Research Communication

Supplementation with Apple Enriched with

L-Arginine May Improve Metabolic Control and
Survival Rate in Alloxan-Induced Diabetic Rats

Andrea Escudero1
Guillermo Petzold2
Jorge Moreno2
Marcelo Gonzalez3
Julio Junod2
Claudio Aguayo4
Jesenia Acurio1
Carlos Escudero1*

Vascular Physiology Laboratory and Group of Investigation in Tumor

Angiogenesis (GIANT), Department of Basic Sciences, Universidad del
Bo-Bo, Chillan, Chile

Departament of Food Engineering, Universidad del Bo Bo, Chillan, Chile

Vascular Physiology Laboratory, Department of Physiology, Faculty of

 n, Concepcion, Chile
Biological Sciences, University of Concepcio

Department of Clinical Biochemistry and Immunology, Faculty of

 n, Concepcion, Chile
Pharmacy, University of Concepcio

Abstract
Supplementation with L-arginine or fresh food with high content
of this amino acid is associated with favorable effects in the
metabolic control of diabetes. We aimed to determine whether
supplementation with apples enriched with L-arginine offer additional benefits compared to L-arginine by itself in a preclinical
study of diabetes. This study combines food-engineer technologies with in vivo and in vitro analysis. In vitro experiments
show that cells derived from non-diabetic animals and exposed
to high glucose (25 mM, 12 H) and cells isolated from alloxaninduced diabetic animals exhibited a reduction (
50%) in the Larginine uptake. This effect was reverted by L-arginine pretreatment (12 H) in both the normal and diabetes-derived cells. In
preclinical studies, normoglycemic (n 5 25) and diabetic groups
(n 5 50) were divided into subgroups that received either L-argi-

nine (375 mg/kg per 10 days) or apple enriched with L-arginine

or vehicle (control). In a preliminary analysis, supplementation
with L-arginine by itself (50%) or apple enriched with L-arginine
(100%) improve survival rate in the diabetic group compared to
control (0%) at the end of the follow up (17 days). This phenomenon was associated with a partial but sustained high plasma
level of L-arginine, as well as plasma concentration of nitrites
and insulin in the L-arginine or apple 1 L-arginine groups after
supplementation. Apple 1 L-arginine supplementation in diabetic animals induced the highest and longest effects in the
level of these three markers among the studied groups. Therefore, apple enriched by L-arginine offers more benefits than L-arC 2013 BioFactors,
ginine by itself in this preclinical study. V
00(00):000000, 2013

Keywords: L-arginine supplementation; diabetes; functional foods;

L-arginine transport; preclinical trial

1. Introduction
L-Arginine

is a cationic amino acid, identify as one of the most

metabolically versatile [1] and involved in several processes
C 2013 International Union of Biochemistry and Molecular Biology, Inc.
V

Volume 000, Number 000, Month/Month 2013, Pages 000-000

*Address for correspondence to: Dr. Carlos Escudero, MD, PhD. Vascular
Physiology Laboratory, Group of Investigation in Tumor Angiogenesis
(GIANT), Department of Basic Sciences, Universidad del Bo-Bo. Phone:
+56-42-463256; E-mail: cescudero@ubiobio.cl.
Received 3 December 2012; accepted 12 February 2013
DOI: 10.1002/biof.1103
Published online in Wiley Online Library
(wileyonlinelibrary.com)

BioFactors

such as protein synthesis, insulin secretion and it is the substrate for nitric oxide (NO) synthesis, among others effects
[2,3]. Likewise many other cell types [see for instance Refs.
4,5], L-arginine uptake in vascular smooth muscle cells from
rat aorta occur via a family of carrier proteins identify as systems L and y [6], a mechanism associated with both control
of plasma level of L-arginine and physiological actions which
include NO synthesis and vascular tone regulation [4,7]. Moreover, several studies [for details see Ref. 5] have described
high L-arginine uptake in human endothelium and smooth
muscle cells derived from diabetic patient and/or cell exposed
to high glucose. Nonetheless, L-arginine metabolism in diabetes is not totally understood, but it has been described that

BioFactors

this disease is associated with reduced L-arginine plasma levels in about 50% compared to control [8,9].
L-Arginine supplementation either by itself [1016] or
using fresh food with high content of L-arginine [17,18]
improves metabolic control of the supplemented diabetic animals. Moreover, similar beneficial effects have been described
in patients with type 2 diabetes [19]. Despite these observations, supplementation of L-arginine is not always traduced
into a continue elevation of plasma levels due to high catabolism. In fact, it is known that the more L-arginine is introduced, the more it is destroyed [20]. Then, approaches aimed
to enhance L-arginine availability appear as exceptional
options for preventing and/or control metabolic alteration in
diabetes.
Food-engineer processes such as vacuum impregnation
and ohmic heating have been used for generating apple
enriched with bioactive molecules such as vitamin E, calcium,
and selenium [21] which may offer functional foods with clinical applications. Despite there are no studies regarding
L-arginine incorporation in apples, it might offer a suitable
strategy for enhancing the compliance of oral administration
of this amino acid, reducing intestine breakdown, and favoring
L-arginine supply for its metabolic actions. In addition, this
strategy could enhance the potential beneficial effects
described for apple consumption [2225]. Using in vivo and in
vitro models of diabetes, our aims were: (1) to determinate
whether supplementation with apples enriched with L-arginine
offers additional benefits compared to L-arginine by itself in
the metabolic control and survive of alloxan-induced diabetes;
and (2) to examine whether L-arginine supplementation
enhances itself uptake in cells exposed to high glucose or isolated from diabetic animals.

