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The most common genetic cause of permanent neonatal diabetes mellitus is activating mutations in KCNJ11, which can usually be treated using oral sulfonylureas (SUs) instead of insulin injections, although some mutations are SU unresponsive. In this work, we provide a report of the pancreatic islet endocrine cell composition and area in a patient with an SU-unresponsive KCNJ11 mutation (p.G334D), in comparison with age-matched controls.Pancreatic autopsy tissue sections from a 2-year-old female child diagnosed with KCNJ11-related diabetes at 4 days of age and 13 age-matched controls were stained with insulin, glucagon, somatostatin, pancreatic polypeptide, and Ki67 antibodies to determine islet endocrine cell composition and area. β-cell ultrastructure was assessed by electron microscopic (EM) analysis. The patient's pancreas (sampling from head to tail) revealed insulin-positive cells in all regions. The pancreatic β-cell (insulin) area was significantly reduced compared with controls: 0.50% ± 0.04% versus 1.67% ± 0.20%, respectively (P0.00001). There were no significant differences in α-cell (glucagon) or δ-cell (somatostatin) area. EM analysis revealed secretory granules with a dense core typical of mature β-cells as well as granules with a lighter core characteristic of immature granules.Our results suggest that mechanisms exist that allow preservation of β-cells in the absence of insulin secretion. It remains to be determined to what extent this reduction in β-cells may be reversible.

Diabetes (T1DM and T2DM) is characterized by a deficit in β-cell mass. A broader understanding of human β-cell replication mechanism is thus important to increase β-cell proliferation for future therapeutic interventions. Here, we show that p27 (Kip1), a CDK inhibitor, is expressed abundantly in isolated adult human islets and interacts with various positive cell cycle regulatory proteins including D-type cyclins (D1, D2 and D3) and their kinase partners, CDK4 and CDK6. Also, we see interaction of cyclin E and its kinase partner, CDK2, with p27 suggesting a critical role of p27 as a negative cell cycle regulator in human islets. Our data demonstrate interaction of p27 with GSK-3 in β-cells and show, employing rodent β-cells (INS-1), isolated human islets and purified β-cells derived from human islets, that siRNA-mediated depletion of GSK-3 or p27 or 1-AKP / BIO - mediated GSK-3 inhibition results in increased β-cell proliferation. We also see reduction of p27 levels following GSK-3 inactivation or depletion. Our data show that serum induction of quiescent INS-1 cells leads to sequential phosphorylation of p27 on its S10 and T187 residues with faster kinetics for S10 corresponding with the decreased levels of p27. Altogether our findings indicate that p27 levels in β-cells are stabilized by GSK-3 and thus p27 down regulation following GSK-3 depletion / inactivation plays a critical role in promoting β-cell replication.

Pancreatic beta-cell response to glucose stimulation is governed by tightly regulated signaling pathways which have not been fully characterized. A screen for novel signaling intermediates identified Pim3 as a glucose-responsive gene in the beta-cell, and here, we characterize its role in the regulation of beta-cell function. Pim3 expression in the beta-cell was first observed through microarray analysis on glucose-stimulated murine insulinoma (MIN6) cells where expression was strongly and transiently induced. In the pancreas, Pim3 expression exhibited similar dynamics and was restricted to the beta-cell. Perturbation of Pim3 function resulted in enhanced glucose-stimulated insulin secretion, both in MIN6 cells and in isolated islets from Pim3(-/-) mice, where the augmentation was specifically seen in the second phase of secretion. Consequently, Pim3(-/-) mice displayed an increased glucose tolerance in vivo. Interestingly, Pim3(-/-) mice also exhibited increased insulin sensitivity. Glucose stimulation of isolated Pim3(-/-) islets resulted in increased phosphorylation of ERK1/2, a kinase involved in regulating beta-cell response to glucose. Pim3 was also found to physically interact with SOCS6 and SOCS6 levels were strongly reduced in Pim3(-/-) islets. Overexpression of SOCS6 inhibited glucose-induced ERK1/2 activation, strongly suggesting that Pim3 regulates ERK1/2 activity through SOCS6. These data reveal that Pim3 is a novel glucose-responsive gene in the beta-cell that negatively regulates insulin secretion by inhibiting the activation of ERK1/2, and through its effect on insulin sensitivity, has potentially a more global function in glucose homeostasis.

The islet of Langerhans is a highly vascularized micro-organ consisting of not only ß-cells but multiple cell types such as α-, delta-, pancreatic polypeptide- and epsilon-cells that work together to regulate glucose homeostatis. We have recently proposed a new model of the neonatal islet formation in mice by a process of fission following contiguous endocrine cell proliferation in the form of branched cord-like structures in embryos and newborns. There exist large stretches of interconnected islet structures along large blood vessels in the neonatal pancreas, which, upon further development, segregate into smaller fragments (i.e., islets) that eventually become more spherical by internal proliferation as seen in the adult pancreas. α-cells span these elongated islet-like structures in the developing pancreas, which we hypothesize represent sites of fission and facilitate the eventual formation of discrete islets. The α-cells express both prohormone convertase 2 and 1/3 (PC 2 and PC 1/3, respectively), which resulted in the processing of the proglucagon precursor into glucagon-like peptide 1, thereby leading to local production of this important ß-cell growth factor. Furthermore, while α-cells in the adult basically only express PC 2, significant activation of PC 1/3 is also observed in mouse models of insulin resistance such as pregnant, ob/ ob, db/db and prediabetic NOD mice, which may be a common mechanism in proliferating ß-cells. Our study suggests an important role of α-cells for ß-cell proliferation and further for the endocrine cell network within an islet.

The C-terminal segment of the human insulin receptor α-chain (designated αCT) is critical to insulin binding as has been previously demonstrated by alanine scanning mutagenesis and photo-cross-linking. To date no information regarding the structure of this segment within the receptor has been available. We employ here the technique of thermal-factor sharpening to enhance the interpretability of the electron-density maps associated with the earlier crystal structure of the human insulin receptor ectodomain. The αCT segment is now resolved as being engaged with the central β-sheet of the first leucine-rich repeat (L1) domain of the receptor. The segment is α-helical in conformation and extends 11 residues N-terminal of the classical αCT segment boundary originally defined by peptide mapping. This tandem structural element (αCT-L1) thus defines the intact primary insulin-binding surface of the apo -receptor. The structure, together with isothermal titration calorimetry data of mutant αCT peptides binding to an insulin minireceptor, leads to the conclusion that putative “insulin-mimetic” peptides in the literature act at least in part as mimics of the αCT segment as well as of insulin. Photo-cross-linking by novel bifunctional insulin derivatives demonstrates that the interaction of insulin with the αCT segment and the L1 domain occurs in trans , i.e., these components of the primary binding site are contributed by alternate α-chains within the insulin receptor homodimer. The tandem structural element defines a new target for the design of insulin agonists for the treatment of diabetes mellitus.

