International Journal of

Environmental Research
and Public Health

Article
Loss of Skeletal Muscle Mass and Intracellular Water as
Undesired Outcomes of Weight Reduction in Obese
Hyperglycemic Women: A Short-Term Longitudinal Study
Jolanta Zalejska-Fiolka 1 , Anna Birková 2, * , Tomasz Wielkoszyński 3 , Beáta Hubková 2, * , Beata Szlachta 1 ,
Rafał Fiolka 4 , Urszula Błaszczyk 1 , Aleksandra Kuzan 5 , Andrzej Gamian 6 , Mária Mareková 2
and Michał Toborek 7

1 Department of Biochemistry, Faculty of Medical Science in Zabrze, Medical University of Silesia,

40-055 Katowice, Poland; [email protected] (J.Z.-F.); [email protected] (B.S.);
[email protected] (U.B.)
2 Department of Clinical and Medical Biochemistry, Pavol Jozef Šafárik University, 040 11 Košice, Slovakia;
[email protected]
3 Higher School of Strategic Planning, 41-303 Dabrowa
˛ Górnicza, Poland; [email protected]
4 Doctoral School, Faculty of Medical Science in Zabrze, Medical University of Silesia, 40-055 Katowice, Poland;
[email protected]
5 Department of Medical Biochemistry, Wrocław Medical University, 50-367 Wrocław, Poland;
[email protected]
6



Institute of Immunology and Experimental Therapy, 53-114 Wrocław, Poland; [email protected]

 7 Department of Biochemistry and Molecular Biology, University of Miami School of Medicine,
Citation: Zalejska-Fiolka, J.; Birková, Miami, FL 33136, USA; [email protected]
A.; Wielkoszyński, T.; Hubková, B.; * Correspondence: [email protected] (A.B.); [email protected] (B.H.); Tel.: +421-55-234-3447 (A.B.);
Szlachta, B.; Fiolka, R.; Błaszczyk, U.; +421-55-234-3365 (B.H.)
Kuzan, A.; Gamian, A.; Mareková,
M.; et al. Loss of Skeletal Muscle Abstract: The current study is focused on the influence of hyperglycemia on weight loss in obese
Mass and Intracellular Water as premenopausal women. Specifically, the study evaluated the impact of a six-month individualized
Undesired Outcomes of Weight low-calorie diet combined with moderate exercise on weight reduction and glucose metabolism in
Reduction in Obese Hyperglycemic obese women with normoglycemia compared to obese women with moderate hyperglycemia. The
Women: A Short-Term Longitudinal results indicated that patients with normoglycemia achieved a successful weight loss, which was
Study. Int. J. Environ. Res. Public
connected to a decrease in adipose tissue and reflected by diminished content of visceral fat area (VFA)
Health 2022, 19, 1001. https://
and percent body fat. In contrast, weight reduction in patients with hyperglycemia was connected not
doi.org/10.3390/ijerph19021001
only to the loss of VFA but also to undesired decrease in skeletal muscle mass as well as intracellular
Academic Editors: Eusebio Chiefari and total body water. These unfavorable outcomes were observed despite normalization of glucose
and Amy Elizabeth Rothberg metabolism reflected by statistically significant lowering glucose, fructosamine, advanced glycation
Received: 23 November 2021
end-products, and HOMA-IR levels. Overall, the obtained results indicate the importance of the
Accepted: 14 January 2022 measurement of the carbohydrate profile in obese women and the need for an early introduction of
Published: 17 January 2022 weight reduction strategies before the development of hyperglycemia.

Publisher’s Note: MDPI stays neutral

Keywords: anthropometric parameters; bioimpedance; extracellular body water (ECW); hyper-
with regard to jurisdictional claims in
glycemia; intracellular body water (ICW); total body water (TBW); free fat mass (FFM); visceral fat
published maps and institutional affil-
area (VFA); weight loss
iations.

Copyright: © 2022 by the authors. 1. Introduction

Licensee MDPI, Basel, Switzerland. Background: The prevalence of overweightness and obesity is increasing rapidly,
This article is an open access article especially in developing countries, and affects approximately 650 million people world-
distributed under the terms and wide [1]. In Poland, approximately 61% of men and 50% of women are overweight or
conditions of the Creative Commons
obese [2]. There is also an equally high rate of increase in the incidence of Type 2 diabetes
Attribution (CC BY) license (https://
(T2D) worldwide. The World Health Organization Global Report on diabetes indicates that
creativecommons.org/licenses/by/
the number of adults living with T2D has almost quadrupled to 422 million people since
4.0/).

Int. J. Environ. Res. Public Health 2022, 19, 1001. https://doi.org/10.3390/ijerph19021001 https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2022, 19, 1001 2 of 15