2. Materials and Methods

This study includes two areas of research, food-engineer and
experimental biology, which in turn include in vitro and in
vivo models.

2.1. Smooth Muscle Cell Culture

Thoracic aortas from normal and diabetic animals (see below)
were dissected and cleaned of surrounding tissue in sterile
buffer phosphate (PBS, in mM: NaCl 13.7, KCl 2.7, Na2HPO4
0.9, KH2PO4 1.8, pH 7.4, 4 C). Then, it was denuded of endothelium by gentle rubbing of intima surface and then cutting
in small pieces (<0.5 cm). Explants were located in dishes precoated with gelatin (1%) for 2 H. Subsequently, culture medium (M199) containing 5 mM D-glucose, 10% new born calf serum (NBCS), 5% fetal calf serum (FCS), 3.2 mM L-glutamine,
and 100 U/mL penicillinstreptomycin (primary culture medium, PCM) was added and cells were maintained under standard conditions (37 C, 5% CO2) until confluence. Confluent cells
were trypsinized (trypsin/EDTA 0.25/0.2%, v/w, 3 min, 37 C)
and plated into 96 wells plaque for L-arginine transport assays.

Prior to assays (12 H), cells were exposed to PCM containing

2% of serum. Cells were used in passage 2.

2.2. L-Arginine Transport

Overall L-arginine transport was measured as described [26].
Briefly, primary culture of smooth muscle cells were exposed
to high D-glucose (25 mM) and/or L-arginine (100 lM) during
12 H, and then to Krebs solution (in mmol/L NaCl 131, KCl 5.6,
NaHCO3 25, NaH2PO4 1, Hepes 20, CaCl2 2.5, MgCl2 1; pH 7.4)
during additional 30 Min before transport assays. Transport of

L-arginine (1 Min, 37 C) was initiated by addition of Hepes-buffered Krebs solution containing unlabelled L-arginine (0.1
1000 lmol/L) and L-[3H]arginine (4 lCi/mL). It was calculated
the kinetic parameters of arginine influx by nonlinear regression analysis. Therefore, saturable L-arginine transport kinetic
parameters Vmax and Km were calculated from data where the
non-saturable, linear uptake of L-arginine was subtracted from
transport data and fitted to the Michaelis-Menten hyperbola
assuming a single saturable transport system.

2.3. Vacuum Impregnation and Ohmic Heating

For food-engineering, apples (cv. Granny Smith) were selected
for producing a functional food enriched with L-arginine by
vacuum impregnation (VI) and ohmic heating (OH) as previously described with slight modifications [27]. Briefly, apples
pieces (1 cm2) were located into an impregnation tank and
immersed in an isotonic solution prepared with apple juice
supplemented with calcium chloride (11%, w/v). For impregnation experiments, a single concentration of L-arginine 1% (w/v)
diluted in the isotonic juice was used. An isotonic solution
without L-arginine was considered as a control. The processing
time was 180 Min for vacuum impregnation (VI) and VI followed by ohmic heating (VI OH). In the VI treatments, a vacuum pulse was applied for 5 Min at 50 mb at the beginning of
the process. After this step, the atmospheric pressure was
restored to complete the process time. For the OH treatments,
samples were immersed in a concentric cylinder tank (3.7 cm
and 19 cm in diameter) made of stainless steel with a plastic
bottom connected to a generator by two electrodes. The isotonic solution, with or without L-arginine, was subject to an
alternating current at 60 Hz and 100 V generating electrical
field strength of 13.0 V/cm. The temperature was measured
with Teflon-coated thermocouples (CPSS-116-24-PFA). Temperature was controlled during the OH treatments with a
water bath and orbital shaking at 100 rpm (Barnstead/LabLine MaxQ 2000, Iowa, USA).

2.4. HPLC for L-Arginine Quantification

L-Arginine concentration in apple enriched by this amino acid
and in plasma was measured as previously described [28] with
small modifications. In brief, the chromatography equipment
consisted of a Hitachi (Model LachRom) separation module
coupled with a fluorescence detector (model F1050) and auto
sampler (Merck, Germany). For instruments control and data
acquisition, VariantStart software from WatersV was used.
R

