The GLP-1 agonist, exendin-4, stimulates LH secretion in female sheep.

Our previous studies showed that microinjection into the median eminence of the sheep of glucagon-like peptide- 1 (GLP-1) or its receptor agonist exendin-4 stimulates luteinising hormone (LH) secretion, but it is unknown whether the same effect may be obtained by systemic administration of the same. The present study measured the response in terms of plasma LH concentrations to intravenous (iv) infusion of exendin-4. A preliminary study showed that infusion of 2 mg exendin-4 into ewes produced a greater LH response in the follicular phase of the oestrous cycle than the luteal phase. Accordingly, the main study monitored plasma LH levels in response to either 0.5 mg or 2 mg exendin-4 or vehicle (normal saline) delivered by jugular infusion for 1 h in the follicular phase of the oestrous cycle. Blood samples were collected at 10 min intervals before, during and after infusion. Both doses of exendin-4 increased mean plasma LH concentrations and increased LH peripheral pulse amplitude. There was no effect on inter-pulse interval or timing of the preovulatory LH surge. These doses of exendin-4 did not alter plasma insulin or glucose concentrations. Quantitative PCR of the gastrointestinal tract samples from a population of ewes confirmed the expression of the preproglucagon gene (GCG). Expression increased aborally and was greatest in the rectum. It is concluded that endogenous GLP-1, most likely derived from the hindgut, may act systemically to stimulate LH secretion. The present data suggest that this effect may be obtained with levels of agonist that are lower than those functioning as an incretin.

Hexapeptides from mammalian inhibitory hormone hunt activate and inactivate nematode reproduction.

BACKGROUND: Biopurification has been used to disclose an evolutionarily conserved inhibitory reproductive hormone involved in tissue mass determination. A (rat) bioassay-guided physicochemical fractionation using ovine materials yielded via Edman degradation a 14-residue amino acid (aa) sequence. As a 14mer synthetic peptide (EPL001) this displayed antiproliferative and reproduction-modulating activity, while representing only a part of the native polypeptide. Even more unexpectedly, a scrambled-sequence control peptide (EPL030) did likewise. METHODS: Reproduction has been investigated in the nematode Steinernema siamkayai, using a fermentation system supplemented with different concentrations of exogenous hexapeptides. Peptide structure-activity relationships have also been studied using prostate cancer and other mammalian cells in vitro, with peptides in solution or immobilized, and via the use of mammalian assays in vivo and through molecular modelling. RESULTS: Reproduction increased (x3) in the entomopathogenic nematode Steinernema siamkayai after exposure to one synthetic peptide (IEPVFT), while fecundity was reduced (x0.5) after exposure to another (KLKMNG), both effects being dose-dependent. These hexamers are opposite ends of the synthetic peptide KLKMNGKNIEPVFT (EPL030). Bioactivity is unexpected as EPL030 is a control compound, based on a scrambled sequence of the test peptide MKPLTGKVKEFNNI (EPL001). EPL030 and EPL001 are both bioinformatically obscure, having no convincing matches to aa sequences in the protein databases. EPL001 has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Peptides up to a 20mer have also been shown to inhibit the proliferation of human cancer and other mammalian cells in vitro, with reproductive upregulation demonstrated previously in fish and frogs, as well as nematodes. EPL001 encodes the sheep neuroendocrine prohormone secretogranin II (sSgII), as deduced on the basis of immunoprecipitation using an anti-EPL001 antibody, with bespoke bioinformatics. Six sSgII residues are key to EPL001's bioactivity: MKPLTGKVKEFNNI. A stereospecific bimodular tri-residue signature is described involving simultaneous accessibility for binding of the side chains of two specific trios of amino acids, MKP & VFN. An evolutionarily conserved receptor is conceptualised having dimeric binding sites, each with ligand-matching bimodular stereocentres. The bioactivity of the 14mer control peptide EPL030 and its hexapeptide progeny is due to the fortuitous assembly of subsets of the novel hormonal motif, MKPVFN, a default reproductive and tissue-building OFF signal.

Onset of normal cycles in postpartum anovulatory dairy cattle treated with kisspeptin.

