International Journal of
Molecular Sciences
Article
Short-Term Physiological Effects of a Very
Low-Calorie Ketogenic Diet: Effects on Adiponectin
Levels and Inflammatory States
Vincenzo Monda 1,† , Rita Polito 2,3,† , Annarita Lovino 3 , Antonio Finaldi 3 , Anna Valenzano 3 ,
Ersilia Nigro 2 , Gaetano Corso 3 , Francesco Sessa 3 , Alessio Asmundo 4 , Nunzio Di Nunno 5 ,
Giuseppe Cibelli 3 and Giovanni Messina 3, *
1
2
3
4
5
*
†
Dipartimento di Medicina Sperimentale, Sezione di Fisiologia Umana e Unità di Dietetica e Medicina dello
Sport, Università degli Studi della Campania “Luigi Vanvitelli”, 80138 Naples, Italy;
vincenzo.monda@unicampania.it
Dipartimento di Scienze e Tecnologie Ambientali Biologiche Farmaceutiche, Università della
Campania (Luigi Vanvitelli), 81100 Caserta, Italy; rita.polito@unicampania.it (R.P.);
nigro@ceinge.unina.it (E.N.)
Dipartimento di Medicina Clinica e Sperimentale, Università di Foggia, 71100 Foggia, Italy;
annaritalovino@libero.it (A.L.); a.finaldi@gmail.com (A.F.); anna.valenzano@unifg.it (A.V.);
gaetano.corso@unifg.it (G.C.); francesco.sessa@unifg.it (F.S.); giuseppe.cibelli@unifg.it (G.C.)
Dipartimento di Scienze biomediche, odontoiatriche e delle immagini morfologiche e funzionali,
sezione di Medicina Legale, Università di Messina, 98122 Messina, Italy; alessio.asmoundo@unime.it
Univesità del Salento, 73100 Lecce, Italy; nunzio.dinunno@unisalento.it
Correspondence: giovanni.messina@unifg.it; Tel.: +39-881588095
These authors contributed equally to this work.
Received: 1 April 2020; Accepted: 30 April 2020; Published: 2 May 2020
Abstract: Adipose tissue is a multifunctional organ involved in many physiological and metabolic
processes through the production of adipokines and, in particular, adiponectin. Caloric restriction
is one of the most important strategies against obesity today. The very low-calorie ketogenic
diet (VLCKD) represents a type of caloric restriction with very or extremely low daily food energy
consumption. This study aimed to investigate the physiological effects of a VLCKD on anthropometric
and biochemical parameters such as adiponectin levels, as well as analyzing oligomeric profiles and
cytokine serum levels in obese subjects before and after a VLCKD. Twenty obese subjects were enrolled.
At baseline and after eight weeks of intervention, anthropometric and biochemical parameters, such
as adiponectin levels, were recorded. Our findings showed a significant change in the anthropometric
and biochemical parameters of these obese subjects before and after a VLCKD. We found a negative
correlation between adiponectin and lipid profile, visceral adipose tissue (VAT), C-reactive protein
(CRP), and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), which confirmed
the important involvement of adiponectin in metabolic and inflammatory diseases. We demonstrated
the beneficial short-term effects of a VLCKD not only in the treatment of obesity but also in the
establishment of obesity-correlated diseases.
Keywords: very low-calorie ketogenic diet (VLCKD); adipose tissue (AT); adiponectin; cytokines;
inflammatory diseases; visceral adipose tissue (VAT); C-reactive protein (CRP); lipid profile
1. Introduction
Adipose tissue (AT) is a multifunctional organ involved in many physiological and metabolic
processes. It is not only a site for energy storage but also an endocrine organ, composed of adipocytes,
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www.mdpi.com/journal/ijms
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and it is also populated by several immune cells such as T lymphocytes and macrophages. As a result of
excessive expansion of AT mass, a high-fat diet, lipolysis activation, and non-shivering thermogenesis
recruit and activate numerous immune cells.
Through the production of adipokines and, in particular, adiponectin, AT is involved in many
metabolic and inflammatory functions, as well as thermoregulation. Moreover, literature data
demonstrate that obese people have a higher incidence of immune and autoimmune diseases [1,2].
In the obese condition, there is an accumulation of visceral adipose tissue (VAT) in the abdominal
area of the body, which is extremely dangerous for health. Furthermore, white visceral fat adipocytes
are particularly active in the release of adipokines, hormones involved in several metabolic and
inflammatory processes, as well as in the normal homeostasis of many organs and tissues. Among
these, adiponectin is the most abundant product from AT. It constitutes about 0.1% of total serum
proteins. Adiponectin circulates as oligomers of different molecular weight, low molecular weight
(LMW), medium molecular weight (MMW), and high molecular weight (HMW), which are the most
biologically active. Through the direct or indirect release of this adipocytokine, visceral fat controls
appetite and energy balance, immunity, angiogenesis, insulin sensitivity, and lipid metabolism [3].
