INVITED REVIEW
Role of C-peptide in human physiology
John
Wahren1,
Karin
Ekberg1,
Jan
Johansson2,
Mikael
Henriksson2,
Aladdin
Pramanik2,
Bo-Lennart
Johansson1,
Rudolf
Rigler2, and
Hans
Jörnvall2
1 Department of Surgical Sciences, Section of
Clinical Physiology, Karolinska Hospital, SE-171 76 Stockholm; and
2 Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden
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ABSTRACT |
The C-peptide of proinsulin is important
for the biosynthesis of insulin but has for a long time been considered
to be biologically inert. Data now indicate that C-peptide in the
nanomolar concentration range binds specifically to cell surfaces,
probably to a G protein-coupled surface receptor, with subsequent
activation of Ca2+-dependent intracellular signaling
pathways. The association rate constant, Kass, for
C-peptide binding to endothelial cells, renal tubular cells, and
fibroblasts is ~3 · 109
M
1. The binding is stereospecific, and
no cross-reaction is seen with insulin, proinsulin, insulin growth
factors I and II, or neuropeptide Y. C-peptide stimulates
Na+-K+-ATPase and endothelial nitric oxide
synthase activities. Data also indicate that C-peptide administration
is accompanied by augmented blood flow in skeletal muscle and skin,
diminished glomerular hyperfiltration, reduced urinary albumin
excretion, and improved nerve function, all in patients with type 1 diabetes who lack C-peptide, but not in healthy subjects. The
possibility exists that C-peptide replacement, together with insulin
administration, may prevent the development or retard the progression
of long-term complications in type 1 diabetes.
sodium-potassium-adenosine 5'-triphosphatase; endothelial
nitric oxide synthase; renal function; autonomic nerve function; G
protein
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INTRODUCTION |
DURING THE COURSE OF INSULIN SYNTHESIS, C-peptide is
cleaved from proinsulin, stored in secretory granules, and eventually released into the bloodstream in amounts equimolar with those of
insulin (45, 52, 53). C-peptide has an essential function in the
synthesis of insulin in that it links the A and B chains in a manner
that allows correct folding and interchain disulfide bond formation.
When C-peptide is removed from proinsulin by proteolytic processing,
the COOH-terminal part of insulin's B-chain becomes exposed and free
to assume an appropriate conformation for effective interaction with
the insulin receptor (51). A schematic representation of the proinsulin
molecule and its components is given in Fig. 1.

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Fig. 1.
Linear representation of human proinsulin indicating amino acid
sequence of C-peptide and showing position of COOH-terminal
pentapeptide, which mimics action of C-peptide in assays of binding and
Na+-K+-ATPase activity.
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After the discovery of the mode of insulin biosynthesis, several early
studies addressed the question of possible physiological effects of
C-peptide. Insulin-like effects on blood glucose levels and on glucose
disposal after glucose loading were looked for but not found (14, 17).
Rat C-peptide was, however, found to diminish glucose-stimulated
insulin release in rats both in vivo and in vitro (6, 57, 58, 63, 64),
whereas the corresponding findings in other animals were less
conclusive (8, 32). C-peptide was also reported to inhibit
arginine-stimulated glucagon release from the isolated perfused rat
pancreas (63) and to inhibit fat-stimulated gastric inhibitory
polypeptide secretion by intestinal cells (6). Despite
these reported effects, it became generally accepted that C-peptide
possesses little or no biological activity and has no other role than
its participation in insulin synthesis (33), a role that is emphasized
by its name, "connecting peptide." Recently, new data have been
presented demonstrating specific binding of C-peptide to cell
surfaces in a manner that suggests the presence of G protein-coupled
membrane receptors. C-peptide may thereby stimulate specific
intracellular processes, influencing renal and nerve function in
C-peptide-deficient type 1 diabetes patients. This contribution aims to
review these new findings and to examine the possible physiological
role of C-peptide.
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C-PEPTIDE BINDING |
The classic manner in which bioactive peptides exert their effects is
via specific binding as ligands to receptors. In such binding, a
limited region of the ligand serves as "active site," effecting
the binding to the receptor. This segment of the peptide is frequently
well conserved across species borders. The binding process can usually
be studied via binding of a labeled peptide in a radioligand assay. In
the case of proinsulin C-peptide, the lack of the two basic
concepts, a conserved active site and an established ligand assay, has
long hampered the recognition of C-peptide as a bioactive hormone per
se. Instead, nonreceptor membrane interactions have been suggested to
explain some C-peptide effects (19). Recently, however, binding studies
using new technology have established a typical receptor interaction
for C-peptide (44). Consequently, it now appears that C-peptide can be
recognized as a receptor ligand; it is just that some properties were
difficult to define initially and that additional or multiple effects
may exist. The molecular interactions that currently form the basis of
a receptor concept are outlined below.
