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Processing of proglucagon into glucagon-like peptide-1 (GLP-1) and GLP-2 in intestinal L cells is mediated by the prohormone convertase 1/3 (PC1/3) while PC2 is responsible for the synthesis of glucagon in pancreatic alpha cells. While GLP-1 is also produced by alpha cells, the identity of the convertase involved in its synthesis is still unsettled. It also remains to be determined whether all alpha cells produce the incretin. The aims of this study were first, to elucidate the identity of the proconvertase responsible for GLP-1 production in human alpha cells, and second, to ascertain whether the number of glucagon cells expressing GLP-1 increase during diabetes. To answer these questions, sections of pancreas from donors' non-diabetic controls, type 1 and type 2 diabetes were processed for double-labelled immunostaining of glucagon and GLP-1 and of each hormone and either PC1 or PC2. Stained sections were examined by confocal microscopy. It was found that all alpha cells of islets from those three groups expressed GLP-1 and PC2 but not PC1/3. This observation supports the view that PC2 is the convertase involved in GLP-1 synthesis in all human glucagon cells and suggests that the regulation of its activity may have important clinical application in diabetes.

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 β-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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

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., Competing Interests: The authors declare no conflicts of interest.

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 Furin fl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furin fl/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 Furin fl/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., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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 (Ca 2+ ) 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 Ca 2+ signals can govern myogenesis. Polycystin-2 (PC2 or TRPP1) is a nonselective cation channel permeable to Ca 2+ . It can interact with intracellular calcium channels to control Ca 2+ 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 Ca 2+ 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 Ca 2+ oscillations, and did not develop depolarization-evoked Ca 2+ 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 Ca 2+ signals and metabolic pathways.

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., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 β-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 β-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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The C 2 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., Competing Interests: The authors declare no competing interest.

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 β-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/β-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 β-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., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Insulin is first produced in pancreatic β-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 β-cells process proinsulin using prohormone convertase 1/3 (PC1/3) and then prohormone convertase 2 (PC2), this finding has not been verified in human β-cells. Immunofluorescence with validated antibodies revealed that there was no detectable PC2 immunoreactivity in human β-cells and little PCSK2 mRNA by in situ hybridization. Similarly, rat β-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, β-cells had elevated PC2 immunoreactivity, suggesting that aberrant PC2 expression may contribute to impaired proinsulin processing in β-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, β-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 β-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., (© 2020 by the American Diabetes Association.)

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., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: All authors declare that they have no conflict of interest.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Metabolic reprogramming is a feature of cancer cells and crucial for tumor growth and metastasis. Interferon-γ (IFNγ) is a cytokine that plays a pivotal role in host antitumor immunity. However, little is known about the roles of metabolic reprogramming in immune responses. Here, we show that colon cancer cells reprogram metabolism to coordinate proper cellular responses to IFNγ by downregulating mitochondrial pyruvate carrier (MPC)1 and 2 via STAT3 signaling. Forced overexpression of MPC promote the production of reactive oxygen species and enhance the apoptosis induced by IFNγ in colon cancer cells. Moreover, inhibiting STAT3 sensitize the antitumor efficacy of IFN-γ against colon cancer cells. Our findings present a previously unrecognized mechanism that colon cancer manipulate to resist IFNγ mediated antitumor immunity that have implications for targeting a unique aspect of this disease., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Although CD8 + T-cell-mediated autoimmune β cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by β cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known β cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8 + T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by β cells and for the development of T cell biomarkers and tolerogenic vaccination strategies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Obesity and type 2 diabetes mellitus (T2DM) are characterized by insulin resistance and impaired glucagon-like peptide-1 (GLP-1) secretion/function. Lipotoxicity, a chronic elevation of free fatty acids in the blood, could affect insulin-signaling in many peripheral tissues. To date, the effects of lipotoxicity on the insulin receptor and insulin resistance in the intestinal L-cells need to be elucidated. Moreover, recent observations indicate that L-cells may be able to process not only GLP-1 but also glucagon from proglucagon. The aim of this study was to investigate the effects of chronic palmitate exposure on insulin pathways, GLP-1 secretion and glucagon synthesis in the GLUTag L-cell line. Cells were cultured in the presence/absence of palmitate (0.5 mM) for 24 h to mimic lipotoxicity. Palmitate treatment affected insulin-stimulated GLP-1 secretion, insulin receptor phosphorylation and IRS-1-AKT pathway signaling. In our model lipotoxicity induced extracellular signal-regulated kinase (ERK 44/42) activation both in insulin stimulated and basal conditions and also up-regulated paired box 6 (PAX6) and proglucagon expression ( Gcg ). Interestingly, palmitate treatment caused an increased glucagon secretion through the up-regulation of prohormone convertase 2. These results indicate that a state of insulin resistance could be responsible for secretory alterations in L-cells through the impairment of insulin-signaling pathways. Our data support the hypothesis that lipotoxicity might contribute to L-cell deregulation.

Neuropeptides are derived from large and inactive proteins which require endoproteolytic processing for the biosynthesis of the bioactive peptides. The maturation of pro-neuropeptide to neuropeptide is believed to be performed by ortholog pro-protein convertase EGL-3 in Caenorhabditis elegans (C. elegans). Furthermore, ortholog of Cathepsin L, CPL-1 are found in C. elegans and can potentially cleave paired basic amino acids at the N-terminal suggesting the presence of both pathways. The objective of this study was to decipher the role of EGL-3 in the proteolysis of FMRF amide-related peptides (FLPs) or neuropeptide-like proteins (NLPs) using synthetic surrogate peptides based on a universal enzymatic cleavage pattern published by Schechter and Berger and used widely in enzymology. The results show evidence that proteolysis controls FLP-21 and NLP-8 related neuropeptide levels in C. elegans. Surrogate peptides were degraded rapidly when exposed to C. elegans S9 fractions leading to the formation of specific peptide fragments related to EGL-3 and CPL-1 pathway. The results suggest that CPL-1 pathway does not compensate for the loss of the EGL-3 pathway. Proteolysis of pro-neuropeptides associated to FLP-21 and NLP-8 in elg-3 mutants are severely hampered leading to a lack of mature bioactive neuropeptides.

