Gary L. Pittenger

Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters.

Induction of islet neogenesis by cellophane wrapping (CW) reverses streptozotocin-induced (STZ) diabetes. Administration of Ilotropin, a protein extract isolated from CW pancreata, causes recapitulation of normal islet ontogeny and reverses STZ diabetes, reducing mortality by 50%. We investigated the hypothesis that a novel gene encoding a constituent of Ilotropin was expressed in the hamster pancreas undergoing islet neogenesis. Islet neogenesis associated protein (INGAP) is a product of a novel gene expressed in regenerating hamster pancreas. Northern blot analysis showed a strong single transcript of 850 bp at 1 and 2 d after CW that disappeared by the 6th day and was absent from untreated control pancreata. INGAP gene is expressed in acinar cells, but not in islets. Western blot analysis demonstrated the presence of INGAP in Ilotropin but not in extracts from control pancreata. A synthetic pentadecapeptide, corresponding to a region unique to INGAP, stimulated a 2.4-fold increase in [3H]thymidine incorporation into hamster duct epithelium in primary culture and a rat pancreatic duct cell line but had no effect on a hamster insulinoma tumor cell line. A portion of human INGAP gene was cloned and appears to be highly homologous to the hamster gene. This data suggests that the INGAP gene is a novel pancreatic gene expressed during islet neogenesis whose protein product is a constituent of Ilotropin and is capable of initiating duct cell proliferation, a prerequisite for islet neogenesis.

A pentadecapeptide fragment of islet neogenesis-associated protein increases beta-cell mass and reverses diabetes in C57BL/6J mice.

Objective:The objective of this study was to demonstrate that islet neogenesis-associated protein (INGAP) peptide, a pentadecapeptide containing the biologically active portion of native INGAP, increases functional β-cell mass in normal animals and can be used therapeutically to reverse hyperglycemia in streptozotocin-induced diabetes. Summary Background Data:INGAP, a 175 amino acid pancreatic acinar cell protein, has been suggested to be implicated in β-cell mass expansion. Methods:In the first part of this study, normoglycemic hamsters were administered either 500 &mgr;g INGAP peptide (n = 30) or saline (n = 20) intraperitoneally daily and sacrificed after 10 or 30 days of treatment. Blood glucose and insulin levels were measured, and a histologic and morphometric analysis of the pancreas was performed to determine the effect of INGAP peptide on the endocrine pancreas. In the second part of the study, 6- to 8-week-old C57BL/6J mice (n = 8) were administered multiple low doses of the β-cell toxin streptozotocin (STZ) inducing insulitis and hyperglycemia. The mice were then injected with INGAP peptide (n = 4) or saline (n = 4) for 39 days and sacrificed at 48 days. Two additional groups of diabetic mice were administered either a peptide composed of a scrambled sequence of amino acids from INGAP peptide (n = 5) or exendin-4 (n = 5), an incretin that has been associated with amelioration of hyperglycemia. Results:Islet cell neogenesis was stimulated in INGAP-treated hamsters by 10 days. At 30 days, the foci of new endocrine cells had the appearance of mature islets. There was a 75% increase in islet number, with normal circulating levels of blood glucose and insulin. Administration of INGAP peptide to diabetic mice reversed the diabetic state in all animals, and this was associated with increased expression of PDX-1 in duct cells and islet cell neogenesis with a reduction of insulitis in the new islets. Diabetic mice treated with exendin-4 or a scrambled INGAP peptide did not revert from hyperglycemia. Conclusion:Because there is a deficiency of β-cell mass in both type-1 and type-2 diabetes, INGAP peptide stimulation of fully functional neoislet differentiation may provide a novel approach for diabetes therapy.

Nerve Growth Factor and Diabetic Neuropathy

Neuropathy is one of the most debilitating complications of both type 1 and type 2 diabetes, with estimates of prevalence between 50–90% depending on the means of detection. Diabetic neuropathies are heterogeneous and there is variable involvement of large myelinated fibers and small, thinly myelinated fibers. Many of the neuronal abnormalities in diabetes can be duplicated by experimental depletion of specific neurotrophic factors, their receptors or their binding proteins. In experimental models of diabetes there is a reduction in the availability of these growth factors, which may be a consequence of metabolic abnormalities, or may be independent of glycemic control. These neurotrophic factors are required for the maintenance of the neurons, the ability to resist apoptosis and regenerative capacity. The best studied of the neurotrophic factors is nerve growth factor (NGF) and the related members of the neurotrophin family of peptides. There is increasing evidence that there is a deficiency of NGF in diabetes, as well as the dependent neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) that may also contribute to the clinical symptoms resulting from small fiber dysfunction. Similarly, NT3 appears to be important for large fiber and IGFs for autonomic neuropathy. Whether the observed growth factor deficiencies are due to decreased synthesis, or functional, e.g. an inability to bind to their receptor, and/or abnormalities in nerve transport and processing, remains to be established. Although early studies in humans on the role of neurotrophic factors as a therapy for diabetic neuropathy have been unsuccessful, newer agents and the possibilities uncovered by further studies should fuel clinical trials for several generations. It seems reasonable to anticipate that neurotrophic factor therapy, specifically targeted at different nerve fiber populations, might enter the therapeutic armamentarium.

