Acute myocardial ischemia and reperfusion injury (IRI) underly the detrimental effects of coronary heart disease on the myocardium. Despite the ongoing advances in reperfusion therapies, there remains a lack of effective therapeutic strategies for preventing IRI. Growth hormone secretagogues (GHS) have been demonstrated to improve cardiac function, attenuate inflammation and modulate the autonomic nervous system (ANS) in models of cardiovascular disease. Recently, we demonstrated a reduction in infarct size after administration of hexarelin (HEX), in a murine model of myocardial infarction. In the present study we employed a reperfused ischemic (IR) model, to determine whether HEX would continue to have a cardioprotective influence in a model of higher clinical relevance. Myocardial ischemia was induced by transient ligation of the left descending coronary artery (tLAD) in C57BL/6 J mice followed by HEX (0.3 mg/kg/day; n = 20) or vehicle (VEH) (n = 18) administration for 21 days, first administered immediately prior-to reperfusion. IR-injured and sham mice were subjected to high-field magnetic resonance imaging to assess left ventricular (LV) function, with HEX-treated mice demonstrating a significant improvement in LV function compared with VEH-treated mice. A significant decrease in interstitial collagen, TGF-β1 expression and myofibroblast differentiation was also seen in the HEX-treated mice after 21 days. HEX treatment shifted the ANS balance towards a parasympathetic predominance; combined with a significant decrease in cardiac troponin-I and TNF-α levels, these findings were suggestive of an anti-inflammatory action on the myocardium mediated via HEX. In this model of IR, HEX appeared to rebalance the deregulated ANS and activate vagal anti-inflammatory pathways to prevent adverse remodelling and LV dysfunction. There are limited interventions focusing on IRI that have been successful in improving clinical outcome in acute myocardial infarction (AMI) patients, this study provides compelling evidence towards the translational potential of HEX where all others have largely failed.

Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE‐GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss‐CRE heterozygous mice with CRE‐activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80‐90% of tdTomato positive neurones in the ARC were co‐labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato‐labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre‐optic areas and to the lateral hypothalamus.

Key points Loss of function of the melanocortin 4 receptor (MC4R) results in hyperphagia, obesity and increased growth. Despite knowing that MC4Rs control food intake, we are yet to understand why defects in the function of the MC4R receptor contribute to rapid linear growth. We show that hyperphagia following germline loss of MC4R in male mice promotes growth while suppressing the growth hormone–insulin‐like growth factor‐1 (GH–IGF‐1) axis. We propose that hyperinsulinaemia promotes growth while suppressing the GH–IGF‐1 axis. It is argued that physiological responses essential to maintain energy flux override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. AbstractDefects in melanocortin‐4‐receptor (MC4R) signalling result in hyperphagia, obesity and increased growth. Clinical observations suggest that loss of MC4R function may enhance growth hormone (GH)‐mediated growth, although this remains untested. Using male mice with germline loss of the MC4R, we assessed pulsatile GH release and insulin‐like growth factor‐1 (IGF‐1) production and/or release relative to pubertal growth. We demonstrate early‐onset suppression of GH release in rapidly growing MC4R deficient (MC4RKO) mice, confirming that increased linear growth in MC4RKO mice does not occur in response to enhanced activation of the GH–IGF‐1 axis. The progressive suppression of GH release in MC4RKO mice occurred alongside increased adiposity and the progressive worsening of hyperphagia‐associated hyperinsulinaemia. We next prevented hyperphagia in MC4RKO mice through restricting calorie intake in these mice to match that of wild‐type (WT) littermates. Pair feeding of MC4RKO mice did not prevent increased adiposity, but attenuated hyperinsulinaemia, recovered GH release, and normalized linear growth rate to that seen in pair‐fed WT littermate controls. We conclude that the suppression of GH release in MC4RKO mice occurs independently of increased adipose mass, and is a consequence of hyperphagia‐associated hyperinsulinaemia. It is proposed that physiological responses essential to maintain energy flux (hyperinsulinaemia and the suppression of GH release) override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. Implications of these findings are likely to extend beyond individuals with defects in MC4R signalling, encompassing physiological changes central to mechanisms of growth and energy homeostasis universal to hyperphagia‐associated childhood‐onset obesity.

