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23
Проблеми екологц та медицини
ного церулоплазмина и экспрессии мРНК TNFa в тканях печени этих животных.
Таким образом, полученные данные свидетельствуют о протективном эффекте метформина на углеводный, липидный обмен, а также уровень экспрессии провоспалительных факторов, связанных с NFkB-сигнальным путем в тканях печени мышей, которые находились на диете с высоким содержанием фруктозы.
Литература
1. Влияние метформина на продукцию провоспалительных цитокинов и инсулинорезистентность (NFkB-сигнальный путь) / Лавренко А. В. [и др.] // Проблемы эндокринологии. - 2012. - №2. - С. 25 - 28
2. Кайдашев 1.П. Актива^я NF-kB при метаболiчному синдромi / I. П. Кайдашев // Фiзiол. Журн. -2012. - Т.58, №1. - С. 93-101
3. Колб В. Г., Калашников В. С. Клиническая биохимия. -Минск: Беларусь,1976. - С. 219 - 220
4. Куценко Л. А. Место церулоплазмина среди белков острой фазы как маркера системного воспаления / Л. А. Куценко, И. П. Кайдашев // Лабораторна дiагностика. - 2011. - 3(57). -С. 59 - 68
5. Лавренко А. В. 4Метформин и пиоглитазон как средства борьбы с системным воспалением низкой интенсивности / А. В. Лавренко, Н. И. Винник, С. М. Расин, М. С. Расин, И. П. Кайдашев // Проблеми екологп та медицини. - 2012. - Т. 16., №3-4. - С. 3 - 8.
6. Activation 15 of the AMP activated protein kinase by short-chain fatty acids is the main mechanism underlying the beneficial effect of a high fiber diet on the metabolic syndrome / Hu GX, Chen GR, Xu H, Ge RS, Lin J. // Med Hypotheses. -2010. - Vol.74(1). - P. 123-126.
7. Arkan M. C. 3Hevener AL,Greten FR, et al. IKK-beta links inflammation to obesity-induced insulin resistance / M. C. Arkan, A. L. Hevener, F. R. Greten [et all.] // Nat.Med. -2005. - N 11. - P. 191-198.
8. Basciano H. Fructose, insulin resistance, and metabolic dyslipidemia [Електронний ресурс] / H. Basciano, L. Federico, A. Khosrow //Nutrition & Metabolism. - 2005. -2:5. - Режим доступу до журн.: doi:10.1186/1743-7075-2-5.
9. Ferguson L. R. Is the dietary fibre concept becoming too hard to digest? / L. R. Ferguson // British Journal of Nutrition. - 2008. - N 100. - P. 693-694
10. Health benefits of dietary fiber / James W Anderson, Pat Baird, Richard H Davis [et all.] // Nutrition Reviews®. - Vol. 67(4). - P. 188-205.
11. Kelley G. L. High dietary fructose induced a hepatic stress response resulting in cholesterol and lipid dysregulation / G. L. Kelley, G. Allan, S. Azhar // Endocrinology. - 2004. -N 145. - P. 548 - 555.
12. Lazar M. A. 16 The tumoral side of insulin resistance / M. A. Lazar // Nat Med. - 2006. - N.12. - P.43 -44.
13. Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB / D. Cai, M. Yuan, D. F. Frantz [et all.] // Nat Med. - 2005. - Vol. 11. -Vol. 183-190
14. Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state / M. Foretz [et all.] // The Journal of Clinical Investigation. - Vol.120, N.7. - P. 2355 - 2369.
15. Metformin therapy in a hyperandrogenic anovulatory mutant murine model with polycystic ovarian syndrome characteristics improves oocyte maturity during superovulation / Sabatini M. E. Et all // Journal of Ovarian Research. -2011. Vol. - 4. - P. 8.
16. Poss 1A. P. A high fructose diet impairs spatial memory in male rats / A. P. Poss [et all.] // Neurobiol Learn Men. -2009. - Vol. 92, N. 3. - P. 410 - 416.
17. Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta / Yuan M., Konstantopoulos N., Lee J. [et all.] // Science. - 2001. -Vol. 293(5535). - P. 1673-1677
18. Schmittgen T. D., Livak K. J. Analyzing real-time PCR data by the comparative Ct method / T. D. Schmittgen, K. J. Livak // NATURE PROTOCOLS. - 2008. - Vol.3, N.6. - P. 1101 - 1108.