1980 [3] and is expected to increase to 693 million by 2045 [4]. The disease is characterized
by high blood glucose and insulin levels due to tissue insulin resistance. There is a strong
association between obesity and T2D, with more than 80% of obese individuals affected by
this disease.
Health risks associated with obesity differ substantially depending on the distribution
of fat tissue. In contrast to the accumulation of peripheral subcutaneous fat, excessive
visceral fat (i.e., abdominal obesity) is strongly associated with obesity-related complica-
tions, including insulin resistance, hyperinsulinemia, hyperglycemia, dyslipidemia, and
hypertension [5]. Waist circumference, an estimate of abdominal obesity, is positively
associated with insulin resistance and the progression from impaired glucose tolerance to
T2D [6,7]. Therefore, an intervention that specifically targets abdominal obesity and insulin
resistance has important implications for diabetes prevention [8].
While abdominal obesity is more common in men, it is associated with more health
risks in women. A larger waist circumference and waist-to-hip ratio (WHR) conferred
a greater risk of cardiovascular disorders (CVDs) in women than in men. In addition,
the WHR ratio was more strongly associated with the risk of CVDs than the body mass
index (BMI) in women [9,10]. Several studies have also demonstrated that T2D affects
women disproportionately [11]. Women with T2D have poorer glycemic control and, when
treated, are less likely to reach the desired normalization of glycated hemoglobin (HbA1c)
compared to men [12–14].
Weight loss programs that employ effective and safe tools are critical to prevent the
development of T2D, cardiovascular diseases, and various other diseases, including can-
cer [15]. The foundation of a successful weight loss strategy should be lifestyle changes,
including high-quality, appropriate, and individualized diet and increased physical activ-
ity [16,17]. Indeed, a personalized diet combined with regular check-ups is considered to
be an effective strategy to improve adherence to the recommendations and avoid body
weight regain, helping with permanent lifestyle changes and gaining greater long-term
benefits [18]. Adding physical activity to a hypocaloric, individualized diet is important to
reduce fat mass only and preserve muscle mass during weight loss [19].
Visceral fat mass is closely related to the metabolic dysregulations in obesity, and
excess of visceral fat leads to the development of insulin resistance and fat accumulation
in internal organs such as liver, heart, pericardium, skeletal muscles, vascular walls, or
blood vessels, which then trigger to the pathophysiology of metabolic diseases. Therefore,
a decrease in fat mass during body mass reduction is associated with the improvement
of metabolic alterations [20–23]. On the other hand, a loss of fat-free mass, including
muscle mass, could be associated with unfavorable results including development of
certain pathologies such as sarcopenia [21,24].
The mechanisms participating in a decrease in muscle mass during weight reduction
are not fully understood. Evidence indicates that a short-term (up to 21 days) calorie
restriction (30–40% energy deficit/day) can decrease the postprandial rate of muscle protein
synthesis and reduce the basal rate of muscle protein synthesis [25]. On the other hand,
it was indicated that a prolonged moderate calorie restriction and 5–10% weight loss
increased the rate of muscle protein synthesis [26]. Overall, the loss of muscle mass
during a prolonged moderate calorie restriction is believed to be mediated by increased
muscle proteolysis rather than suppressed muscle protein synthesis, and it is viewed as
an undesirable effect. It appears that increased physical activity during weight loss may
protect against this unfavorable process [27]. The results of the current study indicate that
other factors that affect the type of tissue being reduced during weight loss should also
be taken into account. One of such factors could be untreated hyperglycemia/impaired
glucose tolerance.
The objective of the present study was to evaluate the impact of hyperglycemia
on weight loss in premenopausal and hyperglycemic women with abdominal obesity.
We hypothesized that elevated glucose levels and impaired glucose tolerance in obese
women on personalized diets and exercise programs can affect weight reduction and loss
Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 3 of 18

Int. J. Environ. Res. Public Health 2022, 19, 1001

on personalized diets and exercise programs can affect weight reduction and loss of 3non- of 15

fat tissues. Due to gender-related metabolic differences, we focused our research on

premenopausal women. While obese women with normoglycemia achieved a successful
of non-fat
weight loss,tissues.
which Due to gender-related
was connected metabolic
to a decrease differences,
in adipose tissuewe
andfocused our
reflected byresearch
dimin-
on premenopausal
ished women.
content of visceral While
fat area (VFA), obese women
weight with normoglycemia
reduction in obese women achieved a suc-
with hypergly-
cessfulwas
cemia weight loss, which
associated waswith
not only connected
the lossto
of aVFA
decrease in adipose
but also with an tissue and decrease
undesired reflected
byskeletal
in diminished
musclecontent
massofasvisceral
well as fat area (VFA),
intracellular weight
and total reduction
body water. in obese women with
The conducted re-
hyperglycemia was associated not only with the loss of VFA but also with
search shows that hyperglycemia/impaired glucose tolerance may be one of the factors an undesired de-
crease in skeletal muscle mass as well as intracellular and
causing the loss of free fat mass tissue during weight reduction. total body water. The conducted
research shows that hyperglycemia/impaired glucose tolerance may be one of the factors
causing
2. Methodsthe loss of free fat mass tissue during weight reduction.
2.1. Study Design
2. Methods
The study
2.1. Study Designwas a prospective, one-center study. Patient recruitment and weight re-
duction program
The study was started in October one-center
a prospective, 2016 and lasted
study.12 Patient
monthsrecruitment
overall. All volunteers
and weightde- re-
clared
ductiontheir willingness
program to in
started participate
October in theand
2016 6-month
lastedweight reduction
12 months program
overall. that con-
All volunteers
sisted of atheir
declared personalized low-calorie
willingness dietaryinprogram
to participate combined
the 6-month weightwith physicalprogram
reduction activity and
that
aconsisted
continuous health education program that was focused on maintenance
of a personalized low-calorie dietary program combined with physical of a balanced
activity
and
and individualized low-calorie
a continuous health diet.
education Every month,
program that was patients
focusedtook part in follow-up
on maintenance visits.
of a balanced
The study was approved by the Ethics Committee of the Medical University
and individualized low-calorie diet. Every month, patients took part in follow-up visits. of Silesia
in Katowice,
The studyPoland (No. KNW/0022/KB1/19/I/16).
was approved by the Ethics Committee In accordance with
of the Medical the provisions
University of
of Silesia
the abovementioned
in Katowice, application
Poland (No. to the ethics committee,
KNW/0022/KB1/19/I/16). each participant
In accordance with the of the study
provisions of
gave informed consent to participate in the experiment and to publish the results
the abovementioned application to the ethics committee, each participant of the study gave of the
research.
informed consent to participate in the experiment and to publish the results of the research.

2.2. Participants
2.2. Participants
Thirty-five obese
Thirty-five obese women
womenout out of
of aa total
total of
of 300
300 patients
patients from
from the
the Metabolic
Metabolic Clinic
Clinic in
in
Miasteczko Śl askie,
˛ Poland qualified for the
Miasteczko Śląskie, Poland qualified for the study (Figure study (Figure 1). All patients entered
All patients entered the the
weight reduction
weight reduction program
program based
based on
on the
the reports
reports indicating
indicating improving
improving lipid
lipid and
and glucose
glucose
levels after
levels after weight
weight lossloss [28].
[28]. All
Allwomen
womenwere were diagnosed
diagnosedasas obese
obeseon on the
the basis
basis ofof medical
medical
examination(body
examination (bodymass massindex
index(BMI),
(BMI),body
bodyfatfatmass
mass(BFM),
(BFM),andandvisceral
visceralfat
fattissue
tissue(VFA).
(VFA).
All women
All women were
were characterized
characterized by BMI indicating obesity.

