Association of common variants of TCF7L2 and PCSK2 with gestational diabetes mellitus in West Bengal, India.

The genetic etiology of gestational diabetes mellitus (GDM) was suggested to overlap with type-2 diabetes(T2D). Transcription factor 7-like 2 (TCF7L2) and Proprotein Convertase Subtilisin/Kexin type 2 (PCSK2) are T2D susceptibility genes of the insulin synthesis/processing pathway. We analyzed associations of TCF7L2 and PCSK2 variants with GDM risk and evaluated their potential impact on impaired insulin processing in an eastern Indian population. The study included 114 GDM (case) and 228 non-GDM pregnant women (control). rs7903146, rs4132670, rs12255372 of TCF7L2, and rs2269023 of PCSK2 were genotyped by PCR-RFLP, and genotype distributions were compared between case and control. Fasting serum proinsulin and C-peptide levels were measured by ELISA and the Proinsulin/C-peptide ratio was considered an indicator of proinsulin conversion. Significantly higher frequency of risk allele (T) of rs12255372 (p = 0.02, OR = 2.0, 95%CI = 1.11-3.64) and rs4132670 (p = 0.002, OR = 2.26, 95%CI = 1.32-3.87) of TCF7L2 was found in GDM cases than non-GDM controls; TT genotype was associated with significantly increased disease risk. In rs7903146 (TCF7L2) and rs2269023 (PCSK2), although the frequency of risk allele (T) was not significantly higher in cases than controls, an association of TT for both variants remained significant with higher GDM risk in the recessive model. Increased serum pro-insulin and proinsulin:c-peptide ratio was found in GDM than non-GDM women and the phenomenon showed significant association with careers of risk alleles for TCF7L2 variants. In conclusion, TCF7L2 and PCSK2 variants are related to GDM risk in the studied population and hence may serve as potential biomarkers for assessing the disease risk. TCF7L2 variants contribute to impaired insulin processing.

Top-Down Proteomics of Mouse Islets With Beta Cell CPE Deletion Reveals Molecular Details in Prohormone Processing.

Altered prohormone processing, such as with proinsulin and pro-islet amyloid polypeptide (proIAPP), has been reported as an important feature of prediabetes and diabetes. Proinsulin processing includes removal of several C-terminal basic amino acids and is performed principally by the exopeptidase carboxypeptidase E (CPE), and mutations in CPE or other prohormone convertase enzymes (PC1/3 and PC2) result in hyperproinsulinemia. A comprehensive characterization of the forms and quantities of improperly processed insulin and other hormone products following Cpe deletion in pancreatic islets has yet to be attempted. In the present study we applied top-down proteomics to globally evaluate the numerous proteoforms of hormone processing intermediates in a ß-cell-specific Cpe knockout mouse model. Increases in dibasic residue-containing proinsulin and other novel proteoforms of improperly processed proinsulin were found, and we could classify several processed proteoforms as novel substrates of CPE. Interestingly, some other known substrates of CPE remained unaffected despite its deletion, implying that paralogous processing enzymes such as carboxypeptidase D (CPD) can compensate for CPE loss and maintain near normal levels of hormone processing. In summary, our quantitative results from top-down proteomics of islets provide unique insights into the complexity of hormone processing products and the regulatory mechanisms.

Replication Activities of Major 5' Terminally Deleted Group-B Coxsackievirus RNA Forms Decrease PCSK2 mRNA Expression Impairing Insulin Maturation in Pancreatic Beta Cells.

Emergence of 5' terminally deleted coxsackievirus-B RNA forms (CVB-TD) have been associated with the development of human diseases. These CVB-TD RNA forms have been detected in mouse pancreas during acute or persistent experimental infections. To date, the impact of the replication activities of CVB-TD RNA forms on insulin metabolism remains unexplored. Using an immunocompetent mouse model of CVB3/28 infection, acute and persistent infections of major CVB-TD populations were evidenced in the pancreas. The inoculation of mice with homogenized pancreases containing major CVB-TD populations induced acute and chronic pancreatic infections with pancreatitis. In the mouse pancreas, viral capsid protein 1 (VP1) expression colocalized with a decrease in beta cells insulin content. Moreover, in infected mouse pancreases, we showed a decrease in pro-hormone convertase 2 (PCSK2) mRNA, associated with a decrease in insulin plasmatic concentration. Finally, transfection of synthetic CVB-TD50 RNA forms into cultured rodent pancreatic beta cells demonstrated that viral replication with protein synthesis activities decreased the PCSK2 mRNA expression levels, impairing insulin secretion. In conclusion, our results show that the emergence and maintenance of major CVB-TD RNA replicative forms in pancreatic beta cells can play a direct, key role in the pathophysiological mechanisms leading to the development of type 1 diabetes.