Use of Apple Enriched with L-Arginine in Preclinical Trial

For standard solutions, L-arginine grade HPLC (Sigma-Aldrich,

MO, USA) diluted in PBS was used for generating a standard
curve from 0 to 20 lM. Both samples and standards were subjected to solid phase extraction (SPE) using Oasis MXC SPE
columns (Water, MA, USA), which were preconditioned and
treated according with manufacture instruction. Analytes were
eluted in tubes with 1.0 mL of concentrated ammonia/water/
methanol (10/40/50, v/v). The solvent was removed by evaporation in a temperature of 6080 C. The residue was dissolved in
0.250 mL HCL (10 mM) and subsequently 0.250 mL of the derivatization solution was added [ortho-phthaldialdehyde (OPA,
7.5 mM) and 3-mercaptopropionic acid (11.5 mM) in potassium borate buffer (200 mM), pH 9.5]. After mixing, the samples were transferred to autosampler vials that were placed in
the alliance separations module with the temperature of the
sample compartment set a 7 C.
Chromatography was performed using a stationary phase
composed by a Symmetry C18 column (3.9  150 mm; 5 lm
particle size: 100 A pore size) with a 10  3 mm guard column
packed with the same stationary phase (Waters, USA). Mobile
phase A consisted of 50 mM potassium phosphate buffer (pH
6.5), containing 8.7% acetonitrile and mobile phase B was acetonitrile/water (50/50, v/v). Separation was performed under
isocratic condition with 100% mobile phase A at a flow rate of
1.1 mL/min and a columns temperature of 30 C. After elution
of the last analyte, strongly retained compounds were quickly
eluted by a robust solvent flush (100% B from 11 to 13 Min).
Between 13 and 16 Min the gradient returned to initial conditions, resulting in a total run time of 16 Min. An injection
volume of 20 lL was used. Fluorescence was measured at
excitation and emission wavelengths of 340 and 455 nm,
respectively. After elution of arginine, the gain of the detector
was switched to a 10-fold higher sensitivity. Peaks were quantified on the basis of peak area.

2.5. Diabetic Induction and Animals Groups

The Ethical Committee of the Universidad del Bio Bio approved
this preclinical study. Seventy-five female rats (weight 230 6
50 g) were included and they were divided into two groups:
control (n 25) and diabetic (n 50). A single intra-peritoneal
injection of alloxan (120 mg/kg; Sigma-Aldrich, MO, USA) was
used for induction of diabetes (day 1). Only rats having a blood
glucose concentration above 200 mg/dL after 3 days of induction (day 4) were kept in the protocol and randomized for
experiments. Animals were maintained in individual cages and
bred under conventional conditions (12 H day/night) with food
and water ad libitum. In this last regard, a pilot study was
conducted in order to quantify the volume of drinking water
and apple juice consumption in normal and diabetic animals.
As expected, diabetic animals drank more water than control
(78.5 6 2.2 mL/day, range 7188 mL/day versus 43.3 6
2.1 mL/day, range 3151 mL/day, P < 0.001). No preference
for apple juice was observed neither control nor in diabetic
groups. To ensure administration of L-arginine (see below),
water consumption was taken into account, therefore, volume

Escudero et al.

for liquids were homologated to 45 mL. Liquids were reposed

everyday. In addition, between 8 and 10 AM, and every 2 days
during all the study until sacrifice, capillary blood sampling
were taken from the tail and used for glycemic control (AccuChek Active, Roche, Germany).

2.6. Randomization and L-Arginine Supplementation

Both diabetic and non-diabetic (normal) groups were randomized into three subgroups. One subgroup received L-arginine
(375 mg/kg), the second a nutritional supplement (apple
L-arginine, ALA) with the equivalent dosage of L-arginine as
a drinking liquid for 10 days. Thus, for preparation of apple
L-arginine beverage, pieces of apples enriched with L-arginine
(10 mg/g) were homogenated (Ultraturex, Daihan Scientific Co
Ltda, Korea) in drinking water considering a final volume of
45 mL and 375 mg/kg of L-arginine. If any residue was present, it was added to the next dose. The third subgroup
received drinking tap water and was used as a control. Supplementation started at day 7 after diabetes induction. Each
subgroup consists of at least six animals.
After 3 (i.e., day 10) or 10 days of supplementation (i.e.,
day 17), animals were sacrificed using an overdose of phenobarbital (50 mg/kg, intra peritoneal) and blood samples were
obtained by intracardiac puncture, as well as aortic rings
were extracted for further process. Blood samples were maintained in ice during all procedures and kept at 80 C until
HPLC analysis. In addition, aortic rings were maintained in
PBS at 4 C and immediately used for isolation of aortic smooth
muscle cells as described previously.

2.7. L-arginine Absorption

In parallel experiments, diabetic rats supplemented with
L-arginine (n 15) or apple L-arginine (n 15) during
3 days as described above (i.e., day 10) were used for determining the time course of plasma levels of L-arginine after a
single oral dose (375 mg/kg) of either L-arginine by itself or
included in apples, respectively.

2.8. Nitric Oxide Plasma Levels

To estimate L-arginine bioavailability, plasma level of nitrite
(i.e., NO metabolites) was measured by the Griess reaction
using a commercially available kit (Promega, USA) as previously described [29]. In brief, 40 lL of same plasma samples
derived from supplemented-animals as described for L-arginine absorption were collected for nitrite quantification. A
spectrophotometer (Phomo, Autobio) was used to read the
samples. It was set at 540 nm. The limit of detection of the
Griess assay was 1.5 lM.

2.9. Dots Blotting

Plasmatic proteins (100 lg) were transferred into nitrocellulose membranes (BioRad Laboratories, Hertfordshire, UK) and
probed with primary rabbit anti-insulin (1:500; Santa Cruz
Biotechnology, Santa Cruz, CA, USA) antibody. Membranes
were rinsed in Tris buffer saline Tween (TBS/T) and incubated

BioFactors

(1 H) in TBS-T/0.2% BSA containing horseradish peroxidaseconjugated goat anti-rabbit antibody. Proteins were detected
by enhanced chemiluminescence and quantified by densitometry. Red-Ponceau staining was used as loading control.

