Waljit S. Dhillo

Post-embryonic ablation of AgRP neurons in mice leads to a lean, hypophagic phenotype

Agouti‐related protein (AgRP) and neuropeptide Y (NPY) are colocalized in arcuate nucleus (arcuate) neurons implicated in the regulation of energy balance. Both AgRP and NPY stimulate food intake when administered into the third ventricle and are up‐regulated in states of negative energy balance. However, mice with targeted deletion of either NPY or AgRP or both do not have major alterations in energy homeostasis. Using bacterial artificial chromosome (BAC) transgenesis we have targeted expression of a neurotoxic CAG expanded form of ataxin‐3 to AgRP‐expressing neurons in the arcuate. This resulted in a 47% loss of AgRP neurons by 16 weeks of age, a significantly reduced body weight, (wild‐type mice (WT) 34.7±0.7 g vs. transgenic mice (Tg) 28.6±0.6 g, P<0.001), and reduced food intake (WT 5.0±0.2 vs. Tg 3.6±0.1 g per day, P<0.001). Transgenic mice had significantly reduced total body fat, plasma insulin, and increased brown adipose tissue UCP1 expression. Transgenic mice failed to respond to peripherally administered ghrelin but retained sensitivity to PYY 3‐36. These data suggest that postembryonic partial loss of AgRP/NPY neurons leads to a lean, hypophagic phenotype.

The Gut Hormones PYY3-36 and GLP-17-36 amide Reduce Food Intake and Modulate Brain Activity in Appetite Centers in Humans

Summary Obesity is a major public health issue worldwide. Understanding how the brain controls appetite offers promising inroads toward new therapies for obesity. Peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) are coreleased postprandially and reduce appetite and inhibit food intake when administered to humans. However, the effects of GLP-1 and the ways in which PYY and GLP-1 act together to modulate brain activity in humans are unknown. Here, we have used functional MRI to determine these effects in healthy, normal-weight human subjects and compared them to those seen physiologically following a meal. We provide a demonstration that the combined administration of PYY3-36 and GLP-17-36 amide to fasted human subjects leads to similar reductions in subsequent energy intake and brain activity, as observed physiologically following feeding.

The kisspeptin system of the human hypothalamus: sexual dimorphism and relationship with gonadotropin-releasing hormone and neurokinin B neurons.

Kisspeptin signaling via the kisspeptin receptor G‐protein‐coupled receptor‐54 plays a fundamental role in the onset of puberty and the regulation of mammalian reproduction. In this immunocytochemical study we addressed the (i) topography, (ii) sexual dimorphism, (iii) relationship to gonadotropin‐releasing hormone (GnRH) neurons and (iv) neurokinin B content of kisspeptin‐immunoreactive hypothalamic neurons in human autopsy samples. In females, kisspeptin‐immunoreactive axons formed a dense periventricular plexus and profusely innervated capillary vessels in the infundibular stalk. Most immunolabeled somata occurred in the infundibular nucleus. Many cells were also embedded in the periventricular fiber plexus. Rostrally, they formed a prominent periventricular cell mass (magnocellular paraventricular nucleus). Robust sex differences were noticed in that fibers and somata were significantly less numerous in male individuals. In dual‐immunolabeled specimens, fine kisspeptin‐immunoreactive axon varicosities formed axo‐somatic, axo‐dendritic and axo‐axonal contacts with GnRH neurons. Dual‐immunofluorescent studies established that 77% of kisspeptin‐immunoreactive cells in the infundibular nucleus synthesize the tachykinin peptide neurokinin B, which is known to play crucial role in human fertility; 56 and 17% of kisspeptin fibers in the infundibular and periventricular nuclei, respectively, contained neurokinin B immunoreactivity. Site‐specific co‐localization patterns implied that kisspeptin neurons in the infundibular nucleus and elsewhere contributed differentially to these plexuses. This study describes the distribution and robust sexual dimorphism of kisspeptin‐immunoreactive elements in human hypothalami, reveals neuronal contacts between kisspeptin‐immunoreactive fibers and GnRH cells, and demonstrates co‐synthesis of kisspeptins and neurokinin B in the infundibular nucleus. The neuroanatomical information will contribute to our understanding of central mechanisms whereby kisspeptins regulate human fertility.