Figure
Figure 1.
1. The
Theflowchart
flowchartof
ofthe
thestudy
study design.
design.

On the first visit, all patients were evaluated for biochemical markers, including the
carbohydrate and lipid profiles. Out of 35 participants involved in the program, 15 women
Int. J. Environ. Res. Public Health 2022, 19, 1001 4 of 15

were normoglycemic and 20 were hyperglycemic, and none of them were taking any
medications known to affect glucose and lipid metabolism. Inclusion criteria for study
participants were body mass index (BMI) >30 kg/m2 , no pharmacological treatment af-
fecting lipid and glucose metabolism, and informed consent to participate in the study.
In addition, study exclusion criteria were lack of consent to participate in the study, se-
vere hepatic, renal, respiratory, or circulatory insufficiency, alterations of consciousness,
treatment-resistant depression, chronic alcohol abuse, pregnancy, history of a severe ner-
vous system injury, implanted cardiac pacemaker, and pharmacological treatment affecting
glucose and lipid metabolism.

2.3. Classification of Patients into Study Groups

Study participants were categorized into two groups based on their plasma glucose
levels when entering the study:
Group NG: The normoglycemic group, normoglycemic patients (15 females) with a
fasting glucose level <100 mg/dL.
Group HG: The hyperglycemic group, hyperglycemic patients (20 females) with a
fasting glucose level above reference values >100 mg/dL.

2.4. The First and Follow-Up Visit Plan, Diet Composition and Physical Activity Intervention,
Variables, and Procedures to Avoid Study Bias
The patients were advised to attend one personalized consultation per month for six
months. On the first visit, the following data were collected: (a) history of obesity, (b) food
preferences and food habits, (c) number and quality of meals consumed per day, (d) number
and quality of liquids consumed per day, (e) health problems, (f) biochemical parameters
from a recent blood test, and (g) physical activity. The questionnaire revealed that patients
ate unhealthily and irregularly as well as led sedentary lifestyles before starting the weight
loss program.
During the first visit performed by a doctor and a dietitian (1 h/patient), the following
parameters were measured based on performance protocols: (a) height; (b) body weight
and body composition (InBody S10, Biospace, Cerritos, CA, United States); and (c) the
individual basic metabolic rate (BMR). Based on this information, the total daily caloric
demand (DCD) and daily energy deficit (calculated as 15% of the total energy of DCD)
were determined for each patient.
One week after the first consultation, all patients received personalized instructions in
an easily readable table about nutrition and diet composition according to recommendations
by the Polish National Food and Nutrition Institute (Polish acronym, IŻŻ) and the World
Health Organization, including (a) a seven-day menu adapted to taste preferences of each
individual, (b) recommendations of food and liquid quantities and the numbers of meals
per day (five times a day and no unhealthy snacks), (c) list of recommended and non-
advised food products, and (d) the list of vegetables and fruits with glycemic index (IG)
with recommendation that one portion could be eaten as a snack per day if necessary. The
participants were advised to implement physical activity for a minimum of 30–40 min three
times per week. The personalized diet was established for each patient and contained all
the necessary ingredients, as shown in Table 1.
Every month, during the follow-up visits also performed by a doctor and a dietitian,
patients were weighed, and their waist and hip circumferences were measured. Patients
were motivated to continue the program and received support in the form of training in
calculating calorie intake and the quality of food products. Weight reduction was monitored
until achieving a healthy body weight or reduction by 5–15% over initial weight and lasted
six months on average.
During the first and the last visit, fasting blood was collected from the vein of the
elbow flexion in quantity of 7 mL (5 mL to obtain serum and 2 mL to obtain plasma),
according to approval by the Ethics Committee application. Obtained data were collected
in the Department of Biochemistry Faculty of Medical Science in Zabrze Medical University
Int. J. Environ. Res. Public Health 2022, 19, 1001 5 of 15

of Silesia, Katowice, Poland. To avoid bias, all the obtained data was anonymized. For
biochemical tests and statistical analysis, the samples were numbered (Figure 1).

Table 1. General diet composition employed in the present study and assembled according to healthy
nutrition recommendations of the Polish National Food and Nutrition Institute and the World Health
Organization (WHO).

Nutrient % of Total Energy Intake Note

Limiting intake of free sugars to less than
Carbohydrates 45–55%
10% of total energy intake.
Limiting intake of saturated fatty acids to
less than 10% of total energy intake, and
intake of 3–6% mono- and polyunsaturated
Fats 25–35%
fatty acids in the form of vegetable oils and
fish oils; omega-3: omega6 1:4 (5);
trans-fatty acids <2% of total energy.
Proteins 15–25% Animal: vegetable sources in 1:2 ratio.
Fiber About 1%
Vegetable 500 g/day in portions Low and medium glycemic index (IG).
Fruits 200/300 g/day Low and medium glycemic index (IG).
Clean water ≥1.5–2 L/day
Salt <4 g/day

2.5. Biochemical Analysis

All procedures followed the good laboratory practice (GLP). Fasting blood samples
were collected to the serum clot (5 mL) and EDTA (2 mL) tubes (Vacutainer, Becton Dickin-
son) during the first and the last visit. After clotting (ca. 30 min), blood was centrifuged
(10 min, 3000 rpm), and the aliquoted serum samples were frozen in −80 degrees Celsius.
The biochemical analyses were carried out in blinded samples by experienced scientific
and technical staff of the Department of Biochemistry. Individual biochemical markers
(except HbA1c) were measured in one repetition and one analytical series after sample
collection was completed for all patients. All analytical methods were under continuous
intralaboratory quality control and met the criteria of the external (interlaboratory) controls
organized by the Central Center for Quality Testing in Laboratory Diagnostics in Łódź
(Łódź, Poland) and the Labquality (Helsinki, Finland).
Serum glucose, fructosamine, and glycated hemoglobin (HbA1c) were determined
using a biochemical analyzer (Miura 200 DA) (I.S.E. S.r.l., Guidonia Montecelio, Italy).
Glucose concentration was assayed by the glucose oxidase method. The fructosamine
assay was based on nitro blue tetrazolium reduction method (both reagents from Bisys-
tems, Barcelona, Spain). HbA1c was determined in EDTA whole blood samples by the
latex-enhanced immunoturbidimetry method (Human GmbH, Wiesbaden, Germany) and
serum insulin by the INS-IRMA kits (KIP1251-KIP1254, DIA Source Immuno Assays S.A.,
Louvain, Belgium). Repeatability (within-run precision coefficients of variation) for glu-
cose, fructosamine, HbA1c, and insulin were 0.6%, 2.1%, 1.4%, and 4.1%, respectively.
Reproducibility (between-run precision coefficients of variation) for the abovementioned
parameters were 1.6%, 3.9%, 3.1%, and 7.7%, respectively. The homeostatic model assess-
ment for insulin resistance (HOMA-IR) was calculated as (fasting insulin concentration
(µIU/m) × fasting glucose concentration (mg/dL))/405.
Concentrations of the advanced glycation end-products (AGE) in serum were deter-
mined in the Department of Medical Biochemistry, Wrocław Medical University, Poland
by the competitive ELISA method according to Indyk et al. [29]. Briefly, 96-well MaxiSorp
plates (Nunc) were coated with synthetic high molecular mass AGE adduct (HMW-AGE)
based on myoglobin, obtained by high-temperature microwave synthesis, and incubated
Int. J. Environ. Res. Public Health 2022, 19, 1001 6 of 15

