Leptin signaling through STAT3 and STAT5 pathways is not required for fertility in mice. A Singireddy, G Anderson. Centre for Neuroendocrinology and Department of Anatomy, Otago School of Medical Sciences, University of Otago, Dunedin.The peptide hormone leptin is critical for the regulation of body weight as well as fertility. Lack of leptin or leptin receptors leads to obesity and infertility. The long-form leptin receptor (LepRb) mediates leptin actions in the hypothalamus through multiple signaling pathways, including Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. Previous studies have shown that deletion of STAT3 or STAT5 from the brain results in an obese phenotype, suggesting that these pathways are critical for body weight regulation. However, their involvement in leptin-mediated regulation of fertility remains unclear.The present study investigates the roles of STAT3 and STAT5 in leptin modulation of fertility.A Cre-loxP transgenics approach was used to delete STAT3 (n = 6), the closely related STAT5 (n = 7), or both STAT3 and STAT5 (n = 9) specifically from leptin receptor expressing cells to investigate their role in the regulation of fertility. Immunohistochemical analysis confirmed the inability of leptin to induce phosphorylation of STAT3 and STAT5 in mutant mice. Body weight and reproductive function were measured in all mutant mice and their littermate controls.Knocking out STAT3 or both STAT3 and STAT5 from LepR expressing cells, but not STAT5 alone led to obesity (P < 0.05, two-way ANOVA), suggesting that leptin-specific STAT3 signaling is crucial for body weight regulation. All STAT3 and STAT5 single knockout (KO) mice exhibited normal puberty onset and fertility compared to control littermates. Surprisingly, STAT3 and STAT5 double KO mice also exhibited normal puberty onset and subsequent fertility (mean difference ± SEM, litters born control 4.1 ± 0.5; litters born double KO 4.0 ± 0.3, P > 0.05, student's t-test).These results suggest that while STAT3 signaling is crucial for the regulation of body weight, neither STAT3 or STAT5 is required for regulation of fertility by leptin.Sympathetic modulation of cardiac function in diabetes - paired in vivo and ex vivostudy. 1A Thaung, 2C Baldi, 1D Schwenke, 1R Lamberts. 1Department of Physiology, Otago School of Medical Sciences, 2Departmentof Medicine, Dunedin School of Medicine, University of Otago, Dunedin.The burden of type 2 diabetes mellitus (T2DM) is increasing globally. The majority of T2DM patients suffer from diabetes-related cardiovascular disease, such as heart failure. The literature hints at augmented sympathetic nerve activity (SNA) as being accountable for this increased risk of cardiovascular disease. Prior findings on cardiac SNA in T2DM were interpreted based on data obtained from recording of muscle SNA, renal SNA, or plasma noradrenaline spillover. Central control of SNA is differentially regulated to different organs. Therefore, it is important to assess SNA to the heart in diabetes. The aims of our study were i) to measure the sympathetic nerve input to the heart in vivo and ii) to assess β-adrenergic receptor (β-AR) responsiveness of the heart to the nerve input ex vivo.Cardiac SNA was directly recorded in Zucker Diabetic Fatty rats (T2DM model) and their nondiabetic littermates in vivo (urethane anesthesia, 1.5 g/kg, intraperitoneal). Cardiac responsiveness to dobutamine (β-AR agonist, 10 μM) was assessed using the Langendorff-perfused isolated heart retrieved from the same animal ex vivo.Preliminary data suggests that sympathetic drive to the heart in diabetic rats is elevated (mean difference ± SEM, diabetic SNA = 0.90 ± 0.53 μV.s, n = 5; nondiabetic SNA 0.66 ± 0.14 μV.s, n = 5).Cardiac responsiveness to dobutamine indicated a reduced response in diabetic rats (diabetic left ventricular pressure = 124 ± 12; nondiabetic left ventricular pressure 186 ± 16 mmHg).This study is the first to directly record cardiac SNA in diabetes. In addition, the paired in vivo and ex vivo approach in the same animal provides the ability to distinguish between the central regulation of cardiac function and the intrinsic functional capacity of the heart in diabetes, respectively. This provides a unique opportunity to understand how the sympathetic nervous system regulates cardiac function in diabetes.Reduced cardiac function and β-adrenergic receptor responsiveness in the isolated human diabetic myocardium. H-Y Wang1, J Baldi2, P Saxena3, S Coffey2,4, M Williams2,4, R Lamberts1. 