Chronologically scheduled snacking with high-protein products within the habitual diet in type-2 diabetes patients leads to a fat mass loss: a longitudinal study

Abstract

Obesity is the most relevant overnutrition disease worldwide and is associated to different metabolic disorders such as insulin resistance and type-2 diabetes. Low glycemic load foods and diets and moderately high protein intake have been shown to reduce body weight and fat mass, exerting also beneficial effects on LDL-cholesterol, triglyceride concentrations, postprandial glucose curve and HDL-cholesterol levels. The present study aimed at studying the potential functionality of a series of low glycemic index products with moderately high protein content, as possible coadjuvants in the control of type-2 diabetes and weight management following a chronologically planned snacking offer (morning and afternoon).
The current trial followed a single group, sequential, longitudinal design, with two consecutive periods of 4 weeks each. A total of 17 volunteers participated in the study. The first period was a free living period, with volunteers' habitual ad libitum dietary pattern, while the second period was a free-living period with structured meal replacements at breakfast, morning snack and afternoon snack, which were exchanged by specific products with moderately high protein content and controlled low glycemic index, following a scheduled temporal consumption. Blood extractions were performed at the beginning and at the end of each period (free-living and intervention). Parameters analysed were: fasting glucose, insulin, glycosylated hemoglobin, total-, HDL- and LDL-cholesterol, triglyceride, C - reactive protein and Homocysteine concentrations. Postprandial glucose and insulin were also measured. Anthropometrical parameters were monitored each 2 weeks during the whole study.
A modest but significant (p = 0.002) reduction on body weight (1 kg) was observed during the intervention period, mainly due to the fat mass loss (0.8 kg, p = 0.02). This weight reduction was observed without apparently associated changes in total energy intake. None of the biochemical biomarkers measured was altered throughout the whole study.
Small changes in the habitual dietary recommendations in type-2 diabetes patients by the inclusion of specific low-glycemic, moderately high-protein products in breakfast, morning and afternoon snacks may promote body weight and fat-mass loss, without apparently altering biochemical parameters and cardiovascular risk-related factors.
Trial registered at clinicaltrials.gov NCT01264523.

Full text

RESEARC H Open Access
Chronologically scheduled snacking with
high-protein products within the habitual diet in
type-2 diabetes patients leads to a fat mass loss:
a longitudinal study
Santiago Navas-Carretero, Itziar Abete, M Angeles Zulet and J Alfredo Martínez
*
Abstract
Background: Obesity is the most relevant overnutrition disease worldwide and is associated to different metabolic
disorders such as insulin resistance and type-2 diabetes. Low glycemic load foods and diets and moderately high
protein intake have been shown to reduce body weight and fat mass, exerting also beneficial effects on LDL-
cholesterol, triglyceride concentrations, postprandial glucose curve and HDL-cholesterol levels. The present study
aimed at studying the potential functionality of a series of low glycemic index products with moderately high
protein content, as possible coadjuvants in the control of type-2 diabetes and weight management following a
chronologically planned snacking offer (morning and afternoon).
Methods: The current trial followed a single group, sequential, longitudinal design, with two consecutive periods
of 4 weeks each. A total of 17 volunteers participated in the study. The first period was a free living period, with
volunteers’habitual ad libitum dietary pattern, while the second period was a free-living period with structured
meal replacements at breakfast, morning snack and afternoon snack, which were exchanged by specific products
with moderately high protein content and controlled low glycemic index, following a scheduled temporal
consumption. Blood extractions were performed at the beginning and at the end of each period (free-living and
intervention). Parameters analysed were: fasting glucose, insulin, glycosylated hemoglobin, total-, HDL- and LDL-
cholesterol, triglyceride, C - reactive protein and Homocysteine concentrations. Postprandial glucose and insulin
were also measured. Anthropometrical parameters were monitored each 2 weeks during the whole study.
Results: A modest but significant (p = 0.002) reduction on body weight (1 kg) was observed during the
intervention period, mainly due to the fat mass loss (0.8 kg, p = 0.02). This weight reduction was observed without
apparently associated changes in total energy intake. None of the biochemical biomarkers measured was altered
throughout the whole study.
