Nutrition, Metabolism & Cardiovascular Diseases (2011) 21, 197e205

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/nmcd

Insulin-mimetic action of conglutin-g, a lupin seed

protein, in mouse myoblasts
I. Terruzzi a, P. Senesi b, C. Magni c, A. Montesano a, A. Scarafoni c,
L. Luzi a,b,*, M. Duranti c

a
Nutrition-Metabolism Unit, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy
b
Dipartimento di Sport, Nutrizione e Salute. Facolta` di Scienze motorie. Universita` degli Studi di Milano, Italy
c
Dipartimento di Scienze Molecolari Agroalimentari, Universita` degli Studi di Milano, Italy

Received 19 February 2009; received in revised form 7 July 2009; accepted 7 September 2009

KEYWORDS Abstract Background and aims: Lupin seed is referred to as an antidiabetic product in tradi-
Lupinus albus; tional medicine. Conglutin-g, a lupin seed glycoprotein, was found to cause a significant
Conglutin-g; plasma glucose reduction when orally administered to rats in glucose overload trials. Conglu-
Insulin signalling; tin-g was identified as being responsible for the claimed biological activity, and the aim of this
Insulin action work was to envisage its hypothetical insulin-mimetic cellular mechanism of action. Insulin is
responsible for proteosynthesis control through IRS/AKT/P70S6k/PHAS1 pathways modulation,
glucose homeostasis through PKC/Flotillin-2/caveolin-3/Cbl activation and muscle differentia-
tion/hypertrophy via muscle-specific MHC gene transcription control.
Methods and results: To assess whether conglutin-g modulates the same insulin-activated kinases,
myoblastic C2C12 cells were incubated after 72 h of differentiation with 100 nM insulin or 0.5 mg/
mL (w10 mM) conglutin-g. Metformin-stimulated cells were used as a positive control. The effect on
the above mentioned pathways was evaluated after 5, 10, 20 and 30 min. In the control cells medium
insulin, conglutin-g and metformin were not added. We demonstrated that insulin or conglutin-g
cell stimulation resulted in the persistent activation of protein synthetic pathway kinases and
increased glucose transport, glut4 translocation and muscle-specific gene transcription regulation.
Conclusions: Our results indicate that conglutin-g may regulate muscle energy metabolism, protein
synthesis and MHC gene transcription through the modulation of the same insulin signalling pathway,
suggesting the potential therapeutic use of this natural legume protein in the treatment of diabetes
and other insulin-resistant conditions, as well as the potential conglutin-g influence on muscle cells
differentiation and regulation of muscle growth.
2009 Elsevier B.V. All rights reserved.

* Corresponding author. Nutrition-Metabolism Unit, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy. Tel.: 39 02 2643
3738; fax: 39 02 2643 3407.
E-mail address: livio.luzi@unimi.it (L. Luzi).

