Digested wheat gluten inhibits binding between leptin and its receptor
Jnsson et al. BMC Biochemistry
Digested wheat gluten inhibits binding between leptin and its receptor
Tommy Jnsson 0
Ashfaque A Memon 0
Kristina Sundquist 0
Jan Sundquist 0
0 Center for Primary Health Care Research, Lund University/Region Skane, Skane University Hospital , Malmo , Sweden
Background: Leptin resistance is considered a primary risk factor for obesity. It has been hypothesized that dietary cereal grain protein could cause leptin resistance by preventing leptin from binding to its receptor. Non-degraded dietary wheat protein has been found in human serum at a mean level of 41 ng/mL. Here, we report our findings from testing whether enzymatically digested gluten from wheat prevents leptin from binding to the leptin receptor in vitro. Gluten from wheat was digested with pepsin and trypsin under physiological conditions. Pepsin and trypsin activity was removed from the gluten digest with a 10 kDa spin-filter or by heat treatment at 100C for 30 min. Binding to the leptin receptor of leptin mixed with gluten digest at a series of concentrations was measured using surface plasmon resonance technology. Results: Binding of the gluten digest to the leptin receptor was not detected. Spin-filtered gluten digest inhibited binding of leptin to the leptin receptor, with 50% inhibition at a gluten digest concentration of ~10 ng/mL. Heat-treated gluten digest did not inhibit leptin binding. Conclusions: Digested wheat gluten inhibits binding of leptin to the leptin receptor, with half-maximal inhibition at 10 ng/mL. The inhibition is significant at clinically relevant concentrations and could therefore serve as a novel pathway to investigate to understand the molecular basis of leptin resistance, obesity and associated disorders.
Gluten; Leptin; Leptin resistance; Obesity
Leptin is a 16-kDa polypeptide secreted by white adipose
tissue into the circulation, as recently reviewed by Zhou
et al. . Circulating leptin levels are proportional to
body fat mass and fluctuate in accordance with changes
in nutritional states. The leptin concentration serves as a
key adiposity signal for the brain, where leptin binds to
and activates the leptin receptor. Leptin is important in
regulating satiety, weight and energy homeostasis. Most
obese patients have high levels of circulating leptin,
indicating an acquired state of leptin resistance, defined by
the reduced ability of leptin to suppress appetite and
weight gain . Leptin resistance is considered a primary
risk factor for the pathogenesis of overweight and
obesity , which in turn is closely associated with various
metabolic disorders including dyslipidemia,
cardiovascular disease, stroke, insulin resistance and type 2 diabetes.
Several mechanisms have been proposed to explain
leptin resistance, including impaired leptin transport, leptin
signaling and leptin-targeted neural circuits .
It has been hypothesized that dietary cereal grain
protein could cause leptin resistance by preventing leptin
from binding to the leptin receptor . Briefly, the
hypothesis rests on the following propositions: (I) The
global pattern of varying prevalence of diseases of affluence,
such as obesity, cardiovascular disease and diabetes,
suggests that some environmental factor specific to agrarian
societies could initiate these diseases; (II) A diet based on
cereal grain could be such an environmental factor;
(III) Leptin resistance is also associated with diseases of
affluence and could be a sign of insufficient adaptation
to a diet based on cereal grain; (IV) Cereal grain proteins
have sufficient properties (i.e. they are unique, are
present in human food, are heat-stable, are resistant to
gastro-intestinal breakdown, enter the human circulation,
and bind to cell surfaces and receptors) to cause leptin
resistance by inhibiting binding of leptin to the leptin
receptor. The hypothesis is supported by a recent study
on human genetic adaptation by Segurel et al., which
showed positive selection of protective variants of the
leptin receptor with regard to type 2 diabetes from the
Neolithic period onward . This indicates the onset of
evolutionary pressure on the leptin receptor when
significant amounts of cereal grain were adopted as food.
