# Intracellular Signalling by C-Peptide

Authors: Claire E.  Hills; Nigel J. Brunskill
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes.  Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na+/K+ ATPase, increased eNOS transcription, and activation of  the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes.


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## Body

## 1. Introduction

Historically proinsulin C-peptide has been
regarded  as an inert byproduct of insulin synthesis and processing. In the last
decade, however, an enlarging body of evidence has emerged from several
laboratories reporting both in vitro
and in vivo effects of C-peptide of great potential relevance to the pathophysiology and treatment of
diabetes. Observations that C-peptide may exhibit characteristics of a peptide
hormone beneficially affecting nerve, renal and microvascular functions in
diabetic animals and in patients with diabetes [1–7],
have stimulated interest in the molecular
mechanisms by which cell signals elicited by C-peptide are transduced into
altered cell and organ behaviour.


## 2. A C-Peptide Receptor

Clearly if C-peptide has peptide hormone-like actions, it is necessary to postulate the existence of a receptor.
Indeed, specific and displaceable binding of125I-labelled C-peptide
was first demonstrated by Flatt et al. [8] who derived a curvilinear Scatchard plot of specific C-peptide binding to
pancreatic islet B-cells.Subsequently, using retro sequence and
all-D-amino acid C-peptide
enantiomers, Ido et al. [9] suggested
that C-peptide-induced improvements in nerve function and vascular permeability
blood flow in diabetes through increases in Na+,K+-ATPase
activity did not result from C-peptide binding in a stereospecific manner to a
receptor. Instead it was hypothesised that biological activity of C-peptide was
dependent on poorly defined membrane interactions that took place due to
structural features related to the C-peptide sequence, but independent of its
direction or chirality. A midportion sequence of C-peptide, largely conserved,
and comprising a high proportion of nonpolar amino acids flanking a C16 proline
was implicated in this activity.Other investigators have also examined
C-peptide fragment bioactivity using Na+,K+-ATPase
activity in rat renal tubular segments as a readout [10]. Two segments of
C-peptide demonstrated functional importance. The rat C-peptide carboxy
terminal pentapeptide, EVARQ, elicited 100% of the activity of intact C-peptide
whereas the remaining portion of the molecule, lacking this 5 amino acid
sequence, was totally inactive. In this rat system, the terminal pentapeptide
of human C-peptide (EGSLQ) elicited 75% activity; and notably the glutamic acid
at position 1 and the glutamine at position 5 are generally conserved in
mammals. Similar well-defined C-terminal
functional sequences are also found in gastrin and cholecystokinin. Overall,
the behaviour of the C-termimal pentapeptide in these studies was typical of a
peptide ligand interacting with a specific receptor. In contrast, several C-peptide
midregion sequences were able to partially recapitulate the activity of the
intact molecule but exhibited rather different properties. Activity relating to
this region was not seen with the des-(27–31)-C-peptide, and several nonnatural
D-amino acid containing sequences
showed some activity. The activity of these segments decreased if they were
greater than 9 amino acids in length. This behaviour is not reminiscent of
peptide-receptor interactions, but similar to the nonspecific type interactions
of C-peptide with plasma membranes postulated by Ido et al. [9].How the balance of activity provided by
these two regions of the C-peptide molecule is manifest in vivo is not well understood. Very recent work suggests that
efficient activation of signalling pathways requires the presence of conserved
glutamic acid residues at positions 3, 11, and 27 of C-peptide, and the
presence of helix-promoting residues in the N-terminal segment [11]. Taken
together with the data relating to its carboxy-terminus,
the overall picture now emerging of the structure-activity relationship of the
C-peptide molecule is one of a tripartite structure where the terminal sections
are involved in functional interactions, with the midregion forming a joining
segment [11].Analysis of rhodamine-labelled human
C-peptide binding to a variety of human-cell membranes has now been carried out
using highly sensitive fluorescence correlation microscopy, allowing
measurement of membrane interactions at the single-molecule level in sub-fL
volumes [12].  High-affinity specific
binding of C-peptide was observed with an association rate constant of ∼3 nM;
half-maximal occupation of binding sites was seen at 0.3 nM C-peptide; and full
occupation at 0.9 nM C-peptide. The maximal number of binding sites,
approximately 1000–1500 per cell,
was found on human renal tubular cell membranes. Binding could be displaced by
excess intact C-peptide and by the C-terminal pentapeptide, but not by a
randomly assembled scrambled C-peptide, insulin, and insulin-like growth factor (IGF)-I or IGF-II.
Importantly, binding of C-peptide could also be largely inhibited by pertussis
toxin (PTX) pretreatment of cells [12]. Overall, these results strongly support
the existence of a specific GTP-binding protein-coupled receptor (GPCR) for
C-peptide, linked either to the G-protein α-subunit Gαi or Gαo, which interacts with the C-terminal penatpeptide
region of the C-peptide molecule.Attempts to identify a C-peptide receptor
by gene-cloning strategies or using proteomic approaches have, to date, been
unsuccessful. Luzi et al. recently reported their inability to isolate a C-peptide receptor either by screening a
human lung fibroblast λ phage cDNA expression library or by proteomic
analysis of proteins co-immunoprecipitated from C-peptide treated human
fibroblasts using anti-C-peptide antibodies [13]. This information was
published in the context of a short-debate paper and therefore methodological
description was by necessity brief. Certainly this data does not prove that a
receptor does not exist, and it remains perfectly possible however that
alternative methodologies may successfully identify a C-peptide receptor in the
future. Effort in this area is well justified and urgently needed.