Loss of hypothalamic Furin affects POMC to proACTH cleavage and feeding behavior in high-fat diet-fed mice.

OBJECTIVE: The hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding. METHODS: POMC-Cre+/- mice were bred with Furinfl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furinfl/fl mice were used as controls. POMCFurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMCFurKO mice and controls. The in-vitro processing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN. RESULTS: In control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMCFurKO mice were hyperphagic and had increased body weight compared to Furinfl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMCFurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMCFurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway. CONCLUSIONS: These results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions.

Hypothalamic CREB Regulates the Expression of Pomc-Processing Enzyme Pcsk2.

BACKGROUND: The hypothalamic proopiomelanocortin (Pomc) neurons act as first-order sensors of systemic energy stores, providing signals that regulate caloric intake and energy expenditure. In experimental obesity, dietary saturated fatty acids affect Pomc endopeptidases (PCs), resulting in the abnormal production of the neurotransmitters α-melanocyte-stimulating hormone (α-MSH) and ß-endorphin, thus impacting energy balance. The cAMP response element-binding protein (CREB) is one of the transcription factors that control the expression of Pomc endopeptidases; however, it was previously unknown if dietary fats could affect CREB and consequently the expression of Pomc endopeptidases. METHODS: Here, we used single-cell RNA sequencing analysis, PCR, immunoblot, ELISA and immunofluorescence histological assays to determine the impact of a high-fat diet (HFD) on the expression and function of hypothalamic CREB and its impact on the melanocortinergic system. RESULTS: The results indicate that CREB is expressed in arcuate nucleus Pomc neurons and is activated as early as nine hours after the introduction of a high-fat diet. The inhibition of hypothalamic CREB using a short-hairpin RNA lentiviral vector resulted in increased diet-induced body-mass gain and reduced energy expenditure. This was accompanied by reduced expression of the Pomc endopeptidases, protein convertase 2, which are encoded by Pcsk2, and by the loss of the high-fat-diet-induced effect to inhibit the production of α-MSH. CONCLUSIONS: This study provides the first evidence for the involvement of CREB in the abnormal regulation of the hypothalamic Pomc endopeptidase system in experimental obesity.

Polycystin-2 (PC2) is a key determinant of in vitro myogenesis.

The development of skeletal muscle (myogenesis) is a well-orchestrated process where myoblasts withdraw from the cell cycle and differentiate into myotubes. Signaling by fluxes in intracellular calcium (Ca2+) is known to contribute to myogenesis, and increased mitochondrial biogenesis is required to meet the metabolic demand of mature myotubes. However, gaps remain in the understanding of how intracellular Ca2+ signals can govern myogenesis. Polycystin-2 (PC2 or TRPP1) is a nonselective cation channel permeable to Ca2+. It can interact with intracellular calcium channels to control Ca2+ release and concurrently modulates mitochondrial function and remodeling. Due to these features, we hypothesized that PC2 is a central protein in mediating both the intracellular Ca2+ responses and mitochondrial changes seen in myogenesis. To test this hypothesis, we created CRISPR/Cas9 knockout (KO) C2C12 murine myoblast cell lines. PC2 KO cells were unable to differentiate into myotubes, had impaired spontaneous Ca2+ oscillations, and did not develop depolarization-evoked Ca2+ transients. The autophagic-associated pathway beclin-1 was downregulated in PC2 KO cells, and direct activation of the autophagic pathway resulted in decreased mitochondrial remodeling. Re-expression of full-length PC2, but not a calcium channel dead pathologic mutant, restored the differentiation phenotype and increased the expression of mitochondrial proteins. Our results establish that PC2 is a novel regulator of in vitro myogenesis by integrating PC2-dependent Ca2+ signals and metabolic pathways.

