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Journal of Diabetes and Metabolic Disorders logoLink to Journal of Diabetes and Metabolic Disorders
. 2020 Sep 5;19(2):1205–1214. doi: 10.1007/s40200-020-00626-w

Anti-inflammatory and antidiabetic effects of grape-derived stilbene concentrate in the experimental metabolic syndrome

Anatoly Kubyshkin 1, Alina Shevandova 1, Vitalina Petrenko 1,✉, Irina Fomochkina 1, Leya Sorokina 1, Alexander Kucherenko 1, Andrey Gordienko 2, Natalia Khimich 2, Evgenia Zyablitskaya 2, Tatiana Makalish 2, Leonid Aliev 1, Natalia Kornienko 1, Ivan Fomochkin 1
PMCID: PMC7843874  PMID: 33553024

Abstract

Aims

This study aimed to investigate the carbohydrate and lipid dynamics, associated inflammation markers and the effectiveness of a grape-derived stilbene concentrate (GDSC) treatment in experimental metabolic syndrome (MetS).

Methods

The study was carried out on 40 male 12-weeks of age Wistar rats. The MetS was induced using the fructose model (feeding with 60%-solid fructose diet for 24 weeks). Rats with induced MetS were treated with polyphenolic GDSC, which was obtained by water-alcohol extraction of Vitis vinifera grapevine (Ressfood LLC, Russia).

Results

The experimentally induced MetS development leads to classic MetS signs, including abdominal obesity, hyperglycemia, high lipid levels and heart damage. The expression of glucose transporter type 4 (GLUT4) and peroxisome proliferator-activated receptor-γ (PPAR-γ) had greater dynamics than biochemical measurements. The development of the associated inflammatory reactions was confirmed by the increased level of Toll-like receptor type 4 (TLR4) and C-reactive protein (CRP) compared to control levels. The use of the GDSC had positive dynamics in carbohydrate and lipid levels, inflammatory marker, also prevented associated inflammation and heart damage.

Keywords: Metabolic syndrome, Molecular markers, PPAR-γ, TLR 4, GLUT 4, CRP, Associated inflammation, Grape-derived stilbene concentrate, Polyphenols

Introduction

According to Worldwide Definition of International Diabetes Federation (IDF), the metabolic syndrome (MetS) includes several risky factors such as abdominal obesity, hyperglycemia, hypercholesterolemia and arterial hypertension. Currently, MetS is one of the most widespread pathology across the globe. The scale of this problem has taken on an especially alarming character in industrialized countries, since a significant part of the population of these countries is exposed to several risk factors such as lack of physical activity, fast-food diet, chronic stress, etc. For example, in the US, nearly 35% of adults, and 50% of those older than age 60, have MetS [1]. MetS has pro-inflammatory, thrombogenic, and atherogenic ability, which increases the risk of life-threatening pathologies - type 2 diabetes, heart attack, stroke and neoplasms [2, 3]. MetS is a multifactorial disease with polymorphism and gene expression disorders, results in the violation of carbohydrate and lipid metabolism and immunological reactivity [4].

One of the key elements of MetS is insulin resistance. Its occurrence is associated with the polymorphism of the genes responsible for the synthesis of peroxisome proliferator-activated receptor type γ (PPAR-γ) proteins. These proteins are involved in the transmission of the insulin signal [5, 6]. Transduction disturbance is realized at the post-receptor level. At the same time, hyperinsulinemia can compensate disorders of the insulin realization for a long time. As the syndrome progresses, β-cells are depleted and insulin resistance is complicated by insulin production insufficiency [7]. This suggests that hyperglycemia, which is considered one of the main MetS criteria, occurs later than some molecular changes of MetS. According to the results of earlier investigation, toll-like receptors (TLRs) are the main trigger of immune response and have a key role in the MetS pathogenesis. They identify typical bacterial antigens in infectious diseases, the lipopolysaccharides (LPS) of intestinal bacteria, endogenous ligands such as free fatty acids, triglycerides (TGs), and trigger the innate and acquired immunity. Saturated fatty acids also can play the role of TLR4 ligands [8]. It has been shown that experimental mice obesity induced by mice feeding with rich fat diet increases the TLR2 and TLR4 expression in adipocytes and hepatocytes [9]. It has also been shown that, although mutation in the TLR4 encoding gene increases the risk of atherosclerosis and coronary heart disease, but decreases the concentration of circulating pro-inflammatory substances such as C-reactive protein (CRP), fibrinogen, cytokines, adhesins etc. As a result, TLR4 activation leads to subclinical systemic inflammation [10].