Radioligand binding.
The first study describing interactions between C-peptide and cell
membranes appeared in 1986. Binding of tyrosylated
125I-labeled rat C-peptide 1 was examined by use
of cultured rat islet tumor cells (10). Evidence for specific binding
of C-peptide was reported, and a Scatchard plot of the data suggested a
nonlinear course. The demonstration of C-peptide binding provided
support for the earlier observations that C-peptide influences the
function of the islet cells (6, 57, 63, 64). Interpretation of the results was complicated, however, by the ongoing
secretion of C-peptide from the cells during the binding studies and by the existence of two different rat C-peptides (55). There is a lack of other studies describing C-peptide binding to cell membranes by use of the radioligand binding technique; only one report for skeletal muscle cell membranes, with negative result, is available (68). Relatively few binding sites per unit cell surface area (10) may
have contributed to the difficulties in demonstrating cellular binding of C-peptide.
Binding evaluated via
Na+-K+-ATPase
stimulation.
Intracellular effects of C-peptide have been examined by using fresh
preparations of proximal segments from the rat nephron, a well defined
experimental model (1, 5). Addition of homologous C-peptide
to the tubular segments was found to increase the intrinsic Na+-K+-ATPase activity in a
concentration-dependent manner (42) (Fig. 2). Moreover, pretreatment of the tubular
segments with pertussis toxin completely blocked this effect. These and
other observations indicate that a G protein interacting with a
ligand-activated receptor may be involved in a C-peptide signal
transduction pathway. Pertussis toxin treatment, known to affect the
-subunit of Gi proteins, may thus interfere with the
interaction between the G protein and a loop region of a
membrane-spanning receptor (15), which in turn could abolish the
C-peptide effects. Although the definition of a receptor is lacking,
the Na+-K+-ATPase studies (42, 43) have
established that C-peptide is capable of eliciting molecular
interactions in cellular systems and that these interactions can be
measured, albeit indirectly. The above results support the notion that
C-peptide effects are mediated via a receptor and that G proteins may
be involved in the signal transduction pathway.

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Fig. 2.
Stimulation of Na+-K+-ATPase activity (means ± SE) in renal tubular segments from rats by homologous C-peptide in
different concentrations. Data are from Ref. 42.
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Fragment activities.
Further studies of the Na+-K+-ATPase activity
of rat renal tubular segments involved a set of C-peptide fragments,
essentially covering the entire length of C-peptide in different
subsets and analogs (43). Two sets of rat C-peptide fragments were
found to elicit stimulatory effects on
Na+-K+-ATPase activity, suggesting the presence
of two different types of interactive sites. One was localized to the
COOH-terminal part of the molecule, with maximal activity from the
COOH-terminal pentapeptide segment, and the other to the midsegment of
C-peptide, with maximal activity from the segment corresponding to
positions 11-19 (43).
The results for the COOH-terminal pentapeptide were characteristic of a
receptor-ligand interaction. This pentapeptide gave full replacement of
the entire C-peptide activity; the remaining part of the molecule,
des-(27
31)-C-peptide, was without effect in this assay, and the
effects were residue specific (43). Consequently, the pentapeptide data
supported the notion of a specific C-peptide receptor that recognizes
the COOH-terminal pentapeptide segment. Notably, the residues at
positions 1 (Glu) and 5 (Gln) of the pentapeptide are conserved in most
mammalian species (Table 1). The above
results for C-peptide fragment stimulation of
Na+-K+-ATPase were all obtained in studies
involving rat tubular segments (43), but similar findings have also
been made for rat pancreatic islets (60).
C-peptide fragments from the midsegment behaved in a different manner.
Their replacement activity was incomplete (maximum 80% of that for the
intact molecule), remaining parts of C-peptide did not lack activity in
the assay, and their fragment activities were not residue specific
(fragments with other or nonnatural D-amino acid residues
were also partly active) (43). The data for the midsegment fragments
suggested the existence of a second "site" with nonspecific
interactions in a receptor-atypical manner, apparently less effective
than the receptor-like interactions for the COOH-terminal pentapeptide
(43).
In conclusion, the combined C-peptide fragment data support the concept
of a traditional receptor binding of C-peptide, localized to its
COOH-terminal segment, but they also indicate the possibility of
secondary effects via other mechanisms and other segments of C-peptide.
Nonreceptor interactions.
