H. Jörnvall

Isolation of glucagon-37 (bioactive enteroglucagon/oxyntomodulin) from porcine jejuno-ileum

A peptide isolated from porcine gut according to its glucagon‐like activity in liver (bioactive enteroglucagon) has been characterized immunologically, biologically and chemically: its potency relative to pancreatic glucagon in interacting with an antiglucagon antibody, hepatic glucagon‐binding sites and hepatic adenylate cyclase was ∼100%, 20% and 10%, respectively. In contrast, it is ∼20‐times more potent than glucagon in oxyntic glands, justifying the term ‘oxyntomodulin’. Chemically, it consists in the 29 amino acid‐peptide glucagon elongated at its C‐terminal end by the octapeptide Lys—Arg—Asn—Lys—Asn—Asn—Ile &—Ala; accordingly, it is called ‘glucagon‐37’

C-peptide is a bioactive peptide

-ATPase.Nerveconductionvelocity.RedbloodcelldeformabilityAbbreviationsBB/Wor BioBreeding/WorcestereNOS endothelial nitric oxide synthaseICAM-1 intracellular adhesion molecule 1MAPK mitogen-activated protein kinaseNCV nerve conduction velocityDuring the past decade, reports from several laboratorieshave focused on the physiological effects of C-peptide.Experimental data and clinical studies suggest that thatC-peptide is a biologically active peptide. Clinical studiesshow that C-peptide administration in type 1 diabetespatients, who lack the peptide, results in amelioration ofdiabetes-induced renal and nerve dysfunction. Molecularstudiesdemonstratebindingtocellmembranes,activationofintracellular signalling pathways, and specific end effects ofimportance for vascular endothelial function. These findingshave prompted the hypothesis that C-peptide deficiency intype 1 diabetes may contribute to the development ofmicrovascular complications, and that C-peptide replace-ment, together with regular insulin therapy, may be benefi-cial in the treatment or prevention of these complications. InthepresentarticlewearguethecaseinfavourofC-peptideasa biologically active peptide based on in vivo data and invitro findings, as summarised in Table 1.Soon after the discovery of insulin biosynthesis in 1967,and the identification of C-peptide and its role in promotingthe correct folding of proinsulin, researchers began toinvestigate whether C-peptide had any insulin-like effects.However,nonewerefound,andinterestinthepeptidefocusedinsteadonitsuseasamarkerofendogenousinsulinsecretion.Interest in a physiological role for C-peptide persisted, andreceived support from the clinical observation that patientswith type 1 diabetes, who continue to maintain a smallendogenous beta cell activity, are less prone to develop long-term complications and have fewer episodes of hypoglycae-mia than those who become totally C-peptide deficient [1, 2].It was also noted that islet or pancreas transplantation intype 1 patients, with restoration of both insulin and C-peptidesecretion, often results in amelioration of the functional andstructural abnormalities that accompany diabetic neuropathyand nephropathy [3, 4]. These considerations gave rise to aseries of studies involving the administration of a replace-ment dose of C-peptide to type 1 diabetes patients.Beneficial effects on renal function and structure intype 1 diabetesClinical studies Glomerular hyperfiltration, a risk factor forthe development of nephropathy, is an early abnormality intype 1 diabetes that is not corrected by insulin therapy. This

Characterization of an avian gastric (proventricular) peptide having sequence homology with the porcine gastrin-releasing peptide and the amphibian peptides bombesin and alytesin

Bombesin, a peptide with 14 amino acid residues, was isolated from the skin of an European frog [I ] and has been the subject of many recent investigations due to its diverse and potent pharmacological effects on the mammalian gastro-intestinal tract, pancreas, and central nervous system (reviewed [2,3]). We have reported the sequence of a 27 residue porcine gastrin-releas~g peptide (GRP) with marked C-terminal homology with bombesin [4]. Subsequent investigations indicate that natural porcine GRP and synthetic bombesin on intravenous administration, share the property of elevating plasma gastro-intestinalpancreatic hormone levels [Sf and that a synthetic replicate of GRP and bombesin on intracranial administration produce similar pharmacological effects [6]. Immunological evidence indicated that the avian proventriculus was an abundant source of bombesin-like immunoreactivity [7-91. We therefore began an investigation of extracts of the chicken proventriculus and report here the isolation and amino acid sequence of a 27 residue chicken proventricular peptide having marked sequence homology with the porcine GRP and the amphibian peprides bombesin and alytesin.