Small fiber neuropathy is associated with the metabolic syndrome.

BACKGROUND The etiology of sensory neuropathies is often not found. We tested the hypothesis that the metabolic syndrome (MS) may be associated with painful neuropathy, in the absence of frank diabetes. METHODS Clinical and quantitative neuropathy assessments were performed on 10 neuropathy patients with MS, 20 with MS with type 2 diabetes (10 recent onset and 10 of >5 years in duration), and 10 healthy, age-matched subjects. Intraepidermal nerve fiber density (IENF) and mean dendrite length (MDL) were determined by quantitative immunofluorescence on skin biopsies using antibody to PGP 9.5. RESULTS In metabolic syndrome, MDL was reduced and correlated negatively with sensory symptoms, signs, HDL-C, and sural nerve amplitude. The strongest inverse metabolic correlate of the metabolic syndrome neuropathy was HDL-C, which also correlated negatively with sensory symptoms, signs, and sural nerve amplitudes. The strongest metabolic correlate of diabetic sensorimotor neuropathy was HbA1c, which was associated with reduced IENF in patients with >5 years in duration of disease as well as reduced peroneal nerve amplitudes. CONCLUSIONS These data indicate that metabolic features, including HDL-C in metabolic syndrome, are associated with small fiber neuropathy and that MDL is an early marker of sensory neuropathy in patients with MS. Reductions in IENF reflect a longer duration of diabetes, with hyperglycemia leading to a sensorimotor neuropathy. These findings support the possibility that there is a sensory neuropathy with reduced intraepidermal nerve fiber length, which cosegregates with features of metabolic syndrome.

Clinical features and morphological characterization of 10 patients with noninsulinoma pancreatogenous hypoglycaemia syndrome (NIPHS).

Objective  Noninsulinoma pancreatogenous hypoglycaemia syndrome (NIPHS), characterized by postprandial neuroglycopaenia, negative prolonged fasts and negative perioperative localization studies for insulinoma, but positive selective arterial calcium stimulation tests and nesidioblastosis in the gradient‐guided resected pancreas, is a rare hypoglycaemic disorder of undetermined aetiology. We analysed the clinical, morphological and immunohistological features to further clarify the aetiology and pathogenesis of this rare disease.

Differential hypertrophy and atrophy among all types of cutaneous innervation in the glabrous skin of the monkey hand during aging and naturally occurring type 2 diabetes

Diabetic neuropathy (DN) is a common severe complication of type 2 diabetes. The symptoms of chronic pain, tingling, and numbness are generally attributed to small fiber dysfunction. However, little is known about the pathology among innervation to distal extremities, where symptoms start earliest and are most severe, and where the innervation density is the highest and includes a wide variety of large fiber sensory endings. Our study assessed the immunochemistry, morphology, and density of the nonvascular innervation in glabrous skin from the hands of aged nondiabetic rhesus monkeys and from age‐matched monkeys that had different durations of spontaneously occurring type 2 diabetes. Age‐related reductions occurred among all types of innervation, with epidermal C‐fiber endings preferentially diminishing earlier than presumptive Aδ‐fiber endings. In diabetic monkeys epidermal innervation density diminished faster, became more unevenly distributed, and lost immunodetectable expression of calcitonin gene‐related peptide and capsaicin receptors, TrpV1. Pacinian corpuscles also deteriorated. However, during the first few years of hyperglycemia, a surprising hypertrophy occurred among terminal arbors of remaining epidermal endings. Hypertrophy also occurred among Meissner corpuscles and Merkel endings supplied by Aβ fibers. After longer‐term hyperglycemia, Meissner corpuscle hypertrophy declined but the number of corpuscles remained higher than in age‐matched nondiabetics. However, the diabetic Meissner corpuscles had an abnormal structure and immunochemistry. In contrast, the expanded Merkel innervation was reduced to age‐matched nondiabetic levels. These results indicate that transient phases of substantial innervation remodeling occur during the progression of diabetes, with differential increases and decreases occurring among the varieties of innervation. J. Comp. Neurol. 501:543–567, 2007.