In a report by Ido et al. (19), it was suggested that C-peptide effects
may be elicited via a mechanism that defies the general rule that
peptide hormones act by binding to stereospecific receptors. Human
C-peptide was found to exert beneficial effects on vascular and neural
dysfunction induced by experimental diabetes in rats. These effects
were seen not only with the native C-peptide but also with its
D-enantiomer and with reverse-sequence C-peptide (19). The
authors suggested that the effects of C-peptide may be independent
of chirality and peptide bond direction and exerted via mechanisms
similar to those of amphipathic antimicrobial peptides. The latter
peptides elicit their effects by nonchiral interactions with membranes,
resulting in the formation of ion channels and interference with
phospholipase A activity (2). Ido et al. suggested that the
glycine-rich segment in the central region of C-peptide (positions
13-17) was important for such biological activity (47). As
discussed above, this conclusion agrees with the second
site deduced from the fragment studies involving
determinations of Na+-K+-ATPase activity (43).
The central region of C-peptide is largely achiral and reasonably well
conserved in mammalian species (Table 1). It should be noted, however,
that the C-peptide molecule differs considerably from that of
antimicrobial peptides, and that the negatively charged C-peptide may
not easily interact with cell membranes to form cation-selective
channels. Thus direct measurements under in vitro conditions have
recently demonstrated that C-peptide fails to bind to lipid vesicles
(16) and that its secondary structure is not altered in the presence of
lipid membranes (16). Consequently, the nonreceptor effects, although observed in two different assays, testing vascular function (19) and
Na+-K+-ATPase stimulation (43), are not only
not ascribable to receptor interactions but also not explainable in
terms of membrane interactions in general. Furthermore, in the study by
Ido et al., heterologous C-peptide was used in a supraphysiological
concentration (100 nM), and the effects required 2-3 days to
become evident, which complicates the evaluation of those findings. It
can be concluded that C-peptide midsegment effects may exist but do not
appear to involve receptor or membrane interactions and do not
interfere with conclusions regarding the receptor-like interactions of
the pentapeptide COOH-terminal segment.
Fluorescence correlation spectroscopy.
New evidence that C-peptide binds to specific cell surface receptors
has recently been reported (44). Thus fluorescence correlation
spectroscopy (FCS) has been applied to the evaluation of
C-peptide-membrane interactions. In FCS, the Brownian movements of a
fluorophore-marked ligand are observed after excitation by a sharply
focused laser beam (9, 38). The small volume element (<1 · 10
15 l) in
which the measurements are performed can be shifted from the incubation
medium containing the labeled ligand to the membrane surface of cells
growing in culture. From the autocorrelation function of the
fluctuations in fluorescence intensity, it is possible to characterize
the diffusion time of the labeled ligand when it is free in the
incubation medium or bound to a cell membrane (9, 38). Compared with
the radioligand binding method, the FCS technique has the advantage of
higher detection sensitivity and an improved signal-to-noise ratio
combined with submicron resolution (9, 38).
In the case of C-peptide, labeling for FCS was achieved by attachment
of tetramethyl-rhodamine to the NH2-terminal end of the
molecule. Specific binding of human C-peptide could be demonstrated for
cultured human renal tubular cells, skin fibroblasts, and saphenous
vein endothelial cells (44). The association rate constant
(Kass) for C-peptide binding was
~3 · 109
M
1, and the specificity of the binding
was evidenced by the consistent displacement of bound C-peptide by
excess unlabeled C-peptide. Addition of a peptide with the same amino
acid composition as human C-peptide but with the residues arranged in
random sequence (scrambled C-peptide), or of D-enantio
C-peptide, failed to displace bound C-peptide, demonstrating the
stereospecific nature of the binding. In contrast, addition of excess
COOH-terminal pentapeptide competitively displaced bound C-peptide,
indicating that the COOH-terminal segment is involved in the binding
process as measured by FCS, in agreement with the ability of the
pentapeptide to stimulate Na+-K+-ATPase
activity (43). Proinsulin, which includes the pentapeptide segment,
failed to elicit displacement of bound C-peptide, suggesting that the
free COOH-terminal end of the segment is required for binding.
Likewise, addition of insulin, insulin-like growth factor (IGF)-I,
IGF-II, or neuropeptide Y (NPY) was not accompanied by displacement of
bound C-peptide, indicating absence of cross-reactions with these
hormones and their membrane receptors. Labeled insulin bound to cell
membranes was not displaced by excess concentrations of C-peptide (44,
68). In the case of proinsulin, the finding is in contrast to reports
that C-peptide may bind with low affinity to a proinsulin receptor (22,
23). Finally, preliminary FCS evidence indicates the presence of
species specificity; at physiological concentrations, rat C-peptide
fails to bind to human cells (unpublished observation).