2.10. Statistical Analysis

Values are mean 6 SEM, where n indicates number of animals. Data was analyzed using one-way analysis of variance
(ANOVA) followed by Bonferroni t-test. Kapplan-Meyer analysis was performed for estimating the survival percentage in
supplemented and non-supplemented animals in the control
and diabetic groups. Mantel-Cox Test was used for comparing
the differences in survival percentages among the studied
groups. The statistical software GraphPad Instat 3.01 and
GraphPad Prism 5.00 (GraphPad Software Inc., California,
USA) were used. P < 0.05 was considered statistically
significant.

3. Results
3.1. L-Arginine Transport
L-arginine transport was adjusted to a double MichaelisMenten equation for both the non-diabetic and diabetic
animals (Fig. 1). It was found kinetic transport curves for
medium affinity (Km, 282 to 369 lM, see Table 1) and transport capacity (Vmax/Km) between 5 to 28  103 pmol/lg pro-

FIG 1

tein/min/lM in normal and diabetic cells in absence or presence of high D-glucose (Figs. 1A and 1C) and/or L-arginine
(Figs. 1B and 1D). Eadie-Hofstee analyses in cells exposed or
not to high D-glucose or L-arginine were linear in either normal or diabetic derived cells (r2 0.90 to 0.97, P < 0.05, in all
cases).
D-Glucose and/or L-arginine incubation (12 H) elevated the
kinetic parameters for L-arginine transport, but differences did
not reach statistical significance in either normal or diabetic
derived cells (Table 1). However, trends observed in the kinetic
curves for L-arginine transport were further investigated using
a single L-arginine concentration (i.e., 250 lM, Fig. 2). ANOVA
analysis of L-arginine uptake in normal and diabetes-derived
cells was statistically significant (P < 0.005, in both groups).
Post hoc analysis showed that normal cells exposed to high
D-glucose exhibit a reduction in the L-arginine uptake (55% 6
4%) compared to normal cells without any treatment (i.e., control). This effect was reverted by L-arginine co-incubation. In
addition, cells derived from diabetic animals exhibited a
reduction in L-arginine uptake (54% 6 7%) compared to control. Contrary to normal cells, high D-glucose increased the
uptake of this amino acid in diabetes-derived cells, reaching
values observed in control (2.9 6 0.4 pmol/lg protein/min).
In addition, pre-incubation with L-arginine by itself exhibited a
slightly (P 0.09) recover of reduced uptake of this
amino acid observed in diabetic-derived cells without any
treatment.

Kinetic parameters for saturable transport of L-arginine. Vascular smooth muscle cells (VSMC) isolated from normoglycemic
(A and B) or diabetic animals (C and D) were used for L-arginine transport using L-3H arginine (4 lCi/mL) and increasing concentrations of unlabeled L-arginine (01000 lM). Cells were preincubated in absence (Basal h) or presence of high glucose
(25 mM, n) and high glucose L-arginine (~, 100 lM) or L-arginine alone (~) for 12 H as described in Methods. Values are
means 6 S.E.M. n 5 each group.

Use of Apple Enriched with L-Arginine in Preclinical Trial

TABLE 1

Kinetic parameters for saturable transport of

L-arginine

Vmax
pmol/lg
protein/
min

Km (lM)

Vmax/Km
pmol/lg
protein/
min/lM

Basal

3.6 6 0.8

354 6 73

0.010 6 0.004

Glucose

1.9 6 0.9

328 6 93

0.005 6 0.002

Glucose L-arginine

5.5 6 1.6

282 6 69

0.019 6 0.008

L-arginine

3.5 6 1.5

284 6 89

0.012 6 0.002

Basal

2.6 6 1.0

349 6 109

0.007 6 0.002

Glucose

8.7 6 3.5

327 6 97

0.026 6 0.011

Glucose L-arginine

8.5 6 3.1

337 6 93

0.025 6 0.009

10.5 6 6.6

369 6 144

0.028 6 0.013

Normal

FIG 2

Diabetes

L-arginine

Vascular smooth muscle cells (VSMC) isolated from normoglycemic

(Normal) or diabetic animals (Diabetes) were used for analysis of the
kinetic parameters for L-arginine transport using L-3H arginine (4 lCi/
ml) and increasing concentrations of unlabeled L-arginine (01000 lM)
in absence (Basal) or presence of high glucose (Glucose, 25 mM) and
high glucose L-arginine (100 lM) or L-arginine alone for 12 hours as
described in Methods. Values are means 6 S.E.M. n 5 each group.

3.2. L-Arginine Levels in Plasma and Apples

L-arginine plasma level was significantly reduced in diabetic
rats (67.1 6 8.2 lM) compared to control (108.5 6 1.9 lM, P <
0.05). On the other hand, L-arginine concentration in fresh
apple was very low (Table 2). After VI or VI OH, L-arginine
content in the apple was increased (73 6 3 and 150 6 5 fold,
respectively). Final concentration of L-arginine in the apple
was 10.1 6 1.1 mg of L-arginine per gram of apple.

L-arginine uptake related to middle affinity transport

system. Vascular smooth muscle cells (VSMC) isolated from normoglycemic (h) or diabetic animals
(n) were used for analysis of the L-arginine uptake
using L-3H arginine (4 lCi/mL) and a single unlabeled
L-arginine concentration (250 lM), in absence () or
presence (12 H, ) of high glucose (25 mM) or L-arginine (100 lM). Values are in means 6 S.E.M n 3
per group. *P < 0.05 vs. Normal (baseline), P < 0.05
vs. diabetics (baseline), and P < 0.05 vs. corresponding value in normoglycemic group.

