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Metabolic Profile Changes in the Testes of Mice with Streptozotocin
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Induced Diabetes Mellitus as Detected by 1H NMR
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C. Mallidis1,2*, B. D. Green3, D. Rogers1, I. M. Agbaje1, J. Hollis3, M. Migaud4,
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E. Amigues4, N. McClure1, R. A. Browne3
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1Obstetrics
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2Basic
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3Human
and Gynaecology, School of Medicine,
Medical Sciences/Anatomy, School of Medicine,
Nutrition and Health Group, School of Biological Sciences,
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4Division
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Chemical Engineering, Queen's University Belfast
of Organic and Medicinal Chemistry, School of Chemistry and
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* Corresponding Author:
Dr Con Mallidis
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Obstetrics and Gynaecology,
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Institute of Clinical Sciences
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Grosvenor Road
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Belfast BT12 6BJ
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United Kingdom
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Tel: + 44 28 90 63 2556
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Fax: + 44 28 90 32 8247
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Email: c.mallidis@qub.ac.uk
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Short Title: Metabolomics of the STZ diabetic mouse testis
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Keywords: Diabetes, Testis, Metabolites, Spermatogenesis
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ABSTRACT
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Contrary to the traditional view, recent studies suggest that diabetes mellitus
29
has an adverse influence on male reproductive function. Our aim was to
30
determine the affect of diabetes on the testicular environment by identifying
31
and then assessing perturbations in small molecule metabolites. Testes were
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obtained from control and streptozotocin induced diabetic C57BL/6 mice, two,
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four and eight weeks post treatment. Diabetic status was confirmed by HbA1c,
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non fasting blood glucose, physiological condition and body weight. Protein
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free, low molecular weight, water soluble extracts were assessed using 1H
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NMR spectroscopy. Principal Component Analysis of the derived profiles was
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used to classify any variations and specific metabolites were identified based
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on their spectral pattern. Characteristic metabolite profiles were identified for
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control and diabetic animals with the most distinctive being from mice with the
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greatest physical deterioration and loss of bodyweight. Eight streptozotocin
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treated animals did not develop diabetes and displayed profiles similar to
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controls. Diabetic mice had decreases in creatine, choline and carnitine and
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increases in lactate, alanine and myo-inositol. Betaine levels were found to be
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increased in the majority of diabetic mice but decreased in two animals with
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severe loss of body weight and physical condition. The association between
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perturbations in a number of small molecule metabolites known to be
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influential in sperm function, with diabetic status and physiological condition,
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adds further impetus to the proposal that diabetes influences important
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spermatogenic pathways and mechanisms in a subtle and previously
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unrecognised manner.
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INTRODUCTION
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Beyond its well established role in erectile dysfunction (Kalter-Leibovici et al.
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2005), an association between diabetes mellitus (DM) and abnormalities of
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male reproductive function has, for many years, been contentious. The current
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prevailing scepticism owes as much to the paucity in both number and scale
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of relevant studies, as to the dearth of any definitive correlations.
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entirely on the comparison of data obtained from routine semen analysis, the
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overall findings of previous studies are conflicting and inconclusive (Ali et al.
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1993, Handelsman et al. 1985, Niven et al. 1995, Vignon et al. 1991). It is not
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surprising, therefore, that DM is largely ignored as a relevant factor in male
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fertility assessment by the majority of fertility specialists (Agbaje et al. 2007).
Based
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Alarm at the increasing incidence of both types 1 & 2 DM in the industrialized
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world, especially amongst young people before and during their reproductive
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years, has coincided with concerns over an apparent worldwide decrease in
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particularly male fertility (Hamilton & Ventura. 2006). This has been reported
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as declining sperm counts and decreasing semen quality (Jensen et al. 2002,
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Morgan. 2003, de Kretser. 1996).
