Highfat simple carbohydrate feeding impairs central ...

21/09/2016

Highfat simple carbohydrate feeding impairs central and peripheral monoamine metabolic pathway triggering the onset of metabolic syndrome in …

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ORIGINAL ARTICLE Year : 2016 | Volume : 64 | Issue : 5 | Page : 923933

Highfat simple carbohydrate feeding impairs central and peripheral monoamine metabolic pathway triggering the onset of metabolic syndrome in C57Bl/6J mice Serena S D’Souza, Asha Abraham Fr George Albuquerque Pai Cell and Molecular Biology Laboratory, Department of Postgraduate Studies and Research in Biotechnology, St Aloysius College (Autonomous), Mangalore, Karnataka, India Correspondence Address: Asha Abraham Fr George Albuquerque Pai Cell and Molecular Biology Laboratory, Department of Postgraduate Studies and Research in Biotechnology, St Aloysius College (Autonomous), Mangalore 575 003, Karnataka India

Abstract Background: Previous studies have shown disturbances in an individual monoamine pathway but have not studied metabolic pathways at the onset and progression of metabolic syndrome (MetS). Aims, Settings, and Design: The aim of this study was to investigate the effect of highfat simple carbohydrate (HFSC) diet on central (hypothalamic) and peripheral (plasma and urine) monoamine metabolic pathways during the development of metabolic syndrome in C57BL/6J mice. Materials and Methods: Monoamines were analyzed in the 1 st, 2 nd, 3 rd, 4 th, and 5 th month after feeding mice the HFSC diet or the control diet using the high performance liquid chromatography (HPLC) system (Shimadzu, Japan). Data was statistically analyzed (by Student's ttest) using Graph Pad Instat Version 3.1. Post statistical analysis, Bonferroni correction was applied to the results of 2 nd, 3 rd, 4 th, and 5 th month in order to calculate the correct error in the study. Results: Significantly lower hypothalamic, plasma, and urine dopamine, and higher hypothalamic and plasma levels of norepinephrine and normetanephrine levels were observed in the HFSC diet fed C57BL/6J mice as compared to the control diet fed C57BL/6J mice after 5 months of feeding. No consistent changes were observed in other brain regions. The turnover ratio indicated that the lower dopamine levels in the HFSC diet fed C57BL/6J mice was due to the increased formation of norepinephrine and homovanillic acid. Conclusion: HFSC diet impairs the central and peripheral dopaminergic and noradrenergic pathways in mice as evidenced by the disturbances in their hypothalamic, plasma, and urine levels and this might be one of the early factors contributing towards the development of the MetS.

How to cite this article: D’Souza SS, Abraham A. Highfat simple carbohydrate feeding impairs central and peripheral monoamine metabolic pathway triggering the onset of metabolic syndrome in C57Bl/6J mice.Neurol India 2016;64:923933

How to cite this URL: D’Souza SS, Abraham A. Highfat simple carbohydrate feeding impairs central and peripheral monoamine metabolic pathway triggering the onset of metabolic syndrome in C57Bl/6J mice. Neurol India [serial online] 2016 [cited 2016 Sep 21 ];64:923933 Available from: http://www.neurologyindia.com/text.asp?2016/64/5/923/190261

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Introduction

An individual afflicted with any three of the five risk factors (central obesity, low highdensity lipoprotein, impaired http://neurologyindia.com/printarticle.asp?issn=00283886;year=2016;volume=64;issue=5;spage=923;epage=933;aulast=D%92Souza

