Sex-specific effects of low protein diet on in utero programming of renal G-protein coupled receptors.

Intrauterine growth restriction (IUGR) is an important risk factor for development of hypertension, diabetes and the metabolic syndrome. Maternal low protein (LP) intake during rat pregnancy leads to IUGR in male and female offspring, although females may be resistant to the development of effect. Current evidence suggests that changes in the renin-angiotensin system (RAS) in utero contribute to this programmed hypertension, via sex-specific mechanisms. The previously orphaned G-protein coupled receptor (GPR91) was identified as a central player in the development of hypertension in adult mice, through a RAS-dependent pathway. However, whether the GPR91 pathway contributes to fetal programming is unknown. Furthermore, the nature of involvement of downstream modulators of the RAS including Gqα/11α and GαS has not been investigated in IUGR-LP rats. Therefore, we postulated that renal GPR91, in conjunction with RAS, is differentially impacted in a sex-specific manner from LP-induced IUGR rats. Pregnant Wistar rats were fed control (C, 20% protein) or LP (8% protein) diet until embryonic day 19 (E19) or postnatal d21. At E19, GPR91 protein and mRNA were increased in both male and female LP kidneys (P<0.05), whereas renin and angiotensin converting enzyme (ACE) were only increased in males (P=0.06 and P<0.05, respectively). On d21, AT1R and Gqα/11α were increased in LP males, while in LP females, AT2R protein was elevated and renin expression was decreased (P<0.05). This study demonstrates that in IUGR-LP rats, up regulation of GPR91 in fetal kidney is mirrored by increased ACE and renin in males. These in utero alterations, when combined with postnatal increases in AT1R-Gqα/11α specifically in male offspring, may predispose to the development of hypertension.

Lineage tracing and resulting phenotype of haemopoietic-derived cells in the pancreas during beta cell regeneration.

AIMS: Transplantation of bone marrow-derived haemopoietic stem cells following streptozotocin (STZ) treatment to induce pancreatic beta cell loss in mice causes the partial regeneration of beta cell mass, with many haemopoietic cells demonstrating endothelial cell markers. This study used genetically tagged haemopoietic lineage-derived cells to determine how endogenous cells are mobilised following beta cell loss and subsequent replacement. METHODS: A double transgenic mouse model, Vav-iCre; R26R-enhanced yellow fluorescent protein (YFP), was used where only haemopoietic lineage cells expressed the Vav1 gene promoter allowing expression of the YFP reporter gene. Between postnatal days 2 and 4 mice were injected with STZ or vehicle (control) and body weight and glycaemia were monitored. Mice were killed between days 10 and 130, and the pancreases were examined by immunofluorescence microscopy. RESULTS: YFP-expressing cells infiltrated the pancreas at all ages, being present around newly forming islets at the pancreatic ducts, and within larger islets. Small numbers of YFP-positive cells (<5%) co-stained for the macrophage markers F4/80 or Mac1, for cytokeratin 19, or for the transcription factor pancreatic and duodenal homeobox 1 (PDX-1), but no co-localisation was seen with insulin or other endocrine hormones. Within islets approximately 30% of YFP-positive cells co-stained for the endothelial cell marker CD31, and following STZ the number of haemopoietic-derived cells, and the proportion that were CD31-positive, both significantly increased after 21 and 40 days, coincident with a partial replacement of beta cells. CONCLUSIONS: Our results suggest that following beta cell loss endogenous haemopoietic-lineage cells contribute to intra-islet angiogenesis, which supports a partial recovery of beta cell mass.

Effects of atorvastatin on the regeneration of pancreatic {beta}-cells after streptozotocin treatment in the neonatal rodent.

To investigate the role of statins in beta-cell regeneration a model of streptozotocin (STZ)-induced beta-cell injury was used in the neonatal rat. We hypothesized that beta-cell growth and regeneration would increase following treatment with atorvastatin and that this would be associated with intraislet vasculogenesis. Pregnant Wistar rats were gavaged with 20 or 40 mg/kg atorvastatin for 21 days commencing on gestation day 15. Atorvastatin was detected in the circulation of the offspring. On postnatal day 4, the pups were given either a control or STZ (70 mg/kg ip) injection. beta-Cell mass had partially recovered by postnatal day 44 following STZ treatment, and atorvastatin (20 mg/kg) significantly increased beta-cell mass in both STZ-treated and control animals. An increase in the numbers of small islets at postnatal day 44 was seen in STZ-treated animals following atorvastatin, suggestive of neogenesis, and glucose tolerance was improved. Treatment with atorvastatin caused an increase in the numbers of intraislet endothelial cells at postnatal day 14 and the percentage of endothelial cells undergoing DNA synthesis, suggesting that angiogenesis had preceded the increase in beta-cell mass. The results indicate that functional beta-cell mass was expanded with atorvastatin in both control and STZ-treated neonatal rats and suggests a novel effect of a statin in promoting islet plasticity.