Prohormone convertase 2 (PC2) functions in the generation of neuropeptides from their precursors. A quantitative peptidomics approach was used to evaluate the role of PC2 in the processing of peptides in a variety of brain regions. Altogether, 115 neuropeptides or other peptides derived from secretory pathway proteins were identified. These peptides arise from 28 distinct secretory pathway proteins, including proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, and many others. Forty one of the peptides found in wild type mice were not detectable in any of the brain regions of PC2 knockout mice, and another twenty four peptides were present at levels ranging from 20–79% of wild type levels. Most of the other peptides were not substantially affected by the mutation, with levels ranging from 80–120% of wild type levels, and only three peptides were found to increase in one or more brain regions of PC2 knock-out mice. Taken together, these results are consistent with a broad role for PC2 in neuropeptide processing, but with functional redundancy for many of the cleavages. Comparison of the cleavage sites affected by the absence of PC2 confirms previous suggestions that sequences with a Trp, Tyr and/or Pro in the P1′ or P2′ position are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.

While diabetic patients often present with comorbid depression, the underlying mechanisms linking diabetes and depression are unknown. The Wistar Kyoto (WKY) rat is a well-known animal model of depression and stress hyperreactivity. In addition, the WKY rat is glucose intolerant and likely harbors diabetes susceptibility alleles. We conducted a quantitative trait loci (QTL) analysis in the segregating F(2) population of a WKY x Fischer 344 (F344) intercross. We previously published QTL analyses for depressive behavior and hypothalamic-pituitary-adrenal (HPA) activity in this cross. In this study we report results from the QTL analysis for multiple metabolic phenotypes, including fasting glucose, post-restraint stress glucose, postprandial glucose and insulin, and body weight. We identified multiple QTLs for each trait and many of the QTLs overlap with those previously identified using inbred models of type 2 diabetes (T2D). Significant correlations were found between metabolic traits and HPA axis measures, as well as forced swim test behavior. Several metabolic loci overlap with loci previously identified for HPA activity and forced swim behavior in this F(2) intercross, suggesting that the genetic mechanisms underlying these traits may be similar. These results indicate that WKY rats harbor diabetes susceptibility alleles and suggest that this strain may be useful for dissecting the underlying genetic mechanisms linking diabetes, HPA activity, and depression.

Using a focal cerebral ischemia model in rats, brain ischemia-induced changes in expression levels of mRNA and protein, and activities of proprotein convertase 2 (PC2) in the cortex were examined. In situ hybridization analyses revealed a transient upregulation of the mRNA level for PC2 at an early reperfusion hour, at which the level of PC2 protein was also high as determined by immunocytochemistry and western blotting. When enzymatic activities of PC2 were analyzed using a synthetic substrate, a significant decrease was observed at early reperfusion hours at which levels of PC2 protein were still high. Also decreased at these reperfusion hours were tissue levels of dynorphin-A(1-8) (DYN-A(1-8)), a PC2 substrate, as determined by radioimmunoassay. Further examination of PC2 protein biosynthesis by metabolic labeling in cultured neuronal cells showed that in ischemic cells, the proteolytic processing of PC2 was greatly attenuated. Finally, in mice, an intracerebroventricular administration of synthetic DYN-A(1-8) significantly reduced the extent of ischemic brain injury. In mice those lack an active PC2, exacerbated brain injury was observed after an otherwise non-lethal focal ischemia. We conclude that brain ischemia attenuates PC2 and PC2-mediated neuropeptide processing. This attenuation may play a role in the pathology of ischemic brain injury.

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and alpha-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg(36)Arg(37) and Arg(73)Arg(74) sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, alpha-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys(53)Lys(54)), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.

OBJECTIVE— Insulin gene (INS) mutations have recently been described as a cause of permanent neonatal diabetes (PND). We aimed to determine the prevalence, genetics, and clinical phenotype of INS mutations in large cohorts of patients with neonatal diabetes and permanent diabetes diagnosed in infancy, childhood, or adulthood. RESEARCH DESIGN AND METHODS— The INS gene was sequenced in 285 patients with diabetes diagnosed before 2 years of age, 296 probands with maturity-onset diabetes of the young (MODY), and 463 patients with young-onset type 2 diabetes (nonobese, diagnosed RESULTS— We identified heterozygous INS mutations in 33 of 141 probands diagnosed at CONCLUSIONS— We conclude that INS mutations are the second most common cause of PND and a rare cause of MODY. Insulin gene mutation screening is recommended for all diabetic patients diagnosed before 1 year of age.

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val(A3), which projects within a crevice between the A- and B-chains. Engineered receptor alpha-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a Pap(B25)-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and alpha-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (Gly(A3) and Ser(A3)) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of Aba(A3) and allo-Thr(A3) analogs are reduced at least 10-fold; the activity of Thr(A3)-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (Aba(A3)) or by altered polarity (allo-Thr(A3) and Thr(A3)). Our results provide evidence that Val(A3), a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the alpha-subunit in a hormone-receptor complex.

We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo . Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with β cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially β cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the Ins2 gene, leading to loss of β cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder.

The insulin receptor is a homodimer composed of two alphabeta half receptors. Scanning mutagenesis studies have identified key residues important for insulin binding in the L1 domain (amino acids 1-150) and C-terminal region (amino acids 704-719) of the alpha subunit. However, it has not been shown whether insulin interacts with these two sites within the same alpha chain or whether it cross-links a site from each alpha subunit in the dimer to achieve high affinity binding. Here we have tested the contralateral binding mechanism by analyzing truncated insulin receptor dimers (midi-hIRs) that contain complementary mutations in each alpha subunit. Midi-hIRs containing Ala(14), Ala(64), or Gly(714) mutations were fused with Myc or FLAG epitopes at the C terminus and were expressed separately by transient transfection. Immunoblots showed that R14A+FLAG, F64A+FLAG, and F714G+Myc mutant midi-hIRs were expressed in the medium but insulin binding activity was not detected. However, after co-transfection with R14A+FLAG/F714G+Myc or F64A+FLAG/F714G+Myc, hybrid dimers were obtained with a marked increase in insulin binding activity. Competitive displacement assays revealed that the hybrid mutant receptors bound insulin with the same affinity as wild type and also displayed curvilinear Scatchard plots. In addition, when hybrid mutant midi-hIR was covalently cross-linked with (125)I(A14)-insulin and reduced, radiolabeled monomer was immunoprecipitated only with anti-FLAG, demonstrating that insulin was bound asymmetrically. These results demonstrate that a single insulin molecule can contact both alpha subunits in the insulin receptor dimer during high affinity binding and this property may be an important feature for receptor signaling.