0939-4753/$ - see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.numecd.2009.09.004
198
Introduction
White lupin seed conglutin-g is a mono-glycosylated protein
consisting of two disulphide bonded subunits of 30 and
17 kDa (amino acid sequence available at TrEMBL, accession
number: Q9FSH9). This 47 kDa monomeric unit undergoes
a pH-dependent reversible association to tetramer at
neutral to slightly alkaline pH values [1,2]. Conglutin-g in
its native conformation is unusually resistant to proteolysis
by trypsin [3]. Conglutin-g was found to interact with
insulin in vitro with a Kd around 7  105 M and, most
importantly, to significantly reduce plasma glucose in
rodents in doses ranging from 30 to 120 mg/kg body weight
[4]. Therefore, this protein was identified as the respon-
sible molecule for the claimed anti-diabetic properties of
lupin seeds in traditional medicine. Since insulin-binding to
its own receptor causes a series of phosphorylation/de-
phosphorylation reactions, [5] which lead the insulin signal
from the receptor to the final metabolic and myogenic
pathways (Fig. 1), we firstly hypothesize that the effect of
conglutin-g on blood glucose is due to an insulin-mimetic
effect of the protein at the level of the intracellular
pathway insulin receptor/IRS-1/PI-3-kinase, eventually
leading to the recruitment and translocation of GLUT4
(Fig. 1). Secondly, as insulin promotes muscle anabolism,
we hypothesize the activation of a protein synthetic and
myogenic process by conglutin-g.
The aim of this work was to test the effect of conglutin-g
in an in vitro model of mouse myoblasts, assessing the
modulation of muscle-specific genes and the phosphoryla-
tion/activation of intracellular kinases involved in the
insulin signalling cascade. Our results indicate that con-
glutin-g shares, with insulin, common effects on the
intracellular kinases tested in this work, suggesting
a possible therapeutic indication as an insulin-mimetic
agent.
Methods
Materials
Anti-actin (I-19), anti-AKT (C-20), antiecaveolin-3 (A-3),
anti4e (P-2), antieflotillin-2 (H-90), anti-IRS1 (H-165),
anti-p-IRS1 (Tyr 632), anti-myogenin, anti-PI3-Kinase p85a
(Z-8), anti-PKC (H-300), anti ea-tubulin (TU-16), anti-
phospho-ERK (E-4), anti-MHC, anti-phospho-Cbl (Tyr700)
and anti-phosho-p70S6 kinase (Thr421/Ser 424), anti-
PHAS1(P-2), monoclonal or polyclonal primary antibodies
and the peroxidase-conjugated secondary antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
U.S.A.). All other reagents were purchased from Sigma
Chem. Co. (St. Louis, MO, U.S.A.). Mouse C2C12 myoblastic
cells were purchased from the European Collection of
Animal Cell Cultures (ECACC).
Purification of lupin conglutin-g
Conglutin-g was purified using a combination of anion
and cation exchange chromatography [1]. For the estima-
tion of purified conglutin-g concentrations, 4 optical
I. Terruzzi et al.
measurements at 280 nm were made. The extinction coef-
ficient of 1 for a solution of 1 mg/ml was used, according
to ref. [2].
Experimental protocol
C2C12 myoblasts were cultured at 37  C (5% CO2) in
a growth medium (GM) containing DMEM, 20% (v/v) fetal
bovine serum, 1% penicillinestreptomycin and 1% L-gluta-
mine. Seventy per cent confluent myoblasts (Ctu) were
differentiated in DMEM supplemented with 1% of horse
serum and antibiotics (DM) for 72 h. Cells used as control
(Ct) were maintained in DM for the duration of the exper-
iment. Other three groups of cells were placed in DM with
the selective addition respectively of insulin (I; 100 nM),
conglutin-g (Cg; 10 mM) and metformin (Mf; 400 mM) and
lysed at 0, 5, 10, 20 and 30 min after the stimuli addition.
For immunofluorescence analysis differentiated C2C12
myoblasts were stimulated with insulin, conglutin-g and
metformin for 30 min. In differentiated cells immuno-
stained with anti-MHC, the DAPI stained nuclei per myo-
tube, myotubes length and diameter were determined and
expressed as average.
Electrophoretic techniques and immunoblotting
analysis
Conglutin-g SDS-PAGE was performed in NuPAGE Novex
Bis-Tris 10% gels by using a XCell SureLock Mini-Cell
(Invitrogen, Milan, Italy). SeeBlue Plus2 Prestained Stan-
dard and SimplyBlue SafeStain (Invitrogen, Milan, Italy)
were used. For protein blot analysis, the gel was trans-
ferred to nitrocellulose transfer membrane (Protran,
Whatman  Schleicher & Schuell) by blotting according to
Towbin et al. [6] on a Transblot Electrophoretic Transfer
Cell (Bio-Rad, Milan, Italy). The membrane was blocked
with 3% fish gelatin for 2 h and washed three times with
0.25% fish gelatin solution both in PBS buffer (10 mM Na
phosphate, pH 7.4, containing 150 mM NaCl), then soaked
for 2 h in PBS buffer containing rabbit anti-conglutin-g in
the ratio 1,500/1 (v/v). The antiserum was prepared and
immuno-affinity purified as previously described [7]. The
bands were revealed by using horseradish peroxidase
conjugate with goat-antirabbit antiserum 2,000/1 (v/v)
(Bio-Rad, Milan, Italy) and hydrogen peroxide with
4-chloronaphtol as substrate. C2C12 myofibers were
homogenized as described [8]. Aliquots of 30 mg superna-
tant proteins from the different samples were resolved by
8% (p-IRS1 Tyr 632, IRS1, MHC, myogenin and p-CBL), 10%
(PI3K p85, AKT1, p-p70 S6K and PKC), 12% (eIF-4E, p-ERK1,
p-ERK2, caveolin-3 and flottilin-2) and 15% (PHAS1) SDS-
PAGE. Electrophoresed proteins were transferred as
described [8] and the membranes incubated with species-
specific secondary antibodies. Immunoreactive bands
were visualized by an enhanced chemiluminescence
method (Amersham Pharmacia Biotech, Piscataway, NJ,
USA). Total cell lysates of 1e2 mg of protein were immu-
noprecipitated with 10 ml antibodies against p-IRS (Tyr
632). The immunoprecipitated samples were subjected to
SDS-PAGE and Western Blot analysis, which was conducted
as described previously.
Conglutin-g and insulin action 199