The recent investigations reveal several pathogenetic links between metabolic disorders associated with obesity and low-grade inflammation development. It is caused by pro-inflammatory secretion of adipocytes, which are overloaded by triglycerides. It causes activation of immune cells with pro-inflammatory functions. In addition, the cells of the white adipose tissue secretes pro-inflammatory substances: interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and monocyte chemoattractant protein-1 [11]. Moreover, lipid-overloaded hypertrophic adipocytes can cause associated inflammation in adipose tissue independently of adipocyte inflammation as a result of defects in glucose transporter type 4 (GLUT4) trafficking to the plasma membrane [12]. Expression of GLUT4 gene has a very narrow range - between 2 to 3-fold, but has a meaningful effect on glucose homeostasis in whole organism, indicating a GLUT4 control as a major homeostatic tool in various physiological conditions or in long-term adaptation to pathology, particularly in obesity [13].

Recent research prove that metabolic disorders are related to subclinical inflammation. They demonstrate the TLR4 signaling in the cells of intestinal epithelium is a starting point in the MetS development by the metabolic pathways activation of microbiota in host organism [14]. The results of another investigation indicate that TLR4 may have a key role in the starting of steatosis, afterwards its exhaustion causes an increase of fatty acid oxidation in the mice liver, preventing TGs accumulation [15].

MetS has complicated and multidirectional pathogenesis with various pathogenetic mechanisms. Respectively, an effective approaches to treatment should have multidirectional effects. Nowdays, the use of plant extracts medications containing trans-resveratrol, proanthocyanidins, stilbenes, and flavonoids is regarded as one of forward-looking ways of pathogenetic correction of MetS [16, 17]. Grape-derived stilbene concentrate (GDSC) is a novel polyphenol substance, full of stilbenes, especially trans-resveratrol and ε-viniferin. A number of studies have revealed the anti-inflammatory, antidiabetic, lipid-lowering, antioxidant and antiproliferative effects of trans-resvertarol [18–20]. One of its mechanism of action is the inhibition of TLR 4/nuclear factor κB (NF-κB)/transcription activators (STAT) intracellular signaling. Furthermore it can reduce cell apoptosis through TLR 4-myeloid differentiation factor 88 (MγD88)-NF-κB cellular pathway [21].

Also it was found that polyphenols have antidiabetic effect and increase the GLUT4 and PPAR-γ expression, which improves insulin sensitivity and metabolic characteristics [22].

The relationship between the resveratrol intake and metabolic characteristics in MetS was established in the latest meta-analysis of 16 controlled studies. Also combining the results of studies indicated significant effect of resveratrol on decreasing weight, TGs, systolic blood pressure and it can improve lipids rate [23].

This study aims to research the dynamics of the carbohydrate and lipid parameters, the associated inflammation markers in an experimental MetS model, and to assess the effectiveness of its treatment with GDSC.

Methods

Animal model and experimental design

The experiments were perfomed on 40 white male rats Wistar of the SPF category (280–290 g, aged 12 weeks) (Laboratory Animal Nursery Pushchino, Russia). The protocol No.1 for the use of animals was reviewed and approved by the V.I.Vernadsky Crimean Federal University Ethics Committee (17th Jan, 2018) and it was in accordance with the European Communities Council Directive, 1986. Rats were housed in cages each containing five animals with a controlled room temperature (20 ± 2 °C), relative humidity (60 ± 5%) and a regulated light cycle (12 h light/dark) with free access to food and water. After 10 days of acclimation on laboratory chow, the rats were randomly divided into 4 groups (n = 10 in each group): the control group of healthy animals with a control diet (5001 Lab Rodent Diet, 0.30% kcal from fructose), rats with experimental model of MetS (high-fructose diet for 24 weeks), rats with diet-induced MetS treated with GDSC (from the 14th to the 24th week), and rats with induced MS treated with GDSC (from the 19th to the 24th week) (Fig. 1).