## 3. C-Peptide and the Na+,k+-Atpase

Na+,K+-ATPase is an ubiquitous membrane-associated protein complex that uses energy from the
hydrolysis of ATP to drive the counter-transport of sodium and potassium across
the plasma membrane. It is widely accepted that impaired Na+,K+-ATPase
activity is present in a variety of cell types in diabetes and contributes to
the pathogenesis of diabetic complications [14–17]. The first indications that
C-peptide may regulate the activity of intracellular enzymes came from studies
of the Na+,K+-ATPase.The kidney tubule is a particularly rich
source of Na+,K+-ATPase and Ohtomo et al. described activation of this
enzyme by rat C-peptide in rat kidney tubules at low to high nanomolar
concentrations [18]. This effect was abolished by PTX and, being inhibited by
the Ca2+-calmodulin-dependent protein phosphatase 2B (calcineurin) inhibitor FK506, appeared dependent on intracellular Ca2+ concentration. Interestingly the stimulatory effect of C-peptide on Na+,K+-ATPase
in these experiments was substantially augmented by neuropeptide Y (NPY).Extending these findings, the same group of
investigators studied the ability of C-peptide fragments to activate Na+,K+-ATPase
in rat kidney proximal tubule segments [10]. Altogether 36 different peptides
and amino acids corresponding to various regions of intact C-peptide were
investigated. Carboxy-terminal tetra- and penta-peptides elicited full activity
whilst midregion peptides elicited only partial activity at best. These results
were in broad agreement with those of Ido et al. [9] (discussed above) and those of the C-peptide binding
studies published the following year [12], and, overall, they supported the
nascent concept of C-peptide binding to a specific receptor with subsequent
activation of downstream effector enzymes.Motivated by these observations of
C-peptide-induced increases in Na+,K+-ATPase activity and
associated improvements in nervous system and renal function, it was
hypothesised that the low C-peptide levels and reduced Na+,K+-ATPase
activity in erythrocyte membranes observed in type 1 diabetes may be related.
Accordingly, it was found that in patients with type 1 diabetes and complete
C-peptide deficiency, erythrocyte activity Na+,K+-ATPase
was consistently lower than healthy controls [19]. In type 2 diabetes,
erythrocyte Na+,K+-ATPase was significantly lower in
insulin-treated patients than in those treated with oral hypoglycaemic agents.
Indeed the fasting C-peptide level was the only variable studied that independently
correlated with Na+,K+-ATPase activity [19]. Subsequently,
it was shown that infusion of C-peptide into patients with type 1 diabetes
resulted in an increase in plasma cGMP and erythrocyte membrane Na+,K+-ATPase
activity. A C-peptide dose response was evident in these studies where maximal
effects were observed with achieved plasma C-peptide levels of ∼3.5 nM [20].  Similar
improvements in rat nerve Na+,K+-ATPase
activity and function have also been observed after exogenous administration of
C-peptide [9].The mechanism(s) by which C-peptide
activates Na+,K+-ATPase has been examined. Using isolated
rat kidney medullary thick ascending limb tubules (MTALs) C-peptide was found
to activate Na+,K+-ATPase at physiological concentrations
[21]. In addition, treatment of MTALs with C-peptide resulted in
phosphorylation of the Na+,K+-ATPase α-subunit and translocation of the Ca2+-dependent protein
kinase C (PKC)-α to the membrane, an index of its
activation. Both of these latter effects were blocked by an inhibitor of PKC [21].Overall, the studies of Na+,K+-ATPase
activity have delineated a stimulatory signalling action of C-peptide at
physiological concentrations dependent on intracellular Ca2+ and
PKC, and sensitive to PTX suggesting the presence of a GPCR for C-peptide.
Replacement of C-peptide in diabetic animals and patients with diabetes has a
salutary effect on Na+,K+-ATPase activity in a variety of
tissues affected by diabetic complications.Prompted by this evidence linking
C-peptide to the Na+,K+-ATPase, and mindful that
vasopressin also stimulates Na+,K+-ATPase activity,
Maestroni et al. [22] studied
C-peptide effects on a recently described vasopressin receptor; the
vasopressin-activated calcium mobilising receptor (VCAM-1). They found that in
human skin fibroblasts and human mesangial cells, C-peptide increased
expression of VCAM-1 at both RNA and protein levels [22]. The effect was
maximal with 1 nM C-peptide and inhibited by PTX. Although no direct link
between C-peptide-induced increases in VCAM-1 and enhanced Na+,K+-ATPase
activity was studied, enhancement of vasopressin action via upregulation of
VCAM-1 provides a further mechanism for C-peptide action relating to the Na+,K+-ATPase.