The efficacy of a long-acting synthetic derivative of kisspeptin (Kp) to initiate normal oestrous cycles was tested in 24 mixed-aged, Holstein-Friesian cows that were 18-25 days postpartum on the day of treatment (D0). Groups of eight cows received saline (Sal) vehicle by intramuscular injection at 8:00 and 16:00 h (Sal-Sal), Kp at 8:00 h and vehicle at 16:00 h (Kp-Sal) or Kp on both occasions (Kp-Kp). The Kp dose was 15 nmol per 60 kg body weight. The ovaries of the cows were examined daily by ultrasonography between D4 and D14. Blood samples were collected from a tail vessel at 0, 2, 4, 8, 10 and 12 h relative to the time of the first injection for luteinizing hormone (LH) and follicle-stimulating hormone assay. Additional samples were collected daily from D4 until D14 and D19, 22, 26 and 29 for progesterone assay. LH surge-like responses were observed in cows treated with Kp at 8:00 h. Ovulation was consistently induced by Kp within 48 h when a dominant ovarian follicle of at least 10 mm in diameter was observed (8/14) but in no cases (6/14) during a new wave of ovarian follicular development comprising follicles <10 mm in diameter. The subsequent ovulatory cycle was of normal length in most cases as compared with short 8- to 12-day cycles observed in spontaneously ovulating cows. We conclude that Kp treatment can induce ovulation in postpartum dairy cows, with ensuing oestrous cycles of normal length, if administered when a mature dominant follicle is present in the ovaries. LAY SUMMARY: Cow fertility is important for efficient, profitable dairy farming. Cows that take too long after calving to become fertile are problematic. We tested a synthetically made, long-acting hormone called kisspeptin (Kp) to advance the time that cows become fertile after calving. Twenty-four dairy cows that had been calved for 3-4 weeks were used. One group of eight cows received an injection of Kp at the morning milking, another eight cows received Kp at both the morning and afternoon milking, while the last group of eight cows served as untreated controls. Kp treatment caused a desirable hormone response from the cows' brain. Normal oestrous cycles resulted, but only when a mature follicle was present in the ovary. Further study is required to analyse whether the use of a long-acting Kp drug could be used as an effective treatment for stimulating dairy cows to become more fertile after calving.

Kiss1 expression in the hypothalamic arcuate nucleus is lower in dairy cows of reduced fertility†.

We tested the hypothesis that divergent genetic merit for fertility of dairy cows is due to aberrant reproductive neuroendocrine function. The kisspeptin status of non-pregnant cows of either positive (POS) or negative (NEG) breeding values (BVs) for fertility was studied in three groups (n = 8), based on their previous post-partum period: POS cows, which had spontaneous ovarian cycles (POS-CYC) and NEG cows, which either cycled (NEG-CYC) or did not cycle (NEG-NONCYC). Ovarian cycles were synchronized, blood samples were taken to define endocrine status, and the animals were slaughtered in an artificial follicular phase. The brains and the pituitary glands were collected for quantitative polymerase chain reaction (qPCR) and in situ hybridization of hypothalamic GNRH1, Kiss1, TAC3, and PDYN and pituitary expression of LHB and FSHB. Gonadotropin releasing hormone (GnRH) and kisspeptin levels were quantified in snap frozen median eminence (ME). GNRH1 expression and GnRH levels in the ME were similar across groups. Kiss1 expression in the preoptic area of the hypothalamus was also similar across groups, but Kiss1 in the arcuate nucleus was almost 2-fold higher in POS-CYC cows than in NEG groups. TAC3 expression was higher in POS-CYC cows. The number of pituitary gonadotropes and the level of expression of LHB and FSHB were similar across groups. We conclude that the lower levels of Kiss1 and TAC3 in NEG cows with low fertility status and may lead to deficient GnRH and gonadotropin secretion.

Glucagon-like peptide-1 control of GnRH secretion in female sheep.

