insulin and glucagon levels in the culture medium decreased rapidly during the first 7 days of culture, but then remained stable for at least 31 days. The hysterotomy-derived ICC responded to 10 mmol/l theophylline plus 20 mmol/l glucose by a 12.2 +/- 3.1 (SEM, N = 7) fold increase in insulin release, as compared with a 5.4 +/- 0.9 fold response of the prostaglandin ICC (N = 16; P less than 0.02). Despite the low proportion of B-cells, (pro)insulin biosynthesis accounted for 10% of the total protein biosynthesis in low (2 mmol/l) glucose. In conclusion, the yield and viability of the ICC were clearly better, if prostaglandin had not been used for the induction of the abortion.

Abortion, Induced↗