3.3. L-Arginine Supplementation

We characterize the survival proportions in normal (n 13)
and diabetic (n 8) animals without supplementation and
under spontaneous evolution. Seventy percent of diabetic animals die after 5 days post-alloxan injection, and maximum
survive were 13 days in this group (n 1). According to protocol, glycemia was significantly higher in diabetic animals compared to normal (P < 0.001, Fig. 3A). Comparing the weight at
the beginning of the study, diabetic animals presented a progressive drop reaching statistical significance since day 5 until
the last day of survive. Thus, maximal weight decrease was
33% 6 2% at day 8, compared to day 1 (P < 0.001) (Fig. 3B).
At the beginning of the supplementation (day 7), glucose
plasma levels were 87 6 5 mg/dL (n 25) and 442 6 57 mg/dL
(n 20) in the normoglycemic and diabetic groups, respectively.
In a preliminary study, supplementation with L-arginine (LA,
n 6) or apple enriched with L-arginine (ALA, n 6) did not

Food-engineering process for generate apple enriched with L-arginine

TABLE 2

Fresh apple

Controla

VI

VI - OH

[Arg] (mg/g)

0.064 6 0.011

0.055 6 0.016

4.668 6 0.825*,

10.14 61.11*,,

Weight (g)

0.782 6 0.032

0.967 6 0.048

0.974 6 0.020

0.809 6 0.041

Fresh apple cubes were subjected to vacuum impregnation (VI, 50 mbar, 5 min) and VI plus ohmic heating (VI-OH, 100 V, 30  C) in isotonic solution. L-arginine concentration in the final product was quantified by HPLC and weight of the apple cubes were assessed in each analysis. a, correspond to description of Control. Control corresponds to apples exposed to VI without L-arginine. Values are in Mean 6 S.E.M, n 7.
* P < 0.05 vs. fresh apple.

P < 0.05 vs. control.

P < 0.05 vs VI.

Escudero et al.

BioFactors

FIG 3

Glycemia and weight control in alloxan-induced diabetes. Animals were divided into two groups normoglycemia (n 13; Normal, h) and diabetes (n 8;
Diabetic, n). A single intraperitoneal injection of
alloxan (120 mg/kg) was used for diabetes induction.
Cutting blood glucose level was >200 mg/dL (A) Glycemia and (B) Weight was monitoring every 48 H
during the entire follow up (17 days). Values are in
means 6 S.E.M. *P<0.05 vs. Normal.

change the survival rate in non-diabetic animals (100% survive

in all groups). Whereas in diabetic animals, high mortality rate
observed in animals without supplementation was reverted by Larginine (n 6) or ALA (n 6) (Fig. 4A). In fact, at the end of
the follow up (day 17), 50% and 100% of the animals remain
alive in L-arginine and ALA groups, respectively; whereas in
non-supplemented diabetic animals, there were no animals able
to finish the study (Chi2 8.8; df 2, P 0.01).

3.4. L-Arginine Supplementation and Metabolic

Findings
After diabetes induction, insulin level was significantly reduced
in diabetic animals (Fig. 4B) compared to controls. Supplementation with L-arginine did not change the relative insulin level
in non-diabetic animals; whereas an increase (1.7 6 0.3 fold)
at 10 days of supplementation was observed in the normoglycemic ALA group. Furthermore, in the diabetic group, supplementation with L-arginine or ALA revert the reduction of
insulin observed in animals without supplementation (ANOVA
0.009). Thus, in the L-arginine group, it was found an

increase (24 6 6 fold) in the relative level of insulin at 3 days

of supplementation, but this effect was not maintained until
the end of follow up (10 days of supplementation). Likewise, in
the ALA group, the elevation of insulin was 9.4 6 7.2 (P
0.08) and 20.1 6 6.7-fold (P < 0.01) at 3 and 10 days of supplementation, respectively.
In non-diabetic animals neither L-arginine nor ALA supplementation, changed the weight (Fig. 5A) or glycemia (Fig.
5C) during the entire follow up. Interestingly, in diabetic animals, L-arginine supplementation avoids weight lose observed
in animals without supplementation (Fig. 5B). In fact, animals
that received L-arginine presented a continue recovery in their
weight reaching similar weightiness that non-diabetic animals
at the end of follow up. On the other hand, diabetic animals
that received ALA presented a significant and progressive
reduction in their weight, reaching a 40% 6 3% of reduction
at the end of follow up (day 17) respect to their initial weight
(Fig. 5B). This reduction observed in the ALA group was
even higher than reduction observed in diabetic animals without supplementation (P < 0.001). A difference of
71 g was
observed between diabetic animals supplemented with L-arginine (196 6 14 g) compared to which received ALA (125 6
14 g, P < 0.05). As presented in Fig. 5D, behavior of glycemia
in diabetic rats was similar among the studied groups.
Plasma levels of L-arginine after supplementation were
measured in order to describe whether this strategy could
revert L-arginine deficiency in our animal model (Table 3). In
non-diabetic animals, despite slightly variations, supplementation with either L-arginine or ALA, did not change significantly the plasma levels of L-arginine. On the other hand,
diabetic animals supplemented for 3 days with either L-arginine (12% 6 1.6%) or ALA (11% 6 0.5%), showed a partial
recovery in the plasmatic L-arginine level; an effect that was
maintained until the end of supplementation (10 days) only in
animals that received ALA (8% 6 1.2%).