Adiponectin has pleiotropic functions on different target tissues through the presence of its
receptors, AdipoR1, AdipoR2, and T-cadherin in liver, muscle, and adipose tissue, where it positively
affects homeostasis and metabolism of glucose and fatty acids [4,5]. As reported by Yamauchi et al.,
the expression of AdipoR1/R2 mediates increased AMP kinase and PPARα ligand activities, as well as
fatty-acid oxidation and glucose uptake by adiponectin [5]. Adiponectin increases insulin sensitivity
and reduces hepatic neo-glucogenesis. Furthermore, many studies reported that adiponectin could be
an early marker assisting in the evaluation of the initial stages of a worsening glucose metabolism. The
measurement of adiponectin concentration would aid in the identification of high-risk individuals
with glycated hemoglobin (HbA1c) concentrations above a particular threshold, independently of
serum glucose concentration. It is well known that glycated hemoglobin is a long-term marker of
glucose metabolism; for these reasons, these two factors may be useful in the prevention of the initial
stage of diabetes [6]. Among the inflammation markers, C-reactive protein (CRP) is an acute phase
reactant marker of inflammation correlated with cardiac injury; in addition, it is strongly related to
adiposity and insulin sensitivity. Literature data report that adiponectin and CRP serum levels are
negatively correlated in type 2 diabetes and obesity [6]. Moreover, numerous studies, both in vitro
and in vivo, also characterized the anti-inflammatory, anti-atherogenic, and anti-angiogenic effects of
adiponectin [7–9].
The anti-inflammatory effects of adiponectin include both the suppression of pro-inflammatory
cytokine production, such as TNF-α and IL-6, C protein, and growth factors, and modulation of the
expression of anti-inflammatory cytokines such as IL-10 in monocytes and macrophages. On the
other hand, TNF-α and other inflammatory markers (IL-6, C-reactive protein, SAA, tPA, MCP-1)
and glucocorticoids suppress adiponectin production and regulate its levels. The anti-inflammatory
cytokines, such as IL-10, positively correlate to adiponectin levels, and the production of this cytokine
is stimulated by adiponectin [3,7]. Furthermore, this adipokine is involved in obesity outcome; in
particular, it is strongly reduced in the serum of obese subjects. Moreover, it improves the oxidation of
free fatty acids in muscle, preventing the growth of free fatty acids and triglycerides as a result of a
high-fat diet. Numerous studies, both in vitro and in vivo, also characterized the anti-atherogenic and
anti-angiogenic effects of this protein [7–9]. Furthermore, it is well known that adiponectin levels are
strongly modified by diets such as the Mediterranean diet or the DASH diet [10].
In this scenario, caloric restriction represents one of the most important strategies against obesity.
Caloric restriction reduces or slows the onset of diseases related to obesity, inducing a considerable
weight loss and having beneficial and anti-inflammatory effects, reducing the production of free
radicals, and favoring higher resistance to stress and prolonging lifespan. A very low-calorie ketogenic
diet (VLCKD) represents a type of caloric restriction with very or extremely low daily food energy
consumption (800 kilocalories per day or less). The VLCKD is a dietetic regimen that mimics fasting by
Int. J. Mol. Sci. 2020, 21, 3228
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restricting carbohydrates with a moderate increase in protein intake, which is proposed to achieve
rapid weight loss. This diet is a particular ketogenic diet that was shown to be effective, at least in the
short to medium term, as a tool to fight obesity [11]. This diet has various beneficial effects on numerous
organs and tissues, inducing weight loss, reducing blood insulin levels, and cardiovascular risk factors,
increasing mitochondriogenesis, and modulating neurotransmitter activity, as well as improving
vascular density in the brain. The ketogenic diet also has an important role as a signaling mediator,
driver of protein post-translational modification, and modulator of inflammation and oxidative stress.
Several studies reported that VLCKD in the short term is able to reduce visceral adipose tissue and
ameliorate lipid profile, in addition to reducing cardiovascular risk factors. [12,13]. The VLCKD is
more effective in inducing weight loss compared with a standard low-calorie diet, presenting higher
patient compliance; in fact, the ketone bodies increase satiety. The mechanism on which this satiety
effect is based is complex and depends on the relationship that is established with several hormones
and metabolites, mainly on a peripheral level [14]. The production of ketone bodies activates the
ventromedial nucleus of the hypothalamus, which is directly related to satiety, and which varies
throughout the day according to the intake of fats. As a consequence of this satiating effect, changes in
body composition characterized by weight loss are produced, related to lower resistance to insulin and
a low atherogenic lipid panel. Meanwhile, an increase in lean mass is shown; therefore, weight loss
would be mainly based on a lower amount of body fat [14].
In light of these literature data, this study aimed to investigate the physiological effects of a VLCKD
on anthropometric and biochemical parameters such as pro-inflammatory and anti-inflammatory
cytokines, as well as on total adiponectin levels and its oligomeric profile. Monitoring was performed
before and after eight weeks of VLCKD.