Obesity and Hyperphagia With Increased Defective ACTH: A Novel POMC Variant.

OBJECTIVE: Patients with pro-opiomelanocortin (POMC) defects generally present with early-onset obesity, hyperphagia, hypopigmentation and adrenocorticotropin (ACTH) deficiency. Rodent models suggest that adequate cleavage of ACTH to α-melanocortin-stimulating hormone (α-MSH) and desacetyl-α-melanocortin-stimulating hormone (d-α-MSH) by prohormone convertase 2 at the KKRR region is required for regulating food intake and energy balance. METHODS: We present 2 sisters with a novel POMC gene variant, leading to an ACTH defect at the prohormone convertase 2 cleavage site, and performed functional studies of this variant. RESULTS: The patients had obesity, hyperphagia and hypocortisolism, with markerly raised levels of ACTH but unaffected pigmentation. Their ACTH has reduced potency to stimulate the melanocortin (MC) 2 receptor, explaining their hypocortisolism. CONCLUSION: The hyperphagia and obesity support evidence that adequate cleavage of ACTH to α-MSH and d-α-MSH is also required in humans for feeding control.

Obesity, POMC, and POMC-processing Enzymes: Surprising Results From Animal Models.

Peptides derived from proopiomelanocortin (POMC) are well-established neuropeptides and peptide hormones that perform multiple functions, including regulation of body weight. In humans and some animals, these peptides include α- and ß-melanocyte-stimulating hormone (MSH). In certain rodent species, no ß-MSH is produced from POMC because of a change in the cleavage site. Enzymes that convert POMC into MSH include prohormone convertases (PCs), carboxypeptidases (CPs), and peptidyl-α-amidating monooxygenase (PAM). Humans and mice with inactivating mutations in either PC1/3 or carboxypeptidase E (CPE) are obese, which was assumed to result from defective processing of POMC into MSH. However, recent studies have shown that selective loss of either PC1/3 or CPE in POMC-expressing cells does not cause obesity. These findings suggest that defects in POMC processing cannot alone account for the obesity observed in global PC1/3 or CPE mutants. We propose that obesity in animals lacking PC1/3 or CPE activity depends, at least in part, on deficient processing of peptides in non-POMC-expressing cells either in the brain and/or the periphery. Genetic background may also contribute to the manifestation of obesity.

A survey of the mouse hindbrain in the fed and fasted states using single-nucleus RNA sequencing.

OBJECTIVE: The area postrema (AP) and nucleus tractus solitarius (NTS) located in the hindbrain are key nuclei that sense and integrate peripheral nutritional signals and consequently regulate feeding behaviour. While single-cell transcriptomics have been used in mice to reveal the gene expression profile and heterogeneity of key hypothalamic populations, similar in-depth studies have not yet been performed in the hindbrain. METHODS: Using single-nucleus RNA sequencing, we provide a detailed survey of 16,034 cells within the AP and NTS of mice in the fed and fasted states. RESULTS: Of these, 8,910 were neurons that group into 30 clusters, with 4,289 from mice fed ad libitum and 4,621 from overnight fasted mice. A total of 7,124 nuclei were from non-neuronal cells, including oligodendrocytes, astrocytes, and microglia. Interestingly, we identified that the oligodendrocyte population was particularly transcriptionally sensitive to an overnight fast. The receptors GLP1R, GIPR, GFRAL, and CALCR, which bind GLP1, GIP, GDF15, and amylin, respectively, are all expressed in the hindbrain and are major targets for anti-obesity therapeutics. We characterise the transcriptomes of these four populations and show that their gene expression profiles are not dramatically altered by an overnight fast. Notably, we find that roughly half of cells that express GIPR are oligodendrocytes. Additionally, we profile POMC-expressing neurons within the hindbrain and demonstrate that 84% of POMC neurons express either PCSK1, PSCK2, or both, implying that melanocortin peptides are likely produced by these neurons. CONCLUSION: We provide a detailed single-cell level characterisation of AP and NTS cells expressing receptors for key anti-obesity drugs that are either already approved for human use or in clinical trials. This resource will help delineate the mechanisms underlying the effectiveness of these compounds and also prove useful in the continued search for other novel therapeutic targets.