Preincubation of cells with pertussis toxin was found to abolish
FCS-measurable binding of C-peptide at physiological concentrations (44), in agreement with the findings from the
Na+-K+-ATPase data (42, 43). The FCS binding
results before and after treatment of the cells with pertussis toxin
are consistent with a typical allosteric mechanism of signal
transduction: before pertussis toxin treatment there was a
high-affinity interaction between C-peptide and its membrane receptor,
reflecting one configuration of the receptor. After pertussis toxin,
the FCS data indicated only a small component of low-affinity
interaction between C-peptide and the receptor, which then most
likely has assumed a new configuration secondary to the effect of
pertussis toxin on the
-subunit of the G protein (15).
It has been a consistent finding that effects of C-peptide
cannot be demonstrated in normal animals or healthy subjects (14, 17,
19, 21, 28, 64, 65); it is only in animals with experimental diabetes
or patients with type 1 diabetes and, consequently, no or very low
C-peptide plasma concentrations, that specific effects have been
observed (11, 25-31, 35, 37, 50, 65). This may have to
do with the binding characteristics for C-peptide (Fig.
3). Half-saturation of C-peptide binding to
renal tubular cells determined by FCS was found to occur already at a
concentration of 0.3 nM, and full saturation was seen at ~0.9 nM
(44). Thus it is likely that, in healthy humans, receptor saturation is
reached already at the ambient physiological C-peptide concentration
(0.5-1.5 nM), so that no additional biological activity can be
expected from a further increase in C-peptide concentration. This may
explain why no C-peptide effects were seen in the early studies
involving healthy humans and animals (14, 17, 64). For a further
understanding of the detailed nature of the C-peptide binding and its
physiological effects, the receptor structure will have to be
determined. Moreover, the FCS results quantitate the binding affinity
to that applicable to physiological C-peptide levels and provide a
C-peptide binding assay.

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Fig. 3.
Binding of rhodamine-labeled human C-peptide to human renal tubular
cells as evaluated by fluorescence correlation spectroscopy (FCS) in
concentration range of 0-5 nM. Association rate constant
(Kass) for C-peptide binding was
3.3 · 109 M 1. Values
are means ± SE shown as %. Data are from Ref. 44.
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The combined C-peptide binding data from the five different approaches
outlined above provide new insights into C-peptide membrane
interactions. There is evidence of stereospecific binding of C-peptide
to a cell surface receptor, which most likely is G protein coupled. The
binding occurs in the low nanomolar concentration range, and the
COOH-terminal pentapeptide appears to mediate the effects. This
interaction fits the classic ligand-receptor concept. In addition,
there are indications of another type of nonspecific, nonchiral
membrane interaction localized to the midsegment of C-peptide, which will require further evaluation for detailed understanding.
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SIGNAL TRANSDUCTION AND CELLULAR EFFECTS |
As mentioned above, rat C-peptide was found to elicit a
concentration-dependent stimulation of
Na+-K+-ATPase activity at nonsaturating
Na+ concentrations (42) (Fig. 2). Rat C-peptides 1 and 2, which differ by two amino acid residues (55), were largely
equipotent in stimulating Na+-K+-ATPase
activity (43), and scrambled C-peptide had no detectable effect.
Subsequent reports have demonstrated that C-peptide stimulates Na+-K+-ATPase activity also in rat sciatic
nerve (19, 54) and granulation tissue (19), pancreatic islets (61), and
red blood cells (4). C-peptide also ameliorates the impaired
deformability of red blood cells from type 1 diabetes patients (35).
This effect was abolished after pretreatment of the erythrocytes with
ouabain, compatible with C-peptide effects being mediated via
stimulation of Na+-K+-ATPase activity (35),
known to be reduced in red blood cells from patients with type 1 diabetes (7).