The role of glucagon-like peptide-1 (GLP-1) on gonadotropin-releasing hormone (GnRH) secretion was investigated in ovariectomised (OVX) ewes, in which GnRH and luteinising hormone (LH) secretion had been restrained by treatment with oestrogen and progesterone. Guide tubes for microinjection were placed above the median eminence (ME) and the animals were allowed to recover for 1 month. Jugular venous blood samples were taken via cannulae at 10 min intervals. Vehicle (50 nL) was injected into the ME at 2 h, followed by injection of GLP-1 ((7-36)-amide - 0.5 or 1 nmol) or its receptor agonist, exendin-4 (0.5 nmol) at 4 h (n = 5). Plasma LH levels were quantified as a surrogate measure of GnRH secretion. GLP-1 microinjection into the ME elicited a large amplitude LH pulse in jugular plasma, the effect was greater at the higher dose. Exendin-4 microinjection caused a large, sustained increase in plasma LH levels. To determine how GLP-1 might exert an effect on GnRH secretion, we employed double labelled in situ hybridisation, with RNAScope, for co-localisation of the GLP-1 receptor (GLP-1R) in GnRH, Kisspeptin and NPY cells in the hypothalami of three ewes in the luteal phase of the estrous cycle. GLP1R expression was clearly visible but the receptor was not expressed in GNRH1 or NPY expressing neurons and was visualised in <5% of KISS1 expressing neurons. We conclude that GLP-1 may act at the level of the secretory terminals of GnRH neurons in the ME to stimulate GnRH secretion, the pathway through which such effect is manifested remains unknown.

Effect of sex and sex steroids on brown adipose tissue heat production in humans.

OBJECTIVE: Retrospective studies suggest that women have more active brown adipose tissue (BAT) than men, but little is known of the effect of fluctuating sex steroids across the menstrual cycle on thermogenesis in women. DESIGN: To characterise the effects of sex and sex steroids on BAT activity we recruited healthy weight men (n = 14) and women at two stages of the menstrual cycle (luteal, n = 9; follicular, n = 11). METHODS: Infrared thermography measured supraclavicular temperature to index BAT thermogenesis in response to both cold (immersion of one hand in water at 15°C) and meal (Ensure, 10 kcal/kg body weight) stimuli. RESULTS: Adaptive BAT temperature responses were greater (P < 0.05) in women than men, irrespective of stage of menstrual cycle. Whereas during cold exposure, the increase in BAT temperature was abrogated (P < 0.05) in women during follicular phase compared to men and women during luteal phase. Plasma concentrations of progesterone, 17ß-estradiol, testosterone and cortisol were measured. Regression analyses demonstrated that baseline BAT temperature was positively correlated (P < 0.05) with progesterone levels, but was inversely associated (P < 0.05) with cortisol concentration. Both cold- and meal-induced changes in BAT temperature mildly correlated (P = 0.07; P < 0.05) with 17ß-estradiol levels, but not with testosterone concentrations. CONCLUSIONS: Baseline supraclavicular temperature is elevated in women during the luteal phase of the menstrual cycle, which correlated with elevated progesterone concentrations. Women exhibited greater thermogenic responses than men, irrespective of the state of the menstrual cycle, which was associated with plasma levels of 17ß-estradiol. We conclude that sex steroids may regulate BAT thermogenesis in healthy adults.

Resilience of Small Ruminants to Climate Change and Increased Environmental Temperature: A Review.

Climate change is a major global threat to the sustainability of livestock systems. Climatic factors such as ambient temperature, relative humidity, direct and indirect solar radiation and wind speed influence feed and water availability, fodder quality and disease occurrence, with production being most efficient in optimal environmental conditions. Among these climatic variables, ambient temperature fluctuations have the most impact on livestock production and animal welfare. Continuous exposure of the animals to heat stress compromises growth, milk and meat production and reproduction. The capacity of an animal to mitigate effects of increased environmental temperature, without progressing into stress response, differs within and between species. Comparatively, small ruminants are better adapted to hot environments than large ruminants and have better ability to survive, produce and reproduce in harsh climatic regions. Nevertheless, the physiological and behavioral changes in response to hot environments affect small ruminant production. It has been found that tropical breeds are more adaptive to hot climates than high-producing temperate breeds. The growing body of knowledge on the negative impact of heat stress on small ruminant production and welfare will assist in the development of suitable strategies to mitigate heat stress. Selection of thermotolerant breeds, through identification of genetic traits for adaption to extreme environmental conditions (high temperature, feed scarcity, water scarcity), is a viable strategy to combat climate change and minimize the impact on small ruminant production and welfare. This review highlights such adaption within and among different breeds of small ruminants challenged by heat stress.

