The impacT of hydroxyciTric acid on The lipid meTabolism profile under experimenTal insulin resisTance syndrome of syrian hamsTers

The syndrome of insulin resistance (Ir) is one of the leading reasons for the increased risk of cardiovascular diseases and their complications. Among the key components of Ir are obesity and dyslipidemia. hydroxycitric acid (hCA), an inhibitor of a key enzyme of lipogenesis ATp citrate lyase (ACLy) is a promising obesity treatment agent. The aim of this work was to investigate the effect of hCA on lipid and lipoproteins content in the blood serum, as well as lipid content and activity of some lipid metabolism enzymes in the liver of hamsters with Ir. Ir was modeled by keeping animals on high-fat diet with addition of fructose. Lipid content was determined by using standard reagent kits, the level of lipoproteins, the activity of glucose 6-phosphate dehydrogenase and ACLY – spectrophotometrically, lysosomal lipase activity – fluorimetrically. Development of hyperlipidemia and atherogenic dyslipidemia, lipid accumulation in the liver, activation of lysosomal lipase and ACLy and reduction of glucose 6-phosphate dehydrogenase activity were shown under Ir. The treatment by hCA reduces the manifestations of hyperlipidemia, but enhances the lipid accumulation in the liver.

The syndrome of insulin resistance (Ir) is one of the leading reasons for the increased risk of cardiovascular diseases and their complications.Among the key components of Ir are obesity and dyslipidemia.hydroxycitric acid (hCA), an inhibitor of a key enzyme of lipogenesis ATp citrate lyase (ACLy) is a promising obesity treatment agent.The aim of this work was to investigate the effect of hCA on lipid and lipoproteins content in the blood serum, as well as lipid content and activity of some lipid metabolism enzymes in the liver of hamsters with Ir.Ir was modeled by keeping animals on high-fat diet with addition of fructose.Lipid content was determined by using standard reagent kits, the level of lipoproteins, the activity of glucose 6-phosphate dehydrogenase and ACLY -spectrophotometrically, lysosomal lipase activity -fluorimetrically.Development of hyperlipidemia and atherogenic dyslipidemia, lipid accumulation in the liver, activation of lysosomal lipase and ACLy and reduction of glucose 6-phosphate dehydrogenase activity were shown under Ir.The treatment by hCA reduces the manifestations of hyperlipidemia, but enhances the lipid accumulation in the liver.k e y w o r d s: ATp-citrate lyase, hydroxycitric acid, insulin resistance, dyslipidemia, hepatic lipidosis.

I
nsulin resistance syndrome (one of the key meta bolic syndrome components) is the most common systemic disease characterized by disturbance of all types of metabolism, and high risk of complications associated with cardiovascular system impairment.Great social significance of this disease is that it leads to early disability and high mortality.Some of the key components of insulin resistance are obesity and dyslipidemia [1].
ATP-citrate lyase (ACLY) is a key lipogenesis enzyme that converts citrate into acetyl-CoA in the cytoplasm; acetyl-CoA is an initial precursor that after connection of carboxyl group gives a malonyl-CoA for the fatty acids biosynthesis.Cytosolic citrate is a derivative of the mitochondrial tricarboxylic cycle, that is why ACLY catalyzes a critical reaction which connects glucose catabolism and lipid synthesis [2].
Lipogenesis is a metabolic pathway that is coordinately regulated in the response to nutritional and hormonal stimuli.It has been shown that dysregulation of lipogenesis significantly contributes to the occurrence of dyslipidemia and metabolic disorders [3].At the same time, the metabolic contribution of each separate lipogenic enzyme in lipid metabolism and also in whole body energy homeostasis is poorly understood [4][5][6].
According to the literature, the amount of both acetyl-CoA and malonyl-CoA is markedly decreased in liver, irrespective of dietary fat intake, and also the level of triacylglycerol and free fatty acids drop in the blood with ACLY knockout.On this background the liver lipid range also changes [2].
Among the drugs and functional foodstuffs that are used to the obesity treatment, the popularity of products from Garcinia Cambogia (garcinia gummi-gutta L.) is coming up now; hydroxycitric acid (HCA) is an active substance of mentioned Garcinia products.The proposed mechanism of HCA action lies in a competitive ACLY inhibition, but the experimental studies of Garcinia drugs are isolated cases [7,8].
Based on the above, the exploration of ACLY activity and lipid metabolism profile using the hydroxycitric acid appears to be topical.