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The combination of decreased fertility and increased DM spurred us to
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examine potential links between the two phenomena. With the availability of
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new techniques which go beyond the limited information obtained by routine
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semen analysis, there is now an opportunity to re examine, and better
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evaluate the effects of diabetes on male reproductive function. In a recent
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study, we reported a significant increase in the number of sperm with
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fragmented nuclear DNA (nDNA) in men with type 1 DM compared to sperm
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from non diabetic controls (Agbaje et al. 2007). This measure, rarely included
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in routine assessment of the male, has been associated with decreased
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embryo quality, lower implantation rates and possibly the onset of some
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childhood cancers in offspring (Lewis & Aitken. 2005). Interestingly, one of the
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few studies that has examined reproductive outcome of couples with a male
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diabetic partner, noted a significantly higher miscarriage rate compared to a
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control population (Babbott et al. 1958).
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DM is a chronic metabolic disease associated with a wide range of
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complications affecting most organ systems. It is characterized by changes in
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blood glucose, plasma lipids, triglycerides and ketones and the measurement
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of specific metabolites is the mainstay of the clinical diagnosis. However,
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beyond the diagnostically recognized metabolic aberrations, human DM has
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more recently been associated with alterations in plasma and urinary levels of
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a number of previously unsuspected metabolites including betaine, carnitine,
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creatinine, catecholamines (such as adrenaline and noradrenaline) as well as
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a number of vitamins (Abdel-Aziz et al. 1975, Bjorgaas et al. 1997, Dellow et
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al. 1999).
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Until recently, detailed studies of the complexity of these metabolite
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changes/imbalances and their relationship to detrimental mechanisms (e.g.
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increased oxidative stress) present in DM and other conditions have been
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confined to changes in bodily fluids, rather than whole tissues. With the
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advent of high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy
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coupled with pattern recognition, known as NMR metabolomics, a tool now
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exists for the rapid and reproducible acquisition of metabolite profiles. In
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conjunction with recognition statistics (multivariate data analysis), this
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powerful new technique provides the means to go beyond the traditional
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single biomarker assessment, instead obtaining a metabolic profile of a
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normal or pathophysiological state. NMR metabolomics has been successfully
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employed to study disparate conditions such as coronary heart disease
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(Brindle et al. 2002), vasospasm (Dunne et al. 2005) and progressive
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neurological diseases (Kaddurah-Daouk. 2006). Variations of the technique
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have also been employed to obtain a biochemical profile of rat testicular
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extracts (Griffin et al. 2000), the in vivo metabolite profile of the rat testis
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(Yamaguchi et al. 2006) and differentiate between different forms of human
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azoospermia based on the metabolic profile of seminal plasma (Hamamah et
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al. 1998).
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The aims of this study were: 1) to determine the profiles of the small
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metabolite molecules of the testis, using NMR spectroscopy; 2) to assess any
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alterations in metabolite balance resulting from the instigation and
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maintenance of DM, and 3) to identify those metabolites most affected and
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thus provide some insight into the mechanisms by which DM may influence
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male reproductive function.
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MATERIALS AND METHODS
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Animals
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All experiments were conducted with the approval of the Animal Ethics
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committee of the Queen’s University of Belfast (QUB) and in compliance with
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the UK Animals (Scientific Procedures) Act 1986.
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Male C57BL/6 mice (5–6 weeks old), initially weighing 18-24g, were randomly
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assigned to one of three time points: 2 week (acute), 4 week and 8 week
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(chronic), each comprising treatment (n= 41) and control groups (n= 37).
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Animals were rendered diabetic by a single intraperitoneal injection of STZ
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(160mg/kg of body weight in 0.1M citrate buffer) whilst control animals were
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sham injected with an equivalent dose of the drug vehicle (i.e. 0.1M citrate
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buffer). Induction of DM was confirmed a week after treatment by blood
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glucose measurement, from tail pricks, using an Ascensia Esprite2
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glucometer (Bayer, UK). Physical condition, non-fasting blood glucose and
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body weight were noted weekly. Any animals showing severe signs of illness
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were given a 1ml intra peritoneal injection of saline and their diet was soaked
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in drinking water. The mice were randomly assigned to cages and maintained
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under standard conditions at the Laboratory Supply Unit, RVH, provided with
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food and water ad libitum and kept on a 12 hour light/dark cycle at 23C.