Although it has been reported that oxytocin stimulates lipolysis in adipocytes, changes in the expression of oxytocin receptor (OTR) mRNA in adipogenesis are still unknown. The present study aimed to investigate the expression of OTR mRNA during adipocyte differentiation and fat accumulation in adipocytes. OTR mRNA was highly expressed in adipocytes prepared from mouse adipose tissues compared to stromal‐vascular cells. OTR mRNA expression was increased during the adipocyte differentiation of 3T3‐L1 cells. OTR expression levels were higher in subcutaneous and epididymal adipose tissues of 14‐week‐old male mice compared to 7‐week‐old male mice. Levels of OTR mRNA expression were higher in adipose tissues at four different sites of mice fed a high‐fat diet than in those of mice fed a normal diet. The OTR expression level was also increased by refeeding for 4 h after fasting for 16 h. Oxytocin significantly induced lipolysis in 3T3‐L1 adipocytes. In conclusion, a new regulatory mechanism is demonstrated for oxytocin to control the differentiation and fat accumulation in adipocytes via activation of OTR as a part of the hypothalamic‐pituitary‐adipose axis.

Ghrelin, a gut hormone originating from the post-translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS-R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin-O-acyltransferase (GOAT), conferring selective binding to the GHS-R1a receptor via acylated ghrelin. Complete loss of preproghrelin-derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl-ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat(-/-) mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat(-/-) mice compared to age-matched wild-type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH-dependent liver genes. Of interest, circulating levels of insulin-like growth factor (IGF)-1 were elevated in goat(-/-) mice. This rise in circulating levels of IGF-1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF-1 release in goat(-/-) mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of altered GH secretory patterning remains unclear, we propose that this may contribute to sustained IGF-1 release and growth in goat(-/-) mice.

Growth hormone secretagogues (GHS) have been shown to possess cardioprotective properties in ischaemic heart disease models. This study aimed to determine whether hexarelin, a synthetic GHS, preserves cardiac function in models of myocardial infarction (MI). MI was induced by permanent ligation of the left descending coronary artery (LAD) in C57BL/6J mice (n = 44). In a second group (n = 39), the LAD was transiently ligated, followed by reperfusion. Vehicle or hexarelin was administered at 0.3 mg/kg/day subcutaneously. Treated and sham mice were subjected to magnetic resonance imaging using a T1-weighted late gadolinium enhancement sequence (LGE) at 9.4 Tesla (T) to measure ventricular function and tissue characteristics at 24 h and 21 days post-LAD ligation. Infarct size and area-at-risk was confirmed using ex-vivo analysis at 16.4T using a 3D T1/T2*-weighted sequence using iron nanoparticles and LGE. Hexarelin-treated mice demonstrated a significant improvement (P < 0.05) in ejection fraction (EF) compared with vehicle mice at 24 h in both the permanent ligation (44.7% vs 37.1%) and reperfused (56.9% vs 49.8%) groups. After 21 days there were no significant differences in EF between sham and hexarelin-treated mice in the reperfused group, hexarelin mice showed a significant improvement in EF compared to vehicle mice (65.1% vs 56.7%). A decreasing trend in infarct size was observed in hexarelin-treated mice acutely; experiments are being completed to determine chronic infarct size. These results indicate that GHS may preserve ventricular function in models of MI and are the first to demonstrate a potential therapeutic effect of GHS against myocardial reperfusion injury in vivo.

Pathological changes associated with obesity are thought to contribute to GH deficiency. However, recent observations suggest that impaired GH secretion relative to excess calorie consumption contributes to progressive weight gain and thus may contribute to the development of obesity. To clarify this association between adiposity and GH secretion, we investigated the relationship between pulsatile GH secretion and body weight; epididymal fat mass; and circulating levels of leptin, insulin, non-esterified free fatty acids (NEFAs), and glucose. Data were obtained from male mice maintained on a standard or high-fat diet. We confirm the suppression of pulsatile GH secretion following dietary-induced weight gain. Correlation analyses reveal an inverse relationship between measures of pulsatile GH secretion, body weight, and epididymal fat mass. Moreover, we demonstrate an inverse relationship between measures of pulsatile GH secretion and circulating levels of leptin and insulin. The secretion of GH did not change relative to circulating levels of NEFAs or glucose. We conclude that impaired pulsatile GH secretion in the mouse occurs alongside progressive weight gain and thus precedes the development of obesity. Moreover, data illustrate key interactions between GH secretion and circulating levels of insulin and reflect the potential physiological role of GH in modulation of insulin-induced lipogenesis throughout positive energy balance.