2. Results
2.1. Anthropometric and Biochemical Futures of VLCKD Obese Patients
The anthropometric and biochemical parameters of the VLCKD obese participants before and
after eight weeks of nutritional intervention are reported in Table 1 weight, body mass index (BMI),
fat mass (FM), and visceral adipose tissue (VAT) were statistically reduced in obese participants after
the VLCKD. Moreover, biochemical parameters such as glycemic and lipid profiles were strongly
ameliorated in these participants after the diet. Interestingly, the adiponectin levels in participants
statistically increased after the diet, while CRP levels strongly decreased. As widely demonstrated, we
found a sexual dimorphism in our study population. In particular, in VLCKD female obese participants,
adiponectin levels were higher compared to male VLCKD obese participants both before and after the
diet intervention (Table 2). For both males and females, we registered a strong increase in adiponectin
levels after eight weeks of the VLCKD. Furthermore, adiponectin negatively correlated with VAT,
with CRP (Figure 1), and with glycated hemoglobin and lipidic profile, but positively correlated
with HDL-cholesterol (Figure 2). In addition, we found a strong reduction of TNF-α serum levels in
participants after the VLCKD; on the contrary, IL-10 serum levels were statistically increased after the
diet intervention. With regard to IL-6 serum levels, we found no strong differences between the two
groups of participants (Table 1). TNF-α and IL-10 strongly correlated to adiponectin serum levels in
participants before and after VLCKD (Figure 3). All these results showed that there is an improvement
in anthropometric and biochemical parameters in a short period of diet intervention.
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Table 1. Body composition and biochemical features of very low-calorie ketogenic diet (VLCKD) of
participants before and after weight-loss.
VLCKD Obese Participants
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T0
p-Value
T1
4 of 12
Sex male/female
10/10
10/10
1541.55
± 141.6
927.79
± 104.9
<0.001
AgeVAT (g)
48 ±
8.2
ns
Height FM
(m) (g)
1.67
± 0.1 ± 1432.5
ns
39,208.77
27,377.03 ± 1217.4
<0.001
WeightFFM
(kg) (g)
91.33
± 17.1
78.73
± 13.3± 1670.6
<0.001
48,789.57±
1712.3
48,093.68
ns
2)
32.19
±
4.78
27.76
±
3.6
<0.001
BMI (kg/m
BMD
1225.57 ± 21.2
1229.31 ± 21.4
ns
VAT (g)
1541.55 ± 141.6
927.79 ± 104.9
<0.001
TotalFM
cholesterol
(mg/dL)
220.13
±
50.7
173.91
±
32.9
<0.05
(g)
39,208.77 ± 1432.5
27,377.03 ± 1217.4
<0.001
HDL
55.13
± 11.1
± 9.1
ns
FFM
(g) (mg/dL)
48,789.57 ±
1712.3
48,093.6847.76
± 1670.6
ns
LDL
(mg/dL)
141.83
±
36.4
107.57
±
27.7
<0.05
BMD
1225.57 ± 21.2
1229.31 ± 21.4
ns
Total cholesterol
(mg/dL)
± 50.7 ± 125.2
173.91
± 32.9
<0.05
Triglycerides
(mg/dL) 220.13135.54
83.25
± 26.1
<0.05
HDL
(mg/dL)(mg/dL)
55.13 ± 96.68
11.1 ± 4.6
47.7693.09
± 9.1 ± 3.3
ns
Glycemia
<0.05
LDL (mg/dL)
141.83 ± 36.4
107.57 ± 27.7
<0.05
HGB (g/dL)
14.13 ± 1,3
13.83 ± 0.9
ns
Triglycerides (mg/dL)
135.54 ± 125.2
83.25 ± 26.1
<0.05
(%)
ns
GlycemiaHba1c
(mg/dL)
96.68 ± 5.65
4.6 ± 0.3
93.095.38
± 3.3± 0.3
<0.05
Insulinemia
<0.05
HGB (g/dL) (U/mL)
14.13 ±10.53
1,3 ± 7.1
13.835.37
± 0.9± 3.7
ns
Hba1c
(%) (mg/dL)
5.65 ± 0.3
5.38 ±
0.3 ± 1.1
ns
Uric acid
4.86 ± 1.0
5.27
ns
Insulinemia
(µU/mL)
10.53
±
7.1
5.37
±
3.7
<0.05
Total protein (g/dL)
7.30 ± 0.4
7.13 ± 0.4
ns
Uric acid
(mg/dL)(U/L)
4.86 ± 21.27
1.0 ± 5.9
5.2723.31
± 1.1 ± 11.4
ns
AST-GOT
<0.05
Total protein (g/dL)
7.30 ± 0.4
7.13 ± 0.4
ns
ALT-GPT (U/L)
26.51 ± 14.8
26.06 ± 16.2
<0.05
AST-GOT (U/L)
21.27 ± 5.9
23.31 ± 11.4
<0.05
Gamma
GT (U/L)
<0.05
ALT-GPT
(U/L)
26.51 ±31.19
14.8 ± 19.8
26.0615.31
± 16.2± 5.4
<0.05
CRP
(mg/mL)
0.89 ± 0.1
<0.05
Gamma
GT (U/L)
31.19 ± 19.8
15.310.48
± 5.4± 0.1
<0.05
CRP
(mg/mL) (g/mL)
0.89 ± 0.1
0.4825.55
± 0.1 ± 1.3
<0.05
Adiponectin
10.8 ± 1.2
<0.001
Adiponectin
(µg/mL)