3.5. L-Arginine Bioavailability

To estimate whether L-arginine impregnation in apples could
change the absorption and bioavailability or this amino acid, a
time course of L-arginine, nitrite, and insulin levels were analyzed in animals supplemented during 3 days and who
received a new single oral dose of L-arginine or ALA. Plasma
level of L-arginine was significantly elevated (1 to 4 H after) in
animals that received ALA compared to those receiving
L-arginine by itself (Fig. 6A). After the first hour of L-arginine
administration in either L-arginine or ALA groups, it was
observed a significant reduction compared with respective initial (time 0) measurement, which was partially recovered at
second hour. After this period, a continue drop was observed
in both L-arginine and ALA groups.
Plasma levels of nitrites were slightly (P 0.08) elevated
in diabetic animals without treatment compared to normoglycemic animals (Fig. 6B). After, L-arginine supplementation
during 3 days as described above, diabetic animals which
received L-arginine exhibited an elevation in the nitrite plasma

Use of Apple Enriched with L-Arginine in Preclinical Trial

FIG 4

FIG 5

Escudero et al.

Effect of L-arginine supplementation in the survival rate and insulinemia in alloxan-induced diabetes. In (A) Kapplan-Meier analysis
in diabetic animals divided into three groups: without supplementation (n 8, n) or supplemented with either L-arginine (LA, n
6, ~) or apple L-arginine (ALA, n 6, ~) during 10 days. Diabetes induction (alloxan, 120 mg/kg) was considered as day 1. Confirmation of diabetes (> 200 mg/dL) was performed at day 4, and supplementation started at day 7. Entire follow up was 17 days.
In (B) plasma proteins (100 lg) from normal (h) and diabetic (n) animals with () or without () supplementation of L-arginine (LA)
or apple L-arginine (ALA) during 3 or 10 days were subjected to dot-blot assays to estimate insulin relative levels. Upper panel
show a representative image for three independent experiments. Ponceau stain was used as loading control. In the bottom is presented a densitometry of insulin/Ponceau ratio. Values are in means 6 S.E.M. n 3 animals per group. In (A) Wilcoxon Test Chi2
8.8 and P 0.01. In (B) *P < 0.05 vs. normal (baseline), P < 0.05 vs. diabetics (baseline), and P < 0.05 vs. corresponding value in
normoglycemic group.

Weight and glycemia in animals supplemented with L-arginine. Normoglycemic (A and C) and diabetic (B and D) animals were
divided into three groups: without supplementation (h) or supplemented with either L-arginine (~) or apple L-arginine (~) during 10 days. Diabetes induction (alloxan, 120 mg/kg) was considered as day 1. Confirmation of diabetes (>200 mg/dL) was performed at day 4, and supplementation started at day 7. Entire follow up was 17 days. Weight (A and B) and glycemia (C and D)
were monitored every 48 H during the entire follow up. Values are normalized to 1, considering respective value at the beginning
of the study. Means 6 S.E.M. n 613 per group. In (B) and (D), *P < 0.05 vs. respective value at diabetes confirmation (Day 4).

BioFactors

L-arginine

plasma level in animals supplemented with L-arginine

TABLE 3

(3 Days)

Normal (lM)
Diabetics (lM)

(10 Days)

Basal

LA

ALA

LA

ALA

108.5 6 1.9

78.6 6 6.1

114.3 6 3.1

92.7 6 18.9

94.0 6 8.5

79.6 6 17.4

78.6 6 6.1

62.4 6 11.8*

75.7 6 14.0

67.0 6 8.2*

Normoglycemic (Normal) and diabetic (Diabetics) animals were divided into three groups: without supplementation (basal) or supplemented
with either L-arginine (LA) or apple L-arginine (ALA) during 10 days. L-arginine plasma levels were measured after 3 or 10 days of supplementation. Values are in means 6 S.E.M. n 34 per group.
* P < 0.05 vs basal condition in normoglycemic animals.

levels after 2 H, which was reverted in the third and fourth

hours after single dose administration. This behavior was different in diabetic animals that received ALA, since elevation of
nitrites was observed as early as the first hour and maintained
until the third hour. Elevation of nitrite in the L-arginine group
at the second hour after single dose was associated with an elevation (6.1 6 0.3 fold) in the insulin plasma level (Figs. 6C and
6D). Nevertheless, a shift in the elevation of insulin level was
observed in the group of ALA compared with L-arginine group;
since, it was observed a progressive enhancement of insulin levels reaching significance at the third hour in respect to time 0.

4. Discussion
Preliminary results indicate that L-arginine supplementation
improve the survive of alloxan-induced diabetic rats; a phenomenon associated with partial recovery of plasma level of
L-arginine toward the normality, as well as increase in insulin
and nitrite plasma level and cellular uptake of this amino
acid. Moreover, the use of apple enriched with L-arginine in
diabetic animals, may offer additional benefits since its effects
are longer than the observed with L-arginine by itself, leading
a better survival rate in those animals. The results also

availability in diabetic animals. Diabetic animals supplemented with either L-arginine (LA, ~) or apple L-arginine
(ALA,~) during 3 days and whose received a new single dose of either L-arginine or apple L-arginine were used for
determinate L-arginine bioavailability. In (A) L-arginine plasma levels immediately after new single dose (time 0) and after 1
to 4 H. B: Nitrite plasma level in normoglicemic (white bar, n 4) and diabetic animals (black bar, n 4) without supplementation. In addition, plasma levels of supplemented animals as in (A), were used for nitrite measurement. C: Representative image of dot-blot for insulin plasma level (insulin) and Ponceau staining in samples from 0 to 4 H after single dose
administration of L-arginine as described about. D: Fold of increase in the densitometry of insulin/Ponceau ratio. Means 6
S.E.M. n 15 per group in the supplemented ones. In (B), n 3 in the non-supplemented normal (N) or diabetic (D)
groups. In (A), (B), and (C) *P < 0.05 vs. respective value at time 0. P < 0.05 vs. respective value in animals receiving Larginine by itself.

