Proceedings of the New Zealand Society of Endocrinology

Annual Meeting held as part of the

MedSciNZ Congress

6-9 Dec 2005

Millennium Hotel, Queenstown, New Zealand

Contents:

NZ38 2005 Nancy Sirett lecture: Incessant ovulation, inflammation and epithelial ovarian carcinogenesis J.S. Fleming

NZ39 Endocrine disruption of sperm production in rats by a high phytoestrogen diet S. Assinder, R. Davis, M. Fenwick, A. Glover

NZ40 Poly(C) binding proteins, -CP1 and -CP2 mediates stabilization of hTERT mRNA by autocrine hGH B. Starling Emerald, Y. Chen, T. Zhu, Z. Zhu, P.D. Gluckman, K. Lee, P.E. Lobie

NZ41 Gene expression changes during cardiac development and the development of cardiac hypertrophy in NPR-1 knockout mice L.J. Ellmers, N.J.A. Scott, J. Piuhola, C.M. Frampton, A.M. Richards, V.A. Cameron

NZ42 Mechano-growth factor (MGF) stimulates expression of brain natriuretic peptide (BNP) in vivo and in vitro V.A. Carpenter, K.G. Matthews, G.P. Devlin, J.A. Jensen, S.P. Stuart, T.J. Watson, P.H. Goldspink, S.Y. Yang, J.V. Conaglen, J.J. Bass, G. Goldspink, C.D. McMahon

NZ43 An impaired antioxidant mechanism is associated with skeletal muscle wasting in cancer-bearing rats F. Jeanplong, S.J. Falconer, J.M. Oldham, K.G. Matthews, C.D. McMahon

NZ44 GH induces a post-translational decrease in mature myostatin in male mice J.M. Oldham, S.J. Falconer, F. Jeanplong, M. Sharma, R. Kambadur, H.K. Smith, H.W. Davey, R.J. Wilkins, D.F. Gerrard, J.C. Conaglen, J.J. Bass, C.D. McMahon

NZ45 Myostatin null mice lose more muscle mass during unloading, but recover faster than wild-type mice K.G. Matthews, J.M. Oldham, S.J. Falconer, F. Jeanplong, H.K. Smith, J.J. Bass, C.D. McMahon

NZ46 Vaccination against myostatin does not increase post-natal muscle development in sheep T.S. Gray, S.J. Falconer, F. Jeanplong, S.P. Stuart, T. Watson, C.J. Berry, G.D. Nicholas, V.K. Siriett, E.J. Plummer, A.S. Hennebry, M. Sharma, R. Kambadur, C.D. McMahon

NZ47 Metabolic risk factors for diabetes in pacific island teenagers A.M. Grant, K.A. McAuley, R.W. Taylor, F. Taungapeau, S.M. Williams, M. Waldron, A. Duncan, M. Harper, A. Goulding

NZ48 The influence of obesity on mortality in people with wild type-2 diabetes in the UK R.A. Lawrenson, H.E. Mulnier, H.E. Seaman, V.S. Raleigh, S.S. Soedamah-Muthu, H.M. Colhoun

NZ49 Long term effects of popular dietary approaches on weight loss and features of insulin resistance K.A. McAuley, K.J. Smith, R.W. Taylor, R.T. McLay, S.M. Williams, V.L. Farmer, J.I. Mann

NZ50 Weighty problems of diabetic patients enrolled on the Otago diabetes register K.J. Coppell, K. Anderson, C. Booker

NZ51 Outcomes after seven years for a cohort of individuals with wild type 2 diabetes: a retrospective observational study C.S. Booker, K.J. Coppell, K. Anderson, S.M. Williams

NZ52 Adiposity rebound and early growth S.M. Williams

NZ53 A novel model for studying the effects of pigmentation genes on body weight C.E. Duchesnes, J. Naggert, M. Tatnell, N. Beckman, M. McGregor, R. Elliott, K.G. Mountjoy

NZ54 How is the arrival of food signaled to nutrition-sensitive tissues? An investigation in rat mammary tissue K.W. Stewart, G.J.S. Cooper, S.R. Davis

NZ55 Determining optimal approaches for successful maintenance of weight loss K.S. Whiteford, K.A. McAuley, J.I. Mann, R.W. Taylor, A. Chisholm, S. Vorgers, S. Williams

NZ56 A novel anti-obesity drug permanently resets body weight Òset-pointÓ K.G. Mountjoy, M. Tercel, C.E. Duchesnes, R. Marnane, M. Tatnell, W.A. Denny, W.R. Wilson

NZ57 Chronic prolactin infusion induces central leptin resistance in pseudopregnant rats R.A. Augustine, S.J. Bunn, D.R. Grattan

NZ58 Mice lacking a-MSH develop adrenal glands, but are obese, and retain the aberrant fat metabolism seen in POMC null mice J.L. Costa, S.L. Forbes, M.B. Brennan

NZ59 Changes in sex steroid levels induce SOCS expression and prolactin secretion during late pregnancy F.J. Steyn, G.M. Anderson, D.R. Grattan

NZ60 A mouse model of hyperprolactinaemia-induced infertility D.R. Grattan, G.M. Anderson, A.E. Herbison

NZ61 Prolactin feedback may be important for enkephalin induction in tuberoinfundibular dopaminergic neurons during late pregnancy and lactation I.C. Kokay, J.M. Pilcher, D.R. Grattan NZ62 Placental lysophospholipids Ð the missing link J. Keelan

NZ62 Placental lysophospholipids - the missing link J. Keelan

 

 

Abstracts:

NZ38 2005 NANCY SIRETT LECTURE

INCESSANT OVULATION, INFLAMMATION AND EPITHELIAL OVARIAN CARCINOGENESIS
J.S. Fleming
Eskitis Institute of Cell and Molecular Therapies, School of Biomolecular and Biomedical Sciences, Griffith University, Nathan Campus, Nathan, QLD 4111, Australia

Epithelial ovarian cancer (EOC) is often a lethal disease because in many cases early symptoms go undetected and there are few specific biomarkers for the early stages of the disease. EOC comprises a highly heterogeneous collection of cancers, which includes tumours of low malignant potential, serous cystadenomas, mucinous and clear cell carcinomas, all of which are likely to originate from a number of epithelial cell types and a variety of progenitor lesions, including cells lining ovarian inclusion cysts. None of the major epidemiological hypotheses explains all we now know about ovarian carcinogenesis. A picture is emerging of at least two pathways; a slower development of high grade EOC via transformation of the cells lining inclusion cysts (Type 1) or a more direct and aggressive development of invasive EOC, directly from the OSE or other epithelial source (Type 2). The key to determining the origins of EOC is an understanding of the very early changes in these two types of carcinogenic pathway. The arrival of laser-capture microdissection and expression profiling by microarray technologies offers the promise of defining these pathways more accurately, as well as providing us with the tools for earlier diagnosis. Results of more recent gene profiling studies are encouraging, because they appear to be starting to identify changes in the expression of gene families and pathways that have occurred during differentiation of the precursor cell to EOC. Our future focus must be to more thoroughly classify the different histotypes by their specific biomarkers and to compare each EOC subtype to a broader range of precursor cells, for example, by using LCM to provide pure reference standard cell populations.