Fig. 1.

Fig. 1

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Design of experimental study

Randomization of animals

Randomized block was provided. According to the design of the experiment, all animals were divided into subgroups, which were analyzed by weight, age and glucose level. A preliminary statistical analysis of the selected subgroups homogeneity, normality and p-differences between subgroups was carried out. All subgroups were homogeneous and hadn’t any significant differences.

Diet

The MetS was induced using the fructose diet with a 60% fructose content of solid feed (Oriental Yeast Co., Ltd., Tokyo) – fructose-fed rats (FFR) (Research Diets, USA) [24, 25]. Control group rats had standard solid feed. Both diets had enough vitamins and minerals to keep the rodents healthy. Except for biweekly overnight fasts, animals had ad libitum access to food and always had ad libitum access to water. The physiological state of the rats was monitored every day. Body weight and food intake were recorded every two weeks.

Experimental grape-derived stilbene concentrate

An experimental grape-derived stilbene concentrate (GDSC) was used for treatment of MetS. It contains stilbenes, catechins, trans-resveratrol, proanthocyanidins and other substances, which have antioxidant, metabolic and anti-inflammatory effects. The experimental grape-derived stilbene concentrate (Ressfood LLC, Russia), obtained by water-alcohol extraction of the Vitis vinifera grapevine, was used for MetS treatment in one group starting from the 14th to the 24th week and in another group from the 19th to the 24th week of feeding. GDSC includes (g/L): gallic acid – 0.158, (+)-D-cateсhin – 0.379, (−)-epicatechin – 0.446, trans-resveratrol – 0.070, ε-viniferin – 0.749, oligomeric proanthocyanidins – 2.569, polymeric proanthocyanidins – 17.329, non-identified stilbene – 0.697, total stilbenes – 1.519, and total phenols by high-performance liquid chromatography – 22.41. The safe effective dose of trans-resveratrol according to the Food and Drug Administration (FDA) and toxicology research is 2 mg/kg per day [26]. The trans-resveratrol dose in GDSC was 0.071 g/L (0.071 mg/mL), so the total daily volume of GDSC, which was pre-dissolved in water, was 7 mL by oral tube for each rat.

Sample collection and analysis

Once every 2 weeks, animals were subjected to an overnight fast before fasting blood glucose measurements (described below). Food was taken away 1–2 h before the starting of the dark cycle, and returned after blood glucose measurements, approximately 12 h later. Fasting blood glucose was obtained via tail prick and measured using a Freestyle glucometer (Abbot Laboratories, Chicago, IL).

Blood samples from animals were collected by tail vein blood sampling during experiments and by cardiac puncture at the end of the experiment. After standing at room temperature for 30 min, the blood was centrifuged at 4 °C and the plasma was recovered. Plasma aliquots for biochemical and lipidomic analyses were collected into Eppendorf tubes and then frozen with a liquid nitrogen and stored at −80 °C for further analysis. The cholesterol and TGs levels were detected using commercially available kits (BioAssay Systems, Inc. USA).

On 24th weeks of experiment, following a 12 h fasting period, all of the rats in each experimental group were euthanized by an intraperitoneal ketamine/xylazine injection and were decapitated. Abdominal fat was extracted from the mesentery of the small intestine and was weighed (2140Adventurer AR electronic lab scale). Abdominal aortae and hearts were also harvested.

Inflammatory markers measurement

The TLR 4, GLUT 4, PPAR-γ (ng/mL), and C-reactive protein (CRP) levels (mg/mL) were measured in plasma by Enzyme-linked immunosorbent assays (ELISA) using commercial ELISA kits (Cusabio Biotech Co, Ltd) according to the manufacturer’s instructions. A detection range was of 0.626 ng/mL-40 ng/mL, a sensitivity was of 0.157 ng/mL, assay time of 1–5 h, sample volume was of 50–100 μL, and a detection wavelength was of 450 nm.