L-Arginine

FIG 6

Use of Apple Enriched with L-Arginine in Preclinical Trial

suggest that apple enriched with L-arginine might improve the

absorption of this amino acid enhancing its bioavailability necessary for generating nitric oxide and/or induce a recovery in
the insulin release. In conclusion, we created for the first time
a functional food (i.e., apple enriched by L-arginine), which
offers more benefits than L-arginine itself at least in this preclinical study.
Alloxan-induced b-pancreatic cell damage is a model of
type 1 diabetes that has been used for this propose for many
decades [10]. Then, the phenotype of this model has been well
characterized. For instance, in rats, it has been described a
reduction in survival rate specially in doses over 200 mg/kg
[10,14], weight lose, polydipsia hyperglycemia, hypertriglyceridemia, hypercholesterolemia, high levels of free fatty acids,
acidosis, low L-arginine plasma level, and death associated to
reduced levels of insulin [1012,18]. It is also well known that
spontaneous recovery of alloxan-induced damage is observed
after 34 days after diabetes induction, which tend to normalize at 1215 days after induction [13,14]. In this report, we
conducted a study for 17 days after diabetes induction, where
basically this phenotype was confirmed.
In concordance with previous publications in diabetic animals [8,9] or in patients with type 2 diabetes [30]; we found a
reduction in plasma level of L-arginine in alloxan-induced diabetes. Causes for this reduction have been related to hepatic
metabolism in both gluconeogenesis and urea synthesis pathways observed after insulin deficiency [8], but the underling
mechanisms are still unclear. Considering that metabolism of
this amino acid require L-arginine uptake, we conducted in
vitro experiments in order to mimic the effect of supplementation in vivo. In primary culture of aortic smooth muscle cells,
results suggest the presence of medium affinity system for Larginine uptake (i.e., system y, Km, 282 to 369 lM), corroborating previous publications by other authors [31,32]. Despite
we did not conducted further identification of L-arginine membrane transporters in our study, it is well described in this cell
model, the presence of at least three members of system y,
named cationic amino acid transport (CAT) type 1 (CAT-1),
type 2A (CAT-2A) and type 2B (CAT-2B) [32]. In general, kinetic parameters were not significantly affected in cells
exposed to high glucose or L-arginine levels; but these experiments might include more than one transporter. Then, in
order to exclude CAT-2A participation (Km 2,150 to 5,200 lM)
and to emphasize the participation of CAT-1 (Km 70 to 250
lM) and/or CAT-2B (Km 38 to 380 lM) [see for instance Ref.
33] we further analyzed the L-arginine uptake in a single concentration (i.e., 250 lM). In this experimental condition, our
results suggest that diabetes (or high glucose levels in cells
derived form normoglycemic animals) exhibit a reduced L-arginine uptake, probably via CAT-1 and/or CAT-2B. In this
regard, our results differ from previous studies where high
glucose exposition was associated to an increased L-arginine
transport in human umbilical vein endothelial cells (HUVEC)
[34,35] or in retinal pigment epithelium [36]. Differences are
attributed to cell type, as well as experimental conditions,

Escudero et al.

since longer (hours) rather than rapid (minutes) exposition

was used in our study. Nevertheless, response to high glucose
levels differs in normal and diabetic cells, showing an increase
instead a reduction, in L-arginine uptake in diabetes-derived
cells. Mechanisms for these differences are unclear, but it is
intriguing that co-presence of high glucose and L-arginine, a
condition probable observed during in vivo experimentation,
might potentiate L-arginine uptake. Furthermore, reduced Larginine uptake observed in either normal cell exposed to high
glucose or diabetes-derived cell is reverted by L-arginine preincubation (12 H), suggesting that extracellular L-arginine
might induce a trans-stimulation of L-arginine uptake in
smooth muscle cells. In a macro point of view, these results
might be interpreted as one of the mechanism associated to
beneficial effect of L-arginine supplementation tending to normalize L-arginine utilization in the muscle. Clearly, more studies are required in this issue.
Interestingly, several reports [1018] have described that
L-arginine supplementation (mainly as arginine-HCL) or including in natural products [17,18] is beneficial for improving metabolic control in animal models of diabetes. Thus, it has been
described that L-arginine supplementation normalizes plasma
glucose level and vascular function in streptozocin-induced
diabetic rats [12], or improve metabolic control (i.e., reducing
glucose and lipids levels) [10], or prevent b cell damage [37] or
produce b cell regeneration in alloxan-induced diabetes [11].
Accordingly, L-arginine supplementation in type 2 diabetic
patients improve control of glycemia by increasing insulin sensitivity [19,38]. Moreover, L-arginine supplementation reduces
both inflammatory and endothelial dysfunction markers in
non-diabetic subjects with cardiovascular diseases [39] as well
as decrease the oxidative stress generated by hyperglucemia
[19,40]. Therefore, L-arginine supplementation may offer a relative new approach to prevent and/or recover b cell damage
and improve metabolic control in diabetic patients.
Our study confirms that L-arginine supplementation
increase insulin levels in diabetic animals, but dynamic of this
increase differ between L-arginine and apple L-arginine.
Thus, in the L-arginine group the robust increase (
25-fold)
observed after 3 days of supplementation, was not maintained
until the end of the follow up, when a less increase was
observed (
6 fold). On the other hand, supplementation with
apple L-arginine, showed a gradual increase in
10 and 21fold at 3 and 10 days of supplementation, respectively. In addition, time course of insulin release was also different in
animals that received ALA compared to L-arginine by itself.
Thus, in the ALA group it was observed a progressive elevation in the insulin levels reaching approximately fourfold
increase at the third hour compared to time 0; however, in
animals receiving L-arginine by itself, this enhancement was
higher (approximately sixfold), rapid and non-maintained in
time. These results can be connected with plasma level of
L-arginine, nitric oxide synthesis, and survival percentage in
the supplemented groups. Firstly, reduced plasma level of
L-arginine detected in animals with diabetes was reverted with