Comparative transcriptome analysis of eyestalk from the white shrimp Litopenaeus vannamei after the injection of dopamine.

Dopamine (DA) is a crucial neuroendocrine-immune factor regulating the stress response of Litopenaeus vannamei. To understand the regulatory mechanisms of DA in L. vannamei, the eyestalks of L. vannamei with injection of DA (10-6 mol/shrimp) at 3 and 12 h were chosen to perform transcriptome analysis in this study. Furthermore, quantitative real-time PCR (RT-PCR) method was used to validate the accuracy of transcriptome data and analyze the expression pattern of candidate differentially expressed genes (DEGs) at different time points (0, 3, 6, and 12 h) after DA injection. The transcriptome data showed that 79,434 unigenes were generated. Therein 204 and 434 DEGs were obtained at 3 and 12 h respectively. Besides, the results of enriched pathways showed that the DEGs were involved in GnRH signaling pathway (ko04912) dopaminergic synapse (ko04728), glutamatergic synapse (ko04724), synapse (GO:0045202), synaptic vesicle transport (GO:0048489). Moreover, the Pearson's correlation coefficient (R) of 13 candidate DEGs between transcriptome sequencing and RT-PCR was 0.948, which confirmed the reliability and the accuracy of the transcriptome sequencing results. Furthermore, the results of interaction analysis uncovered 4 pairs of DEGs between eyestalks and hemocytes. Therefore, these results revealed that DA promoted the sensitivity of eyestalk to gonadal related hormones, induced the expression of neuroendocrine factor, enhanced the synaptic behavior and neural signal transduction, regulated immune systems and antioxidation, inhibited the visual function, and promoted the molting. These findings will benefit to foster the understanding on the effects of biogenic amines on neuroendocrine-immune (NEI) networks of crustacean, and supply a substantial material and foundation for further researching of the NEI response.

Protein restriction during pregnancy impairs intra-islet GLP-1 and the expansion of ß-cell mass.

We evaluated whether protein restriction during pregnancy alters the morphometry of pancreatic islets, the intra-islet glucagon-like peptide-1 (GLP-1) production, and the anti-apoptotic signalling pathway modulated by GLP-1. Control non-pregnant (CNP) and control pregnant (CP) rats were fed a 17% protein diet, and low-protein non-pregnant (LPNP) and low-protein pregnant (LPP) groups were fed a 6% protein diet. The masses of islets and ß-cells were similar in the LPNP group and the CNP group but were higher in the CP group than in the CNP group and were equal in the LPP group and the LPNP group. Both variables were lower in the LPP group than in the CP group. Prohormone convertase 2 and GLP-1 fluorescence in α-cells was lower in the low-protein groups than in the control groups. The least PC2/glucagon colocalization was observed in the LPP group, and the most was observed in the CP group. There was less prohormone convertase 1/3/glucagon colocalization in the LPP group than in the CP group. GLP-1/glucagon colocalization was similar in the LPP, CP and CNP groups, which showed less GLP-1/glucagon colocalization than the LPNP group. The mRNA Pka, Creb and Pdx-1 contents were higher in islets from pregnant rats than in islets from non-pregnant rats. Protein restriction during pregnancy impaired the mass of ß-cells and the intra-islet GLP-1 production but did not interfere with the transcription of genes of the anti-apoptotic signalling pathway modulated by GLP-1.

Hypothalamic extended synaptotagmin-3 contributes to the development of dietary obesity and related metabolic disorders.

The C2 domain containing protein extended synaptotagmin (E-Syt) plays important roles in both lipid homeostasis and the intracellular signaling; however, its role in physiology remains largely unknown. Here, we show that hypothalamic E-Syt3 plays a critical role in diet-induced obesity (DIO). E-Syt3 is characteristically expressed in the hypothalamic nuclei. Whole-body or proopiomelanocortin (POMC) neuron-specific ablation of E-Syt3 ameliorated DIO and related comorbidities, including glucose intolerance and dyslipidemia. Conversely, overexpression of E-Syt3 in the arcuate nucleus moderately promoted food intake and impaired energy expenditure, leading to increased weight gain. Mechanistically, E-Syt3 ablation led to increased processing of POMC to α-melanocyte-stimulating hormone (α-MSH), increased activities of protein kinase C and activator protein-1, and enhanced expression of prohormone convertases. These findings reveal a previously unappreciated role for hypothalamic E-Syt3 in DIO and related metabolic disorders.