Expression of genes for Kisspeptin (KISS1), Neurokinin B (TAC3), Prodynorphin (PDYN), and gonadotropin inhibitory hormone (RFRP) across natural puberty in ewes.

Expression of particular genes in hypothami of ewes was measured across the natural pubertal transition by in situ hybridization. The ewes were allocated to three groups (n = 4); prepubertal, postpubertal and postpubertally gonadectomized (GDX). Prepubertal sheep were euthanized at 20 weeks of age and postpubertal animals at 32 weeks. GDX sheep were also euthanized at 32 weeks, 1 week after surgery. Expression of KISS1, TAC3, PDYN in the arcuate nucleus (ARC), RFRP in the dorsomedial hypothalamus and GNRH1 in the preoptic area was quantified on a cellular basis. KISS1R expression by GNRH1 cells was quantified by double-label in situ hybridization. Across puberty, detectable KISS1 cell number increased in the caudal ARC and whilst PDYN cell numbers were low, numbers increased in the rostral ARC. TAC3 expression did not change but RFRP expression/cell was reduced across puberty. There was no change across puberty in the number of GNRH1 cells that expressed the kisspeptin receptor (KISS1R). GDX shortly after puberty did not increase expression of any of the genes of interest. We conclude that KISS1 expression in the ARC increases during puberty in ewes and this may be a causative factor in the pubertal activation of the reproductive axis. A reduction in expression of RFRP may be a factor in the onset of puberty, removing negative tone on GNRH1 cells. The lack of changes in expression of genes following GDX suggest that the effects of gonadal hormones may differ in young and mature animals.

Comparative Assessment of Thermotolerance in Dorper and Second-Cross (Poll Dorset/Merino × Border Leicester) Lambs.

The objective of this study was to compare the thermotolerance of second-cross (SC; Poll Dorset × Merino × Border Leicester) and Dorper lambs. Dorper and SC lambs (4-5 months of age) were subjected to cyclic heat stress (HS) (28-40 °C). The temperature was increased to 38-40 °C between 800 and 1700 h daily and maintained at 28 °C for the remainder of the day (30-60% relative humidity (RH)) in climatic chambers for 2 weeks (n = 12/group), with controls maintained in a thermoneutral (TN) (18-21 °C, 40-50% RH) environment (n = 12/group). Basal respiration rate (RR), rectal temperature (RT) and skin temperature (ST) were higher (p < 0.01) in SC lambs than in Dorpers. HS increased RR, RT and ST (p < 0.01) in both genotypes, but the levels reached during HS were lower (p < 0.01) in Dorpers. HS increased (p < 0.01) water intake to a greater extent in SC lambs, while feed intake was reduced (p < 0.05) by HS in SC lambs but not in Dorpers. HS increased (p < 0.01) blood urea nitrogen and creatinine in SC lambs only. Plasma non-esterified fatty acid concentrations were reduced (p < 0.05) by HS in SC lambs but increased (p < 0.05) in Dorpers. There was no effect of HS on pO2, cHCO3- and cSO2, but higher (p < 0.01) blood pH and lower (p < 0.01) pCO2 were recorded under HS in both genotypes. Blood electrolytes and base excess were reduced (p < 0.01) under HS, while a genotype difference (p < 0.05) was only observed in blood K+ and hemoglobin concentrations. Basal plasma prolactin concentrations were lower (p < 0.01) in Dorpers but were elevated at a similar level during HS (p < 0.01) in both genotypes. Dorper lambs are more resilient to HS than SC lambs. Future research should focus on confirming whether the better heat tolerance of Dorpers is translated to better returns in terms of growth performance and carcass traits over the summer months.

Genetic Selection for Thermotolerance in Ruminants.

Variations in climatic variables (temperature, humidity and solar radiation) negatively impact livestock growth, reproduction, and production. Heat stress, for instance, is a source of huge financial loss to livestock production globally. There have been significant advances in physical modifications of animal environment and nutritional interventions as tools of heat stress mitigation. Unfortunately, these are short-term solutions and may be unsustainable, costly, and not applicable to all production systems. Accordingly, there is a need for innovative, practical, and sustainable approaches to overcome the challenges posed by global warming and climate change-induced heat stress. This review highlights attempts to genetically select and breed ruminants for thermotolerance and thereby sustain production in the face of changing climates. One effective way is to incorporate sustainable heat abatement strategies in ruminant production. Improved knowledge of the physiology of ruminant acclimation to harsh environments, the opportunities and tools available for selecting and breeding thermotolerant ruminants, and the matching of animals to appropriate environments should help to minimise the effect of heat stress on sustainable animal genetic resource growth, production, and reproduction to ensure protein food security.

