Precursor cells in mouse islets generate new beta-cells in vivo during aging and after islet injury.

Whereas it is believed that the pancreatic duct contains endocrine precursors, the presence of insulin progenitor cells residing in islets remain controversial. We tested whether pancreatic islets of adult mice contain precursor beta-cells that initiate insulin synthesis during aging and after islet injury. We used bigenic mice in which the activation of an inducible form of Cre recombinase by a one-time pulse of tamoxifen results in the permanent expression of a floxed human placental alkaline phosphatase (PLAP) gene in 30% of pancreatic beta-cells. If islets contain PLAP(-) precursor cells that differentiate into beta-cells (PLAP(-)IN(+)), a decrease in the percentage of PLAP(+)IN(+) cells per total number of IN(+) cells would occur. Conversely, if islets contain PLAP(+)IN(-) precursors that initiate synthesis of insulin, the percentage of PLAP(+)IN(+) cells would increase. Confocal microscope analysis revealed that the percentage of PLAP(+)IN(+) cells in islets increased from 30 to 45% at 6 months and to 60% at 12 months. The augmentation in the level of PLAP in islets with time was confirmed by real-time PCR. Our studies also demonstrate that the percentage of PLAP(+)IN(+) cells in islets increased after islet injury and identified putative precursors in islets. We postulate that PLAP(+)IN(-) precursors differentiate into insulin-positive cells that participate in a slow renewal of the beta-cell mass during aging and replenish beta-cells eliminated by injury.

Abrogation of protein convertase 2 activity results in delayed islet cell differentiation and maturation, increased alpha-cell proliferation, and islet neogenesis.

To date, the role of pancreatic hormones in pancreatic islet growth and differentiation is poorly understood. To address this issue, we examined mice with a disruption in the gene encoding prohormone convertase 2 (PC2). These mice are unable to process proglucagon, prosomatostatin, and other neuroendocrine precursors into mature hormones. Initiation of insulin (IN) expression during development was delayed in PC2 mutant mice. Cells containing IN were first detected in knockout embryos on d 15 of development, 5 d later than in wild-type littermates. However, the IN(+) cells of d 15 PC2 mutant mice coexpressed glucagon, as did the first appearing beta-cells of controls. In addition, lack of PC2 perturbed the pattern of expression of transcription factors presumed to be involved in the determination of the mature alpha-cell phenotype. Thus, in contrast to controls, alpha-cells of mutant mice had protracted expression of Nkx 6.1 and Pdx-1, but did not express Brn-4. Islets of adult mutant mice also contained cells coexpressing insulin and somatostatin, an immature cell type found only in islets of the wild-type strain during development. In addition to the effects on islet cell differentiation, the absence of PC2 activity resulted in a 3-fold increase in the rate of proliferation of proglucagon cells during the perinatal period. This increase contributed to the development of alpha-cell hyperplasia during postnatal life. Furthermore, the total beta-cell volume was increased 2-fold in adult mutants compared with controls. This increase was due to islet neogenesis, as the number of islets per section was significantly higher in knockout mice compared with wild-type mice, whereas both strains had similar rates of IN cell proliferation. These results indicate that hormones processed by PC2 affected processes that regulate islet cell differentiation and maturation in embryos and adults.

Detrimental effect of protracted hyperglycaemia on beta-cell neogenesis in a mouse murine model of diabetes.

AIMS/HYPOTHESIS: Previous studies have shown that new beta cells differentiate from intra-islet precursors in pancreatic islets of mice in which diabetes is induced by injecting a high dose of the beta-cell toxin streptozotocin. Moreover, the re-establishment of euglycaemia by insulin therapy 1 day after streptozotocin treatment improved the process of regeneration. We sought to assess whether a 1-week delay in the restoration of euglycaemia would affect beta-cell regeneration. METHODS: Adult CD-1 mice were injected with 200 mg/kg of streptozotocin. One group of mice remained hyperglycaemic throughout the experiment while a second group became normoglycaemic following the administration of insulin therapy 1 week after the injection of streptozotocin. Pancreata removed at different times after treatment were processed for visualization ofbeta precursor-cell markers and insulin by confocal microscopy. RESULTS: New beta cells appeared in islets of streptozotocin-treated mice after restoration of normoglycaemia. Like islets of streptozotocin mice in which blood glucose concentrations were rapidly restored, islets of mice that became normoglycaemic 1 week after streptozotocin treatment also had two potential insulin precursor cell types. Protracted hyperglycaemia however, had several harmful effects on insulin cell neogenesis, such as a reduction in the number of euglycaemic mice with successful beta-cell regeneration and a decrease in the number and survival of the newly differentiated insulin-containing cells. CONCLUSION/INTERPRETATION: These results indicate that islets gradually lose their regenerative potential when they are exposed to high circulating glucose concentrations for an extended period of time.