Support also comes from animal studies, which show
that dietary components can induce leptin resistance in
rats in relatively short periods of time at normal body
weight and leptin levels . Dall et al. demonstrated
that cereal grain protein in the form of digested wheat
gliadin caused a 20% increase in weight gain when
injected into non-obese diabetic (NOD) mice in a
prediabetic state , possibly indicating the induction of
leptin resistance. Dall et al. also demonstrated that
digested wheat gliadin caused a dose-dependent
increase in insulin secretion when incubated with rat
insulinoma cells and rat islets, which was possibly caused
by inhibition of current through ATP-sensitive K+ (KATP)
channels . These findings could be indirect evidence
that digested wheat gliadin actually inhibits leptin binding,
since leptin was previously reported to cause the opposite
effect in insulinoma cells . Further support comes from
human clinical studies, in which El-Shebini et al. found
that indices of leptin resistance are improved by replacing
bread with vegetables in otherwise similar hypocaloric
diets and with similar weight loss . Another study by
Ryberg et al. on the effects of a diet without cereal
grains also showed significant effects on leptin . Our
previous dietary intervention study comparing effects
of diets with and without cereal grains showed a strong
correlation between relative change in leptin and cereal
grain intake .
In this study, we tested the last proposition of the
above hypothesis, which is that cereal grain proteins
inhibit leptin binding. Cereal grain proteins have already
been reported to bind to a receptor by Lammers et al.,
who found that cereal grain peptides derived from
enzymatic digestion of wheat gliadin with the gut enzymes
pepsin and trypsin under physiological conditions bind
to the chemokine receptor CXCR3 expressed in mouse
and human intestinal epithelia and laminae propriae,
leading to zonulin release and increased intestinal
permeability . Kamikubo et al. reported that wheat
germ agglutinin binds to the leptin receptor in vitro and
inhibits binding of leptin to the leptin receptor .
Wheat germ agglutinin is found in common wheat flour
but not in human blood . We chose to examine
cereal grain protein from wheat, which is the main
source of vegetable protein in human food. The main
protein component of wheat is gluten, which is the
cohesive and elastic mass that remains after starch has
been removed from cereal grain flour by rinsing with
water. More specifically, wheat gluten is a composite of
several kinds of proteins, such as gliadins (molecular
weight ~30 kDa) and glutenins (molecular weight
~3090 kDa). Gluten intake has increased greatly over the
last hundred years and has accelerated during the last
few decades [14,15]. This increase is largely due to
breeding of gluten-rich cereal grain varieties and most
recently by the use of extra gluten in baking and food
processing to make dough easier to work and bread
fluffier . Soares et al. found that a gluten-free diet
reduces leptin, adiposity, inflammation and insulin
resistance in mice despite a similar energy intake . Chirdo
et al. reported the presence of non-degraded wheat
gliadin in human serum (at a mean level of 41 ng/mL) ,
as previously also reported for other dietary proteins by
Husby et al. [18,19].
Here, we used surface plasmon resonance (SPR)
technology to monitor the interaction between leptin and
the leptin receptor and its inhibition by enzymatically
digested gluten from wheat.
Digestion of gluten
To mimic physiological conditions in the human intestine,
gluten from wheat was digested according to the protocol
of De Ritis et al.  with slight modifications. 100 g of
gluten from wheat (Sigma-Aldrich: G5004) was digested
in 1 L of 0.2 N HCl (pH 1.8) containing 2 g of pepsin
(Sigma-Aldrich: P6887) at 37C for 2 hours. The pH was
checked periodically and adjusted to 1.8 with HC1 or
NaOH as necessary. The pH was then adjusted to 8.0 with
2 N NaOH. The pepsin-digested gluten was further
digested by addition of 2 g of trypsin (Sigma-Aldrich:
T4799). The resulting pepsin- and trypsin-digested gluten
was vigorously stirred at 37C for 4 h. The pH was
checked periodically and adjusted to pH 8.0 with HC1 or
NaOH as necessary.
Pepsin and trypsin removal
Pepsin (molecular weight ~40 kDa) and trypsin
(molecular weight ~25 kDa) were removed from the gluten
digest by either spin-filtering through a 10 kDa filter or
heat-treatment at 100C for 30 min followed by
centrifugation at 13000 g for 10 min. The gluten digest
concentration after filtering or centrifugation was determined from
the absorbance at 280 nm, assuming an absorbance of 1 at
1 mg/mL. To check whether any pepsin or trypsin activity
remained, 1.8 g/mL leptin (recombinant human leptin,
R&D Systems) was incubated in gluten digest
(spin-filtered only or spin-filtered and heat-treated) for 1 h or 24 h
at 37C. The samples were resolved by SDS PAGE
(Invitrogen) and were blotted using the iBlot Gel Transfer
Stacks, PVDF, mini kit (Invitrogen) according to the
manufacturers instructions. After washing, the blots were
incubated with anti-leptin HRP-conjugated antibody (HyTest
Ltd, cat. # 2LE1C) at 4C overnight. The antibody was used
at a 1:1000 dilution relative to the stock concentration in
the product (HyTest Ltd, cat. # 2LE1C). Immunoreactive
bands were detected using ECL reagents (GE Healthcare
All SPR experiments were performed using the Biacore
3000 system (Biacore AB, Uppsala, Sweden) and Sensor
Chips coated with carboxylated dextran (CM5, GE
Healthcare). The flow rate was 10 L/min throughout
immobilization and all experiments. Immobilization of
human leptin receptor/Fc chimera (R&D Systems
389LR) was performed using amine coupling with HBS-EP
running buffer (10 mM HEPES, 3.4 mM EDTA,
150 mM NaCl, 0.005% Tween 20, pH 7.4). The Sensor
Chip surface was activated by injecting a solution of
0.05 M NHS and 0.2 M EDC in water (mixed just prior
to injection) for 7 min, followed by a short buffer rinse.
