Breakthroughs and Views

Molecular effects of proinsulin C-peptideq

Jan Johansson,a,* Karin Ekberg,b Jawed Shafqat,a Mikael Henriksson,a

Alexander Chibalin,b John Wahren,b and Hans J€oornvalla

a Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Swedenb Department of Surgical Sciences, Karolinska Hospital, SE-171 76 Stockholm, Sweden

Received 10 June 2002

Abstract

The proinsulin C-peptide has been held to be merely a by-product in insulin biosynthesis, but recent reports show that it elicits

both molecular and physiological effects, suggesting that it is a hormonally active peptide. Specific binding of C-peptide to the

plasma membranes of intact cells and to detergent-solubilised cells has been shown, indicating the existence of a cell surface receptor

for C-peptide. C-peptide elicits a number of cellular responses, including Ca2þ influx, activation of mitogen-activated protein (MAP)

kinases, of Naþ,Kþ-ATPase, and of endothelial NO synthase. The pentapeptide EGSLQ, corresponding to the C-terminal five

residues of human C-peptide, mimics several of the effects of the full-length peptide. The pentapeptide displaces cell membrane-

bound C-peptide, elicits transient increase in intracellular Ca2þ concentration and stimulates MAP kinase signalling pathways and

Naþ,Kþ-ATPase. The Glu residue of the pentapeptide is essential for displacement of the full-length C-peptide, and free Glu can

partly displace bound C-peptide, suggesting that charge interactions are important for receptor binding. Many C-peptide effects,

such as phosphorylation of MAP-kinases ERK 1 and 2, stimulation of Naþ,Kþ-ATPase and increases in intracellular calcium

concentrations are inhibited by pertussis toxin, supporting interaction of C-peptide with a G-protein-coupled receptor. However, all

C-peptide effects cannot be explained in this manner, and it is possible that additional interactions are involved. Combined, the

available observations show that C-peptide is biologically active and suggest a molecular model for its physiological effects. � 2002Elsevier Science (USA). All rights reserved.

The proinsulin C-peptide has long been consideredmerely a by-product of insulin biosynthesis. Thus, it isof structural importance in the folding of proinsulin byproviding a spacer sequence that can be removed oncefolding is completed [1,2]. Early on, C-peptide was ex-amined for possible metabolic effects, but none was thenestablished, see [2,3]. The observation that C-peptidevaries considerably between species with regard to itsamino acid sequence and chain-length has been taken asan additional indication for lack of C-peptide biologicalactivity. As a consequence, C-peptide has generally beenheld to lack an active, hormone-like role. However, newfindings in diabetic patients and animal models of dia-betes, correlated with defined molecular effects, havechanged this view [4–13]. Here, we review the current

understanding of the molecular mechanisms involved inC-peptide action and the structural properties ofC-peptide in relation to cell binding and activity. Wealso briefly summarise the in vivo effects of C-peptide.

C-peptide primary and secondary structures

Human C-peptide has 31 residues; it lacks basicresidues and contains five acidic residues (Fig. 1). Inwater, C-peptide is devoid of detectable stable sec-ondary structure, but the N-terminal 11 residues forman a-helical structure in 95% aqueous trifluoroethanol[14]. The region of positions 13–25, containing five Glyresidues, is in parts lacking in some mammals, and hasnot been reported to have a stable secondary structure.The C-terminal five residue segment (encompassingpositions 27–31), which contains two highly conservedresidues, has been found to possess biological activityof its own, and is also unstructured in trifluoroethanol[14].

Biochemical and Biophysical Research Communications 295 (2002) 1035–1040

www.academicpress.com

BBRC

qAbbreviations: CP, proinsulin C-peptide; eNOS, endothelial NO

synthase; MAP, mitogen-activated protein; NMDA, N-methyl-D-

aspartate; NO, nitric oxide; PI3-K, phosphoinositide 3-kinase.* Corresponding author. Fax: +46-8-337462.

E-mail address: jan.johansson@mbb.ki.se (J. Johansson).