GABAB receptors and glucose homeostasis: evaluation in GABAB receptor knockout mice.

GABA has been proposed to inhibit insulin secretion through GABAB receptors (GABABRs) in pancreatic beta-cells. We investigated whether GABABRs participated in the regulation of glucose homeostasis in vivo. The animals used in this study were adult male and female BALB/C mice, mice deficient in the GABAB1 subunit of the GABABR (GABAB(-/-)), and wild types (WT). Blood glucose was measured under fasting/fed conditions and in glucose tolerance tests (GTTs) with a Lifescan Glucose meter, and serum insulin was measured by ELISA. Pancreatic insulin content and islet insulin were released by RIA. Western blots for the GABAB1 subunit in islet membranes and immunohistochemistry for insulin and GABAB1 were performed in both genotypes. BALB/C mice preinjected with Baclofen (GABABR agonist, 7.5 mg/kg ip) presented impaired GTTs and decreased insulin secretion compared with saline-preinjected controls. GABAB(-/-) mice showed fasting and fed glucose levels similar to WT. GABAB(-/-) mice showed improved GTTs at moderate glucose overloads (2 g/kg). Baclofen pretreatment did not modify GTTs in GABAB(-/-) mice, whereas it impaired normal glycemia reinstatement in WT. Baclofen inhibited glucose-stimulated insulin secretion in WT isolated islets but was without effect in GABAB(-/-) islets. In GABAB(-/-) males, pancreatic insulin content was increased, basal and glucose-stimulated insulin secretion were augmented, and impaired insulin tolerance test and increased homeostatic model assessment of insulin resistance index were determined. Immunohistochemistry for insulin demonstrated an increase of very large islets in GABAB(-/-) males. Results demonstrate that GABABRs are involved in the regulation of glucose homeostasis in vivo and that the constitutive absence of GABABRs induces alterations in pancreatic histology, physiology, and insulin resistance.

Taurine supplement in early life altered islet morphology, decreased insulitis and delayed the onset of diabetes in non-obese diabetic mice.

AIMS/HYPOTHESIS: We hypothesised that nutritional taurine, which is important for the development of the endocrine pancreas and reduces cytokine-induced apoptosis in pancreatic beta cells, would prevent or delay the onset of autoimmune diabetes, if given early in life to the non-obese diabetic (NOD) mouse. METHODS: Pregnant NOD mice received a diet supplemented with taurine throughout gestation or until weaning, and the pancreas of the offspring was examined using immunohistochemistry. This was done at postnatal day 14 and after 8 weeks (assessment of insulitis). The animals were also monitored until they became diabetic. RESULTS: At 14 days, pancreatic islet mass was significantly greater in animals treated with taurine than in controls. This finding was associated with a greater incidence of islet cell proliferation and a lower incidence of apoptosis. At age 8 weeks the number of islets manifesting insulitis was reduced by more than half, and the area of insulitis was reduced by 90%. Taurine treatment delayed the mean onset time of diabetes from 18 to 30 weeks in females, and from 30 to 38 weeks in males, while 20% of treated females remained free of diabetes after one year. CONCLUSIONS/INTERPRETATION: Taurine supplementation in early life altered islet development, reduced insulitis and delayed the onset of diabetes in NOD mice.

Low-protein diet during early life causes a reduction in the frequency of cells immunopositive for nestin and CD34 in both pancreatic ducts and islets in the rat.