 

NZ39

ENDOCRINE DISRUPTION OF SPERM PRODUCTION IN RATS BY A HIGH PHYTOESTROGEN DIET
S. Assinder, R. Davis, M. Fenwick, A. Glover
Department of Anatomy and Structural Biology, University of Otago, Dunedin, NZ

Daily sperm production is regulated by apoptosis of developing germ cells. Increased germ cell apoptosis is induced by testosterone withdrawal or disruption of estrogen action. It is hypothesized that phytoestrogens increase apoptosis of developing germ cells, decreasing sperm production. This study aimed to investigate the effects of chronic dietary phytoestrogen exposure of the adult male rat on spermatogenesis and germ cell apoptosis. Sixteen male Wistar rats used in this study were offspring of females maintained on a low phytoestrogen diet prior to conception through to weaning. After weaning juveniles were fed the same diet into adulthood. Eight males were transferred to a high phytoestrogen diet for 24 days and subsequently testes were collected from all animals. Daily sperm production was significantly decreased in the high phytoestrogen fed animals. Stereological assessment of testes determined the number of spermatogonia and spermatocytes to be similar but post-meiotic spermatids to be significantly decreased in testes of high phytoestrogen fed rats. Immunohistochemistry determined that the high phytoestrogen
fed rats had significantly more active caspase-3 labeled seminiferous tubules. Furthermore, real-time PCR determined that expression of pro-apoptotic p53 was significantly increased whilst the anti-apoptotic Bcl-2 was significantly decreased in the high phytoestrogen group. These changes were accompanied by a significant decrease in estrogen receptor a. Androgen receptor and estrogen receptor b expression were not significantly different between the two treatment groups, nor were plasma gonadotrophin levels or testicular testosterone concentrations. In conclusion, exposure of the adult male rat to dietary phytoestrogens increases germ cell apoptosis, decreasing daily sperm production. This is probably mediated by disruption of steroid receptor expression.

 

NZ40

POLY(C) BINDING PROTEINS, -CP1 AND -CP2 MEDIATES STABILIZATION OF hTERT mRNA BY AUTOCRINE hGH
B. Starling Emerald1, Y. Chen2, T. Zhu2, Z. Zhu3, P.D. Gluckman1, K. Lee3, P.E. Lobie1
1Liggins Institute, University of Auckland, 2-6 Park Avenue, Private Bag 92019, Auckland, NZ
2Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore
3Department of Medicine, National University of Singapore 117609, Republic of Singapore

In the human mammary epithelial cell human growth hormone (hGH) gene expression is increased in proliferative lesions and the highest level of hGH gene expression occurs in cases of metastatic mammary carcinoma. To define the role and the molecular mechanism of hGH action in human mammary gland morphogenesis and neoplastic progression, we have analyzed the consequences of forced expression of hGH in human mammary epithelial cells. We herein demonstrate that autocrine hGH production in human mammary carcinoma cells results in increased telomerase activity as a result of specific upregulation of telomerase catalytic subunit (hTERT) mRNA and protein. This increase in hTERT gene expression is not due to increased transcriptional activation of the hTERT promoter but is the result of increased stability of hTERT mRNA exerted by CU rich cis-regulatory sequences present in the 3’UTR of TERT mRNA. Autocrine hGH upregulates two poly(C) binding proteins, _CP1 and _CP2, which bind to these cis-regulatory elements and stabilize hTERT mRNA. We have therefore demonstrated that post-transcriptional modulation of the level of hTERT mRNA is one mechanism for regulation of cellular telomerase activity.

 

NZ41

GENE EXPRESSION CHANGES DURING CARDIAC DEVELOPMENT AND THE DEVELOPMENT OF CARDIAC HYPERTROPHY IN NPR-1 KNOCKOUT MICE
L.J. Ellmers1, N.J.A. Scott1, J. Piuhola1,2, C.M. Frampton1, A.M. Richards1, V.A. Cameron1
1Cardioendocrine Research Group, Christchurch School of Medicine and Health Sciences, Christchurch, NZ
2Department of Pharmacology and Toxicology, Biocenter Oulu, University of Oulu, Finland

Atrial (ANP) and brain natriuretic peptide (BNP) protect the heart against the adverse changes during cardiac remodeling. The Npr-1 receptor mediates both ANP and BNP bioactivity, and mice lacking the Npr-1 gene exhibit cardiac remodeling. We have observed that Npr-1 knockout (KO) mice have decreased survival during embryogenesis, suggesting previously unrecognized roles for natriuretic peptides in fetal cardiac development. Cardiac anatomy and gene expression profiles were compared using cDNA microarray and quantitative real-time PCR of Npr-1 KO and wild-type (WT), male and female mice at embryonic 12.5 and 15.5 days post-coitum (dpc), neonatal day one, and 8-week and 6-month old adult mice (n=6 per group). Cardiac contractile responses to elevated ventricular stretch were studied in isolated adult hearts. Embryo Npr-1 KO mice had significantly enlarged hearts from 15.5 dpc (p<0.05). The mean arterial pressure was significantly increased by 32mmHg (p<0.001) in adult KO mice, and heart weight to body weight ratios were increased significantly (p<0.01) in all KO groups except in 6-month old males. Microarray analysis indicated altered gene expression of at least 3,000 genes in embryonic hearts (p<0.05), and 1460 genes differentially regulated (p<0.05) in adult KO versus WT mice. Differentially expressed genes included those involved in cardiac structure, developmental axis formation, transcriptional regulation, cell proliferation and hypertrophy, calcium signaling, structural proteins involved in muscle contraction, fibrosis and cell ion channels. These results demonstrate that natriuretic peptide signaling interacts with gene pathways regulating cardiac development during embryogenesis and the progression of cardiac hypertrophy in adult mice.