## 4. The Effect of C-Peptide on Endothelial  Nitric Oxide Synthase (ENOS)

One consistent observation following
in vivo administration of
C-peptide in type 1 diabetes is augmentation of microvascular blood flow to
tissues and organs including muscle, skin, and kidney [23]. The possibility
that these effects could be mediated by C-peptide regulation of nitric oxide
(NO) was first provided by the finding that increased glucose utilisation in
streptozotocin diabetic rats stimulated by C-peptide was sensitive to
inhibition of NOS by coadministration of N-monomethyl-l-arginine
(L-NMMA) [24]. This finding was in agreement with earlier studies that
described an NO-dependent pathway for glucose transport and metabolism by
muscle tissue [25, 26]. Subsequently, it was demonstrated that that C-peptide
mediated arteriolar dilatation was dependent on NO [27, 28].Details of the
mechanism underlying C-peptide’s ability to increase NO production have been
provided by Wallerath et al. [29].
Working with a bovine aortic endothelial cell model (BAEC), these investigators
reported that at the physiological postprandial concentration of 6.6 nM,
C-peptide stimulated NO release consequential upon a rise in intracellular Ca2+.
These authors speculated that C-peptide signalling resulted in activation of
the Ca2+-sensitive endothelial NOS (eNOS) following increased Ca2+ influx, thus explaining in large
part the vasodilatory effects of C-peptide observed in vivo [29].Other workers have
demonstrated upregulation of eNOS expression by C-peptide. In BAEC, enhanced NO
release stimulated by C-peptide was accompanied by upregulation of eNOS gene
transcription [30]. This effect appeared to be dependent on the upstream phosphorylation
and activation of extracellular signal-regulated mitogen activated protein
kinase (ERK).Activation of the NO
system by C-peptide signalling may also have other consequences. In C-peptide,
injected rats expression of eNOS is increased as is basal aortic NO production.
Reduced cell surface
expression of the adhesion molecules P-selectin and ICAM-1 on the
microvascular endothelium was observed and as a result leukocyte/endothelial
interactions were attenuated [31].