Aims/hypothesis: Enteroendocrine K and L cells are pivotal in regulating appetite and glucose homeostasis. Knowledge of their distribution in humans is sparse and it is unknown whether alterations occur in type 2 diabetes. We aimed to evaluate the distribution of enteroendocrine K and L cells and relevant prohormone-processing enzymes (using immunohistochemical staining), and to evaluate the mRNA expression of the corresponding genes along the entire intestinal tract in individuals with type 2 diabetes and healthy participants., Methods: In this cross-sectional study, 12 individuals with type 2 diabetes and 12 age- and BMI-matched healthy individuals underwent upper and lower double-balloon enteroscopy with mucosal biopsy retrieval from approximately every 30 cm of the small intestine and from seven specific anatomical locations in the large intestine., Results: Significantly different densities for cells positive for chromogranin A (CgA), glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, peptide YY, prohormone convertase (PC) 1/3 and PC2 were observed along the intestinal tract. The expression of CHGA did not vary along the intestinal tract, but the mRNA expression of GCG, GIP, PYY, PCSK1 and PCSK2 differed along the intestinal tract. Lower counts of CgA-positive and PC1/3-positive cells, respectively, were observed in the small intestine of individuals with type 2 diabetes compared with healthy participants. In individuals with type 2 diabetes compared with healthy participants, the expression of GCG and PYY was greater in the colon, while the expression of GIP and PCSK1 was greater in the small intestine and colon, and the expression of PCSK2 was greater in the small intestine., Conclusions/interpretation: Our findings provide a detailed description of the distribution of enteroendocrine K and L cells and the expression of their products in the human intestinal tract and demonstrate significant differences between individuals with type 2 diabetes and healthy participants., Trial Registration: NCT03044860.

Several recent studies have suggested that compounds known as endocrine-disrupting chemicals (EDCs) can promote obesity by serving as ligands for nuclear receptors, including the peroxisome proliferator-activated receptor γ (PPARγ) and the glucocorticoid receptor (GR). Thiazolidinedione insulin sensitizers, which act as ligands for PPARγ, also interact with and regulate the activity of the mitochondrial pyruvate carrier (MPC). We evaluated whether several EDCs might also affect MPC activity. Most of the EDCs evaluated did not acutely affect pyruvate metabolism. However, the putative endocrine disruptors tributyltin (TBT) and tolylfluanid (TF) acutely and markedly suppressed pyruvate metabolism in isolated mitochondria. Using mitochondria isolated from brown adipose tissue in mice with adipocyte-specific deletion of the MPC2 protein, we determined that the effect of TF on pyruvate metabolism required MPC2, whereas TBT did not. We attempted to determine whether the obesogenic effects of TF might involve MPC2 in adipose tissue. However, we were unable to replicate the published effects of TF on weight gain and adipose tissue gene expression in wild-type or fat-specific MPC2 knockout mice. Treatment with TF modestly enhanced adipogenic gene expression in vitro but had no effect on GR activation or phosphorylation in cultured cells. These data suggest that TF may affect mitochondrial pyruvate metabolism via the MPC complex but also call into question whether this compound affects GR activity and is obesogenic in mice., (Copyright © 2018 Endocrine Society.)

Equine pituitary pars intermedia dysfunction (PPID) is characterized by hyperplasia of the pars intermedia (PI) melanotrophs of the pituitary gland (PG), and increased production of proopiomelanocortin (POMC). POMC is cleaved by prohormone convertase 1 (PC1) to produce adrenocorticotropic hormone (ACTH), and further processing of ACTH by PC2 to produce alpha-melanocyte stimulating hormone (α-MSH) and corticotropin-like intermediate peptide (CLIP). High plasma ACTH concentrations in horses with PPID might be related to reduced conversion of ACTH to α-MSH by PCs. The hypothesis of this study was that PC1 and PC2 expression in the pituitary gland are altered in PPID, resulting in an abnormal relative abundance of POMC derived proteins. The objectives of this study were to identify the partial sequences of equine POMC, PC1, and PC2 mRNAs; and to determine whether the expression of POMC, PC1, and PC2 mRNAs in whole pituitary extracts, and POMC-protein in the cavernous sinus blood of horses are altered in PPID. We confirmed (RT-PCR and sequencing) that the partial sequences obtained match the corresponding regions of predicted equine POMC, PC1 and PC2 sequences. The expression (quantification by RT-qPCR) of POMC, PC1 and PC2 mRNAs were found upregulated in the pituitary of horses with PPID. Plasma (measured using RIA/ELISA) ACTH and α-MSH were elevated in PPID horses. These results indicate distinct differences in gene and protein expression of POMC and its intermediates, and processing enzymes in PPID. It provides evidence to support the notion that local, pituitary-specific inadequacies in prohormone processing likely contribute to equine PPID.