 

NZ42

MECHANO-GROWTH FACTOR (MGF) STIMULATES EXPRESSION OF BRAIN NATRIURETIC PEPTIDE (BNP) IN VIVO AND IN VITRO
V.A. Carpenter2, K.G. Matthews1, G.P. Devlin2, J.A. Jensen2, S.P. Stuart1, T.J. Watson1, P.H. Goldspink3, S.Y. Yang4, J.V. Conaglen2, J.J. Bass5, G. Goldspink4, C.D. McMahon1
1AgResearch Ltd, Hamilton, NZ
2Waikato Clinical School, Hamilton, NZ
3University of Illinois, Chicago, USA
4Royal Free and University College Medical School, London, UK
5Neuren Pharmaceuticals, Auckland, NZ

Serum concentrations of BNP are acutely increased after myocardial infarction (MI) and may act to unload the infarcted heart. The stimulus for BNP secretion is not known, but expression is induced by mechanical stretch. Interestingly, expression of MGF, a splice-variant of IGF-I, is also induced by mechanical stretch. We have previously shown that MGF improves the ventricular ejection fraction after MI. Therefore, we hypothesized that MGF induces expression and secretion of BNP, which, in turn, may have beneficial effects. In experiment 1, MI was induced by occluding the left circumflex coronary artery of sheep. Ewes received either saline, IGF-I, MGF E domain alone, or full MGF. Left ventricular function was studied before and after MI. Blood samples were collected for BNP assay. In experiment 2, H9C2 cardiomyocytes were treated with 100 and 300 ng of MGF for 30, 60, 120 and 180 min. After treatment, mRNA was extracted and reverse transcribed. PCR was then used to detect BNP mRNA. Cardiac ejection fraction was reduced by 40% (P<0.001) at d 1 in all sheep, but at d 6 improved by 4% in sheep treated with MGF (E or full peptide; at least P<0.05). Serum BNP concentration was increased in ewes treated with the E domain of MGF (P<0.05) and BNP mRNA increased in MGF-treated H9C2 cells. We conclude that the E domain of MGF stimulates BNP expression, which may have beneficial effects in the post-infarct heart.

 

NZ43

AN IMPAIRED ANTIOXIDANT MECHANISM IS ASSOCIATED WITH SKELETAL MUSCLE WASTING IN CANCER-BEARING RATS
F. Jeanplong, S.J. Falconer, J.M. Oldham, K.G. Matthews, C.D. McMahon
Functional Muscle Genomics, AgResearch Ltd, NZ

Progressive and uncontrolled wasting of skeletal muscle is termed cachexia and is often associated with cancer. Cachexia is associated with the overproduction of reactive oxygen species (ROS), which increase muscle protein oxidation, and proteolysis through the activation of the ATP-dependent ubiquitin-proteasome system. Antioxidant defenses may be overrun by ROS leading to a metabolic imbalance and a subsequent catabolic state. However, the underlying cellular mechanism that controls oxidative stress in cachectic skeletal muscle is poorly understood. We hypothesized that the induction of key antioxidant enzymes like superoxide dismutases (SODs) and catalase, implicated in the regulation of ROS-mediated muscle damage, are impaired in cancer cachexia. Male rats (3 months) were injected i.p. with the AH130 tumor and killed at day 0, 2, 4 and 6 (n=6 per group). At death, hind limb muscles (biceps femoris, gastrocnemius, plantaris, quadriceps femoris and soleus) were excised and weighed. All muscles lost between 15-20% of their mass compared to controls by day 6 (P<0.05). Messenger RNA levels of mitochondrial and cytosolic SOD were unchanged but the expression of extracellular SOD decreased (P<0.05) by day 6, as determined by real-time RT-PCR. Catalase mRNA expression was not altered except for a tendency for higher values at day 6. Using microarray gene expression profiling we have previously shown that type I collagen mRNA decreased up to 5-fold (P<0.05) at day 7 in skeletal muscles of AH130 tumor-bearing rats. These data suggest that the impaired regulation of SODs results in the accumulation of ROS in muscle cells and the extracellular matrix causing oxidative damage and possible fragmentation of type I collagen in AH130 cancer rats.

 

NZ44

GH INDUCES A POST-TRANSLATIONAL DECREASE IN MATURE MYOSTATIN IN MALE MICE
J.M. Oldham1, S.J. Falconer1, F. Jeanplong1, M. Sharma1, R. Kambadur1, H.K. Smith2, H.W. Davey3, R.J. Wilkins4, D.F. Gerrard3, J.C. Conaglen5, J.J. Bass6, C.D. McMahon1
1AgResearch Ltd, Hamilton, NZ
2University of Auckland, Auckland, NZ
3University of Otago, Dunedin, NZ
4University of Waikato, Hamilton, NZ
5Waikato Clinical School, Hamilton, NZ
6Neuren Pharmaceuticals, Auckland, NZ

Previous studies have shown that GH acts via the signal transducer Stat5B to regulate sexually dimorphic growth in mice. Myostatin is an inhibitor of skeletal muscle development and we have shown that the abundance of mature myostatin protein is lower in male than in female mice at maturity suggesting that myostatin also regulates sexually dimorphic growth. Furthermore, myostatin mRNA is higher in male than in female mice suggesting that there is a post-translational reduction in mature myostatin protein in males. Our objective was to determine if GH reduces the abundance of mature myostatin and increases myostatin mRNA in hypophysectomised male mice. Hypophysectomised mice were injected i.p. with 50 mg of GH and killed at 0, 30, 60 and 120 min. Skeletal muscles (Gastrocnemius and Q. femoris) were excised and protein and mRNA extracted. Myostatin mRNA, determined using real-time PCR, was reduced (P<0.05) in vehicle injected hypohysectomised mice, but was restored to that of sham operated controls 120 min after injection of GH. In contrast, abundance of mature myostatin protein was higher in vehicle injected hypophysectomised mice and was reduced to that of sham operated controls 120 min after injection of GH. We further showed that expression of myostatin mRNA was higher in male Stat5B-/- and wild-type mice, but only wild-type males have a lower abundance of mature myostatin protein. These data suggest that GH induces transcription of myostatin via a non-Stat5B-mediated pathway, while inducing a post-translation decrease in mature myostatin protein via Stat5B.