1Department of Physiology, Otago School of Medical Sciences, 2Department of Medicine, Dunedin School of Medicine, University of Otago,3Department of Cardiothoracic Surgery, 4Department of Cardiology, Dunedin Hospital, Dunedin.The prevalence of type 2 diabetic mellitus is closely associated with cardiovascular complications. Despite unknown aetiologies, it is established that patients with preserved ejection fraction (EF) are still vulnerable to heart failure. Until now, little is known about the specific functional effects of diabetes on a heart with preserved EF and hence this study aimed to address this by investigating the functional parameters of the heart from these diabetic patients. It is hypothesised that isolated cardiac muscles from diabetic patients will have reduced functional parameters both at basal conditions, and after β-adrenoceptor (β-AR) stimulation mimicking a physiological stress response.Using isolated cardiac muscles obtained from right atrial appendages of patients undergoing coronary artery bypass grafting, functional characteristics of non-diabetic (n = 8) and diabetic myocardium (n = 6) were compared. Samples from patients who had acute coronary artery disease and reduced EF (< 40%) were excluded. Contractile and relaxation parameters of both cohorts were first determined under basal conditions, then in response to a β-AR agonist (dobutamine, 0.1 to 10 μM).Compared to non-diabetics, diabetic muscles had a slower rate of maximum contraction (+dF/dtmax) (287 ± 55 vs. 220 ± 19 mN/mm2/s, P < 0.05, Student's t-test) and relaxation (-dF/dtmax) (-188 ± 25 vs. -130 ± 14 mN/mm2/s, P < 0.05) with prolonged relaxation times under basal conditions. The diabetic muscles were also less responsive to β-AR stress response, as shown by the lesser increase in developed tension, +dF/dtmax and -dF/dtmax, and smaller reduction relaxation times. These results suggest that cardiac muscles from diabetic patients contract less forcefully, as well as having a prolonged relaxation time under basal conditions and during β-AR activated stress.In conclusion, diabetic human atrial myocardium from patients with preserved EF has systolic and diastolic dysfunctional characteristics, and reduced β-AR responsiveness.Colocalisation of estrogen receptor alpha with leptin receptor-expressing cells in the hypothalamus of the mouse using transgenics. J Kim, G Anderson. Centre for Neuroendocrinology and Department of Anatomy, Otago School of Medical Sciences, University of Otago, Dunedin.Leptin and 17β-estradiol (E2) are two seemingly distinct hormones that are critical regulators of both energy balance and reproduction. E2 has similar anorexigenic effects to that of leptin, and both hormones exert their effects in many overlapping hypothalamic regions to regulate nutrition and fertility. Estrogen receptor alpha (ERα) has been suggested to interact with the leptin signaling pathway, modulating leptin sensitivity. Technical difficulties of leptin receptor (LepR) immunohistochemistry, identifying hypothalamic cells that are both leptin and E2responsive has been inconclusive. Using two different, highly specific transgenic mouse models, the project aims to characterise hypothalamic cells that are both E2 and leptin-responsive.This study utilised transgenic mice that express green fluorescent protein (GFP) from the LepR promoter, or in neuropeptide-Y (NPY) neurons, which are known to be leptin-responsive. Brains were harvested from euthanized mice, the preoptic area and entire hypothalamus were sliced into 30μm coronal sections. A subcutaneous injection of leptin (1mg/kg) was administered and phosphorylated STAT3, a marker of LepR activation, was colocalised with GFP; confirming that the GFP expressing cells are indeed leptin responsive. Following validation of the transgenic model, we used immunohistochemistry to determine if ERα colocalises with the GFP expressing Leptin responsive cells.A low percentage of GFP positive LepR cells co-expressing ERα were observed in the arcuate nucleus (24%), ventromedial hypothalamus (25%), and ventral premamillary nucleus (35%). However, high coexpression was recorded at the medial preoptic area (88%), and moderate to high amounts at the rostral dorsomedial hypothalamus (64%) and lateral hypothalamus (62%). We then explored the possibility that E2 and leptin may selectively interact in specific cell types, however we observed no colocalisation of ERα with NPY-positive neurons.This project has provided a reliable identification of LepR-ERα colocalisation and explores the mechanisms underlying the anorexigenic effects of E2. Furthermore, it identifies the potential leptin responsive cells that may be involved in the link between nutrition and fertility.Triadin decreases the propensity for Store Overload Induced Calcium Release (SOICR). M Deo, J McLay, P Jones. Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.Arrhythmia occurs in a number of heart diseases including heart failure (HF). It has been shown that Store Overload Induced Calcium Release (SOICR) is a common mechanism underlying many types of arrhythmia. SOICR occurs due to the inappropriate opening of the cardiac ryanodine receptor (RyR2) once calcium in the sarcoplasmic reticulum (SR) reaches a certain threshold. RyR2 forms a large macromolecular complex with other proteins. One such protein is triadin; which is known to be lost from the RyR2 complex in HF. In the present study, we aim to characterise the effect of triadin on the threshold for SOICR to determine whether the loss of triadin could be responsible for the arrhythmias associated with HF.Single cell fluorescence imaging in HEK 293 cells stably expressing RyR2 with or without transfection of triadin, using the luminally targeted calcium indicator protein D1ER, showed that the expression of triadin increased the threshold for SOICR (86.2% ± 0.03%, n = 77, p < 0.001, mean % of maximum store ± S.E.M, unpaired t-test) as compared to RyR2 alone (79.5% ± 0.78%, n = 54), suggesting the presence of triadin inhibits SOICR. This was confirmed by measuring the occurrence of SOICR (increases in cytosolic calcium) using the cytosolic calcium dye Fluo-4-AM. This showed that SOICR occurs less readily in cells expressing RyR2 and triadin (65.3% ± 1.8%, n = 21, p < 0.01, at 1mM extracellular calcium) than in cells expressing RyR2 alone (75.0% ± 2.5%, n = 22).These results provide evidence that the presence of triadin reduces the propensity for SOICR by increasing the threshold that SR calcium must reach to trigger SOICR. Conversely, this suggests that the loss of triadin in HF is arrhythmogenic. Therefore, stabilising the interaction of triadin with RyR2 in patients susceptible to arrhythmias is likely to be therapeutic.Macrophage and T cell distribution in autoimmune inflammation of the gut. E Dunn1, E Taylor1, M Schultz2, G Butt3, R Kemp1. 1Department of Microbiology and Immunology, Otago School of Medical Sciences, 2Dunedin School of Medicine,3Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin.The autoimmune diseases Inflammatory Bowel Disease (IBD), Spondyloarthropathy (SpA) and Type 1 Diabetes Mellitus (T1DM) have epidemiological, symptomatic and genetic overlap. Many patients with IBD develop SpA and overlapping genetic loci exist between IBD and T1DM patients. This genetic and symptomatic crossover between IBD, SpA, and T1DM suggests a role for the immune system in linking these diseases. The aim of this study was to establish methods for intestinal T cell and macrophage analysis with which to investigate the pathophysiological crossover between intestinal inflammation in patients with IBD, SpA and T1DM.Intestinal tissue biopsies are collected from healthy or diseased patients, dissociated, then cells labelled with cell-specific antibodies and analysed using flow cytometry. Methods were initially optimised using healthy donors.Analysis of different areas of the intestinal tract of healthy individuals using these techniques revealed increased T cell frequencies in the terminal ileum (TI) compared to the colon (24.9 ± 3.4% and 9.2 ± 2.1%, respectively, mean ± SEM, n = 5, P < 0.001, One-way-ANOVA with Tukey's posthoc test). Further analysis of TI mucosal tissue from IBD patients revealed increases in inflammatory (IL-17+) (IBD, 1.1 ± 0.4%; control, 0.23 ± 0.04%, n = 3, P < 0.05, unpaired Student's t-test) and CD8+ regulatory (FoxP3+CD25Hi) T cells (IBD, 0.95 ± 0.20%; control, 0.16 ± 0.06%, n = 3, P < 0.01). This illustrates the efficacy of these methods in analysing healthy and inflamed environments and that IBD mucosa harbours distinct immune populations. Macrophages have also been identified by these methods, and their phenotypes in intestinal mucosa of IBD, SpA and T1DM patients will now be characterised.Knowledge of the presence and function of innate and adaptive immune cell populations will provide insight into the linkages between IBD, SpA and T1DM, and show how the immune balance has been altered to favour disease progression.Improved atrial relaxation in human type 2 diabetes mellitus. S Lingam1,P Saxena2, S Coffey3, 4, M Williams3, 4, J Baldi4, R Lamberts1, P Jones1. 1Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, 2Department of Cardiothoracic Surgery, 3Department of Cardiology, Dunedin Hospital, Dunedin,4Department of Medicine, Dunedin School of Medicine, University of Otago, Dunedin.Type 2 diabetes mellitus (T2DM) is characterised by impaired relaxation of the heart that can eventually lead to heart failure. The cardiac relaxation pathway is governed by the combined activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and phospholamban (PLB). SERCA2a actively resequesters free cytosolic Ca2+ to the sarcoplasmic reticulum, promoting cardiac relaxation, whereas PLB inhibits this SERCA2a activity, thereby reducing cardiac relaxation. Several type 1 diabetes mellitus (T1DM) animal and human studies and a few T2DM animal studies have found decreased SERCA2a activity and increased PLB expression in the left ventricle of the heart, indicating this is a mechanism which impairs cardiac relaxation in diabetes. The present study investigates the protein expression of SERCA2a and PLB in humans with T2DM.To examine the level of SERCA2a and PLB protein expression, we carried out western blotting analysis in human atrial appendages from T2DM (n = 5) and non-diabetic (n = 15) patients with preserved contractile function, undergoing coronary artery bypass surgery (CABG). Blot analysis (mean ± SEM, unpaired t-test) revealed similar total SERCA2a protein levels between T2DM and non-diabetic patients (T2DM 0.98 ± 0.2, non-diabetics 0.80 ± 0.1,P = 0.88). There was, however, a significant decrease in PLB protein levels in T2DM patients compared to non-diabetic patients (T2DM 1.33 ± 0.4, non-diabetics 2.20 ± 0.4, P = 0.02), resulting in a decreased PLB:SERCA2a ratio in T2DM patients compared to non-diabetic patients (T2DM 2.00 ± 0.8, non-diabetics 5.60 ± 1.5, P = 0.17).Thus, in contrast to previous studies in diabetic animals (T1DM and T2DM) and humans (T1DM), our current data in T2DM patients, without contractile dysfunction, indicate increased SERCA2a activity, suggesting improved atrial relaxation. This suggests that in the pre-failing diabetic heart SERCA2A activity is increased as a compensatory mechanism against other diabetic changes that reduce cardiac relaxation.Characterisation of a tamoxifen-inducible STAT5 knockout mouse. P Gustafson, S Bunn, D Grattan. Department of Anatomy, Otago School of Medical Sciences, University of Otago, Dunedin.Prolactin synthesis and release is regulated by activation of the hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. While experiments using a genetic knockout (KO) suggest that signal transducer and activator of transcription 5 (STAT5) mediates this activation, a developmental loss of TIDA neurons compromises this conclusion. Therefore, this projected aimed to characterise an inducible STAT5 KO model in which neuronal development is preserved.Transgenic mice were generated using the tamoxifen-inducible LoxP-Cre recombinase (Cre) system to delete STAT5 from adult females (0.2mg tamoxifen per day for 5 days, intraperitoneal). Wildtype (WT) and inducible STAT5 KO mice were treated with prolactin (0.2mg for 20 min, intraperitoneal) before being euthanized. Neural tissue was collected and 30μm coronal sections were taken through the hypothalamus. Cre, tyrosine hydroxylase (a marker for the TIDA neurons) and phosphorylated STAT5 (pSTAT5) expression was detected using immunohistochemistry.TIDA neuron number in STAT5 KO mice (122 ± 6, mean ± SEM, n=7) was not significantly different to WT control mice (112 ± 6, n=12; P=0.26, Student's t-test). Tamoxifen induced Cre nuclear translocation in approximately 50% of these neurons. The percentage of TIDA neurons expressing pSTAT5 in STAT5 KO mice (55 ± 6%, n=8) was not significantly different from inducible-Cre control mice (51 ± 1, n=3). Tamoxifen initially suppressed prolactin levels from 199 ± 86ng/ml to 24 ± 3ng/ml in STAT5 KO mice. Levels recovered back to that of untreated WT control mice (250 ± 34ng/ml) after three weeks but then fell again after four (44 ± 11ng/ml) and six (55 ± 13ng/ml) weeks (P=0.0011, one-way ANOVA with Dunnett's posthoc test).TIDA neuron number was preserved in STAT5 KO mice. Tamoxifen did not delete STAT5 from TIDA neurons but the complex temporal profile of prolactin levels in the KO suggests STAT5 deletion may have occurred in another neuronal population that regulate prolactin secretion.\r\n