Background and Aims: An intra-islet incretin system has been recently suggested to operate through modulation of the expression and activity of proconvertase 1/3 and 2 (PC1/3, PC2) in pancreatic alpha-cell accounting for local release of GLP-1. Little is known, whether this alpha-cell activity can be affected by the metabolic alterations occurring in type 2 diabetes, such as hyperglycemia, hyperlipidemia or hyperglucagonemia., Materials and Methods: AlphaTC1/6 cells from a mice pancreatic cell line were incubated in the presence of two glucose (G) concentration (5.5 and 16.7 mM) for 16 h with or without free fatty acid, IL6 or glucagon. GLP-1 secretion was measured by ELISA and expression of PC1/3 and PC2 by RT-PCR and western blot; cell viability was determined by MTT method, Reactive Oxygen Species generation (ROS) by H2DCFDA fluorescence and apoptosis by Annexin staining and Propidium Iodine (PI) fluorescence., Results: Upon 16.7G incubation, GLP-1 secretion (total and active) was significantly increased in parallel with a significant increment in PC1/3 expression, a slight increase in cell viability and ROS generation and by a decrement in PC2 expression with no change in cell apoptosis. When cells were incubated at 5.5mM glucose with FFA, also an increment in GLP-1 secretion and PC1/3 expression was observed together an increment in ROS generation, a decrement in cell viability, and a modest increment in apoptosis. When incubated with 16.7mM glucose with FFA, the increment in GLP-1 secretion was reduced to basal, accompanied by an increment in apoptosis and ROS generation. This was also observed with IL-6, but in this case, no modification in ROS generation or apoptosis was observed when compared to 16.7mM glucose. The presence of glucagon did not modify any of the parameters studied., Conclusion: These data suggest that under hyperglycemic, hyperlipidemia or inflammatory conditions, alpha cells can increase expression PC1/3 and activate GLP-1 secretion, which may contribute protecting both alpha and beta-cells from glucose and lipotoxicity, while this effect seems to be lost in the presence of both pathophysiological conditions.

Although autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple kidney cysts, the most frequent cause of death in ADPKD patients is cardiovascular disease. ADPKD is linked to mutations in PKD1 or pkd2, the genes that encode for the proteins polycystin 1 and polycystin 2 (PC1 and PC2, respectively). The cardiovascular complications have been assumed to be a consequence of renal hypertension and activation of renin/angiotensin/aldosterone (RAAS) pathway. However, the expression of PC1 and PC2 in cardiac tissue suggests additional direct effects of these proteins on cardiac function. We previously reported that zebrafish lacking PC2 develop heart failure, and that heterozygous Pkd2 +/- mice are hypersensitive to acute β-adrenergic receptor (βAR) stimulation. Here, we investigate the effect of cardiac stress (prolonged continuous βAR stimulus) on Pkd2 +/- mice. After βAR stimulation for 7 days, wild-type (WT) mice had increased left ventricular mass and natriuretic peptide (ANP and BNP) mRNA levels. The WT mice also had upregulated levels of PC2 and chromogranin B (CGB, an upstream regulator of BNP). Conversely, Pkd2 +/- mice had increased left ventricular mass, but natriuretic peptide and CGB expression levels remained constant. Reversal of the increased cardiac mass was observed in WT mice 3 days after cessation of the βAR stimulation, but not in Pkd2 +/- mice. We suggest that cardiac stress leads to upregulation of the PC2-CGB-BNP signaling axis, and this pathway regulates the production of cardio-protective natriuretic peptides. The lack of a PC2-dependent cardio-protective function may contribute to the severity of cardiac dysfunction in Pkd2 +/- mice and in ADPKD patients.

Dysregulated glucagon secretion is a hallmark of type 2 diabetes (T2D). To date, few effective therapeutic agents target on deranged glucagon secretion. Family with sequence similarity 3 member D (FAM3D) is a novel gut-derived cytokine-like protein, and its secretion timing is contrary to that of glucagon. However, the roles of FAM3D in metabolic disorder and its biological functions are largely unknown. In the present study, we investigated whether FAM3D modulates glucagon production in mouse pancreatic alpha TC1 clone 6 (αTC1-6) cells. Glucagon secretion, prohormone convertase 2 (PC2) activity, and mitogen-activated protein kinase (MAPK) pathway were assessed. Exogenous FAM3D inhibited glucagon secretion, PC2 activity, as well as extracellular-regulated protein kinase 1/2 (ERK1/2) signaling and induced MAPK phosphatase 1 (MKP1) expression. Moreover, knockdown of MKP1 and inhibition of ERK1/2 abolished and potentiated the inhibitory effect of FAM3D on glucagon secretion, respectively. Taken together, FAM3D inhibits glucagon secretion via MKP1-dependent suppression of ERK1/2 signaling. These results provide rationale for developing the therapeutic potential of FAM3D for dysregulated glucagon secretion and T2D.

Tachykinin and opioid neuropeptides play a fundamental role in pain transmission, modulation and inhibition. The proteolysis control of endogenous tachykinin and opioid neuropeptides has a significant impact on pain perception. The role of proprotein convertases (PCs) in the proteolysis of proneuropeptides was previously established but very few studies have shown the direct impact of PCs on the regulation of specific tachykinin and opioid peptides in the central nervous system. There is an increasing interest in the therapeutic targeting of PCs for the treatment of pain but it is imperative to assess the impact of PCs on the pronociceptive and the endogenous opioid systems. The objective of this study was to determine the relative concentration of targeted neuropeptides in the spinal cord of WT, PC1 -/+ and PC2 -/+ animals to establish the impact of a restricted PCs activity on the regulation of specific neuropeptides. The analysis of tachykinin and opioid neuropeptides were performed on a HPLC-MS/MS (High-Resolution Quadrupole-Orbitrap Mass Spectrometer). The results revealed a significant decrease of Dyn A (p<0.01), Leu-Enk (p<0.001), Met-Enk (p<0.001), Tach 58-71 (p<0.05), SP (p<0.01) and NKA (p<0.001) concentrations in both, PC1 -/+ and PC2 -/+ animals. Therefore, the modulation of PCs activity has an important impact on specific pronociceptive peptides (SP and NKA), but the results also shown that endogenous opioid system is hindered and consequently it will affect significantly the pain modulatory pathways. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