Perennial Ryegrass Alkaloids Increase Respiration Rate and Decrease Plasma Prolactin in Merino Sheep under Both Thermoneutral and Mild Heat Conditions.

A study was undertaken to determine the effects of feeding two levels of perennial ryegrass alkaloids (nil vs. moderate) under two climatic conditions. Alkaloids were fed via endophyte-infected perennial ryegrass seed and hay. Twenty-four Merino ewe weaners (six months, initial BW 32 ± 1.7 kg) were used in a study that lasted for 21 days after 14 days of adaptation. Sheep were fed either a control or alkaloid (Alk, 110 µg/kg LW ergovaline and 75 µg/kg LW lolitrem B) supplemented diet. Sheep were exposed to either constant thermoneutral (TN, 21-22 °C, 49% RH) or mildly heated (HS, 33 °C 1000-1500 h, 28% relative humidity) conditions. Dietary Alk and HS reduced dry matter intake (DMI) (p < 0.001, p = 0.02, respectively) with the combination of both reducing DMI by 42%. Reductions in DMI resulted in a lower daily gain in the Alk treatment (p < 0.001). Feed digestibility was reduced in the combined treatment (p = 0.03). Rectal temperature, respiration rate, and skin temperature increased in the Alk treatment. Plasma prolactin concentrations were decreased by Alk and increased by mild HS. The data indicate that production is compromised in the presence of Alk and mild HS, with this effect being exacerbated by a combination of both.

Lipopolysaccharide reduces gonadotrophin-releasing hormone (GnRH) gene expression: role of RFamide-related peptide-3 and kisspeptin.

RFamide-related peptide (RFRP)-3 reduces luteinising hormone (LH) secretion in rodents. Stress has been shown to upregulate the expression of the RFRP gene (Rfrp) with a concomitant reduction in LH secretion, but an effect on expression of the gonadotrophin-releasing hormone (GnRH) gene (Gnrh1) has not been shown. We hypothesised that lipopolysaccharide (LPS)-induced stress affects expression of Rfrp, the gene for kisspeptin (Kiss1) and/or Gnrh1, leading to suppression of LH levels in rats. Intracerebroventricular injections of RFRP-3 (0.1, 1, 5 nmol) or i.v. LPS (15µgkg-1) reduced LH levels. Doses of 1 and 5 nmol RFRP-3 were then administered to analyse gene expression by in situ hybridisation. RFRP-3 (5 nmol) had no effect on Gnrh1 or Kiss1 expression. LPS stress reduced GnRH and Kiss1 expression, without affecting Rfrp1 expression. These data indicate that LPS stress directly or indirectly reduces Gnrh1 expression, but this is unlikely to be due to a change in Rfrp1 expression.

Brown adipose tissue thermogenesis in polycystic ovary syndrome.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is associated with increased obesity with a greater propensity to weight gain and a lack of sustainable lifestyle interventions. Altered brown adipose tissue (BAT) thermogenesis is a potential contributor to obesity in PCOS. BAT activity and modulation have not been studied in PCOS. This observational study explored BAT thermogenesis and its associations in women with and without PCOS. PARTICIPANTS AND METHODS: Cutaneous temperature was recorded from supraclavicular (indicator of BAT activity) and upper arm regions using dataloggers (SubCue, Calgary, Canada) in a cross-sectional substudy, nested within a randomized control trial, of community-recruited premenopausal women with (n = 47, Rotterdam diagnostic criteria) and without (n = 11) PCOS. RESULTS: Complete temperature data were available in 44 PCOS (mean age: 30.0 ± 6.2, mean BMI: 29.3 ± 5.5) and 11 non-PCOS (mean age: 33.0 ± 7.0, mean BMI: 25 ± 3) women. Women with PCOS had lower supraclavicular skin temperature compared to controls overall (33.9 ± 0.7 vs 34.5 ± 1, P < 0.05) and during sleep (34.5 ± 0.6 vs 35.2 ± 0.9, P < 0.001). In the PCOS group, supraclavicular skin temperature overall and over sleep and waking hours correlated inversely with testosterone (r = -0.41 P < 0.05, r = -0.485 P < 0.01 and r = -0.450 P < 0.01 respectively). Testosterone levels explained approximately 15%, 30% and 20% of the variability in supraclavicular skin temperature overall and over sleep and waking hours in women with PCOS, respectively. CONCLUSION: Women with PCOS have lower BAT activity compared to controls. BAT thermogenesis is negatively associated with androgen levels in PCOS.