The C-peptide effect on Na+-K+-ATPase activity
of renal cells was inhibited by pretreatment of the cells with
pertussis toxin (42), as outlined above. The results of
Ohtomo et al. (42) also indicate that C-peptide activates
Ca2+-dependent intracellular signaling pathways. Exposure
of cultured proximal convoluted tubular cells to homologous C-peptide
in the concentration range 10
11-10
9 M
resulted in rapid and consistent increments of the intracellular Ca2+ concentration. It is noteworthy that the C-peptide
concentration required for increasing the intracellular
Ca2+ concentration (42) was less than that needed for
stimulation of Na+-K+-ATPase activity in renal
tubule segments (Fig. 2) (42). This is most likely related to the fact
that the tubule segment, but not the cultured cells, underwent a
preparation procedure involving freezing, thawing, and collagenase
treatment, possibly resulting in interference with cell membrane
structures (42). When the cultured renal tubule cells were maintained
in a calcium-free medium, exposure to C-peptide failed to increase
intracellular Ca2+ levels. Moreover, addition of FK506, a
specific inhibitor of calcium/calmodulin-dependent protein phosphatase
2B (PP2B), resulted in complete inhibition of the stimulatory effect of
C-peptide. PP2B is of major importance in the regulation of
Na+-K+-ATPase activity in tubular cells because
of its ability to convert the phosphorylated, inactive form of
Na+-K+-ATPase to its dephosphorylated, active
form (1, 18). The simplified, overall signal transduction pattern that
emerges is thus that C-peptide activates a membrane receptor coupled to
a pertussis toxin-sensitive G protein, with subsequent activation of
Ca2+-dependent signaling pathways and stimulation of PP2B,
resulting in increased Na+-K+-ATPase activity
(Fig. 4).

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Fig. 4.
Available data are compatible with the hypothesis that C-peptide binds
to cell membrane receptors coupled to a pertussis toxin-sensitive G
protein. The G protein activates Ca2+ channels, resulting
in an increased intracellular Ca2+ concentration and
activation of both endothelial nitric oxide synthase (eNOS) and
Ca2+-calmodulin-dependent protein phosphatase 2B (PP2B).
PP2B subsequently converts the phosphorylated form of
Na+-K+-ATPase into its dephosphorylated, active
form.
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The possibility exists that C-peptide may act in synergism with other
hormones. This is suggested by the finding that the dose-response curve
for C-peptide and Na+-K+-ATPase activity in
renal tubular segments was shifted more than two orders of magnitude to
the left, indicating increased C-peptide effectiveness, in the presence
of subthreshold concentrations of NPY (42). NPY is released after
activation of the sympathetic adrenergic system and is known to act
synergistically with norepinephrine (59). Tissue levels of NPY are
upregulated in animals with experimental diabetes and C-peptide
deficiency (62). The results may suggest that C-peptide effects are
dependent on sympathetic adrenergic activity. It is noted that, with
regard to stimulation of Na+-K+-ATPase
activity, no synergistic interaction was observed between C-peptide and
insulin (42). In contrast, the smooth muscle-relaxing effect exerted by
C-peptide (human) on rat muscle arterioles was potentiated by the
presence of a low insulin concentration (24). Further experimental work
will be required for a better understanding of these phenomena.
Administration of C-peptide to type 1 diabetes patients is accompanied
by circulatory responses: it results in increased blood flow in
skeletal muscle at rest (29) and during exercise (28), augmented
capillary blood cell velocity and redistribution of skin microvascular
blood flow (11), and increased renal blood flow (31). Again, no effects
are observed in healthy subjects (28) or animals (21). C-peptide has
also been found to increase blood flow, capillary filtration
coefficients, and the permeability surface-area product in the isolated
perfused rat hindquarter, primarily indicating recruitment of
capillaries (36). The cellular mechanism underlying this vasodilator
effect of C-peptide has not been fully established, but preliminary
evidence suggests that C-peptide, via an increase in the intracellular
Ca2+ concentration, stimulates endothelial nitric
oxide synthase (eNOS) activity (30, 34, 37) (Fig. 4). Thus
C-peptide (human) was found to increase NO release from
bovine aortic endothelial cells in a concentration-dependent manner.
The C-peptide-induced nitric oxide (NO) release was abolished by the
addition of an NO synthase inhibitor (34). Further support for the
notion that C-peptide administration may be accompanied by augmented NO
formation is provided by the observation that forearm blood flow
increments induced by C-peptide in type 1 diabetes patients can be
inhibited by a NO synthase blocker (30). In agreement with the above
findings, it has been reported that C-peptide induces a
concentration-dependent dilatation of rat skeletal muscle arterioles
and that this occurs via a NO-mediated mechanism (24).
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C-PEPTIDE AND RENAL FUNCTION |
Patients with type 1 diabetes frequently develop glomerular
hyperfiltration early in the course of their disorder (39, 40). Adequate insulin therapy does not correct this phenomenon (46). In
contrast, patients with type 2 diabetes, in whom insulin and C-peptide
levels are within or above the normal range, do not show glomerular
hyperfiltration or hypertrophy (12, 48). These considerations prompted
studies of possible C-peptide effects on renal function in type 1 diabetes patients (31). A group of young patients without signs of late
diabetic complications were studied under euglycemic conditions.