0006-291X/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.PII: S0006 -291X(02 )00721 -0

C-peptide binding to cells

Binding of rat C-peptide 1 to cultured rat islet tumorcells, predominantly composed of insulin-secretingb-cells, was demonstrated using a radioligand bindingtechnique [15]. In contrast, human C-peptide was re-ported not to bind to crude cell membranes preparedfrom skeletal muscle, using the same technique [16].Proinsulin binding to cells has been reported, and humanC-peptide was able to compete with proinsulin, but notwith insulin, for binding to IM-9 lymphoblasts [17–19].Moreover, human C-peptide has been recently shown tobind to cell membranes from human renal tubular cells,skin fibroblasts, and saphenous vein endothelial cellsusing fluorescence correlation spectroscopy [20–22].Saturation of the C-peptide binding to the cell surfacewas then reported at low nanomolar concentrations.Addition of an excess of unlabelled C-peptide displacesthe bound C-peptide, but all-D-amino acid C-pep-tide, scrambled C-peptide, insulin, proinsulin, IGF-I, orIGF-II do not compete for binding, demonstrating thespecificity and the chiral nature of C-peptide binding tocell membranes.The detergent CHAPS can release C-peptide-binding

macromolecules from cells, which suggests that bindingcomponents can be purified and characterised [21].However, detection of binding required strict control ofconditions and short handling times, which complicatedinterpretations and further purification attempts.

Cellular effects of C-peptide

Effects on intracellular Ca2þ levels. C-peptide in-creases the intracellular concentration of calcium [23–25]. This is also true for the C-terminal pentapeptide ofC-peptide [25]. Addition of the calcium chelator EGTAto the medium abolishes the effects of C-peptide on in-tracellular calcium levels, suggesting that the increase ismediated by influx of extracellular calcium [23]. Like-wise, addition of pertussis toxin prevents the effects onintracellular calcium levels of both full-length C-peptideand of the C-terminal pentapeptide [25], which suggest

that G-protein-dependent effects are upstream of theinflux of calcium. It appears worthwhile to further ex-plore the effects of specific inhibitors on the C-peptide-elicited calcium increase to find out what signallingpathways are involved.Effects on mitogen-activated protein (MAP) kinase.

C-peptide has been found to induce phosphorylation ofthe MAP-kinases ERK 1 and 2 of a mouse embryonicfibroblast cell line (Swiss 3T3), while reverse-sequenceC-peptide and all-D-amino acid C-peptide did not stim-ulateMAP-kinase phosphorylation [26].Moreover, ERK1 and 2 phosphorylations were also observed in humanrenal tubular cells upon incubation with homologousC-peptide [27]. Preincubation of the cells with pertussistoxin or with PD98059, aMAP-kinase kinase 1 inhibitor,abolished the stimulatory effect on ERK phosphoryla-tion. Combined, these data suggest that C-peptide acti-vates a MAP-kinase-dependent signalling pathway.Effects on Naþ,Kþ-ATPase activity. A stimulatory ef-

fect of C-peptide on Naþ,Kþ-ATPase activity has beendemonstrated in both in vitro and in vivo studies. Thus,incubation of rat renal tubular segments with homolo-gous C-peptide in the concentration range 10–100 nMelicited a rise in Naþ,Kþ-ATPase activity as measured byhydrolysis of [32P]ATP [23,28]. The increase in activitywas concentration-dependent and could be blocked bypretreatment of the tubular segments with pertussis toxinor addition of by FK 506, a specific inhibitor of the Ca2þ-calmodulin-dependent protein phosphatase 2B. The lat-ter enzyme has an important function in rat renal tubularcells by converting the phosphorylated, low-activityform of Naþ,Kþ-ATPase to its dephosphorylated, activeform. In addition, incubation of the tubular segments ina Ca2þ free medium was found to completely abolish theC-peptide signal [23]. Human renal tubular cells in pri-mary culture have also been studied with regard toNaþ,Kþ-ATPase activity after exposure to humanC-peptide [27]. Oubain-sensitive uptake of 86Rbþ wasthen used as a measure of Naþ,Kþ-ATPase activity. Aconcentration-dependent stimulation was seen in therange 1–10 nM of C-peptide, and the effect was abolishedby pretreatment with pertussis toxin.Red blood cells frompatientswith type 1diabetes show

reduced Naþ,Kþ-ATPase activity [29] and have impaireddeformability. The latter phenomenon is reversible byexposure of the red blood cells to C-peptide [30]. TheC-peptide effect appears to be mediated via Naþ,Kþ-ATPase, because pretreatment of the cells with ouabain, aspecific inhibitor of Naþ,Kþ-ATPase, abolished the effectof C-peptide on the red cell deformability. Rat C-peptide2 given in replacement doses to diabetic BB/Wor rats, andhuman C-peptide administered in supra-physiologicaldoses to streptozotocin diabetic rats, have been found tostimulate Naþ,Kþ-ATPase activity in the sciatic nerveand to partially correct the diabetes-induced reduction inNaþ,Kþ-ATPase activity [6,8].