Conclusions: Small changes in the habitual dietary recommendations in type-2 diabetes patients by the inclusion
of specific low-glycemic, moderately high-protein products in breakfast, morning and afternoon snacks may
promote body weight and fat-mass loss, without apparently altering biochemical parameters and cardiovascular
risk-related factors.
Trial Registration: Trial registered at clinicaltrials.gov NCT01264523.
* Correspondence: jalfmtz@unav.es
Department of Nutrition, Food Science, Physiology and Toxicology,
University of Navarra, Irunlarrea 1, 31008 Pamplona, Spain
Navas-Carretero et al.Nutrition Journal 2011, 10:74
http://www.nutritionj.com/content/10/1/74
© 2011 Navas-Carretero et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

Introduction
Type-2 diabetes prevalence in adults has grown in the
last years in many societies, accompanying the high inci-
dence of obesity-related and other cardiovascular risk
factors [1]. Indeed, obesity is the most relevant overnu-
trition disease worldwide, being more dramatic than a
self-esteem problem or an aesthetic issue, since it is
associated to different metabolic disorders such as cor-
onary diseases, hypertension, certain tumors, dislipide-
mia, biliary disorders, immunodeficiencies and insulin
resistance [2].
Different studies have shown the efficacy of low-fat
diets on weight reduction, which has been associated
to an improvement in overweight-related chronic
pathological conditions [3,4]. However, the concomi-
tant high carbohydrate content in some weight-loss
diets may imply the intake of foods with an elevated
glycemic index, which leads to alterations in appetite
directly associated to weight regain [5]. Furthermore,
these commonly sugar-rich diets may exert negative
effects on lipid profile [6] and insulin resistance [7]. In
addition, there is increasing evidence about the key
role of the quantity and quality of carbohydrates in
diets, on the risk of developing chronic diseases [7-11].
Moreover, diets with a high glycemic load have been
related to a higher risk of developing diabetes [6],
while studies carried out with foods and diets of low
glycemic load and high protein content have been
associated with a reduction on LDL-cholesterol and
triglyceride concentrations, as well as a lower post-
prandial glucose curve and an increase in HDL-choles-
terol levels [7-14].
A number of trials have also demonstrated that diets
with a low glycemic load increase satiety, reduce energy
intake and produce a raised thermogenic effect [15,16],
favoring weight loss and subsequently reducing cardio-
vascular disease and diabetes manifestations, although
this apparently higher efficacy of diets with this macro-
nutrient approach remains as a debate issue [17].
Additional investigations have shown that meals and
diets with high protein content may be effective to com-
bat the most important metabolic alterations observed
in diabetic patients [7,12,18-20]. Additionally, some stu-
dies have demonstrated beneficial effects associated to
dietary interventions based on specific breakfast patterns
[12,19,20].
The present study aimed at researching the potential
functionality of a series of low glycemic index products
with a moderately high protein content, as possible
coadjuvants in the control of type 2-diabetes and weight
management within a nutritionally balanced dietary pat-
tern, following a chronologically planned snacking offer
(morning and afternoon), as well as the influence on
biomarkers of the metabolic syndrome and cardiovascu-
lar risk manifestations.
Methods
The study protocol was approved by the Clinical
Research Ethics Committee of the University of Navarra
(reference: 078/2009), and was registered at clinicaltrials.
gov (identifier: NCT01264523). The full trial protocol
can be accessed in Clinical Trials website (clinicaltrials.
gov) by introducing the identifier. Recruitment took part
during February - March 2010 and the study finished in
June 2010. Written informed consent was given by all
the volunteers participating in the intervention.
Taking into account the design of the present study as
a single group longitudinal intervention, the authors
have tried to fulfill the CONSORT 2010 guidelines [21],
except for those points where it was considered non-
applicable, such as blinding.
Study design
The present trial was developed at the facilities of the
University of Navarra with 17 participants, and followed
a longitudinal design, with two consecutive periods of 4
weeks each. The first period, control phase, was a free
living diet and status, where the patients followed their
habitual ad libitum dietary pattern (following the
recommendations of their physician), while the second
periodconsistedonaninterventionphasewithstruc-
tured meal replacements, where the volunteers’habitual
breakfast, morning snack and afternoon snack, were
exchanged by specific products, provided by the
researchers, with a moderately high protein content and
a controlled low glycemic index, following a scheduled
temporal consumption. Therefore, the measurements
and evaluation in the first period are considered as con-
trol values in relation to the intervention phase.