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Samples
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Animals were sacrificed at the specified time points by carbon dioxide
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inhalation. Immediately, they were weighed and blood samples were obtained
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by cardiac puncture for final glucose estimation and determination of total
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glycated haemoglobin (HbA1c) percentage using A1cNOW kits (Metrika Inc,
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CA, USA). In each case the left testis was quickly excised, external fat
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removed and snap frozen by immersion in liquid nitrogen. Samples were
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assigned Ds codes and stored at -80ºC. Mice with glucose levels > 15 mM
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and HbA1c > 7% were considered diabetic.
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Preparation for 1H NMR analysis
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Testes were individually lyophilized, a 6 mm steel ball bearing placed into
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each tube and the tissue vigorously milled for 10 minutes using a minimix
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standard shaker (Merris Engineering, Maidenhead, Berkshire). One ml
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H2O:CH3OH (60:40) solution was then added to each vial and the milling
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process repeated for a further 10 minutes. Upon completion, the steel ball
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bearings were removed, the samples centrifuged at 16,162 g for 15 minutes
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and the supernatant decanted. The supernatants were dried for 14 h in a
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vacuum concentrator at room temperature. The subsequent dried material
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was dissolved in 650 µl of 0.1 M phosphate buffer (pH 7.0), in
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containing 1 mM sodium trimethylsilyl-2,2,3,3-tetradeuteroproprionate (TSP)
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(Sigma Aldrich, UK) and any insoluble material removed by centrifugation
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(16,162 g for 15 minutes). Finally, 600 µl of the remaining supernatant was
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transferred to a 5 mm diameter NMR tube.
2H O,
2
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Metabolomics
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Samples were analyzed at 303º K and spun at 20 Hz using a Bruker 300 MHz
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spectrometer. Technical reproducibility of the instrument’s set up was
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confirmed by running technical replicates for 24 of the sample extracts. One-
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dimensional spectra were acquired across an 8 kHz spectral width giving 32K
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data points using the NOESY pulse sequence and sixty four transients
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acquired. Spectral processing was conducted using an ACDLabs NMR
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Processor v9.0 (ACD Labs, Toronto, Canada). The summed transients were
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multiplied by a 0.5 Hz apodization factor prior to Fourier transformation;
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chemical shifts were referenced to the TSP resonance ( = 0.0), and baseline
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correction was performed manually. Peak assignments were made by
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reference to published work on NMR metabolite identification (Fan. 1996) and
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analysis of rat testis (Griffin et al. 2000, Yamaguchi et al. 2006). Metabolite
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identities were confirmed by spiking samples with the suspected compounds,
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acquiring further spectra and demonstrating co-resonance of the peaks from
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the sample and the added compound.
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Data Analysis
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Data reduction was carried out by manually integrating the regions of the
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spectra between 0.50 ppm and 9.50 ppm, where possible, to individual peaks.
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The regions which contained the water, methanol and acetone resonance
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signals were excluded. The data were normalized to the total spectral integral
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in the NMR spectrum, mean-centered and evaluated by principal components
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analysis (PCA) using Simca-P+ v11.0 (Umetrics, Umeå, Sweden).
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RESULTS
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STZ induced Diabetes Mellitus
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The STZ treated animals (responders) had significantly higher HbA1c
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percentages and basal glucose levels compared to those of the control mice
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(Table 1). The extent of the differences, increased with the progression of
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disease with the largest differences being seen in two animals (Ds 70 – 2
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week and Ds 11 – 4 week) who displayed the greatest deterioration in
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physical condition (sick responders). As expected, a small number of mice (n
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= 7), treated with STZ did not develop DM (non responders) as reflected by
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their percentage HbA1c and basal plasma glucose values which did not differ
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from control values. The only exception being one animal (Ds 30) which had a
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high HbA1c percentage but a weight gain and basal blood glucose (across a
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number of weeks) similar to control animals.