Subcutaneous Injection of Kisspeptin-54 Acutely Stimulates Gonadotropin Secretion in Women with Hypothalamic Amenorrhea, But Chronic Administration Causes Tachyphylaxis

BACKGROUND Kisspeptin is a critical regulator of normal reproductive function. A single injection of kisspeptin in healthy human volunteers potently stimulates gonadotropin release. However, the effects of kisspeptin on gonadotropin release in women with hypothalamic amenorrhea (HA) and the effects of repeated administration of kisspeptin to humans are unknown. AIM The aim of this study was to determine the effects of acute and chronic kisspeptin administration on gonadotropin release in women with HA. METHODS We performed a prospective, randomized, double-blinded, parallel design study. Women with HA received twice-daily sc injections of kisspeptin (6.4 nmol/kg) or 0.9% saline (n = 5 per group) for 2 wk. Changes in serum gonadotropin and estradiol levels, LH pulsatility, and ultrasound measurements of reproductive activity were assessed. RESULTS On the first injection day, potent increases in serum LH and FSH were observed after sc kisspeptin injection in women with HA (mean maximal increment from baseline within 4 h after injection: LH, 24.0 +/- 3.5 IU/liter; FSH, 9.1 +/- 2.5 IU/liter). These responses were significantly reduced on the 14th injection day (mean maximal increment from baseline within 4 h postinjection: LH, 2.5 +/- 2.2 IU/liter, P < 0.05; FSH, 0.5 +/- 0.5 IU/liter, P < 0.05). Subjects remained responsive to GnRH after kisspeptin treatment. No significant changes in LH pulsatility or ultrasound measurements of reproductive activity were observed. CONCLUSION Acute administration of kisspeptin to women with infertility due to HA potently stimulates gonadotropin release, but chronic administration of kisspeptin results in desensitization to its effects on gonadotropin release. These data have important implications for the development of kisspeptin as a novel therapy for reproductive disorders in humans.

Gut hormones and appetite control

The gastrointestinal tract is the largest endocrine organ in the body. It secretes more than 20 different peptide hormones, which serve both a local regulatory function and provide a means by which the gut can regulate appetite and satiety. As the worldwide prevalence of obesity reaches epidemic proportions, the importance of delineating the mechanisms which regulate food intake becomes even more urgent. There is now a substantial body of work in both rodent and human models demonstrating the effects of these peptides on appetite and work is underway to therapeutically manipulate the gut-brain axis for the treatment of obesity. In addition, it may also be possible to use our understanding of the entero-endocrine system to treat calorie-deficient states.

Twice‐Weekly Administration of Kisspeptin‐54 for 8 Weeks Stimulates Release of Reproductive Hormones in Women With Hypothalamic Amenorrhea

Kisspeptin is a novel therapeutic target for infertility. A single kisspeptin‐54 (KP‐54) injection acutely stimulates the release of reproductive hormones in women with hypothalamic amenorrhea (HA), a commonly occurring condition characterized by absence of menstruation; however, twice‐daily administration of KP‐54 results in tachyphylaxis. We determined the time course of desensitization to twice‐daily KP‐54 injections, compared the effects of twice‐daily and twice‐weekly administration regimens of KP‐54, and studied the effects of long‐term twice‐weekly administration of KP‐54 on the release of reproductive hormones in women with HA. When KP‐54 was administered twice daily, responsiveness to luteinizing hormone (LH) diminished gradually, whereas responsiveness to follicle‐stimulating hormone (FSH) was nearly abolished by day 2. Twice‐weekly KP‐54 administration resulted in only partial desensitization, in contrast to the complete tolerance achieved with twice‐daily administration. Women with HA who were treated with twice‐weekly KP‐54 injections had significantly elevated levels of reproductive hormones after 8 weeks as compared with treatment with saline. No adverse effects were observed. This study provides novel pharmacological data on the effects of KP‐54 on the release of reproductive hormones in women with HA.