The age‐associated decline in growth hormone ( GH) secretion may be a consequence of the reduction in the number of GH ‐releasing hormone ( GHRH ) positive neurones. However, it remains unclear whether an alteration in the number or distribution of somatostatin ( SST ) neurones contributes to this change. In the present study, we characterised the role of SST in modulating the change in pulsatile GH secretion in male C 57 B l/6 J mice throughout puberty and into early adulthood. We assessed pulsatile GH secretion in mice at 4, 8 and 16 weeks of age. These ages correspond to early pubertal, early adulthood and adulthood, respectively. We show an elevation in peak, total and pulsatile GH secretion coinciding with periods of rapid linear growth. Using in situ hybridisation and morphometric methods, we mapped the distribution of S st m RNA expression within the mouse brain relative to this change in pulsatile GH secretion. The results obtained show that altered pulsatile GH secretion in male mice from 4–16 weeks of age does not coincide with a significant change in the number of S st m RNA expressing neurones or an abundance of S st m RNA expression throughout the arcuate nucleus ( ARC ) and periventricular nucleus ( P e V ). Rather, we observed a progressive decline in S st m RNA expressing neurones within subnuclei of the paraventricular nucleus at this time. We conclude that structural changes in S st expression within the P e V and ARC may not reflect the observed decline in pulsatile GH secretion in mice from puberty into early adulthood.

Current methodology to monitor pulsatile LH release in mice is limited by inadequate assay sensitivity, resulting in the need for collection of large blood volumes. Thus, assessment of pulsatile LH secretion in mice remains highly challenging, and observations are limited to adult mice. To address this, we developed a highly sensitive ELISA for assessment of mouse LH concentrations in small fractions of whole blood. We demonstrate that this assay is capable of reliably detecting LH down to a theoretical limit of 0.117 ng/mL in a 2-μL fraction of whole blood. Using an established frequent blood collection procedure, we validated the accuracy of this method by determining the pulsatile LH secretion in early-adult (10 weeks old) C57BL6/J male mice. Data demonstrate regular pulsatile release of LH, with peaks in LH secretion rarely exceeding 3 ng/mL. Moreover, assessment of LH release in Gpr54 knockout mice demonstrates the lack of pulsatile LH release after the loss of kisspeptin-mediated pubertal maturation. We next determined age-associated changes in pulsatile LH secretion by assessment of LH secretion in prepubertal (28 days old) C57BL6/J male mice and repeated assessment in the same mice in adulthood (120 days old). Data demonstrate that the rise in total LH secretion in mice after pubertal maturation occurs along with an overall rise in the pulsatile LH secretion rate. This was coupled with a significant increase in the number of LH secretory events (number of pulses). In addition, we observed a decrease in the clearance (increased half-life) and a decrease in the regularity (approximate entropy) of LH release. This method will be of wide general utility within the field of reproductive biology.

The transition between puberty and adulthood is accompanied by a slowing in linear growth. Although GH is a key factor that drives somatic development into adulthood, early adulthood coincides with a reduction in circulating levels of GH. To this extent, a pathological decline in postpubertal GH secretion is detrimental to attainment of peak lean muscle mass and bone mass and promotes adiposity and increases susceptibility to the development of obesity in adulthood. Here we characterized pulsatile GH secretion in C57BL/6J mice at 12 and 16 wk of age. Deconvolution analysis of these measures reveals a reduction in pulsatile GH secretion between 12 and 16 wk of age. Dietary intervention with high-fat feeding at 8 wk of age results in a significant increase in adiposity, the development of glucose intolerance, and hyperinsulinemia. We show the exacerbation of the age-associated decline in pulsatile GH secretion in high-fat-fed mice after 4 wk of dietary intervention (at 12 wk of age), and a further suppression of pulsatile GH secretion by 8 wk of dietary intervention (at 16 wk of age). Suppressed pulsatile secretion of GH did not coincide with an elevation in circulating free fatty acids. Rather, we observed increased hepatic triglyceride content and an eventual decrease in circulating levels of IGF-I. Given the established role of GH in maintaining healthy aging, we anticipate that an advancing of the age-associated decline in pulsatile GH secretion as a consequence of dietary-induced weight gain may have long-term ramifications on adult health.