10.8 ± 1.2
25.55 278
± 1.3± 9.2
<0.001
TNF-α
(pg/mL)
345 ± 6.5
<0.05
TNF-α (pg/mL)
345 ± 6.5
278 ± 9.2
<0.05
IL-10 (pg/mL)
117 ± 7
168 ± 8.8
<0.001
IL-10 (pg/mL)
117 ± 7
168 ± 8.8
<0.001
IL-6
(pg/mL)
236±
4.4
232
±
5
ns
IL-6 (pg/mL)
236 ± 4.4
232 ± 5
ns
Body mass index (BMI); visceral adipose tissue (VAT); fat mass (FM); fat-free mass (FFM); bone
Body mass index (BMI); visceral adipose tissue (VAT); fat mass (FM); fat-free mass (FFM); bone mineral density (BMD);
mineral density
(BMD);
hemoglobin
hemoglobin
(Hba1c);
aspartate
hemoglobin
(HGB); glycated
hemoglobin
(Hba1c);(HGB);
aspartate glycated
aminotransferase
(AST); alanine
aminotransferase
(ALT);
C-reactive protein
(CRP);
not significant
(ns). Some (ALT);
of the data
are the same
as in (CRP);
Table 1 of
our
previously
aminotransferase
(AST);
alanine
aminotransferase
C-reactive
protein
not
significant
published paper [15].
(ns). Some of the data are the same as in Table 1 of our previously published paper [15].
Table 2. Adiponectin serum levels in male and female VLCKD obese participants.
Table 2. Adiponectin serum levels in male and female VLCKD obese participants.
Adiponectin Levels (µg/mL)
Adiponectin Levels (g/mL)
Female
Female
9.23 ±9.23
0.7 ± 0.7
12.4412.44
± 1.07± 1.07
23.67 ±
1.6 ± 1.6
27.3 ±
1.33± 1.33
23.67
27.3
Male Male
VLCKD
obese
participants
VLCKD
obese
participants
T0 T0
VLCKD
obese
participants
T1 T1
VLCKD
obese
participants
p-Value
p-Value
<0.05
<0.05
<0.05
<0.05
Figure1.1.Adiponectin
Adiponectinnegatively
negatively correlated
correlated to
to VAT
VAT and
and CRP
CRP in
in VLCKD
Figure
VLCKD participants
participants before
beforeand
andafter
after
diet.There
Thereisisaanegative
negative correlation
correlation between
between VAT,
VAT, adiponectin
adiponectin serum
serum levels,
levels, adiponectin,
diet.
adiponectin, and
andCRP
CRP
serumlevels
levelsininVLCKD
VLCKDparticipants
participants(A,B).
(A,B).
serum
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Figure 2. Adiponectin
Adiponectin negativelycorrelated
correlated toglycated
glycated hemoglobinand
and lipidicprofile
profile butpositively
positively
Figure
Figure2.
2. Adiponectin negatively
negatively correlated to
to glycated hemoglobin
hemoglobin and lipidic
lipidic profile but
but positively
correlated
to
HDL-cholesterol
in
VLCKD
participants
before
and
after
diet.
There
was
a
negative
correlated
correlated to
to HDL-cholesterol
HDL-cholesterol in
in VLCKD
VLCKD participants
participants before
before and
and after
afterdiet.
diet. There
There was
was aa negative
negative
correlation
between
glycemic
and
lipid
profile
and
adiponectin
serum
levels
(A,B,D,E).
On the
correlation
andand
lipidlipid
profile
and adiponectin
serum serum
levels (A,B,D,E).
On the contrary,
correlationbetween
betweenglycemic
glycemic
profile
and adiponectin
levels (A,B,D,E).
On the
contrary,
there
was
a
positive
correlation
between
adiponectin
and
HDL-cholesterol
(C).
there
was there
a positive
between adiponectin
and HDL-cholesterol
(C).
contrary,
was correlation
a positive correlation
between adiponectin
and HDL-cholesterol
(C).
Figure
Adiponectincorrelated
correlatedtotoTNF-
TNF-αand
and
IL-10
serum
levels
in our
participants
before
and
Figure3.
3. Adiponectin
IL-10
serum
levels
in our
participants
before
and after
Figure 3. Adiponectin correlated to TNF- and IL-10 serum levels in our participants before and after
after
There
a negative
correlation
between
TNF-αand
andadiponectin
adiponectinserum
serum levels
levels (A). On
diet.diet.
There
waswas
a negative
correlation
between
TNF-α
On the
the
diet. There was a negative correlation between TNF-α and adiponectin serum levels (A). On the
contrary,
there
was
a
positive
correlation
between
adiponectin
and
IL-10
(B).
contrary, there was a positive correlation between adiponectin and IL-10 (B).
contrary, there was a positive correlation between adiponectin and IL-10 (B).