BioFactors

both L-arginine and apple L-arginine supplementation at

third day, but at the 10th day only animals, which received
apple L-arginine preserved this benefit. Secondly, in the preliminary analysis, survival percentage in diabetic animals was
much better (100% of survive) in those which received apple
L-arginine compared to L-arginine by itself (50% of survive) at
the end of the follow up. Third, L-arginine, nitric oxide and
insulin plasma levels were consistently more elevated in ALA
compared to L-arginine by itself, suggesting a better availability of this amino acid in the ALA group. In this last regard,
since this amino acid is highly metabolized in the intestine
reducing its half-life after supplementation [41], it is feasible
that impregnation in apple can offer a protection for breakdown and improve L-arginine availability in plasma. More
studies are needed for elucidating this issue.
Using food-processing methods, we created for the first
time a functional food (i.e., apple) enriched with L-arginine. In
this regard, food-processing methodologies has previously
used for impregnation of calcium, vitamin E, and probiotic
among other bioactive molecules in apples [see for instance
Refs. 21,42] offering new products able to use in pre-clinical
and clinical application with an exceptional compliance. Our
results indicate that after the food-engineer processing,
enrichment of apple with L-arginine was
160-fold compared
with fresh fruit, reaching a final concentration of
10 mg/g of
apple. In general terms, estimating a 70 kg male as a reference and 100 g of apple enriched with L-arginine, this functional food would cover at least 2% of the requirements of
L-arginine in human (0.8 to 2 g/kg/day) [43].
On the other hand, we also found a significant and progressive weight lose in animals which received apple L-arginine. This reduction was higher compared to diabetic animals
without supplementation, and it represent one-third of the
weight reached by animals supplemented with L-arginine by
itself. Reasons for this reduction are unclear, but recently
Mcknight et al. [41] reviewed data available in the literature
showing that dietary L-arginine supplementation reduces adiposity in several human and animal models of obesity and type
2 diabetes mellitus, a phenomenon associated to reduction in
the growth of white adipose tissue.
Contrarily to previous reports [10,11,14] showing reduction in glycemia after L-arginine supplementation in alloxaninduced diabetes, we found no significant changes in respect
to diabetic animals without supplementation. Differences are
related with experimental design, since in our study (a) only
animals which present a glucose level > 200 mg/dL were
included, and (b) supplementation started after 7 days postdiabetes induction. In this regard, other studies have described
that pretreatment [10] or immediate initiation (i.e., after diabetes diagnosis) [11,16] of L-arginine supplementation was associated with recovery of normoglycemia in diabetic animals.
This discrepancy is particularly important, since a spontaneous recovery toward normoglycemia has been well described
in this animal model after 3 to 4 days post-diabetes induction.
Actually, in our study we found a significant reduction in glu-

10

cose plasma level in diabetic animals with or without supplementation at 7 to 10 days after induction, suggesting that lack
of differences in glycemia observed in our study could be due
to late initiation of L-arginine supplementation. Nevertheless,
one of the fortresses in our study may be that we described a
clearer phenomenon associated directly to L-arginine supplementation rather than a combination with spontaneous
recovery.
In conclusion L-arginine supplementation improves the
survive of alloxan-induced diabetic rats; a phenomenon associated with partial recover of L-arginine plasma level toward the
normality, as well as increase in nitric oxide and insulin level,
which were also associated with augmentation of cellular
uptake of this amino acid. Moreover, we created by the first
time a functional food that may offers additional benefits for
control of diabetes, than L-arginine or apple consumption by
itself. Taken all this evidences into account, L-arginine supplementation and specially foods enriched with this amino acid,
like the apple L-arginine described in this study, holds great
promise as a safe and cost-effective nutrient for improving
metabolic profile in diabetes.

Acknowledgements
The authors would like to thank all research staff at Vascular
Physiology Laboratory and the Group of Investigation in Tumor
Angiogenesis (GIANT), Universidad del Bio Bio for their technical support. They appreciate the editorial assistance of Mr.
Cristian Celis. Fondecyt Regular 1100684, Fondecyt Initiation
11100192, Conicyt 79112027, DIUBB 122109 GI/EF financed
this study. A. Escudero holds post-graduate fellowships from
the Universidad del Bio Bio.

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