Pre-metazoan origin of neuropeptide signalling

Neuropeptides are a diverse class of signalling molecules in metazoans. They occur in all animals with a nervous system and also in neuron-less placozoans. However, their origin has remained unclear because no neuropeptide shows deep homology across lineages and none have been found in sponges. Here, we identify two neuropeptide precursors, phoenixin and nesfatin, with broad evolutionary conservation. By database searches, sequence alignments and gene-structure comparisons we show that both precursors are present in bilaterians, cnidarians, ctenophores and sponges. We also found phoenixin and a secreted nesfatin precursor homolog in the choanoflagellate Salpingoeca rosetta. Phoenixin in particular, is highly conserved, including its cleavage sites, suggesting that prohormone processing occurs also in choanoflagellates. In addition, based on phyletic patterns and negative pharmacological assays we question the originally proposed GPR-173 (SREB3) as a phoenixin receptor. Our findings indicate that signalling by secreted neuropeptide homologs has pre-metazoan origins and thus evolved before neurons.

Elevated islet prohormone ratios as indicators of insulin dependency in islet transplant recipients

Autologous pancreatic islet transplantation is an established therapy for patients with chronic pancreatitis. However, the long-term transplant outcomes are modest. Identifying indicators of graft function will aid the preservation of transplanted islets and glycemic control. To this end, we analyzed beta cell prohormone peptide levels in a retrospective cohort of total pancreatectomy autologous islet transplant patients (n=28). Proinsulin-to-C-peptide (PI/C) and proIAPP-to-total IAPP (proIAPP/IAPP) ratios measured at 3 months post-transplant were significantly higher in patients who remained insulin dependent at 1 year follow-up. In a mouse model of human islet transplantation, recipient mice that later became hyperglycemic displayed significantly higher PI/C ratios than mice that remained normoglycemic. Histological analysis of islet grafts showed reduced insulin- and proinsulin-positive area, but elevated glucagon-positive area in grafts that experienced greater secretory demand. Increased prohormone convertase 1/3 was detected in glucagon-positive cells, and glucagon-like peptide 1 (GLP-1) area was elevated in grafts from mice that displayed hyperglycemia or elevated plasma PI/C ratio, demonstrating intra-islet incretin production in metabolically challenged human islet grafts. These data indicate that in failing grafts, alpha cell prohormone processing is likely altered, and incomplete beta cell prohormone processing may be an early indicator of insulin dependency.

Altered islet prohormone processing: A cause or consequence of diabetes?

Peptide hormones are first produced as larger precursor prohormones that require endoproteolytic cleavage to liberate the mature hormones. A structurally conserved but functionally distinct family of nine prohormone convertase enzymes (PCs) are responsible for cleavage of protein precursors of which PC1/3 and PC2 are known to be exclusive to neuroendocrine cells and responsible for prohormone cleavage. Differential expression of PCs within tissues define prohormone processing; whereas glucagon is the major product liberated from proglucagon via PC2 in pancreatic α-cells, proglucagon is preferentially processed by PC1/3 in intestinal L cells to produce glucagon-like peptides 1 and 2 (GLP-1, GLP-2). Beyond our understanding of processing of islet prohormones in healthy islets, there is convincing evidence that proinsulin, proIAPP, and proglucagon processing is altered during prediabetes and diabetes. There is predictive value of elevated circulating proinsulin or proinsulin : C-peptide ratio for progression to type 2 diabetes and elevated proinsulin or proinsulin : C-peptide is predictive for development of type 1 diabetes in at risk groups. After onset of diabetes, patients have elevated circulating proinsulin and proIAPP and proinsulin may be an autoantigen in type 1 diabetes. Further, preclinical studies reveal that α-cells have altered proglucagon processing during diabetes leading to increased GLP-1 production. We conclude that despite strong associative data, current evidence is inconclusive on the potential causal role of impaired prohormone processing in diabetes, and suggest that future work should focus on resolving the question of whether altered prohormone processing is a causal driver or merely a consequence of diabetes pathology.