Fig. 1. Proinsulin C-peptide amino acid sequence. The upper line

shows the amino acid sequence of human C-peptide and the bars

correspond to the number of alternative amino acids found at each

position in 20 mammalian C-peptide amino acid sequences (J€oornvall et

al., unpublished).

1036 J. Johansson et al. / Biochemical and Biophysical Research Communications 295 (2002) 1035–1040

Effects on endothelial nitric oxide synthase (eNOS)activity. In a study using bovine aortic cells and a re-porter cell assay it could be demonstrated that exposureof cells to C-peptide resulted in stimulation of eNOSactivity and release of NO in a concentration- and time-dependent manner [24]. Addition of an eNOS blockingsubstance (L-NAME) or a calcium-binding agent in-hibited the C-peptide effect, suggesting that the effect ofC-peptide on eNOS is mediated via an increase in in-tracellular Ca2þ [24]. In keeping with these observations,C-peptide is reported to elicit a dilation of skeletalmuscle arterioles via a NO-mediated mechanism [31].Moreover, leukocyte–endothelium interactions inducedin rat mesenteric venules by both thrombin andL-NAME are diminished by C-peptide [32]. C-peptideadministration is associated with an increase in eNOSmRNA levels and a marked increase in basal NO releasefrom aortic endothelial cells. C-peptide may thus act asan inhibitor of leukocyte/endothelium interactions byinhibiting endothelial cell adhesion molecules viamaintenance of NO release from endothelial cells [32].In support of this hypothesis, C-peptide exerts cardio-protective effects against leukocyte-mediated reperfusioninjury in the isolated rat heart, most likely due to re-duced leukocyte adherence to vascular endotheliumsecondary to stimulation of NO production by the en-dothelium [33].Replacement administration of C-peptide to patients

with type 1 diabetes results in a concentration-depen-dent rise in muscle blood flow [34]. The C-peptide in-duced rise in muscle blood flow is seen both at rest andduring physical exercise and can be blocked by additionof L-NAME [7,35]. C-peptide administration is alsoreported to result in increases in blood flow to the kid-neys, the skin, and the nerves in the diabetic state[5,9,36].

Activity of C-peptide fragments and regions

Some of the conserved C-peptide residues (Fig. 1) arethe consequence of the requirements for folding andprocessing of proinsulin. The biological effects and cellbinding of C-peptide are likely to be mediated through aspecific region(s) of the peptide, and its sequence con-servation may partly be a reflection of this. Attemptshave been made to locate the activities and properties ofC-peptide with respect to its sequence. The rat C-termi-nal pentapeptide (EVARQ) was found to possess 100%of the Naþ,Kþ-ATPase stimulatory activity of the intactmolecule, while the remaining segment, des-(27–31)C-peptide, failed to elicit detectable activity [28]. Fur-thermore, human C-peptide bound to renal tubular cellmembranes was effectively displaced by a 1000-foldmolar excess of the human pentapeptide EGSLQ [20],indicating that the C-terminal fragment is involved in a

specific binding process. The pentapeptide EGSLQ, butnot des-(27–31)C-peptide, elicits an increase in intracel-lular calcium [25] and causes phosphorylation of MAP-kinases in human renal tubular cells [27]. Thus, fourdifferent approaches demonstrate a molecular effect ofthe intact C-peptide and a similar effect of its C-terminalpentapeptide. This supports the view that the C-terminalsegment constitutes an active site of C-peptide.An internal fragment corresponding to positions