Feeding a low-protein (LP) diet to pregnant and lactating rats impairs pancreatic islet mass and insulin release in the offspring, leading to glucose intolerance as adults. We hypothesized that an LP diet changes the number of pancreatic endocrine precursor cells or cells supporting endocrine cell neogenesis. Pregnant rats were given LP (8% protein) or a control (20% protein) diet from conception until postnatal d 21. Cells containing nestin, CD34, or c-Kit were quantified in pancreata of the offspring. Stellate cells immunoreactive for nestin were seen to be adjacent to ductal epithelium and were resident within the islets. These were proliferative and immunonegative for cytokeratin 20, fibronectin, tyrosine hydroxylase, pancreatic duodenal homeobox 1, Nk homeodomain transcription factor 6.1, or insulin, but expressed vimentin. Approximately 20% of islet nestin-positive cells also expressed the endothelial cell marker platelet endothelial cell adhesion molecule-1. Both ducts and islets also contained CD34- and c-Kit-positive cells with similar morphology to those expressing nestin. Offspring from rats fed the LP diet had significantly less nestin/CD34-positive cells and reduced expression of nestin mRNA. Within islets, there was an associated decrease in cell proliferation and in cells immunopositive for pancreatic duodenal homeobox 1. Nestin-positive cell number within islets correlated positively with the percent area of beta-cells. Supplementation of pregnant and lactating rats with taurine reversed the deficits in mean islet area and nestin-positive cells caused by the LP diet within the islets of the offspring. Nutritional programming of postnatal beta-cell mass may involve an altered abundance of cells expressing nestin and/or CD34, which may limit endocrine cell development.

Taurine supplementation to a low protein diet during foetal and early postnatal life restores a normal proliferation and apoptosis of rat pancreatic islets.

AIMS/HYPOTHESIS: In our previous studies a low protein diet (8% vs 20%) given during foetal and early postnatal life induced abnormal development of the endocrine pancreas; beta-cell mass and islet-cell proliferation were reduced while apoptosis was increased. Taurine, an important amino acid for development was also reduced in maternal and foetal plasma of protein deficient animals. In this study we aim to evaluate the role of taurine in the alterations observed in rats after a low protein diet. METHODS: Four groups of rats were given either a control, a low protein, or control and low protein diets with 2.5% taurine in the drinking water. Diets were given to gestating and lactating mothers and to their pups until day 30. Beta and endocrine cell masses were analysed as well as DNA synthesis and apoptosis after taurine supplementation in foetuses and pups. We also investigated insulin like growth factor-II (IGF-II), inducible nitric oxide synthase (iNOS), and Fas by immunohistochemistry. RESULTS: In foetuses and neonates nourished with a low protein diet, taurine supplementation restored normal DNA synthesis and apoptosis. This led to adequate beta and endocrine cell mass in pups. In islet cells, immunoreactivity was increased for IGF-II, reduced for Fas and unchanged for iNOS after taurine supplementation. CONCLUSION/INTERPRETATION: Taurine supplementation to a low protein diet in foetal and early postnatal life prevents the abnormal development of the endocrine pancreas. The mechanisms by which taurine acts on DNA synthesis and apoptosis rate of endocrine cells involve IGF-II, Fas regulation but not iNOS.

Glucocorticoid receptor expression is altered in pancreatic beta cells of the non-obese diabetic mouse during postnatal development.

Glucocorticoids play a crucial role in the regulation of carbohydrate metabolism and in the immune response, and can influence the development of diabetes in certain animal models including autoimmune type 1 diabetes in the non-obese diabetic (NOD) mouse. In these animals, the onset of destructive autoimmune pancreatic changes (insulitis) occurs at around 3 weeks of age. Moreover, the incidence of diabetes is significantly higher in females compared to males. However, the underlying mechanisms for this sex-specificity are unknown. Therefore, the present study was undertaken to examine the expression of the glucocorticoid receptor (GR) in pancreatic islets of Langerhans of the NOD mouse during the first 3 weeks of postnatal development. Immunohistochemistry was used to determine pancreatic GR expression and to identify insulin-secreting beta cells in postnatal (1-, 2-, and 3-week-old) NOD mice. Age-matched NOD.SCID mice (immunodeficient animals with the same NOD genetic background) were used as control animals. In both strains, regardless of sex or age, GR staining was found predominantly in the cytoplasm of beta cells but was also present in other cell types within the islets. At all ages, the percentage of islet cells containing GR was similar between male and female animals of the same strain. In control mice, the percentage of islet cells containing GR increased progressively from 80% at 1 week of age to 100% at 3 weeks of age. In marked contrast, in the NOD mice, the proportion of islets containing GR decreased from 95% at week 1 to only 60% at 3 weeks of age. We conclude that sex-specific differences in the incidence of diabetes are not associated with altered pancreatic GR expression in NOD mice during early postnatal development. However, the distinct and remarkable decrease in islet GR levels at 3 weeks of age may contribute to the onset of insulitis, and potentially to the ontology of diabetes in NOD mice, as a result of the loss of protective immunosuppressive effects of glucocorticoids.

A long-term high-carbohydrate diet causes an altered ontogeny of pancreatic islets of Langerhans in the neonatal rat.