 

NZ45

MYOSTATIN NULL MICE LOSE MORE MUSCLE MASS DURING UNLOADING, BUT RECOVER FASTER THAN WILD-TYPE MICE
K.G. Matthews, J.M. Oldham, S.J. Falconer, F. Jeanplong1, H.K. Smith, J.J. Bass, C.D. McMahon
AgResearch Ltd, Hamilton, NZ
The University of Auckland, Auckland, NZ
Neuren Pharmaceuticals, Auckland, NZ

Myostatin null (Mstn-/-) mice lose more muscle mass than wild-type controls during hind-limb suspension (HLS). Our objective was to determine if Mstn-/- mice would also recover muscle mass faster than wild-type controls when reloaded after HLS. Eighteen Mstn-/- and 18 wild-type mice were subjected to HLS and killed at days 0, 2 and 7 of suspension (n=6 per group). A further group of 18 mice for each genotype were subjected to HLS for seven days, then re-loaded for 2, 4 and 7 days (n=6 per group). At death, muscles in the hind-limb (biceps femoris, gastrocnemius, soleus, plantaris and quadriceps femoris) were excised and weighed. Expression of myogenic regulatory factors (MyoD, Myf5, myogenin), ubiquitin ligases (atrogin-1, MuRF-1) and the proteasome subunit RC2 were assessed using real-time PCR. The mass of all dissected muscles was reduced to a greater extent (at least P<0.01) in Mstn-/- mice during HLS and recovered at a faster rate on reloading, when compared with controls. Expression of MRFs was similar in Mstn-/- and controls apart from day 2 of HLS when expression in Mstn-/- mice was 0.5-7% of controls (P<0.001). Expression of atrogin-1 and MuRF-1 was persistently lower in Mstn-/- mice that controls during HLS and reloading. These data suggest that impaired satellite cell function, rather than induction of the ubiquitin-proteasome pathway contributes to the extensive atrophy observed in Mstn-/- mice, while faster recovery of muscles with reloading results from activation of the larger pool of satellite cells in Mstn-/- muscles.

 

NZ46

VACCINATION AGAINST MYOSTATIN DOES NOT INCREASE POST-NATAL MUSCLE DEVELOPMENT IN SHEEP
T.S. Gray, S.J. Falconer, F. Jeanplong, S.P. Stuart, T. Watson, C.J. Berry, G.D. Nicholas, V.K. Siriett, E.J. Plummer, A.S. Hennebry, M. Sharma, R. Kambadur, C.D. McMahon
Functional Muscle Genomics, AgResearch Ltd, NZ

Myostatin inhibits development of skeletal muscle and this effect is most pronounced in Mstn-/- mice and in cattle in which naturally occurring mutations render the protein inactive. The objective of the current study was to determine if blockade of myostatin can increase post-natal development of skeletal muscle in sheep. Twenty Finn x Romney sheep were allocated to receive vehicle (Freunds adjuvant) or mature myostatin administered intramuscularly (400 mg in Freunds complete adjuvant) at weaning (three months), then at monthly intervals (200 mg in Freunds incomplete adjuvant) to seven months of age. Sheep were weighed every two weeks and a blood sample collected by venepuncture. Sheep were killed, the hot carcass weight was recorded and four skeletal muscles (B. femoris, Q. femoris, Gastrocnemius, Semitendinosus) were excised and weighed. Sheep vaccinated against myostatin developed an antibody titre that was 15000 fold higher than controls (P<0.001) and was maintained for the last three months of the study. However, vaccination against myostatin did not alter growth rate or body mass. Furthermore, muscle mass was either unchanged or, in the case of Semitendinosus, reduced (P<0.05). In addition, myostatin mRNA was quantified using real-time PCR and found to be reduced (P<0.05) in the Semitendinosus of vaccinated sheep compared with controls. These data are consistent with non-specific immunopotentiation, whereby antibodies prolonged, rather than neutralised the activity of myostatin. These data suggest that vaccination against myostatin is ineffective at increasing post-natal muscle development in intact, male sheep during the linear growth phase.

 

NZ47

METABOLIC RISK FACTORS FOR DIABETES IN PACIFIC ISLAND TEENAGERS
A.M. Grant1, K.A. McAuley2, R.W. Taylor3, F. Taungapeau4, S.M. Williams5, M. Waldron3, A. Duncan3, M. Harper3, A. Goulding1
1Department of Medical and Surgical Sciences, University of Otago, Dunedin, NZ
2The Edgar National Centre for Diabetes Research, University of Otago, Dunedin, NZ
3Department of Human Nutrition, University of Otago, Dunedin, NZ
4Otago Pacific Peoples Health Trust, NZ
5Department of Preventive and Social Medicine, University of Otago, Dunedin, NZ

Type 2 diabetes is prevalent in Pacific Islanders but little is known regarding risk factors for this disorder in adolescents. We are studying risk factors for metabolic syndrome in 80 Pacific Islanders (40 males, 40 females) aged 15-18 yr. Results obtained on the first 38 participants will be presented. Heights, weights, waist girths and blood pressure were measured. Body mass index (BMI) was calculated. Fasting bloods were analysed for glucose, insulin and lipids, and an oral glucose tolerance test was performed. Body composition was evaluated by dual energy-x-ray absorptiometry. High adiposity (BMI and fat percentage) was present in 31and 26 subjects, respectively. Three participants had BMI values > 40. Two girls had fasting blood glucose levels above 7.0 mmol/l (international criteria for type 2 diabetes); l7 subjects had elevated fasting insulin levels and 5 participants reported acanthosis nigricans. Two subjects had raised systolic blood pressure. These results confirm worldwide concerns regarding the rising incidence of Type 2 diabetes in overweight adolescents (1). There is an urgent need to implement strategies to reduce diabetes risk in young Pacific Islanders living in NZ
(1) Viner RM, Sega TY, Lichtarowicz-Krynska E, & Hindmarsh P (2005) Archives of Disease in Childhood 90: 10-14.

 

NZ48

THE INFLUENCE OF OBESITY ON MORTALITY IN PEOPLE WITH TYPE-2 DIABETES IN THE UK
R.A. Lawrenson1, H.E. Mulnier2, H.E. Seaman2, V.S. Raleigh2, S.S. Soedamah-Muthu3, H.M. Colhoun4
1Waikato Clinical School, University of Auckland, NZ
2University of Surrey, Guildford, UK
3University Medical Center Utrecht, Urtecht, Holland
4Conway Institute of Biomolecular and Biomedical Research, University College, Dublin, Ireland

Death certificates under-report diabetes as a contributory factor, resulting in unreliable estimates of mortality due to diabetes. The influence of obesity on mortality in type 2 diabetes is not well-documented. We aimed to study all cause mortality and the influence of obesity and smoking on mortality in patients with type 2 diabetes in a large cohort selected from the General Practice Research Database (GPRD).Methods. A cohort of 44,230 patients with type 2 diabetes aged 35 to 89 years in 1992 was identified. A comparison group of 219,797 matched by year of birth and sex with no record of diabetes at any time was randomly selected. Hazards ratios (HRs) for all-cause mortality during the period January 1992 – October 1999 were calculated using the Cox Proportional Hazards Model. The effects of body mass index (BMI), smoking and duration of diabetes on all-cause mortality amongst people with diabetes was assessed (n=28,725) using logistic regression.Results The HR for all-cause mortality in type 2 diabetes compared with no diabetes was 1.93 (95% CI 1.89,1.97). The HR in men was 1.77 (95% CI 1.72,1.83) and in women 2.13 (95% CI 2.06,2.20). The HR decreased with increasing age. In the multivariate analysis which included only those with diabetes the HR for all-cause mortality amongst smokers was 1.50 (95% CI 1.41,1.61), and using patients with a BMI 20-24 as the reference group, in those with a BMI 25-29 the HR was 0.97 (95% CI 0.91,1.03), BMI 30-34 HR 1.13 (95% CI 1.04,1.22), BMI 35-54 the HR was 1.43 (95% CI 1.28,1.59) and for those with a BMI 15-19 the HR was 1.38 (95% CI 1.18,1.61). Conclusions Patients with type 2 diabetes have almost double the mortality rate compared with those without. The relative risk decreases with age. In people with type 2 diabetes, obesity and smoking both contribute to the risk of all-cause mortality supporting doctrines to stop smoking and lose weight.