Epitope mapping of an uncertain endogenous antigen implies secretogranin II peptide splicing.

Background: The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma. This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. Four purification runs gave Edman N-terminal sequences relating to 1MKPLTGKVKEFNNI 14. This is bioinformatically obscure and has been resistant to molecular biological investigation. The sequence was synthesized as the peptide EPL001, against which was raised a goat polyclonal antiserum, G530. Used in an antigen capture campaign, G530 pointed to the existence of a novel derivative of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. The proposed SgII derivative was dubbed SgII-70, yet the sequence commonality between SgII and EPL001 is essentially NNI. Methods: Immunohistochemical (IHC) labelling with G530 is reported within rat, mouse and human cerebrovasculature and in glandular elements of the mouse intestine. Epitope mapping involved IHC peptide preabsorption, allied to deductive bioinformatics and molecular modelling in silico. Results: G530 is deemed monoepitopic in regard to both its synthetic antigen (EPL001) and its putative endogenous antigen (SgII related). The epitope within EPL001 of the anti-EPL001 antibody is inferred to be the contiguous C-terminal 9KEFNNI 14. This is so because the G530 blockade data are consistent with the epitope in the mammalian endogenous antigen being part contiguous, part non-contiguous KE·F·NNI, ex hypothesi. The observed immunostaining is deduced to be due to pre-SgII-70, which has a non-C-terminal NNI, and SgII-70, which has an N-terminal MLKTGEKPV/N and a C-terminal NNI (these two motifs being in the reverse order in the SgII parent protein). Conclusion: The present data are consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII-related epitope. SgII is apparently subject to peptide splicing, as has been reported for the related chromogranin A.

Exercise counteracts the homeostatic decrease in thermogenesis caused by caloric restriction in sheep.

Caloric restriction causes a homeostatic reduction in thermogenesis. We aimed to determine whether exercise could counteract this. We studied four groups of normal-weight ewes ( n = 5), including control sedentary fed ad libitum, exercise fed ad libitum (30 min/d, 5 d/wk), diet-restricted (70% of ad libitum food intake), and combined diet and exercise. Temperature probes implanted in sternal and retroperitoneal adipose tissue and skeletal muscle measured thermogenesis. After the 4-wk intervention, hypothalami were collected for in situ hybridization, and fat and muscle biopsies were collected for real-time PCR and Western blotting. Combined diet and exercise reduced adiposity ( P < 0.05). Caloric restriction alone reduced overnight temperatures in sternal and retroperitoneal fat ( P < 0.05), which was counteracted by exercise ( P < 0.05). Exercise did not induce expression of cellular markers of browning in adipose tissue. There was no effect of diet or exercise on skeletal muscle thermogenesis. Combined diet and exercise increased the expression of neuropeptide Y and agouti-related protein in the hypothalamic arcuate nucleus ( P < 0.05), consistent with reduced adiposity. Gene expressions of key hypothalamic appetite-regulating peptides were not associated with altered thermogenesis. We demonstrate that exercise counteracts the inhibitory effect of caloric restriction to restore thermogenesis in adipose tissue of sheep.-Fuller-Jackson, J.-P., Clarke, I. J., Rao, A., Henry, B. A. Exercise counteracts the homeostatic decrease in thermogenesis caused by caloric restriction in sheep.

Continuous Kisspeptin Restores Luteinizing Hormone Pulsatility Following Cessation by a Neurokinin B Antagonist in Female Sheep.