GH deficiency has been found in subjects with amyotrophic lateral sclerosis (ALS). Disrupted endocrine function could contribute to the progressive muscle loss and hypermetabolism seen in ALS. It is not possible to study all the elements of the GH-IGF-I axis in ALS patients. Consequently, it remains unclear whether dysfunctional GH secretion contributes to disease pathogenesis and why GH and IGF-I directed treatment strategies are ineffective in human ALS. The hSOD1(G93A) transgenic mouse model is useful for the detailed investigation of the pathogenesis of ALS. We report that symptomatic male hSOD1(G93A) transgenic mice exhibit a deficiency in GH secretion similar to that seen in human ALS. Further characterization of the GH-IGF-I axis in hSOD1(G93A) mice reveals central and peripheral abnormalities that are not found in wild-type age-matched controls. Specifically, we observe aberrant endogenous pulsatile GH secretion, reduced pituitary GH content, and decreased circulating levels of IGF-I, indicating global GH deficiency in hSOD1(G93A) mice. Furthermore, a reduction in the expression of the IGF-I receptor α-subunit in skeletal muscle and lumbar spinal cords of hSOD1(G93A) mice suggests impaired IGF-I signaling within these tissues. This is the first account of disrupted GH secretion in a transgenic mouse model of ALS. These observations are essential for the development of effective GH and IGF-I targeted therapies in ALS.

Aims Adiponectin is an important adipokine whose levels are decreased in obesity despite increases in adipocyte mass. Studies in animal models implicate adiponectin as an insulin sensitizer in skeletal muscle and liver. Thiazolidinediones (TZDs) are insulin sensitizers and ligands for peroxisome proliferator-activated ? receptors (PPAR gamma) and these receptors are expressed in beta cells where their activation promotes cell survival. We hypothesize that adiponectin promotes beta cell survival by activating PPAR gamma. Methods We used MIN6 cells to investigate the effect of adiponectin on PPAR gamma expression, beta-cell proliferation, insulin synthesis and insulin secretion. Results We demonstrate that MIN6 cells contain adiponectin receptors and that adiponectin activates PPAR gamma mRNA and protein expression. This increase in PPAR gamma expression is blocked by the PPAR gamma antagonist, GW9662, indicating a transcriptional feedback loop involving PPAR gamma activation of itself. Adiponectin causes a significant increase in insulin content and secretion and this occurs also via PPAR gamma activation due to the inhibitory effect of GW9662. Adiponectin also promotes MIN6 cell proliferation, however, this effect is independent of PPAR gamma activation. Conclusions Our results identify novel roles for the adipokine, adiponectin, in beta-cells function. Adiponectin upregulates PPAR gamma expression, insulin content and insulin secretion through PPAR gamma-dependent mechanisms. Reductions in circulating adiponectin levels in obese individuals could therefore result in negative effects on beta-cell function and this may have direct relevance to beta-cell dysfunction in type 2 diabetes.

Fasting results in the mobilization of adipose stores and the elevation of levels of free fatty acids (FFA). In humans, this process is driven by a release in GH. Little is known regarding the role of GH in modulating this process during early stages of fasting in the mouse. Confirmation of the role of GH in modulating FFA release in the fasting mouse is of particular importance given the frequent use of mouse models to study metabolic mechanisms. Here, we correlate the initial release of FFA throughout fasting in mice with pulsatile GH secretion. Observations illustrate the rapid release of FFA in response to food withdrawal. This does not correlate with a rise in GH secretion. Rather, we observed a striking loss in pulsatile secretion of GH throughout the first 6 h of fasting, suggesting that GH does not modulate the initial release of FFA in the mouse in response to fasting. This was confirmed in GH receptor knockout mice, in which we observed a robust fasting-induced rise in FFA. We further illustrate the dynamic relationship between the orexigenic and anorexigenic hormones ghrelin and leptin during fasting in the mouse. Our findings show an initial suppression of leptin and the eventual rise in circulating levels of acyl-ghrelin with fasting. However, altered acyl-ghrelin and leptin secretion occurs well after the rise in FFA and the suppression of GH secretion. Consequently, we conclude that although acyl-ghrelin and leptin may modulate the physiological response to drive food intake, these changes do not contribute to the initial loss of pulsatile GH secretion. Rather, it appears that the suppression of GH secretion in fasting may occur in response to an elevation in fasting levels of FFA or physiological stress. Observations highlight a divergent role for GH in modulating FFA release between man and mouse.