## 5. Stimulation of Mitogen Activated Protein Kinases (MAPKS) by C-Peptide

The MAPKs are evolutionary conserved enzymes that link
cell-surface receptors or chemical and physical stresses to key regulatory
targets within cells [32–34]. MAPKs phosphorylate multiple targets on serine
and threonine and, as a result, control many critical cell functions such as
growth, gene expression, and cell survival and adaptation. The MAPK family includes the ERKs 1 and 2,
c-Jun N-terminal kinases (JNK1, JNK2, JNK3), p38s (p38α, p38β, p38γ, p38δ), and ERK5.In recent years, several reports have described the
ability of C-peptide to induce phosphorylation and activation of members of the
MAPK family. The initial impetus for study in this area is developed from the earlier observations
of synergy between C-peptide and NPY signalling in the activation of Na+,K+-ATPase
[18]. NPY is well known to activate MAPK [35, 36] and this finding therefore
prompted Kitamura and colleagues to study possibility of MAPK mediated C-peptide
signalling in the mouse embryonic fibroblast cell line, Swiss 3T3  [37]. Using both immunoblotting of
phosphorylated ERK1 and ERK2 with phosphospecific antisera and an in vitro
kinase assay as  readouts of ERK
activation, these workers showed that incubation of 3T3 cells with C-peptide resulted
in brisk activation of ERK evident at a concentration as low as 1 pM and
maximal with 1 nM C-peptide [37]. This stimulatory effect was also seen with
NPY, and activation of ERK by both NPY and C-peptide was abolished by PTX.
Neither retrosequenced nor all D-amino
acid human C-peptide stimulated ERK. Not all cell types tested by these workers
responded to C-peptide with ERK activation. No effect was seen in 3T3-L1 cells,
L6E9 muscle cells, HepG2 hepatoma cells, NG108.15
neuroblastoma cells, or C6 glioma cells [37].Activation of ERK is involved in eNOS gene
transcription, an event previously also shown to be increased by C-peptide (see
above). In an attempt to link these phenomena, studies were performed in LEII
mouse lung capillary endothelial cells to examine the ability of C-peptide to
activate transcription factors [38]. In this cell type, C-peptide stimulated
both p38 and ERK MAPK activities, but not c-Jun N-terminal kinase. By
comparison, insulin was only able to activate ERK but not p38 MAPK. In addition,
C-peptide activated the cAMP response element (CRE)-binding protein (CREB)/activating
transcription factor-1 (ATF-1) in a p38 but not ERK-dependent manner, thereby
causing binding of these transcription factors to CRE. Consequently, it was
confirmed that enhanced eNOS transcription in BAECs following C-peptide
treatment was indeed MAPK-dependent [39]. Therefore differences in C-peptide
responses between various cell types clearly exist. In BAECs, ERK activation is
required for eNOS transcription, whereas transcription factor activation in
LEII cells follows C-peptide stimulation of p38.Loss of renal tubular cells is a prominent feature of
diabetic nephropathy. It is therefore of significance that tubular cell growth
and survival may be modulated by C-peptide signalling. In the opossum kidney
(OK) immortalised proximal tubular cell line C-peptide also potently activates
ERK, maximally at a concentration of 300 pM and declining thereafter with a
bell-shaped dose-response curve [40]. Furthermore, C-peptide also induced
activation of Akt in OK cells. This event was sensitive to wortmannin and
indicative of phosphatidylinositide-3-kinase (PI-3-kinase) activation. The dose-response
curve for Akt activation revealed a maximal effect at a C-peptide concentration
of 5 nM, remaining constant thereafter up to a C-peptide concentration of 100
nM, and thus being quite distinct from that for ERK activation. In addition,
C-peptide caused an influx of Ca2+ into the OK cells upon which a
consequent translocation and activation of PKCα were absolutely dependent [40]. These
findings are in agreement with the earlier studies of Tsimaratos et
al. that showed PKC-induced activation of kidney Na+,K+-ATPase
in C-peptide exposed tubular segments [21] (see above). Most importantly, these
signalling events had a functional consequence with significant enhancement of
proliferation seen in C-peptide treated cells [40]. All of these events were
sensitive to PTX again providing evidence of the likely presence of a GPCR for
C-peptide.Most recently C-peptide has been found to promote
translocation of the low molecular weight GTP-binding protein, Rho A, from the
cytoplasm to the membrane of human kidney proximal tubular cells [41]. This
effect was completely reliant on the upstream activation of phospholipase C
(PLC). In fact, in these cells the activation of ERK, JNK, as well as PKC-ε and -δ was each sensitive to PLC inhibition,
indicating an obligate dependency on upstream PLC activation by C-peptide. Once
more, all stimulatory effects of C-peptide were PTX sensitive [41].The molecular mechanisms by which
C-peptide activates MAPK can now be described with some precision. The signal
transduction pathway involves: (i) C-peptide binding to a PTX sensitive GPCR; (ii)
the activation of PLC; (iii) subsequently increased diacylglycerol and
intracellular Ca2+ levels stimulate several PKC isoforms; (iv) PKC-dependent
activation and translocation of RhoA to plasma membrane; and (v)
phosphorylation and activation of MAPKs.