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fasting blood glucose, high triglycerides, and hypertension) is said to have the metabolic syndrome (MetS).[1] Earlier, MetS was considered to be an inadvertent consequence of aging because it was seen mainly in adults and aged individuals. However, MetS is now being diagnosed in children and adolescents as well.[2] Twenty four and 65% of obese women as well as 43 and 78% of obese men from the Netherlands and Finland, respectively, were reported to have MetS in an agestandardized study with elevated blood pressure being the most frequently occurring factor contributing to the prevalence of MetS.[3] The high prevalence of MetS has important health implications. It is associated with increased risk of coronary heart disease and is a strong predictor of type 2 diabetes.[4] It is also associated with increased risk for chronic kidney disease,[5] cognition problems,[6] and reproductive health problems such as the polycystic ovary syndrome.[7] Children with MetS were found to have lower grasping power as compared to non MetS children.[8] Current therapies include diet, exercise, and agents indicated for the treatment of individual components of the syndrome. The presentday drug therapies include drugs for hypertension, such as diuretics and acetylcholine esterase inhibitors; drugs to manage the low values of highdensity lipoprotein cholesterol and the high values of lowdensity lipoprotein cholesterol; and, insulin sensitive drugs such as metformin and pioglitazone. The side effects of these drugs give rise to newer problems in addition to the existing ones. The drawbacks of these therapies are that they aim at treating the symptoms and not the core problem. Thus, there is a need to systematically study the syndrome in its entirety to gain valuable insights to ameliorate the syndrome. Lately, monoamines have been implicated in the development of obesity and diabetes. Phenylalanine and tyrosine are precursors for the dopaminergic and noradrenergic neurotransmitters. The pathway is described in [Figure 1].[9] Briefly, phenylalanine is a precursor for tyrosine (catalyzed by phenylalanine hydroxylase) and tyrosine, a precursor for dopamine (catalyzed by tyrosine hydroxylase). Dopamine is further metabolized to norepinephrine and homovanillic acid. Dopamine is also metabolized intraneuronally (possibly due to reuptake within the neuron) as well as extraneuronally (that is after it is released from the neuron) by monoamine oxidase and catecholOmethyl transferase to homovanillic acid.[10] Thus, homovanillic acid measurement reflects a combined measure of both dopamine release and metabolism. Norepinephrine is also metabolized to epinephrine by phenylethanolamineN methyl transferase, and to normetanephrine by catecholOmethyl transferase.{Figure 1} Alterations in the dopaminergic and noradrenergic pathways have a role to play in the occurrence of individual components of MetS such as hyperglycemia and dyslipidemia. Compulsive eating, as evidenced by obesity, is proposed to be due to the “reward deficiency syndrome” due to reduction in the dopamine D2 receptors.[11] Dopamine deficiency in obese individuals was found to be associated with prolonged pathological eating.[12] Studies in Dbh−/− (dopamineβhydroxylase deficient) mice show that central norepinephrine and epinephrine are required to regulate blood glucose levels.[13] High levels of norepinephrine were found to contribute to hypertension and low epinephrine levels were associated with dyslipidemia.[14],[15] Thus, it is clear that monoamines play a vital role in the carbohydrate and lipid metabolism. Most of the reports have studied these monoamines once the condition has already been established. These studies are carried out in models or subjects suffering from one of these abnormalities and not in the MetS per se. Moreover, these studies are limited to one monoamine measurement at a time (such as dopamine or norepinephrine), which does not provide a clear picture as to what may be the cause for the overall observed result. Thus, in our study, we have induced MetS using a dietbased approach in male C57BL/6J mice and have analyzed the monoamine metabolic pathway in different regions of the brain as well as plasma and urine during the onset and progression of the syndrome.

Experimental Procedures

Animals used Pathogenfree 2monthold male and female C57BL/6J mice were procured from the National Institute of Nutrition (NIN, Hyderabad, India). They were maintained at 25 ± 2°C with a 12 h light and dark cycle and housed in polypropylene cages. They were used as the breeder stock on attaining maturity. One month old male C57BL/6J mice obtained by breeding were used for the experimental study. The male mice were chosen for the study as hormonal variations associated with estrous cycle affect many energy balance related pathways.[16],[17],[18] In addition, the body composition and body fat distribution differs in male and female subjects, wherein the female ones have a higher percentage of body fat and less abdominal fat as compared to males.[19] The experimental mice http://neurologyindia.com/printarticle.asp?issn=00283886;year=2016;volume=64;issue=5;spage=923;epage=933;aulast=D%92Souza