New Findings: What is the central question of this study? The antidiabetic effects of thiazolidinedione (TZD) drugs may be mediated in part by a molecular interaction with the constituent proteins of the mitochondrial pyruvate carrier complex (MPC1 and MPC2). We examined the ability of a mutant mouse strain expressing an N-terminal truncation of MPC2 (Mpc2Δ16 mice) to respond to TZD treatment. What is the main finding and its importance? The response of Mpc2Δ16 mice to TZD treatment was not significantly different from that of wild-type C57BL6/J control animals, suggesting that the 16 N-terminal amino acids of MPC2 are dispensable for the effects of TZD treatment. Rosiglitazone and pioglitazone are thiazolidinedione (TZD) compounds that have been used clinically as insulin-sensitizing drugs and are generally believed to mediate their effects via activation of the peroxisome proliferator-activated receptor γ (PPARγ). Recent work has shown that it is possible to synthesize TZD compounds with potent insulin-sensitizing effects and markedly diminished affinity for PPARγ. Both clinically used TZDs and investigational PPARγ-sparing TZDs, such as MSDC-0602, interact with the mitochondrial pyruvate carrier (MPC) and inhibit its activity. The MPC complex is composed of two proteins, MPC1 and MPC2. Herein, we used mice expressing a hypomorphic MPC2 protein missing 16 amino acids in the N-terminus (Mpc2Δ16 mice) to determine the effects of these residues in mediating the insulin-sensitizing effects of TZDs in diet-induced obese mice. We found that both pioglitazone and MSDC-0602 elicited their beneficial metabolic effects, including improvement in glucose tolerance, attenuation of hepatic steatosis, reduction of adipose tissue inflammation and stimulation of adipocyte browning, in both wild-type and Mpc2Δ16 mice after high-fat diet feeding. In addition, truncation of MPC2 failed to attenuate the interaction between TZDs and the MPC in a bioluminescence resonance energy transfer-based assay or to affect the suppression of pyruvate-stimulated respiration in cells. Collectively, these data suggest that the interaction between TZDs and MPC2 is not affected by loss of the N-terminal 16 amino acids nor are these residues required for the insulin-sensitizing effects of these compounds., (© 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.)

Despite substantial advances in the research exploring the pathogenesis of Type 1 Diabetes (T1D), the pathophysiological mechanisms involved remain unknown. We hypothesized in this study that interferon alpha (IFNα) participates in the early stages of T1D development by triggering endoplasmic reticulum (ER) stress. To verify our hypothesis, human islets and human EndoC-βH1 cells were exposed to IFNα and tested for ER stress markers, glucose stimulated insulin secretion (GSIS) and insulin content. IFNα treatment induced upregulation of ER stress markers including Binding immunoglobulin Protein, phospho-eukaryotic translation initiation factor 2α, spliced- X-box binding protein-1, C/EBP homologous protein and activating transcription factor 4. Intriguingly, IFNα treatment did not impair GSIS but significantly decreased insulin production in both human islets and EndoC-βH1 cells. Furthermore, IFNα decreased the expression of both proinsulin convertase 1 and proinsulin convertase 2, suggesting an altered functional state of the beta cells characterized by a slower proinsulin-insulin conversion. Pretreatment of both human islets and EndoC-βH1 cells with chemical chaperones 4-phenylbutyric acid and tauroursodeoxycholic acid completely prevented IFNα effects, indicating an ER stress-mediated impairment of insulin production. We demonstrated for the first time that IFNα elicits ER stress in human beta cells providing a novel mechanistic role for this virus-induced cytokine in the development of T1D. Compounds targeting molecular processes altered in ER-stressed beta cells could represent a potential therapeutic strategy to prevent IFNα-induced beta cell dysfunction in the early onset of T1D., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Aims/hypothesis: A contributor to beta cell failure in type 2 diabetes and islet transplants is amyloid formation by aggregation of the beta cell peptide, islet amyloid polypeptide (IAPP). Similar to the proinsulin processing pathway that generates insulin, IAPP is derived from a prohormone precursor, proIAPP, which requires cleavage by prohormone convertase (PC) 1/3 and PC2 in rodent pancreatic beta cells. We hypothesised that loss of PC2 would promote beta cell death and dysfunction in a rodent model of human beta cell proIAPP overexpression., Methods: We generated an islet transplant model wherein immune-deficient mouse models of diabetes received islets expressing amyloidogenic human proIAPP and lacking PC2, leading to restoration of normoglycaemia accompanied by increased secretion of human proIAPP. Blood glucose levels were analysed for up to 16 weeks in transplant recipients and grafts were assessed for islet amyloid and beta cell number and death., Results: Hyperglycaemia (blood glucose >16.9 mmol/l) returned in 94% of recipients of islets expressing human proIAPP and lacking PC2, whereas recipients of islets that express human proIAPP and normal PC2 levels remained normoglycaemic for at least 16 weeks. Islet graft failure was accompanied by a ∼20% reduction in insulin-positive cells, yet the degree of amyloid deposition and beta cell apoptosis was similar to those of controls expressing human proIAPP with functional PC2 levels., Conclusions/interpretation: PC2 deficiency in transplanted mouse islets expressing human proIAPP promotes beta cell loss and graft failure. Our data suggest that impaired NH 2 -terminal processing and increased secretion of human proIAPP promote beta cell failure.