Blood extractions were performed at recruitment, if
the volunteers didn’t have a recent blood analysis (dur-
ing the last three months), and on week 0 (beginning of
the free living period), week 4 (end of free-living period
and beginning of the intervention period) and week 8
(end of the intervention period). Anthropometrical para-
meters were also measured on weeks 0, 2, 4, 6 and 8 of
the trial.
The primary outcomes of the current intervention
were body-weight and fat mass, while glucose and lipid
metabolism, as well as selected cardiovascular risk bio-
markers were established as secondary outcomes.
Volunteers were asked to maintain their physical
activity throughout the whole intervention. An impor-
tant increase/decrease of physical activity compared to
the baseline estimation was considered an exclusion cri-
teria. Physical activity was estimated with a 24 h recall
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at the beginning and the end of the nutritional
intervention.
Products assayed
The products employed for the intervention trial (Ener-
zona
©
) were supplied by Equipe Enervit. These products
are manufactured following a characteristic 40-30-30
energy distribution, with 40% of energy provided by pro-
teins, 30% energy provided by carbohydrates and 30%
energy provided by lipids. Additionally, all products are
of low glycemic index (under 55 units). The variety of
products assayed (Table 1), which volunteers had to
consume all of them, with alternate options for each
day, consisted on bars (27 g) with different flavors
(orange, coconut, vanilla, cocoa and yoghourt), milk
shakes (50 g) to dissolve in 150 mL semi-skimmed milk
(chocolate, strawberry-yoghourt and cappuccino flavors),
salted snacks (25 g packs, black olives and mediterra-
neanstyle),biscuits(50g/8units,coconut,cocoaand
oat) and minirock snacks (25 g/pack, soy and chocolate
chips).
Study volunteers
Subjects participating in the nutritional intervention had
to fulfill the following inclusion criteria: to be type-2
diabetes diagnosed patients, aged between 45 and 75
years old, following the dietary recommendations pre-
scribed by their primary care physician, and eventually
treated only with metformin (stable dosage during at
least three months).
Exclusion criteria, which were controlled by a specifi-
cally trained physician, were to have a BMI under 22 or
over 35 kg/m
2
, to follow a pharmacological treatment
with other drugs but metformin, or being already insulin-
dependent, to have other concomitant pharmacological
treatments for weight loss, hormonal substitutive therapy,
altered thyroid function, etc. without an stable dosage (at
least three months prior the beginning of the study).
Additional exclusion criteria were to suffer from com-
plications due to type 2 diabetes (microangiopathy, poly-
neuropathy, cardiopathy, hepatic and renal impairments,
etc) or having a recent (within the three months prior
to the beginning of the study) uncontrolled diagnostic
of hypercholesterolemia and/or hypertryglyceridemia.
Sample size calculation
Assuming a maximum loss of 0.6 kg/week, and expect-
ing a total weight loss during the intervention period of
2.4 ± 2.5 kg compared to the free-living period, for an a
value of 0.05 (5%) and an statistical power of 80%, the
number of participants needed was estimated at 13
volunteers. Assuming an expected 20% drop-out during
the trial, the minimum sample size required was estab-
lished at 16 volunteers.
Anthropometrical measures
Body weight, and body composition status were mea-
sured by a bioimpedance equipment (Tanita SC-330,
Tanita corp, Japan), waist and hip circumferences were
measured with a commercial measure tap following vali-
dated protocols [22]. Measures were taken with the par-
ticipants in a fasting state of at least 8 hours.
Glucose metabolism determinations
Fasting glycosylated hemoglobin levels were assessed at
the Clinica Universidad de Navarra (Pamplona, Spain),
Table 1 Substitution guidelines during the intervention period of the volunteers’habitual breakfast, morning and
afternoon snack.