Neonatal rats fed a high-carbohydrate (HC) formula by gastrostomy are hyperinsulinemic but normoglycemic. We determined whether HC formula altered pancreatic islet cell ontogeny. Rats were reared from d 4 on an HC formula or a high-fat formula, or were allowed to suckle naturally, and the pancreata were examined histologically from animals < or =24 d of age. The mean area of individual islets was reduced, but islet number was increased in HC rats compared with mother-fed or high fat-fed animals, which were similar. Islets from HC animals were relatively deficient in alpha cells and had a greater incidence of islet cells with fragmented DNA, indicative of apoptosis. Ductal epithelium, a source of new islets by neogenesis, had a greater incidence of cells staining immunopositive for proliferating cell nuclear antigen, a marker of cell replication, and a lower incidence of apoptosis. The islet cell mitogen and survival factor, IGF-II, had a reduced mRNA expression in whole pancreas from HC animals. The relative area of islet cells demonstrating IGF-II immunoreactivity was reduced in HC-fed rats versus controls, although a greater percentage of ductal epithelial cells were immunopositive. HC formula alters islet cell ontogeny by affecting islet size and number, which may be linked to an altered IGF-II expression.

Ontogeny of fibroblast growth factors in the early development of the rat endocrine pancreas.

Pancreatic islet ontogeny involves endocrine cell neogenesis from ductal epithelium and islet expansion by cell replication, balanced by apoptotic deletion of endocrine cells which, in rat, is pronounced in the neonate. Fibroblast growth factors (FGF) are involved in tissue morphogenesis, and we examined the distribution and ontogeny of several FGF within rat pancreas from late fetal life until weaning. Islet cell replication (immunohistochemistry for proliferating cell nuclear antigen) did not change, but a transient increase in ductal epithelial cell replication existed between postnatal days (pnd) 10 and 14. Immunoreactive FGF-1 was found mainly in alpha cells of islets, and FGF-2 immunoreactivity and mRNA throughout the islets, their distribution increasing with age. Both FGF-1 and -2 were also located in ductal epithelium, being maximally distributed at pnd 10-14, coincident with increased cell replication, and when mRNA transcripts encoding FGF-1 (4.4 kb) and FGF-2 (7 kb) were relatively increased in pancreata. FGF-4 and -6 immunoreactivities were localized strongly within islets and ductal cells. In contrast, immunoreactive FGF-7 was associated with pancreatic mesenchyme and intra-and extraislet endothelial cells, and mRNA abundance was transiently increased between pnd 4 and 12, suggesting a role in the initiation of endocrine cell neogenesis. Exogenous FGF-7 was fivefold more potent than FGF-1 or -2 in stimulating DNA synthesis within isolated rat islets. Multiple FGF are expressed within defined compartments of developing pancreas and may contribute to endocrine cell neogenesis and islet function.

Increased and persistent circulating insulin-like growth factor II in neonatal transgenic mice suppresses developmental apoptosis in the pancreatic islets.

In rats, a proportion of pancreatic beta-cells are deleted by apoptosis in the second week of postnatal life and replaced by endocrine cell neogenesis from pancreatic ductal epithelium. This coincides with a reduction in pancreatic insulin-like growth factor II (IGF-II) expression, and IGF-II has been shown to act as a beta-cell survival factor in vitro. To examine whether IGF-II regulates beta-cell apoptosis in vivo, an IGF-II transgenic mouse model was used in which mouse IGF-II is overexpressed in skin, gut, and uterus driven by a keratin promoter, so that circulating IGF-II is retained postnatally. Mice were killed between postnatal days 7 and 26, and the pancreas was examined histologically. Apoptotic cells were visualized by the terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling method, and proliferating cells were examined by immunohistochemistry for proliferating cell nuclear antigen. In nontransgenic mice, serum IGF-II was absent by 26 days, but mean (+/-SEM) values were 45+/-9 ng/ml (n = 5) in transgenic animals. A 2- to 3-fold rise in islet cell apoptosis was seen in normal animals between days 11 and 16, but this was substantially decreased in IGF-II transgenic mice (day 11; control, 12+/-1%; transgenic, 6+/-1%; P < 0.01; n = 5). Consequently, islets from IGF-II transgenic mice had a significantly greater mean area from days 11-16, but the proportions of beta- and alpha-cells and circulating insulin levels were not changed. Islet cell DNA synthesis was increased in transgenic mice on days 13 and 16. The total islet number per section did not alter. The results show that a persistent presence of circulating IGF-II postnatally alters endocrine pancreatic ontogeny in the mouse and largely prevents the wave of developmental apoptosis that precipitates beta-cell turnover in neonatal life.