Regeneration of pancreatic beta cells from intra-islet precursor cells in an experimental model of diabetes.

We previously reported that new beta cells differentiated in pancreatic islets of mice in which diabetes was produced by injection of a high dose of the beta cell toxin streptozotocin (SZ), which produces hyperglycemia due to rapid and massive beta cell death. After SZ-mediated elimination of existing beta cells, a population of insulin containing cells reappeared in islets. However, the number of new beta cells was small, and the animals remained severely hyperglycemic. In the present study, we tested whether restoration of normoglycemia by exogenous administered insulin would enhance beta cell differentiation and maturation. We found that beta cell regeneration improved in SZ-treated mice animals that rapidly attained normoglycemia following insulin administration because the number of beta cells per islet reached near 40% of control values during the first week after restoration of normoglycemia. Two presumptive precursor cell types appeared in regenerating islets. One expressed the glucose transporter-2 (Glut-2), and the other cell type coexpressed insulin and somatostatin. These cells probably generated the monospecific cells containing insulin that repopulated the islets. We conclude that beta cell neogenesis occurred in adult islets and that the outcome of this process was regulated by the insulin-mediated normalization of circulating blood glucose levels.

Expression pattern for adrenomedullin during pancreatic development in the rat reveals a common precursor with other endocrine cell types.

Adrenomedullin is an alpha-amidated 52-amino acid peptide involved in many physiological actions, among others the regulation of insulin secretion. Using immunohistochemical methods, we found that adrenomedullin immunoreactivity first appears at day 11.5 of embryonic development in the rat, coinciding with the appearance of pancreatic glucagon. The early appearance of adrenomedullin in the developing pancreas may indicate an active involvement in either the morphogenesis of the organ or its endocrine/paracrine/autocrine hormone regulation during intrauterine life. We also investigated the pattern of colocalizations of adrenomedullin with the other pancreatic hormones. At some point during development all the cell types express adrenomedullin, progressively evolving towards the adult pattern where only the pancreatic polypeptide cells contain a strong immunoreactivity for adrenomedullin. At this point the remaining cells of the islet are, in general, weakly stained. This sequential and time-dependent expression of adrenomedullin suggests a tight regulation similar to that observed for other modulatory substances responsible for embryonic morphogenesis.

Islet injury induces neurotrophin expression in pancreatic cells and reactive gliosis of peri-islet Schwann cells.

Pancreatic islets are enveloped by a sheath of Schwann cells, the glial cells of the peripheral nervous system (PNS). The fact that Schwann cells of the PNS become reactive and express nerve growth factor (NGF) and other growth factors following axotomy suggested the possibility that peri-islet Schwann cells could become activated by islet injury. To test this hypothesis, we examined two animal models of islet injury. The first model was mice and rats injected with streptozotocin (SZ), a specific beta-cell toxin. The second model was NOD mice, a strain in which beta cells are deleted by an autoimmune process. We found that peri-islet Schwann cells became reactive following islet injury and began to express increased levels of NGF and the neurotrophin receptor p75. Lesions to the pancreas also markedly induced NGF expression by exocrine and endocrine cells. Neurotrophin expression was not unique to adult tissues since pancreatic cells transiently expressed p75, the NGF receptor Trk A, and NGF during development. These observations suggest that NGF could play an important role in pancreas during embryogenesis and in processes leading to repair following islet injury in adults.

Differentiation of new insulin-producing cells is induced by injury in adult pancreatic islets.

The ability of the adult pancreas to generate new insulin (beta) cells has been controversial because of difficulties in unequivocally identifying the precursor population. We recently determined that beta cells were generated during development from precursors that expressed the homeodomain-containing transcription factor pancreas duodenum homeobox gene-1 (PDX-1). To investigate whether PDX-1+ stem cells are present in adult pancreas, we examined two animal models of diabetes. One model was produced by injecting adult mice with streptozotocin (SZ), a toxin that produces hyperglycemia due to rapid and massive beta cell death. After SZ-mediated elimination of existing IN+/PDX-1+ cells, a population of somatostatin (SOM)+/PDX-1+ cells, a cell type thought to represent an embryonic islet precursor cell, appeared in islets. The appearance of SOM+/PDX-1+ cells was followed in time by the differentiation to SOM+/IN+/PDX-1+ cells. SOM+/PDX-1+ cells also appeared in islets of nonobese diabetic mice, a strain of mice in which beta cell destruction is immune-mediated. Our findings establish the existence of PDX-1+ beta cell precursors in the adult pancreas and indicate that their differentiation is induced by islet injury.