Breakfast (choose alternatively one option each day)
Females Males
•3 bars •4 bars
or or
•2 bars plus a glass (250 mL) of semi-skimmed milk •3 bars plus a glass (250 mL) of semi-skimmed milk
or or
•1 milk shake (disolved in 150 mL semi-skimmed milk) •1 milk shake (disolved in 150 mL semi-skimmed milk) + 4 biscuits
or or
•1 glass (250 mL) of semi-skimmed milk plus 8 biscuits •1 glass (250 mL) of semi-skimmed milk plus 8 biscuits plus 1 bar
Morning and afternoon snack (choose alternatively one option each day)
Females Males
•1 bar •1 bar
or or
•1 salted snack (25 g) •1 salted snack (25 g)
or or
•1 Minirock (25 g - soy and chocolate snacks) pack •1 Minirock (25 g - soy and chocolate snacks) pack
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by a high pressure liquid chromatography (HPLC) meth-
odology [23]. Serum glucose was measured in an autoa-
nalyser Pentra C-200 (HORIBA ABX, Madrid, Spain)
and insulin concentrations were determined by an
enzyme-linked immunosorbent assay (ELISA) kit (Mer-
codia, Uppsala, Sweden) in a Triturus autoanalyser (Gri-
fols SA, Barcelona, Spain). Insulin resistance was
estimated by the Homeostasis Model Assessment Index
(HOMA-IR), which was calculated as stated in the fol-
lowing formula [24]: HOMA-IR = [glucose (mmol/L) ×
insulin (µU/ml)]/22, 5
Postprandial serum glucose and insulin levels were
also measured at 30, 60 and 120 minutes after the con-
sumption of both the habitual breakfast and the test
breakfast with 40-30-30 products at weeks 4 and 8,
respectively.
Lipid metabolism variables
Total cholesterol, HDL-cholesterol and triglyceride
serum concentrations were measured in an autoanalyser
Pentra C-200 (HORIBA ABX, Madrid, Spain). LDL-cho-
lesterol levels were calculated following the Friedewald
formula [25].
Inflammatory markers
C - reactive protein concentrations were analysed by an
ELISA assay (Inmunodiagnostics, MA, USA) in a Tri-
turus autoanalyser (Grifols SA, Barcelona, Spain).
Homocysteine was determined in an autoanalyser Pentra
C-200 (HORIBA ABX, Madrid, Spain).
Dietary intake and satiety assessment
During the free-living period and during the nutritional
intervention period, participants filled two 72 h dietary
records, in which they must declare all the foods and
quantities they had eaten in each period [26]. These
questionnaires were further analysed with the DIAL
software (Alce Ingenieria, Madrid, Spain).
Satiety was measured through self-reported question-
naires previously validated [27], based on a Visual Ana-
logue Scale (VAS). Volunteers filled a total of four
questionnaires during the postprandial glucose curves
(before, having breakfast, 30, 60 and 120 minutes after
having breakfast) at weeks 4 and 8.
Statistics
The differences on variables between the beginning and
the end of each period were analysed by a paired t-test,
while the analysis of differences between both periods
(free-living vs. intervention) was performed through an
independent measures t-test. Postprandial glucose and
insulin concentrations were analysed through a repeated
measures ANOVA. Values of p < 0.05 were considered
as statistically significant. All the statistical analysis were
performed with the SPSS 15.1 software for Windows
(SPSS Inc, Chicago, USA).
Results
Adherence to the nutritional intervention
Fifty-two subjects suffering from type-2 diabetes demon-
strated an interest in participating on the study. Once at
the Metabolic Unit, staff explained to them the com-
plete protocol of the present nutritional intervention, 12
subjects declined the invitation to participate, and
another 23 patients were excluded for not fulfilling the
inclusion criteria: 11 were on another pharmacological
treatment but not metformin, 6 subjects had a recent
diabetes diagnostic and treatment was not yet estab-
lished and 5 subjects were not within the requested age
range, while another subject exceeded BMI criteria.
Finally, 17 selected volunteers started the study, from
which 2 subjects withdrew before the final visit, whose
baseline anthropometrical characteristics of the partici-
pants are reported (Table 2).
Changes in anthropometrical parameters
Initial BMI on volunteers was of 28.6 kg/m
2
.Body
weight remained unchanged during the free-living per-
iod (from week 0 to week 4), while a statistically signifi-
cant decrease of about 1 kg was detected (Figure 1a)
during the intervention period (week 4-week 8). Inter-
estingly this change was observed without apparently
associated changes in total energy intake (Table 3). In
fact, energy intake was slightly higher in the intervention
period (ns). This weight reduction was associated to a
marked (p < 0.02) fat mass loss (0.8 kg), without statisti-
cally significant changes in fat-free mass (Figure 1b).