 

NZ49

LONG TERM EFFECTS OF POPULAR DIETARY APPROACHES ON WEIGHT LOSS AND FEATURES OF INSULIN RESISTANCE
K.A. McAuley1, K.J. Smith2, R.W. Taylor2, R.T. McLay2, S.M. Williams3, V.L. Farmer1, J.I. Mann1,2
1Edgar National Centre for Diabetes Research, University of Otago, Dunedin, NZ
2Department of Human Nutrition, University of Otago, Dunedin, NZ
3Department of Preventive and Social Medicine, University of Otago. Dunedin, NZ

High carbohydrate-high fibre diets are recommended for weight loss and for treating and preventing diseases such as diabetes and cardiovascular disease. There is, however, widespread interest in alternative dietary approaches. We report a randomised trial comparing high fat and high protein diets with the conventional approach. Ninety-three overweight insulin resistant women received advice following randomisation to high fat (HF), high protein (HP) or high carbohydrate (HC) dietary regimes, to achieve weight loss followed by weight maintenance over 12 months. Weight, body composition and measures of carbohydrate and lipid metabolism were investigated. Retention rates were 93% for HP and 75% for both HC and HF. Features of the metabolic syndrome improved in all groups during the first 6 months, but to a greater extent on HF and HP than HC (1). During the second 6 months, the HF group had increases in waist circumference (mean difference 4.4 cm [95% CI 3.0, 5.8]), fat mass (2.3 kg [1.5, 3.1]), triglycerides (0.28 mmol/L [0.09, 0.46]) and 2-hour glucose (0.70 mmol/L [0.22, 1.18]). Overall, there was substantial sustained improvement in waist circumference, triglycerides and insulin in the HP group and sustained but more modest changes on HC (2). HP and HC approaches appear to be appropriate options for insulin resistant individuals. HF diets are not advisable in the long term.
(1) McAuley KA, Hopkins CM et al. (2005) Diabetologia 48(1): 8-16.
(2) McAuley, KJ Smith et al. (September 13, 2005) International Journal of Obesity. Advance online publication;doi:10.1038/sj.ijo.0803075

 

NZ50

WEIGHTY PROBLEMS OF DIABETIC PATIENTS ENROLLED ON THE OTAGO DIABETES REGISTER
K.J. Coppell1,2, K. Anderson1 and C. Booker2
1Otago Diabetes Project, Dunedin, NZ
2Edgar National Centre for Diabetes Research, University of Otago, Dunedin, NZ

To describe weight and weight-related problems amongst type 2 diabetic patients enrolled on the Otago Diabetes Register. Data were extracted from the Otago Diabetes Register, established in 1998 to monitor diabetes care in the Otago region, NZ Diabetes-related data for enrolled patients were collected annually from general practices. For the 2004 review year 1,013 type 2 diabetic patients aged <55 years at diagnosis were compared with 1,704 diagnosed at >55 years. Overall, since 1998 mean weight increased significantly for both men and women, from 86.9 to 89.0kg (p<0.001) and 77.9 to 78.5kg (p<0.001), respectively. In 2004, 59% of patients aged <55 years at diagnosis were obese (BMI>30) compared with patients aged >55 years at diagnosis (39%). Risks of hypertension, dyslipidaemia, ischaemic heart disease and retinopathy were higher amongst obese compared with normal weight patients in both age groups. (Table 1) The risks were highest for obese patients aged <55 years at diagnosis.

Table 1: Risk of hypertension, dyslipidaemia and selected diabetes complications amongst obese compared with normal weight type 2 diabetic patients aged <55 or >55 years at diagnosis, 2004

<55 years at diagnosis (n=597)

>55 years at diagnosis (n=656)

OR

95% CI

p-value

OR

95% CI

p-value

Hyertension	3.39	2.00-5.74	<0.001	1.76	1.27-2.43	0.001
Dyslipidaemia	1.62	0.97-2.72	0.066	1.43	1.05-1.94	0.023
CVA/TIA	1.07	0.46-2.51	0.872	1.02	0.66-1.58	0.919
IHD	2.56	1.36-4.80	0.004	1.31	0.96-1.78	0.088
PVD	1.91	0.76-4.80	0.166	0.86	0.52-1.42	0.560
Retinopathy	1.82	1.00-3.30	0.050	1.05	0.70-1.57	0.817
Periph neuropathy	1.58	0.73-3.40	0.243	1.01	0.62-1.63	0.976
Overt nephropathy	2.57	0.83-7.98	0.104	0.81	0.39-1.69	0.580

  Adjusted for age, gender and duration of diabetes

Obesity is more prevalent amongst diabetic patients diagnosed at a young age. These patients are at high risk of complications. Avoidance of obesity offers the most likely approach to prevent early onset type 2 diabetes and its complications.

 

NZ51

OUTCOMES AFTER SEVEN YEARS FOR A COHORT OF INDIVIDUALS WITH TYPE-2 DIABETES: A RETROSPECTIVE OBSERVATIONAL STUDY
C.S. Booker1, K.J. Coppell1,2, K. Anderson2, S.M. Williams3
1Edgar National Centre for Diabetes Research, University of Otago, Dunedin, NZ
2Otago Diabetes Project, Dunedin, NZ
3Department of Preventive and Social Medicine, University of Otago, Dunedin, NZ

The Otago Diabetes Register was established in 1998 as part of a regional project to improve diabetes care in Otago, NZ Data including diabetes complications, clinical measures and biochemistry tests were collected annually from general practice records for enrolled patients. Death certificate information was also added to the register. The aim of this study was to describe outcomes after seven years for the cohort of 1478 patients with Type 2 diabetes who enrolled in 1998. Mean (SD) age at enrolment was 67.0 (11.8) years and mean duration of diabetes was 8.2 (7.6) years. By 2004, 22.4% were deceased and 5.8 % had moved out of the region. Data for 1,023 patients who were alive and resident in Otago during the seven years of follow-up were analysed. Amongst these individuals diabetes medication increased in intensity; the proportion using diet only therapy decreased from 29.3% to 18.0%, and the proportion using both insulin and an oral hypoglycaemic agent increased from 4.0% to 13.5%. Most clinical and biochemical measures improved over the 7 years, probably reflecting the increased use of antihypertensive and lipid lowering medication, while mean (SD) HbA1c increased from 7.3 (1.8)% to 7.7 (1.5)%. As expected the prevalence of diabetes complications increased with increasing duration of diabetes. This descriptive study confirms the progressive nature of diabetes, and that the prevalence of complications increases with duration of diabetes. The continuing decline in glycaemic control observed in those remaining alive throughout follow-up presents an ongoing challenge for diabetes management.