Coupling was achieved by injecting 100 l of human
leptin receptor/Fc chimera in 10 mM sodium acetate
buffer (pH 5.0) into three flow cells at concentrations
of 1, 3 and 10 g/mL. One channel received no
receptor to serve as a blank control. All four flow cells were
blocked by injection of 70 l of 1 M ethanolamine
(pH 8.5), followed by buffer flow for at least 2 h.
Association of human leptin (R&D Systems: 398-LP) was
monitored by injection of 30 nM leptin for 10 min, and
its dissociation was monitored by buffer flow for up to
15 h. The instrument reports the amount bound per
surface area in RU (1 RU = 1 ng/mm2), as derived from
the angle of minimum total internal reflection of the
incident light. The experiment was repeated three times for a
series of 30 nM leptin samples with increasing
concentrations of gluten digest (heat-treated or spin-filtered)
ranging from 0.9 ng/mL to 450 g/mL. In these experiments,
the association of leptin was monitored for 10 min and
dissociation for 5 min. Dissociation was followed by
regeneration by injection of 25 mM glycine/HCl (pH 2.5) for
The dissociation phase data were fitted using a single
I I0:ekoff:t R
The value obtained for koff was then used during
fitting of the association phase data to obtain an estimate
of kon with the following equation:
I Rmax:c:kon: 1 ec:kon koff:t = c:kon koff
The inhibition data were analyzed by finding the
plateau value at the end of each injection of leptin alone or
leptin plus gluten digest.
Attempts to monitor binding of gluten digest to the leptin
receptor suffered from too low a signal-to-noise ratio and
were therefore inconclusive. Instead, we studied binding
to the leptin receptor of leptin alone and leptin mixed with
gluten digest at a series of concentrations. Leptin alone
was found to interact with the leptin receptor with high
affinity (koff = 4.3.10-4 s-1, kon = 6.4.105 M-1 s-1, KD = 0.6
nM, Figure 1A,B), in line with earlier findings . The
heat-treated gluten digest did not inhibit binding of leptin
to the leptin receptor. However, the spin-filtered gluten
digest reduced binding of leptin to the leptin receptor in a
concentration-dependent manner. Examples of SPR
sensorgrams for a few concentrations of spin-filtered gluten
digest are shown in Figure 1A, with the relative
concentration of bound leptin at the plateau value shown for all
concentrations in Figure 1C (see below). While
experiments with non-purified proteolytic gluten digest would
suffer from potential digestion of the receptor on the chip,
we here evaluated two methods for protease inactivation/
removal. While heat treatment followed by centrifugation
seems to remove all protease activity (leptin bound with
an undiminished signal during repeated injections), it also
removed all leptin-inhibiting activity from the gluten
digest. The use of molecular weight filters was successful and
seems to remove all protease activity while leaving
leptininhibiting activity intact in the remaining filtrate. The final
leptin binding plateau value obtained in the absence or
presence of the filtrated gluten digest is plotted versus the
logarithm of the gluten digest concentration (Figure 1C).
The gluten digest was found to dose-dependently inhibit
binding of leptin to the leptin receptor. A sigmoidal curve
was obtained, with half-maximal inhibition of leptin
binding at a gluten digest concentration of ~10 ng/mL.
Figure 1 SPR analysis of binding of leptin to the leptin receptor and its inhibition by the gluten digest. A) Examples of sensorgrams
recorded during the injection of 30 nM leptin alone (black) or in the presence of 0.0022 (blue), 0.0045 (green), 0.018 (orange) or 0.3 (red) g/mL gluten
digest over a Sensor Chip with immobilized leptin receptor-Fc chimera. The pink dashed line is a fit to the black line using equation 2 (see Methods).