Introduction: Among ICD recipients, AF predicts appropriate device therapy, but mechanisms underlying ventricular proarrhythmic are unclear. We hypothesised that irregular ventricular activation alters ion channel expression and cellular electrophysiology. Methods: mRNA expression of ion channels and accessory subunits was examined in neonatal rat ventricular myocytes (NVCMs) paced either regularly or irregularly for 24 hours to simulate ventricular activation in sinus rhythm (SR) and AF respectively. Relevant transmembrane currents were measured after 96 hours of regular vs irregular pacing using whole cell patch clamp approach. LV myocardium from end-stage heart failure (ESHF) patients in SR and chronic AF undergoing cardiac transplantation also underwent mRNA expression studies as guided by results from the NVCM model. Results: Irregular pacing induced a 48% increase in NVCM mRNA expression of KCNA4. However, there was no difference in Ito current density across pacing strategies. Irregular pacing induced trivial changes in NVCM mRNA expression for the Na channel SCN5A (11% reduction), and the Ca channels CACNA1C (19% increase), and CACNA1H (8% decrease). No differences were observed for ion channels CACNA1G, KCNQ1, KCNH2, KCND3, KCNJ2, and KCNJ11; accessory subunits KCNE1, KCNE2, KCNIP2, and ABCC9; and gap junction subunits GJA1 and GJA5. There were no differences in mRNA expression of SCN5A, CACNA1C, CACNA1H, or KCNA4 in LV myocardium from ESHF patients in AF vs SR. Conclusion: In an acute model, irregular ventricular activation associated with AF induces modest changes in mRNA expression of some ion channels, but fails to account for differences in electrophysiologic phenotype among patients with HF.

Measures of pulsatile GH secretion require frequent collection and analysis of blood samples at regular intervals. Due to blood volume constraints, repeat measures of circulating levels of GH in mice remain challenging. Consequently, few observations exist in which the pulsatile pattern of GH secretion in mice have been characterized. To address this, we developed a technique for the collection and analysis of circulating levels of GH at regular and frequent intervals in freely moving mice. This was achieved through the development of a sensitive assay for the detection of GH in small (2 μl) quantities of whole blood. The specificity and accuracy of this assay was validated following guidelines established for single-laboratory validation as specified by the International Union of Pure and Applied Chemistry. We incorporated an established method for tail-clip blood sample collection to determine circulating levels of GH secretion in 36 whole blood samples collected consecutively over a period of 6 h. Resulting measures were characterized by peak secretion periods and interpulse stable baseline secretion periods. Periods characterized by elevated whole blood GH levels consisted of multicomponent peaks. Deconvolution analysis of resulting measures confirmed key parameters associated with pulsatile GH secretion. We show a striking decrease in pulsatile GH secretion in mice after 12-18 h of fasting. This model is necessary to characterize the pulsatile profile of GH secretion in mice and will significantly contribute to current attempts to clarify mechanisms that contribute to the regulation of GH secretion.

Background: Despite adequate rate control, combination of AF with heart failure (HF) promotes HF progression and lethal arrhythmia. The role of ventricular irregularity remains ill-defined. We previously showed irregular pacing to induce a hypertrophic gene and protein expression profile in neonatal rat ventricular cardiomyocytes (NVCMs). Here, we examined cellular and molecular mechanisms underlying apparent alterations in LV contractile and electrical function induced by co-incident AF.

Obesity is associated with reduced levels of growth hormone (GH) and the disruption of pulsatile GH secretion. This results in relative GH deficiency. It is likely that a regulatory relationship between GH secretion and adipose tissue exists as the secretion of GH recovers to normal levels after a reduction in body weight. This report characterise the expression and interaction of adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) and adiponectin, respectively, in regulating the activity of GH secreting cells. Polymerase chain reaction analysis of the GH3 cell line, rat anterior pituitary gland and isolated somatotroph cells from transgenic GFP expressing mice confirmed the expression of both AdipoR1 and AdipoR2 in GH secretory cells. Because GH cells expressed both receptors, it is likely that the measured increase in GH secretion, observed in primary cultured rat pituitary cells after 30 min of incubation with full-length murine adiponectin, was mediated by a direct receptor regulated process. Adiponectin induced an increase in intracellular Ca2+ through both the influx of extracellular Ca2+ and the release of intracellular Ca2+ stores resulting in the secretion of GH. Furthermore, results confirm that this increase in GH secretion depended mainly on an increase in Ca2+ influx through L-type Ca2+ channels. It is concluded that adiponectin directly regulates GH secretion from somatotrophs by binding to either adiponectin receptor, and that this is mediated via a similar process observed after the stimulation of GH secretion by GH-releasing hormone.