Figure 1 Intracellular pathways of insulin signaling. Ins: insulin; IRS-1: insulin Receptor Substrate 1; PI3K: phosphatidylinositol
3-kinase; AKT also called PKB: protein kinase B; p70S6K: p70 ribosomal protein S6 kinase; PHAS1 also called 4EBP1: eukaryotic
initiation factor 4E-binding protein-1; eIF4E/4G/4A: eukaryotic initiation factor13 4E/4G/4A; PKC: protein kinase C; Flo-2: flotillin-
2; Glut4: glucose transporter 4; Cav-3: caveolin-3; CBL: tyrosine phosphorylated protein of the protooncogenes c-Cbl and Cbl-b;
CAP: Cbl associated protein; ERK1/2: extracellular signal-regulated kinases 1/2.

The membrane was stripped and reprobed with an
antibody to actin or tubulin to confirm equal protein
loading per sample. Quantitative measurement of immu-
noreactive bands was performed by densitometric analysis
using the Scion Image software (Scion Corporation, Fred-
erick, MD, USA).
Immunofluorescence analysis
For immunofluorescence, cells were fixed in 4% para-
formaldehyde, permeabilized with 0.2% Triton X-100, and
blocked with PBS containing 1% bovine serum albumin. Cells
were then immunostained with anti-MHC rhodamine
conjugated and nuclei revealed with DAPI staining.
lupin protein extract. Two main bands of Mr around 30 and
16e17 kDa were visible. The sizes of these bands fitted with
those of the large and small conglutin-g subunits. A minor
band of 47 kDa was attributed to the uncleaved conglutin-g
precursor (Duranti, unpublished results). This protein
preparation was judged sufficiently homogenous to be
suitable for cell trials. The protein blot with specific anti-
bodies (Fig. 2B) confirmed the identity of the proteins. The
lack of mobility differences between the purified conglutin-
g and the one in the total protein extract suggested that no
modification to the covalent continuity of the protein had
occurred during the purification procedure.
Analyses of insulin signaling proteins