B) Example of a sensorgram recorded during buffer flow after complete injection of 30 nM leptin (black). The pink dashed line is a fit to the data using
equation 1 (see Methods). The two vertical lines occur during the extended time dissociation when the machine switches between its two pumps.
C) Plot of relative intensity at the final leptin binding plateau during injection of 30 nM leptin versus gluten digest concentration. The error bars represent
the standard deviation of three measurements.
No binding of gluten digest to the leptin receptor was
detected. This may indicate that digested gluten was too
small to detect when bound to the leptin receptor or
that digested gluten instead bound to leptin. Regardless,
the gluten digest may inhibit leptin binding directly by
obstructing the binding site on leptin or the leptin
receptor, or indirectly by causing a conformational change
in leptin or the leptin receptor that disturbs their ability
to bind one another.
Comparison with findings from other studies
Lammers et al. showed that gluten digest caused
concentration-dependent displacement of the CXCR3
receptor ligand, with 50% ligand displacement with a
gluten digest concentration of 1 mg/mL . The much
lower gluten digest concentration of 10 ng/mL needed
in our study for half-maximal inhibition of leptin
binding could be due to sensitivity differences in the studies
respective binding model. It could also be due to
concentration and/or activity differences between the
inhibiting substances in the respective gluten digests.
Limitations of the present study
This is an in vitro study and more research has to be
performed to clarify the possible clinical relevance of
our observations. Also, the study examined effects of
wheat gluten, thus leaving other wheat proteins and all
other cereal grain proteins for future studies.
Furthermore, protease activity was removed from the gluten
digest by spin-filtering through a 10 kDa filter. This will
have removed larger possibly active substances from the
gluten digest. Such substances can be examined in future
Research and clinical implications
The concentrations at which digested wheat gluten
inhibited leptin in our study are in the same range as the
concentrations previously reported for gliadin and other
dietary proteins in human serum [17-19], thus making
cereal grain proteins clinically relevant as a possible
cause of leptin resistance and obesity. Our findings
warrant further research, not only on the effects of proteins
from wheat and other cereal grains on leptin signalling,
but also on the effects of other dietary proteins on other
receptors, structures and functions in the body.
To assess the clinical implications of the study results,
we should consider previous findings on the relationship
between serum leptin and body fat mass in humans, which
was found to be a strong linear or quadratic correlation
(R = 0.86, P < 0.0001 for the linear correlation and R =
0.85, P < 0.001 for the quadratic correlation), as measured
by underwater weighing or bioelectric impedance analysis
[22,23]. Also, another study showed that there was a linear
relationship between serum leptin and cerebrospinal fluid
leptin in lean individuals (R = 0.41, P < 0.05) . Such
correlations are of course not certain indications of a
causal connection and most certainly oversimplify the
mechanisms causing obesity. However, if there were a
causal linear relationship between leptin level and body fat
mass, a tentative 50% reduction in binding of leptin to
the leptin receptor due to continual intake of cereal
grain proteins would lead to a doubling of body fat
mass. Furthermore, for an adult with 20% body fat
mass, a doubling of body fat mass would increase body
mass index (BMI) by 20%. This is the difference between
current mean BMI among Swedish adults of ~25 kg/m2
and a healthier ~21 kg/m . A corresponding BMI
improvement would probably also reduce obesity-associated
metabolic disorders such as dyslipidemia, cardiovascular
disease, stroke, insulin resistance and type 2 diabetes in
Digested wheat gluten inhibits binding of leptin to the
leptin receptor, with half-maximal inhibition at 10 ng/mL. The
inhibition is significant at clinically relevant concentrations
and could therefore serve as a new pathway to investigate
to understand the molecular basis of leptin resistance,
obesity and associated disorders.
TJ conceived the study, participated in the design of the study and wrote
the article. AM, KS, JS, SO, AN, MB and SL participated in the design and
execution of the study and the drafting of the article, as well as revising it
for important intellectual content. All authors read and approved the final
The authors are grateful to Anna Hedelius for technical assistance and to
Dr De Vincenzi and Dr Silano at Istituto Superiore di Sanit in Rome, Italy, for
kindly sending us their wheat digest for initial experiments. The study was
funded by Skne University Hospital Foundations and Endowments
(SUS stiftelser och donationer) and Dr P Hkanssons stiftelse.
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