Leptin signaling through STAT3 and STAT5 pathways is not required for fertility in mice. A Singireddy, G Anderson. Centre for Neuroendocrinology and Department of Anatomy, Otago School of Medical Sciences, University of Otago, Dunedin.The peptide hormone leptin is critical for the regulation of body weight as well as fertility. Lack of leptin or leptin receptors leads to obesity and infertility. The long-form leptin receptor (LepRb) mediates leptin actions in the hypothalamus through multiple signaling pathways, including Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. Previous studies have shown that deletion of STAT3 or STAT5 from the brain results in an obese phenotype, suggesting that these pathways are critical for body weight regulation. However, their involvement in leptin-mediated regulation of fertility remains unclear.The present study investigates the roles of STAT3 and STAT5 in leptin modulation of fertility.A Cre-loxP transgenics approach was used to delete STAT3 (n = 6), the closely related STAT5 (n = 7), or both STAT3 and STAT5 (n = 9) specifically from leptin receptor expressing cells to investigate their role in the regulation of fertility. Immunohistochemical analysis confirmed the inability of leptin to induce phosphorylation of STAT3 and STAT5 in mutant mice. Body weight and reproductive function were measured in all mutant mice and their littermate controls.Knocking out STAT3 or both STAT3 and STAT5 from LepR expressing cells, but not STAT5 alone led to obesity (P < 0.05, two-way ANOVA), suggesting that leptin-specific STAT3 signaling is crucial for body weight regulation. All STAT3 and STAT5 single knockout (KO) mice exhibited normal puberty onset and fertility compared to control littermates. Surprisingly, STAT3 and STAT5 double KO mice also exhibited normal puberty onset and subsequent fertility (mean difference ± SEM, litters born control 4.1 ± 0.5; litters born double KO 4.0 ± 0.3, P > 0.05, student's t-test).These results suggest that while STAT3 signaling is crucial for the regulation of body weight, neither STAT3 or STAT5 is required for regulation of fertility by leptin.Sympathetic modulation of cardiac function in diabetes - paired in vivo and ex vivostudy. 1A Thaung, 2C Baldi, 1D Schwenke, 1R Lamberts. 1Department of Physiology, Otago School of Medical Sciences, 2Departmentof Medicine, Dunedin School of Medicine, University of Otago, Dunedin.The burden of type 2 diabetes mellitus (T2DM) is increasing globally. The majority of T2DM patients suffer from diabetes-related cardiovascular disease, such as heart failure. The literature hints at augmented sympathetic nerve activity (SNA) as being accountable for this increased risk of cardiovascular disease. Prior findings on cardiac SNA in T2DM were interpreted based on data obtained from recording of muscle SNA, renal SNA, or plasma noradrenaline spillover. Central control of SNA is differentially regulated to different organs. Therefore, it is important to assess SNA to the heart in diabetes. The aims of our study were i) to measure the sympathetic nerve input to the heart in vivo and ii) to assess β-adrenergic receptor (β-AR) responsiveness of the heart to the nerve input ex vivo.Cardiac SNA was directly recorded in Zucker Diabetic Fatty rats (T2DM model) and their nondiabetic littermates in vivo (urethane anesthesia, 1.5 g/kg, intraperitoneal). Cardiac responsiveness to dobutamine (β-AR agonist, 10 μM) was assessed using the Langendorff-perfused isolated heart retrieved from the same animal ex vivo.Preliminary data suggests that sympathetic drive to the heart in diabetic rats is elevated (mean difference ± SEM, diabetic SNA = 0.90 ± 0.53 μV.s, n = 5; nondiabetic SNA 0.66 ± 0.14 μV.s, n = 5).Cardiac responsiveness to dobutamine indicated a reduced response in diabetic rats (diabetic left ventricular pressure = 124 ± 12; nondiabetic left ventricular pressure 186 ± 16 mmHg).This study is the first to directly record cardiac SNA in diabetes. In addition, the paired in vivo and ex vivo approach in the same animal provides the ability to distinguish between the central regulation of cardiac function and the intrinsic functional capacity of the heart in diabetes, respectively. This provides a unique opportunity to understand how the sympathetic nervous system regulates cardiac function in diabetes.Reduced cardiac function and β-adrenergic receptor responsiveness in the isolated human diabetic myocardium. H-Y Wang1, J Baldi2, P Saxena3, S Coffey2,4, M Williams2,4, R Lamberts1. 