DARPP-32 promoter directs transgene expression to renal thick ascending limb of loop of Henle.

DARPP-32, a dopamine- and adenosine 3',5'-cyclic monophosphate (cAMP)-regulated inhibitor of protein phosphatase-1, is highly colocalized with neuronal and nonneuronal D1-type receptors. DARPP-32 concentration is enriched in the renal outer medulla and in the medium-size spiny neurons of the brain. In the ascending limb of the loop of Henle, DARPP-32 is phosphorylated following stimulation by dopamine and other first messengers, and in this form inhibits the activity of the Na(+)-K(+)-adenosinetriphosphatase pump. For functional analysis of the DARPP-32 promoter in the kidney, we characterized the murine gene. There are two groups of transcription start sites utilized in the brain, but the proximal set appears to be preferentially used in the kidney. In four of four lines of mice carrying a DARPP-32/lacZ transgene with 2.1 kb of 5'-flanking DNA, adult kidney lacZ transgene expression mimicked that of endogenous DARPP-32. There was no ectopic expression in peripheral organs. We conclude that the sequences necessary for direction of DARPP-32 expression to the medullary thick ascending limb are contained within this 2.1-kb fragment.

A transgene coding for a human insulin analog has a mitogenic effect on murine embryonic beta cells.

We have investigated the mitogenic effect of three mutant forms of human insulin on insulin-producing beta cells of the developing pancreas. We examined transgenic embryonic and adult mice expressing (i) human [AspB10]-proinsulin/insulin ([AspB10]ProIN/IN), produced by replacement of histidine by aspartic acid at position 10 of the B chain and characterized by an increased affinity for the insulin receptor; (ii) human [LeuA3]insulin, produced by the substitution of leucine for valine in position 3 of the A chain, which exhibits decreased receptor binding affinity; and (iii) human [LeuA3, AspB10]insulin "double" mutation. During development, beta cells of AspB10 embryos were twice as abundant and had a 3 times higher rate of proliferation compared with beta cells of littermate controls. The mitogenic effect of [AspB10]ProIN/IN was specific for embryonic beta cells because the rate of proliferation of beta cells of adults and of glucagon (alpha) cells and adrenal chromaffin cells of embryos was similar in AspB10 mice and controls. In contrast to AspB10 embryos, the number of beta cells in the LeuA3 and "double" mutant lines was similar to the number in controls. These findings indicate that the [AspB10]ProIN/IN analog increased the rate of fetal beta-cell proliferation. The mechanism or mechanisms that mediate this mitogenic effect remain to be determined.

Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny.

The XlHbox 8 homeodomain protein of Xenopus and STF-1, its mammalian homolog, are selectively expressed by beta cells of adult mouse pancreatic islets, where they are likely to regulate insulin expression. We sought to determine whether the expression of the homeobox protein/s during mouse embryonic development was specific to beta cells or, alternatively, whether XlHbox 8/STF-1 protein/s were initially expressed by multipotential precursors and only later became restricted to the insulin-containing cells. With two antibodies, we studied the localization of STF-1 during murine pancreatic development. In embryos, as in adults, STF-1 was expressed by most beta cells, by subsets of the other islet cell types and by mucosal epithelial cells of the duodenum. In addition, most epithelial cells of the pancreatic duct and exocrine cells of the pancreas transiently contained STF-1. We conclude that in mouse, STF-1 not only labels a domain of intestinal epithelial cells but also provides a spatial and temporal marker of endodermal commitment to a pancreatic and subsequently, to an endocrine beta cell fate. We propose a model of pancreatic cell development that suggests that exocrine and endocrine (alpha, beta, delta and PP) cells arise from a common precursor pool of STF-1+ cells and that progression towards a defined monospecific non-beta cell type is correlated with loss of STF-1 expression.

Insulin expression in pancreatic islet cells relies on cooperative interactions between the helix loop helix factor E47 and the homeobox factor STF-1.

The development of endocrine cell types within the pancreas is thought to involve the progressive restriction of pluripotential stem cells, which gives rise to the four major cell types: insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-expressing cells. The mechanism by which these peptide hormone genes are induced and then either maintained or repressed during development is unknown, but their coexpression in early precursor cells suggests the involvement of common regulatory factors. Here we show that the somatostatin transcription factor STF-1 is also a principal regulator of insulin expression in beta-cells of the pancreas. STF-1 stimulates the insulin gene by recognizing two well defined islet-specifying elements on the insulin promoter and by subsequently synergizing in trans with the juxtaposed helix-loop-helix protein E47. Within the STF-1 protein, an N-terminal trans-activation domain functions cooperatively with E47 to stimulate insulin transcription. As truncated STF-1 polypeptides lacking the N-terminal activation domain strongly inhibit insulin promoter activity in beta-islet cells, our results suggest that the specification of islet cell types during development may be in part determined by the expression of STF-1 relative to other islet cell factors.