ENGLISH VERSION: INFLUENCE OF METFORMIN ON LIPID METABOLISM AND CHRONIC INFLAMMATION IN THE LIVER TISSUES OF MICE ON A DIET RICH IN FRUCTOSE
Shlykova O.A., Mikityuk M.V., Bobrova N.L.., Izmailova O.V., Mamontova T.V., Baranova A.F., Vesnina L.E., KaidashevI.P. Higher State Educational Establishment of Ukraine "Ukrainian Medical Stomatological Academy", Poltava
The effect of metformin on lipid metaboiism and the expression of pro-inflammatory factors in the liver tissue of mice that were on a diet rich in fructose (DRF) (60 g fructose /100 g of food) has been studied. The concentrations of total cholesterol (TC), triglycerides (TG), ceruloplasmin (CP), glucose in serum and mRNA expression of inhibitor of NF-kB (IkBa), mRNA of tumor necrosis factor a (TNFa) have been determined in homogenates of iiver tissues in 3-months-aged mice of BALBc line weighing 25-30 g. It has been demonstrated that increasing the content of fructose in the diet leads to increased concentrations of glucose, TC and TG in the blood of mice. Manifestations of systemic inflammatory response become stronger, as evidenced by the increase in CP in the blood serum and the expression level of mRNA of TNFa in iiver tissues of animals that were on a DRF. Metformin in the dose of 50 mg / kg per day ^ reduced the production of triglycerides in the blood serum of mice that were on a DRF; it contributed to the normaiization of hepatic gluconeogenesis. Administering metformin caused anti-inflammatory effect by lowering serum CP and expression of mRNA of TNFa in iiver tissues of these animals. Thus, the data suggest the protective effect of metformin on iipid metaboiism and the expression of pro-inflammatory factors associated with NF-kB-signaiing pathway in the iiver tissues of mice which were on a DRF.
Keywords: diet rich in fructose, metformin, expression, proinflammatory factors.
Numerous studies display that diet rich in fructose oxidative stress, impaired glucose tolerance, insulin re-(DRF) causes various pathological changes, including sistance, type 2 diabetes, liver conditions, hypertension
* To cite this Shlykova O.A., Mikityuk M. V., Bobrova N.L., at all. Influence of metformin on lipid metabolism and chronic inflammation in the liver tissues of mice on a diet rich in fructose / /Problemy ekologii ta medytsyny. - 2014. - Vol 18, № 3-4. - P. 67-70.
Tom 18. N 3-4 2014 p.
and other cardiovascular diseases [16]. It is shown that lipid metabolism in the development of stress of hepato-cytes in animals on DRF occurs in the result of fructose metabolism overload [11]. It is assumed that the increased production of lipids in the liver enhances mito-chondrial beta-oxidation of fatty acids, generation of peroxidation products that stimulate IkB-kinase (IKK), and therefore the activation of NFkB [7]. It is known that molecular mechanisms of chronic low-level systemic inflammation (CSI) are closely associated with the activation of proinflammatory nuclear transcription factors (NTF), primarily NFkB [5].
In recent years, the concept of permanent NF-kB activation (long-term and low-intensity) has been formulated as a possible model of pathological process that determines the relationship between insulin resistance, chronic inflammation, hypertension, endothelial dysfunction and dyslipidemia (I.P. Kaidashev, 2012). In this regard, the search for methods to reduce the transcriptional activity of NF-kB (diet, exercise, therapeutic drugs) is relevant, since it will eliminate the molecular basis of the metabolic / insulin resistance syndrome and reduce the risk of cardiovascular diseases [2]. Previous studies have shown that activation of metformin reduces NFkB, possibly by inhibiting the activation of IKK [1].
The aim of the research is to study the influence of metformin on blood glucose, lipid metabolism and the level of expression of proinflammatory factors in the liver tissues of mice that were on a diet rich in fructose.
Materials and methods of the research
The experiment was performed on mice of BALBc line aged 3 months, weighing 25-30 g, kept in polypropylene cages under controlled conditions of 12 hours light / 12 hours dark cycle. Animals were on a standard diet (60% of vegetable amylum) and with unlimited access to water for one week. The study was conducted in accordance with authorization of the Bioethics Commission of Higher State Educational Establishment of Ukraine "Ukrainian Medical Stomatological Academy".
In a week after acclimation period, the animals were divided into four equal groups: group 1 - mice on a standard diet and water; group 2 - mice on a standard diet and water, which were administered metformin (per os) in a dose of 50 mg / kg from the 85th day of the experiment for 49 days [14, 15]; group 3 - mice on a DRF (60 g fructose / 100 g of food); group 4 - mice on a DRF, which were administered metformin (per os) in a dose of 50 mg / kg from the 85th day of the experiment for 49 days.