1Department of Physiology, Otago School of Medical Sciences, 2Department of Medicine, Dunedin School of Medicine, University of Otago,3Department of Cardiothoracic Surgery, 4Department of Cardiology, Dunedin Hospital, Dunedin.The prevalence of type 2 diabetic mellitus is closely associated with cardiovascular complications. Despite unknown aetiologies, it is established that patients with preserved ejection fraction (EF) are still vulnerable to heart failure. Until now, little is known about the specific functional effects of diabetes on a heart with preserved EF and hence this study aimed to address this by investigating the functional parameters of the heart from these diabetic patients. It is hypothesised that isolated cardiac muscles from diabetic patients will have reduced functional parameters both at basal conditions, and after β-adrenoceptor (β-AR) stimulation mimicking a physiological stress response.Using isolated cardiac muscles obtained from right atrial appendages of patients undergoing coronary artery bypass grafting, functional characteristics of non-diabetic (n = 8) and diabetic myocardium (n = 6) were compared. Samples from patients who had acute coronary artery disease and reduced EF (< 40%) were excluded. Contractile and relaxation parameters of both cohorts were first determined under basal conditions, then in response to a β-AR agonist (dobutamine, 0.1 to 10 μM).Compared to non-diabetics, diabetic muscles had a slower rate of maximum contraction (+dF/dtmax) (287 ± 55 vs. 220 ± 19 mN/mm2/s, P < 0.05, Student's t-test) and relaxation (-dF/dtmax) (-188 ± 25 vs. -130 ± 14 mN/mm2/s, P < 0.05) with prolonged relaxation times under basal conditions. The diabetic muscles were also less responsive to β-AR stress response, as shown by the lesser increase in developed tension, +dF/dtmax and -dF/dtmax, and smaller reduction relaxation times. These results suggest that cardiac muscles from diabetic patients contract less forcefully, as well as having a prolonged relaxation time under basal conditions and during β-AR activated stress.In conclusion, diabetic human atrial myocardium from patients with preserved EF has systolic and diastolic dysfunctional characteristics, and reduced β-AR responsiveness.Colocalisation of estrogen receptor alpha with leptin receptor-expressing cells in the hypothalamus of the mouse using transgenics. J Kim, G Anderson. Centre for Neuroendocrinology and Department of Anatomy, Otago School of Medical Sciences, University of Otago, Dunedin.Leptin and 17β-estradiol (E2) are two seemingly distinct hormones that are critical regulators of both energy balance and reproduction. E2 has similar anorexigenic effects to that of leptin, and both hormones exert their effects in many overlapping hypothalamic regions to regulate nutrition and fertility. Estrogen receptor alpha (ERα) has been suggested to interact with the leptin signaling pathway, modulating leptin sensitivity. Technical difficulties of leptin receptor (LepR) immunohistochemistry, identifying hypothalamic cells that are both leptin and E2responsive has been inconclusive. Using two different, highly specific transgenic mouse models, the project aims to characterise hypothalamic cells that are both E2 and leptin-responsive.This study utilised transgenic mice that express green fluorescent protein (GFP) from the LepR promoter, or in neuropeptide-Y (NPY) neurons, which are known to be leptin-responsive. Brains were harvested from euthanized mice, the preoptic area and entire hypothalamus were sliced into 30μm coronal sections. A subcutaneous injection of leptin (1mg/kg) was administered and phosphorylated STAT3, a marker of LepR activation, was colocalised with GFP; confirming that the GFP expressing cells are indeed leptin responsive. Following validation of the transgenic model, we used immunohistochemistry to determine if ERα colocalises with the GFP expressing Leptin responsive cells.A low percentage of GFP positive LepR cells co-expressing ERα were observed in the arcuate nucleus (24%), ventromedial hypothalamus (25%), and ventral premamillary nucleus (35%). However, high coexpression was recorded at the medial preoptic area (88%), and moderate to high amounts at the rostral dorsomedial hypothalamus (64%) and lateral hypothalamus (62%). We then explored the possibility that E2 and leptin may selectively interact in specific cell types, however we observed no colocalisation of ERα with NPY-positive neurons.This project has provided a reliable identification of LepR-ERα colocalisation and explores the mechanisms underlying the anorexigenic effects of E2. Furthermore, it identifies the potential leptin responsive cells that may be involved in the link between nutrition and fertility.Triadin decreases the propensity for Store Overload Induced Calcium Release (SOICR). M Deo, J McLay, P Jones. Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.Arrhythmia occurs in a number of heart diseases including heart failure (HF). It has been shown that Store Overload Induced Calcium Release (SOICR) is a common mechanism underlying many types of arrhythmia. SOICR occurs due to the inappropriate opening of the cardiac ryanodine receptor (RyR2) once calcium in the sarcoplasmic reticulum (SR) reaches a certain threshold. RyR2 forms a large macromolecular complex with other proteins. One such protein is triadin; which is known to be lost from the RyR2 complex in HF. In the present study, we aim to characterise the effect of triadin on the threshold for SOICR to determine whether the loss of triadin could be responsible for the arrhythmias associated with HF.Single cell fluorescence imaging in HEK 293 cells stably expressing RyR2 with or without transfection of triadin, using the luminally targeted calcium indicator protein D1ER, showed that the expression of triadin increased the threshold for SOICR (86.2% ± 0.03%, n = 77, p < 0.001, mean % of maximum store ± S.E.M, unpaired t-test) as compared to RyR2 alone (79.5% ± 0.78%, n = 54), suggesting the presence of triadin inhibits SOICR. This was confirmed by measuring the occurrence of SOICR (increases in cytosolic calcium) using the cytosolic calcium dye Fluo-4-AM. This showed that SOICR occurs less readily in cells expressing RyR2 and triadin (65.3% ± 1.8%, n = 21, p < 0.01, at 1mM extracellular calcium) than in cells expressing RyR2 alone (75.0% ± 2.5%, n = 22).These results provide evidence that the presence of triadin reduces the propensity for SOICR by increasing the threshold that SR calcium must reach to trigger SOICR. Conversely, this suggests that the loss of triadin in HF is arrhythmogenic. Therefore, stabilising the interaction of triadin with RyR2 in patients susceptible to arrhythmias is likely to be therapeutic.Macrophage and T cell distribution in autoimmune inflammation of the gut. E Dunn1, E Taylor1, M Schultz2, G Butt3, R Kemp1. 1Department of Microbiology and Immunology, Otago School of Medical Sciences, 2Dunedin School of Medicine,3Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin.The autoimmune diseases Inflammatory Bowel Disease (IBD), Spondyloarthropathy (SpA) and Type 1 Diabetes Mellitus (T1DM) have epidemiological, symptomatic and genetic overlap. Many patients with IBD develop SpA and overlapping genetic loci exist between IBD and T1DM patients. This genetic and symptomatic crossover between IBD, SpA, and T1DM suggests a role for the immune system in linking these diseases. The aim of this study was to establish methods for intestinal T cell and macrophage analysis with which to investigate the pathophysiological crossover between intestinal inflammation in patients with IBD, SpA and T1DM.Intestinal tissue biopsies are collected from healthy or diseased patients, dissociated, then cells labelled with cell-specific antibodies and analysed using flow cytometry. Methods were initially optimised using healthy donors.Analysis of different areas of the intestinal tract of healthy individuals using these techniques revealed increased T cell frequencies in the terminal ileum (TI) compared to the colon (24.9 ± 3.4% and 9.2 ± 2.1%, respectively, mean ± SEM, n = 5, P < 0.001, One-way-ANOVA with Tukey's posthoc test). Further analysis of TI mucosal tissue from IBD patients revealed increases in inflammatory (IL-17+) (IBD, 1.1 ± 0.4%; control, 0.23 ± 0.04%, n = 3, P < 0.05, unpaired Student's t-test) and CD8+ regulatory (FoxP3+CD25Hi) T cells (IBD, 0.95 ± 0.20%; control, 0.16 ± 0.06%, n = 3, P < 0.01). This illustrates the efficacy of these methods in analysing healthy and inflamed environments and that IBD mucosa harbours distinct immune populations. Macrophages have also been identified by these methods, and their phenotypes in intestinal mucosa of IBD, SpA and T1DM patients will now be characterised.Knowledge of the presence and function of innate and adaptive immune cell populations will provide insight into the linkages between IBD, SpA and T1DM, and show how the immune balance has been altered to favour disease progression.Improved atrial relaxation in human type 2 diabetes mellitus. S Lingam1,P Saxena2, S Coffey3, 4, M Williams3, 4, J Baldi4, R Lamberts1, P Jones1. 1Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, 2Department of Cardiothoracic Surgery, 3Department of Cardiology, Dunedin Hospital, Dunedin,4Department of Medicine, Dunedin School of Medicine, University of Otago, Dunedin.Type 2 diabetes mellitus (T2DM) is characterised by impaired relaxation of the heart that can eventually lead to heart failure. The cardiac relaxation pathway is governed by the combined activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and phospholamban (PLB). SERCA2a actively resequesters free cytosolic Ca2+ to the sarcoplasmic reticulum, promoting cardiac relaxation, whereas PLB inhibits this SERCA2a activity, thereby reducing cardiac relaxation. Several type 1 diabetes mellitus (T1DM) animal and human studies and a few T2DM animal studies have found decreased SERCA2a activity and increased PLB expression in the left ventricle of the heart, indicating this is a mechanism which impairs cardiac relaxation in diabetes. The present study investigates the protein expression of SERCA2a and PLB in humans with T2DM.To examine the level of SERCA2a and PLB protein expression, we carried out western blotting analysis in human atrial appendages from T2DM (n = 5) and non-diabetic (n = 15) patients with preserved contractile function, undergoing coronary artery bypass surgery (CABG). Blot analysis (mean ± SEM, unpaired t-test) revealed similar total SERCA2a protein levels between T2DM and non-diabetic patients (T2DM 0.98 ± 0.2, non-diabetics 0.80 ± 0.1,P = 0.88). There was, however, a significant decrease in PLB protein levels in T2DM patients compared to non-diabetic patients (T2DM 1.33 ± 0.4, non-diabetics 2.20 ± 0.4, P = 0.02), resulting in a decreased PLB:SERCA2a ratio in T2DM patients compared to non-diabetic patients (T2DM 2.00 ± 0.8, non-diabetics 5.60 ± 1.5, P = 0.17).Thus, in contrast to previous studies in diabetic animals (T1DM and T2DM) and humans (T1DM), our current data in T2DM patients, without contractile dysfunction, indicate increased SERCA2a activity, suggesting improved atrial relaxation. This suggests that in the pre-failing diabetic heart SERCA2A activity is increased as a compensatory mechanism against other diabetic changes that reduce cardiac relaxation.Characterisation of a tamoxifen-inducible STAT5 knockout mouse. P Gustafson, S Bunn, D Grattan. Department of Anatomy, Otago School of Medical Sciences, University of Otago, Dunedin.Prolactin synthesis and release is regulated by activation of the hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. While experiments using a genetic knockout (KO) suggest that signal transducer and activator of transcription 5 (STAT5) mediates this activation, a developmental loss of TIDA neurons compromises this conclusion. Therefore, this projected aimed to characterise an inducible STAT5 KO model in which neuronal development is preserved.Transgenic mice were generated using the tamoxifen-inducible LoxP-Cre recombinase (Cre) system to delete STAT5 from adult females (0.2mg tamoxifen per day for 5 days, intraperitoneal). Wildtype (WT) and inducible STAT5 KO mice were treated with prolactin (0.2mg for 20 min, intraperitoneal) before being euthanized. Neural tissue was collected and 30μm coronal sections were taken through the hypothalamus. Cre, tyrosine hydroxylase (a marker for the TIDA neurons) and phosphorylated STAT5 (pSTAT5) expression was detected using immunohistochemistry.TIDA neuron number in STAT5 KO mice (122 ± 6, mean ± SEM, n=7) was not significantly different to WT control mice (112 ± 6, n=12; P=0.26, Student's t-test). Tamoxifen induced Cre nuclear translocation in approximately 50% of these neurons. The percentage of TIDA neurons expressing pSTAT5 in STAT5 KO mice (55 ± 6%, n=8) was not significantly different from inducible-Cre control mice (51 ± 1, n=3). Tamoxifen initially suppressed prolactin levels from 199 ± 86ng/ml to 24 ± 3ng/ml in STAT5 KO mice. Levels recovered back to that of untreated WT control mice (250 ± 34ng/ml) after three weeks but then fell again after four (44 ± 11ng/ml) and six (55 ± 13ng/ml) weeks (P=0.0011, one-way ANOVA with Dunnett's posthoc test).TIDA neuron number was preserved in STAT5 KO mice. Tamoxifen did not delete STAT5 from TIDA neurons but the complex temporal profile of prolactin levels in the KO suggests STAT5 deletion may have occurred in another neuronal population that regulate prolactin secretion.\r\n