Heart and abdominal aorta histology

Heart and abdominal aorta histological studies was carried out with visible light and scanning electron microscopy (SEM). Hearts from each rats groups (n = 4 per group) were fixed in 10% neutral-buffered formalin, embedded in a single paraffin blocks (cut = 6 μm), stained with hematoxylin and eosin. Heart and abdominal aorta sections were examined by an independent experienced observer blinded to the treatment of the tissue. Laboratory equipment such as: LOGOS hybrid histological processor (Millestone, Italy) and Leica techniques (Germany) - LEEC LTD cutting station, modular microtome RM 2255 with automatic rotation of the filling center EG 1150, DM2000 laboratory microscope, Bond-Max immunohistostainer, an Aperio CS2 digital scanner were used. Scanning electron microscopy was carried out using a scanning electron microscope REM - 106 in high vacuum mode with an accelerating voltage of 20 kV. The pre-dried samples of heart were vacuum sprayed using a conductive coating in the form of a 20 nm layer of gold. DFC 495 Leica camera and lens N Plan 40x or N Plan 10 SL for imaging were used.

Statistical analysis

Statistical analysis was carried out using the Statistica 10.0 program using parametric (Student’s T test) and non-parametric (Wilcoxon’s W-test) tests or analysis of variance with post-tests for multiple comparisons, as indicated in the figure legends. Significance was assigned to p ≤ 0.05.

All measurements and studies were carried out using measuring instruments that passed metrological calibration and auxiliary equipment that was certified at the Center for Collective Use of Scientific Equipment (Molecular Biology) of the Medical Academy named after S.I. Georgievsky (structural unit) V.I. Vernadsky Crimean Federal University.

Results

The experimentally induced MetS development leads to classic MetS signs, including abdominal obesity, hyperglycemia, hypercholesterolemia, and hypertriglyceridemia. Abdominal obesity, which is the main MetS criterion, was found. Abdominal fat in the MetS group was 149.6% higher than in the control group (p < 0.001). Starting from the 14th week, GDSC treatment of MetS caused a 53.6% (p < 0.001) abdominal fat reduction compared to the MetS group without correction. GDSC treatment starting from the 19th week caused a 40.0% abdominal fat reduction compared to the MetS group without correction (Fig. 2).

Fig. 2.

Fig. 2

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Abdominal fat. 1: MetS group without treatment; 2: MetS group treated with GDSC beginning the 14th week; 3: MetS group treated with GDSC beginning the 19th week; 4: control group. * - p < 0.001 сompared to control. # - p < 0.001 сompared to MetS group without treatment

Using this experimental MetS model in rats we found out that hyperglycemia development took a long time. Most of the time, the glucose level in the blood of the FFR was even lower than the control values. The lowest value of this parameter was detected after 4 weeks of investigation. At that time point, it was 23.0% (p < 0.01) lower compared to that of controls. Only after the 18th week the glucose level began to rise. By the 24th week the average blood glucose value in the induced MetS rats reached its maximum value – 6.8 mmol/L, which was 17.2% (p < 0.001) higher than in the control group. At the same time, the average glucose level in the treated GDSC group starting from 14th week decreased to 5.0 mmol/L, which was 36.0% (p < 0.001) lower than the blood glucose levels in the induced MetS group without treatment. GDSC treatment of induced MetS rats beginning the 19th week was less effective; the glucose level in that group decreased by 13.0% (p < 0.05) compared to the blood glucose levels in the induced MetS group without treatment (Fig. 3).

Fig. 3.

Fig. 3

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Glucose levels in MetS after early and late experimental treatment with GDSC. MetS vs Control: ** p < 0.01, *** p < 0.001. MetS+ GDSC since 14 week vs Control: ## p < 0.01, ### p < 0.001. MetS+ GDSC since 19 week vs Control: ++ p < 0.01, MetS+ GDSC since 14 week vs MetS: ••• p < 0.001. MetS+ GDSC since 19 week vs MetS: ⊗ p < 0.05

The results of these studies showed that the experimental MetS development was accompanied with significant disorders of lipid metabolism. The total cholesterol level rose and reached a statistically significant increase (15.8%; p < 0.01) until the 24th week of fructose feeding. The use of GDSC prevented the cholesterol level increase. The cholesterol level was 28.0% (p < 0.01) lower in the GDSC treatment group starting from the 14th week than in induced MetS rats without GDSC treatment. In rats which GDSC treatment began in the 19th week, the cholesterol level was 17.3% (p < 0.05) lower than in induced MetS rats without GDSC treatment.