Neonatal beta-cell apoptosis: a trigger for autoimmune diabetes?

In neonatal rodents, the beta-cell mass undergoes a phase of remodeling that includes a wave of apoptosis. Using both mathematical modeling and histochemical detection methods, we have demonstrated that beta-cell apoptosis is significantly increased in neonates as compared with adult rats, peaking at approximately 2 weeks of age. Other tissues, including the kidney and nervous system, also exhibit neonatal waves of apoptosis, suggesting that this is a normal developmental phenomenon. We have demonstrated that increased neonatal beta-cell apoptosis is also present in animal models of autoimmune diabetes, including both the BB rat and NOD mouse. Traditionally, apoptosis has been considered a process that does not induce an immune response. However, recent studies indicate that apoptotic cells can do the following: 1) display autoreactive antigen in their surface blebs; 2) preferentially activate dendritic cells capable of priming tissue-specific cytotoxic T-cells; and 3) induce the formation of autoantibodies. These findings suggest that in some circumstances physiological apoptosis may, in fact, initiate autoimmunity. Initiation of beta-cell-directed autoimmunity in murine models appears to be fixed at approximately 15 days of age, even when diabetes onset is dramatically accelerated. Taken together, these observations have led us to hypothesize that the neonatal wave of beta-cell apoptosis is a trigger for beta-cell-directed autoimmunity.

A low protein diet alters the balance of islet cell replication and apoptosis in the fetal and neonatal rat and is associated with a reduced pancreatic expression of insulin-like growth factor-II.

A programmed turnover of pancreatic beta cells occurs in the neonatal rat involving a loss of beta cells by apoptosis, and their replacement by islet cell replication and neogenesis. The timing of apoptosis is associated with a loss of expression of a survival factor, insulin-like growth factor-II (IGF-II), in the pancreatic islets. Offspring from rats chronically fed a low protein isocalorific diet (LP) exhibit a reduced pancreatic beta cell mass at birth and a reduced insulin secretion in later life. This study therefore investigated the impact of LP on islet cell ontogeny in the late fetal and neonatal rat, and any associated changes in the presence of IGFs and their binding proteins (IGFBPs). Pregnant Wistar rats were fed either LP (8% protein) or normal (C) (20% protein) chow from shortly after conception until the offspring were 21 days postnatal (PN). Bromo-deoxyuridine (BrdU) was administered 1 h before rats were killed and pancreata removed from animals between 19.5 days fetal life and postnatal day 21. Offspring of rats given LP diet had reduced birthweight, pancreatic beta cell mass, and pancreas insulin content, with smaller islets compared with control fed animals, which persisted to weaning. Histological analysis showed that islets from pups given LP diet had a lower nuclear labeling index with BrdU in the beta cells, although, paradoxically, more beta cells showed immunoreactivity for proliferating cell nuclear antigen (PCNA). Because PCNA is present in G1 as well as S phase of the cell cycle, we quantified the number of beta cells immunopositive for cyclin D1, a marker of G1, and NEK2, an indicator of cells in G2 and mitosis. More beta cells in islets from LP-fed animals contained cyclin D1, but less contained NEK2 than did those in controls. This suggests that the beta cell cycle may have a prolonged G1 phase in LP-fed animals in vivo. Offspring of rats given C diet had a low rate of islet cell apoptosis detected by the TUNEL method in fetal and neonatal life (1-2%), with a transient increase to 8% at PN day 14. Offspring of rats receiving LP diet demonstrated a significantly greater level of islet cell apoptosis at every age, rising to 15% at PN 14. IGF-II mRNA was quantified in whole pancreas and was significantly reduced in LP-fed animals at ages up to PN day 10. IGF-II immunoreactivity within the islets of LP-fed rats was also less apparent, but no changes were seen in immunoreactive IGF-I or IGFBPs-2 to -5. These findings show that LP diet changes the balance of beta cell replication and apoptosis in fetal and neonatal neonatal life, which may involve an altered length of beta cell cycle, and contribute to the smaller islet size and impaired insulin release seen in later life. A reduced pancreatic expression of IGF-II may contribute to the lower beta cell proliferation rate and increased apoptosis seen in the fetus and neonate after feeding LP diet.

Insulin-like growth factors prevent cytokine-mediated cell death in isolated islets of Langerhans from pre-diabetic non-obese diabetic mice.

Interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) contribute to the initial stages of the autoimmune destruction of pancreatic beta cells. IL-1beta is released by activated macrophages resident within islets, and its cytotoxic actions include a stimulation of nitric oxide (NO) production and the initiation of apoptosis. Insulin-like growth factors (IGFs)-I and -II prevent apoptosis in non-islet tissues. This study investigated whether IGFs are cytoprotective for isolated islets of Langerhans from non-obese diabetic mice (NOD) mice exposed to cytokines. Pancreatic islets isolated from 5-6-week-old, pre-diabetic female NOD mice were cultured for 48 h before exposure to IL-1beta (1 ng/ml), TNF-alpha (5 ng/ml), IFN-gamma (5 ng/ml) or IGF-I or -II (100 ng/ml) for a further 48 h. The incidence of islet cell apoptosis was increased in the presence of each cytokine, but this was significantly reversed in the presence of IGF-I or -II (IL-1beta control 3.5+/-1.6%, IL-1beta 1 ng/ml 27.1+/-5.8%, IL-1beta+IGF-I 100 ng/ml 4.4+/-2.3%, P<0.05). The majority of apoptotic cells demonstrated immunoreactive glucose transporter 2 (GLUT-2), suggesting that they were beta cells. Islet cell viability was also assessed by trypan blue exclusion. Results suggested that apoptosis was the predominant cause of cell death following exposure to each of the cytokines. Co-incubation with either IGF-I or -II was protective against the cytotoxic effects of IL-1beta and TNF-alpha, but less so against the effect of IFN-gamma. Exposure to cytokines also reduced insulin release, and this was not reversed by incubation with IGFs. Immunohistochemistry showed that IGF-I was present in vivo in islets from pre-diabetic NOD mice which did not demonstrate insulitis, but not in islets with extensive immune infiltration. Similar results were seen for IGF-binding proteins (IGFBPs). These results suggest that IGFs protect pre-diabetic NOD mouse islets from the cytotoxic actions of IL-1beta, TNF-alpha and IFN-gamma by mechanisms which include a reduction in apoptosis.

Overexpression of insulin-like growth factor-II in transgenic mice is associated with pancreatic islet cell hyperplasia.

We have used an insulin-like growth factor (IGF)-II transgenic mouse model in which mouse IGF-II is widely overexpressed, resulting in increased fetal size and selective organ overgrowth, to investigate the effects on the development of the endocrine pancreas. Fetuses examined on day 19.5-20 of gestation had significantly elevated circulating levels of IGF-II, compared with control mice. The pancreatic islets in transgenic animals were of irregular shape and had a mean area five times greater than in controls, whereas the mean number of islets per tissue section was not altered. The size of individual endocrine cells was not altered. Although the islets in animals expressing the IGF-II transgene were considerably larger, immunohistochemistry for insulin and glucagon showed that the relative proportion of beta-cells was significantly less, and that of alpha-cells was higher. Normal islet morphology was disrupted, with alpha-cells appearing in small groups within the islets, as well as on the periphery, whereas beta-cells were often seen at the edge of the islets. Twice as many islet cells (21.9% vs. 11.4%) were involved in cell replication, detected by the presence of immunoreactive proliferating cell nuclear antigen, in pancreata from transgenic mice vs. controls, whereas the number of cells undergoing apoptosis was significantly reduced. Abundant IGF-II messenger RNAwas found within the islets of transgenic animals by in situ hybridization, and the relative area of islets demonstrating immunoreactive IGF-II was significantly greater. Immunoreactive IGF-I was much less abundant and was further reduced in islets of transgenic animals. The area of islets immunopositive for IGF binding protein-2 was unaltered. Despite the presence of islet hyperplasia, circulating insulin levels and serum glucose levels were not significantly different between transgenic and control mice. These results show that an overexpression of IGF-II in fetal life has a profound effect on islet morphology and causes islet hyperplasia while reducing the attrition of islet cells by apoptosis.

Cellular distribution and ontogeny of insulin-like growth factors (IGFs) and IGF binding protein messenger RNAs and peptides in developing rat pancreas.