2.2. Adiponectin Oligomerization State Analysis in Serum by Western Blotting Ting
2.2. Adiponectin
Adiponectin Oligomerization
Oligomerization State
State Analysis
Analysis in
in Serum
Serum by
by Western
Western Blotting
Blotting Ting
Ting
2.2.
The distribution of serum adiponectin in our VLCKD participants was analyzed by western
The distribution
distribution of
of serum
serum adiponectin
adiponectin in our
our VLCKD
VLCKD participants
participants was
was analyzed
analyzed by western
western
The
blotting
ting analysis (Figure
4). Three bands in
corresponding
to HMW (≥250
kDa), MMWby
(180 kDa),
blotting
ting
analysis
(Figure
4).
Three
bands
corresponding
to
HMW
(≥250
kDa),
MMW
(180
kDa),
blotting ting analysis (Figure 4). Three bands corresponding to HMW (≥250 kDa), MMW (180 kDa),
and LMW
LMW (70
(70 kDa)
kDa) oligomers
oligomers were
were evident
evident in
in the
the serum
serum from
from our
our participants;
participants; Figure
Figure 2A
2A shows
shows aa
and
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and LMW (70 kDa) oligomers were evident in the serum from our participants; Figure 2A shows
blot
of of
thethe
different
oligomers
(HMW,
MMW,
andand
LMW)
present
in the
of two
arepresentative
representative
blot
different
oligomers
(HMW,
MMW,
LMW)
present
in serum
the serum
of
VLCKD
participants
at
baseline
(T0)
and
after
eight
weeks
of
VLCKD
(T1).
The
densitometric
two VLCKD participants at baseline (T0) and after eight weeks of VLCKD (T1). The densitometric
evaluation of
ofthe
theadiponectin
adiponectinoligomers
oligomers
serum
from
all participants
compared
before
and eight
after
evaluation
in in
serum
from
all participants
compared
before
and after
eight
weeks
of
nutritional
intervention
confirmed
that
HMW,
MMW,
and
LMW
oligomers
increased
weeks of nutritional intervention confirmed that HMW, MMW, and LMW oligomers increased after
afterdietary
the dietary
intervention
(Figure
(p < 0.05).
In particular,
HMW
adiponectin
oligomers,
which
the
intervention
(Figure
2B) (p2B)
< 0.05).
In particular,
HMW
adiponectin
oligomers,
which
are
are
the
most
biologically
active,
were
statistically
increased
in
VLCKD
participants
after
the
period
the most biologically active, were statistically increased in VLCKD participants after the period of
of diet
intervention.
diet
intervention.
Figure
profile after
Figure 4.
4. Improvement
Improvement of
of adiponectin
adiponectin oligomeric
oligomeric profile
after eight
eight weeks
weeks of
of VLCKD.
VLCKD. Adiponectin
Adiponectin
oligomers,
analyzed
by
Western
blotting
ting,
increased
in
the
adiponectin
from
oligomers, analyzed by Western blotting ting, increased in the adiponectin from VLCKD
VLCKD participants
participants
after
weeks of
of VLCKD
VLCKD (T1).
(T1). (A)
(A)A
Arepresentative
representative blot
blot of
ofdifferent
different oligomers
oligomers of
of adiponectin
adiponectin (high,
(high,
after eight
eight weeks
medium, and low molecular weight (HMW, MMW, and LMW)) in the serum of two VLCKD participants
medium, and low molecular weight (HMW, MMW, and LMW)) in the serum of two VLCKD
at baseline (T0) and after eight weeks of VLCKD intervention (T1). (B) Graphical representation of pixel
participants at baseline (T0) and after eight weeks of VLCKD intervention (T1). (B) Graphical
quantization of all VLCKD participants at baseline (T0) and eight weeks after VLCKD intervention (T1).
representation of pixel quantization of all VLCKD participants at baseline (T0) and eight weeks after
VLCKD intervention (T1).
3. Discussion
The VLCKD is increasingly promoted as a strategy to fight obesity. Although the VLCKD is
3. Discussion
effective for weight loss and weight control, a comprehensive determination of its relationship with
The VLCKD
is increasinglychanges,
promoted
as a strategy
tothe
fight
obesity. Although
is
biochemical
and physiological
in particular
with
adipokines
producedthe
by VLCKD
AT, is still
effective
for weight This
loss is
and
control,
comprehensive
determination
its relationship
with
largely
unexplored.
theweight
first study
that aevaluated
total adiponectin
serumoflevels
using an ELISA
biochemical
and
physiological
changes,
in
particular
with
the
adipokines
produced
by
AT,
is
still
test and its oligomeric profile using Western blotting analysis. Furthermore, the Western blotting
largely unexplored.
This is themethod
first study
that evaluated
totalresults
adiponectin
serum
using
analysis
is a semi-quantitative
to support
the previous
obtained
by thelevels
ELISA
test. an
ELISA
test
and
its
oligomeric
profile
using
Western
blotting
analysis.