PCSK2 expression in neuroendocrine tumors points to a midgut, pulmonary, or pheochromocytoma-paraganglioma origin.

Neuroendocrine tumors (NETs) are often diagnosed from the metastases of an unknown primary tumor. Specific immunohistochemical (IHC) markers indicating the location of a primary tumor are needed. The proprotein convertase subtilisin/kexin type 2 (PCSK2) is found in normal neural and neuroendocrine cells, and known to express in NETs. We investigated the tissue microarray (TMA) of 86 primary tumors from 13 different organs and 9 metastatic NETs, including primary tumor-metastasis pairs, for PCSK2 expression with polymer-based IHC. PCSK2 was strongly positive in all small intestine and appendiceal NETs, the so-called midgut NETs, in most pheochromocytomas and paragangliomas, and in some of the typical and atypical pulmonary carcinoid tumors. NETs showing strong positivity were re-evaluated in larger tumor cohorts confirming the primary observation. In the metastases, the expression of PCSK2 mirrored that of the corresponding primary tumors. We found negative or weak staining in NETs from the thymus, gastric mucosa, pancreas, rectum, thyroid, and parathyroid. PCSK2 expression did not correlate with Ki-67 in well-differentiated NETs. Our data suggest that PCSK2 positivity can indicate the location of the primary tumor. Thus, PCSK2 could function in the IHC panel determined from screening metastatic NET biopsies of unknown primary origins.

Mof regulates glucose level via altering different α-cell subset mass and intra-islet glucagon-like peptide-1, glucagon secretion.

BACKGROUND: Males absent on the first (Mof) is implicated in gene control of diverse biological processes, such as cell growth, differentiation, apoptosis and autophagy. However, the relationship between glucose regulation and Mof-mediated transcription events remains unexplored. We aimed to unravel the role of Mof in glucose regulation by using global and pancreatic α-cell-specific Mof-deficient mice in vivo and α-TC1-6 cell line in vitro. METHODS: We used tamoxifen-induced temporal Mof-deficient mice first to show Mof regulate glucose homeostasis, islet cell proportions and hormone secretion. Then we used α-cell-specific Mof-deficient mice to clarify how α-cell subsets and ß-cell mass were regulated and corresponding hormone level alterations. Ultimately, we used small interfering RNA (siRNA) to knockdown Mof in α-TC1-6 and unravel the mechanism regulating α-cell mass and glucagon secretion. RESULTS: Mof was mainly expressed in α-cells. Global Mof deficiency led to lower glucose levels, attributed by decreased α/ß-cell ratio and glucagon secretion. α-cell-specific Mof-deficient mice exhibited similar alterations, with more reduced prohormone convertase 2 (PC2)-positive α-cell mass, responsible for less glucagon, and enhanced prohormone convertase 1 (PC1/3)-positive α-cell mass, leading to more glucagon-like peptide-1 (GLP-1) secretion, thus increased ß-cell mass and insulin secretion. In vitro, increased DNA damage, dysregulated autophagy, enhanced apoptosis and altered cell fate factors expressions upon Mof knockdown were observed. Genes and pathways linked to impaired glucagon secretion were uncovered through transcriptome sequencing. CONCLUSION: Mof is a potential interventional target for glucose regulation, from the aspects of both α-cell subset mass and glucagon, intra-islet GLP-1 secretion. Upon Mof deficiency, Up-regulated PC1/3 but down-regulated PC2-positive α-cell mass, leads to more GLP-1 and insulin but less glucagon secretion, and contributed to lower glucose level.

Molecular characterization and spatiotemporal expression of prohormone convertase 2 in the Pacific abalone, Haliotis discus hannai.