Aging is accompanied by declines in memory performance, and particularly affects memories that rely on hippocampal-cortical systems, such as episodic and explicit. With aged populations significantly increasing, the need for preventing or rescuing memory deficits is pressing. However, effective treatments are lacking. Here, we show that the level of the mature form of insulin-like growth factor 2 (IGF-2), a peptide regulated in the hippocampus by learning, required for memory consolidation and a promoter of memory enhancement in young adult rodents, is significantly reduced in hippocampal synapses of aged rats. By contrast, the hippocampal level of the immature form proIGF-2 is increased, suggesting an aging-related deficit in IGF-2 processing. In agreement, aged compared to young adult rats are deficient in the activity of proprotein convertase 2, an enzyme that likely mediates IGF-2 posttranslational processing. Hippocampal administration of the recombinant, mature form of IGF-2 rescues hippocampal-dependent memory deficits and working memory impairment in aged rats. Thus, IGF-2 may represent a novel therapeutic avenue for preventing or reversing aging-related cognitive impairments., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Durable antibody production after vaccination or infection is mediated by long-lived plasma cells (LLPCs). Pathways that specifically allow LLPCs to persist remain unknown. Through bioenergetic profiling, we found that human and mouse LLPCs could robustly engage pyruvate-dependent respiration, whereas their short-lived counterparts could not. LLPCs took up more glucose than did short-lived plasma cells (SLPCs) in vivo, and this glucose was essential for the generation of pyruvate. Glucose was primarily used to glycosylate antibodies, but glycolysis could be promoted by stimuli such as low ATP levels and the resultant pyruvate used for respiration by LLPCs. Deletion of Mpc2, which encodes an essential component of the mitochondrial pyruvate carrier, led to a progressive loss of LLPCs and of vaccine-specific antibodies in vivo. Thus, glucose uptake and mitochondrial pyruvate import prevent bioenergetic crises and allow LLPCs to persist. Immunizations that maximize these plasma cell metabolic properties might thus provide enduring antibody-mediated immunity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous big dynorphin (BDyn) and dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild-type (WT), PC1(-/+) and PC2(-/+) animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments dynorphin 1-19, dynorphin 1-13, dynorphin 1-11 and dynorphin 1-7, and C-terminal processing of Dyn A generates dynorphin 1-13, dynorphin 1-11 and dynorphin 1-7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, the proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even the partial inhibition of PC1 or PC2 may impair the endogenous opioid system., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The physiological role of the TGR5 receptor in the pancreas is not fully understood. We previously showed that activation of TGR5 in pancreatic β cells by bile acids induces insulin secretion. Glucagon released from pancreatic α cells and glucagon-like peptide 1 (GLP-1) released from intestinal L cells regulate insulin secretion. Both glucagon and GLP-1 are derived from alternate splicing of a common precursor, proglucagon by PC2 and PC1, respectively. We investigated whether TGR5 activation in pancreatic α cells enhances hyperglycemia-induced PC1 expression thereby releasing GLP-1, which in turn increases β cell mass and function in a paracrine manner. TGR5 activation augmented a hyperglycemia-induced switch from glucagon to GLP-1 synthesis in human and mouse islet α cells by GS/cAMP/PKA/cAMP-response element-binding protein-dependent activation of PC1. Furthermore, TGR5-induced GLP-1 release from α cells was via an Epac-mediated PKA-independent mechanism. Administration of the TGR5 agonist, INT-777, to db/db mice attenuated the increase in body weight and improved glucose tolerance and insulin sensitivity. INT-777 augmented PC1 expression in α cells and stimulated GLP-1 release from islets of db/db mice compared with control. INT-777 also increased pancreatic β cell proliferation and insulin synthesis. The effect of TGR5-mediated GLP-1 from α cells on insulin release from islets could be blocked by GLP-1 receptor antagonist. These results suggest that TGR5 activation mediates cross-talk between α and β cells by switching from glucagon to GLP-1 to restore β cell mass and function under hyperglycemic conditions. Thus, INT-777-mediated TGR5 activation could be leveraged as a novel way to treat type 2 diabetes mellitus., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Understanding the role of hypothalamic neuropeptides and hormones in energy balance is paramount in the search for approaches to mitigate the obese state. Increased hypothalamic-pituitary-adrenal axis activity leads to increased levels of glucocorticoids (GC) that are known to regulate body weight. The axis initiates the production and release of corticotropin-releasing hormone (CRH) from the paraventricular nucleus (PVN) of the hypothalamus. Levels of active CRH peptide are dependent on the processing of its precursor pro-CRH by the action of two members of the family of prohormone convertases 1 and 2 (PC1 and PC2). Here, we propose that the nutrient sensor sirtuin 1 (Sirt1) regulates the production of CRH post-translationally by affecting PC2. Data suggest that Sirt1 may alter the preproPC2 gene directly or via deacetylation of the transcription factor Forkhead box protein O1 (FoxO1). Data also suggest that Sirt1 may alter PC2 via a post-translational mechanism. Our results show that Sirt1 levels in the PVN increase in rats fed a high fat diet for 12 weeks. Furthermore, elevated Sirt1 increased PC2 levels, which in turn increased the production of active CRH and GC. Collectively, this study provides the first evidence supporting the hypothesis that PVN Sirt1 activates the hypothalamic-pituitary-adrenal axis and basal GC levels by enhancing the production of CRH through an increase in the biosynthesis of PC2, which is essential in the maturation of CRH from its prohormone, pro-CRH., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Human islet amyloid polypeptide (h-IAPP) is a peptide hormone that is synthesized and cosecreted with insulin from insulin-secreting pancreatic β-cells. Recently, h-IAPP was proposed to be the main component responsible for the cytotoxic pancreatic amyloid deposits in patients with type 2 diabetes mellitus (T2DM). Since the causative factors of IAPP (or amylin) oligomer aggregation are not fully understood, this review will discuss the various forms of h-IAPP aggregation. Not all forms of IAPP aggregates trigger the destruction of β-cell function and loss of β-cell mass; however, toxic oligomers do trigger these events. Once these toxic oligomers form under abnormal metabolic conditions in T2DM, they can lead to cell disruption by inducing cell membrane destabilization. In this review, the various factors that have been shown to induce toxic IAPP oligomer formation will be presented, as well as the potential mechanism of oligomer and fibril formation from pro-IAPPs. Initially, pro-IAPPs undergo enzymatic reactions to produce the IAPP monomers, which can then develop into oligomers and fibrils. By this mechanism, toxic oligomers could be generated by diverse pathway components. Thus, the interconnections between factors that influence amyloid aggregation (eg, absence of PC2 enzyme, deamidation, reduction of disulfide bonds, environmental factors in the cell, genetic mutations, copper metal ions, and heparin) will be presented. Hence, this review will aid in understanding the fundamental causative factors contributing to IAPP oligomer formation and support studies for investigating novel T2DM therapeutic approaches, such as the development of inhibitory agents for preventing oligomerization at the early stages of diabetic pathology.