One of the important MetS criteria is the TGs level in the blood. The results of our investigations showed that by the 24th week of experimental MetS development, the TGs level increased by 38.0% (р < 0.01) compared to levels in the controls. The TGs levels in the groups with early and late treatment were 35.7% (p < 0.001) and 34.7% (p < 0.001) lower than in induced MetS rats without GDSC treatment, respectively (Fig. 4).

Fig. 4.

Fig. 4

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Cholesterol and TGs levels in MS after early and late experimental treatment with GDSC. * - compared to control: ** p < 0.01. # - compared to MetS group without treatment: # p < 0.05, ## p < 0.01, ### p < 0.001

It is well known that the MetS development is accompanied by heart disease. So the next stage of our research was to examine the morphology of the heart and blood vessels. It has been shown that MetS modeling causes heart and blood vessels damage, including stasis and perivascular edema, myocardial bundles rip, endothelial damage, loosening of the aortic wall, destruction of the elastic skeleton and soaking of the subendothelial intima layer with lipid inclusions (Figs. 5 and 6A1–2, B1–2).

Fig. 5.

Fig. 5

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40х. А. Fragments of the abdominal aortae from the males in the control group. Paraffin cut. Stained with hematoxylin and eosin. A1 Vessel wall. A2 The elastic framework of the aorta (arrows). В. MS 24th. Fragments of aortae from males with hyperglycemia. Paraffin cut. Stained with hematoxylin and eosin. В1 Loosening of the vessel wall, soaking of the subendothelial intima layer with lipid inclusions (arrow). В2 Damage to the endothelium in the aortic wall (arrow)

Fig. 6.

Fig. 6

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40х A. Heart fragments from the males in the control group. Paraffin cut. Stained with hematoxylin and eosin. A1. Myocardium of the left ventricle large. A2. Myocardium of the atrium. B. MS 24th. Fragments of the heart from males with hyperglycemia. Paraffin cut. Stained with hematoxylin and eosin. В1. Myocardium of the left ventricle. The phenomena of stasis and perivascular edema with the release of blood cells from the lumen of blood vessels. В2. Endocardium, myocardium. Cleavage of cardiomyocyte bundles (thin arrow), endothelial damage (thick arrow)

Treatment of experimental MetS with GDSC had a positive effect on the heart and aorta morphologically. This was confirmed by saved heart and vessels structure, saved myofibril striation and abundant myocardial vascularization compared to MetS group without treatment (Fig. 7a, b).

Fig. 7.

Fig. 7

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Scanning electron microscopy of cardiac tissue from rats with MetS (a) and MetS+ GDSC beginning in the 14th week (b). А: Ripped myocardial fibers. B: Saved cardiomyocyte myofibril striation. Abundant myocardial vascularization

Before the development of biochemical disorders, by the 14th week of feeding, the GLUT4 concentrations had already changed significantly – it was 250.1% higher than in the control group (p < 0.001).

By the 24th week of feeding the experimental MetS modeling was accompanied with the increase of the main transmembrane glucose transporter GLUT4 – it was 580.0% higher than control values (p < 0.001). In the 14th week GDSC treated group the GLUT4 level was 1150.4% higher than in control group (p < 0.001) and was 86.9% higher compared to the induced MetS group without GDSC treatment (p < 0.01). In experimental group with GDSC treatment starting from 19th week, the GLUT4 concentration was 57,6% higher compared to levels in the MetS group without treatment (p < 0.01) and was 77.3% higher than in the 14th week GDSC treated group (p < 0.001) (Fig. 8).

Fig. 8.

Fig. 8

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GLUT4 and PPAR-γ levels in MetS following early and late experimental treatment with GDSC. Compared to control: ** p < 0.01, *** p < 0.001. Compared to MetS group without treatment: # p < 0.05, ## p < 0.01, ### p < 0.001. MetS+GDSC since 14 week vs MetS+GDSC since 19 week: • p < 0.05, •• p < 0.01

One of the key roles in maintaining energy homeostasis belongs to PPARs, a group of hormone-dependent nuclear transcription factors. The level of PPAR-γ receptor expression correlates reliably with the sensitivity of adipocytes to insulin. Induction of experimental MetS caused a compensatory 17.0% increase in the PPAR-γ concentration. Experimental GDSC treatment of MetS beginning with the 14th week of the experiment caused an 11.0% increase in PPAR-γ level compared to the MetS group without treatment. Starting from 19th week late onset treatment caused a 51.2% decrease in the PPAR-γ level compared to the MetS group without GDSC treatment and caused 67,2% decrease compared to the 14th week GDSC treated group (p < 0.05) (Fig. 8).