## 6. C-Peptide Activation of Pi-3-Kinase

The PI 3-kinases are a family of enzymes
that phosphorylate the hydroxyl group at the 3 positions of the inositol ring of phosphatidyinositol. PI-3-kinases
regulate a remarkably diverse range of cellular functions, including growth,
proliferation, survival, differentiation, motility, and intracellular
trafficking [42]. The PI-3-kinases are also a key component of insulin
signalling pathways and this is of substantial interest in diabetes. Many of
these functions of PI-3-kinase are related to their ablilty to activate protien
kinase B (Akt) [42].Robust C-peptide activation of PI-3-kinase has now
been demonstrated by several authors in various cell types. As a result, it is
known that physiological concentrations of C-peptide can regulate several
aspects of PI-3-kinase signalling pathways in OK cells [40], Swiss 3T3
fibroblasts [37], SH-SY5Y neuroblastoma cells [43], human CD4+T
cells [44] L6 myoblasts [45]. As a direct consequence, it is now
recognised that stimulation of PI-3-kinase by C-peptide acting alone is responsible for (i)
enhancement of neuronal and kidney tubular cell proliferation [40, 43]; (ii)
increased T cell migration [44]; (iii) stimulation of peroxisome proliferators-activated
receptor-γ (PPARγ) in kidney tubule cells and associated gene transcription [46]; and (iv)
upregulated glycogen synthesis in skeletal muscle cells [45].