Islet-cell regeneration in the diabetic hamster pancreas with restoration of normoglycaemia can be induced by a local growth factor(s)

SummaryPartial pancreatic duct obstruction in the hamster leads to the induction of endocrine-cell differentiation and new islet formation. We prepared cytosolic extracts from the partially obstructed pancreas and identified one, which when administered i.p., produced significant increases in the incorporation of tritiated thymidine by ductular and islet cells, as well as a corresponding increase in islet mass. In this study, we evaluate the ability of this extract to reverse streptozotocin diabetes mellitus. Hamsters were treated i. p. twice daily for 7 weeks with either 0.9% NaCl (saline) (n=10) or a cytosol extract (n=10) prepared previously from partially obstructed hamster pancreata. All animals in the cytosol group survived vs only 60% of the saline group (p=0.02). Random blood glucose levels were greater than 22.2 mmol/l in 90% of the saline group vs 40% in the cytosol group (p<0.05). Pancreatic tissue from the surviving saline animals and from persistently hyperglycaemic cytosol-treated animals, showed intra-cytoplasmic vacuolation of islet cells, a characteristic lesion of sustained hyperglycaemic states. Vacuolation was not observed in normoglycaemic extract treated animals. Islets in hyperglycaemic animals demonstrated a profound decrease or absence of immunoreactive insulin, compared to an abundance of immunoreactive beta cells in cytosol-treated animals that reverted to normoglycaemia. In this group, single cells or nests of cells stained for insulin or glucagon cells were identified in ductal epithelium in association with cells budding from the duct. Morphometric analysis of pancreata in reverted cytosol-treated animals showed a new population of small islets compared with saline controls and an increased islet mass. In summary, streptozotocin diabetes can be reversed by new islet formation induced by local pancreatic growth factors, the exact nature of which remains to be determined.

Intramuscular injection of islet neogenesis-associated protein peptide stimulates pancreatic islet neogenesis in healthy dogs.

Objectives: Diabetes is a serious health problem. It has been proposed that islet neogenesis from pancreatic progenitor cells may restore insulin secretion in diabetic mammals. Islet neogenesis- associated protein (INGAP) stimulates islet neogenesis; therefore, we hypothesized that it would stimulate islet neogenesis in dogs. Methods: Forty nondiabetic beagle dogs were randomly divided into 4 groups. Group 1 received daily intramuscular injections of vehicle, whereas the other 3 groups received daily INGAP104-118 injections of 0.5, 1.5, or 10 mg/kg. After 30 days, pancreatic tissues were collected, and RNA and histological sections were analyzed. Results: In dogs treated with 1.5 mg/kg INGAP104-118, there was a significant (P < 0.001) increase in the percentage of insulin-positive cells (P < 0.001) and insulin gene expression. There was a trend to increased insulin-positive cells and gene expression with treatments of 0.5 and 10 mg/kg peptide. Protein gene product 9.5-positive cells were increased with treatment. Conclusions: These results indicate that INGAP stimulates cells in the pancreatic duct epithelium of healthy dogs (putative islet progenitor cells) to develop along a neuroendocrine pathway and form new islets in response to INGAP peptide. The INGAP might be an effective therapy for diabetes.

Cloning genomic INGAP: a Reg-related family member with distinct transcriptional regulation sites.

The protein product of hamster islet neogenesis-associated protein (INGAP) cDNA induces new pancreatic islet development. Manipulation of this process provides a new therapeutic strategy for the treatment of diabetes. As regulators of INGAP gene expression are unknown over 6 kb of hamster genomic INGAP has been cloned. Sequence analysis has identified a 3 kb 5-prime region with core promoter elements that is rich in transcription factor binding sites and six exons for the coding region. Analysis of promoter activity reveals stimulus-responsive DNA elements which have been identified though deletion analysis. Comparison of transcription factor binding sites in INGAP to the related gene RegIIIdelta exposes potential sites for differential gene regulation.

The Partial Isolation and Characterization of Ilotropin, a Novel Islet-Specific Growth Factor

In this series of studies, we have presented evidence for a novel, pancreatic islet-specific growth factor, which we call ilotropin. Ilotropin is acid stable, heat stable, ethanol-precipitable, and sensitive to trypsin digestion. It appears to have a molecular weight between 29 - 44,000, and preliminary data not presented here suggests that it has a relatively basic pI. Unlike many other growth factors, ilotropin does not bind to heparin. Ilotropin is distinguishable from most of the known growth factors on the basis of at least one of the characteristics established in these studies. The apparent molecular weight of 29 - 44,000 eliminates all but the larger growth factors such as PDGF and hepatic growth factor. The fact that ilotropin is acid stable rules out identity with hepatic growth factor, and its lack of binding to heparin and apparent basic pI rules out identity with PDGF. Thus, the combination of characteristics described in these studies eliminates most of the known growth factors as candidates for the role of ilotropin. Certain growth factor precursor molecules (e.g. TGF-a) and several interleukins and cytokines (e.g. pro-IL-1 and melanocyte growth factor) also fall into this molecular weight range. Whether these proteins might be related to ilotropin or play a role in its biological activity remains to be determined. Current studies of ilotropin include further purification to homogeneity, determination of the peptide sequence of ilotropin, and development of an in vitro bioassay using trophic responses of primary cultures of pancreatic duct cells as an indicator of ilotropin activity. With purified material we ought to be able to identify the cells of origin and the target cells for the action of ilotropin, and establish assays to determine the relationship to failure of beta-cell regeneration that accompanies diabetes. Ultimately we hope that ilotropin may lead to new ways of approaching aspects of the problems presented in pancreatic beta-cell failure.