 

NZ52

ADIPOSITY REBOUND AND EARLY GROWTH
S.M. Williams
Department of Preventive and Social Medicine, University of Otago, Dunedin, NZ

The timing of adiposity rebound, or the age in childhood at which body mass index (BMI) reaches its nadir, is a recognised risk factor for obesity in later life. As rebound occurs when the ratio of the log transformed values of weight velocity and height velocity is 2 its timing depends on one or both of these. Data from a cohort of children born in Dunedin in 1972-73, seen at 2 yearly intervals between ages 3 and 11y showed that the mean age of adiposity rebound was 6.6 y (sd=1.10) for boys and 6.0 y (sd=1.21) for girls. Correlations between the timing of rebound and estimated weight and height velocity at age 3 y after adjusting for sex were –0.48 and –0.00, showing that early rebound depended on higher weight velocity, measured in percentage terms, rather than height velocity. The results also showed that weight velocity at age 3 y was more variable than height velocity, suggesting that it was less well controlled. Restraining early weight gain could delay adiposity rebound and prevent obesity in early adulthood.

 

NZ53

A NOVEL MODEL FOR STUDYING THE EFFECTS OF PIGMENTATION GENES ON BODY WEIGHT
C.E. Duchesnes1,2, J. Naggert4, M. Tatnell1, N. Beckman5, M. Mc Gregor5, R. Elliott5, K.G. Mountjoy1,2
1Department of Physiology, The University of Auckland, Auckland, NZ
2Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, NZ
5Diatranz NZ Ltd, Auckland, NZ
4The Jackson Laboratory, Bar Harbor, USA

A very large, yellow coat coloured mouse arose spontaneously during a programme of selection for body size in a breeding colony at Ruakura, NZ in the 1980s. We have established a colony and named the strain NZ Ginger (NZG). A comparison of NZG mice with other strains revealed that their body weights at 50 days exceed those of obese dominant agouti (AVY) mice: NZG male 41.77 ± 3.50 and female 32.85 ± 2.29; AVY male 29.86 ± 3.50 and female 28.11 ± 3.18; and the increased body weight (BW) appears to be attributable to increased muscle mass. Furthermore, NZG mice fed a high fat diet (45% of calories from fat) for 18 weeks are susceptible to developing obesity with partial insulin resistance. Cross breeding of NZG with CAST/Ei mice to map genes contributing to BW and pigmentation, demonstrated that BW of NZG mice correlates with coat colour and both BW and coat colour are polygenic phenotypes. Fine mapping narrowed our genome scan to the pink-eyed gene locus and expression studies of genes located within this locus revealed that only pink-eyed expression was impaired in NZG mice. Pink-eyed gene is expressed in the skin and brain of C57/BL6 but not NZG mice. The absence of pink-eyed, a pigmentation gene, in NZG mice most likely contributes to the ginger coat colour and we are investigating whether it also contributes to increased BW. We propose NZG mice as a model to understand the molecular relationships between pigmentation genes and BW.

 

NZ54

HOW IS THE ARRIVAL OF FOOD SIGNALLED TO NUTRITION-SENSITIVE TISSUES? AN INVESTIGATION IN RAT MAMMARY TISSUE
K.W. Stewart1, G.J.S. Cooper2, S.R. Davis3
1School of Science & Primary Industries, Waikato Institute of Technology, NZ
2School of Biological Sciences, The University of Auckland, Auckland, NZ
3AgResearch Ruakura, Hamilton, NZ

Milk production during lactation has been demonstrated to be acutely sensitive to availability of food in many mammals such as ruminants and rodents. Although this has been known for many years, the pathways by which nutritional status is signalled to the mammary glands and the metabolic processes targeted by these pathways have not been clearly identified. In this study, both in vivo and in vitro methods were used to examine nutrient signalling in rat mammary tissue. Accurate methods for measuring blood flow and arteriovenous difference in glucose were applied for the first time to the inguinal mammary glands of anaesthetised rats at peak lactation to investigate mechanisms by which the return of food to 18 h-food-deprived rats was signalled to the mammary glands to stimulate rapid restoration of milk production. After 1 h of refeeding, glucose supply to the glands had returned to the fed level but glucose uptake had increased to a level 60% higher (n=7). In further in vitro experiments, treatment with a gut extract and insulin both enhanced uptake of glucose by acini from food-deprived lactating rats. The major conclusions from these studies were that insulin and a factor from the gut are likely to play roles in signalling to the mammary gland that nutrient supply has been restored after re-feeding of food-deprived rats. However, neither the glucose supply itself nor the major nerve supplying the mammary gland appeared to mediate this signalling.

 

NZ55

DETERMINING OPTIMAL APPROACHES FOR SUCCESSFUL MAINTENANCE OF WEIGHT LOSS
K.S. Whiteford1, K.A. McAuley1, J.I. Mann1, R.W. Taylor1, A. Chisholm1, S. Vorgers1, S. Williams2
1Department of Human Nutrition, University of Otago, Dunedin, NZ
2Department of Preventive and Social Medicine, University of Otago, Dunedin, NZ

Although short term weight loss is often achievable in overweight individuals, long term maintenance is generally poor. Information is urgently required regarding optimum macronutrient composition and method of delivery for dietary regimes aimed at weight maintenance. A 2x2 factorial study design was used to determine the most cost effective programme and most appropriate macronutrient composition in order to maintain weight loss over a 2-year period. Two hundred women who had recently lost at least 5% of initial body weight were randomised into one of two support programmes. One programme provided intensive health professional support in combination with regular circuit training classes. The other programme provided brief and frequent ‘weigh-ins’ and support by a research nurse with peer group support meetings. Participants were also randomised to one of two diets with varying macronutrient composition. One was a high carbohydrate, high fibre, low glycaemic index diet, which promoted fruit, vegetables, wholegrain breads and cereals and legumes. The other was relatively high in monounsaturated fat and protein and low in glycaemic load, which promoted fruit, vegetables and moderate intakes of nuts, avocado, olive oil, fish, lean poultry and lean red meat. Baseline and 1-year data will be presented.