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All control mice gained weight throughout the course of the study, with the
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increases ranging from 113 % (DS 37; week 2) to 168 % (Ds 72; week 8) of
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their initial weight. No changes in either HbA1c or basal glucose levels were
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observed. Amongst the STZ diabetic animals, end weights ranged from 61%
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to 97 % (week 2), 70% to 123% (week 4) and 78% to 116% (week 8) of their
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initial weight. In all cases weight gain was significantly less than that of the
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control animals. .Percentage HbA1c levels were significantly correlated (P <
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0.001) with weight changes within each STZ treated group (week 2: r = -0.79;
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week 4: r = -0.75 week 8: r = -0.80).
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No differences in spematogenic activity or testicular architecture were
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discernable on histological examination of the contralateral testis (data not
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shown)
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NMR Metabolite Profiles
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An NMR spectra of the polar metabolite extract of a mouse testis showing the
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internal standard TSP at ( = 0.0 ppm) and the most abundant metabolites is
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presented in Figure 1. The most prominent resonances arose from alanine,
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betaine creatine, choline, carnitine, lactate, leucine and myo inositol. Only
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trace levels of glucose were observed.
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Principal component analysis (PCA) plot (Figure 2), representing each testis
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extract by a single point, revealed a distinct separation between the control
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non diabetic (Figure 2B) and the STZ-diabetic mice (Figure 2C).
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metabolite profiles of the STZ non-responders (Table1) were similar to those
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of the non diabetic control animals and clustered within the same region of the
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PCA plot (Figure 2C). The metabolic perturbations of the diabetic animals
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varied in degree but clearly separated them from the control group. Principal
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component 1 (PC1), accounted for the majority of the variation (48 %), and
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principal component 2 (PC2) accounted for 22 % of the variation. The tight
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clustering of the control mice in the PCA analysis confirmed that the observed
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separation along PC1 and PC2 was not attributable to bias during the
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experiment due to harvest date or cage.
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The
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The mice with the greatest weight loss, regardless of duration of treatment;
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had the most perturbed metabolite profiles. These STZ treated mice were
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observed further to the right of the PCA plot separating along PC1 notably Ds
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70, Ds 57, Ds 17, Ds 8, Ds 10, Ds 28 and Ds 33 (week 2), Ds 11, Ds 25,
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(week 4) and Ds 59, Ds 77, Ds 38 and Ds 15, (week 8). The perturbations
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found in these STZ treated diabetic mice (separating to the right along PC1),
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included lower creatine, choline and carnitine levels (Figure 3), with the
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corresponding resonances, normalized to the total spectral integral, 11.9%
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17.3% and 13.1 lower than the control group respectively and increased
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lactate, alanine and myo-inositol of 39.4 % 43.7 %, 46.8 % respectively.
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Betaine, which was largely responsible for the separation along PC2 (Figure
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2C), differed from the other metabolites in that STZ responders showed both
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increased and decreased levels (Figure 4 ). In the mice within each 2, 4 and 8
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week group that showed weight gain, which included the STZ non responders,
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betaine levels were similar to those observed for the control animals. For the
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majority of STZ treated mice that showed weight loss, betaine levels
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increases up to approximately 130% of control values. However, for the most
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cachexic mice betaine levels were lower that control values most evident in
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the 8 week group.
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DISCUSSION
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The prevailing view amongst clinicians and researchers has been that DM has
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little, if any, affect on male reproductive function. This is based, in reality, on
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the lack of definitive correlations between DM and impaired sperm
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quantity/quality.
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challenged by information obtained using techniques other than the previously
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reported routine light microscopic semen analysis. A significant increase has
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been reported with DM in the percentage damaged sperm nuclear DNA
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(Agbaje et al. 2007). This previously unreported factor is of importance for
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and beyond spermatogenesis and fertilization (Morris et al. 2002). We have
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also found heightened levels of the receptor for advanced glycation end
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products (RAGE), a group of heterogenous compounds implicated in an
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increasing number of diabetic complications, in the testis, epididymis and
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sperm of diabetic men (Mallidis et al. 2007). These findings suggest that the
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influence of DM may be subtle and consequently not reflected by changes in
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traditional sperm parameters such as concentration, motility and morphology.