C-peptide infusion for 3 h at a rate sufficient to raise its
concentration to physiological levels (~0.9 nM) decreased the
glomerular filtration rate (GFR) by 7%, and renal plasma flow
increased modestly. Infusion of C-peptide to reach higher
concentrations (~2.1 nM) was not accompanied by further changes in
GFR or renal plasma flow. Even though the observed effect was modest,
it did establish a significant renal effect of C-peptide (31).
The influence of C-peptide on glomerular hyperfiltration, functional
reserve capacity, and renal protein leakage have been examined in
streptozotocin diabetic rats (50). Administration of C-peptide (human)
for 90 min was accompanied by diminished glomerular hyperfiltration
(-20%), improved functional reserve as evidenced by augmented GFR
after glycine loading, and a marked reduction (
70%) in protein
leakage compared with diabetic control animals. The specificity of the
C-peptide effects was evident from the observation that infusion of
scrambled C-peptide had no effect. Further evaluation of renal
C-peptide effects in patients with type 1 diabetes has been extended to
include more prolonged administration. In a double-blind, randomized
study, patients with type 1 diabetes received by subcutaneous pump
infusion for 4 wk either insulin plus equimolar amounts of C-peptide or
insulin alone (27). In the C-peptide-treated group, GFR decreased on average by 6%, whereas no changes in GFR were seen in the group receiving insulin only. Moreover, a significant reduction in the level
of urinary albumin excretion was seen in the C-peptide group but not in
the group given insulin only (27).
The above findings in patients have been further explored in a study
involving C-peptide administration for 3 mo (25). The aim was to
evaluate the possibility that C-peptide administration may reduce the
level of microalbuminuria in patients with early signs of diabetic
nephropathy. Patients were studied in a double-blind, randomized,
crossover design and received C-peptide plus insulin for 3 mo
and insulin only for 3 mo. All patients were normotensive and had
urinary albumin excretion rates between 25 and 220 µg/min before the
study. During the C-peptide study period, urinary albumin excretion
decreased progressively to significantly lower values than those found
during the control period (Fig. 5). The
albumin excretion had decreased by ~40% at the end of the 3-mo
period, whereas no significant change occurred during the control
period. A similar response was seen in the urinary
albumin-to-creatinine ratio. The patients remained normotensive
throughout the study, and glycemic control improved slightly but to the
same extent in the two treatment groups. Thus the diminished albumin
excretion during C-peptide administration could not be ascribed to a
reduction in arterial blood pressure or an amelioration of blood
glucose control (25). The mechanism underlying the beneficial effect of
C-peptide on renal function in diabetes is not known. However, it is
possible that C-peptide may have exerted a direct effect on the
glomerular handling of albumin, as suggested by the studies of renal
function in animals with experimental diabetes (50). As discussed
above, C-peptide has the capacity to stimulate both renal
Na+-K+-ATPase (42, 43) and eNOS (34), and it
may be hypothesized that C-peptide can influence glomerular membrane
permeability and transport, as well as regional blood flow of the
kidney, possibly leading to improvements in renal function in the
diabetic state.

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Fig. 5.
Urinary albumin excretion expressed as % of basal value in patients
with type 1 diabetes during treatment with C-peptide plus insulin
(hatched columns) and insulin alone (open columns). Data were
calculated from geometric means of urinary albumin excretion.
Differences between the 2 treatments were significant after 2 mo
(P < 0.05) and 3 mo (P = 0.01). Data are from Ref.
25.
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In summary, the present evidence demonstrates that C-peptide has the
capacity to diminish glomerular hyperfiltration and reduce urinary
albumin excretion in both experimental and type 1 clinical diabetes.
Studies involving C-peptide administration of longer duration will be
required to determine whether C-peptide may have a role in the
prevention and treatment of diabetic nephropathy.
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C-PEPTIDE AND NERVE FUNCTION |
Diabetic neuropathy is an important clinical feature of type 1 diabetes. Either the peripheral or the autonomic nerves, or both, may
be involved. Autonomic nerve dysfunction is found in nearly 40% of the
patients, even though just a few present with clear-cut symptoms. The
etiology of diabetic neuropathy is not fully understood. In addition to
the toxic effects of elevated blood glucose levels, the possible
influence of vascular dysfunction involving the vasa nervorum has been
implicated (56). Reduced levels of endoneurial
Na+-K+-ATPase (19) and diminished nerve blood
flow (3, 20) are reported for the diabetic state. The effect of
C-peptide on nerve function in diabetes has been evaluated in animal
studies (streptozotocin-diabetic rats). C-peptide (human) prevented the
decreased caudal motor nerve conduction velocity (MNCV) in diabetic
rats but did not affect MNCV in healthy control rats (19). In the same
study, C-peptide administration prevented the diabetes-induced
reduction of sciatic nerve Na+-K+-ATPase
activity. Similar results have been reported for spontaneously diabetic
insulin-deficient BB/W rats (54). Administration of homologous
C-peptide for 2 mo resulted in significant improvements in MNCV and
nerve Na+-K+-ATPase levels compared with
untreated controls.