for 4 h at 37 ◦ C. The plates were then blocked with 10% skim milk for 18 h at 4 ◦ C. Di-
luted serum samples were subsequently incubated overnight at 55 ◦ C with proteinase K
from Tritirachium album (0.3 mg/mL, Sigma Aldrich, St. Louis, MI, USA) and heated for
20 min at 116 ◦ C to denature the proteinase, cooled, centrifuged for 15 min at 15,000× g,
and the supernatants were diluted twice with phosphate-buffered saline (PBS). Next, the
samples were incubated with noncommercial monoclonal antibodies IgE anti-MAGE, and
100 µL of the mixtures were distributed to wells of the HMW-AGE-coated plate, followed
by incubation at room temperature for 1.5 h. In parallel, synthetic low molecular mass
AGE (LMW-AGE) solutions were incubated with antibody and distributed to wells for
the preparation of a standard curve. The LMW-AGE was obtained by high-temperature
microwave synthesis and isolated by gel filtration on the HW-40S and P2 bed. After incuba-
tion, plates were washed three times in PBS with 0.05% Tween 20 (PBST). Next, the reaction
was carried out with secondary, horseradish peroxidase (HRP)-conjugated, anti-mouse IgE
polyclonal antibody diluted 1:7000 (AP21482HR-N, OriGene Technologies, Inc., Rockville,
MD, USA) for 2.5 h at room temperature, followed by washing the plates again three times
with PBST. The HRP enzymatic activity bound to solid phase was assayed by a substrate
solution containing o-phenylenediamine and hydrogen peroxide (Sigma Aldrich, St. Louis,
MI, USA) in citrate buffer (100 µL/well). Absorbance was measured at 450 nm in the
EnSpire Manager microplate reader. Concentrations of the AGE-10 were calculated from
the standard curve prepared. Repeatability and reproducibility of this method were 4.5%
and 7.9%, respectively. The methodology has been patented by Institute of Immunology
and Experimental Therapy of the Polish Academy of Science [30].

2.6. Body Mass Composition Using Bioimpedance Analysis

Bioimpedance was selected for body mass composition measurement as a fast and
efficient method with high accuracy [31]. Body mass composition parameters, such as arm
circumference (AC), arm muscle circumference (AMC), visceral fat area (VFA), percent body
fat (PBF), free fat mass (FFM), skeletal muscle mass (SMM), protein (an indicator of body
nutrition), bone mineral content (BMC), total body water (TBW), intracellular body water
(ICW), and extracellular body water (ECW) were analyzed during the first and the last visit
(twice for each patient) using a professional body composition analyzer by bioelectrical
impedance spectroscopy (InBody S10, Biospace, Cerritos, CA, USA). Resistance (R) of arms,
trunk, and legs was measured after 3 h of fasting. To avoid measurement errors, the results
are automatically calculated on the basis of 30 impedance measurements using six different
frequencies (1 kHz, 5 kHz, 50 kHz, 250 kHz, 500 kHz, 1000 kHz) of each of the five parts of
the body (right and left upper limb, torso, right and left lower limb) with an eight-polar
tactile-electrode impedance meter, two of each which were in contact with the middle finger
and the thumb on each arm and around the ankle of each foot. Each time, the same person
performed the analysis for a given patient. Because it was a short-term study (6 months)
conducted on adult women, aging and growth were not considered factors affecting the
results. The device does not require calibration before measurement. The device certificates
guarantee reliability, reproducibility, and calibration: the InBodyS10 analyzer had the
following certificates: International ISO 9001: 2015; International ISO 13485: 2016; Medical
certificate EN60601-1; Medical certificate EN60601-1-2 and CE MDD (Directive 93/42/EEC).
Because body position, hydration status, consumption of food and beverages, ambient
air and skin temperature, and recent physical activity affect bioimpedance results, we
created the measurement protocol for standardization and calibration. The bioimpedance
measurement protocol included (a) age, sex, height, body mass; (b) longitudinal follow-up
was performed at the same slot, and the menstrual cycle was considered (the study was not
conducted during menstruation period; (c) fasting 3 h was required; (d) no alcohol intake
for 12 h; (e) no medications that affect the water balance measurement (hormones, diuretics,
steroids); (f) no metallic accessories or magnetic targets; (g) the patient had to remove
heavy clothes and jewelry; (h) the study was not conducted after abruptly standing up;
(i) the room temperature was 20–25 degrees C during measurement. After half an hour, the
Int. J. Environ. Res. Public Health 2022, 19, 1001 7 of 15

analysis was performed for each patient who came to the clinic, and all the subjects were
always in the upright position and had to be standing for at least 5 min before taking the
measurement. The repeatability of the measurements expressed as percentage coefficients
of variation was determined on the basis of 12 measurements performed on the same
person in one day. All the assessed parameters were characterized by the coefficients of
variation below 3%.