Reproducibility and statistics analysis
All experiments were performed three times, with similar
results. Data are expressed as means  SE. The unpaired t-
test was used to compare AUC (area Under the Curve)
calculated for the time courses. Differences between
groups were considered statistically significant if P  0.05.
Results
Homogeneity of lupin conglutin-g
Fig. 2A shows the SDS-PAGE pattern under reducing condi-
tions of the purified conglutin-g as compared to the total
In our previous work, we focused our investigation on the
insulin-modulated pathways involved in protein anabolism,
glucose homeostasis, gene expression and mRNA translation
regulation, in different tissues [8e10]. We monitored the
ability of both insulin and conglutin-g to stimulate the same
pathways in vitro. Metformin, the first-line oral anti-dia-
betic drug, was used as a positive control. Fig. 3a and A
illustrate typical patterns of IRS-1observed on western blots
of C2C12 myotube at different times of stimulus by con-
glutin-g, insulin or metformin (time course). Compared to
the time course of control cells, insulin maximally stimu-
lated the IRS-1 protein content within 20 min by w32%, but
lupin protein stimulation was even greater (w35%) at
10 min (p  0.007). Metformin exerted a gradual increase of
the amount of IRS-1, which was maximal at 30 min (w76%).
200
Figure 2 Panel A: SDS-PAGE pattern under reducing condi-
tions of the purified conglutin-g (lane: 1) as compared to the
total lupin protein extract (lane: 2). M Z marker proteins.
Panel B: protein blot with specific anti-conglutin-g antibodies
on the purified conglutin-g (lane: 1) and the total protein
extract (lane: 2). M Z marker proteins. See text for experi-
mental details.
The tyrosine phosphorylation of IRS-1 generates docking
sites for several SH2-containing proteins. Among these, the
predominant partner seems to be the p85 regulatory
subunit of the PI3K 3-kinase. We used anti-phospho-Tyr IRS-
1 antibody to measure IRS-1 phosphorylation and we found
that, unlike control and metformin, both conglutin and
insulin are able to maintain Tyr IRS1 phosphorylation until
the end of the experiment as shown in Fig. 3a.
Insulin enhanced p85-PI3 kinase concentration (Fig. 3B)
within 5 min (w62%), and the effect was persistent up to
the end of the experiment (w56%) while the metformin
effect was negligible. The effect of conglutin-g was slower
but robust, with a peak effect at 30 min (w40%). Fig. 3C
shows the modulation of Akt-1, a downstream target of PI
3-kinase involved in the insulin-signalling pathway leading
to glucose utilization, glycogen and protein synthesis, and
CAP dependent translation. Insulin and conglutin-g
increased the concentration of Akt-1 protein at 20 min
(w13% and 33% respectively), while metformin reached the
top concentration at 5 min (w20%). The differences in Akt-
1 protein content between the control and the conglutin-g
was significant with p  0.03.
Protein synthesis pathway
As seen in Fig. 3D, insulin induced a p70S6 K phosphoryla-
tion within 5 min (w40%) increasing up to w94% in 30 min.
Conglutin-g exerted a maximum stimulus on p70 S6 kinase
phosphorylation within 5 min (67%), which persisted but
gradually decreased until 30 min (w50%, p  0.02).
I. Terruzzi et al.
Metformin exerted an activation of p70S6K phosphorylation
within 10 min (w46%) then decreased until 30 min (w32%).
The activation of PHAS1 is associated with different
residues of phosphorylation that produce isoforms of the
enzymes slowly migrating on SDS-PAGE. The blots appear as
two distinct bands: a rapidly migrating band (a) and the
more slowly migrating bands (b) representing the more
active form of PHAS1. We evaluated the shift in the enzyme
mobility as indexes of insulin and conglutin-g effect on
PHAS1 phosphorylation. Fig. 3E and F show that the inten-
sities of a fast migrating (PHAS-1A) and b slowly migrating
(PHAS-1B) bands, were higher in conglutin-g, insulin and
metformin-stimulated cells with respect to the control.
(PHAS-1A conglutin-g vs control, p  0.02; PHAS-1A con-
glutin-g vs control , p  0.003). Phosphorylation of 4E-BP1
frees eIF-4E, allowing the assembly of the active complex
eIF-4F and the consequent binding of mRNA to the 40S
ribosomal subunit. Thus, the increment of 4E-BP1 phos-
phorylation or eIF-4E content indicates a stimulated
translation and an activation of the initial events in protein
biosynthesis.
Fig. 3G represents the amount of eIF4E after insulin or
conglutin-g stimulation in C2C12 myotube. Insulin
enhanced eIF4E protein concentration with a persistent
effect from 5 to 20 min (w45%). This effect decreased but
persisted until the end of the experiment (w30%). Con-
glutin-g stimulated eIF4E protein concentration in 5 min
(w21%) and the effect increased up to 30 min (w68%,
p  0.03). In contrast, metformin was not able to increase
eIF4E enzyme concentration, except a weak increment at
30 min.
Glucose transport pathway
PKC, a phosphatidylinositol (PI) 3-kinase downstream
kinase, plays a very important role in activating glucose
transport, but a separate PI3K-independent pathway,
involving the insulin-stimulated CBL phosphorylation, is
required to promote the GLUT4 translocation to the plasma
membrane. Expression of flotillin-2 and caveolin-3 is
necessary for the activation of this pathway. In our study
conglutin-g increased the PKC concentration (Fig. 4A) at
approximately the same values at 10 (w28%) and 20 min
(w26%), then decreasing to w20% at the end of the
experiment. These data, compared to control, were
significant with p  0.04. Insulin effect was earlier (5 min)
than conglutin-g and reached a maximum at 20 min
(w32%). Metformin was able to stimulate protein concen-
tration more than the other two stimuli. Flotillin-2 protein
concentration, increased from 5 to 30 min in all the tested
conditions, as represented in Fig. 4B, with a peak at 10 min
by insulin (w60%) and conglutin-g (w52%; p  0.03). The
differences in protein content between the control group
and the conglutin-g group was significant with (p  0.03.).
Metformin did not exert any increase of flotillin-2 concen-
tration. Caveolin-3 protein concentration was increased by
both conglutin-g and insulin with a maximum stimulus at
10 min (w22%) and 30 min (w34%) respectively, as repre-
sented in Fig. 4C. Metformin exerted a greater stimulation
with respect to the other stimuli, which was up and above
70% toward the end of the experiment. The data presented
in Fig. 4D show conglutin-g and insulin ability to maximally
phosphorylate Cbl at 10 min (w64%) and 30 min (w56%)
Conglutin-g and insulin action 201