The standard diet comprised the grain mixture (wheat, oats, barley, and millet), maize, sunflower seeds, peas, corn granules, nuts, and dried fruit. Mineral component were: calcium, phosphorus, potassium, sodium. Vitamins: A, B1, B2, D3, E, N. The nutrient content: crude protein - 11%; crude fat - 4.0%; crude fiber - 10.0%; calcium - 0.15%, phosphorus - 0 1%; moisture - 10%.
On the 134th day of the experiment, after 12-hour fasting period, the concentration of glucose in blood serum of animals was determined before and in 120 minutes after the glucose load (2 g / kg) (test the glucose tolerance). The concentration of glucose (GL) was determined in blood from the tail vein using "GLYUKOFOT" glucometer with standard indicator strips "GEMOGLAN" (PIE "NORM", Ukraine).
On the 135th day of the experimental period, the animals were euthanized by cervical dislocation. Blood was collected in dry plastic vials. Serum was separated by centrifugation at 400 g for 10 minutes. In blood serum,
concentration of total cholesterol (TC) and triglycerides (TG) using the reagents kit (LACHEMA, Czech Republic), the concentration of ceruloplasmin (CP) by enzymatic oxidation of p-phenyldiamine were determined [3]. The sampling of liver tissue was performed and homogenised on ice in refrigerated 0.1M phosphate buffer, pH 7.4. In liver homogenate, the content of RNA messenger (mRNA), NF-kB inhibitor (IkBa) and mRNA of tumor necrosis factor a (TNFa), isolated using the reagents kit "Ribo-sol-B" (AmpliSens, Russia), were determined. For obtaining cDNA in the reverse transcription reaction, primer oligo (dT) 18 and reverse transcriptase M-MuLV (SibEnzyme, Russia) were used. Gene expression was analyzed by PCR method in "real time", using oligonu-cleotide primers for cDNA IkBa: 5'-TGAAGGACGAGGAGTACGAGC-3'; 5'-TTCGTGGATGATTGCCAAGTG-3' and TNFa: 5'-CCCTCACACTCAGATCATCTTCT-3'; 5'-GCTACGACGTGGGCTACAG-3', in the presence of the dye SYBR Green I ("Synthol", Russia), using the detecting amplifier DT-322 ("DNA Technology", Russia) via relative quantitative analysis. The gene of glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene: 5'-AACTTTGGCATTGTGGAAGG-3'; 5'-GGATGCAGGGATGATGTTCT-3'.
For data analysis, relative Ct method with the calculation formula ACt was used [18].
The results were processed statistically using the program STATISTICA 6.0. ("StatSoft", USA). The data are presented as the sample average (M), the sample standard deviation (S / D). The critical level of significance when testing the statistical hypotheses was set at 0.05.
Results and discussion
As our research has shown, the gradual increase in body weight of mice in the studied groups during the trial period was observed. Table 1 displays that the final average body weight of mice (g) in group 1 was 32,9 ± 2,0; in groups 2, 3 and 4: 34,7 ± 2,33; 29,9 ± 5,29; 31,1 ± 2,08, respectively. It should be noted that the average value of the final weight of mice in groups did not differ significantly from each other.
Table 1.
Change in body weight in the studied groups of animals (M ±
SD).
Parameter Groups
1 2 3 4
Initial body weight, g 28,4±2,2 31,2±1,56 25,9±2,1 29,2±1,13
Body weight in 84 days, g 32,6±2,8 33,7±0,77 28,8±3,2 30,3±1,36
Body weight in 134 days, g 32,9±2,0 34,7±2,33 29,9±5,29 31,1 ±2,08
When determining the glucose tolerance, a statistically significant increase in concentration of GL (in fasting state) in the blood of mice on DRF was observed, unlike the mice of group 1 (p = 0.0059) (Fig.1). Under the influence of metformin, glucose concentration in the blood serum of mice from group 4 was reduced in comparison with group 3 (p = 0.052) (Figure 1)
npoSAeMH eKOAorii Ta MeanuHHH
Figure 1. Glucose level in groups of studied animals in fasting state and in 120 min after the glucose load (2 g / kg).
Note: * - p <0.05 as compared with group 1.
In our studies, the lipogenesis processes in liver cells varied under the influence of DRF, as evidenced by the increase in TC to 3.05 ± 0.57 mmol / l and triglyceride to 2.15 + 0.42 mmol / l in the serum of group 3 as compared with mice of group 1, who were on a standard diet with no added fructose (p <0.05) (Table 2).
Table 2.
Changes in the level of total cholesterol and triglycerides in the serum of studied animals (M + SD).