Food intake and satiety assessment
Total energy intake did not greatly differ between the
free-living and the nutritional intervention period (table
3), while protein intake significantly increased during
the dietary snacking substitution with 40-30-30 pro-
ducts. Caloric profile in breakfast, morning and after-
noon snack switched from a typical distribution around
Table 2 Anthropometrical baseline characteristics of the
subjects who completed the study (n = 15).
VARIABLE MEAN SD
Weight (kg) 82.5 12.7
BMI (kg/m
2
) 28.6 4.3
Waist circumference (cm) 102.0 10.7
Hip circumference (cm) 102.7 9.8
Fat mass (%) 29.5 8.1
Fat-free mass (kg) 58.4 6.9
Water (%) 50.7 2.9
There were no differences in anthropometrical features at the beginning of
the second phase of the study compared to the values shown.
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15-55-30 (% protein-% carbohydrate-% lipid of the total
energy intake) in the free-living period to the 40-30-30,
during the intervention period. Glycemic index was also
evaluated in the breakfast, morning snack and afternoon
snack, and no significant differences were observed
between both periods (table 3), despite that a global
trend to increase was found as a consequence of 40-30-
30 products consumption. No changes were observed in
relation to physical activity during the intervention per-
iod (data not shown).
The analysis of the VAS questionnaire during the
postprandial glucose curve, revealed no differences on
hunger, satiety, satisfaction, intake and thirst (not
shown), between the habitual breakfast or the 40-30-30
80,00
81,00
82,00
83,00
84,00
85,00
02468
TIme (weeks)
Weight (kg)
a
.
0,00
10,00
20,00
30,00
40,00
50,00
60,00
70,00
Fat Mass Fat-free mass
Kg
Week 4 Week 8
b.
ns
* p = 0,002
p< 0.02
Figure 1 Anthropometrical changes during the study. The free living period corresponds to week 0 - week 4, while the nutritional
intervention with 40-30-30 products corresponds to weeks 4-8. 1a: Body weight evolution. 1b: Fat mass Vs Fat-free mass.
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Table 3 Changes in macronutrient intake between the free-living and the intervention period
FREE-LIVING PERIOD (WEEK 0 TO 4) INTERVENTION PERIOD (WEEK 4 TO 8)
Daily intake Breakfast Morning
snack
Lunch Afternoon
snack
Dinner Daily intake Breakfast Morning
snack
Lunch Afternoon
snack
Dinner
Energy (kcal) 1710 ± 354 300 ± 168 93 ± 86 713 ± 115 110 ± 86 493 ± 223 1815 ± 335 346 ± 59 111 ± 54 722 ±
154
111 ± 66 524 ±
238
Carbohydrates
(g)
152 ± 52
(36)
39 ± 24
(52)
12 ± 11
(52)
51 ± 21
(29)
12 ± 11
(44)
38 ± 23
(31)
145 ± 48
(32)
34 ± 6
(39)
10 ± 4
(36)
53 ± 29
(30)
10 ± 6
(36)
38 ± 18
(29)
- Sugars (g) 73 ± 25 23 ± 17 8 ± 8 17 ± 7 7 ± 7 18 ± 8 72 ± 16 26 ± 6 7 ± 4 15 ± 7 7 ± 5 15 ± 9
Glycemic Index 51 ± 10 43 ± 22 37 ± 21 58 ± 9 40 ± 10 40 ± 10
Fiber (g) 20 ± 8 4 ± 3 1 ± 1 9 ± 6 1 ± 1 5 ± 2 22 ± 7 4 ± 1 1 ± 1 10 ± 5 1 ± 1 5 ± 2
Proteins (g) 78 ± 16* (18) 11 ± 6*
(15)
4±5*
(17)
37 ± 7
(21)
4±4
(15)
21 ± 13
(17)
100 ± 15*
(22)
25 ± 5*
(29)
7±3*
(25)
36 ± 9
(20)
7±5
(25)
24 ± 11
(18)
Lipids (g) 74 ± 13 (39) 10 ± 6
(30)
2±2
(19)
33 ± 6
(42)
4±3
(33)
24 ± 12
(44)
79 ± 15
(39)
12 ± 2
(31)
4±1
(32)
35 ± 9
(44)
4±2
(32)
26 ± 11
(45)
Values are Mean ± SD. Values within parentheses concern % of energy supplied by the respective macronutrients
* Significant differences (p < 0.05) between the free-living period and the intervention period
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breakfast (Figure 2), while the values obtained during
the 120 minutes postprandial state were as expected.
