Michelle L. Johnson

Glucagon-like peptide-1 (GLP-1) increases in plasma and colon tissue prior to estrus and circulating levels change with increasing age in reproductively competent Wistar rats

HIGHLIGHTSIncreased GLP‐1 levels were found in plasma and colon tissue during proestrus.Reduced stomach contents were recorded on the morning of estrus.High GLP‐1 at proestrus may indicate increased satiety leading into estrus.Plasma and colon PYY unchanged during estrous cycle; possible differential PYY/GLP‐1 secretion.Increasing age affects GLP‐1 regulatory control in reproductively competent females. ABSTRACT There is a well‐documented association between cyclic changes to food intake and the changing ovarian hormone levels of the reproductive cycle in female mammals. Limited research on appetite‐controlling gastrointestinal peptides has taken place in females, simply because regular reproductive changes in steroid hormones present additional experimental factors to account for. This study focussed directly on the roles that gastrointestinal‐secreted peptides may have in these reported, naturally occurring, changes to food intake during the rodent estrous cycle and aimed to determine whether peripheral changes occurred in the anorexigenic (appetite‐reducing) hormones peptide‐YY (PYY) and glucagon‐like peptide‐1 (GLP‐1) in female Wistar rats (32–44 weeks of age). Total forms of each peptide were measured in matched fed and fasted plasma and descending colon tissue samples for each animal during the dark (feeding) phase. PYY concentrations did not significantly change between defined cycle stages, in either plasma or tissue samples. GLP‐1 concentrations in fed plasma and descending colon tissue were significantly increased during proestrus, just prior to a significant reduction in fasted stomach contents at estrus, suggesting increased satiety and reduced food intake at this stage of the cycle. Increased proestrus GLP‐1 concentrations could contribute to the reported reduction in food intake during estrus and may also have biological importance in providing the optimal nutritional and metabolic environment for gametes at the potential point of conception. Additional analysis of the findings demonstrated significant interactions of ovarian cycle stage and fed/fasted status with age on GLP‐1, but not PYY plasma concentrations. Slightly older females had reduced fed plasma GLP‐1 suggesting that a relaxation of regulatory control of this incretin hormone may also take place with increasing age in reproductively competent females.

Plasma Ghrelin Concentrations Were Altered with Oestrous Cycle Stage and Increasing Age in Reproductively Competent Wistar Females

Changes in appetite occur during the ovarian cycle in female mammals. Research on appetite-regulatory gastrointestinal peptides in females is limited, because reproductive changes in steroid hormones present additional experimental factors to control for. This study aimed to explore possible changes in the orexigenic (appetite-stimulating) gastrointestinal peptide hormone ghrelin during the rodent oestrous cycle. Fed and fasted plasma and stomach tissue samples were taken from female Wistar rats (32–44 weeks of age) at each stage of the oestrous cycle for total ghrelin quantification using radioimmunoassay. Sampling occurred during the dark phase when most eating takes place in rats. Statistical analysis was by paired-samples t-test, one-way ANOVA on normally distributed data, with Tukey post-hoc tests, or Kruskal-Wallis if not. GLM univariate analysis was used to assess main effects and interactions in ghrelin concentrations in the fed or fasted state and during different stages of the ovarian cycle, with age as a covariate. No consistent fed to fasted ghrelin increases were measured in matched plasma samples from the same animals, contrary to expectations. Total ghrelin concentrations did not significantly change between cycle stages with ANOVA, in either fed or fasted plasma or in stomach tissue. This was despite significantly decreased fasted stomach contents at oestrus (P = 0.028), suggesting decreased food intake. There was however a significant interaction in ghrelin plasma concentrations between fed and fasted proestrus rats and a direct effect of age with rats over 37 weeks old having lower circulating concentrations of ghrelin in both fed and fasted states. The biological implications of altered ghrelin plasma concentrations from 37 weeks of age are as yet unknown, but warrant further investigation. Exploring peripheral ghrelin regulatory factor changes with increasing age in reproductively competent females may bring to light potential effects on offspring development and nutritional metabolic programming.