Furthermore,
the
Western
The main differences between low-calorie and ketogenic diets regard macronutrient composition.
blotting
analysis
is is
a semi-quantitative
method to support
the previous
results obtained
by the ELISA
A
low-calorie
diet
a balanced diet characterized
by 45–55%
carbohydrates,
15–25% proteins,
and
test.
25–35% fat, with the additional dose of 30 g of fiber in the form of fruit and vegetables [15]. On the
The the
main
differences
between
low-calorie(<50
and
ketogenic
diets regard
macronutrient
contrary,
ketogenic
diet is low
in carbohydrates
g daily
from vegetables)
and lipids
(only 10 g
composition.
A
low-calorie
diet
is
a
balanced
diet
characterized
by
45%–55%
carbohydrates,
15%–
of olive oil per day) [16].
25% In
proteins,
andanthropometric
25%–35% fat, with
the additional
dose of in
30VLCKD
g of fiber
in the form
of fruit
and
this study,
and biochemical
parameters
participants
before
and after
vegetables
[15].
On
the
contrary,
the
ketogenic
diet
is
low
in
carbohydrates
(<50
g
daily
from
eight weeks of diet intervention were investigated, focusing on the effects of this diet on total adiponectin
vegetables)
and lipids
(onlyThe
10 results
g of olive
oil per
day) study
[16]. showed that the eight-week intervention
and
its oligomeric
profile.
of the
present
In
this
study,
anthropometric
and
biochemical
parameters
in VLCKD
participants
before and
with the VLCKD produced significant weight loss in our participants,
decreasing
pro-inflammatory
after
eight
weeks
of
diet
intervention
were
investigated,
focusing
on
the
effects
of
this
diet
on total
cytokine production, increasing adiponectin serum levels, and improving metabolic profile. To support
adiponectin
and itsstudies
oligomeric
profile.
The results
ofofthe
present study
showed In
that
the eight-week
our
results, several
reported
beneficial
effects
a ketogenic
diet [17–20].
addition,
VLCKD
intervention
with
the
VLCKD
produced
significant
weight
loss
in
our
participants,
decreasing
prois advantageous in increasing satiety despite a negative energy balance, and sustaining basal energy
inflammatory
cytokine
production,
increasing
adiponectin
serum
levels,
and
improving
metabolic
expenditure despite body weight loss due to a sparing of fat mass [20–22]. Interestingly, as reported by
profile.etToal.,
support
our results,
studies
reported
beneficial
a ketogenic
diet [17–20].
Zhang
a ketogenic
diet in several
combination
with
exercise
reducedeffects
PPARγofand
lipid synthetic
genes,
In addition, VLCKD is advantageous in increasing satiety despite a negative energy balance, and
sustaining basal energy expenditure despite body weight loss due to a sparing of fat mass [20–22].
Interestingly, as reported by Zhang et al., a ketogenic diet in combination with exercise reduced
Int. J. Mol. Sci. 2020, 21, 3228
7 of 12
as well as enhancing the PPARα and lipid β-oxidation gene program in the liver compared to those
in a ketogenic diet without exercise [23]. On the contrary, some studies reported that any beneficial
effects are only transient [24]. In addition, Ellenbroek et al. supported that a long-term ketogenic
diet (22 weeks) caused dyslipidemia, a pro-inflammatory state, signs of hepatic steatosis, glucose
intolerance, and a reduction in beta and alpha cell mass, without weight loss in mice; however, the
induction of ketosis and the response to ketosis in man and mouse are quite different, and 22 weeks is
a very long period for a mouse that could be compared to several years in human beings [23,25]. The
strength of this study is that, in a short period, the VLCKD changed the anthropometric and metabolic
profile of our participants in a statistically significant manner. It is well known that VAT is dangerous
to health, generating chronic low inflammation and leading to an imbalance in the function of adipose
tissue; for these reasons, the inverse correlation between adiponectin and VAT and pro-inflammatory
cytokines such as TNF-α is very important [26–28]. Gustafson and colleagues reported that, among the
fat storage compartments in the body, VAT was found to be an important source of pro-inflammatory
adipokines such as TNF-α and IL-6, and it was associated with an increased risk for atherosclerosis,
more so than subcutaneous fat [27]. Moreover, significant changes in VAT, as well as in inflammatory
and adipose tissue activity biomarkers, were reported, suggesting that the VLCKD, in the short term,
can be considered very important in the deregulation of the balance between abdominal fat and the
production of pro-inflammatory mediators. We also tested adiponectin, whose expression is strongly
increased after eight weeks of VLCKD. Adiponectin is the most abundant circulating adipokine,
and plasmatic levels, together with free fatty acids (FFA), are statistically inversely related to body
fat, abdominal visceral fat, and glucose and lipid metabolism. This adipokine, through its HMW
oligomers, which are the most biologically active, strongly increased in our participants after the diet
intervention. Adiponectin exerts an anti-inflammatory effect and modulates insulin sensitivity by
stimulating glucose utilization and fatty acid oxidation. On the contrary, elevated FFA was linked
with the development of insulin resistance, defects in insulin secretion, nonalcoholic fatty liver disease,
and metabolic syndrome [28]. As reported by Nigro et al., serum adiponectin levels are reduced in
obese and diabetic subjects and are considered as a marker of various metabolic diseases, as well as of
improvement of metabolic activity [29]. Furthermore, the results of the present study show a negative
correlation between adiponectin and glycated hemoglobin; these findings, in agreement with Okoro et
al., confirm the strong involvement of adiponectin in metabolic syndrome and in the establishment
of type 2 diabetes [29–31]. However, the molecular mechanism underlying the cause-and-effect
relationship between hypoadiponectinemia and insulin resistance is not yet fully clear [32]. Indeed,
in vivo and in vitro studies suggested that adiponectin has an antidiabetic and hypoglycemic effect [33],
activating hepatic insulin receptor and promoting pancreatic beta-cell function [33–35].