Although it is clear that β-cell pathology, in particular disordered insulin secretion, is a key underlying pathogenetic feature of most forms of diabetes, complex interactions of the islets with other organs, such as brain, liver, gut, and several peripheral tissues, are also essential for the normal integration of metabolism. It is this area that has been explored in the Seventh Annual Servier-IGIS Meeting, which was held last Spring in St. Jean Cap Ferrat in Southern France. The focus of these meetings, since their inception in 2000, has been the β-cell and the mechanisms underlying its development and function as the source of insulin, the most essential regulator of the blood glucose level. The familiar pathways of glucose, lipid, and amino acid metabolism in humans and other mammals are, of course, fundamental to almost all organisms, except perhaps for the most highly specialized bacteria, so it is not surprising that insulin-like molecules and the insulin/IGF receptor signaling system are well conserved features of all metazoans that have been studied. With the rise of multicellular organisms in evolution came the need to regulate and coordinate metabolism and growth in order to maintain both the constancy of the internal environment (homeostasis) and also to respond to the external environment. One of the most prominent environmental stimuli had to be the availability of nutrients and fuels for survival and growth. The insulin-like hormones, insulin and IGF, appear to have evolved, along with a panoply of other regulatory substances, to fine-tune the efficient uptake, storage, and utilization of nutrients for either energy production or growth. Appropriately, the symposium opened with an introductory lecture by Leopold, reviewing our current knowledge of the control of metabolism and growth in the fruit fly, Drosophila melanogaster , by insulin-like peptides and an insulin signaling pathway that is remarkably similar …

The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor. This study investigates the processing of proGIP in PC1/3 and PC2 null mice and in cell lines using adenovirus-mediated overexpression. Supporting a role for PC1/3 in proGIP processing, we found co-localization of GIP and PC1/3 but not PC2 in intestinal sections by immunohistochemistry, and analysis of intestinal extracts from PC1/3-deficient animals demonstrated severely impaired processing to GIP, whereas processing to GIP was unaltered in PC2-deficient mice. Accordingly, overexpression of preproGIP in the neuroendocrine AtT-20 cell line that expresses high levels of endogenous PC1/3 and negligible levels of PC2 resulted in production of GIP. Similar results were obtained after co-expression of preproGIP and PC1/3 in GH4 cells that express no PC2 and only low levels of PC1/3. In addition, studies in GH4 cells and the alpha-TC1.9 cell line, expressing PC2 but not PC1/3, indicate that PC2 can mediate processing to GIP but also to other fragments not found in intestinal extracts. Taken together, our data indicate that PC1/3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells.

Pro-vasopressin and pro-oxytocin are prohormones processed in the neurointermediate lobe pituitary to form the biologically active peptide hormones, arginine vasopressin (AVP) and oxytocin. Neurointermediate lobe pituitaries from normal (+/+), heterozygous (+/−), PC2-Null (−/−), PC1/3-Null and oxytocin-Null mice were analyzed by SELDI-TOF mass spectroscopy for the peptide hormone products, AVP, oxytocin and neurophysin I and II. Molecular ion species with masses characteristic of oxytocin, AVP, neurophysin I and II, i.e. 1009.41, 1084.5, 9677 and 9679 daltons respectively, were identified in all but the oxytocin-Null mice by comparison with synthetic standards or by C-terminal sequence analysis. Other ion species were found specifically in PC2-Null, heterozygote or normal mice. The results indicate that, in mice, both PC1/3 or PC2 enzyme activity are capable, but not required to correctly process pro-vasopressin or pro-oxytocin to their constituent active peptide hormones.

Five years already! We, the members of the International Group on Insulin Secretion (IGIS), are very happy that the Servier-IGIS Symposium on insulin secretion and islet pathophysiology, which we first organized in 2000, has, beyond our expectations, become a traditional, awaited, and much appraised event. The pleasant venue and climate of the French Riviera in early spring undoubtedly contribute to the attractiveness of the symposium. However, we trust that the major reason for its success is the high quality of the presentations and scientific exchanges. These standards extend to the rapid publication of most contributions as refereed and edited review or original articles in a series of supplements to Diabetes (1–4). Both the symposium and the publication are made possible by a generous, unrestricted educational grant from Les Laboratoires Servier (Paris). This fifth edition focused on therapeutic approaches of type 2 diabetes and their impact on islet function. Although their hypoglycemic properties had already been recognized in 1942, sulfonamides (carbutamide) started to be used in the treatment of type 2 diabetes in 1955 (5). If millions of diabetic patients have successfully been treated with sulfonylureas for 50 years, it is because these compounds luckily hit a key regulatory site of insulin secretion, the ATP-sensitive K+ channel (KATP channel) that was to be discovered about 30 years later (6,7) and whose structure was identified only 10 years ago (8,9). Several sessions of the symposium were dedicated to this target, its role in various tissues, and the perspective of designing novel drugs with high selectivity. Alternative ways to stimulate deficient insulin secretion were discussed as well as the impact that treatment, whether directly targeting β-cells or not, may have on islet function and progression of diabetes. Whenever possible and relevant, the contributors bridged our …

Confocal imaging of GFP-tagged secretory granules combined with the use of impermeant extracellular dyes permits direct observation of insulin packaged in secretory granules, trafficking of these granules to the plasma membrane, exocytotic fusion of granules with the plasma membrane, and eventually the retrieval of membranes by endocytosis. Most such studies have been done in tumor cell lines, using either confocal methods or total internal reflectance microscopy. Here we compared these methods by using GFP–syncollin or PC3–GFP plus rhodamine dextrans to study insulin granule dynamics in insulinoma cells, normal mouse islets, and primary pancreatic beta cells. We found that most apparently docked granules did not fuse with the plasma membrane after stimulation. Granules that did fuse typically fused completely, but a few dextran-filled granules lingered at the membrane. Direct recycling of granules occurred only rarely. Similar results were obtained with both confocal and total internal reflection microscopy, although each technique had advantages for particular aspects of the granule life cycle. We conclude that insulin exocytosis involves a prolonged interaction of secretory granules with the plasma membrane, and that the majority of exocytotic events occur by full, not partial, fusion.

We investigated the proteolytic processing of mouse pro-GHRH [84 amino acids (aa)] by furin, PC1/3, PC2, and PC5/6A. We created six point mutations in the N- and C-terminal cleavage sites, RXXR decreased and RXRXXR decreased, respectively. The following results were obtained after transient transfection/cotransfection and metabolic pulse-chase labeling studies in several neuroendocrine cells. 1) Furin was the most efficient convertase in cleaving the N-terminal RXXR/RXRR site to generate intermediate I, 12-84aa, whereas PC1/3 was the most potent in processing the C-terminal RXRXXR site to yield mature GHRH, 12-53aa. 2) Both PC1/3 and PC5/6A also processed the N-terminal site but less efficiently than furin. 3) PC2 was much weaker in cleaving the C-terminal site relative to PC1/3 to generate mature GHRH. 4) The Q10R mutant was significantly more susceptible to furin cleavage at the N-terminal site than the wild-type pro-GHRH. And 5) the N- and C-terminal P1 Arg residues, R11 and R54, respectively, were essential for mature GHRH production. We also showed localization of the GHRH immunoreactive peptides in Golgi and secretory granules in neuroendocrine cells by an immunofluorescence assay. We conclude that the efficient production of mature GHRH from pro-GHRH is a stepwise process mediated predominantly by furin at the N-terminal cleavage site followed by PC1/3 at the C terminus.

