The observation that the two active forms of proprotein convertase 1/3 (PC1/3) were differentially expressed in beta cells of normal islets raised the possibility that this heterogeneity is lost during type 1 diabetes (T1D) progression. To test this hypothesis, the expression of the convertase was evaluated by confocal microscopy in sections of human pancreas of autoantibody positive (AA+) and T1D donors and compared with that of control. Islets of T1D pancreas were comprised of beta cells expressing either low or high PC1/3 levels and all islets of a pancreatic section contained only one beta cell type. Pancreata of AA+ donors contained either of these two classes of islets intermixed with normal islets comprised of beta cells with heterogeneous PC1/3 expression. This alteration affected the expression of proinsulin and insulin, which in most AA+ and T1D donors were lower than in controls. The present results indicate that the heterogeneity of PC1/3 expression is lost in all beta cells in a subset islets of AA+ donors and in all islets of T1D donors. These findings suggest that the heterogeneity of PC1/3 expression is a biomarker of human beta cell health and that its loss coincides with the initial stages of T1D.

Proprotein convertases (PCs) are involved in the pathogenesis of various diseases, making them promising drug targets. Most assays for PCs have been performed with few standard substrates, regardless of differences in cleavage efficiencies. Derived from studies on substrate-analogue inhibitors, 11 novel substrates were synthesized and characterized with five PCs. H-Arg-Arg-Tle-Lys-Arg-AMC is the most efficiently cleaved furin substrate based on its kcat/KM value. Due to its higher kcat value, acetyl-Arg-Arg-Tle-Arg-Arg-AMC was selected for further measurements to demonstrate the benefit of this improved substrate. Compared to our standard conditions, its use allowed a 10-fold reduction of the furin concentration, which enabled Ki value determinations of previously described tight-binding inhibitors under classical conditions. Under these circumstances, a slow-binding behavior was observed for the first time with inhibitor MI-1148. In addition to furin, four additional PCs were used to characterize these substrates. The most efficiently cleaved PC1/3 substrate was acetyl-Arg-Arg-Arg-Tle-Lys-Arg-AMC. The highest kcat/KM values for PC2 and PC7 were found for the N-terminally unprotected analogue of this substrate, although other substrates possess higher kcat values. The highest efficiency for PC5/6A was observed for the substrate acetyl-Arg-Arg-Tle-Lys-Arg-AMC. In summary, we have identified new substrates for furin, PC1/3, PC2, and PC7 suitable for improved enzyme-kinetic measurements.

PCSK1 encodes an enzyme required for prohormone maturation into bioactive peptides. A striking number of SNPs and rare mutations in PCSK1 are associated with a range of clinical phenotypes. Infants bearing two copies of a catalytically inactivating mutation, such as G209R, exhibit life-threatening chronic diarrhea and subsequently develop systemic endocrinopathies. Using CRISPR/Cas9 technology, we have engineered a mouse model bearing a G209R missense mutation in exon 6 of the murine Pcsk1 locus. Most pups homozygous for the G209R mutation succumbed by day 2, and surviving pups were severely dwarfed. In homozygous (but not heterozygous) pups, blood glucose levels were significantly lower, accompanied by elevated plasma insulin-like immunoreactivity and accumulation of large quantities of unprocessed proinsulin in the pancreas. Peptide hormone processing was also aberrant in G209R mouse pituitary, with mature ACTH levels markedly reduced in homozygotes, accompanied by a significant accumulation of POMC. We also observed a significant reduction in PC1/3 protein in the brains of G209R homozygous mice by Western blotting, while PC2 levels remained unaffected. Most likely due to the continued presence of PC2, pituitary and brain levels of α-MSH were not impaired. Analysis of intestinal cell types indicated a modest reduction of enteroendocrine cells in G209R homozygotes. We suggest that the G209R Pcsk1 mouse model recapitulates many of the dramatic neonatal deficiencies of human patients with this homozygous mutation.

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 β-melanocyte-stimulating hormone (MSH). In certain rodent species, no β-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.