Isolation and characterization of a novel transcription factor that binds to and activates insulin control element-mediated expression.

Pancreatic beta-cell-type-specific transcription of the insulin gene is principally regulated by a single cis-acting DNA sequence element, termed the insulin control element (ICE), which is found within the 5'-flanking region of the gene. The ICE activator is a heteromeric complex composed of an islet alpha/beta-cell-specific factor associated with the ubiquitously distributed E2A-encoded proteins (E12, E47, and E2-5). We describe the isolation and characterization of a cDNA for a protein present in alpha and beta cells, termed INSAF for insulin activator factor, which binds to and activates ICE-mediated expression. INSAF was isolated from a human insulinoma cDNA library. Transfection experiments demonstrated that INSAF activates ICE expression in insulin-expressing cells but not in non-insulin-expressing cells. Cotransfection experiments showed that activation by INSAF was inhibited by Id, a negative regulator of basic helix-loop-helix (bHLH) protein function. INSAF was also shown to associate in vitro with the bHLH protein E12. In addition, affinity-purified INSAF antiserum abolished the formation of the activator-specific ICE-binding complex. Immunohistochemical studies indicate that INSAF is restricted in terms of its expression pattern, in that INSAF appears to be detected only within the nuclei of islet pancreatic alpha and beta cells. All of these data are consistent with the proposal that INSAF is either part of the ICE activator or is antigenically related to the specific activator required for insulin gene transcription.

Mammalian adrenal chromaffin cells coexpress the epinephrine-synthesizing enzyme and neuronal properties in vivo and in vitro.

Adrenal chromaffin cells and neurons of the sympathetic ganglia are derived from common precursors in the neural crest. The phenotype of the sympathoadrenal progenitor cell is unknown, but adult chromaffin cells are distinguished by the expression of phenylethanolamine-N-methyltransferase (PNMT) and the lack of neurofilament (NF) and neuritic processes. Mature neurons have processes and express NF, but are PNMT-. We hypothesize that embryonic adrenal cells are multipotential. This implies that the cells can coactivate all the traits characteristic of mature sympathetic neurons and chromaffin cells and then selectively extinguish expression of either the chromaffin or the neuronal traits, depending on the environment. We further asked whether this repression is plastic and can be environmentally modified in adult chromaffin cells. We demonstrate that, in vivo, embryonic (e-) rat adrenal cells coexpress PNMT and the intermediate- and high-molecular-weight neurofilaments at e-15.5 (21%), e-16.5 (40%), and e-20.5 (23%). When cultured in complete or glucocorticoid-depleted media for 5 to 14 days, 20% of adult bovine chromaffin cells which remain PNMT+ reexpress NF and extend NF+ and PNMT+ processes. Both the expression of NF and the extension of neurites are inhibited by the addition of 10(-7) M dexamethasone to complete media. We conclude that the embryonic adrenal medullary cells simultaneously express traits of mature chromaffin cells and neurons and that the phenotypes remain labile in the adult mammalian chromaffin cell. In addition, coexpression of PNMT, NF, and neurite extension are not mutually exclusive in either the embryonic or adult adrenal chromaffin cell.

XIHbox 8, an endoderm-specific Xenopus homeodomain protein, is closely related to a mammalian insulin gene transcription factor.

The cis-acting sequences that mediate insulin gene expression exclusively in pancreatic islet beta-cells are localized within the 5'-flanking region between nucleotides -340 and -91. We have identified an evolutionarily conserved, A+T-rich element at -201/-196 basepairs in the rat insulin II gene that is essential for efficient expression in beta-cells. Affinity-purified antibody to the XIHbox 8 protein super-shifted the major beta-cell-activator factor complex binding to the -201/-196 element. XIHbox 8 is a Xenopus endoderm-specific homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and developing pancreas. Antibody to XIHbox 8 specifically interacts with a 47-kilodalton protein present in this DNA complex. Immunohistochemical studies revealed XIHbox 8-like proteins within the nucleus of almost all mouse islet beta-cells and a subset of islet alpha- and beta-cells. These results are consistent with the proposal that an XIHbox 8-related homeoprotein of 47 kilodalton is required for expression of the mammalian insulin gene in beta-cells. Experiments conducted with antiserum raised to somatostatin transcription factor-1 (STF-1), a recently isolated mammalian XIHbox 8-related homeoprotein, indicate that the STF-1 protein is the mammalian homolog of Xenopus XIHbox 8.