Changes in biochemical biomarkers
Blood analysis did not evidence statistically significant
changes in any biochemical measurement during the
whole study (table 4). Equally, postprandial glucose and
insulin responses were similar in both periods (Figure 3).
Discussion
The present trial reports the benefits of including within
the habitual diet, different moderately high protein pro-
ducts (40% Carbohydrates-30% protein-30% lipids) for
weight management, following a chronologically sched-
uled pattern. Thus it has been demonstrated that, within
a free-living diet without dietary or energy restrictions,
the substitution of a single meal by products with higher
protein content resulted in a weight loss, due mainly to
a fat mass reduction. In this sense, the results obtained
with this work are in accordance with data previously
reported [26,28-31].
Ad libitum diets with high protein intake have been
considered as useful approaches for effective weight loss
and later maintenance [11,28,29]. Indeed, some long-
term studies with no calorie restriction and programmed
macronutrient distribution have resulted in a more
effective weight loss and maintenance than conventional
macronutrient distribution energy-restricted diets
[11,30,31]. In a recent study, an isocaloric diet with a
moderately high content in protein led to a body weight
and fat mass reduction after 10 weeks [26], without
affecting fat-free mass and maintaining glucose and lipid
profile, which matches with the outcomes of the current
intervention.
The short duration of the present trial together with a
total modest increase of the total protein intake (from
a
b
c
d
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
10,00
0 30 60 120
Time (min)
Score
Habitual Breakfastl
40-30-30 Snack
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0 30 60 120
Time (min)
Score
Habitual Breakfast
40-30-30 Snack
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
03060120
Time (min)
Score
Habitual Breakfast
40-30-30 Snack
0,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
0 30 60 120
Time (min)
Score
Habitual Breakfast
40-30-30 Snack
Figure 2 VAS postprandial questionnaire results. 2(a). Hunger score; 2(b). Satiety score; 2(c). Satisfaction score; 2(d) Intake score.
Table 4 Changes in biochemical determinations between
the beginning and the end of the study.
Baseline Final p
Glucose (mg/dL) 159.2 ± 62.2 156.7 ± 59.4 ns
Insulin (mU/L) 10.2 ± 5.1 10.0 ± 4.9 ns
HOMA-IR 3.65 ± 2.00 3.43 ± 1.84 ns
Glycosylated Hemoglobin (%) 7.0 ± 1.3 7.2 ± 1.5 ns
Total cholesterol (mg/dL) 177.0 ± 37.4 176.9 ± 32.8 ns
HDL-cholesterol (mg/dL) 42.7 ± 9.0 42.5 ± 8.1 ns
LDL-cholesterol (mg/dL) 92.0 ± 37.2 102.9 ± 25.7 ns
Tryglycerides (mg/dL) 178.5 ± 103.6 157.7 ± 92.8 ns
AST (UI/L) 31.8 ± 22.2 26.9 ± 16.1 ns
ALT (UI/L) 36.6 ± 23.5 31.6 ± 19.5 ns
Uric acid (mg/dL) 5.8 ± 1.3 6.1 ± 1.5 ns
Homocysteine (μmol/L) 22.7 ± 4.4 23.3 ± 4.4 ns
C-reactive protein (mg/L) 13.6 ± 15.1 9.8 ± 10.7 ns
Values are Mean ± SD. No significant differences were observed.
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18 to 22% of caloric intake) may have played a role on
the slight, although statistically significant, weight and
fat mass reduction (approximately 1 kg). However, a
period of 4 weeks is a good predictor of body fat
changes with a dietary approach [32,33]. Indeed, when
analyzing meals separately, the total protein content in
breakfast, morning and afternoon snack has been
increased in a range from 50 to 95% during the inter-
vention compared to the free-living period, which
seemed to be enough to produce a body weight reduc-
tion without caloric restriction, and gives support about
the importance on timing of energy consumption, meal
100,0
125,0
150,0
175,0
200,0
03060120
Time (min)
Glucose concentration (mg/dL)
Habitual B reakfast
40-30-30 Snack
a.