## 7. Effects of C-Peptide on Transcription Factors

Given that many of the changes in cell phenotype that
take place as a result of signalling by bioactive molecules are mediated by
altered gene and protein expression, it is not surprising that C-peptide
regulates activity of several key transcription factors.Extending their observations of C-peptide activation
of MAPKs Kitamura et al. described phosphorylation and activation of CREB,
ATF-1, and ATF-2 in LEII cells [38]. Indeed 1 nM C-peptide and phorbol ester, a
positive control, were equipotent in this regard. CREB and ATF proteins are
transcription factors that bind when activated to specific response elements,
CRE, in DNA thereby regulating transcription. Gel mobility shift assays clearly
showed the binding of CREB to CRE in C-peptide treated cells. In the latter
study, specific genes subject to regulation were not identified. However using
neuroblastoma cells, it was reported that C-peptide treatment enhanced
expression and translocation of nuclear factor-κB (NF-κB) and expression of the Bcl2 protein—an important
mediator of the antiapoptotic effects governed by NF-κB [43]. Control of NF-κB by C-peptide in Swiss 3T3 has also been
demonstrated. In these cells, 1 nM C-peptide activated transcription of
cyclooxygenase-2 (COX-2) via NF-κB consequent upon upstream activation of PKC [47]. COX-2, a cytokine
inducible gene, is the rate-limiting enzyme in the conversion of arachidonic
acid to prostaglandin, but the potential consequences of its upregulation by
C-peptide remain unclear.We performed detailed studies of transcription factor
activation in OK proximal tubular cells [46, 48], comparing C-peptide
effects to those of insulin and focussing on PPARγ and NF-κB. A member of the nuclear hormone
receptor family, PPARγ is the target for the insulin-sensitising
thiazolidinediones currently used as therapeutic agents in the treatment of
type 2 diabetes [49]. The expression of PPARγ may also be regulated by
insulin [50]. Using transient transfection of
a peroxisome proliferator response element (PPRE)-luciferase reporter construct,
we showed that both C-peptide and insulin transactivated PPRE via PPARγ [46].
C-peptide (EC50 4 nM) was more potent than insulin (EC50 10 nM) in this regard, but both
agents evoked phosphorylation of PPARγ similarly via activation of PI-3-kinase. One consequence of PPARγ activation by C-peptide
and insulin was enhanced transcription of the
prototypic PPARγ regulated gene, CD36 [46]. Clearly,
therefore, there is a degree of overlap between insulin and C-peptide
signalling with both agent’s effects being directed via PI-3-kinase towards
PPARγ. However, only the effects of C-peptide were
attenuated by PTX and therefore C-peptide must be signalling through a receptor
system fundamentally distinct from that of insulin. These data indicate an
important novel mechanism whereby C-peptide and insulin may interact to
regulate glycaemia and the expression of PPAR regulated genes such as those involved
in metabolic control and inflammation.The next step was to establish proof of principle
that C-peptide had the capacity to act as a protective agent in diabetic
nephropathy. To this end, we investigated whether C-peptide could counteract
adverse effects precipitated by the administration of TNF-α to OK proximal tubular cells [48].
TNF-α is recognised as a major player in the development of diabetic
nephropathy and may contribute to tubular cell apoptosis and tubular atrophy
prominently observed in diabetic nephropathy [51–55]. TNF-α is a pleiotropic peptide cytokine, capable of
eliciting a wide spectrum
of cellular responses including differentiation, proliferation, inflammation,
and cell death via interaction with two members of the TNF receptor family,
TNF-R1 and TNF-R2 [54]. Predominantly produced by monocytes/macrophages but
also by T and B-lymphocytes and glomerular mesangial cells [55, 56], TNF-α binding to TNF-R1 may simultaneously trigger apoptotic pathways by
recruitment of death effector adaptor molecules with subsequent activation of
caspase cascades, and antiapoptotic pathways by a pathway involving TNF receptor-associated factor2 (TRAF2) and nuclear
factor-κB (NF-κB). Integration of these events determines the eventual cellular
response to TNF-α stimulation. In particular NF-κB stimulates transcription of antiapoptotic factors that modulate the
caspase cascade, and thus NF-κB activity acts as a checkpoint in a
cell’s decision to survive or apoptose in response to a given stimulus.When applied to OK cells, TNF-α markedly reduced viability and induced apoptosis [48]. This
was completely prevented by pretreatment with insulin or C-peptide. At the same
time both insulin and C-peptide activated NF-κB as judged by luciferase reporter assay. Insulin demonstrated a typical
sigmoidal dose-response of NF-κB activation, maximal at an applied
concentration of 100 nM. On the other hand, C-peptide displayed completely
different bell-shaped curve of NF-κB stimulation, maximal with a 5 nM applied concentration. As in previous
studies, PTX blocked only the C-peptide effect. However in these studies, the
presence of a Gαi-linked GPCR was revealed using an additional
technique whereby C-peptide but not insulin was shown to stimulate GTPγS binding to Gαi in OK cell membranes. In this work, the ability of
C-peptide to prevent TNF-α-induced apoptosis appeared to be due to
its ability to induce the expression, via NF-κB activation, of survival genes such as TRAF2 [48].This study provided evidence for the ability of C-peptide to regulate the expression of
beneficial genes in the face of pathophysiological stimuli relevant to the development
and pathology of diabetic nephropathy. Again, C-peptide appeared to be acting
via Gαi, and although C-peptide shared some signalling
properties with insulin it clearly possessed its own unique signalling
capabilities.