Improved timed-mating, non-invasive method using fewer unproven female rats with pregnancy validation via early body mass increases

For studies requiring accurate conception-timing, reliable, efficient methods of detecting oestrus reduce time and costs, whilst improving welfare. Standard methods use vaginal cytology to stage cycle, and breeders are paired-up using approximately five proven females with proven males to achieve at least one conception on a specific day. We describe an alternative, fast, consistent, non-invasive method of timed-mating using detection of lordosis behaviour in Wistar and Lister-Hooded rats that used unproven females with high success rates. Rats under reverse lighting had body masses recorded pre-mating, day (d) 3–4, d8, d10 and d18 of pregnancy. Using only the presence of the oestrus dance to time-mate females for 24 hours, 89% of Wistar and 88% of Lister-Hooded rats successfully conceived. We did not observe behavioural oestrus in Sprague-Dawleys without males being present. Significant body mass increases following mating distinguished pregnant from non-pregnant rats, as early as d4 of pregnancy (10% ± 1.0 increase cf. 3% ± 1.2). The pattern of increases throughout gestation was similar for all pregnant rats until late pregnancy, when there were smaller increases for primi- and multiparous rats (32% ± 2.5; 25% ± 2.4), whereas nulliparous rats had highest gains (38% ± 1.5). This method demonstrated a distinct refinement of the previous timed-mating common practice used, as disturbance of females was minimised. Only the number required of nulli-, primi- or multiparous rats were mated, and body mass increases validated pregnancy status. This new breeding management method is now established practice for two strains of rat and has resulted in a reduction in animal use.

A Bayesian view of murine seminal cytokine networks

It has long been established that active agents in seminal fluid are key to initiating and coordinating mating-induced immunomodulation. This is in part governed by the actions of a network of cytokine interactions which, to date, remain largely undefined, and whose interspecific evolutionary conservation is unknown. This study applied Bayesian methods to illustrate the interrelationships between seminal profiles of interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p70), IL-13, IL-17, eotaxin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-gamma, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP-1) alpha, MIP-1beta, regulated on activation normal T cell expressed and secreted (RANTES), tumour necrosis factor (TNF)-alpha, leptin, inducible protein (IP)-10 and vascular endothelial growth factor (VEGF) in a rat model. IL-2, IL-9, IL-12 (p70), IL-13, IL-18, eotaxin, IFN-gamma, IP-10, KC, leptin, MCP-1, MIP-1alpha and TNF-alpha were significantly higher in serum, whilst IL-1beta, IL-5, IL-6, IL-10, IL-17, G-CSF and GM-CSF were significantly higher in seminal fluid. When compared to mouse profiles, only G-CSF was present at significantly higher levels in the seminal fluid in both species. Bayesian modelling highlighted key shared features across mouse and rat networks, namely TNF-alpha as the terminal node in both serum and seminal plasma, and MCP-1 as a central coordinator of seminal cytokine networks through the intermediary of KC and RANTES. These findings reveal a marked interspecific conservation of seminal cytokine networks.

Sex differences in gut satiety hormones peptide-YY and glucagon-like peptide-1 in pups raised in larger lactation litter sizes

Introduction: Previous studies have established that the size of litters during lactation influences body size and adiposity in male rat pups. Levels of gut hormones such as appetite-stimulating ghrelin and appetite-reducing peptide-YY (PYY) and glucagon-like peptide-1 (GLP-1) have not been examined in this model despite an increased susceptibility to obesity into adulthood. This study aimed to establish whether changes in gut appetite hormones, resulting from lactation litter size, could contribute to the increased risk of adulthood obesity. Methods: Male and female littermates from small (n=4), control (n=8) and large (n=12) litter sizes were studied at weaning age (25 days), with litter sizes adjusted prior to 1 day postpartum; pups remained with the dams throughout the study. Stomach ghrelin and colonic PYY and GLP-1 were quantified using radioimmunoassay. Stomachs were emptied, blotted dry and weighed. Body length was taken from between the front paws to the anus. Results and Discussion: No differences were found in stomach ghrelin levels between the pups, but levels of the gut satiety hormones PYY and GLP-1 were found to be altered in colon tissue. In descending colon, PYY and GLP-1 concentrations were significantly lower in male, but not female, pups from the large litters. Physically, both the male and female pups fed in the large litters were significantly shorter and lighter and had significantly lighter stomach tissue. We suggest that the significantly smaller male pups may have had reduced satiety as a mechanism to achieve ‘catch-up growth’ into adulthood. It is unclear whether females may show similar adaptations at a later stage. This study further demonstrated differences between young males and females, with differences evident even before sexual maturation. This work highlights the importance of standardising litter size as early as possible, especially in studies of appetite control.