0,0
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
50,0
0 30 60 120
Time (min)
Insul in concentrati on (mU/L)
Habitual B reakfast
40-30-30 Snack
b.
Figure 3 Postprandial glucose and insulin curves either with the habitual breakfast and the 40-30-30 snacks, which were not
significantly different. 3a: Postprandial glucose curve. 3b: Postprandial insulin curve.
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frequency and nutrient quality intake for weight mainte-
nance [3,4,8,11,12,34].
The combination of protein content and glycemic
index in a diet could be determinant on body composi-
tion changes [11,35]. In a recent study in Europe [11], it
has been demonstrated the efficacy of high-protein, low-
glycemic index diets on adults for weight maintenance
[11]. In this same study, it was suggested that the iso-
lated effect of protein content or glycemic index in ad
libitum diets did not influence body weight in children,
while the conjunction of these two dietary factors has
been shown as protective against obesity [35]. Likewise,
the current dietary intervention achieved an increase in
protein intake together with a low glycemic index in the
substituted meals, which may explain the observed
improvement in body composition as assessed by bioim-
pedance measurements, a method which has been
recently validated in our research group using Dual X-
Ray Absorptiometry (DXA) as gold standard reference
[22].
For the first time to our knowledge, it has been
demonstrated that the inclusion of some specific meals
in the habitual diet with high-protein low-glycemic
index products may be sufficient for weight manage-
ment, preserving lean mass and helping to decrease fat
mass in type-2 diabetes patients, compared to a control
period of free-living diet. These effects should be
ascribed to the protein induced rise in thermogenesis
[7,11,26,36,37], or even to the increased satiety conse-
quence of high protein ingestion [38,39], although the
assessed satiety scores have not reflected this effect in
the current study.
In relation to the lack of changes concerning glucose
and insulin levels as well as the insulin resistance index,
it may be due to the short intervention period, although
previous studies with similar or even shorter periods
have shown clinically relevant effects with respect to an
improvement on insulin sensitivity [9].
Furthermore, contrasting with other nutritional
intervention studies, our results did not show differ-
ences between the free-living and the intervention per-
iods on lipid metabolism. Several studies have
evidenced that exchanging protein for fat improves
lipid-related cardiovascular risk profile [36,40]. How-
ever, most of these studies used energy-restricted, low-
fat, high-protein diets [37,40-43]. Indeed, all the foods
included in these hypocaloric diets were low fat pro-
ducts, decreasing also saturated fat intake, which could
be one of the main factors involved in the reduction of
total and LDL cholesterol. On the other hand and in
agreement with our findings, several studies comparing
high protein diets have not observed changes in lipid
profile, concretely on total and LDL cholesterol
[7,37,42]. However, a recent study by Morenga and
co-workers [36] found that an ad libitum diet relatively
high in protein improved total cholesterol and low-
density lipoprotein cholesterol in comparison with
standard dietary advice. In this case, the fiber content
of the diet was also relatively high (>35 g per day),
which could counteract the high fat consumption in
this group [36]. It is important to point out that, in
the present study, the driven substitution of specific
single meals by 40-30-30 products, led to a modest
increase in the total dietary daily protein content
(22%), which is lower than the quantities routinely
used in high protein intervention programmes [36].
High protein diets have been also related with reduc-
tions in triglyceride levels [36,37,44,45]. Comparing the
duration of the present nutritional intervention period
to other nutritional programmes, this one may have
been relatively short to achieve significant changes in
triglyceride levels. Indeed, triglyceride levels tended to
belowerattheendofthenutritional intervention per-
iod in spite of not reaching statistical significance.
Moreover, the effect on triglyceride levels has not always
been observed in longer dietary interventions with a
moderately high-protein content [7,46]
A limitation encountered for the implementation of
the current nutritional intervention has been the low
sample size and the limited duration of the intervention,
which can not permit us to generalize the outcomes
obtained without further research. However, it is gener-
allyassumedthatfindingstatistical significance with a
small population is more difficult than when having a
higher sample size. This outcome usually indicates that
there is a real difference between the experimental peri-
ods. In any case, a type II error can not be discarded
[47]. Furthermore, as the study was designed as a longi-
tudinal intervention with two consecutive periods, the
first period is really a control, where the researchers
only performed observational follow-up work. This
approach has been already successfully employed and
published elsewhere [48-51].