Data in the literature confirmed that the ketogenic diet is associated with increases in adiponectin
in obese subjects [31,35]. Sherrier et al. hypothesized that the level of nutritional ketosis may be an
important factor in the regulation of adiponectin expression, since ketones influence AMPK activity
through adiponectin [36]. Furthermore, it is worth mentioning that adipose tissue is the target of many
metabolically active factors, many of which induce the secretion of adiponectin. [36]. On the contrary,
Garaulet et al. reported that adiponectin is related to protection against the metabolic syndrome
but is not involved in the regulation of VLCD-induced improvement of insulin sensitivity [37]. In a
chronic inflammatory state, such as obesity, the physiologic status of the cells presents changes in
adipose tissue altering the production of adipokines [35]. In this scenario, another essential function
of adiponectin and its HMW oligomers is their role in inflammatory and immune responses. We
found a negative correlation between adiponectin and pro-inflammatory cytokines such as TNF-α;
in fact, adiponectin is able to suppress nucleus NF-κB translocation and pro-inflammatory cytokine
expression, including TNF-α, IL-1b, and IL-6 [38–40]. In addition, as we demonstrated, adiponectin
positively correlates with IL-10 serum levels; in fact, it increases the expression of anti-inflammatory
mediators, such as IL-10, and induces the polarization of anti-inflammatory M2 macrophages [41,42].
Furthermore, adiponectin enhances cold-induced browning of subcutaneous adipose tissue through M2
Int. J. Mol. Sci. 2020, 21, 3228
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macrophage proliferation and promotes cell proliferation via the activation of serine/threonine-specific
protein kinase Akt, consequently leading to beige cell activation [43–45]. Moreover, Tsuchida et al.
suggested that adiponectin, through innate immune response-dependent mechanisms, can regulate
insulin sensitivity and energy expenditure. On the other hand, adiponectin and its HMW oligomers are
strongly involved not only in metabolic processes but also in inflammatory and immune responses [45].
In light of this evidence, the negative correlations that we found between adiponectin and lipid profile,
VAT, CPR, and TNF-α confirmed the profound involvement of adiponectin in many metabolic and
inflammatory diseases and, in parallel, also confirmed the beneficial short-term effects of VLCKD
intervention not only in the treatment of obesity but also in the establishment of obesity-correlated
diseases. Indeed, for its anti-inflammatory properties, the ketogenic diet is used as an adjuvant
treatment in the cancer therapy. As suggested by Weber et al., a ketogenic diet creates an unfavorable
metabolic environment for cancer cells and, thus, can be regarded as a promising adjuvant as a
patient-specific multifactorial therapy [15]. The main limitation of this study is related to the small
number of participants; contrariwise, the short time of the observation period can represent a strong
point of the study. Indeed, the short period of VLCKD intervention has beneficial effects not only on
metabolic rate but also on the inflammatory state, improving adiponectin and IL-10 levels, as well as
reducing both TNF-α and IL-6 levels.
4. Materials and Methods
4.1. Participants
Twenty obese subjects (10 females, 10 males), aged between 20 and 60 years (mean 48 ± 10 years),
were enrolled. The study took place at the Laboratory of Physiology, Department of Clinical and
Experimental Medicine, University of Foggia. This study was performed in accordance with the
Declaration of Helsinki and approved by the local ethics committee on 22 May 2018, n◦ 440/DS.
According to the current legislation in Italy, informed consent was signed by all participants, who were
free to leave the study at any moment. Exclusion criteria were as follows: medical history positive for
renal insufficiency, hyperuricemia, severe hepatic insufficiency, type 1–2 diabetes mellitus treated with
insulin, atrioventricular block, heart failure, cardiovascular and cerebrovascular diseases, unbalanced
hypokalemia, hypo-hyperthyroidism, chronic treatment with corticosteroid drugs, severe mental
disorders, neoplasms, pregnancy, and lactation. All participants were highly motivated and none of
them had any previous experience with low-carbohydrate or ketogenic diets.
4.2. Study Protocol
As previously reported, all participants underwent a general medical examination. We recorded
age, height, weight, blood pressure, and laboratory tests, at baseline and after eight weeks [46,47].