Gluconeogenesis is critical for maintenance of euglycemia during fasting. Elevated gluconeogenesis during type 2 diabetes (T2D) contributes to chronic hyperglycemia. Pyruvate is a major gluconeogenic substrate and requires import into the mitochondrial matrix for channeling into gluconeogenesis. Here, we demonstrate that the mitochondrial pyruvate carrier (MPC) comprising the Mpc1 and Mpc2 proteins is required for efficient regulation of hepatic gluconeogenesis. Liver-specific deletion of Mpc1 abolished hepatic MPC activity and markedly decreased pyruvate-driven gluconeogenesis and TCA cycle flux. Loss of MPC activity induced adaptive utilization of glutamine and increased urea cycle activity. Diet-induced obesity increased hepatic MPC expression and activity. Constitutive Mpc1 deletion attenuated the development of hyperglycemia induced by a high-fat diet. Acute, virally mediated Mpc1 deletion after diet-induced obesity decreased hyperglycemia and improved glucose tolerance. We conclude that the MPC is required for efficient regulation of gluconeogenesis and that the MPC contributes to the elevated gluconeogenesis and hyperglycemia in T2D., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pyruvate transport across the inner mitochondrial membrane is believed to be a prerequisite for gluconeogenesis in hepatocytes, which is important for the maintenance of normoglycemia during prolonged food deprivation but also contributes to hyperglycemia in diabetes. To determine the requirement for mitochondrial pyruvate import in gluconeogenesis, mice with liver-specific deletion of mitochondrial pyruvate carrier 2 (LS-Mpc2(-/-)) were generated. Loss of MPC2 impaired, but did not completely abolish, hepatocyte conversion of labeled pyruvate to TCA cycle intermediates and glucose. Unbiased metabolomic analyses of livers from fasted LS-Mpc2(-/-) mice suggested that alterations in amino acid metabolism, including pyruvate-alanine cycling, might compensate for the loss of MPC2. Indeed, inhibition of pyruvate-alanine transamination further reduced mitochondrial pyruvate metabolism and glucose production by LS-Mpc2(-/-) hepatocytes. These data demonstrate an important role for MPC2 in controlling hepatic gluconeogenesis and illuminate a compensatory mechanism for circumventing a block in mitochondrial pyruvate import., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aims/hypothesis: We studied the impact of Roux-en-Y gastric bypass (RYGB) on the density and hormonal gene expression of small-intestinal enteroendocrine cells in obese patients with type 2 diabetes., Methods: Twelve patients with diabetes and 11 age- and BMI-matched controls underwent RYGB followed by enteroscopy ~10 months later. Mucosal biopsies taken during surgery and enteroscopy were immunohistochemically stained for glucagon-like peptide-1 (GLP-1), peptide YY (PYY), cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide (GIP) and prohormone convertase 2 (PC2) and the expression of GCG (encoding preproglucagon), PYY, CCK, GIP, GHRL (encoding ghrelin), SCT (encoding secretin), NTS (encoding neurotensin) and NR1H4 (encoding farnesoid X receptor) was evaluated., Results: The density of cells immunoreactive for GLP-1, CCK and GIP increased in patients after RYGB and the density of those immunoreactive for GLP-1, PYY, CCK and PC2 increased in controls. In both groups, GHRL, SCT and GIP mRNA was reduced after RYGB while PYY, CCK, NTS and NR1H4 gene expression was unaltered. GCG mRNA was upregulated in both groups., Conclusions/interpretation: Numerous alterations in the distribution of enteroendocrine cells and their expression of hormonal genes are seen after RYGB and include increased density of GLP-1-, PYY-, CCK-, GIP- and PC2-positive cells, reduced gene expression of GHRL, SCT and GIP and increased expression of GCG.

Previous studies have shown that the administration of docosahexaenoic acid (DHA) or GW9508, a GPR40/FFA1 (free fatty acid receptor) agonist, facilitates β-endorphin release in the arcuate nucleus of the hypothalamus in mice. However, the mechanisms mediating β-endorphin release induced by GPR40/FFA1 agonists remain unknown. In this study, we focused on the changes in expression of hypothalamic prohormone convertase (PC) 2, which is a calcium-dependent subtilisin-related proteolytic enzyme. The intracerebroventricular injection of DHA or GW9508 significantly increased PC2 protein expression in the hypothalamus. This increase in PC2 expression was inhibited by pretreatment with GW1100, a GPR40/FFA1 antagonist. Furthermore, PC2 protein expression gradually increased over time after complete Freund's adjuvant. These increase in PC2 expression were inhibited by pretreatment with GW1100. However, GW1100 by itself had no effect on PC2 levels. Taken together, our findings suggest that activation of the hypothalamic GPR40/FFA1 signaling pathway may regulate β-endorphin release via PC2, and regulate the endogenous pain control system., (Copyright © 2015 Elsevier B.V. All rights reserved.)