 

NZ56

A NOVEL ANTI-OBESITY DRUG PERMANENTLY RESETS BODY WEIGHT ‘SET-POINT’
K.G. Mountjoy1,2, M. Tercel3, C. Duchesnes1,2, R. Marnane1, M. Tatnell1, W.A. Denny3, W.R. Wilson3 1Department of Physiology, The University of Auckland, Auckland, NZ
2Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, NZ
3Auckland Cancer Society Research Centre, FMHS, The University of Auckland, Auckland, NZ

During routine testing of a class of potential anticancer drugs we observed that one analogue caused animals to initially lose up to 15% of their body weight, and maintain this body weight for the length of the study (160 days post injection). Subsequently, we confirmed similar responses to this drug in 4 different strains of mice (C3HeN, AVY, a/a, NZG) approximately 50-70 days old, following a single ip injection of either DMSO (control) or drug in DMSO. Both food and water intake transiently increased 20-50 days post drug injection and then intake of both was similar between control and drug treated mice despite the drug treated animals weighing significantly less than control mice. Autopsy at 160 days revealed striking reductions in abdominal fat mass, normal histology for liver, pancreas and kidney, markedly reduced serum leptin, normal serum insulin and glucose, and reduced serum triglycerides. In liver and fat tissue we observed induction of several genes involved in fatty acid synthesis (Fasn, PPAR__ Scd1, Dgat1) but no corresponding changes were observed in skeletal muscle. Thus the drug treated mice appear to be making fat and then clearing fat. Paradoxically, the drug treated mice exhibited decreased hypothalamic expression of a gene that decreases appetite (POMC) and increased expression of 2 genes (NPY and AGRP) that increase food intake. These data suggest that the drug has permanently reset body weight ‘set-point’ since the drug treated mice ate as much as control mice despite weighing less than control mice.

 

NZ57

CHRONIC PROLACTIN INFUSION INDUCES CENTRAL LEPTIN RESISTANCE IN PSEUDOPREGNANT RATS
R.A. Augustine, S.J. Bunn, D.R. Grattan
Department of Anatomy and Structural Biology, and Centre for Neuroendocrinology, University of Otago, Dunedin, NZ

Pregnancy in the rat is characterised by hyperphagia, increased fat deposition and a rise in plasma leptin, with central leptin resistance developing at midpregnancy. Pseudopregnant rats are also hyperphagic but do not become leptin resistent, even when given progesterone implants to extend pseudopregnancy beyond the time that leptin resistance develops during pregnancy. Progesterone implants extend the twice-daily prolactin surges observed in both pseudopregnant rats and in the first half of pregnancy. In pregnancy, however, the surges of prolactin are superceded by rising levels of placental lactogen, which inhibits the prolactin surges by negative feedback and then remains elevated throughout the remainder of pregnancy. Therefore, our hypothesis was that chronically high lactogen levels induce changes in the pregnant rat brain that cause leptin resistance. Pseudopregnant rats received subcutaneous progesterone implants, along with an Alzet miniosmotic pump that delivered either aCSF or ovine prolactin (oPRL) into the lateral ventricle. Jugular cannulae were implanted for collection of blood samples (4x daily) and plasma rat prolactin measured by radioimmunoassay. oPRL infusion suppressed endogenous prolactin surges in a manner similar to that caused by placental lactogen during pregnancy. oPRL/aCSF-infused pseudopregnant rats were fasted overnight, injected with aCSF or leptin (4 _g) one hour before lights off, and food intake was measured three and 24 hours later. Leptin treatment significantly reduced food intake in aCSF-treated pseudopregnant rats. By contrast, oPRL-treated pseudopregnant rats did not respond to a central leptin injection, eating equivalent amounts to controls. These data demonstrate that chronic elevation in prolactin induces leptin resistance in pseudopregnant rats, and suggests that the leptin resistance of pregnancy may be induced by prolonged secretion of placental lactogen.

 

NZ58

MICE LACKING a-MSH DEVELOP ADRENAL GLANDS, BUT ARE OBESE, AND RETAIN THE
ABERRANT FAT METABOLISM SEEN IN POMC NULL MICE
J.L. Costa, S.L. Forbes, M.B. Brennan
Department of Biology, University of Denver, USA

Alpha-melanocyte stimulating hormone (_-MSH) is a 13-amino acid peptide which is embedded within the sequence of the 39-amino acid adrenocorticotropin (ACTH). This unique structure, arising in the pro-opiomelanocortin gene and pre-protein, is preserved from the earliest chordate lineages studied (1). Indeed, ACTH and _-MSH have been linked through evolution and play significant roles in coordinating the hypothalamic-pituitary-adrenal (HPA) axis in higher organisms; this role has been indeterminate and undeterminable before the creation of the POMC null mouse, but confounded by their numerous phenotypes (2, 3). The creation of a mouse that possesses functional ACTH but not _-MSH refines this relationship to a degree not previously possible. _-MSH null mice are similar to POMC null mice in that they are obese, hyperphagic, and have aberrant fat metabolism. However, _-MSH null mice, unlike POMC null mice, possess functional adrenal glands and thereby produce corticosterone. These results are the first in the dissection of _-MSH away from ACTH and the other POMC derived peptide hormones, and helps to elucidate their individual functions in the HPA axis.
(1) Takahashi, A., et al., Evolutionary significance of proopiomelanocortin in agnatha and chondrichthyes. (2001) Comp Biochem Physiol B Biochem Mol Biol, 129(2-3): p. 283-9.
(2) Yaswen, L., et al., Obesity in the mouse model of pro-opiomelanocortin deficiency responds to peripheral melanocortin [see comments] (1999) Nat Med, 5(9): p. 1066-70.
(3) Forbes, S., et al., Integrated control of appetite and fat metabolism by the leptin- proopiomelanocortin pathway (2001) Proc Natl Acad Sci U S A, 98(7): p. 4233-7.

NZ59

CHANGES IN SEX STEROID LEVELS INDUCE SOCS EXPRESSION AND PROLACTIN SECRETION DURING LATE PREGNANCY
F.J. Steyn, G.M. Anderson, D.R. Grattan
Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago, Dunedin, NZ

During late pregnancy, tuberoinfundibular-dopaminergic (TIDA) neurons that normally regulate prolactin release via negative feedback become unresponsive to prolactin resulting in hyperprolactinemia. Suppressors of cytokine signalling (SOCS) proteins negatively regulate prolactin signal transduction. During late pregnancy levels of SOCS mRNA expression are significantly elevated, coinciding with decreased TIDA activity and an antepartum prolactin surge; therefore we hypothesise that the loss of ability of prolactin to stimulate TIDA activity may be due to elevated levels of SOCS. The aim of the present study was to determine specifically what hormonal changes lead to the induction of SOCS mRNA. In particular, we aimed to determine whether rising oestradiol and/or the rapid decline in progesterone, characteristic of late pregnancy, induces SOCS expression in the arcuate nucleus. Animals were ovariectomised on day 18 of pregnancy and treated with a regime of either or both estradiol and progesterone subcutaneous implants (designed to mimic late pregnancy hormonal levels). Compared to control animals, the timing of parturition and the antepartum prolactin surge remained normal in oestrogen and progesterone treated animals when progesterone was removed on day 21. In the absence of progesterone, oestrogen significantly advanced parturition and the antepartum prolactin surge. No surge was observed the absence of oestrogen or when progesterone treatment was maintained beyond day 21. Results suggest that the timing of the antepartum prolactin surge is dependent on the presence of oestrogen and absence of progesterone. We are currently using this experimental paradigm to investigate if oestrogen in the absence of progesterone causes the reduction in TIDA activity and the elevation of SOCS mRNA levels normally seen during late pregnancy.