Figure 3 a) representative blot of immunoprecipitated phospho Tyr IRS1. Densitometric analysis of A) insulin receptor substrate
1 protein (IRS-1), B) PI3K-p85 subunit, C) AKT-1, D) p70S6K phosphorylation, E) PHAS-1a phosphorylation, F) PHAS-1 b phosphory-
lation and G) eiF4-E protein content in C2C12 myofibers when not stimulated (Ct) or after insulin (I), conglutin-g (Cg) or metformin
(Mf) stimulation, at the times detailed in the Methods. The histograms depict the balance obtained by calculating the difference
between the densitometric value measured at specific experimental times in the stimulated cells and the densitometric value
measured at the same time in the control cells. The data of three independent experiments are expressed as a percentage in
relation to the amount of target protein at 0 min. Protein quantification was adjusted for the corresponding a-tubulin level and the
data expressed as mean S.D. The differences in IRS-1, AKT-1 and eiF4E protein content between the control and the conglutin-g
were significant, respectively, with *p  0.007, *p  0.03 and *p  0.03. The differences in p70S6 K phosphorylation, PHAS-1a and
PHASb between the control and the conglutin g stimulated cells were significant, respectively, with *p  0.02, *p  0.02 and
*p  0.003.

respectively. Differences among control and conglutin-g
treated cells in Cbl phosphorylation level were significant
(p  0.03). Metformin effect was barely detectable.
ERKs pathway
Neither insulin nor conglutin-g were able to significantly
modify ERK1 and ERK2 phosphorylation with respect to the
control group, as shown in Fig. 4E and F. Conglutin-g
increased the phosphorylation of ERK1 at 20 min, up to 16%,
and up to 27% ERK2 phosphorylation (p  0.03). Insulin
caused a 30% increase of ERK1 activation at 20 min while
ERK2 insulin stimulation never exceeded 28% at 30 min.
Metformin exerted a much greater activation on both ERK1
and ERK2 with respect to both the other two stimuli.
Analyses of muscle-specific gene expression
To evaluate whether conglutin-g, as insulin [11], is able to
trigger gene transcription control, we studied the content
of MHC muscle-specific protein, responsible for a tissue
differentiation process. As shown in Fig. 5A, conglutin-g, as
well as insulin, was able to increase MHC protein content
(Fold Changes Z 4.5, p  0.05 and Fold Changes Z 10.2;
p  0.03 respectively) with respect to the unstimulated
202 I. Terruzzi et al.