FAM20C is a secretory kinase responsible for the phosphorylation of multiple secreted proteins in mammalian cells; it has been shown to phosphorylate serine residues within a variety of different bone proteins. In this work we demonstrate that FAM20C also phosphorylates threonines, specifically those within the N-terminal domain of the neuroendocrine chaperone 7B2. Analysis of the primary sequence of 7B2 revealed that three threonine residues in its N-terminal domain are located within FAM20C consensus motifs: Thr73, Thr99, and Thr111. The individual substitution of Thr73 and Thr111 residues by neutral alanines caused a marked decrease in the total phosphorylation of 7B2. Furthermore, the phosphomimetic substitution of Thr111 by Glu clearly diminished the ability of 7B2 to activate pro-prohormone convertase 2 (PC2) in 7B2-lacking SK-N-MC neuroblastoma cells, suggesting that the phosphorylation of this residue critically impacts the 7B2-proPC2 interaction. However, the phosphomimetic mutation did not alter 7B2's ability to function as an antiaggregant for human islet amyloid polypeptide. FAM20C-mediated phosphorylation of a common alternatively spliced variant of human 7B2 that lacks Ala100 (thus eliminating the Thr99 phosphorylation consensus site) was similar to the Ala-containing protein, but this variant did not activate proPC2 as efficiently as the Ala-containing protein. Although threonines within 7B2 were phosphorylated efficiently, FAM20C was incapable of performing the well-known regulatory threonine phosphorylation of the molecular chaperone binding immunoglobulin protein. Taken together, these results indicate that FAM20C plays a role in 7B2-mediated proPC2 activation by phosphorylating residue Thr111; and that 7B2 function is regulated by alternative splicing.

Background: Tranexamic acid (trans-4-aminomethylcyclohexanecarboxylic acid) is a medicinal amino acid used in skin whitening care. This study examined the effects of tranexamic acid on the melanocyte activation of the skin induced by an ultraviolet (UV) B eye irradiation., Methods: The eye or ear was locally exposed to UVB at a dose of 1.0 kJ/m(2) using a 20SE sunlamp after covering the remaining body surface with aluminum foil., Results: UVB eye irradiation induced melanocyte activation of the skin, similar to that observed following UVB ear irradiation, which was suppressed by the administration of tranexamic acid treatment. The plasma α-melanocyte-stimulating hormone (α-MSH) content was increased by UVB irradiation of the eye; however, the increase in α-MSH was suppressed by tranexamic acid treatment. In addition, UVB eye irradiation induced the up-regulation of prohormone convertase (PC) 2 in the pituitary gland. Meanwhile, the increase in PC2 induced by UVB eye irradiation was suppressed by tranexamic acid treatment., Conclusions: These results clearly indicate that tranexamic acid decreases the expression of PC2, which cleavages from proopiomelanocortin to α-MSH in the pituitary gland, thereby suppressing melanocyte activation., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pyruvate lies at a central biochemical node connecting carbohydrate, amino acid, and fatty acid metabolism, and the regulation of pyruvate flux into mitochondria represents a critical step in intermediary metabolism impacting numerous diseases. To characterize changes in mitochondrial substrate utilization in the context of compromised mitochondrial pyruvate transport, we applied (13)C metabolic flux analysis (MFA) to cells after transcriptional or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC). Despite profound suppression of both glucose and pyruvate oxidation, cell growth, oxygen consumption, and tricarboxylic acid (TCA) metabolism were surprisingly maintained. Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched-chain amino acid oxidation. Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with the use of lipids and amino acids as catabolic and anabolic fuels., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Antagonism of the effects of glucagon as an adjunct therapy with other glucose-lowering drugs in the chronic treatment of diabetes has been suggested to aggressively control blood glucose levels. Antagonism of glucagon effects, by targeting glucagon secretion or disabling the glucagon receptor, is associated with α-cell hyperplasia. We evaluated the influence of total glucagon withdrawal on islets of Langerhans using prohormone convertase-2 knockout mice (PC2-ko), in which α-cell hyperplasia is present from a young age and persists throughout life, in order to understand whether or not sustained glucagon deficit would lead to islet tumorigenesis. PC2-ko and wild-type (WT) mice were maintained drug-free, and cohorts of these groups sampled at 3, 12 and 18 months for plasma biochemical and morphological (histological, immunohistochemical, electron microscopical and image analytical) assessments. WT mice showed no islet tumours up to termination of the study, but PC2-ko animals displayed marked changes in islet morphology from α-cell hypertrophy/hyperplasia/atypical hyperplasia, to adenomas and carcinomas, these latter being first encountered at 6-8 months. Islet hyperplasias and tumours primarily consisted of α-cells associated to varying degrees with other islet endocrine cell types. In addition to substantial increases in islet neoplasia, increased α-cell neogenesis associated primarily with pancreatic duct(ule)s was present. We conclude that absolute blockade of the glucagon signal results in tumorigenesis and that the PC2-ko mouse represents a valuable model for investigation of islet tumours and pancreatic ductal neogenesis., (© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.)

Type 2 diabetic (T2D) patients exhibit fasting relative hyperproinsulinemia owing to pancreatic β-cell dysfunction. To clarify the mechanism underlying this hyperproinsulinemic state, we evaluated the activities of the endopeptidases prohormone convertase (PC) 1/3 and PC2 in T2D patients. Fasting blood levels of intact proinsulin (IPI), total proinsulin (t-PI) and C-peptide were measured simultaneously, and intravenous glucagon loading was performed to investigate the dynamics of circulating proinsulin-related molecules released from pancreatic β-cells in 12 healthy volunteers and 18 T2D patients. Taking advantage of the 95% cross-reactivity between proinsulin and des-31,32-proinsulin (des-31,32-PI) with the human proinsulin radioimmunoassay kit used in this study, we estimated PC1/3 and PC2 activities using the following formulas: des-31,32-PI = (t-PI-IPI)/0.95; PC1/3 activity = des-31,32-PI/IPI; and PC2 activity = C-peptide/des-31,32-PI. C-peptide responses to glucagon were slightly lower among T2D patients. IPI and the IPI/C-peptide ratio were significantly higher in T2D patients (p<0.05 and p<0.01, respectively). There was no difference in des-31,32-PI levels or PC2 activity between the two groups. However, PC1/3 activity was significantly lower in T2D patients than in the control group (p<0.01). We propose that decreased activity of PC1/3 rather than PC2 in pancreatic β-cells is involved in the impaired proinsulin processing, resulting in elevated IPI levels in T2D patients.