The neuroendocrine prohormone convertases 1 and 2 (PC1 and PC2) are expressed in endocrine intestinal L cells and pancreatic A cells, respectively, and colocalize with proglucagon in secretory granules. Mice lacking PC2 have multiple endocrinopathies and cannot process proglucagon to mature glucagon in the pancreas. Disruption of PC1 results in dwarfism and also multiple neuroendocrine peptide processing defects. This study compares the pancreatic and intestinal processing of proglucagon in mice lacking PC1 expression with that in age-matched wild-type controls. Because proglucagon was found to precipitate in acidic extracts, the intestinal processing profile was analyzed in both acidic and neutral extracts by gel filtration, HPLC, and RIA. Supporting a central role for PC2 in glucagon biosynthesis, we found normal processing of proglucagon to glucagon in the pancreas, whereas the intestinal proglucagon processing showed marked defects. Tissue proglucagon levels in null mice were elevated, and proglucagon processing to glicentin, oxyntomodulin, and glucagon-like peptide-1 and -2 (GLP-1 and GLP-2) was markedly decreased, indicating that PC1 is essential for the processing of all the intestinal proglucagon cleavage sites. This includes the monobasic site R(77) and, thereby, production of mature, biologically active GLP-1. We also found elevated glucagon levels, suggesting that factors other than PC1 that are capable of processing to mature glucagon are present in the secretory granules of the L cell. These findings strongly suggest that PC1 is essential for intestinal proglucagon processing in vivo and, thereby, plays an important role in production of the incretin hormone GLP-1 and the intestinotrophic hormone GLP-2.

The subtilisin-like prohormone convertases (PCs) contain an essential downstream domain (P domain), which has been predicted to have a β-barrel structure that interacts with and stabilizes the catalytic domain (CAT). To assess possible sites of hydrophobic interaction, a series of mutant PC3–enhanced GFP constructs were prepared in which selected nonpolar residues on the surface of CAT were substituted by the corresponding polar residues in subtilisin Carlsberg. To investigate the folding potential of the isolated P domain, signal peptide–P domain–enhanced GFP constructs with mutated and/or truncated P domains were also made. All mutants were expressed in βTC3 cells, and their subcellular localization and secretion were determined. The mutants fell into three main groups: ( i ) Golgi/secreted, ( ii ) ER/nonsecreted, and ( iii ) apoptosis inducing. The destabilizing CAT mutations indicate that the side chains of V292, T328, L351, Q408, H409, V412, and F441 and nonpolar fragments of the side chains of R405 and W413 form a hydrophobic patch on CAT that interacts with the P domain. We also have found that the P domain can fold independently, as indicated by its secretion. Interestingly, T594, which is near the P domain C terminus, was not essential for P domain secretion but is crucial for the stability of intact PC3. T594V produced a stable enzyme, but T594D did not, which suggests that T594 participates in important hydrophobic interactions within PC3. These findings support our conclusion that the catalytic and P domains contribute to the folding and thermodynamic stability of the convertases through reciprocal hydrophobic interactions.

The subtilisin-like proprotein convertases PC1/3 (SPC3) and PC2 (SPC2) are believed to be the major endoproteolytic processing enzymes of the regulated secretory pathway. They are expressed together or separately in neuroendocrine cells throughout the brain and dispersed endocrine system in both vertebrates and invertebrates. Disruption of the gene-encoding mouse PC1/3 has now been accomplished and results in a syndrome of severe postnatal growth impairment and multiple defects in processing many hormone precursors, including hypothalamic growth hormone-releasing hormone (GHRH), pituitary proopiomelanocortin to adrenocorticotropic hormone, islet proinsulin to insulin and intestinal proglucagon to glucagon-like peptide-1 and -2. Mice lacking PC1/3 are normal at birth, but fail to grow normally and are about 60% of normal size at 10 weeks. They lack mature GHRH, have low pituitary growth hormone (GH) and hepatic insulin-like growth factor-1 mRNA levels and resemble phenotypically the “little” mouse (Gaylinn, B. D., Dealmeida, V. I., Lyons, C. E., Jr., Wu, K. C., Mayo, K. E. & Thorner, M. O. (1999) Endocrinology 140, 5066–5074) that has a mutant GHRH receptor. Despite a severe defect in pituitary proopiomelanocortin processing to mature adrenocorticotropic hormone, blood corticosterone levels are essentially normal. There is marked hyperproinsulinemia but without impairment of glucose tolerance. In contrast, PC2-null mice lack mature glucagon and are chronically hypoglycemic (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H. & Steiner, D. (1997) Proc. Natl. Acad. Sci. USA 94, 6646–6651). The PC1/3-null mice differ from a human subject reported with compound heterozygosity for defects in this gene, who was of normal stature but markedly obese from early life. The PC1/3-null mice are not obese. The basis for these phenotypic differences is an interesting topic for further study. These findings prove the importance of PC1/3 as a key neuroendocrine convertase.

The neuroendocrine processing endoproteases PC2 and PC1/3 are expressed in the beta cells of the islets of Langerhans and participate in the processing of proinsulin to insulin and C-peptide. We have previously shown that disruption of PC2 (SPC2) expression significantly impairs proinsulin processing. Here we report that disruption of the expression of PC1/3 (SPC3) produces a much more severe block in proinsulin conversion. In nulls, pancreatic and circulating proinsulin-like components comprise 87% and 91%, respectively, of total insulin-related immunoreactivity. Heterozygotes also show a more than 2-fold elevation in proinsulin levels to approximately 12%. Immunocytochemical and ultrastructural studies of the beta cells reveal the nearly complete absence of mature insulin immunoreactivity and its replacement by that of proinsulin in abundant immature-appearing secretory granules. In contrast, alpha cell morphology and glucagon processing are normal, and there is also no defect in somatostatin-14 generation. Pulse-chase labeling studies confirm the existence of a major block in proinsulin processing in PC1/3 nulls with prolongation of half-times of conversion by 7- and 10-fold for proinsulins I and II, respectively. Lack of PC1/3 also results in increased levels of des-64,65 proinsulin intermediates generated by PC2, in contrast to PC2 nulls, in which des- 31,32 proinsulin intermediates predominate. These results confirm that PC1/3 plays a major role in processing proinsulin, but that its coordinated action with PC2 is necessary for the most efficient and complete processing of this prohormone.

Prohormone convertase 2 (PC2) plays an essential role in the processing of proglucagon to mature active glucagon in pancreatic alpha-cells (J Biol Chem 276:27197-27202, 2001). Mice lacking PC2 demonstrate multiple defects, including chronic mild hypoglycemia and dramatic hyperplasia of the pancreatic alpha-cells. To define the contribution of mature glucagon deficiency to the hypoglycemia and alpha-cell hyperplasia, we have attempted to correct the defects by delivery of exogenous glucagon by micro-osmotic pumps. Intraperitoneal delivery of 0.5 microg glucagon/h in PC2(-/-) mice resulted in the normalization of blood glucose concentrations. Islet remodeling through the loss of hyperplastic alpha-cells was evident by day 11 after pump implantation; by 25 days postimplantation, PC2(-/-) islets were indistinguishable from wild-type islets. These rapid changes were brought about by induction of apoptosis in the alpha-cell population. Morphological normalization of islets was also accompanied by marked downregulation of endogenous preproglucagon gene expression, but with little or no change in the level of preproinsulin gene expression. Exogenous glucagon delivery also normalized hepatic expression of the gluconeogenic enzyme PEPCK. These results demonstrate that the lack of mature glucagon in PC2(-/-) mice is responsible for the aberrant blood glucose levels, islet morphology, and gene expression, and they confirm the role of glucagon as a tonic insulin antagonist in regulating glycemia.