2.7. Statistical Analysis

The results are presented before and after the 6-month weight reduction program. The
differences between these values are calculated as delta (∆) and/or percentage change (∆%).
All results are shown as mean ± SD. Statistical analysis was performed with SPSS Statistics
22 (IBM, Armonk, NY, USA). The Kolmogorov–Smirnov test was used for normality testing.
A two-sample t-test was used to determine differences of clinical parameters between
groups and between values within groups before and after dietary intervention, assuming
or not assuming equal variances based on Levene’s test for equality of variances. For
parameters with non-normal distribution, the Mann–Whitney test was used to determine
differences between groups, and the Wilcoxon signed-rank test was used to determine
differences within groups before and after dietary intervention. The Pearson correlation
coefficient expressed the strength and direction of the linear relationship between the
two parameters.
Statistical significance was assumed at p-value of <0.05.

3. Results
3.1. Characteristics of Participants and Study Limitation
Despite the admission of 300 eligible patients to the program, compliance was met
only by 35 women that are included in the study, characteristics of which shows Table 2.
This was due to the following reasons: (a) the majority of originally preselected patients
appeared to be taking medications affecting carbohydrate and lipid metabolism; thus,
they met our exclusion criteria; (b) we experienced patient drop-out of the study (some
patients who started the program stopped attending follow-up visits after one, two, or three
months and unfortunately, these individuals had to be removed from the study group);
(c) despite declaration, some patients did not undertake a prescribed diet and/or physical
activity regimen.

Table 2. Participants initial characteristic data.

Normoglycemic Group (NG) Hyperglycemic Group (HG) NG vs. HG

Parameter (n = 15) (n = 20) p
Before Program After Program Before Program After Program Before Program After Program
Age [year] 51 ± 13.3 47.5 ± 7.8 0.81
Weight [kg] 95.5 ± 21.0 83 ± 14.9 89.7 ± 16.3 80.8 ± 14.7 0.65 0,98
p 0.069 0.057
BMI 36.3 ± 6.7 30.5 ± 4.9 34.8 ± 6.1 31.2 ± 5.8 0.98 0.59
p 0.042 * 0.061
WC (cm) 107 ± 15 94 ± 11 103 ± 13 93 ± 14 0.89 0.51
p 0.016 * 0.027 *
Glucose
94 ± 7.0 85 ± 12.8 119 ± 49.5 92.0 ± 15.5 <0.001 ** 0.067
(mg/dL)
p 0.05 * 0.005 **
Data are expressed as mean ± SD for comparison differences between measurements before and after the weight
reduction program within and between the groups. T-test significance: * p ≤ 0.05; ** p ≤ 0.01.
 Int. J. Environ. Res. Public Health 2022, 19, 1001 8 of 15

3.2. Impact of the Weight Reduction Program on Carbohydrate Metabolism

Obese normoglycemic women had fasting glucose levels at 94 ± 7.0 mg/dL, while fast-
ing glucose levels in hyperglycemic patients was moderately elevated to 119.5 ± 49.5 mg/dL
(p < 0.001) prior to the weight reduction program. A 6-month program resulted in a sig-
nificant decrease in glucose levels in both groups; however, a larger change, by 23%, was
detected in hyperglycemic patients (p = 0.005), and levels reached normoglycemic range
without difference between groups (p = 0.067) (Figure 2A). The initial concentrations of
glycated hemoglobin (HbA1c) were within reference values at ~5.2 (%) in both groups.
There were no significant changes in HbA1c concentrations after weight reduction in
both study groups (Table 3). The initial levels of fructosamine of 353 ± 67.4 (µmoL/L)
were higher in hyperglycemic patients compared to 306 ± 37.9 (µmoL/L) detected in
normoglycemic
Int. J. Environ. Res. Public Health 2022, 19, patients (p = 0.008) (Figure 2B). There were no changes observed
x FOR PEER REVIEW 9 ofafter
18
weight reduction in the normoglycemic group; however, fructosamine levels significantly
decreased to 303.0 ± 42.1 (µmoL/L) in hyperglycemic patients (p = 0.023) (Figure 2B).

Figure 2. Improvement of carbohydrate profile of overweight hyperglycemic women as the result of

the weight reduction program. Normoglycemic and hyperglycemic obese women were placed on
a 6-month personalized weight reduction program consisting of a moderate calorie restriction and
moderate exercise individualized to each patient. Glucose (A), fructosamine (B), advanced glycation
products (C), and HOMA IR (D) were measured at the beginning and the end of the program. Data
are mean ± SD. T-test significance: p ≤ 0.05.

The concentrations of AGE were elevated before the weight reduction program, reach-
ing 3278 ± 833 (µg/mL) in the normoglycemic patients and 3725 ± 815 (µg/mL) in the
hyperglycemic patients. These levels decreased significantly as the result of the pro-
gram in both groups, namely to 2850 ± 345 in the normoglycemic group (p = 0.013) and
to 2870 ± 424 in the hyperglycemic group (p = 0.0043) (Figure 2C). There were no statisti-
cally significant differences between these groups.
The initial concentrations of insulin were 10.2 ± 5.3 (µIU/mL) in the normoglycemic
patients and 9.0 ± 3.4 in the hyperglycemic patients. There were no significant changes
observed as a result of weight reduction in both groups and between the groups (Table 3).
HOMA-IR index exceeded the normal range in both groups before the weight reduction
program and it was 2.3 ± 1.3 in the normoglycemic group and 2.6 ± 1.4 in the hyperglycemic
 Int. J. Environ. Res. Public Health 2022, 19, 1001 9 of 15

group. Following the program, HOMA-IR decreased in both groups, reaching the level of
significance in both groups (p = 0.05 in normoglycemic group and p = 0.025 in hyperglycemic
group) (Figure 2D).

Table 3. Impact of the weight reduction program on glycated hemoglobin (HbA1c) and insulin levels
in obese normoglycemic and hyperglycemic women.

Normoglycemic Group Hyperglycemic Group

NG vs. HG
(NG) (HG)
p
(n = 15) (n = 20)
Before After Before After Before After
Parameter
Program Program Program Program Program Program
HbA1 (%) 5.2 ± 0.8 5.1 ± 0.7 5.2 ± 0.5 5.2 ± 0.6 0.52 0.46
p 0.55 0.43
Insulin
10.2 ± 5.3 8.6 ± 3.7 9.0 ± 3.4 8.8 ± 4.9 0.56 0.94
(µIU/mL)
p 0.34 0.66
Data are expressed as mean ± SD for comparison differences between measurements before and after the weight
reduction program within and between the groups. T-test significance: p ≤ 0.05. Parameters of carbohydrate
metabolism that changed significantly within and/or between the groups as the result of the implemented weight
reduction program are shown in Figure 2A–D.