Figure 4 Densitometric analysis of A) PKC protein content, B) flotillin-2 protein content, C) caveolin-3 protein content, D) Cbl
phosphorylation, E) ERK-1 phosphorylation and F) ERK-2 phosphorylation in C2C12 myofibers when not stimulated (Ct) or after
insulin (I), conglutin-g (Cg) or metformin (Mf) stimulation at the times described in the Methods. The histograms depict the balance
obtained by calculating the difference between the densitometric value measured at specific experimental times in the stimulated
cells and the densitometric value measured at the same time in the control cells. The data of three independent experiments are
expressed as a percentage in relation to the amount of target protein at 0 min. Protein quantification was adjusted for the cor-
responding a-tubulin level and the data expressed as mean S.D. The differences in PKC, flotillin-2 and caveolin-3 protein content
between the control and the conglutin-g were significant, respectively, with *p  0.004, *p  0.03 and *p  0.03. The differences in
Cbl, ERK1 and ERK2 phosphorylation between the control and the conglutin-g stimulated cells were significant with *p  0.03.

undifferentiated muscle cells (Ctu), but metformin was not
able to induce the same gene expression.
Moreover, we studied the protein content of myogenin,
a transcription factor playing a pivotal role in the activation
of the MHC gene. Fig. 5B shows that myogenin concentra-
tion by both insulin and conglutin-g was consistent with
that of MHC protein content and reached maximal peaks at
30 min (w73% and 84% respectively), while, in this case too,
metformin did not show any effect. The myogenin protein
content between the control and the conglutin g was
significant with p  0.02.
To validate the effect of the stimuli on MHC protein
content and myoblasts differentiation and to investigate
whether those stimuli could improve the recruitment of
myoblasts into myotubes, or myotubes hypertrophy, three-
day differentiated C2C12 cells were stimulated with
conglutin-g, insulin and metformin for 30 min. Images of
MHC-positive myotubes, detected by immunofluorescence
(Fig. 5C), superimposed on DAPI stained nuclei (Fig. 5D),
showed that in cells stimulated with metformin, conglutin-
g or insulin, as compared with the unstimulated differen-
tiated muscle cells, the number of nuclei present in
MHC-positive myotubes (17.9 3.4, 23.5 6.1, 27.8 8.1
and 14.5 4.3 respectively as shown in Fig. 5E), myotubes
diameter (cm1.3 0.4, cm1.9 0.4, cm2.3 0.7 and
cm1.2 0.4 respectively as shown in Fig. 5F), length
(cm17.7 3.7, cm18.4 3.0, cm21.6 4.0 and 13.7 2.5
respectively as shown in Fig. 5G) and fusion index
Conglutin-g and insulin action 203

Figure 5 Densitometric analysis of: A) MHC protein content and B) myogenin protein content in C2C12 myofibers when not
stimulated (Ct) or after insulin (I), conglutin-g (Cg) or metformin (Mf) stimulation at the times described in the methods. The
histograms depict the balance obtained by calculating the difference between the densitometric value measured at specific
experimental times in the stimulated cells and the densitometric value measured at the same time in the control differentiated
cells (Ct) for myogenin and in the control undifferentiated cells (Ctu) for MHC. The data of myogenin and MHC of three independent
experiments are expressed as a percentage in relation to the amount of target protein at 0 min and as Fold Changes respectively.
Protein quantification was adjusted for the corresponding a-tubulin level and the data expressed as mean S.D. The differences in
MHC and myogenin protein content between the control and the conglutin-g stimulated cells were significant., respectively , with
*p  0.05 and *p  0.02. C2C12 myoblasts were differentiated for 72 h and treated as described in the method section. C) MHC in
myotubes of control and stimulated with Mf, Cg and I respectively, was detected by immunofluorescence (red). D) Images of MHC-
positive myotubes were laid upon DAPI stained nuclei images. E) The nuclei present in MHC-positive myotubes were counted and
the average graphically represented. The difference between the control and the conglutin-g stimulated cells was significant with
*p  0.04. F) The diameters of MHC-positive myotubes were measured and the average, expressed in cm, were graphically rep-
resented. The difference between the control and the conglutin-g stimulated cells was significant with *p  0.03. G) The length of
MHC-positive myotubes was measured and the average, expressed in cm, is graphically represented. The difference between the
control and the conglutin-g stimulated cells was significant with *p  0.05. H) The number of DAPI stained nuclei present in MHC-
positive cells/number of total DAPI stained nuclei per microscopic field (fusion index) was determined and expressed as
percentage. The difference between the control and the conglutin-g stimulated cells was significant with *p  0.04. The data of
three independent experiments are expressed as mean S.D.