During the study, dietary adherence was measured daily, between 2:00 and 4:00 p.m., by capillary
blood ketone assessment, (GD40 Delta test strips, TaiDoc Technology Co., Taiwan). Nutritional ketosis
was defined as a blood ketone (b-hydroxybutyrate) level >0.5 mmol/L. Body weight was recorded at
the same time of the day, to the nearest 0.1 kg (SECA 711, Hamburg, Germany) with the participants
wearing light clothing, and height was recorded to the nearest 0.1 cm (SECA 213, Hamburg, Germany).
Fasting (12 h) blood samples were collected at 8:00 a.m., and blood samples were centrifuged with the
resultant serum stored at −80 ◦ C until use [47].
4.3. Anthropometric Measurements
Height, weight, BMI, and waist circumference of the 20 obese participants were recorded. Body
weight was measured in a fasting state in the morning with a mechanical balance (±0.1 kg, SECA 700,
Hamburg, Germany). BMI was calculated as body weight divided by height squared (kg/m2 ) with
categories in accordance with the World Health Organization guidelines. To define participants with
alterations in BMI and circumferences, the reference intervals normalized to the age of each participant
Int. J. Mol. Sci. 2020, 21, 3228
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were applied [47]. Body composition was estimated by DXA (Lunar Prodigy DXA, GE Healthcare,
USA), via whole-body scan. VAT was quantified by subtracting subcutaneous fat from total abdominal
fat, reported in grams, using the CoreScan software (GE Healthcare, Madison, WI, USA) [48].
4.4. Biochemical Parameters
Blood human samples were taken after obtaining the informed consent from the patients or control
subjects in accordance with the tenets of the Declaration of Helsinki. The samples were collected after
an overnight fast (12 h), and serum samples were collected. Serum albumin, insulin, C-reactive protein,
glucose, total cholesterol, HDL and LDL cholesterol, and triglycerides were measured. Concentrations
of total adiponectin in serum were measured via an enzyme-linked immunosorbent assay (ELISA)
using a commercial kit (Elabscience, Houston, Texas, USA). Furthermore, we analyzed TNF-α, IL-10,
and IL-6 serum levels via enzyme-linked immunosorbent assay (ELISA) using a commercial kit (BD
Opt EIA for human TNF-α, IL-10, IL-6). All ELISA tests were performed in triplicate, and the protocols
followed were according to the manufacturers’ instructions.
4.5. Western Blotting Ting Analysis
Serum samples from all participants were quantified for total proteins by the Bradford assay
(Bio-Rad, Hercules, CA, USA), and 10 µg of total protein was heated in 1× Laemmli buffer at 95 ◦ C
for 10 min and loaded onto 10% SDS-PAGE gels as previously described [49]. The immunoblots were
developed by ECL (Amersham Biosciences, Piscataway, NJ, USA) with the use of Kodak BioMax
Light film, digitalized with a scanner (1200 dpi), and analyzed by densitometry with ImageJ software
(https://imagej.nih.gov/ij/). Each sample was tested three times in duplicate.
4.6. Diet Intervention
The participants followed the VLCKD according to a commercial weight loss program (Lignaform,
Therascience, 3, rue de l’Industrie, 98000 Principato di Monaco), previously reported in our published
paper [47]. The characteristics of this diet are reported in Table 3.
Table 3. VLCKD characteristics.
Fats (%)
Proteins (%)
Carbohydrates
Carbohydrates from vegetables (g/day)
Total Kcal/day
43
43
14
<50
700–900
4.7. Statistical Analysis
Statistical analyses were performed using the StatView software 5.0.1.0 (SAS Institute, Cary, NC,
USA). All data are presented as means ± SD. A p-value ≤0.05 was used for statistical significance.
Adiponectin, VAT, and CRP serum concentrations were correlated by Pearson’s or Spearman’s rho
tests, according to data distribution. A p-value < 0.05 was considered statistically significant. Multiple
comparisons on Western blotting ting experiments were performed.
Author Contributions: Conceptualization, V.M., R.P., G.C. (Giuseppe Cibelli), and G.M.; methodology, V.M. and
R.P.; validation, G.C. (Giuseppe Cibelli) and G.M.; formal analysis, A.V., F.S., E.N., A.L., and A.A.; data curation
A.F. and N.D.N.; writing—original draft preparation, V.M., R.P., G.C. (Gaetano Corso), and G.M.; writing—review
and editing, G.C. (Giuseppe Cibelli), and G.M.; visualization, G.C. (Giuseppe Cibelli) and G.M. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by Therascience (Monaco), free of charge to the participants. The funding
source had no involvement in the study design, recruitment of participants, study interventions, data collection,
or interpretation of the results.
Int. J. Mol. Sci. 2020, 21, 3228
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Acknowledgments: We wish to thank the Scientific Bureau of the University of Catania for language support.
We wish to thank Antonietta Messina, Ines Villano, Marco Carotenuto, Fiorenzo Moscatelli, Girolamo Di Maio,
Aurora Daniele, and Marcellino Monda of University of Campania, Luigi Vanvitelli for their support.
Conflicts of Interest: The authors declare no conflict of interest.
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