Our research also revealed that induction of experimental MetS is accompanied by the development of an associated inflammatory response. This is based on the increase in the TLR4 concentration by 470.3% compared to that of controls (p < 0.001). GDSC treatment of induced MetS beginning with 14th week caused a 65.1% decrease in the TLR4 concentration compared to induced MetS group without treatment (p < 0.001); the same results were received for the induced MetS group treated with GDSC starting from 19th week - 57.9% decrease in the TLR4 concentration compared to induced MetS rats without treatment (p < 0.001) (Fig. 9).

Fig. 9.

Fig. 9

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TLR4 levels and CRP levels in induced MetS rats following early and late experimental treatment with GDSC. Compared to control: ** p < 0.01, *** p < 0.001. Compared to MetS group without treatment: ## p < 0.01, ### p < 0.001. MetS+ GDSC since 14 week vs MetS+ GDSC since 19 week: ••• p < 0.05, ••• p < 0.01

CRP is one of the systemic inflammatory reaction markers. Our research showed that in the experimental MetS model, this parameter was 150,4% higher compared to control levels (p < 0.001). In GDSC treated group beginning with 14th week CRP level was 76.0% (p < 0.01) lower than in induced MetS rats without GDSC treatment. In group treated with GDSC starting from 19th week of the experiment, the CRP level decreased the most – it was 81.61% lower than in the induced MetS group without GDSC treatment (p < 0.001) (Fig. 9).

Discussion

The results of this study demonstrate that MetS model using the fructose diet with a 60% fructose content of solid feed is absolutely valid. It is proved by the expected trends in the levels of the indicators used as MetS markers. Meanwhile, the metabolic parameters traditionally used as the MetS markers - abdominal obesity, hyperglycemia, hypercholesterolemia and hypertriglyceridemia [27, 28] - showed persuasive trend only by the 24th week. For example, most of the time the glucose levels in the induced MetS group were even lower than control values. Only after the 18th week the glucose level began to rise in comparison to those of the controls. The lower glucose levels in MetS animals vs.controls in the first weeks of the study can be considered as compensatory hyperinsulinemia in response to intense frucose stimulation. It leads to overexpression of the main transmembrane glucose transporter GLUT4, which translocates to the cell surface in response to insulin-enhancing sensitivity [29]. PI3-K activation in insulin-stimulated GLUT4 plays a primary role along with Akt2 in contributing to insulin-mediated GLUT4 redistribution [30]. Again, only after the 18th week the glucose level began to rise in comparison to that of the control, which in our opinion is evidence of the development of insulin resistance. Lipid metabolism disorders also showed typical dynamics during the several months of fructose feeding. Only by the 24th week of the experiment the total cholesterol and TGs levels mildly reached statistically increased levels. In contrast, the GLUT4 concentration had already changed by the 14th week.

In our studies we used GDSC as the experimental treatment for MetS: it is an experimental medicine, which according to information provided by the manufacturer, contains natural phytoalexins, proanthocyanidins, resveratrol, catechin, and other active ingredients. GDSC has a similar polyphenols composition as “Enoant,” “Enoant Premium” and “Fenokor” [31], but additionally has trans-resveratrol, ε-viniferin and a lot of stilbenes. The results of our research showed that GDSC treatment has a positive effect on most of the metabolic, molecular, and morphological markers of experimental MetS.

Nevertheless, the effectiveness of treatment was different, depending on time it began. Of particular note was the difference in efficacy for metabolic markers. Thus, early experimental treatment (from the 14th week) caused a much more impressive decrease in the glucose, cholesterol, and triglycerides levels compared to late treatment starting from the 19th week.