Expression of the trans-active factors that stimulate insulin control element-mediated activity appear to precede insulin gene transcription.

Cell type-specific expression of the major differentiated products of alpha (glucagon) and beta (insulin) cells are regulated by sequences found within their 5'-flanking region. Specific transcription of the insulin gene appears to be principally controlled by a single cis-acting DNA element, termed the insulin control element (ICE). The ICE activator acts in combination with other positive regulatory factors that interact within this region to generate the correct, cell type-specific expression. In the present study, we show that the ICE activator is not only present but is functionally active in the islet glucagon-producing alpha cell line, alpha TC6. Analysis of the expression of various transfected insulin enhancer expression plasmids demonstrated that the insulin enhancer is active in alpha TC6 cells, although at a lower level than in beta cells. The reduced transcription from these constructs appears to be a consequence of the lack of other essential positive regulator(s). The alpha TC6 cells were also shown to display neuronal-like properties. Since islet cells appear to evolve from an alpha-like precursor cell that transiently expresses neuronal cell markers, these results would indicate that the ICE activator factor is induced before transcription of the insulin gene in the developing islet.

Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon and the neuronal proteins tyrosine hydroxylase and neuropeptide Y, but not pancreatic polypeptide.

The early progenitor cells to the pancreatic islets in the mouse have been characterized so as to re-examine their possible lineage relationships to the four islet cell types found in mature islets. Insulin and glucagon were both first expressed at embryonic day 9.5, and many cells coexpressed these two markers, as shown by light and electron microscopic analysis using double-label immunohistochemistry. Incubation of embryonic pancreas with 1% glutaraldehyde, a fixative commonly used by electron microscopists, abolished this reactivity, thereby explaining reported difficulties in detecting these precursor cells. Using antisera specific for neuropeptide Y (NPY) a peptide with considerable homology to pancreatic polypeptide (PP), we show that NPY first appears with insulin and glucagon immunoreactivity at E9.5, and is co-expressed with glucagon in a majority of adult alpha cells. As we have previously reported, PP itself is first detectable immunocytochemically at postnatal day 1 with PP-specific antibodies. However, antibodies raised against bovine PP are shown by dot blotting to recognize NPY with comparable avidity, indicating that a recent report of islet progenitor cells containing PP at E9.5 (Herrera, P. L., Huarte, J., Sanvito, F., Meda, P., Orci, L. and Vassalli, J. D. (1991) Development 113, 1257-1265), actually represents cross-reactivity to NPY. The data support a model in which early precursor cells to the endocrine pancreas co-activate and co-express a set of islet cell hormone and neural genes, whose expression is both selectively increased and extinguished as development proceeds, concomitant with a restriction to the patterns of expression characteristic of mature islet cell types.

Hypertension and a tumor of the glomus jugulare region. Evidence for epinephrine biosynthesis.

Glomus jugulare tumors have been reported to secrete norepinephrine and cause severe hypertension with features similar to pheochromocytoma. In contrast, epinephrine secretion has not been observed in these neoplasms. This has been attributed to the absence of the norepinephrine-methylating enzyme, phenylethanolamine-N-methyltransferase (PNMT), required for epinephrine synthesis. We report a patient with severe hypertension caused by a glomus tumor that secreted norepinephrine and epinephrine. Following selective venous sampling, catecholamines were quantified by radioenzymatic assay. Marked elevations in norepinephrine and epinephrine release were localized to the glomus tumor. The enzymes involved in catecholamine biosynthesis, including PNMT and tyrosine hydroxylase, were identified immunocytochemically in the tumor. The glomus tumor had staining patterns identical to those observed within normal rat glomus cell. Hypertension resolved with resection of the functioning tumor. This is the first report of PNMT in a functioning paraganglioma of the glomus jugulare region. The factors that determine why functional activity is expressed only rarely by paraganglioma remain undefined.

On the origin of pancreatic endocrine cells, proliferation and neoplastic transformation.

In the adult mouse, pancreatic islets contain four islet cell types: alpha, beta, delta and pancreatic polypeptide cells that synthesize glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. The early progenitor cells to the pancreatic islets are multipotential and coactivate all the islet-specific genes from the time they first appear. As development proceeds, expression of islet-specific hormones becomes restricted to the pattern of expression characteristic of mature islet cells. The phenotype of mature islet cells, however, is not stable since different environmental stimuli can induce the reappearance of embryonal traits in mature beta cells.