materials and methods
24 male Syrian hamsters (mesocricetus auratus L.) at the age of 20 weeks at the beginning of experiment were used.Animals have been divided doi: http://dx.doi.org/10.15407/ubj88.03.078 into 3 groups by 8 in each one: 1) intact animals that were kept on a standard diet 2) group of animals with insulin resistance syndrome (IR) − group which diet contained 29% of animal fat [9] with addition of fructose (2.5 g/100 g of body weight per day) [10], 5 weeks (IR group) 3) animals which have been administered intragastrically the HCA (STIFIMOL preparation manufactured by Kyiv Vitamin Factory) at the daily dose of 5 mg/kg body weight starting from 3 rd week of insulin resistance syndrome modelling (IR+ST group).After 5 weeks the animals were decapitated under chloralose-urethane anesthesia.The studies were conducted in accordance with national General ethical principles of animal experiments (Ukraine, 2001), which are consistent with provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986).
The studied objects were the blood serum and liver of laboratory animals.A 25% homogenate and postmitochondrial and mitochondrial fractions were prepared from perfused liver by differential centrifugation.
The development of IR was confirmed by the blood serum level of glucose and insulin in fasting state, calculating insulin resistance index using the algorithm Homeostasis Model Assessment (the data are not included in the article).
Total lipids concentration was determined using a standard kit (Eagle Diagnostics, USA) in a reaction with vanillic reagent.The content of triacylglycerols was determined using a standard enzymatic glycerol oxidase assay kit (KONE Oy, Finland).Total cholesterol content was determined by standard enzymatic cholesteroloxidase kit (Boehringer Mannheim GmbH diagnostica, Germany).The content of free fatty acids (FFA) was determined using colo rimetric method [11].
Lipoproteins of blood serum were fractionated by disc electrophoresis in vertical polyacrylamide gel plates (160×140×2 mm) [12].α-lipoprotein, pre-β-and β-lipoproteins, the amount of which is called apo-B-containing lipoproteins were identified at the electropherograms.Stained by black sudan 10 lipoproteins were eluted, the light absorption of eluate was measured spectrophotometrically at 595 nm [13].
Statistical analysis of the data was performed using Statistica software package (StatSoft Inc., USA, version 6.0).The significance of differences between groups was assessed by non-parametric Mann-Whitney test.

results and discussion
Keeping animals on the described diet for 5 weeks led to insulin resistance development, which was confirmed by insulin resistance index increasing 2.6 times in the parallel experiment (3.02 vs 1.78 in the intact animals).
The obtained data are the evidence that high energy diet lead to the hyper-and dyslipidemia that are the typical signs of IR (Table 1): the content of total lipids (TL) (mainly due to neutral lipids), lipoproteins (due to apo-B-containing ones) and nonesterified fatty acids are increased in the blood of experimental animals.Level of high-density lipoproteins (HDL) is somewhat reduced in this case.
All these changes have the pronounced proatherogenic character and may point the lipid metabolism shift in animal's organism towards the lipids accumulation and adipose tissue redistribution in favor of the visceral one.The latter fact is confirmed by free fatty acids (FFA) accumulation in blood, at that the main FFA source in metabolic syndrome is lipolysis activation in adipose tissue.The use of HCA significantly reduces all the studied parameters, except FFA and total cholesterol (TC), their content remains increased.Such changes are the indicators of: 1 st , the fatty acids source in the serum is not their synthesis de novo that should be slightly suppressed by HCA action, but the active lipolysis, and 2 nd , more complex cholesterol metabolism regulation exists along with its synthesis from components of non-carbohydrate nature.
The assumption about predominant role of lipolysis activation in FFA release into blood under high-caloric diet condition is supported by the data of lysosomal acid lipase activity in the liver of experimental animals (Table 2).This activity increased 2.35-fold compared to the intact animals and remained high when HCA was used.
As for lipogenesis inhibition that occurs because of decreased carbohydrate-derived substrates flux due to HCA usage, it correlates with a decrease of G-6-PDG activity observed under experimental IR condition and amplified under STIFIMOL action (Table 2).
We obtained data on the decrease of ACLY activity under the STIFIMOL usage (Table 2), and they confirm the assumption about HCA effect realization by virtue of this enzyme inhibition.The consequence of such action should be a switching the FFA synthesis (and, to some extent, cholesterol as well) to non-carbohydrate sources of acetyl-CoA, particularly to FFA resynthesis from food lipids.
It is needed to pay attention to the increase of total lipid level in the liver tissue, particularly tria-

T a b l e 1. Some parameters of lipid metabolism in Syrian hamsters males serum with insulin resistance syndrome (Ir group) and under hydroxycitric acid administration (Ir+ST group) (m ± m, n = 8)
Here and in Table 2: * the differences are substantial as related to intact animals (p ≤ 0.05); ** the differences are substantial as related to IR group (p ≤ 0.05).

Parameters
Group  2).Such effect under IR conditions is probably a result of the FFA utilization that come to the liver from blood in the context of active lipolysis and leads to secretion of lipoproteins (LP) enriched by TAG and serves as additional pro-atherogenic factor.Since a secretion of apo-B-containing LP is apparent ly somewhat reduced at the HCA application (Table 1), TAG accumulation in the liver can lead to lipidosis, and later to hepatic steatosis.Perhaps, more significant LL activation at the HCA administration as compared to model pathology group (Table 2) is an attempt to compensate for TAG accumulation through their degradation and oxidation.The dangerous consequence of HCA usage is a decrease of cholesterol content in liver that does