11–19 of the C-peptide also exhibits some stimulatoryeffects on Naþ,Kþ-ATPase activity [28], and it is re-ported that a C-peptide mid-portion is important fornormalisation of glucose-induced vascular dysfunctionin a rat model [6]. The mid-fragment sequence is highlyvariable between species, and a peptide comprisingamino acid residues 11–19 of C-peptide does not dis-place cell membrane-bound human C-peptide [22]. Thissuggests that the mechanisms of the mid-portion seg-ment are different from those of the C-terminal segment.The possible importance of Glu per se and of Glu-

terminal peptides has been considered [22,37]. C-peptideand other Glu-terminal peptides have been shown toselectively suppress N-methyl-D-aspartate (NMDA)glutamate receptor-mediated secretion of gonadotropin-releasing hormone in rat hypothalamic explants [37].LL-Glutamate stimulates insulin secretion in the ratpancreas via stimulation of NMDA receptors. Thus,C-peptide could be involved in an auto-feedbackmechanism of insulin secretion.The current view is that the C-terminal pentapeptide

segment of C-peptide is important for specific binding tomolecular targets and for evoking biological effects, andthat a Glu residue is involved in these processes.

Molecular mechanisms of C-peptide action

As described above several actions of C-peptide areapparently mediated via G-protein-coupled pathways,but it is possible that further interactions are involved(Fig. 2).(i) Pertussis toxin treatment inhibits C-peptide stim-

ulation of Naþ,Kþ-ATPase activity [23,27], calcium in-flux [25], and activation of MAP kinases [26,27],indicative of receptors coupled to adenylate cyclase,specifically transmembrane G-protein-coupled receptorswith the a-subunit of the Gi=G0 subtype. Interestingly,pertussis toxin not only blocks C-peptide effects but alsointerferes with C-peptide binding to cell membranes [20].Moreover, activation of protein kinase C and phos-phoinositide 3-kinase (PI3-K) is apparently involved inC-peptide induced phosphorylation of MAP kinases[26].(ii) Interactions between C-peptide and receptors with

catalytic activity are indicated by results showing that C-peptide attenuates protein tyrosine phosphatase activity

J. Johansson et al. / Biochemical and Biophysical Research Communications 295 (2002) 1035–1040 1037

[38]. Protein tyrosine phosphatases inactivate the insulinsignalling pathway by dephosphorylation of the insulinreceptor, insulin receptor substrates, and MAP kinases.Hence, C-peptide and insulin might have a synergisticeffect on the insulin signalling pathway at the level of theinsulin receptor. This is further corroborated by therecent finding that C-peptide at physiological concen-trations mimics insulin effects in myoblasts; it activatesinsulin receptor tyrosine kinase, insulin receptor sub-strate-1 tyrosine phosphorylation, PI3-K activity, andMAP kinase phosphorylation [39]. If C-peptide is addedin the presence of high insulin concentrations, no furthereffects are observed, indicating that C-peptide and in-sulin may use the same signalling pathway [39]. Theseauthors suggested that low C-peptide levels enhanceinsulin effects, while at supra-physiological concentra-tions C-peptide blunts insulin effects. However, C-pep-tide, unlike insulin, does not activate Akt (protein kinaseB), suggesting that C-peptide also works via mecha-nisms distinct from those of insulin. C-peptide-inducedstimulation of glycogen synthesis in the myoblasts wasblocked by Wortmannin, an inhibitor of PI3-K activity,but not by pertussis toxin [39]. In contrast to thesefindings, Zierath et al. [16] found that C-peptide stimu-lates glucose transport in human muscle strips, and thatthese effects were not mediated via the insulin receptoror tyrosine kinase activation.(iii) C-peptide interactions with ligand-gated ion

channels coupled to glutamate receptors have beenconsidered because of the capacity of free glutamic acidto partly displace C-peptide from cell membranes, andby the importance of N-terminal Glu in intact C-peptideand its C-terminal pentapeptide in binding and biolog-ical activity [22,37]. The observed effects of C-peptide on