C-peptide stimulates ERK1/2 and JNK MAP kinases via activation of protein kinase C in human renal tubular cells.

Aims/hypothesisAccumulating evidence indicates that replacement of C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, but the molecular mechanisms involved are incompletely understood. C-peptide shows specific binding to a G-protein-coupled membrane binding site, resulting in Ca2+ influx, activation of mitogen-activated protein kinase signalling pathways, and stimulation of Na+, K+-ATPase and endothelial nitric oxide synthase. This study examines the intracellular signalling pathways activated by C-peptide in human renal tubular cells.MethodsHuman renal tubular cells were cultured from the outer cortex of renal tissue obtained from patients undergoing elective nephrectomy. Extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and Akt/protein kinase B (PKB) activation was determined using phospho-specific antibodies. Protein kinase C (PKC) and RhoA activation was determined by measuring their translocation to the cell membrane fraction using isoform-specific antibodies.ResultsHuman C-peptide increases phosphorylation of ERK1/2 and Akt/PKB in a concentration- and time-dependent manner in renal tubular cells. The C-terminal pentapeptide of C-peptide is equipotent with the full-length C-peptide, whereas scrambled C-peptide has no effect. C-peptide stimulation also results in phosphorylation of JNK, but not of p38 mitogen-activated protein kinase. MEK1/2 inhibitor PD98059 blocks the C-peptide effect on ERK1/2 phosphorylation. C-peptide causes specific translocation of PKC isoforms δ and ɛ to the membrane fraction in tubular cells. All stimulatory effects of C-peptide were abolished by pertussis toxin. The isoform-specific PKC-δ inhibitor rottlerin and the broad-spectrum PKC inhibitor GF109203X both abolish the C-peptide effect on ERK1/2 phosphorylation. C-peptide stimulation also causes translocation of the small GTPase RhoA from the cytosol to the cell membrane. Inhibition of phospholipase C abolished the stimulatory effect of C-peptide on phosphorylation of ERK1/2, JNK and PKC-δ.Conclusions/interpretationC-peptide signal transduction in human renal tubular cells involves the activation of phospholipase C and PKC-δ and PKC-ɛ, as well as RhoA, followed by phosphorylation of ERK1/2 and JNK, and a parallel activation of Akt.

Isolation and characterization of bovine vasoactive intestinal peptide (VIP)

The vasoactive intestinal peptide (VIP) in the forms hitherto isolated is composed of 28 amino acid residues. In addition to the properties implied by its name, it possesses a broad spectrum of biological activities, although the physiological function is yet unknown. Originally isolated from upper intestinal tissue, VIP or material showing VIP-like immunoreactivity, has later been found in neural structures throughout the gastrointestinal tract, as well as in certain other areas of the peripheral nervous system, and in parts of the central nervous system. It has also been demonstrated in certain endocrine cells of the gastrointestinal tract [ 11. The structure of the porcine peptide was first determined [2,3]. Chicken VIP was isolated later and its amino acid sequence was found to differ from porcine VIP in four residues, corresponding to an 86% identity [4]. This study reports an improved isolation procedure for VIP, the isolation of bovine VIP and the determination of its complete primary structure.

Cellular internalization of proinsulin C-peptide

Abstract.Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37°C for up to 1 h, and was reduced at 4°C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action.