Parameter Groups
1 2 3 4
TC, mmol / l 1,6 ± 0,38 1,75 ± 0,31 3,05 ± 0,57* 2,6 ± 0,46
TG, mmol / l 1,13 ±0,57 1,3 ± 0,35 2,15 ± 0,42* 1,48 ± 0,23**
Note: * - p <0.05 as compared with group 1;
** - p <0.05 as compared with group 3.
Metformin reduced the intensity of lipogenesis in liver cells of group 4, as evidenced by reduction in triglycerides (1,48 ± 0,23 mmol / l) as compared to group 3 (2,15 ± 0,23 mmol / l) (p = 0.049), the TC level did not significantly change (p = 0.2) (Table 2).
At the same time, according to histological studies, no clinically significant structural changes in liver tissues were observed in the studied groups of mice. The indicators of liver mass coefficient in the studied groups of animal did not change as well, amounting in group 1 - 0,056 ± 0,008, group 2 - 0,054 ± 0,003, group 3 - 0,06 ± 0,009, group 4 - 0,061 ± 0,01.
At the next stage of work, manifestations of systemic inflammatory response were assessed. As markers of the inflammatory process, the level of CP in blood serum and the expression of mRNA of TNFa and iKBa in liver ho-mogenates were determined. In previous studies of I. P. Kaidashev et al., 2008, it was assumed that the contents of CP in serum may be a marker of increased risk of inflammation and metabolic syndrome, and along with other indicators, it will allow assessing the level of systemic inflammatory response [4]. As seen from the data presented in Figure 2, the increase in fructose content in the diet led to a significant increase in the level of ceru-loplasmin in serum of group 3 - 297.65 + 17.07 mg / l as compared with group 1 - 221.9 + 18,5 mg / l (p = 0.0004).
300 250 200 mg/l 150 100 50
12 3 4
Figure 2. Changes in the level of ceruloplasmin in groups of studied animals.
Note: * - p <0.05 as compared with group 1;
** - p <0.05 as compared with group 3.
The expression of mRNA of TNFa and IkBa in liver tissues of mice by increasing the content of fructose in the diet, as well as under exposure to metformin, was studied.
In the group of animals on a DRF, expression of mRNA of TNFa increased and expression of mRNA of IkBa did not virtually change (Table 3).
Under the action of metformin, the manifestations of chronic inflammation reduced due to decrease of ceruloplasmin level in serum of animals from group 4 (246,67 ± 15,41 mg / l) as compared to group 3 (297,65 ± 17,07 mg / L) (p = 0.0044) (Figure 2). Under the influence of metformin, expression of mRNA of TNFa in liver cells decreased. Metformin had virtually no effect on expression of mRNA of IKBa (Table 3).
Table 3.
Changes in the relative mRNA expression of mRNA of TNFa and IkBa in liver tissues of studied animals (M ± SD).
Parameter Groups
1 2 3 4
Expression of TNF-alpha, ACt 14,67 ±0,74 11,62 ±0,78 12,42 ±0,94* 14,75 ±1,04**
Expression of IkB-alpha, ACt 11,2 ±1,04 10,0 ±0,28 10,62 ±0,88 11,2 ±1,97
Note: * - p <0.05 as compared with group 1;
** - p <0.05 as compared with group 3.
In our study it has been demonstrated that the increase of fructose content in the diet leads to increased concentrations of glucose, TC and TG in the blood of mice. Manifestations of systemic inflammatory response are exacerbated, as evidenced by the increase in the level of ceruloplasmin in serum and the level of expression of mRNA of TNFa in liver tissues of animals that were on DRF. At the same time, we observed no increase in body mass and structural changes in the liver tissue of mice by high fructose intake with food.
The data on the changes in the process of lipogenesis in liver cells of mice on a DRF are consistent with studies of H. Basciano et al, 2005, in which it is shown that large admission of fructose to the liver activates the metabolism of glucose and the way of its assimilation, leads to a significant increase in the rate of de novo lipogenesis, the synthesis of triglycerides (TG), which is due to the high flux of glycerol and acyl molecule units of fructose catabolism. These metabolic disorders appear to be one of the reasons underlying the induction of insulin resistance, which is often observed in humans and animals with large consumption of dietary fructose [8].
Том 18. N 3-4 2014 р.
No increase in body weight of mice, as well as the lack of changes in weight and structure of the liver in our 5 studies may be due to the high content of dietary fiber in . the diet of mice. Numerous experimental and clinical studies have shown beneficial effects of a diet high in dietary fiber for metabolic syndrome [6, 9, 10].