NMDA receptors [37] would also fit with an involve-ment of a particular class of glutamate receptors, but itremains possible that C-peptide Glu residues are crucialin any type of receptor interaction. However, the first 26amino acid residues of rat C-peptide, including anN-terminal Glu, lack the stimulatory activity of intactC-peptide on Naþ,Kþ-ATPase activity in rat renaltubular segments [28], and human C-peptide (1–26) hasa poor capacity to displace C-peptide bound to humancell membranes [22].It has also been suggested that the effects of C-peptide

may be mediated by direct membranotropic mechanismsinstead of by classical receptor–ligand interactions [6].However, the hallmarks of traditional pore-formingpeptides are lacking in C-peptide; it shows no tendencyto self-associate and it is overall hydrophilic and nega-tively charged [10]. In line with this, C-peptide was laterfound not to associate in a stable manner with lipidmembranes or micelles [14].The above observations are of interest regarding

possible diversity of C-peptide action, but it should bepointed out that C-peptide concentrations differ widelybetween the experimental systems used. Saturation ofbinding to cells is apparently reached already within thephysiological concentration range [20]. As a conse-quence, receptor-mediated physiological effects arelikely to take place at or below the physiologicalC-peptide concentration (0.5–1:5� 10�9 M), whereassupra-physiological concentrations might induce non-specific effects.In summary, the current view on C-peptide action

(Fig. 2) is that G-protein-coupled receptor(s) areinvolved, since addition of pertussis toxin reducesC-peptide binding to cells and blunts C-peptide effectson signal mediators, as well as enzyme activity. Exper-imental data in support of C-peptide binding to otherreceptor types indicate that additional signal pathway(s)may also be affected, which may converge with aG-protein-coupled receptor pathway at different levels.Notably, relaxin, a member of the insulin-like peptidehormone family, was recently found to work viaG-protein-coupled receptors and adenylate cyclaseactivation, but also possible to elicit effects via a tyrosinekinase pathway [40,41].

In vivo effects of C-peptide replacement therapy

Replacement of C-peptide in rats with streptozotocin-induced diabetes is accompanied by correction ofglomerular hyperfiltration, diminished levels of microal-buminuria, and regression of glomerular hypertrophy[11,12]. Likewise, when C-peptide is administered inreplacement doses to patientswith type 1 diabetes, there issignificant reduction of both glomerular hyperfiltrationand urinary albumin excretion [4,5,13].

Fig. 2. Signal pathways of interest in relation to reported C-peptide

effects. The central (blue) pathway represents C-peptide effects medi-

ated by G-protein-coupled receptor(s) (GPCR), i.e., are blocked by

pertussis toxin [23,25–27]. The right (yellow) pathway represents effects

which appear to be mediated via the insulin receptor and are not in-

hibited by pertussis toxin [39]. The left (orange) pathway represents the

finding that C-peptide can bind to an ion-channel-coupled glutamate

receptor [37], which possibly could result in Ca2þ influx. See text for

further details. PKC, protein kinase C; PI3-K, phosphoinositide

3-kinase; MAPK, mitogen-activated protein kinase; eNOS, endothelial

NO synthase.

1038 J. Johansson et al. / Biochemical and Biophysical Research Communications 295 (2002) 1035–1040

Effects of C-peptide replacement therapy on functionaland structural changes in peripheral nerves have beenstudied in rats that spontaneously develop diabetes.C-peptide administration for two months was found toprevent a major proportion of the nerve conductionvelocity defect and the paranodal swelling that otherwiseoccurred [8]. In patients with autonomic nerve dysfunc-tion, increased heart rate variability during deep breath-ing has been seen following C-peptide administration[13,42]. In addition, preliminary evidence from a studyinvolving C-peptide replacement therapy in type 1diabetes patients without overt symptoms of neuropathyindicate that three months of treatment results in signifi-cant improvement of sensory nerve conduction velocityand vibration thresholds (Ekberg et al., unpublishedobservation). C-peptide replacement therapy in rats isknown to be accompanied by significant improvement innerve Naþ,Kþ-ATPase activity [6,8] and in nerve bloodflow [9]. The relevance of the observed C-peptide effects issupported by the fact that both deficient NO formationand reduced levels of Naþ,Kþ-ATPase are factors ofpathogenetic importance for diabetic neuropathy [43].The present data suggest that C-peptide replacementtherapy together with insulin therapy in type 1 diabetespatients may be beneficial in preventing or retarding thedevelopment of long-term complications.

Acknowledgment

This work was supported by the Juvenile Diabetes Foundation

(project JDFI 1-1999-647).

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