## 8. Is C-Peptide an Insulin Mimetic?

There is no doubt that some components of C-peptide
signalling pathways are shared with those
of insulin. For example, activations of MAPKs and
PI-3-kinase are well described downstream events following insulin-insulin
receptor interactions [57]. In the absence of a fully characterised C-peptide
receptor, and given that C-peptide exhibited some insulin-like actions such as
increasing muscle glucose transport [58, 59], Grunberger et al. wondered
whether C-peptide
signalling may simply be explained by activation of the insulin receptor
signalling system. They found in L6 myoblasts that C-peptide (0.3–3 nM)
activated insulin receptor tyrosine kinase, tyrosine phosphorylation of the
insulin receptor substrate-1 (IRS-1), MAPK, PI-3-kinase, p90 ribosomal S6
kinase (p90RSK) and glycogen synthase kinase-3 (GSK3), and glycogen synthesis
[45]. Submaximal concentrations of insulin and C-peptide were additive in
effect, whereas maximal concentrations were not. C-peptide stimulation of
glycogen synthesis was not abrogated by PTX, although earlier specific steps in
the signalling pathway were not tested in this way [45]. These findings
contrasted with those of Zierath et al. who found no evidence of insulin
receptor activation by C-peptide in human skeletal muscle [59]. Nonetheless
their results led Grunberger et al. to
speculate that, amongst other possibilities, C-peptide per se may activate the insulin receptor [45].There are several lines of evidence against this
however. The most compelling is the description by multiple authors of the
specific PTX sensitivity of C-peptide effects, and most recently the
observation that C-peptide provokes GTPγS binding to Gαi, [48] an observation
compatible with guanine nucleotide exchange occurring on a G-protein after ligation of a GPCR.
Furthermore, the dose-response curves for insulin are typically sigmoidal
whereas those for C-peptide are often bell shaped with a diminution of activity
at higher concentrations. At least in
vitro C-peptide appears more potent on a molar basis than insulin.
Typically C-peptide signalling effects are apparent at midhigh pM
concentrations, maximal at ∼1–5 nM and thereafter decline. In contrast, insulin
responses generally become evident at ∼10 nM increasing up to 100–1000 nM where
they plateau. Finally, using plasmon resonance no interaction of C-peptide with
soluble purified insulin receptor A or insulin-like growth factor receptor was
detected in a very recent study [60].The balance of evidence suggests that C-peptide mediates its own distinct signalling through a
GPCR. There is certainly some overlap between the signalling pathways activated
by C-peptide and insulin but this is a common observation in biological
systems. Crosstalk between GPCR activated signalling and receptor tyrosine
kinase activated pathways occurs by transactivation. This well-described
mechanism of cross-communication between signalling systems enables the cell to
integrate multiple signals derived from its environment.  Transactivation of the epidermal growth
factor receptor following agonist stimulation of Gαi or Gαq coupled GPCRs
represents the paradigm for this system, however similar events have now been described
for the platelet derived growth factor receptor, the insulin-like growth
factor-1 receptor, and IRS-1.


## 9. Summary and Conclusion

In the last 10 years, there has been a
revolution in thinking about C-peptide. There can now be no doubt that
C-peptide truly exerts a variety of biological effects. These effects are
underpinned by solid evidence of cell signalling events (summarised in Table1),
most likely mediated via a specific GPCR. The concentrations of C-peptide
required to elicit signalling outputs are entirely congruent with what is known
about binding affinities of C-peptide for its putative receptor. It should be
acknowledged however that the lack of detailed identification/cloning of a
receptor remains a weakness in the field. Nonetheless, the importance of these
signalling effects cannot be underestimated. The pathways stimulated are
fundamental to cell function and phenotype, impacting on such basic processes
as life and death. The next challenge is to identify the receptor and to bring
C-peptide nearer to the clinical arena in order to test its potential benefits
in ameliorating the devastating consequences and complications of diabetes.Table 1
Signalling elements activated by C-peptide according to cell type
studied (for abbreviations see text).
Cell type studiedSignalling elementKidney tubularMuscleFibroblastsEndothelialErythrocytesNeuroblastomaCD4 + T CellsNerveNa+,K+-ATPase+++↑[Ca2+]i++PKC++NO release++ERK++++p38 MAPK+JNK+PLC+p90RSK+GSK3+PI3K+++++CREB+NF-κB+++PPARγ+



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