3.3. Differential Impact of the Weight Reduction Program on Weight Loss in Normoglycemic and
Hyperglycemic Obese Women
The weight reduction program resulted in a decrease in weight for both normoglycemic
and hyperglycemic patients by 13% (p = 0.069) and 10% (p = 0.057), respectively (Table 4).
Int. J. Environ. Res. Public Health 2022, Concurrent
19, x FOR PEERBMI changes in normoglycemic patients (a decrease by ~16%; p = 0.042) were
REVIEW 12 of 18
statistically significant but not in hyperglycemic patients (a decrease by ~10%; p = 0.061)
(Figure 3A).

Figure 3. Body mass parameters that differentially changed in normoglycemic or hyperglycemic

obese women placed on a 6-month personalized weight reduction consisting of a moderate calorie
restriction and moderate exercise individualized to each patient. Body mass index (A), percent body
fat (B), free fat mass of the trunk (C), protein (D), skeletal muscle mass (E), intracellular body water
(F), and the total body water of the trunk (G) were measured at the beginning and the end of the
program. Data are mean ± SD. T-test significance: p ≤ 0.05.
Int. J. Environ. Res. Public Health 2022, 19, 1001 10 of 15

Table 4. Impact of the weight reduction program on selected body composition parameters in obese
normoglycemic and hyperglycemic women.

Normoglycemic Group (NG) Hyperglycemic Group (HG) NG vs. HG

(n = 15) (n = 20) p
Parameter Before Program After Program Before Program After Program Before Program After Program
Weight (kg) 95.5 ± 21.0 83 ± 14.9 89.7 ± 16.3 80.8 ± 14.7 0.65 0.98
p 0.069 0.057
WC (cm) 107 ± 15 94 ± 11 103 ± 13 93 ± 14 0.89 0.51
p 0.016 * 0.027 *
AC (cm) 38.7 ± 5.8 33 ± 3.8 39.2 ± 6.3 34.8 ± 3.2 0.38 0.16
p 0.012 * 0.003 **
AMC (cm) 29.8 ± 4.5 27.2 ± 2.6 32.2 ± 5.0 29.0 ± 2.5 0.15 0.07
p 0.023 * 0.003 **
VFA (cm2 ) 153.5 ± 35.3 109.2 ± 31.9 134.7 ± 32.3 110.4 ± 28.9 0.28 0.74
p 0.020 * 0.030 *
FFM (kg) 49.6 ± 9.9 49.5 ± 7.4 54.2 ± 6.7 50.6 ± 6.2 0.55 0.97
p 0.49 0.064
BMC (kg) 2.8 ± 0.6 2.8 ± 0.5 3.0 ± 0.4 2.9 ± 0.5 0.93 0.62
p 0.87 0.35
TBW (L) 36.5 ± 7.2 36.4 ± 5.4 39.8 ± 5 37.2 ± 4.6 0.57 0.99
p 0.47 0.068
ECW (L) 14.0 ± 2.7 13.8 ± 2.2 14.5 ± 2.0 14.2 ± 2.0 0.91 0.97
p 0.44 0.21
ECW/TBW 0.384 ± 0.008 0.382 ± 0.011 0.38 ± 0.018 0.38 ± 0.010 0.047 * 0.76
p 0.58 0.20
ECW/TBW
0.384 ± 0.009 0.383 ± 0.010 0.38 ± 0.017 0.38 ± 0.011 0.052 0.83
TR
p 0.78 0.15
Data are expressed as mean ± SD for comparison differences between measurements before and after the weight
reduction program within and between the groups. T-test significance: * p ≤ 0.05; ** p ≤ 0.01. Body parameters
that significantly changed in normoglycemic or hyperglycemic obese women as the result of the implemented
weight reduction program are shown in Figure 3.

Significant decreases in waist circumference (WC) by ~12% (p = 0.016), arm circum-

ference (AC) by ~15%, arm muscle circumference (AMC) by ~9%, visceral fat area (VFA)
by ~29% (Table 4), and percent body fat (PBF) by ~14% (Figure 3B) were observed in the
normoglycemic patients as a result of the weight reduction program. On the other hand,
there were no changes observed in free fat mass (FFM), skeletal muscle mass (SMM), or
total body protein, as well as the parameters connected with water content in the body,
such as intracellular body water (ICW) and total body water (TBW) in these patients
(Figure 3C–G, respectively).
Similar to the normoglycemic patients, the weight reduction program significantly
decreased WC (p = 0.027), AC (p = 0.003), AMC (p = 0.003) (all by 10–11%), and VFA by ~20%
(p = 0.03) in the hyperglycemic patients (Table 4). In contrast to the normoglycemic group,
there also was a significant decrease in FFM of the trunk (FFM TR) by ~7% (p = 0.0045)
(Figure 3C), and undesired diminished values of the protein by ~7.5% (p = 0.032) (Figure 3D),
and SMM by ~8% (p = 0.033) (Figure 3E), without changes in PBF (Figure 3B) in the
hyperglycemic patients as a result of the weight reduction program. Significant decreases
in ICW by ~8% (p = 0.033) (Figure 3F) and TBW of the trunk (TBW TR) by ~9% (p = 0.006)
(Figure 3G) were also observed in the hyperglycemic patients. Overall, these results suggest
that weight change in hyperglycemic patients was driven in a large part by the losses
in SMM, intracellular body water, and the total body water of the trunk. There were no
statistically significant changes found in extracellular body water (ECW) and bone mineral
content (BMC) in both groups and between the groups (Table 4).
Int. J. Environ. Res. Public Health 2022, 19, 1001 11 of 15

3.4. Correlations between Carbohydrate Metabolism and Markers of Body Mass

When analyzing correlations between plasma levels of carbohydrate metabolism with
markers of body mass in normoglycemic patients, only limited associations have been
found (Table 5). Specifically, AGE levels were positively correlated with changes in AC
and AMC. On the other hand, no correlations were observed between glucose, HbA1c,
fructosamine, insulin, and HOMA-IR with AC, AMC, VFA, and PBF. In contrast, strong
positive correlations between AGE and VFA, between AGE and ICW, and between WC
and ICW were observed in hyperglycemic patients, suggesting that changes in ICW could
influence AGE levels and weight (Table 6).