BACKGROUND: The insect neuroendocrine system acts in the regulation of physiology, development and growth. Molecular evolution of this system hence has the potential to allow for major biological differences between insect groups. Two prohormone convertases, PC1/3 and PC2, are found in animals and both function in the processing of neuropeptide precursors in the vertebrate neurosecretory pathway. Whereas PC2-function is conserved between the fly Drosophila and vertebrates, ancestral PC1/3 was lost in the fly lineage and has not been functionally studied in any protostome.
RESULTS: In order to understand its original functions and the changes accompanying the gene loss in the fly, we investigated PC1/3 and PC2 expression and function in the beetle Tribolium castaneum. We found that PC2 is broadly expressed in the nervous system, whereas surprisingly, PC1/3 expression is restricted to specific cell groups in the posterior brain and suboesophageal ganglion. Both proteases have parallel but non-redundant functions in adult beetles\' viability and fertility. Female infertility following RNAi is caused by a failure to deposit sufficient yolk to the developing oocytes. Larval RNAi against PC2 produced moulting defects where the larvae were not able to shed their old cuticle. This ecdysis phenotype was also observed in a small subset of PC1/3 knockdown larvae and was strongest in a double knockdown. Unexpectedly, most PC1/3-RNAi larvae showed strongly reduced growth, but went through larval moults despite minimal to zero weight gain.
CONCLUSIONS: The cell type-specific expression of PC1/3 and its essential requirement for larval growth highlight the important role of this gene within the insect neuroendocrine system. Genomic conservation in most insect groups suggests that it has a comparable individual function in other insects as well, which has been replaced by alternative mechanisms in flies.

UNASSIGNED: Glucagon-like peptide-1 (GLP-1) is secreted from the intestinal L-cells to stimulate insulin secretion in the blood glucose control. Our previous study indicates that Sennoside A (SA) can increase the plasma GLP-1 level in a mouse model of type 2 diabetes. However, the mechanism of SA activity remains largely unknown. This issue was explored in this study.
UNASSIGNED: C57BL/6 mice were randomly divided into four groups: a control group without drug treatment, and the other groups with different SA dosages, respectively. Blood glucose was assayed by oral glucose tolerance test (OGTT). Plasma GLP-1 and insulin were investigated. Colon tissues were collected for mRNA or Western blot analysis. Immunofluorescence staining assays were performed to evaluate the number of β-cells and L-cells. In NCI-H716 cells, extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors were employed to investigate the SA-induced GLP-1 secretion mechanism.
UNASSIGNED: In this work, the SA was found to improve OGTT in mice. Plasma GLP-1 and insulin were markedly elevated by SA at the dosage of 45 mg/kg/day. Meanwhile, the increased phosphorylation status of EKR1/2 and prohormone convertase 1/3 (PC1/3) proteins were observed in the colon of SA-treated mice. The number of L-cells exhibited no change in each group. In the NCI-H716 cells, GLP-1 secretion induced by SA was blocked by the ERK1/2 inhibitor.
UNASSIGNED: The present study provides a direct evidence for the interaction between SA and L cells for induction of GLP-1 secretion. These data suggest that GLP-1 secretion induced by SA is dependent on the ERK1/2 signaling pathway. Therefore, the SA is a new drug candidate for the type 2 diabetes treatment by induction of GLP-1 secretion.

Proglucagon is expressed in both pancreatic alpha cells and intestinal epithelial L cells and is cleaved into glucagon and glucagon-like peptide-1 (GLP-1) by different prohormone convertases (PCs). Recent studies have shown that α-cells can also secrete GLP-1, which may improve islet function. However, little is known about the factors influencing GLP-1 secretion by α cells. In this study, we investigated whether insulin promotes GLP-1 secretion by α cells, as well as the mechanisms underlying this phenomenon.
We cultured the alpha-cell line In-R1-G9 in low- or high-glucose medium in the presence or absence of insulin to determine the influence of glucose concentrations on the actions of insulin. We also treated In-R1-G9 cells with insulin for different times and at different doses. Then GLP-1 and glucagon protein expression levels were estimated. Moreover, ERK and phosphatidylinositol-3-kinase/AKT (PI3K/AKT) pathway activity levels and prohormone convertase expression levels were evaluated to elucidate the mechanism underlying the effects of insulin on GLP-1 secretion by α-cells.
Insulin promoted GLP-1 secretion in a time- and dose-dependent manner under high-glucose conditions. Inhibiting the PI3K/AKT pathway with LY294002 and the Ras/mitogen-activated protein kinase (RAS/MAPK) pathway with PD98059 reduced GLP-1 secretion, respectively, in inhibitor-treated cells compared with insulin-treated cells. Moreover, insulin increased prohormone convertase 1/3 expression levels in the corresponding group of IN-R1-G9 cells compared with the control group of cells.
Insulin facilitates GLP-1 secretion by pancreatic alpha cells by inducing PC1/3 expression under high-glucose conditions, a phenomenon that may be associated mainly with PI3K/AKT pathway activation.