Circulating glucocorticoid levels oscillate with a robust circadian rhythm, yet the physiological relevance of this rhythmicity remains unclear. Here, we show that modulation of circadian glucocorticoid oscillation by enhancing its amplitude leads to anxiolytic-like behavior. We observed that mice with adrenal subcapsular cell hyperplasia (SCH), a common histological change in the adrenals, are less anxious than mice without SCH. This behavioral change was found to be dependent on the higher amplitude of glucocorticoid oscillation, although the total glucocorticoid secretion is not increased in these mice. Genetic and pharmacologic experiments demonstrated that intermediate opioid peptides secreted from SCH activate CXCR7, a β-arrestin-biased G-protein-coupled receptor (GPCR), to augment circadian oscillation of glucocorticoid levels in a paracrine manner. Furthermore, recapitulating this paracrine axis by subcutaneous administration of a synthetic CXCR7 ligand is sufficient to induce anxiolytic-like behavior. Adrenocortical β-arrestin-biased GPCR signaling is a potential target for modulating circadian glucocorticoid oscillation and emotional behavior., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In Huntington disease (HD), hypothalamic neuropeptidergic systems are not equally affected at the peptide and mRNA levels. Because prohormone convertases (PCs) are critically involved in the conversion of propeptides into their active forms, we postulated that a decrease in PC expression may underlie these discrepancies. Therefore, we assessed the expression of PC1/3 and PC2 in the hypothalamic infundibular, suprachiasmatic, and paraventricular nuclei in postmortem tissues of HD patients and controls (n = 9, each) using immunocytochemistry and quantitative reverse transcription polymerase chain reaction. We also assessed PC1/3 and PC2 mRNA expression in the inferior frontal gyrus and colocalization of both PCs with corticotropin-releasing hormone and α-melanocyte-stimulating hormone. In HD patients, PC1/3 and PC2 expression was decreased in the hypothalamic infundibular (both p = 0.046) and paraventricular nuclei (p = 0.031 and p = 0.019). In the suprachiasmatic nucleus, PC1/3 and PC2 expressions were not different between HD and control cases; PC1/3 and PC2 mRNA levels in the inferior frontal gyrus were also not different. None of the PCs was colocalized with corticotropin-releasing hormone, whereas α-melanocyte-stimulating hormone showed colocalization with PC1/3 and PC2. These data suggest that defects in the processing of hypothalamic neuropeptides in HD may partially arise from decreased PC1/3 and PC2 expressions. These changes might contribute to selective neuropathology underlying various clinical manifestations and may provide novel therapeutic targets in HD patients.

Goals: The aim of this report is to delineate the clinical, pathologic, and enteroendocrine (EE) features of prohormone convertase 1/3 (PC1/3) deficiency in children., Background: Prohormone convertases play a pivotal role in the activation of biologically inactive hormones. Congenital defects in the EE axis, such as PC1/3 deficiency, have been rarely reported and their pathophysiological mechanisms are largely unknown., Study: EE function and pathology was evaluated in 4 males (1, 2, 7, and 10 y old) from 2 families with PC1/3 deficiency at a university children's hospital. Clinical course, pathology analysis including immunohistochemistry for PC1/3, PC2, and glucagon-like peptide 1 (GLP-1) and electron microscopy, as well as EE function tests (GLP-1, GLP-2, oral glucose tolerance test) were performed., Results: All (n=4) suffered from congenital severe diarrhea associated with malabsorption. The diarrhea improved during the first year of life and hyperphagia with excessive weight gain (BMI>97th percentile) became the predominant phenotype at an older age. Analysis of the enteroendocrine axis revealed high proinsulin levels (57 to 1116 pmol/L) in all patients, low serum GLP-2 levels, and impaired insulin and GLP-1 secretion after an oral glucose tolerance test at a young age, with improvement in 1 older child tested. Electron microscopy showed normal ultrastructure of enterocytes and EE cells. Immunohistochemistry revealed normal expression of chromogranin A, a marker of EE cells but markedly reduced immunostaining for PC1/3 and PC2 in all patients., Conclusions: PC1/3 deficiency is associated with an age dependent, variable clinical phenotype caused by severe abnormalities in intestinal and EE functions. Serum level of proinsulin can be used as an effective screening tool.

Background: The processing of proprotein convertase (PC)-mediated neuropeptide plays a very important role in carcinogenesis and tumor proliferation., Aim: To investigate proneuropeptide processing mechanism in tumorigenesis and tumor proliferation., Materials and Methods: The expression and processing profiles of PC1, carboxypeptidase E (CPE), PC2, GHRH, or neuropeptide Y (NPY) gene and protein level were investigated between 42 human breast tumor tissues and 21 tumor-adjacent normal tissues., Results: Gene analyses indicated that the proPC1, CPE, or preproNPY gene had higher expression in the breast tumor tissues, whereas the proPC2 or preproGHRH gene showed lower expression in the tissues. Protein analyses showed that the proPC1, PC1, CPE, GHRH, and preproNPY proteins were upregulated in the tumor tissues, whereas the proPC2, PC2, preproGHRH, and NPY proteins were down-regulated in them. The tissue results were highly corroborated with the serum data from the tumor patients and healthy women., Conclusions: The higher PC1 and CPE expressions as well as the transformation of more proGHRH into active GHRH peptide suggest stronger PC1/CPE-mediated neuropeptide processing in the tumor, whereas the lower PC2 expression as well as the transformation of less proNPY into active NPY peptide suggests a weak PC2-mediated processing in it. The alterations of the convertase expressions and processing show that there is a differential proprotein processing system in the tumor, which leads to the abnormal distributions of species, ratio, and concentration of (pro)peptide(s) in the microenvironment of cells. The latter may contribute to cancer progression.

The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, β-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert., (Copyright © 2013 Elsevier Inc. All rights reserved.)