Table 5. Correlation analysis among studied parameters in obese normoglycemic women (n = 15)
before and after the weight reduction program. The correlation is significant: * p ≤ 0.05; ** p ≤ 0.01.

Pearson’s R
∆AC ∆AMC ∆VFA ∆PBF
p
0.97 0.87 0.88 0.74
∆Weight
0.000 ** 0.000 ** 0.000 ** 0.004 **
0.96 0.86 0.90 0.73
∆BMI 0.000 ** 0.000 ** 0.000 ** 0.005 **
0.88 0.85 0.82 0.40
∆WC 0.000 ** 0.000 ** 0.000 ** 0.18
0.22 0.25 −0.16 −0.18
∆Glucose 0.47 0.41 0.59 0.57
0.28 0.30 0.04 0.09
∆HbA1 0.35 0.32 0.88 0.78
0.03 −0.07 −0.15 0.31
∆Fructosamine 0.92 0.83 0.62 0.33
0.58 0.62 0.35 0.39
∆AGEs 0.039 * 0.023 * 0.22 0.19
0.39 0.49 0.20 −0.01
∆Insulin 0.21 0.11 0.52 0.98
0.35 0.44 0.08 −0.08
∆HOMA IR 0.26 0.15 0.81 0.80

Table 6. Correlation analysis among studied parameters in obese hyperglycemic women (n = 20)
before and after the weight reduction program. The correlation is significant: * p ≤ 0.05; ** p ≤ 0.01.

Pearson´s
∆FFM ∆TBW
R ∆AC ∆AMC ∆VFA ∆FFM ∆SMM ∆PROT ∆ICW
TRUNK TRUNK
p
0.66 0.00 0.47 −0.16 −0.07 −0.10 −0.10 −0.08 −0.10
∆Weight
0.004 ** 0.99 0.036 * 0.54 0.80 0.69 0.69 0.75 0.71
0.64 −0.02 0.48 −0.17 −0.08 −0.11 −0.11 −0.10 −0.11
∆BMI 0.005 ** 0.93 0.031 * 0.52 0.77 0.67 0.66 0.72 0.69
0.54 0.07 0.51 −0.19 −0.10 −0.18 −0.18 −0.10 0.82
∆WC
0.024 * 0.80 0.023 * 0.47 0.71 0.50 0.50 0.70 0.001 **
0.43 0.07 0.16 0.00 0.04 0.06 0.07 0.002 0.32
∆Glucose 0.09 0.78 0.49 0.99 0.89 0.83 0.78 0.94 0.28
−0.09 −0.00 −0.11 0.04 −0.08 0.06 0.06 −0.10 0.43
∆HbA1 0.74 0.99 0.63 0.89 0.77 0.83 0.81 0.72 0.15
0.11 −0.13 0.13 −0.09 −0.28 −0.05 −0.03 −0.31 0.06
∆Fructosamine
0.68 0.63 0.59 0.74 0.28 0.86 0.92 0.23 0.86
−0.42 −0.02 0.58 −0.41 −0.45 −0.39 −0.38 −0.46 0.56
∆AGEs
0.10 0.95 0.007 ** 0.10 0.07 0.12 0.13 0.06 0.045 *
−0.17 −0.42 0.10 0.02 −0.23 0.08 0.07 −0.22 0.47
∆Insulin 0.54 0.12 0.69 0.95 0.42 0.79 0.80 0.43 0.13
∆HOMA −0.20 −0.51 0.11 0.11 −0.30 0.18 0.18 −0.34 0.19
IR 0.50 0.061 0.68 0.70 0.30 0.54 0.53 0.24 0.52
Int. J. Environ. Res. Public Health 2022, 19, 1001 12 of 15

4. Discussion
The results of the present study indicate that relatively modest lifestyle changes, such
as moderate energy restriction and physical exercise, can effectively influence weight
reduction and improve glucose tolerance in hyperglycemic obese women. The primary
therapies for weight reduction among individuals with insulin resistance and T2D are diet
and physical exercise, as they promote both weight loss and improve glucose tolerance.
Indeed, it is generally accepted that energy restriction can significantly improve fasting
glucose concentrations and glucose tolerance, even with relatively small changes in body
weight. Such dietary intervention is generally well tolerated by obese individuals when
implemented for a short period of time [32,33]. For example, it was demonstrated that
blood pressure and metabolic parameters, such as lipid metabolism and glucose tolerance,
can all be improved by a moderate 5–10% weight reduction [34]. Exercise, in addition to
facilitating weight loss, enhances biological action of insulin, and exerts a preventive input
on the incidence of T2D [35].
Abdominal obesity caused by visceral fat accumulation strongly correlates with insulin
resistance and a high risk for T2D [36,37]. Therefore, the approaches to improve the glucose
metabolic state are based on burning fat tissue, especially VFA. The personalized strategy
implemented in the present study differs from the majority of the weight loss methodologies
as it favors a long-term intervention in order to achieve and maintain sustainable changes
in weight reduction and developing healthy eating and exercise habits. Moreover, longer
duration of the weight reduction program, such as 6 months implemented in the present
study, does not require severe dietary restrictions, which are usually difficult or even
impossible to maintain for patients for a prolonged period of time.
The present study was performed on women volunteers interested in losing weight
and changing lifestyles through a balanced diet and physical exercise. All patients were
obese (BMI > 30) with visceral fat (VFA > 100) exceeding the reference values. The ques-
tionnaire revealed that patients ate unhealthily and irregularly and led sedentary lifestyles
before starting the weight loss program. Part of the studied pool of patients exhibited
moderately elevated glucose levels, which did not affect HbA1c levels that remained within
normal range. The implemented weight reduction program consisted of a personalized
dietary and exercise program and was effective as all patients lost weight and decreased
several other parameters of body mass. Moreover, there were significant improvements
in glucose, AGE, and HOMA-IR levels, leading to normalization of glucose metabolism.
These outcomes are in line with the literature data. For example, it was demonstrated
that weight loss strategies using dietary, physical activity, or behavioral interventions
produced significant improvements in weight control among people with prediabetes, and
a significant decrease in incidence of diabetes [38].
The observed impact of the personalized weight reduction program was strikingly
different when comparing the normoglycemic and hyperglycemic patients. The most
important data of the present study indicate that the weight reduction program decreased
BMI only in the normoglycemic group but not in hyperglycemic women. This effect was
connected with favorable loss of adipose tissue and reflected by a decrease of VFA and PBF,
without any significant changes in the parameters connected to body water content (e.g.,
ICW) and muscle mass (e.g., body protein content and SMM). We propose that sensitivity
of peripheral tissues to insulin, and thus the greater susceptibility of adipose tissue to
insulin-dependent lipolysis, can be responsible for these changes. On the contrary, weight
loss in patients with hyperglycemia was connected not only to the loss of VFA but also
to non-fat mass loss, such as undesired decreases in SMM, free fat mass (FFM), body
protein mass, ICW, and the total body water of the trunk (TBW TR). These outcomes
could result from the baseline insulin resistance in these patients. Clearly, altered glucose
metabolism as manifested by hyperglycemia at the start of the weight reduction program
had detrimental impact on the entire weight loss process. These observations are consistent
with the notion that the impact of lifestyle intervention on diabetes prevention cannot be
solely ascribed to the body weight reduction [39]. It should also by noted that WC, AC, and
Int. J. Environ. Res. Public Health 2022, 19, 1001 13 of 15