The overall results of this study may have been partly
affected by the fact that the participants in the nutri-
tional intervention were type-2 diabetes patients with
initially controlled dietary treatment. In fact, the glyce-
mic indexes in the breakfast, morning and afternoon
snack during the free-living period were relatively low at
baseline (< 55 units), and similar to the glycemic load
reached with the products assayed. Thus, in another
trial [38], the consumption of a low glycemic index
breakfast during 21 days, compared to a high glycemic
breakfast, led to a significant reduction on fasting glu-
cose levels without affecting other biochemical biomar-
kers in obese subjects. In addition, a benefit on satiety
was also reported, as it increased with the low glycemic
index meal [38], which was not seen in the current trial.
Navas-Carretero et al.Nutrition Journal 2011, 10:74
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These observations suggest that in this population, the
moment/time of consumption may be relevant in inter-
preting the results.
Moreover, when comparing two hypocaloric diets dif-
fering in the glycemic index, beneficial additional effects
were found after weight loss (-5.3% vs. -7.5% change
with the high- or low-glycemic diet, respectively) as well
as in total- and LDL-cholesterol concentrations, where
the decrease was 4-fold higher in the low glycemic
index diet [52].
The reduction of glycemic index in a specific meal
[38] or a diet [39] has also been associated to an
increase in the satiety and a reduction on the voluntary
food intake during the postprandial state. Indeed, the
voluntary food intake may be an 80% higher after the
consumption of a high glycemic vs.alowglycemic
index meal [39]. In this context, it is also in agreement
with previous studies the similarity of the satiety scores
observed between the free-living period and the inter-
vention period in the present trial, as glycemic index
remained unchanged between both periods.
The present results together with those from others
[19,34,38] indicate the evident benefits of nutritional
interventions on selected meals, giving an increasing
importance to chrononutrition and meal frequency
intake. Therefore, this is a good example of translational
research carried out in a limited number of volunteers.
Conclusions
Summing up, the present trial evidences that small
changes in the habitual dietary recommendations in
type-2 diabetes patients by the inclusion of specific low-
glycemic, moderately high-protein products in breakfast,
morning and afternoon snacks may promote body
weight and fat-mass loss, without apparently altering
biochemical parameters and cardiovascular risk-related
factors. The importance of these findings are related to
the novelty of demonstrating that increasing protein
content of selected meals offered in specific moments
(breakfast, morning and afternoon snack) leads to an
improvement in body composition, and it is also impor-
tant to highlight the effect of regulating meal frequency
and timing as a basis for future research concerning
chrononutrition.
Acknowledgements and Funding
The authors are grateful to the volunteers of the study, as well as Mutilva
medical center for the support on volunteers’recruitment, specifically Dr.
Carmen Frauca. They also want to thank clinical and laboratory support to
Blanca Martinez de Morentin, Salome Perez and Veronica Ciaurriz.
The supply of the high-protein, low-glycemic index products from Equipe
Enervit, and the partial funding from Rovi Laboratories SA is gratefully
acknowledged. This study was also funded by the special research line
“Nutrition, Health and Obesity”of the University of Navarra (LE/97).
Authors’contributions
SNC participated in the design of the study, development of the trial,
outcomes measurements, data analysis and drafted the final manuscript.
IA participated in the design of the study, outcomes measurements and
drafted the final manuscript.
MAZ participated in the design of the study, data analysis and helped to
draft the final manuscript.
JAM conceived the study, participated on its design and coordination and
performed a critical review of the final manuscript. He also managed the
funding to carry out the intervention.
All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 11 April 2011 Accepted: 14 July 2011 Published: 14 July 2011
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doi:10.1186/1475-2891-10-74
Cite this article as: Navas-Carretero et al.: Chronologically scheduled
snacking with high-protein products within the habitual diet in type-2
diabetes patients leads to a fat mass loss: a longitudinal study. Nutrition
Journal 2011 10:74.
Navas-Carretero et al.Nutrition Journal 2011, 10:74
http://www.nutritionj.com/content/10/1/74
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