 

NZ60

A MOUSE MODEL OF HYPERPROLACTINAEMIA-INDUCED INFERTILITY
D.R. Grattan1, G.M. Anderson1, A.E. Herbison2
1Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago, Dunedin, NZ
2Centre for Neuroendocrinology and Department of Physiology, University of Otago, Dunedin, NZ

Hyperprolactinaemia causes infertility. While the mechanisms are largely unknown, they involve decreased pulsatile secretion of luteinizing hormone (LH), likely mediated through reduced activity of gonadotrophin-releasing hormone (GnRH) neurons in the hypothalamus. Prolactin action on GnRH neurons has been relatively uninvestigated, however, because of the difficulty in monitoring activity of these neurons in vivo. The aim of the present study was to determine whether prolactin influences gonadotrophin secretion and GnRH neurons in mice, in order develop a model that would allow use of novel transgenic approaches to investigate prolactin effects on GnRH neurons in vivo. Adult female C57/B6-J mice were ovariectomised and treated with a subcutaneous implant maintaining low physiological levels of estradiol (OVX+E). In the first experiment, an osmotic minipump was implanted to deliver ovine prolactin (oPRL) or vehicle into a lateral ventricle. Seven days later, mice were bled and serum LH measured by radioimmunoassay. In a second experiment, mice received twice daily injections of oPRL (50 µg, sc) or vehicle for 48 hours, then were perfusion fixed and brains processed for immunohistochemistry. Phosphorylation of CREB (pCREB) was used as a marker for activation of signaling in GnRH neurons. GnRH and pCREB were detected using specific antibodies. oPRL treatment significantly suppressed LH in OVX+E mice (1.9±0.6 ng/ml compared with 5.0±1.3 mg/ml, P<0.05). Under control conditions, expression of pCREB in GnRH neurons was low (2.2±0.33%). oPRL induced in a 4-fold increase in pCREB expression (9.82±3.70%, p<0.05). These data demonstrate that chronic prolactin acts on GnRH neurons in the hypothalamus and suppresses pituitary gonadotrophin secretion. These data are consistent with the hypothesis that prolactin inhibits reproduction through an action on GnRH neurons.

 

NZ61

PROLACTIN FEEDBACK MAY BE IMPORTANT FOR ENKEPHALIN INDUCTION IN TUBEROINFUNDIBULAR DOPAMINERGIC NEURONS DURING LATE PREGNANCY AND LACTATION
I.C. Kokay, J.M. Pilcher, D.R. Grattan
Department of Anatomy and Structural Biology and Centre for Neuroendocrinology, University of Otago, Dunedin, NZ

The secretion of prolactin from the pituitary gland is primarily controlled by dopamine released from tuberoinfundibular dopaminergic (TIDA) neurons located in the hypothalamus. During late pregnancy and lactation, however, the activity of TIDA neurons is suppressed and the neurons appear to be less responsive to the direct feedback actions of prolactin. This allows a hyperprolactinemic state to develop. Our previous studies have shown that during lactation, there is a marked increase in the number of non-dopaminergic neurons in the arcuate nucleus that express prolactin receptor mRNA. As this is paralleled by a dramatic rise in enkephalin production in TIDA neurons, we investigated by double label in situ hybridisation, the co-expression of PRL-R mRNA in enkephalin mRNA-expressing neurons in groups of non-pregnant, pregnant and lactating rats. In parallel sets of sections, co-expression of enkephalin mRNA in TIDA neurons was quantified. Our results show that in all three groups, the majority (>80 %) of enkephalin-producing cells expressed PRL-R mRNA, and, during late pregnancy and lactation, when circulating levels of prolactin are high, there was a significant increase in the overall number of cells that co-expressed PRL-R and enkephalin mRNA. In these latter two groups, the majority of enkephalin-positive neurons also were double-labelled for TH mRNA, identifying them as TIDA neurons. These results indicate that although TIDA neurons do not respond to the high PRL levels characteristic of late pregnancy and lactation with an increase in dopamine production, they continue to express PRL-Rs. It is possible that PRL feedback may be important for the upregulation of enkephalin observed in these neurons under these conditions.

 

NZ62

PLACENTAL LYSOPHOSPHOLIPIDS – THE MISSING LINK OF PARTURITION?
J. Keelan
Liggins Institute and Department of Pharmacology and Clinical Pharmacology, The University of Auckland, Faculty of Medical and Health Science, Auckland, NZ

The timing of human parturition is thought to be determined primarily by increases in local concentrations of uterotonic factors, such as prostaglandins (PGs), in combination with changes in uterine quiescence and receptivity as term approaches. Pathological disturbance of these processes, for example in the presence of intrauterine inflammation or haemorrhage, results in preterm labour and birth. However, while PGs have been identified as important initiators of labour, the paracrine and autocrine interactions between the uterus and placental tissues are complex and incompletely understood, and the causes of preterm labour are in many cases obscure. Finally, the identity of the mechanism(s) which coordinate fetal maturity with the timely onset of parturition are the subject of considerable debate. Lysophospholipids such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are potent bioactive lipid mediators generated within intrauterine tissues and fluids by the enzyme lysophospholipase-D, which is secreted by placental trophoblast and fetal membrane tissues. Levels of this enzyme in the maternal circulation increase with gestational age as placental size and maturity increases. LPA and S1P, acting via specific receptors within the uterus, induce rho kinase activation and increase myometrial contractility. LPA and S1P also stabilise myometrial oxytocin receptor mRNA and heighten uterine responsiveness to oxytocin, an important uterotonic hormone. S1P increases uterine PGE2 production, and also stimulates chemokine release and leukocyte recruitment / activation. Finally, LPA and S1P are also generated locally by inflammatory activation or platelet activation, processes known to be associated with preterm birth. Lysophospholipids may therefore be the long sought paracrine / endocrine regulators of human parturition, linking placental size & maturity and various pathologies with the onset of both term and preterm labour.