Prohormone convertases (PCs) are subtilisin-like proteases responsible for the intracellular processing of prohormones and proneuropeptides in vertebrates and invertebrates. The full-length PC2 cDNA sequence was cloned from pleuropedal ganglion of Haliotis discus hannai, consisted of 2254-bp with an open reading frame of 1989-bp and encoded a protein of 662 amino acid residues. The architecture of Hdh PC2 displayed key features of PCs, including a signal peptide, a pro-segment domain with sites for autocatalytic activation, a catalytic domain, and a pro-protein domain (P-domain). It shares the highest homology of its amino acid sequence with the PC2 from H. asinina and to lesser extent with that of Homo sapiens and Rana catesbeiana PC2. Sequence alignment analysis indicated that Hdh PC2 was highly conserved in the catalytic domain, including a catalytic triad of serine proteinases of the subtilisin family at positions Asp-195, His-236, and Ser-412. The cloned sequence contained a canonical integrin binding sequence, and four cysteine residues involved in the formation of an intramolecular disulfide link. Phylogenetic analysis revealed that the Hdh PC2 is robustly clustered with the Has PC2. Quantitative PCR assay demonstrated that the Hdh PC2 was predominantly expressed in the pleuropedal ganglion rather than in other examined tissues. Although PC2 mRNA was expressed throughout the gametogenetic cycle of male and female abalone, the expression level was significantly higher in the ripening stage of female abalone. Also, a significantly higher expression was observed in the pleuropedal ganglion and gonadal tissues at a higher effective accumulative temperature (1000°C). In situ hybridization revealed that the PC2 mRNA expressing neurosecretory cells were distributed in the cortex region of the pleuropedal ganglion. According to the results, it can be concluded that pleuropedal ganglion is the highest site of PC2 activity, and this enzyme might be involved in the abalone reproduction process.

Revisiting Proinsulin Processing: Evidence That Human ß-Cells Process Proinsulin With Prohormone Convertase (PC) 1/3 but Not PC2.

Insulin is first produced in pancreatic ß-cells as the precursor prohormone proinsulin. Defective proinsulin processing has been implicated in the pathogenesis of both type 1 and type 2 diabetes. Though there is substantial evidence that mouse ß-cells process proinsulin using prohormone convertase 1/3 (PC1/3) and then prohormone convertase 2 (PC2), this finding has not been verified in human ß-cells. Immunofluorescence with validated antibodies revealed that there was no detectable PC2 immunoreactivity in human ß-cells and little PCSK2 mRNA by in situ hybridization. Similarly, rat ß-cells were not immunoreactive for PC2. In all histological experiments, PC2 immunoreactivity in neighboring α-cells acted as a positive control. In donors with type 2 diabetes, ß-cells had elevated PC2 immunoreactivity, suggesting that aberrant PC2 expression may contribute to impaired proinsulin processing in ß-cells of patients with diabetes. To support histological findings using a biochemical approach, human islets were used for pulse-chase experiments. Despite inhibition of PC2 function by temperature blockade, brefeldin A, chloroquine, and multiple inhibitors that blocked production of mature glucagon from proglucagon, ß-cells retained the ability to produce mature insulin. Conversely, suppression of PC1/3 blocked processing of proinsulin but not proglucagon. By demonstrating that healthy human ß-cells process proinsulin by PC1/3 but not PC2, we suggest that there is a need to revise the long-standing theory of proinsulin processing.

Translation of insulin granule proteins are regulated by PDI and PABP.

Glucose mediated insulin biosynthesis is tightly regulated and shared between insulin granule proteins such as its processing enzymes, prohormone convertases, PC1/3 and PC2. However, the molecular players involved in the co-ordinated translation remain elusive. The trans-acting factors like PABP (Poly A Binding Protein) and PDI (Protein Disulphide Isomerize) binds to a conserved sequence in the 5'UTR of insulin mRNA and regulates its translation. Here, we demonstrate that 5'UTR of PC1/3 and PC2 also associate with PDI and PABP. We show that a' and RRM 3-4 domains of PDI and PABP respectively, are necessary for RNA binding activity to the 5'UTRs of insulin and its processing enzymes.

Hippocampal changes in mice lacking an active prohormone convertase 2.