To determine the role of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in the development of the pancreas, and specifically of the islets of Langerhans, we have examined the cellular distribution and developmental changes in the expression of IGFs and IGFBPs in the pancreas of the fetal and neonatal rat between 19.5 days of gestation and postnatal day 28. This represents a period of substantial growth and restructuring of the beta cell component in islets of this species. IGF-I, IGF-II, and IGFBPs-1 to -6 mRNAs were localized by in situ hybridization, and peptides by immunohistochemistry, in histological sections. IGF-II mRNA was highly expressed in islet cells and some ductal epithelial cells in late fetal and early neonatal life, but was barely detectable by postnatal day 28. IGF-II peptide showed a similar distribution. IGF-I mRNA was barely detected in the fetus or neonate and was localized predominantly in the ductal and acinar tissues after postnatal day 7. IGF-I immunoreactivity was associated with some islet cells in the fetus and neonate, suggesting an endocrine rather than a paracrine source. We performed co-localization studies to assess whether the distribution of IGFs within the pancreas might be due to a sequestration by locally produced IGFBPs. The presence of mRNAs for both IGFBPs-1 and -2 was minimal in the pancreas prior to postnatal day 7, although subsequently IGFBP-1 mRNA was seen in islet cells, while IGFBP-2 mRNA was localized in both islets and acinar tissues. In contrast, both IGFBPs-1 and -2 immunoreactivities were identified in islets from late fetal life, suggesting a circulatory source for these IGFBPs during early pancreatic development. IGFBPs-3 to -5 mRNAs and immunoreactivities were identified within islet cells throughout fetal and neonatal life, with IGFBPs-3 and -5 being mainly associated with the alpha cell-rich islet mantle. The results show a compartmentalization of IGFs within pancreatic tissue, reflecting both paracrine and endocrine sources. The localization and action of IGFs in pancreas likely involves sequestration and distribution by endogenous as well as circulating IGFBPs.

Growth factors and the regulation of fetal growth.

Fetal growth demands a coordinated increase in size of the fetus and the placenta, and both are determined, in part, by locally produced peptide growth factors. The availability of growth factors to individual tissues may be due to local changes in gene expression, but it is also controlled by proteolytic release from extracellular matrix stores. Members of the fibroblast growth factor (FGF) family are stored within basement membranes, while insulin-like growth factors (IGFs) are stored in association with specific binding proteins (IGFBPs). Insulin is a major trophic hormone in utero, and pancreatic beta-cell mass is determined by locally produced IGF-II and members of the FGF family. The mitogenic effects of IGF-II on beta-cells are determined by IGFBPs, which are themselves expressed with a distinct ontogeny within the islets of Langerhans. Overexpression of IGF-II or IGFBP-I can result in nesidioblastosis. Shortly after birth in rodents, many pancreatic beta-cells are destroyed by a process of apoptosis but are simultaneously replaced as a result of beta-cell neogenesis. This process may enrich the pancreas in beta-cells suited to the metabolic demands of postnatal life. The wave of beta-cell apoptosis coincides with a dramatic decrease in the local expression of IGF-II. These events may be functionally linked, because exogenous IGF-II will protect isolated islets from cytokine-induced apoptosis. FGF-2 is also widely expressed within fetal tissues and may be an important regulator of placental angiogenesis. FGF-2 appears in the maternal circulation during pregnancy, with peak values late in the 2nd trimester. It is associated with a circulating binding protein derived from the extracellular domain of the FGFR1 receptor. Levels of FGF-2 in maternal serum correlate positively with fetal size both in the 2nd trimester and at term. The expression of FGF-2 in placenta and its presence in maternal blood are elevated in pregnancies complicated by diabetes and are greatest in diabetic pregnancies associated with retinopathy. Thus, maternal FGF-2 may be a useful indicator of both fetal development and the risk of maternal pathology in pregnancies complicated by diabetes.

Apoptosis in the pancreatic islet cells of the neonatal rat is associated with a reduced expression of insulin-like growth factor II that may act as a survival factor.