ALK7, a Receptor for Nodal, Is Dispensable for Embryogenesis and Left-Right Patterning in the Mouse

ABSTRACT Mesendoderm formation and left-right patterning during vertebrate development depend upon selected members of the transforming growth factor β superfamily, particularly Nodal and Nodal-related ligands. Two type I serine/threonine kinase receptors have been identified for Nodal, ALK4 and ALK7. Mouse embryos lacking ALK4 fail to produce mesendoderm and die shortly after gastrulation, resembling the phenotype of Nodal knockout mice. Whether ALK4 contributes to left-right patterning is still unknown. Here we report the generation and initial characterization of mice lacking ALK7. Homozygous mutant mice were born at the expected frequency and remained viable and fertile. Viability at weaning was not different from that of the wild type in ALK7−/−; Nodal +/− and ALK7 −/−; ALK4 +/− compound mutants. ALK7 and ALK4 were highly expressed in interdigital regions of the developing limb bud. However, ALK7 mutant mice displayed no skeletal abnormalities or limb malformations. None of the left-right patterning abnormalities and organogenesis defects identified in mice carrying mutations in Nodal or in genes encoding ActRIIA and ActRIIB coreceptors, including heart malformations, pulmonary isomerism, right-sided gut, and spleen hypoplasia, were observed in mice lacking ALK7. Finally, the histological organization of the cerebellum, cortex, and hippocampus, all sites of significant ALK7 expression in the rodent brain, appeared normal in ALK7 mutant mice. We conclude that ALK7 is not an essential mediator of Nodal signaling during mesendoderm formation and left-right patterning in the mouse but may instead mediate other activities of Nodal and related ligands in the development or function of particular tissues and organs.

Unordered structure of proinsulin C-peptide in aqueous solution and in the presence of lipid vesicles

Abstract. Proinsulin C-peptide ameliorates renal and autonomic nerve function and increases skeletal muscle blood flow, oxygen uptake and glucose transport in patients with insulin-dependent diabetes mellitus. These effects have in part been ascribed to the stimulatory influence of C-peptide on Na+,K+-ATPase and endothelial nitric oxide synthase. To evaluate the capacity of C-peptide to insert into lipid bilayers and form ion channels, C-peptide secondary structure and membrane interactions were studied with circular dichroism spectroscopy and size exclusion chromatography. C-peptide is shown to lack a stable secondary structure, both when part of proinsulin and when free in aqueous solution, although the N-terminal third of the peptide exhibits an α-helical conformation in trifluoroethanol. Moreover, C-peptide remains disordered in the aqueous solvent in the presence of lipid vesicles, regardless of vesicle composition. In conclusion, C-peptide is unlikely to elicit physiological effects through stable conformation-dependent interactions with lipid membranes.

Isolation and characterization of neuropeptide Y from porcine intestine

The isolation and primary structure of intestinal neuropeptide Y (NPY) is described. The peptide was purified from porcine intestinal extracts using a chemical assay and radioimmunoassay for NPY. The amino acid sequence of this peptide is: Tyr‐Pro‐Ser‐Lys‐Pro‐Asp‐Asn‐Pro‐Gly‐Glu‐Asp‐Ala‐Pro‐Ala‐Glu‐Asp‐LeuAla‐Arg‐Tyr‐Tyr‐Ser‐Ala‐Leu‐Arg‐His‐Tyr‐Ile‐Asn‐Leu‐Ile‐Thr‐Arg‐Gln‐Arg‐Tyr‐NH2. This indicates that the structure of intestinal NPY is identical to the NPY of brain origin.

Separate functional features of proinsulin C-peptide

Abstract.Proinsulin C-peptide influences a number of physiological parameters in addition to its well-established role in the parent proinsulin molecule. It is of interest as a candidate for future co-replacement therapy with insulin for patients with diabetes mellitus type 1, but specific receptors have not been identified and additional correlation with functional effects is desirable. Based on comparisons of 22 mammalian proinsulin variants, we have constructed analogues for activity studies, choosing phosphorylation of mitogen-activated protein kinases (MAPKs) in Swiss 3T3 fibroblasts for functional measurements. In this manner, we find that effective phosphorylation of MAPKs is promoted by the presence of conserved glutamic acid residues at positions 3, 11 and 27 of C-peptide and by the presence of helix-promoting residues in the N-terminal segment. Previous findings have ascribed functional roles to the C-terminal pentapeptide segment, and all results combined therefore now show the importance of different segments, suggesting that C-peptide interactions are complex or multiple.