AMC diminished in both normoglycemic and hyperglycemic patients following the weight
reduction program.
While the loss of ICW in hyperglycemic patients as a result of the weight reduction
program might be puzzling, the patients were encouraged to replace their diets with
healthy alternatives. Thus, changing the diet and assuming a reduction in the consumption
of animal saturated fatty acids (e.g., butter, cream, sausages, red meat) in favor of diet
containing higher levels of healthy unsaturated fatty acids (such as fish, fresh olive oil, nuts,
or seeds) can lead to remodeling of cell membranes and improvement of transmembrane
transport. The effect of this remodeling may be the loss of ICW with no changes in the
amount of extracellular water (ECW). In addition, the observed loss of muscle tissue
(e.g., diminished SMM) could also indirectly contribute to the loss of ICW. The process of
losing this tissue may, in turn, be the result of an intensification of the gluconeogenesis
induced by a calorie-restricted diet. It also appears that water loss observed in patients
with hyperglycemia could influence, at least in part, the observed improvements of the
carbohydrate metabolic state because ICW levels exhibited a significant correlation both
with changes in VFA and with AGE.
The weight reduction program resulted not only in a marked improvement of glucose
levels in hyperglycemic obese women but also in a decrease in the levels of AGE in both
studied groups, reflecting the overall highly beneficial impact of the strategy implemented
in the present study. Lowering the concentration of AGEs is a favorable phenomenon
because of high biological activity of these compounds that results in tissue damage leading
to the development of diabetes and diabetic complications.
The limitations of the presented study are the following:
1. Small study groups for reasons described in the Methods section.
2. The study included only women.
3. The obtained results cannot be applied to the whole population of hyperglycemia
patients, as the study conducted only patients without treatment affecting glucose
and lipid metabolism.
4. The confirmation of the results in a long-term study is required.

5. Conclusions
The present study indicates that weight reduction based on a balanced caloric restric-
tion diet combined with moderate exercise is more efficient in normoglycemic than in
hyperglycemic obese women. Despite improvements in glucose and AGE levels, reduction
in weight in obese women with hyperglycemia involved undesired loss of non-fat tissues,
such as skeletal muscle mass and body protein content, as well as intracellular and total
body water. These results indicate the importance of the measurements of the carbohydrate
profile in overweight and obese women and introducing an early weight reduction program
before the development of hyperglycemia.

Author Contributions: Conceptualization, J.Z.-F.; methodology, T.W., B.S., A.K., A.G. and R.F.;
validation, U.B.; formal analysis, M.M.; investigation, J.Z.-F., T.W. and B.S.; resources, J.Z.-F. and
M.T.; data curation, A.B. and B.H.; writing—original draft preparation, J.Z.-F. and R.F.; writing—
review and editing, A.B. and M.T.; visualization, A.B. and M.T.; supervision, J.Z.-F. and M.T.; project
administration, J.Z.-F.; funding acquisition, J.Z.-F. and M.T. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by the Medical University of Silesia in Katowice (KNW-1-
109/K/8/Z and KNW-1/171/K/9/O) and by the Metropolis GZM Science and Education Support
Fund under the program “Metropolitan Fund for Science Support” (RW/13/2021).
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Ethics Committee of the Medical University of Silesia
in Katowice, Poland (No. KNW/0022/KB1/19/I/16).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Int. J. Environ. Res. Public Health 2022, 19, 1001 14 of 15

Data Availability Statement: The database of aggregated statistics prepared for analysis is stored in
secure, confidential, password-protected storage in the server of the Medical University of Silesia. The
data has been anonymized. Completely deidentified records could be made available to interested
persons/organizations on request at the author’s address [email protected].
Acknowledgments: Special thanks for Sławomir Kasperczyk, Irena Gryc and laboratory staff from
Clinic in Miasteczko Ślaskie
˛ for allowing visiting patients and help in blood collection and for senior
technicians Ewa Chwalińska and Edyta Hudziec from Department of Biochemistry Faculty of Medical
Science in Zabrze Medical University of Silesia in Katowice for help in biochemical analysis.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
AC, arm circumference; AMC, arm muscle circumference; AGE, advanced glycation end-products;
BMI, body mass index; DCD, daily caloric demand; ECW, extracellular body water; FFM, free fat
mass; HbA1c, glycated hemoglobin; HOMA-IR, homeostatic model assessment for insulin resistance;
ICW, intracellular body water; IG, glycemic index; PBF, percent body fat; SMM, skeletal muscle mass;
TBW, total body water; T2D, Type 2 diabetes; VFA, visceral fat area; WC, waist circumference.

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