Data from nerve function studies in patients with type 1 diabetes are
also available. Patients with symptoms of diabetic polyneuropathy were
studied twice under euglycemic conditions and during a 3-h intravenous infusion of either human C-peptide or saline in a double-blind study (26). Plasma concentrations of C-peptide rose to
levels within the physiological range during C-peptide infusion. Heart
rate variability during deep breathing, an indicator of autonomic,
primarily vagal nerve activity, rose markedly (+50%) during C-peptide
infusion but did not change during saline administration (Fig.
6). A significant improvement was also seen
in the brake index during tilting in the patients who showed a reduced
index before the study; no response was observed during saline infusion (26). Indexes of motor or sensory nerve function did not change significantly during C-peptide infusion. Nerve function has also been
evaluated in a subgroup of the patients who received C-peptide for 3 mo
in studies of renal function (25). The patients showed signs of
autonomic nerve dysfunction before the study, and after 3 mo
of C-peptide administration their heart rate variability during deep
breathing had improved by 20%. In contrast, no change or a slight
deterioration was seen in the same patients during the control period
with insulin therapy only. Signs of sensory neuropathy were present
before the study in six of these patients; improved sensory nerve
function, as evidenced by significantly improved temperature threshold
discrimination, was observed after the 3-mo C-peptide treatment but not
after the control period (25). Metabolic control was similar during the
two study periods (25).

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Fig. 6.
Heart rate variability during deep breathing in type 1 diabetes
patients expressed as %variability in R-R interval during expiration
and inspiration. Measurements were made before and after 3 h of iv
infusion of C-peptide or saline. Values are means ± SE. Data are from
Ref. 26.
|
|
In summary, the combined experimental and clinical data provide
evidence that C-peptide administration may ameliorate nerve dysfunction
in type 1 diabetes. The stimulatory effect of C-peptide on nerve
Na+-K+-ATPase activity (19, 54) and eNOS (34)
may provide a background to the findings. The possibility that
C-peptide may be beneficial in the treatment of diabetic neuropathy
warrants further studies involving more prolonged periods of C-peptide
administration. All of the above results relate to experimental and
clinical type 1 diabetes. Whether C-peptide might exert a similar
influence on nerve function in patients with type 2 diabetes, a
disorder characterized by elevated levels of both insulin and C-peptide during its first phase, remains to be determined. However, there is
preliminary evidence to indicate that there may be resistance to the
action of C-peptide in muscle tissue from type 2 diabetes patients
(66).
 |
C-PEPTIDE AND GLUCOSE UTILIZATION |
Early studies of C-peptide effects demonstrated that supraphysiological
concentrations of C-peptide increase and extend the hypoglycemic effect
of insulin in alloxan-diabetic rats (64). At an early stage, a possible
effect of C-peptide on blood glucose levels or the disposal of a
glucose load was also investigated in healthy subjects and type 1 diabetes patients, but with negative results (14, 17). Direct
examination of the influence of C-peptide on glucose transport in
skeletal muscle under in vitro conditions has, however, shown that
human C-peptide is capable of stimulating 3-O-methylglucose
transport in incubated human muscle strips in a concentration-dependent
manner (67). The effect was seen in muscle strips from both healthy
subjects and type 1 diabetes patients and appears to occur via a
mechanism that is independent of the insulin receptor and of receptor
tyrosine kinase activation (68). The effects on glucose transport of
supraphysiological concentrations of insulin and C-peptide were not
additive (68), which raises the possibility that C-peptide may still
stimulate muscle glucose transport via the insulin-stimulated rather
than the exercise-mediated pathway.
The influence of C-peptide on glucose utilization in the in vivo
situation has been studied using the clamp technique in streptozotocin diabetic rats (37, 65). Supraphysiological concentrations of human
C-peptide were found to elicit marked increases in whole body glucose
utilization, whereas scrambled C-peptide had no effect (65).
Physiological concentrations of rat C-peptides 1 and 2 were found to be
equally potent in stimulating whole body glucose utilization in the
diabetic animals, but they had no effect in healthy, nondiabetic rats
(37). A major proportion of the C-peptide-induced stimulation of
glucose utilization could be blocked by treatment with
N-monomethyl-L-arginine (L-NMMA),
suggesting that the effect is elicited through a NO-mediated pathway.