(57% 0.1, 68% 7.4, 74% 0.1 and 47% 0.1 respectively
as shown in Fig. 5H) gradually increased.
Discussion
Since the first findings at the beginning of the 30s, various
authors have attempted the isolation of lupin components
with glycemic lowering properties [12,13]. Our recent data
support the conclusion that the lupin protein conglutin-g is
the active compound responsible for the blood glucose
lowering effect [4]. In the present work we have evaluated
conglutin-g effects on the activation of intracellular
kinases common to the insulin signaling cascade using in
vitro models of mouse myoblasts. We firstly observed that
incubation of cells with conglutin-g causes the activation of
the intracellular IRS-1/PI-3-kinase pathway eventually
involved in glucose homeostasis [5] and protein synthesis
stimulation. The eukaryotic translation initiation factor
eIF4E, released as the result of PHAS-1 phosphorylation,
204
acts on translation initiation and promotes the messenger
RNA export of several genes involved in the cell cycle and
growth [14]. Our present data show that incubation of
mouse myoblasts with conglutin-g stimulates p70 S6 kinase
and eIF-4E activation as shown in Fig. 3D and G. Moreover,
conglutin-g is able to stimulate PHAS-1 activation (Fig. 3E
and F). Phosphorylation of AKT-1 is upstream of the phos-
phorylation of p70 S6 kinase as well as of eIF-4E activation,
and both insulin and conglutin-g show comparable effects
on its phosphorylation with a modest stimulation above
basal at any lt (Fig. 3C).
Taken together, these results suggest that conglutin-g
plays an important role in regulating the level of the
protein synthesis and modulating the activation of this
machinery in the cell. In skeletal muscle cells flotillin-2
and caveolin-3 coordinately modulate the insulin-stimu-
lated translocation of GLUT4, localized in flotillin-2-con-
taining perinuclear domains, to selective domains of
sarcolemma. Upon insulin stimulation, caveolae micro-
domains containing caveolin-3 and the insulin receptor,
move away from the plasma membrane toward the
cytoplasm and temporarily interact with flotillin-2/GLUT4-
containing domains prior to reaching the sarcolemma. The
insulin receptor moves from caveolin-3-to flotillin-2-con-
taining domains. GLUT4, together with flotillin-2, moves
to the sarcolemma, promoted by the insulin-stimulated
tyrosine phosphorylation of CBL [15]. Our results show that
conglutin-g, as insulin, increases both flottilin-2 and
caveolin-3 concentrations (Fig. 4B and C) and CBL phos-
phorylation (Fig. 4D), playing an important role in the
vesicular transport of GLUT4 and in the regulation of
muscle energy metabolism.
The robust insulin-like activity of conglutin-g is difficult
to explain since its primary structure (Swiss-Prot accession
number: Q9FSH9), size and other molecular features are
totally dissimilar to those of insulin. Nonetheless, con-
glutin-g shares with insulin some interaction properties: i.
in vitro, conglutin-g binds to insulin itself; ii. like insulin,
conglutin-g is capable of binding metal ions including
Zn, Cu, Cd, Co in a decreasing order of cell differentiation [28,29] and ERKs inhibition is shown to
affinity [16]. How these findings can be related to the
effects monitored in the present work is not yet under-
stood. In two papers published in the 90s, an unusual
insulin-binding activity of a soybean protein, basic globulin
7S (Bg7S), was also demonstrated. However no study on the
potential bioactivity of this soybean protein was under-
taken [17,18]. Lupin conglutin-g shares great amino acid
sequence identity with soybean Bg7S (Swiss-Prot Accession
number: Q8RVH5) [19] and other incompletely character-
ized plant proteins, such as various endoglucanase inhibi-
tors (TrEMBL Accession numbers: B9VUU9, Q7XJE7,
B9VUV0). The lupin seed hypoglycemic effects and the
presence of conglutin-g-related proteins in other seeds
makes it reasonable to speculate on the existence of