Induction of experimental MetS was accompanied by a 580.0% increase in GLUT 4 levels (a 1150.4% increase in the group treated with GDSC) compared to control values. It is suggested that so impressive an increase in the main transmembrane glucose transporter is a compensatory reaction, which help to keep normal glucose levels in the blood during the initial period of the experiment. Expressive GLUT4 dynamics under the GDSC treatment probably is resulting from improvement of insulin sensitivity, which prevents glucose and lipid problems.

MetS development has been accompanied by changes in PPAR-γ concentration, which modulated the expression of the main transmembrane glucose transporter GLUT4. This was confirmed by the results of our study, which indicate a parallel PPAR-γ and GLUT4 increase in the experimental MetS modeling coupled with early GDSC use beginning with the 14th week of research. In addition, PPAR-γ plays an important role in maintaining the energy homeostasis regulation of the adipocytes differentiation and the lipids accumulation [32, 33].

Markers of the acute phase inflammatory reaction associated with the MetS induction also showed significant elevation. Thus, the CRP level in the induced MetS model was increased more than twice compared to its basal level; and the TLR 4 level showed an 4-fold elevation. In this regard, the use of molecular markers of cellular responses and indicators of the associated inflammatory response for early diagnostics and assessment of MetS severity seems appropriate.

The participation of systemic subclinical inflammation in the MetS pathogenesis has been confirmed by numerous researchers [34–36]. TLRs plays a key role in this process. It has been experimentally proven [37, 38] that the TLR4 in adipocytes can be activated by the action of free fatty acids, followed by the launch of NF-κB signaling pathways. This in turn causes the secretion of monocytic chemoattractant protein-1 (MCP-1), TNF-α, IL-1, IL-6, chemokines, and cytokines. Therefore, it is logically that TLR4 is some link to inflammation activation and its level is in relationship with metabolic problems. The results of our study show that the MetS development causes expressive increase i’n the TLR4 concentration. At the same time, the use of the GDSC causes decreases in the levels of the most dynamic and sensitive inflammatory marker. The same trend was shown by another classical marker for the intensity of systemic inflammatory reaction, CRP, but the decrease was not as dramatic.

The positive influence of GDSC on the biochemical and molecular markers of experimental MetS is confirmed by the morphological results, which showed cardio- and angioprotective effects in the MetS model. Cardiac damage is caused by a change of myocardial substrate metabolism. With the loss of insulin-dependent glucose flux, the predominant cardiomyocyte energy substrate would be fatty acids. The unbalanced glucose-taking in the cardiomyocyte can upset the cytosolic redox state, which leads to cardiac damage [39].

In conclusion, molecular markers such as GLUT4, PPAR-γ, and TLR4 show more expressive dynamics than biochemical ones in induced MetS, which supports the recommendation to use the listed molecular markers for early diagnosis and delivery of efficient treatment. The MetS treatment with the experimental GDSC reduces the severity of metabolic disorders. The multi-vector therapeutic effect of GDSC reduces the severity of the inflammatory process associated with the MetS. It is also worth noting that it is more effective when the treatment is begun as early as possible.

The limitations of the study

This study was limited by a lack of a placebo-controlled group, not large enough samples and lack of monitoring after correction end to estimate lasting of obtained effects.

Funding information

This work was partially supported by the V.I. Vernadsky Crimean Federal University Development Program for 2015–2024.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Anatoly Kubyshkin, Email: kubyshkin_av@mail.ru.

Alina Shevandova, Email: shevandova_a_a@mail.ru.

Vitalina Petrenko, Email: petrenko-vitalina@mail.ru.

Irina Fomochkina, Email: fomochkina_i@mail.ru.

Leya Sorokina, Email: leya.sorokina@mail.ru.

Alexander Kucherenko, Email: aleksandr-kucherenko@bk.ru.

Andrey Gordienko, Email: uu4jey@mail.ru.

Natalia Khimich, Email: natkhimich@mail.ru.

Evgenia Zyablitskaya, Email: evgu79@mail.ru.

Tatiana Makalish, Email: gemini_m@list.ru.

Leonid Aliev, Email: all.spitfire@mail.ru.

Natalia Kornienko, Email: nkornienko50@mail.ru.

Ivan Fomochkin, Email: soniashko@mail.ru.

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