Although circulatory effects of L-NMMA need to be
considered, it is noteworthy that the effect of C-peptide on glucose
utilization also remained blocked when adenosine was co-administered
with L-NMMA, in an attempt to overcome
L-NMMA-induced reductions in blood flow (37).
Data on C-peptide and glucose utilization are also available from
studies in humans. Type 1 diabetes patients have been examined under
euglycemic conditions, by use of the clamp technique and C-peptide
infusion, at two dose levels (31, 49). Whole body glucose turnover
increased by 25% when C-peptide levels were raised to ~0.8 nM, but
no further increase was observed when C-peptide concentrations rose to
~2.1 nM, in agreement with the concept that C-peptide binding to cell
membranes becomes saturated already at ~0.9 nM (44). Direct
measurements of forearm muscle glucose uptake during C-peptide
administration in type 1 diabetes patients (28, 29) have confirmed that
the augmented whole body glucose utilization observed in the euglycemic
clamp study is a consequence of increased muscle glucose uptake rather
than inhibition of hepatic glucose production. The question can be
raised as to whether the observed short-term (2-h) effects of C-peptide
on glucose utilization in type 1 diabetes patients (31, 49) will
translate into lower blood glucose levels and/or diminished insulin
requirements during long-term C-peptide administration. This does not
seem to be the case, because in patients receiving C-peptide plus
insulin for 3 mo, blood glucose levels, indexes of glycemic control,
and insulin doses were all unchanged compared with the same patients
given insulin only (25). In summary, C-peptide's relatively marked stimulatory effect on glucose utilization in in vitro experiments and
animal studies, which may be NO mediated, appears to be less pronounced
in humans and detectable in short-term studies but not during prolonged
administration of C-peptide.
 |
SUMMARY: C-PEPTIDE IS A BIOLOGICALLY ACTIVE PEPTIDE HORMONE |
The currently available information establishes that C-peptide is not
as biologically inert as previously believed. Instead, it now emerges
as an active peptide hormone with potentially important physiological
effects. Even though C-peptide is formed from proinsulin and
co-secreted with insulin, we should consider the possibility that
C-peptide is a separate entity with biochemical and physiological characteristics that are different from those of insulin. New data now
demonstrate the presence of significant C-peptide-cell membrane
interactions, and there is direct evidence of stereospecific binding of
C-peptide to a cell surface receptor, different from that of insulin
and other related hormones (44). The COOH-terminal pentapeptide segment
is essential for binding (44) and constitutes an active site (43) in
similarity to other biologically active but unrelated peptides, such as
gastrin (13) and cholecystokinin (41). The nature of the C-peptide
receptor remains to be determined, but it is most likely G protein
coupled (42, 44). The Kass for C-peptide binding is
~3 · 109
M
1, and saturation of C-peptide binding
occurs already at physiological concentrations (44), which explains why
C-peptide effects have been difficult to observe in the past. These
findings all agree with the classic concept of ligand-receptor
interaction. There is also evidence of another type of interaction
localized to the glycine-rich midsegment of the molecule (19, 43), the
physiological importance of which remains to be established.
In addition to the binding data, the first outline of an intracellular
signal transduction pattern for C-peptide emerges. It involves the
activation of Ca2+-dependent signaling pathways, with
subsequent stimulation of Na+-K+-ATPase (4, 19,
42, 43) and eNOS activities (24, 30, 34). Both of these enzyme systems
are known to show attenuated activities in type 1 diabetes,
particularly in renal and nerve tissue. There is now evidence to
indicate that replacement of C-peptide in type 1 diabetes is
accompanied by improved renal function, as evidenced by correction of
glomerular hyperfiltration (27, 31, 50) and diminished urinary albumin
excretion (25, 27), and amelioration of nerve dysfunction (25, 26).
C-peptide replacement together with insulin administration, a therapy
possibly closer to nature's own way, may thus be beneficial in type 1 diabetes patients.
 |
ACKNOWLEDGEMENTS |
The work discussed in this review has been supported by grants from
the Swedish Medical Research Council (no. 11201), the Swedish Natural
Science Research Council (no. 34115), the Marianne and Marcus
Wallenberg Foundation, the European Commission (Bio4 CT972123), and
Schwarz Pharma (Monheim, Germany).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: J. Wahren,
Section of Clinical Physiology A2:01, Karolinska Hospital, SE-171 76 Stockholm, Sweden (E-mail: john.wahren{at}ks.se).
 |
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