a family of plant proteins, which share some functional
property with mammalian insulin. A striking difference with
insulin is that conglutin-g is highly resistant to digestive
enzymes [3] and this might explain the maintenance of its
activity after absorption [4]. In this respect, further dedi-
cated studies are definitely needed to understand the
metabolic fate of this protein. Nonetheless, the present
findings make this natural protein highly appealing for the
I. Terruzzi et al.
potential treatment of type 1 and 2 diabetes. Moreover, the
present study on the mechanism of action makes conglutin-
g suitable for additional investigation as an insulin-sensi-
tizing compound, opening its possible applications both as
drug or food integrators in obesity and many other insulin-
resistant conditions such as the metabolic syndrome [20],
polycystic ovary [21] and HIV-lipodystrophy [22].
In recent years there have been considerable advances
in the understanding of mechanisms by which insulin
regulates, both in a positive and negative manner, the
expression of genes encoding proteins involved in a wide
variety of biologic activities. In particular, studying the
impact of hyperinsulinemia on myosin heavy chain (MHC)
gene regulation in human skeletal muscle [23], has shown
that insulin exerts a rapid influence at the transcriptional
level for the MHC protein. In this present work we find
that insulin is able to increase, more than six hundred
times, the MHC gene expression with respect to the
control. In addition, the expression of MHC gene results
positively regulated by conglutin-g with respect to the
unstimulated differentiated muscle cells. Overall, our
results demonstrate that MHC genes expressed in muscle
tissues is transcriptionally regulated by both insulin and
conglutin-g, while metformin is unable to produce the
same effect.
The transcription factor myogenin plays a pivotal role in
the activation of muscle-specific gene MHC and is critical
for skeletal muscle development [24e27]. Myogenin is
absent in undifferentiated cells and is over expressed in
myoblasts committed to myogenic fate and in muscle
terminal differentiation. For these reasons we wanted to
assess whether the observed variation in MHC gene
expression corresponded to an adequate expression of the
myogenic marker myogenin. In this work we provide
evidence that both conglutin-g and insulin are able to
enhance mouse C2C12 myoblastic cells differentiation as
shown by an increase in the accumulation of differentiation
markers such as MHC and myogenin.
ERKs are shown to negatively regulate skeletal muscle
enhance myoblasts fusion and the expression of the late
differentiation marker myosin heavy chain [30]. Consis-
tently in our study C2C12 cell differentiation and fusion to
form multinucleated myotubes were accompanied by
a reduction in the activating phosphorylation levels of
ERKs. It is reasonable to assume that the deprivation of
serum mitogens or growth factors leads to inactivation
of the ERKs, but our previous data [8] showed that in human
vascular endothelial cells insulin was able to activate ERKs
even in the absence of serum. Instead, in our cellular
model, insulin and conglutin-g block ERK1/2 activity pre-
venting myoblast proliferation and promoting their differ-
entiation, while metformin is not able to produce the same
effect. The present data indicate that conglutin-g, as
insulin, allows the recruitment of myoblasts, enhancing
their fusion into multinucleated myotubes and promoting
increment of their length and diameter. Our present data
provides elements to hypothesize that conglutin-g as
insulin could influence muscle cell differentiation and
contribute to the regulation of muscle growth. This last
possible effect of conglutin-g opens new posssbilities of
marketing applications as food and sports integrators too.
Conglutin-g and insulin action 205

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