Asma Fikri, Rosemary Smyth and Michael Munday
Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, London, UK
Diabetes Mellitus is a group of metabolic diseases characterized by hyperglycaemia resulting from the deficiency in insulin production and/or insulin resistance, or both. The chronic hyperglycaemia of diabetes is associated with long-term damage of various organs, including eyes, kidneys, liver, and the heart (World Health Organisation & International Diabetes Federation, 2006). Animal models in diabetes research are very common due to the difficulties faced carrying out human studies. Such models of diabetes have successfully revealed molecular mechanisms of pathology and therapeutic intervention, however, no model exhibits all of the major pathogenecities of T2D (Maher et al., 2009; Xu et al., 2013).
Our particular goal is the study of the pre-diabetic state and the slow development of the T2D condition.The aim of this study was to create a non-genetic animal model in which the Metabolic Syndrome (MetSyn) develops as a result of obesity induced by high fat/high carbohydrate feeding, eventually leading to T2D. This would replicate the most common pathway of T2D development in humans. Plasma and urine metabolite profiles were analysed to identify patterns that might be indicative of the gradual development of T2D and thus provide potential biomarkers for the process.
20 male Hanover-Wistar rats were divided into 2 groups (control and treated). The treated group was fed on a special high fat/high carbohydrate diet containing 45% Kcal from fat and 35.5% Kcal from carbohydrates, while the control group was fed on standard chow for 8 weeks. Rats were placed in metabolism cages for the collection of 12 hours fasting urine samples at 0 (before administering the special diet) and at weeks 2, 4, and 8 of the study. 1ml blood samples were collected via tail vein bleeding at the above mentioned timepoints. All animals were weighed five times a week, before and after the 12 hour fast. 1H NMR spectroscopy in combination with multivariate statistical analysis and a two-way ANOVA were applied to explore metabolite variability and significance of changes in urine and plasma samples. Changes in metabolites were compared with previously published literature (Maher et al., 2009; Couto Alves et al., 2009; Griffin et al., 2011; Xu et al., 2013).
At the end of week 8, the mean body weight of treated animals (446.5 g ± 44) was significantly greater than controls (389.4 g ± 40, ***P<0.001). Post-mortem investigations of the treated animals showed a significant increase in adipose tissue surrounding internal organs and subcutaneously. Treated livers were pale and weighed less than the controls'.
Metabolic changes in treated animals due to special diet were identified at as early as week 2 of the study and they became more significant as the study progressed. 1H NMR analysis of plasma samples at the end of the study (week 8) showed a significant increase in plasma phospholipids, 2-oxoglutarate, taurine, succinate, glucose and creatine and a decrease in creatinine (**P<0.01). Furthermore, analysis of urine samples of the same time point showed a very significant increase in creatine, glucose, taurine, 2-oxoglutarate, trimethyl-N-oxide (TMAO), creatinine, lactate and 3-hydroxybutytrate (***P<0.001). On the other hand, formate, hippurate, fumarate, 2-oxoglutarate, citrate, N-methyl nicotinic acid (NMNA) and succinate were significantly decreased (**P<0.01).
These metabolic changes have been observed in other published T2D mdoels and are related to liver and kidney dysfunction, atherosclerosis and disregulated glucose metabolism (Maher et al., 2009; Xu et al., 2013).
In this project, we have investigated a potential model for the development of MetSyn and T2D. 1H NMR analysis revealed interesting patterns of plasma and urinary metabolite changes.These patterns might be potential markers for MetSyn and T2D development.
Keywords: Metabolic Syndrome, Type 2 Diabetes, Metabolomics, Biomarkers, NMR, Animal Model.
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