Recently, however, this clinical position has begun to be
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This present study describes changes in the testicular metabolome following
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the induction by STZ of an experimental type 1 diabetes, and identifies
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perturbations in several important metabolites. Specifically, diabetic mice were
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found to have reductions in carnitine, creatine, and choline and increases in
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lactate, alanine and myo-inositol.
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Carnitine
(
hydroxyl--N-trimethylaminobutyrate)
is
a
water
soluble
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quaternary amine. It has an obligatory role in  oxidation, the mediation of
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long chain fatty acid transport into the mitochondrial matrix (Swamy-Mruthinti
292
and Carter. 1999) and has been found to inhibit oxidative stress (Pignatelli et
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al. 2003) and protect cells from chromosomal aberrations induced by
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hydrogen peroxide (Santoro et al. 2005). It is obtained either from the diet or
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synthesized from lysine and methionine and its concentration in epididymal
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fluid is 2000 fold higher than that in the blood (Enomoto et al. 2002).
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Clinically, due to its association with sperm maturation and the attainment of
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motility (Deana et al. 1989), it has been used primarily as a dietary
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supplement in the treatment of asthenozoospermia (reviewed by (Ng et al.
300
2004)). It has also been found to act as a possible anti-apoptotic agent aiding
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spermatogenic recovery following testicular injury (Ng et al. 2004).
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addition, it has been shown to be an effective treatment against the elevated
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production of reactive oxygen species associated with abacterial prostato-
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vesiculo-epididymitis (Vicari & Calogero. 2001).
In
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Creatine, the most widely used ergogenic supplement, helps maintain ATP
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levels during large fluctuations in energy demand. As a temporary store, it
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transports ATP between the site of energy production and the site of its
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utilization (Lee et al. 1998) and has antioxidant properties (Lawler et al. 2002).
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It is a downstream product of glycine and arginine and is present in high levels
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in the testis where, unlike the sites of major usage (i.e. skeletal and cardiac
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muscle), it is not absorbed from the blood but synthesized. The testis is one of
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the major sources of creatine in the body (Lee et al. 1998). It is thought to
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have a significant role in both male and female germ cell development but its
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precise role in male reproductive function remains undefined, though
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creatinouria has been suggested as a potential biomarker for testicular
317
damage(Draper and Timbrell. 1996).
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Choline has been associated with the epididymis and has been primarily
320
studied for its role in sperm tail function (reviewed by (Sastry &
321
Sadavongvivad. 1978)). It is present in large amounts in the seminal plasma
322
and has been successfully employed as a forensic marker for the presence of
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semen (Noppinger et al. 1987). In contrast, only low levels of choline and its
324
derivatives have been found in the testis (Hinton & Setchell. 1980). Of these,
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acetylcholine has been associated with increased Ca2+ transport in sperm
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(Bray et al. 2005), an effect also shared by carnitine (Deana et al. 1989).
327
328
Considering the noted antioxidant qualities of these three metabolites, either
329
individually (Lawler et al. 2002, Pignatelli et al. 2003) or in combination
330
(Sachan et al. 2005), it is tempting to speculate that their observed reduction
331
contributes to the state and sequelae of oxidative stress in the diabetic testis
332
acting as at least part of the mechanism leading to the increased percentage
333
of nuclear DNA damage reported in diabetic subjects.. For the time being,
334
however, this hypothesis remains speculative and requires further exploration.
335
336
We have also observed elevations of lactate, alanine and myo-inositol.
337
Although no specific role in male reproductive function has been attributed to
338
alanine, the presence of myo-inositol in the testis has been known for over 40
339
years. Myo-inositol is synthesized by Sertoli cells (Robinson & Fritz. 1979)
340
and its levels have been previously found to be significantly increased in the
341
testes of STZ diabetic rats (Rancour & Wells. 1980). However, the same study
342
also reported increases in testicular glucose levels, a finding that was not
343
corroborated by our results. Similarly, lactate, which is used by the germ cells
344
primarily as a substrate for ATP production (Boussouar & Benahmed. 2004)
345
has, in other studies, been found to decrease in the testicular cells of Goto-
346
Kakizaki, STZ and alloxan diabetic models (Amaral et al. 2006, Sharaf et al.