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were divided into two groups, the control fed C57BL/6J mice and the HFSC fed C57BL/6J mice (n = 30 each). These groups were fed with the control feed and the HFSC feed, respectively, at the rate of 5 g/day.[20] The HFSC feed was formulated in reference to the study by Fraulob et al.,[21] with some modifications. The modifications included the addition of sucrose (16%, weight/weight) and reducing the amount of cornstarch (10%, weight/weight). The control feed provided 10% calories from proteins, 6% from fat, and 84% from carbohydrates, whereas the HFSC feed provided 12% calories from proteins, 46% calories from fat, and 42% calories from carbohydrates. The mice were provided with fresh food and water, both ad libitum daily. All the experiments were carried out according to the Committee for the Purpose of Control and Supervision (CPCSEA) guidelines with approval from the Institutional animal ethics committee (Sanction No. SAC/IAEC/110/2011 dt. 30th March, 2011). Sample preparation The mice were sacrificed by cervical dislocation after 1 month, 2 months, 3 months, 4 months, and 5 months of feeding. The brain was dissected into five regions–brain stem, cerebellum, hypothalamus, corpus striatum, and cerebral cortex–on a chilled glass plate.[22] These regions were chosen because the brain stem has been implicated in nutrient sensing, and the hypothalamus in the regulation of food intake as well as satiety. The cerebellum is involved in motor and cognitive functions, the corpus striatum in food reward and cognitive processes, and the cerebral cortex in cognitive decline. These regions were immediately dipped in liquid nitrogen and stored at −80°C until further analysis. The blood, collected from the heart in ethylene diamine tetra acetic acid (EDTA)coated tubes, was centrifuged at 2000 g for 10 min to obtain plasma, which was then stored at −80°C until further analysis. Free flowing urine under no stress was collected and stored in 6N hydrochloric acid at −80°C until further analysis.[23] The protein in the sample was precipitated by adding 0.4 N perchloric acid and centrifuged at 2000 g for 10 min at 4°C (Kubota, Japan). The supernatant was filtered through a 0.22 µm highperformance liquid chromatography (HPLC) grade nylon filter and the filtrate was used for analysis using the HPLC system (Shimadzu, Japan) fitted with an electrochemical detector (Decade II AntecLeyden, the Netherlands).[24] The values of norepinephrine, epinephrine, normetanephrine, dopamine, and homovanillic acid were determined in the HPLC system (Schimadzu, Japan) fitted with C18 reverse phase columns of 5 µm particle size and 250 × 4.6 mm length. The mobile phase consisted of 75 mM sodium dihydrogen orthophosphate, 1 mM sodium octyl sulfonate, 50 mM ethylene diamine tetra acetic acid (EDTA) and 7% acetonitrile. The pH was adjusted to 2.83 with orthophosphoric acid, filtered through the 0.45 µm filter (Millipore) and degassed. A Shimadzu pump (LC20A, Shimadzu, Japan) was used to deliver the solvent at the rate of 1 ml/min. The neurotransmitters were identified by an amperometric detection using an electrochemical detector (Decade II, Antec Leyden, Shimadzu, the Netherlands) with a detection range of 1 pA. 20 µl of the acidified supernatant was injected into the system. The peaks were identified by the relative retention times compared with the external standards and were quantified by manual integration (LC Solution Software, Shimadzu, Japan). Data obtained were statistically analyzed and interpreted. Turnover studies The turnover ratios were calculated in order to obtain a better understanding of the metabolic pathways. These ratios were analyzed from the individual values of monoamines obtained following the HPLC analysis. The product of the reaction was divided by the reactant to obtain the turnover ratio. For example, turnover of dopamine to its products, norepinephrine and homovanillic acid, in the tissues of control fed C57BL/6J mice, was calculated as follows: Turnover of dopamine to norepinephrine (NE/DA) = nmoles of norepinephrine per g wet weight of tissue of control fed C57BL/6J mice/nmoles of dopamine per g wet weight of tissue of control fed C57BL/6J mice. Turnover of dopamine to homovanillic acid (HVA/DA) = nmoles of homovanillic acid per g wet weight of tissue of control fed C57BL/6J mice/nmoles of dopamine per g wet weight of tissue of control fed C57BL/6J mice. The turnover ratio for plasma and urine were calculated as above in per ml of plasma or urine. Statistical analysis The obtained data were statistically analyzed (using the Student's ttest) using Graph Pad Instat Version 3.1. http://neurologyindia.com/printarticle.asp?issn=00283886;year=2016;volume=64;issue=5;spage=923;epage=933;aulast=D%92Souza

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Following the statistical analysis, Bonferroni correction was applied to the 2nd, 3rd, 4th, and 5th month results in order to calculate the correct error in the study. For example, for the 5th month, only those values that were below 0.01 were considered significant (P