Erratum to: Rev Endocr Metab Disord (2010) 11:205-215 DOI 10.1007/s11154-010-9151-3 The following INS gene mutation was numbered incorrectly: In Figure 1, Figure 2, and section 6.1, we refer to the mutation V93L. This should have read as V92L. This does not affect the numbering of the other mutations, which remain correct. The corrected versions of Figures 1 and ​and22 are presented below. We apologize for this error. Fig. 1 Diagrammatic representation of the amino acid sequence of human preproinsulin (signal peptide–green, B-chain–red, C-peptide–orange, A-chain–dark blue) indicating sites of mutations identified in patients with diabetes as ... Fig. 2 Summary of human insulin gene mutations and disease phenotype. The numbers in brackets indicate the number of probands with that specific mutation. PNDM, permanent neonatal diabetes mellitus; TNDM, transient neonatal diabetes mellitus; MODY, maturity-onset ...

Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe(B24) to a 60° rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor's L1-β2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile(A2), Val(A3), Val(B12), Phe(B24), and Phe(B25)) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.

Mice homozygous for a deletion in the gene encoding prohormone convertase 2 (PC2) are generally healthy but have mild hypoglycemia and flat glucose-tolerance curves. Their islets show marked alpha (A)-cell hyperplasia, suggesting a possible defect in glucagon processing (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646–6651). In this report we have examined the biosynthesis and processing of proglucagon in isolated islets from these mice via pulse-chase labeling and find that proglucagon undergoes essentially no processing in chase periods up to 8 h in duration. Only a small percent of cleavage at the sensitive interdomain site (residues 71 and 72) appears to occur. These observations thus conclusively demonstrate the essentiality of PC2 for the production of glucagon in the islet A-cells. Ultrastructural and immunocytochemical studies indicate the presence of large amounts of proglucagon in atypical appearing secretory granules in the hyperplastic and hypertrophic A-cells, along with morphological evidence of high rates of proglucagon secretion in PC2 null islets. These findings provide strong evidence that active glucagon is required to maintain normal blood glucose levels, counterbalancing the action of insulin at all times.

Several mouse strains expressing human islet amyloid polypeptide (IAPP) have been created to study development of islet amyloid and its impact on islet cell function. The tendency to form islet amyloid has varied strongly among these strains by factors that have not been elucidated. Because some beta cell granule components are known to inhibit IAPP fibril formation in vitro, we wanted to determine whether a mouse strain expressing human IAPP but lacking the nonamyloidogenic mouse IAPP is more prone to develop islet amyloidosis. Such a strain was created by cross-breeding a transgenic mouse strain and an IAPP null mouse strain. When fed a fat-enriched diet, male mice expressing only human IAPP developed islet amyloid earlier and to a higher extent than did mice expressing both human and mouse IAPP. Supporting these results, we found that mouse IAPP dose-dependently inhibits formation of fibrils from human IAPP. Female mice did not develop amyloid deposits, although small extracellular amorphous IAPP deposits were found in some islets. When cultivated in vitro, amyloid deposits occurred within 10 days in islets from either male or female mice expressing only human IAPP. The study shows that formation of islet amyloid may be dependent on the environment, including the presence or absence of fibril inhibitors or promoters.

Improperly folded membrane proteins are retained in the endoplasmic reticulum and then diverted to a degradative pathway by a network of molecular chaperones and intracellular proteases. Here we report that mutant insulin proreceptors (Pro 62 ) retained in the early secretory pathway undergo proteolytic cleavage at a tetrabasic concensus site for the subtilisin-like protease furin (SPC 1), generating two unstable proteolytic intermediates of 80/120 kDa corresponding to α (135 kDa) and β (90 kDa) subunits. These are degraded more rapidly than the uncleaved proreceptor protein. Site-directed mutagenesis of the normal RKRR processing site prevented cleavage. Use of inhibitors and furin-deficient cell lines confirmed that furin is responsible for proreceptor cleavage; furin overexpression increased the degradation of mutant but not wild-type receptors. Together, these results suggest that processing and degradation occur sequentially for mutant proreceptors.

Pancreatic beta cells respond to changes in blood glucose by secreting insulin and increasing insulin synthesis. To identify genes used in these responses, we have carried out expression profiling of beta cells exposed to high (25 mM) or low (5.5 mM) glucose by using oligonucleotide microarrays. Functional clustering of genes that averaged a 2.2-fold or greater change revealed large groups of secretory pathway components, enzymes of intermediary metabolism, cell-signaling components, and transcription factors. Many secretory pathway genes were up-regulated in high glucose, including seven members of the endoplasmic reticulum (ER) translocon. In agreement with array analysis, protein levels of translocon components were increased by high glucose. Most dramatically, the alpha subunit of the signal recognition particle receptor was increased over 20-fold. These data indicate that the translocon and ribosome docking are major regulatory targets of glucose in the beta cell. Analysis of genes encoding enzymes of intermediary metabolism indicated that low glucose brought about greater utilization of amino acids as an energy source. This conclusion was supported by observations of increased urea production under low-glucose conditions. The above results demonstrate genome-wide integration of beta-cell functions at the level of transcript abundance and validate the efficacy of expression profiling in identifying genes involved in the beta-cell glucose response.

Insulin was discovered in 1922 as the causative factor for a human metabolic disorder (diabetes mellitus), but it was recognized early that the hormone had a broad phylogenetic distribution. By the mid 1970s, insulin had been isolated and sequenced from all classes of vertebrates, including Agnatha. Also it was discovered that the insulin gene family in vertebrates included two closely related hormones named insulin-like growth factor (IGF)-I and -II. More recently, the application of recombinant DNA techniques have identified insulin-like peptide genes in invertebrates, including insects, molluscs and nematode and these findings clearly establish that insulin is an evolutionarily ancient hormone which is present in all metazoa. Here we briefly review the structure and function of the insulin/IGF gene family in vertebrates and invertebrates. Although these studies are ongoing, it appears that in invertebrates the insulin-like peptides function predominately as mitogenic growth factors that act to promote tissue growth and development. However, in vertebrates the mitogenic growth function has been subsumed by IGF-I and -II while insulin has acquired the function of being primarily a metabolic regulatory hormone. The gene duplication and divergence events necessary for this development probably occurred early during vertebrate evolution in the transition from protochordates, represented by extant amphioxus, to primitive jawless vertebrates, represented by extant lamprey and hagfish.