347
1978). The differences in the reported variations of these particular
348
metabolites in the diabetic testis remain a source of conjecture.
349
350
The greatest variation between diabetic and non diabetic animals was found
351
in the levels of betaine. This compound is supplied mainly from the diet. Both
352
it and choline are intermediates in a pathway involved in the production of
353
creatine. While this pathway predominates in the liver, kidneys and pancreas
354
it is also known to operate in the Sertoli cells (Lee et al. 1998). Accumulating
355
in various cell types during osmotic stress, betaine levels have been found to
356
increase five fold in the urine, whilst remaining unchanged in the blood, of
357
diabetic patients (Dellow et al. 2001). The only reported effect of betaine on
358
male reproductive function is the partial alleviation of the adverse affects on
359
the spermatogenesis of methylenetetrahydrofolate reductase (MTHFR)
360
deficiency in mice. A life long supplementation of betaine was found to
361
increase sperm concentration and fertility, possibly by mediating alterations in
362
the transmethylation pathway, thus maintaining normal methylation levels
363
within germ cells (Kelly et al. 2005). In our diabetic model, betaine was found
364
both to increase and to decrease depending on the severity of the condition.
365
Elevations of betaine occurred in diabetic mice that were of similar body
366
weight to controls, whilst diabetic mice exhibiting cachexia exhibited a
367
decrease in betaine concentration and more severe perturbations in the other
368
metabolites. Whether the imbalance in betaine occurs as part of a global
369
physiological response to hyperglycaemia, is a protective mechanism during
370
the early stages of the disease, or is possibly the result of lipid catabolism
371
remains unclear.
372
373
Overall, STZ treated diabetic mice show a wide distribution on the PCA plot
374
indicating varying degrees of metabolite abnormalities with larger changes
375
associated with worsening glucose homeostasis and decreasing body weight.
376
The severity of diabetes and its complications were reflected in the testis
377
metabolome, where, though all diabetic mice exhibited abnormal metabolic
378
profiles, the severity of the abnormality was likely to be exacerbated by low
379
body-weight and overall physical wellbeing. As has previously been shown,
380
STZ treatment did not result in sufficient beta-cell destruction for diabetes to
381
develop in all mice (Frenkel et al. 1978). The clustering of the metabolic
382
profiles of these STZ non responding animals with those of the control mice
383
provides evidence that the metabolite perturbations noted by the STZ
384
responders were a direct consequence of beta-cell destruction and the
385
ensuing reduction in insulin secretion, and not an effect of the STZ itself.
386
387
All the metabolites identified in this study have been previously shown to be
388
prominent in the metabolomic profiles of the normal testis (Griffin et al. 2000,
389
Yamaguchi et al. 2006). Additionally, the concentrations of the majority of
390
these in blood and urine, have been found to be altered in DM (Abdel-Aziz et
391
al. 1975, Bjorgaas et al. 1997, Dellow et al. 1999). This study shows, for the
392
first time, that these perturbations extend to the testis itself.
393
394
In conclusion, our data indicate that there are important metabolite changes in
395
the diabetic testis.
396
should be considered as a relevant factor in the assessment of male fertility.
This adds further support the argument that diabetes
397
398
ACKNOWLEDGEMENTS
399
The authors thank the staff of the animal facility of the Queens University of
400
Belfast for all their efforts on our behalf. We gratefully acknowledge the
401
financial support of the Fertility Research Trust, Spear Bell Endowment and
402
the Department of Agriculture and Rural Development, Northern Ireland.
403
404
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405
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407
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Figure Legends
553
Figure 1 1H NMR spectrum of polar metabolites extracted from mouse testis.
554
The resonance from the internal standard (TSP) is at 0.00 ppm.
555
Figure 2 Principal component analysis (PCA) scores plot distinguishing
556
testicular metabolite profiles of (A) Controls (blue circles), (B) STZ-treated
557
mice developing diabetes (red circles) and (C) STZ-treated non-responders
558
(green circles). Annotations correspond to metabolite profiles stated in the
559
text.