Pulsatile secretion of the gonadotropin-releasing hormone (GnRH) drives pulsatile secretion of the luteinizing hormone (LH), with evidence that this depends on kisspeptin (Kiss) input to GnRH neurons. Kiss administration causes acute GnRH/LH secretion, and electrophysiological data suggest that Kiss neurons may act in a phasic manner to drive GnRH secretion, but there is not definitive evidence for this. The product of the Kiss-1 gene is proteolytically cleaved to smaller products, and the 10 amino acid C-terminal product (Kiss-10) displays full bioactivity. We have shown previously that continuous delivery of Kiss-10 to anestrous ewes can cause a surge in GnRH secretion and ovulation and increases LH pulse frequency in humans. Here, we tested the hypothesis that continuous Kiss-10 delivery can support pulsatile GnRH/LH secretion in the sheep. Neurokinin B (NKB) provides positive drive to Kiss neurons, so we therefore infused an NKB antagonist (ANT-08) intracerebroventricularly to induce cessation of pulsatile GnRH/LH secretion, with or without concomitant continuous Kiss-10 infusion. ANT-08 suppressed GnRH/LH pulsatility, which was immediately restored with continuous Kiss-10 infusion. These data support the notion that Kiss-10 action is downstream of NKB signaling and that continuous Kiss-10 stimulation of GnRH neurons is sufficient to support a pulsatile pattern of GnRH/LH secretion. This offers further support to the theory that GnRH pulse generation is intrinsic to GnRH neurons and that pulsatile GnRH release can be affected with continuous stimulation by Kiss-10.

Mysterious inhibitory cell regulator investigated and found likely to be secretogranin II related.

In the context of a hunt for a postulated hormone that is tissue-mass inhibiting and reproductively associated, there is described probable relatedness to a granin protein. A 7-8 kDa polypeptide candidate (gels/MS) appeared in a bioassay-guided fractionation campaign involving sheep plasma. An N-terminal sequence of 14 amino acids was obtained for the polypeptide by Edman degradation. Bioinformatics and molecular biology failed to illuminate any ovine or non-ovine protein which might relate to this sequence. The N-terminal sequence was synthesized as the 14mer EPL001 peptide and surprisingly found to be inhibitory in an assay in vivo of compensatory renal growth in the rat and modulatory of nematode fecundity, in line with the inhibitory hormone hypothesis. Antibodies were raised to EPL001 and their deployment upheld the hypothesis that the EPL001 amino acid sequence is meaningful and relevant, notwithstanding bioinformatic obscurity. Immunohistochemistry (IHC) in sheep, rodents and humans yielded staining of seeming endocrine relevance (e.g. hypothalamus, gonads and neuroendocrine cells in diverse tissues), with apparent upregulation in certain human tumours (e.g. pheochromocytoma). Discrete IHC staining in Drosophila melanogaster embryo brain was seen in glia and in neuroendocrine cells, with staining likely in the corpus cardiacum. The search for the endogenous antigen involved immunoprecipitation (IP) followed by liquid chromatography and mass spectrometry (LC-MS). Feedstocks were PC12 conditioned medium and aqueous extract of rat hypothalamus-both of which had anti-proliferative and pro-apoptotic effects in an assay in vitro involving rat bone marrow cells, which inhibition was subject to prior immunodepletion with an anti-EPL001 antibody-together with fruit fly embryo material. It is concluded that the mammalian antigen is likely secretogranin II (SgII) related. The originally seen 7-8 kDa polypeptide is suggested to be a new proteoform of secretogranin II of ∼70 residues, SgII-70, with the anti-EPL001 antibody seeing a discontinuous epitope. The fly antigen is probably Q9W2X8 (UniProt), an uncharacterised protein newly disclosed as a granin and provisionally dubbed macrogranin I (MgI). SgII and Q9W2X8 merit further investigation in the context of tissue-mass inhibition.

Kisspeptin Stimulates Growth Hormone Release by Utilizing Neuropeptide Y Pathways and Is Dependent on the Presence of Ghrelin in the Ewe.