Strengthening the systemic inflammatory response in 6. our study is confirmed by an increased concentration of serum ceruloplasmin and expression of mRNA of TNFa in the liver, and as is known, TNFa is a marker of activation of NFkB. There is evidence to show that hepatic in- 7 sulin resistance induced by NFkB activation is associated with increased expression of TNFa, IL-6 and IL-1 [12].
In our previous studies it has been suggested that 8. metformin affects NFkB-signaling pathway, possibly by changing the activity of IKK [1]. IKK activation is one of the key mediators of insulin resistance, as evidenced by 9. the increased sensitivity to insulin at directed disorders in IKK gene [17]. Considering the fact that in this study the activation of NFkB in the liver of mice may be associated 10 with overexpression of IKK, causing resistance of liver and skeletal muscle to the action of insulin and signs of 11 systemic inflammatory response (increase of serum IL-6) [13], we continued to study the effect of metformin on NFkB-signaling pathway in the experiment.
In our study, metformin at 50 mg / kg reduced the 12 production of triglycerides in liver cells of mice that were 13 on DRF, facilitated normalization of hepatic gluconeo-genesis. Administering metformin has anti-inflammatory effect, by reducing the concentration of serum ceru-loplasmin and expression of mRNA of TNFa in liver tis- 14 sues of these animals.
Thus, the findings suggest that metformin has protective effect on carbohydrate and lipid metabolism, as well 15. as the level of expression of proinflammatory factors associated with NFkB-signaling pathway in liver tissues of mice that were on a diet rich in fructose.
References
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I. P. Kaydashev // Laboratorna diagnostika. - 2011. -3(57). -S. 59 - 68
Lavrenko A. V. Metformin i pioglitazon kak sredstva bor'by s sistemnym vospaleniem nizkoy intensivnosti / A. V. Lav-renko, N. I. Vinnik, S. M. Rasin, M. S. Rasin, I. P. Kaydashev // Problemi ekologii ta medizini. - 2012. - T. 16., №3-4. - S. 3 - 8.
Activation of the AMP activated protein kinase by short-chain fatty acids is the main mechanism underlying the beneficial effect of a high fiber diet on the metabolic syndrome / Hu GX, Chen GR, Xu H, Ge RS, Lin J. // Med Hypotheses. -2010. - Vol.74(1). - P. 123-126. Arkan M. C. Hevener AL,Greten FR, et al. IKK-beta links inflammation to obesity-induced insulin resistance / M. C. Arkan, 3 A. L. Hevener, F. R. Greten [et all.] // Nat.Med. -2005. - N 11. - P. 191-198.
Basciano H. Fructose, insulin resistance, and metabolic dyslipidemia [Elektronniy resurs] / H. Basciano, L. Federico, A. Khosrow // Nutrition & Metabolism. - 2005. - 2:5. -Режим доступу до журн.: doi:10.1186/1743-7075-2-5. Ferguson L. R. Is the dietary fibre concept becoming too hard to digest? / L. R. Ferguson // British Journal of Nutrition. - 2008. - N 100. - P. 693-694 Health benefits of dietary fiber / James W Anderson, Pat Baird, Richard H Davis [et all.] // Nutrition Reviews®. - Vol. 67(4). - P. 188-205.
Kelley G. L. High dietary fructose induced a hepatic stress response resulting in cholesterol and lipid dysregulation / G. L. Kelley, G. Allan, S. Azhar // Endocrinology. - 2004. -N 145. - P. 548 - 555.
Lazar M. A. The tumoral side of insulin resistance / M. A. Lazar // Nat Med. - 2006. - N.12. - P.43 -44. Local 19 and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB / D. Cai, M. Yuan, D. F. Frantz [et all.] // Nat Med. - 2005. - Vol. 11. -Vol. 183-190
Metformin 7 inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state / M. Foretz [et all.] // The Journal of Clinical Investigation. - Vol.120, N.7. - P. 2355 - 2369. Metformin therapy in a hyperandrogenic anovulatory mutant murine model with polycystic ovarian syndrome characteristics improves oocyte maturity during superovulation / Sabatini M. E. Et all // Journal of Ovarian Research. -2011, 4:8
Poss A. P. A high fructose diet impairs spatial memory in male rats / A. P. Poss [et all.] // Neurobiol Learn Men. -2009. - Vol. 92, N. 3. - P. 410 - 416. Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta / Yuan M., Konstantopoulos N., Lee J. [et all.] // Science. - 2001. -Vol. 293(5535). - P. 1673-1677
Schmittgen T. D., Livak K. J. Analyzing real-time PCR data by the comparative Ct method / T. D. Schmittgen, K. J. Livak // NATURE PROTOCOLS. - 2008. - Vol.3, N.6. - P. 1101 - 1108
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