560
Figure 3 Loadings plot of PCA plot shown in Figure 2. Points furthest from the
561
axis of PC 1 and PC 2, respectively, contributed most to the separation
562
observed in the scores plot.
563
Figure 4 Relationship between betaine concentrations and changes in body
564
weight in STZ-treated mice in (A) 2 week, (B) 4 week and (C) 8 week groups.
565
Betaine levels in STZ non-responders were similar to controls. The majority of
566
STZ-responders had elevated levels of betaine. However, a proportion of
567
STZ-responders showed decreased levels of betaine; these mice consistently
568
had amongst the lowest weight gain/greatest weight loss within each group.
Table 1
Initial Body
Weight (g)
Final Body
Weight (g)
Change in Body
Weight (%)
Mean Non
Fasting Weekly
Glucose (mM)
Final Non
Fasting
Glucose (mM)
HbA1C
(%)
2 wk (n=11)
18.44 ± 0.33
23.26 ± 0.46
126.41 ± 2.58
11.7 ± 0.72
15.24 ± 1.27
4.19 ± 0.11
4 wk (n=12)
19.07 ± 0.28
25.12 ± 0.57
132.18 ± 3.45
9.13 ± 0.36
12.45 ± 0.67
4.64 ± 0.14
8 wk (n=14)
19.98 ± 0.35
28.99 ± 0.42
145.54 ± 2.88
7.74 ± 0.26
10.26 ± 0.78
5.14 ± 0.34
2 wk (n=12 )
20.08 ± 0.25b
16.17 ± 0.77c
80.29 ± 3.29c
31.45 ± 1.02c
32.38 ± 0.66c
7.36 ± 0.4c
4 wk (n=10)
21.08 ± 0.49b
20.47 ± 1.37a
96.92 ± 5.80c
28.41 ± 1.79c
30.10 ± 1.85c
9.52 ± 0.35c
8 wk (n=10)
19.96 ± 0.38
19.74 ± 0.73c
99.16 ± 3.96c
29.04 ± 1.39c
33.23 ± 0.07c
11.89 ± 0.39c
2 wk (n=1)
18.70
11.45
64.23
33.30
33.3
8.2
4 wk (n=1)
23.00
16.10
70.00
33.00
33.33
10.80
2 wk (n=1)
19.50
20.70
106.15
20.80
20.10
4.40
4 wk (n=3)
20.70 ± 0.70
26.33 ± 0.38
127.38 ± 2.60
9.17 ± 0.09
9.77± 2.23
6.80 ± 0.95
8 wk (n=3)
22.27 ± 2.99
29.37 ± 1.22
136.69 ± 18.49
11.83 ± 1.17
14.6 ± 2.64
5.53 ± 0.24
Control
STZ
Responders
Sick Responders
Non Responders
a
p=0.008; bp=0.001; cp<0.001
Figure 1
picture.ESP
Creatine
0.008
0.007
TSP
0.006
Normalized Intensity
Creatine
Betaine
0.005
Carnitine
0.004
Choline
0.003
Alanine
myo_Inositol
Acetone
0.002
Lactate
Betaine
B glucose
0.001
Leucine/Isoleucine/Valine
Acetate
0
5.0
4.5
4.0
3.5
3.0
2.5
2.0
Chemical Shift (ppm)
1.5
1.0
0.5
0
Figure 2
Figure 3
27
Figure 4
A
140
Betaine concentration as a percentage of the
controls
1
130
120
110
100
STZ non-responder
90
80
70
60
50
50
60
70
80
90
100
110
End body weight as a perercentage of starting weight
Betaine concentration as a percentage of the
controls
B
140
130
120
110
100
90
STZ non-responders
80
70
60
50
50
60
70
80
90
100
110
120
130
140
End body weight as a perercentage of starting weight
Betaine concentration as a percentage of the
controls
C
130
120
110
100
90
STZ non-responders
80
70
60
50
50
70
90
110
130
150
End body weight as a perercentage of starting weight
2
170