GPR142 agonists are being pursued as novel diabetes therapies by virtue of their insulin secretagogue effects. But it is undetermined whether GPR142\'s functions in pancreatic islets are limited to regulating insulin secretion. The current study expands research on its action.
We demonstrated by in situ hybridization and immunostaining that GPR142 is expressed not only in β cells but also in a subset of α cells. Stimulation of GPR142 by a selective agonist increased glucagon secretion in both human and mouse islets. More importantly, the GPR142 agonist also potentiated glucagon-like peptide-1 (GLP-1) production and its release from islets through a mechanism that involves upregulation of prohormone convertase 1/3 expression. Strikingly, stimulation of insulin secretion and increase in insulin content via GPR142 engagement requires intact GLP-1 receptor signaling. Furthermore, GPR142 agonist increased β cell proliferation and protected both mouse and human islets against stress-induced apoptosis.
Collectively, we provide here evidence that local GLP-1 release from α cells defines GPR142\'s beneficial effects on improving β cell function and mass, and we propose that GPR142 agonism may have translatable and durable efficacy for the treatment of type 2 diabetes.

Variants in Proprotein Convertase Subtilisin/Kexin Type 1 (PCSK1) may be causative for obesity as suggested by monogenic cases and association studies. Here we assessed the functional relevance in experimental studies and the clinical relevance through detailed metabolic phenotyping of newly identified and known PCSK1 variants in children.
In 52 obese children selected for elevated proinsulin levels and/or impaired glucose tolerance, we found eight known variants and two novel heterozygous variants (c.1095 + 1G > A and p.S24C) by sequencing the PCSK1 gene. Patients with the new variants presented with extreme obesity, impaired glucose tolerance, and PCOS. Functionally, c.1095 + 1G > A caused skipping of exon8 translation and a complete loss of enzymatic activity. The protein was retained within the endoplasmic reticulum (ER) causing ER stress. The p.S24C variant had no functional effect on protein size, cell trafficking, or enzymatic activity. The known variants rs6230, rs35753085, and rs725522 in the 5\' end did not affect PCSK1 promoter activity. In clinical association studies in 1673 lean and obese children, we confirmed associations of rs6232 and rs6234 with BMI-SDS and of rs725522 with glucose stimulated insulin secretion and Matsuda index. We did not find the new variants in any other subjects.
We identified and functionally characterized two rare novel PCSK1 variants of which c.1095 + 1G > A caused complete loss of protein function. In addition to confirming rs6232 and rs6234 in PCSK1 as polygenic risk variants for childhood obesity, we describe an association of rs725522 with insulin metabolism. Our results support the contribution of PCSK1 variants to obesity predisposition in children.

PCSK1, encoding prohormone convertase 1/3 (PC1/3), was one of the first genes linked to monogenic early-onset obesity. PC1/3 is a protease involved in the biosynthetic processing of a variety of neuropeptides and prohormones in endocrine tissues. PC1/3 activity is essential for the activating cleavage of many peptide hormone precursors implicated in the regulation of food ingestion, glucose homeostasis, and energy homeostasis, for example, proopiomelanocortin, proinsulin, proglucagon, and proghrelin. A large number of genome-wide association studies in a variety of different populations have now firmly established a link between three PCSK1 polymorphisms frequent in the population and increased risk of obesity. Human subjects with PC1/3 deficiency, a rare autosomal-recessive disorder caused by the presence of loss-of-function mutations in both alleles, are obese and display a complex set of endocrinopathies. Increasing numbers of genetic diagnoses of infants with persistent diarrhea has recently led to the finding of many novel PCSK1 mutations. PCSK1-deficient infants experience severe intestinal malabsorption during the first years of life, requiring controlled nutrition; these children then become hyperphagic, with associated obesity. The biochemical characterization of novel loss-of-function PCSK1 mutations has resulted in the discovery of new pathological mechanisms affecting the cell biology of the endocrine cell beyond simple loss of enzyme activity, for example, dominant-negative effects of certain mutants on wild-type PC1/3 protein, and activation of the cellular unfolded protein response by endoplasmic reticulum-retained mutants. A better understanding of these molecular and cellular pathologies may illuminate possible treatments for the complex endocrinopathy of PCSK1 deficiency, including obesity.