In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. In this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched (IQ) peptides, for methylcoumarin amide-based fluorogenic peptides, and for recombinant rat proenkephalin. IQ peptides exhibited specificity constants (kcat/Km) between 9.4 x 10(4) M-1 s-1 (Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp+ ++; where Abz is ortho-aminobenzoic acid and EDDnp is N-(2, 4-dinitrophenyl)ethylenediamine)) and 0.24 x 10(4) M-1 s-1 (Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino acids were hydrolyzed with specificity constants ranging between 2.0 x 10(3) M-1 s-1 (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 x 10(4) M-1 s-1 (

Insulin-like growth factor-2 (IGF-2) is expressed in most embryonic tissues and is required for normal development during gestation. After birth IGF-2 expression is extinguished in most tissues, but the gene is often reactivated during tumorigenesis. Tumors secrete high molecular weight forms of IGF-2 that result from aberrant post-translational processing of pro-IGF-2. As a first step toward understanding how high molecular weight IGF-2 peptides might contribute to tumor progression, we have characterized the biosynthesis of IGF-2 in a human embryonic cell line. We have found that pro-IGF-2 can initially form two disulfide isomers that undergo rearrangement to a single conformation in vivo. The addition of N-acetylgalactosamine to Ser71, Thr72, Thr75, and Thr139 likely occurs in the cis- Golgi apparatus. Sialic acid addition begins in the trans- Golgi apparatus, but IGF-2 peptides must reach the trans-Golgi network for oligosaccharide maturation to be completed. Endoproteolysis occurs concomitant to or slightly after oligosaccharide maturation. Cleavage was observed only at Arg104, resulting in the secretion of IGF-2-(1-104) and free E-peptide. Proteolysis required basic residues in the P1 (Arg104) and P4 (Arg101) positions, was completely blocked by a furin inhibitor, and was enhanced by coexpression with furin, PACE4, PC6A, PC6B, and LPC. These data suggest that members of the subtilisin-related proprotein convertase family mediate processing of pro-IGF-2 at Arg104. We did not detect the IGF-2 peptides that are most abundant in normal serum, mature IGF-2, and IGF-2-(1-87), in this expression system, which indicates that novel endoproteases are responsible for generating these products.

The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of ≈150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded β-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.

A unique feature of the eukaryotic subtilisin-like proprotein convertases (SPCs) is the presence of an additional highly conserved sequence of approximately 150 residues (P domain) located immediately downstream of the catalytic domain. To study the function of this region, which is required for the production of enzymatically active convertases, we have expressed and characterized various P domain-related mutants and chimeras in HEK293 cells and alpha-TC1-6 cells. In a series of C-terminal truncations of PC3 (also known as PC1 or SPC3), PC3-Thr594 was identified as the shortest active form, thereby defining the functional C-terminal boundary of the P domain. Substitutions at Thr594 and nearby sites indicated that residues 592-594 are crucial for activity. Chimeric SPC proteins with interchanged P domains demonstrated dramatic changes in several properties. Compared with truncated wild-type PC3 (PC3-Asp616), both PC3/PC2Pd and PC3/FurPd had elevated activity on several synthetic substrates as well as reduced calcium ion dependence, whereas Fur/PC2Pd was only slightly decreased in activity as compared with truncated furin (Fur-Glu583). Of the three active SPC chimeras tested, all had more alkaline pH optima. When PC3/PC2Pd was expressed in alpha-TC1-6 cells, it accelerated the processing of proglucagon into glicentin and major proglucagon fragment and cleaved major proglucagon fragment to release GLP-1 and tGLP-1, similar to wild-type PC3. Thus, P domain exchanges generated fully active chimeric proteases in several instances but not in all (e.g. PC2/PC3Pd was inactive). The observed property changes indicate a role for the P domain in regulating the stability, calcium dependence, and pH dependence of the convertases.

The prohormone convertases PC2 (SPC2) and PC3/PC1 (SPC3) are the major precursor processing endoproteases in a wide variety of neural and endocrine tissues. Both enzymes are normally expressed in the islet beta cells and participate in proinsulin processing. Recently we generated mice lacking active PC2 due to a disruption of the PC2 gene (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J. J., Carroll, R. J., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646-6651). Here we report that these PC2 mutant mice have elevated circulating proinsulin, comprising 60% of immunoreactive insulin-like components. Acid ethanol extractable proinsulin from pancreas is also significantly elevated, representing about 35% of total immunoreactive insulin-like components. These increased amounts of proinsulin are mainly stored in secretory granules, giving rise to an altered appearance on electron microscopy. In pulse-chase experiments, the mutant islets incorporate lesser amounts of isotopic amino acids into insulin-related components than normal islets. In both wild-type and mutant islets, proinsulin I was processed more rapidly to insulin, reflecting the preference of both PC2 and PC3 for substrates having a basic amino acid positioned four residues upstream of the cleavage site. The overall half-time for the conversion of proinsulin to insulin is increased approximately 3-fold in the mutant islets and is associated with a 4-5-fold greater elevation of des-31,32 proinsulin, an intermediate that is formed by the preferential cleavage of proinsulin at the B chain-C-peptide junction by PC3 and is C-terminally processed to remove Arg31 and Arg32 by carboxypeptidase E. The constitutive release of newly synthesized proinsulin from both mutant and wild-type islets during the first 1-2 h of chase was normal (

Insulin-like growth factor (IGF) 1 receptor (IGF1R)-mediated signaling plays key roles in growth, development, and physiology. Recent studies have shown that there are two distinct ig f1r genes in zebrafish, termed ig f1ra and ig f1rb. In this study, we tested the hypothesis that zebrafish ig f1ra and ig f1rb resulted from a gene duplication event at the ig f1r locus and that this has led to their functional divergence. The genomic structures of zebrafish ig f1ra and ig f1rb were determined and their loci mapped. While zebrafish ig f1ra has 21 exons and is located on linkage group (LG) 18, zebrafish ig f1rb has 22 exons and mapped to LG 7. There is a strong syntenic relationship between the two zebrafish genes and the human IG F1R gene. Using a MO-based loss-of-function approach, we show that both Igf1ra and Igf1rb are required for zebrafish embryo viability and proper growth and development. Although Igf1ra and Igf1rb demonstrated a large degree of functional overlap with regard to cell differentiation in the developing eye, inner ear, heart, and muscle, they also exhibited functional distinction involving a greater requirement for Igf1rb in spontaneous muscle contractility. These findings suggest that the duplicated zebrafish ig f1r genes play largely overlapping but not identical functional roles in early development and provide novel insight into the functional evolution of the IGF1R/insulin receptor gene family.