Islet cell ontogeny will define adult beta-cell mass and will consist of a balance of islet cell birth and death. We have investigated the ontogeny of factors that may be related to developmental apoptosis in the islets, insulin-like growth factor II (IGF-II) and inducible nitric oxide synthase (iNOS), in pancreata of young Wistar rats. Pancreata were collected from rats of 21 days gestation to 29 days postnatal age. In situ hybridization and immunohistochemistry showed that IGF-II was expressed and present in fetal and neonatal islet cells, but declined rapidly 2 weeks after birth. Little IGF-I was associated with fetal or postnatal islets. Apoptosis in islet cells was visualized by molecular histochemistry for DNA breakage in tissue sections. Apoptosis was low in the fetus, but increased in incidence postnatally so that 13% of islet cells were undergoing apoptosis on postnatal day 14, with the incidence declining thereafter. Immunohistochemistry for iNOS showed that it was expressed within beta-cells and was most abundant 12 days after birth. When islets were isolated from rat pancreata 20-22 days after birth, islet cell viability, DNA synthetic rate, and insulin release were reduced after incubation with interleukin-1beta, tumor necrosis factor, or interferon-gamma. An increased rate of islet cell survival was found after simultaneous incubation with IGF-I or -II. Cytokine-mediated islet cell death involved the induction of apoptosis. Islets isolated from neonatal rats were not killed after exposure to these cytokines at the same concentrations, but cytokine-induced cell death was seen when neonatal islets were incubated with a neutralizing antibody against IGF-II. These experiments show that a peak of islet cell apoptosis that is maximal in the rat pancreas 14 days after birth is temporally associated with a fall in the islet cell expression of IGF-II. IGF-II was shown to function as an islet survival factor in vitro. The induction of islet cell apoptosis in vivo may involve an increased expression of iNOS within beta-cells.

Human placenta and fetal membranes contain peptide YY1-36 and peptide YY3-36.

Extracts of human term amniotic, placental, and chorion/decidua tissue contained, respectively, 4.36 +/- 2.79 (pmol/g wet wt; mean +/- S.E.M.: n = 5). 2.78 +/- 0.5 (n = 5) and 0.68 +/- 0.68 (n = 5) peptide YY (PYY)-like immunoreactivity. Using a specific PYY antiserum, gel filtration chromatography and reverse-phase high performance liquid chromatography (HLPC), amniotic, placental and fetal intestinal tissue extracts were demonstrated to contain PYY-like immunoreactivity consisting of equal amounts of PYY1-36 and PYY3-36. The presence of pancreatic polypeptide was not detected in any of the extracts. Positive immunohistochemical staining for PYY was seen in extravillous trophoblasts in the decidual septa and fetal membranes, the syncytiotrophoblast and cytotrophoblasts, amniotic epithelial cells and in maternal decidual stromal cells. Positive staining for PYY was found at the earliest date examined (9.5 weeks) and remained present throughout pregnancy to term. PYY1-36 and PYY3-36 may play important roles in human pregnancy, acting via endocrine or paracrine mechanisms.

Fibroblast growth factor-2 and fibroblast growth factor receptor-1 mRNA expression and peptide localization in placentae from normal and diabetic pregnancies.

Fibroblast growth factor-2 (FGF-2) is a potent mitogen expressed widely during embryogenesis and in tissues of the human fetus. It is recognized as an endothelial cell mitogen and is angiogenic in vivo. Expression of FGF-2 mRNA has also been shown within the human term placenta, and FGF-2 isolated from placental tissue, suggesting a role in placental growth including angiogenesis. The purpose of this study was to quantify and localize the sites of expression of FGF-2 and its high-affinity receptor, FGFR1, within placentae from normal term human pregnancies (n=8, 39-42 weeks), and pregnancies complicated by pregestational, type 1 diabetes (n=8, 36-40 weeks). Tissues were collected immediately following delivery and were either snap-frozen for RNA isolation, or fixed for either in situ hybridization using a 35S-labelled cRNAs encoding human FGF-2 or FGFR1, or immunocytochemistry using antibodies against human FGF-2 or FGFR1. Northern blot hybridization showed a significantly increased abundance of mRNAs for both FGF-2 and FGFR1 in placentae from diabetic women compared to those from normal women. In normal term placenta FGF-2 mRNA was present at low abundance in fetal villous tissue, in the vascular endothelium of blood vessels, and in the syncytiotrophoblast. FGF-2 mRNA was considerably more abundant in the syncytiotrophoblast and villous tissue of placentae from diabetic patients. Messenger RNA for FGFR1 was similarly distributed to that encoding FGF-2. Immunocytochemistry revealed abundant FGF-2 and FGFR1 peptides in villous vascular endothelial cells, and associated with the cell membranes of stromal tissues in placentae from control pregnancies. Little immunoreactive FGF-2 was present in the syncytiotrophoblast at term. In pregnancies complicated by diabetes intense staining for immunoreactive FGF-2 and for FGFR1 additionally existed in syncytiotrophoblast. The results suggest that FGF-2 acting as an autocrine agent contributes to placental angiogenesis, but may be released from the syncytium into the maternal circulation. Expression is elevated in placentae from diabetic pregnancies, and is particularly associated with the syncytiotrophoblast. This suggests a placental source for the elevated circulating maternal FGF-2 previously described in diabetic pregnancy.