SUN-260 Dual Role of Carboxypeptidase E in Prohormone Processing and a Novel Neurotrophic Factor Mediating Neuroprotection and Cognitive Functions in Hippocampal CA3 Neurons in Mice

Stress causes release of glucocorticoids from the adrenals which then circulate to the brain. High concentrations glucocorticoid from chronic severe stress results in pathophysiology in the brain, including neuronal degeneration, cell death and cognitive dysfunction, leading to diseases such as Alzheimer Disease and Major Depressive Disorders. Neurotrophic/growth factors such as BDNF, NGF and NT3 have been linked to these pathological conditions. Carboxypeptidase E (CPE), a proneuropeptide/prohormone processing enzyme, also named neurotrophic factor-α1(NFα1) is highly expressed in the stress-vulnerable hippocampal CA3 neurons, and was shown to have neuroprotective activity from in vitro studies. Here we investigated if CPE-NFα1 functions in vivo, independent of its enzymatic activity, and the mechanism underlying its action. We generated knock-in mice expressing a non-enzymatic form of CPE, CPE-E342Q, but not wild-type CPE. The CPE-E342Q mice showed significantly decreased neuropeptide content and exhibited obesity, diabetes and infertility due to lack of prohormone processing activity, similar to CPE-KO mice. However, they showed no hippocampal CA3 degeneration, exhibited neurogenesis in the dentate gyrus, and displayed normal spatial learning and memory, similar to CPE wild-type mice, after weaning stress; unlike CPE-KO mice which showed hippocampal CA3 neuronal degeneration and cognitive deficits. Binding studies showed that radiolabeled CPE bound hippocampal cell membrane specifically, in a saturable manner. Binding of CPE and CPE-E342Q to hippocampal neurons activated Erk signaling and pre-treatment with either of these proteins protected neurons against H2O2- or glutamate-induced neurotoxcity by increasing BCL2 expression. In vitro and in vivo inhibitor studies demonstrated that this neuroprotective effect was independent of tyrosine kinase receptor signaling. Taken together, the data provide evidence that CPE-NFα1 is a unique neurotrophic factor which acts through a non-tyrosine kinase receptor to activate Erk-BCL2 signaling to protect hippocampal CA3 neurons against stress-induced neurodegeneration and maintaining normal cognitive functions in mice.

Abnormalities in Proinsulin Processing in Islets from Individuals with Longstanding T1D

ABSTRACTWork by our group and others has suggested that elevations in circulating proinsulin relative to C-peptide is associated with development of Type 1 diabetes (T1D). We recently described the persistence of detectable serum proinsulin in a large majority (95.9%) of individuals with longstanding T1D, including individuals with undetectable serum C-peptide. Here we describe analyses performed on human pancreatic sections from the nPOD collection (n=30) and isolated human islets (n=10) to further explore mechanistic etiologies of persistent proinsulin secretion in T1D. Compared to nondiabetic controls, immunostaining among a subset (4/9) of insulin positive T1D donor islets revealed increased numbers of cells with proinsulin-enriched, insulin-poor staining. Laser capture microdissection followed by mass spectrometry revealed reductions in the proinsulin processing enzymes prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE) in T1D donors. Twenty-four hour treatment of human islets with an inflammatory cytokine cocktail reduced mRNA expression of the processing enzymes PC1/3, PC2, and CPE. Taken together, these data provide new mechanistic insight into altered proinsulin processing in long-duration T1D and suggest that reduced β cell prohormone processing is associated with proinflammatory cytokine-induced reductions in proinsulin processing enzyme expression.

Measurement of Pro-Islet Amyloid Polypeptide (1–48) in Diabetes and Islet Transplants

Context: Islet amyloid is a feature of β-cell failure in type 2 diabetes (T2D) and type 1 diabetes (T1D) recipients of islet transplants. Islet amyloid contains islet amyloid polypeptide (IAPP; amylin), a circulating peptide that is produced in β cells by processing of its precursor, proIAPP1-67, via an intermediate form, proIAPP1-48. Elevated proinsulin to C-peptide ratios in the plasma of persons with diabetes suggest defects in β-cell prohormone processing. Objective: Determine whether plasma levels of precursor forms of IAPP are elevated in diabetes. Design, Setting, and Patients: We developed an immunoassay to detect proIAPP1-48 in human plasma, and we determined the ratio of proIAPP1-48 to mature IAPP in subjects with T1D, T2D, recipients of islet transplants, and healthy controls. Results: The proIAPP1-48 immunoassay had a limit of detection of 0.18 ± 0.06 pM and cross-reactivity with intact proIAPP1-67 <15%. Healthy individuals had plasma concentrations of proIAPP1-48 immunoreactivity of 1.5 ± 0.2 pM and a proIAPP1-48 to total IAPP ratio of 0.28 ± 0.03. Plasma concentrations of proIAPP1-48 immunoreactivity were not significantly different in subjects with T2D but were markedly increased in T1D recipients of islet transplants. Children and adults with T1D had reduced mature IAPP levels relative to age-matched controls but an elevated ratio of proIAPP1-48 to total IAPP. Conclusion: The β cells in T1D and islet transplants have impaired processing of the proIAPP1-48 intermediate. The ratio of proIAPP1-48-to-IAPP immunoreactivity may have value as a biomarker of β-cell stress and dysfunction.