Prohormone convertase 2 (PC2) is essential for the biosynthesis of many neuropeptides, including several of them in hippocampus. In mouse brain, lacking an enzymatically active PC2 (PC2-null) causes accumulation of many neuropeptides in their precursor or intermediate forms. Little is known about how a PC2-null state may affect the function of the hippocampus. In this study, adult PC2-null mice and their wildtype (WT) littermates were subjected to three analyses to determine possible changes associated with PC2-null at physiological, behavioral, and molecular levels, respectively, under normal and stressed conditions. Electrophysiological recordings of hippocampal slices were performed to measure evoked field-excitatory postsynaptic potentials (EPSP), long-term potentiation (LTP), and paired-pulse facilitation (PPF). Morris water maze (MWM) testing was conducted to examine behavioral changes that are indicative of hippocampal integrity. Quantitative mass spectrometry analysis was used to determine changes in the hippocampal proteome in response to a focal cerebral ischemic insult. We found that there were no significant differences in the threshold of evoked EPSPs between PC2-null and WT animals. However, an increase in LTP in both triggering rate and amplitude was observed in PC2-null mice, suggesting that PC2 may be involved in regulating synaptic strength. The PPF, on the other hand, showed a decrease in PC2-null mice, suggesting a presynaptic mechanism. Consistent with changes in LTP, PC2-null mice displayed decreased latencies in finding the escape platform in the MWM test. Further, after distal focal cerebral ischemia, the hippocampal proteomes incurred changes in both WT and PC2-null mice, with a prominent change in proteins associated with neurotransmission, exocytosis, and transport processes seen in the PC2-null but not WT mice. Taken together, our results suggest that PC2 is involved in regulating hippocampal synaptic plasticity, learning, and memory behaviors, as well as the hippocampal response to stresses originating in other regions of the brain.

EGL-3 and EGL-21 are required to trigger nocifensive response of Caenorhabditis elegans to noxious heat.

Caenorhabditis elegans (C. elegans) is a widely used model organism to examine nocifensive response to noxious stimuli, including heat avoidance. Recently, comprehensive analysis of the genome sequence revealed several pro-neuropeptide genes, encoding a series of bioactive neuropeptides. C. elegans neuropeptides are involved in the modulation of essentially all behaviors including locomotion, mechanosensation, thermosensation and chemosensation. The maturation of pro-neuropeptide to neuropeptide is performed by ortholog pro-protein convertases and carboxypeptidase E (e.g. EGL-3 and EGL-21). We hypothesized that C. elegans egl-3 or egl-21 mutants will have a significant decrease in mature neuropeptides and they will display an impaired heat avoidance behavior. Our data has shown that thermal avoidance behavior of egl-3 and egl-21 mutants was significantly hampered compared to WT(N2) C. elegans. Moreover, flp-18, flp-21 and npr-1 mutant C. elegans displayed a similar phenotype. EGL-3 pro-protein convertase and EGL-21 carboxypeptidase E are essential enzymes for the maturation of pro-neuropeptides to active neuropeptides in C. elegans. Quantitative mass spectrometry analyses with egl-3 and egl-21 mutant C. elegans homogenates demonstrated that proteolysis of ProFLP-18 and ProFLP-21 are severely impeded, leading to a lack of mature bioactive neuropeptides. Not only FLP-21 but also FLP-18 related mature neuropeptides, both are ligands of NPR-1 and are needed to trigger nocifensive response of C. elegans to noxious heat.

Neuropeptide signaling regulates the susceptibility of developing C. elegans to anoxia.

Inadequate delivery of oxygen to organisms during development can lead to cell dysfunction/death and life-long disabilities. Although the susceptibility of developing cells to low oxygen conditions changes with maturation, the cellular and molecular pathways that govern responses to low oxygen are incompletely understood. Here we show that developing Caenorhabditis elegans are substantially more sensitive to anoxia than adult animals and that this sensitivity is controlled by nervous system generated hormones (e.g., neuropeptides). A screen of neuropeptide genes identified and validated nlp-40 and its receptor aex-2 as a key regulator of anoxic survival in developing worms. The survival-promoting action of impaired neuropeptide signaling does not rely on five known stress resistance pathways and is specific to anoxic insult. Together, these data highlight a novel cell non-autonomous pathway that regulates the susceptibility of developing organisms to anoxia.