A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene in Cpe(fat)/Cpe(fat) mice affects proinsulin processing. Cell lines derived from the pancreatic beta-cells of Cpe(fat)/Cpe(fat) mice were generated by crossing C57BLKS/J-Cpe(fat)/+ mice with NOD mice expressing the simian virus 40 large T oncogene under the control of the rat insulin II promoter. Two cell lines, designated NIT-2 and NIT-3, were cultured from adenomatous islets obtained from F2 littermates and were compared with the NIT-1 cell line previously developed from mice with wild-type CPE. Electron microscopy of the cultured NIT-2 and -3 cells showed increased numbers of enlarged and electron-lucent granules compared with NIT-1 cells. Pro-CPE, but not the mature form of CPE, is present in NIT-2 and -3 cells, and neither pro-CPE nor CPE are secreted into the medium. Immunocytochemistry shows the pro-CPE to be localized to an endoplasmic reticulum-like structure in NIT-3 cells. Proinsulin is less extensively processed in NIT-2 and -3 cells than in NIT-1 cells, indicating that the Cpe(fat) mutation affects both the endopeptidase and carboxypeptidase reactions. The secretion of insulin/proinsulin from NIT-2 and -3 cells is significantly elevated by secretagogues, indicating that CPE is not required for sorting proinsulin into the regulated pathway.

Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B-chain it appears to be essential for the shift from the alpha-helix B8-B19 to the beta-turn B20-B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.

Circulating monocytes in human peripheral blood are readily available, easily obtained, and can be cultured in vitro. Once plated, the monocytes spontaneously differentiate into macrophages. Undifferentiated human monocytes do not express carboxypeptidaseE (CPE), prohormone convertase3 (PC3/PC1) or proenkephalin (ENK), suggesting that gene induction during differentiation results in the expression of these genes. RT-PCR of human monocyte-derived macrophage (HMDM) mRNA showed detectable levels of ENK mRNA at 48 h after plating, followed by PC3 and CPE mRNAs at 72 h. PC3 expression was confirmed by Western blotting in THP-1 cells. Similarities in expression of enzymes involved in the conversion of neuroendocrine precursors, such as proenkephalin, into functionally mature peptides underscores the close association HMDMs may share with the neuroendocrine system. Because of this, HMDMs may prove to be a valuable, non-surgical source of human tissue for clinical genetic diagnosis of some convertase disorders.© 1997 Federation of European Biochemical Societies.

Insulin-like growth factor I (IGF-I) is required for normal embryonic development and postnatal growth. Like most hormones and growth factors, IGF-I is synthesized as a proprotein that is converted to the mature form by endoproteolysis. Processing of pro-IGF-I to mature IGF-I occurs by cleavage within the unique pentabasic processing motif Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg77. We have previously shown that human embryonic kidney 293 cells process pro-IGF-IA at Arg71 to generate IGF-I-(1-70) and at Arg77 to produce IGF-I-(1-76). Cleavage at each of these sites requires upstream basic residues, indicating that subtilisin-related proprotein convertases (SPCs) may be involved. In order to investigate the identity of the endogenous enzymes involved in maturation of pro-IGF-IA, we have expressed wild-type and mutant pro-IGF-IA in 293 cells and in the furin-deficient Chinese hamster ovary cell line, RPE.40. We have also co-expressed these constructs with SPCs that are thought to play a role in processing precursor proteins in the constitutive pathway: furin, PACE4, PC6A, PC6B, and LPC. The results show that furin is most active at cleaving wild-type and mutant pro-IGF-IA and can cleave these precursors at multiple sites within the pentabasic motif. PC6A and LPC are less active than furin but cleave only at Arg71. PACE4 and PC6B have very little activity on pro-IGF-IA precursors. Wild-type pro-IGF-IA was correctly processed to mature IGF-I in 10 of 10 cell lines that were tested. Since furin, PC6A, and LPC are known to have a broad pattern of tissue distribution and we have demonstrated expression of LPC in RPE.40 cells, our results suggest that these SPCs may be responsible for the endogenous pro-IGF-IA processing activity observed in a wide variety of cell lines.

Insulin secretion and β-cell biology are the main theme of the Servier-IGIS Meetings that have been held yearly since 2000 in St. Jean Cap Ferrat in southern France (1–5). Previous symposia have mostly focused on insulin secretion and its relationship with β-cell defects in type 2 diabetes. Type 1 diabetes has not previously been considered, since it is generally viewed exclusively as an autoimmune disease. However, the pathophysiology of either disease is centered on β-cells, and type 1 and type 2 diabetes share many concerns, especially the goal of preserving or restoring a normal functional β-cell mass. Evidence has accumulated that immune tolerance reflects a permanent cross talk between the different cells involved in immune survey functions and peripheral tissues, as in the case of β-cells in type 1 diabetes. As in most common autoimmune diseases, the problem of the antigenic specificity of the autoimmune reaction that drives the β-cell damage has been one with most unpredictable issues over the past 20 years. Whether the activation of autoimmunity proceeds from intrinsic immune dysregulation or requires the presence of β-cells is now no longer a question. Against all predictions, the search for the “diabetes autoantigen” has resulted in a long list of candidates with rather loose identification criteria. All presently high-ranking β-cell autoantigens are defined as proteins expressed by β-cells, not as β-cell–specific antigens. Indeed, evidence has accumulated to indicate that B- and T-cells recognize many autoantigens, rather than a single autoantigen, during diabetes development and that most autoantigens are not β-cell specific. This makes type 1 diabetes a β-cell disease rather than an antigen-specific immune disease. As for type 2 diabetes, there is overwhelming evidence that it cannot be considered exclusively from the angle of the β-cell. β-Cells are at the center of multiple physiological loops that send …

Insulin is a major autoantigen in islet autoimmunity and progression to type 1 diabetes. It has been suggested that the insulin B-chain may be critical to insulin autoimmunity in type 1 diabetes. INS-IGF2 consists of the preproinsulin signal peptide, the insulin B-chain, and eight amino acids of the C-peptide in addition to 138 amino acids from the IGF2 gene. We aimed to determine the expression of INS-IGF2 in human pancreatic islets and autoantibodies in newly diagnosed children with type 1 diabetes and controls. INS-IGF2, expressed primarily in beta cells, showed higher levels of expression in islets from normal compared with donors with either type 2 diabetes (p = 0.006) or high HbA1c levels (p < 0.001). INS-IGF2 autoantibody levels were increased in newly diagnosed patients with type 1 diabetes (n = 304) compared with healthy controls (n = 355; p < 0.001). Displacement with cold insulin and INS-IGF2 revealed that more patients than controls had doubly reactive insulin-INS-IGF2 autoantibodies. These data suggest that INS-IGF2, which contains the preproinsulin signal peptide, the B-chain, and eight amino acids of the C-peptide may be an autoantigen in type 1 diabetes. INS-IGF2 and insulin may share autoantibody-binding sites, thus complicating the notion that insulin is the primary autoantigen in type 1 diabetes.

Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival1,2. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer’s disease3; aberrant signalling occurs in diverse cancers, exacerbated by crosstalk with the homologous type 1 insulin-like growth factor receptor (IGF1R)4. Despite more than three decades of investigation, the three-dimensional structure of the insulin–insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal β-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone–receptor recognition is novel within the broader family of receptor tyrosine kinases5. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titration calorimetry data that dissect the hormone–insulin receptor interface. Together, our findings provide an explanation for a wealth of biochemical data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogues.