Although kisspeptin is the primary stimulator of gonadotropin-releasing hormone secretion and therefore the hypothalamic-pituitary-gonadal axis, recent findings suggest kisspeptin can also regulate additional neuroendocrine processes including release of growth hormone (GH). Here we show that central delivery of kisspeptin causes a robust rise in plasma GH in fasted but not fed sheep. Kisspeptin-induced GH secretion was similar in animals fasted for 24 hours and those fasted for 72 hours, suggesting that the factors involved in kisspeptin-induced GH secretion are responsive to loss of food availability and not the result of severe negative energy balance. Pretreatment with the neuropeptide Y (NPY) Y1 receptor antagonist, BIBO 3304, blocked the effects of kisspeptin-induced GH release, implicating NPY as an intermediary. Kisspeptin treatment induced c-Fos in NPY and GH-releasing hormone (GHRH) cells of the arcuate nucleus. The same kisspeptin treatment resulted in a reduction in c-Fos in somatostatin (SS) cells in the periventricular nucleus. Finally, blockade of systemic ghrelin release or antagonism of the ghrelin receptor eliminated or reduced the ability of kisspeptin to induce GH release, suggesting the presence of ghrelin is required for kisspeptin-induced GH release in fasted animals. Our findings support the hypothesis that during short-term fasting, systemic ghrelin concentrations and NPY expression in the arcuate nucleus rise. This permits kisspeptin activation of NPY cells. In turn, NPY stimulates GHRH cells and inhibits SS cells, resulting in GH release. We propose a mechanism by which kisspeptin conveys reproductive and hormone status onto the somatotropic axis, resulting in alterations in GH release.

Glucocorticoid treatment facilitates development of a metabolic syndrome in ovariectomized Macaca Mulatta fed a high fat diet.

Metabolic syndrome (MetS) is characterized by a cluster of key features, which include abdominal obesity, insulin resistance, hypertension, and dyslipidemia. The aim of this study was to assess the impact of elevated glucocorticoid levels on the development of MetS in middle-aged female rhesus monkeys (Macaca Mulatta) after ovariectomy. Six female ovariectomized rhesus monkeys (9-13years) were randomly assigned to either a control group (normal diet, n=3) or a group in which MetS was facilitated (n=3). The MetS group fed with HFD (15% fat) and received oral prednisone acetate treatment (50mg/day). After 24months, the GCs treatment was withdrawn with continuation of high-fat feeding for a further 12months. After 24months, the MetS group displayed a significant increase in body weight and abdominal circumference. Additionally, the MetS animals displayed abnormal serum lipids, insulin resistance and impaired glucose tolerance. Histology of liver biopsies indicated marked accumulation of lipid droplets in hepatocytes of MetS animals. Withdrawal of GCs treatment led to recovery from above-mentioned metabolic disorders. Whereas GCs treatment increased leptin expression, it lowered expression of adiponectin and other factors in adipose tissue. Expression of Hydroxy-steroid dehydrogenase-1 and glucose transporter type-4 in the livers of MetS animals were reduced. We conclude that in the context of high fat diet, high levels of exogenous GCs contribute to the development of MetS in non-human primates.

Ontogeny and Thermogenic Role for Sternal Fat in Female Sheep.

Brown adipose tissue acting through a unique uncoupling protein (UCP1) has a critical role in preventing hypothermia in newborn sheep but is then thought to rapidly disappear during postnatal life. The extent to which the anatomical location of fat influences postnatal development and thermogenic function in adulthood, particularly following feeding, is unknown, and we examined both in our study. Changes in gene expression of functionally important pathways (i.e., thermogenesis, development, adipogenesis, and metabolism) were compared between sternal and retroperitoneal fat depots together with a representative skeletal muscle over the first month of postnatal life, coincident with the loss of brown fat and the accumulation of white fat. In adult sheep, implanted temperature probes were used to characterize the thermogenic response of fat and muscle to feeding and the effects of reduced or increased adiposity. UCP1 was more abundant in sternal fat than in retroperitoneal fat and was retained only in the sternal depot of adults. Distinct differences in the abundance of gene pathway markers were apparent between tissues, with sternal fat exhibiting some similarities with muscle that were not apparent in the retroperitoneal depot. In adults, the postprandial rise in temperature was greater and more prolonged in sternal fat than in retroperitoneal fat and muscle, a difference that was maintained with altered adiposity. In conclusion, sternal adipose tissue retains UCP1 into adulthood, when it shows a greater thermogenic response to feeding than do muscle and retroperitoneal fat. Sternal fat may be more amenable to targeted interventions that promote thermogenesis in large mammals.