Insulin and the insulin-like growth factors IGF-1 and IGF-II are found in all vertebrates, and these anabolic peptides share primary and tertiary structural features which suggest that they have evolved from a common ancestral gene. We have proposed that an insulin-like peptide (ILP) cDNA recently cloned from the protochodate amphioxus may represent the ancestral gene in that the deduced sequence of ILP contains features of both insulin and IGF, and it evidently represents a hybrid insulin/IGF molecule. To expand this hypothesis we have cloned the cDNA that encodes the cognate receptor from amphioxus. Primary sequence comparisons show that the ILP receptor is a member of the insulin receptor family, which in mammals includes the insulin receptor (IR), type I IGF receptor (IGF-IR), and IR-related receptor (IRR). In overall amino acid sequence, the ILP receptor is 48.6% identical to the human (h)IR, 47.3% identical to hIGF-IR, and 43.7% identical to hIRR, and this contrasts with the finding that hIR and hIGF-IR share 57.6% identity. Using degenerate oligonucleotide primers, we show by RT-PCR that amphioxus contains only a single member of the insulin receptor gene family. To complement the sequence comparison, we expressed the ILP receptor protein by transfecting the cDNA into 293 cells. Autophosphorylation of the expressed ILP receptor was half-maximally stimulated by a synthetic ILP analog, (B1-Thr)ILP, at a concentration of about 5 x 10(-7) M. Interestingly, autophosphorylation of the ILP receptor was also stimulated by incubation with either mammalian insulin or IGF-I, although equally high concentrations (10(-5) M) of each were required. Based on these results, we propose that, analogously to the ILP gene, the ancestral ILP receptor gene also duplicated and diverged to generate the IR and IGF-IR genes during the evolutionary transition from protochordates to vertebrates. Our results also indicate that the amphioxus ILP receptor contains the basic structural determinants that are necessary for binding and activation by mammalian insulin and IGF-I.

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.

Insulin secretion and β-cell biology are the focus of the Servier-IGIS Symposia that have been held yearly since 2000 in St. Jean Cap Ferrat in southern France (1). This volume, the fourth of a series published in Diabetes (1–3), collects the proceedings of the fourth Servier-IGIS Symposium, which focused on novel factors involved in islet biology and β-cell function. Special emphasis was placed on nuclear receptors, new genes that are associated with type 2 diabetes, and the role of mitochondria and lipid modulators in islet function. This symposium offers a striking example of how better knowledge of β-cell biology and the introduction of genetic tools to explore the pathophysiology of type 2 diabetes complement each other to improve our understanding of glucose homeostasis. Section I details the role of nuclear receptors in islet function, with special emphasis on peroxisome proliferator-activated receptors (PPARs). PPARα was cloned in 1990; related receptors were subsequently cloned, namely PPARδ (or β) and PPARγ. They form heterodimers with retinoid X receptor (RXR); their natural ligands are fatty acids and lipid-derived substrates. These receptors are major regulators of lipid metabolism, linking availability of glucose and lipids and long-term metabolic adaptation. Most importantly, they are targeted by key drugs used in treatment of hyperlipidemias and diabetes, namely fibrates and thiazolidinediones. Thiazolidinediones are synthetic PPARγ agonists developed to improve glucose tolerance by enhancing insulin sensitivity and restoring β-cell function. Patients with dominant-negative PPAR γ mutations develop severe hyperglycemia, and PPAR γ gene variants have been associated with type 2 diabetes. Besides the many actions of PPARγ agonists on the transcription of genes controlling insulin sensitivity in adipose tissue, they increase glucose sensing in liver and in β-cells. In the latter, PPARγ also enhances growth and prevents apoptosis. PPARα (the target of fibrates) controls lipid uptake and oxidation …

Insulin-like growth factor I (IGF-I) is a mitogenic peptide that is produced in most tissues and cell lines and plays an important role in embryonic development and postnatal growth. IGF-I is initially synthesized as a prohormone precursor that is converted to mature IGF-I by endoproteolytic removal of the carboxyl-terminal E-domain. Regulation of the conversion of proIGF-I to mature IGF-I is a potential mechanism by which the biological activity of this growth factor might be modulated. Endoproteolysis of the IGF-I prohormone occurs at the unique pentabasic motif Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg. Recently, a family of enzymes which cleave prohormone precursors at sites containing multiple basic residues has been discovered. The goals of this study were 1) to determine which basic residues in the pentabasic proIGF-I processing site were necessary for proper cleavage and 2) to examine the role that subtilisin-related proprotein convertase 1 (SPC1/furin) might play in proIGF-I processing. We have shown that an expression vector coding for an epitope-tagged proIGF-I directs synthesis and secretion of mature IGF-I-(1-70), extended IGF-I-(1-76), proIGF-I, and N-glycosylated proIGF-I in human embryonic kidney 293 cells. Extended IGF-I-(1-76) is produced by cleavage at Arg77 and requires both Arg74 (P4) and Arg77 (P1). Cleavage at Arg77 does not occur in the SPC1-deficient cell lines RPE.40 and LoVo, suggesting that processing at this site is mediated by SPC1. Mature IGF-I-(1-70) is produced by cleavage at Arg71 and requires both Lys68 (P4) and Arg71 (P1). Lys65 in the P7 position is important for efficient cleavage. SPC1 is not required for processing at Arg71 since this cleavage occurs in RPE.40 and LoVo cells. These data suggest the existence of a processing enzyme which is specific for the Lys-X-X-Arg motif of proIGF-I.

Human, but not mouse, islet amyloid polypeptide (IAPP) is amyloidogenic. Transgenic mice overexpressing human IAPP in the β cells of the islets of Langerhans should be useful in identifying factors important for the deposition of IAPP as insoluble amyloid fibrils. Transgenic mice expressing human IAPP were examined using several experimental models for the production of persistent hyperglycemia, as well as for the overstimulation and/or inhibition of β cell secretion. Obesity was induced by aurothioglucose. Persistent hyperglycemia was produced by long-term administration of glucocorticosteroids or by partial pancreatectomy. Inhibition of normal β cell exocytosis by diazoxide administration, with or without concurrent dexamethasone injections, was carried out to increase crinophagy of secretory granules. The human IAPP gene was also introduced into the db and ob mouse models for diabetes. Finally, isolated islets cultivated in vitro at high glucose concentration were also examined. No amyloid deposits were found in the pancreata of any of the animals, either by light microscopy after Congo red staining or by electron microscopy after immunogold labeling with antibodies specific for human IAPP. Aurothioglucose treatment resulted in increased numbers of granules in the β cell and the appearance of large lysosomal bodies without amyloid. However, islets from db and ob mice expressing human IAPP cultivated in vitro in the presence of glucocorticosteroid and/or growth hormone, were found to contain extracellular amyloid deposits reacting with antibodies to human IAPP. Oversecretion of human IAPP or increased crinophagy are not sufficient for amyloid formation. This indicates that other factors must influence amyloid deposition; one such factor may be the local clearance of IAPP.