Differential patterns of neuronal activation in the brainstem and hypothalamus following peripheral injection of GLP-1, oxyntomodulin and lithium chloride in mice detected by manganese-enhanced magnetic resonance imaging (MEMRI)

We have used manganese-enhanced magnetic resonance imaging (MEMRI) to show distinct patterns of neuronal activation within the hypothalamus and brainstem of fasted mice in response to peripheral injection of the anorexigenic agents glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM) and lithium chloride. Administration of both GLP-1 and OXM resulted in a significant increase in signal intensity (SI) in the area postrema of fasted mice, reflecting an increase in neuronal activity within the brainstem. In the hypothalamus, GLP-1 administration induced a significant reduction in SI in the paraventricular nucleus and an increase in the ventromedial hypothalamic nucleus whereas OXM reduced SI in the arcuate and supraoptic nuclei of the hypothalamus. These data indicate that whilst these related peptides both induce a similar effect on neuronal activity in the brainstem they generate distinct patterns of activation within the hypothalamus. Furthermore, the hypothalamic pattern of signal intensity generated by GLP-1 closely matches that generated by peripheral injection of LiCl, suggesting the anorexigenic effects of GLP-1 may be in part transmitted via nausea circuits. This work provides a framework by which the temporal effects of appetite modulating agents can be recorded simultaneously within hypothalamic and brainstem feeding centres.

Effect of direct injection of melanin-concentrating hormone into the paraventricular nucleus: Further evidence for a stimulatory role in the adrenal axis via SLC-1

Melanin‐concentrating hormone (MCH) is implicated in the control of a number of hormonal axes including the hypothalamic‐pituitary adrenal (HPA) axis. Previous studies have shown that there is evidence for both a stimulatory and an inhibitory action on the HPA axis; therefore, we attempted to further characterize the effects of MCH on this axis. Intracerebroventricular injection of MCH increased circulating adrenocorticotropic hormone (ACTH) at 10 min post injection. Injection of MCH directly into the paraventricular nucleus (PVN) was found to increase both circulating ACTH and corticosterone 10 min after injection. Additionally, MCH was found to increase corticotropin‐releasing factor (CRF) release from hypothalamic explants, and this effect was abolished by the specific SLC‐1 antagonist SB‐568849. Neuropeptide EI, a peptide from the same precursor as MCH was also found to increase CRF release from explants. These results suggest that MCH has a stimulatory role in the HPA axis via SLC‐1, and that MCH exerts its effects predominantly through the PVN CRF neuronal populations

The hypothalamic melanocortin system stimulates the hypothalamo-pituitary-adrenal axis in vitro and in vivo in male rats.

α-Melanocyte-stimulating hormone (α-MSH) is an agonist, and agouti-related protein (Agrp) an endogenous antagonist at the melanocortin 3 and 4 receptors which are found in the central nervous system (CNS). We have examined the effect of α-MSH and Agrp on the hypothalamo-pituitary-adrenal (HPA) axis in vitro and in vivo in male rats. Intraparaventricular nuclear (iPVN) injection of [Nle4,D-Phe7]-α-MSH (NDP-MSH) (a long-acting α-MSH analogue) increased plasma adrenocorticotropic hormone (ACTH) (10 min post-injection: 25.0 ± 3.9 vs. saline 10.9 ± 2.0, p < 0.05) and plasma corticosterone (10 min post-injection: 174.1 ± 14.2 vs. saline 124.7 ± 16.3 ng/ml, p < 0.05). iPVN injection of Agrp(83–132) increased plasma ACTH (24.2 ± 4.0 vs. saline 10.1 ± 1.0 pg/ml, p < 0.01). The combination of NDP-MSH and Agrp(83–132) administered iPVN significantly increased plasma ACTH (10 min post-injection: 21.3 ± 3.8 vs. 10.9 ± 2.0, p < 0.05) and plasma corticosterone (10 min post-injection: 169.0 ± 15.1 vs. saline 124.7 ± 16.3 ng/ml, p < 0.05), but there was no additive effect. Hypothalamic explants treated with α-MSH (100 nM) resulted in a 159 ± 23% increase in corticotropin-releasing hormone (CRH) release (p < 0.01) and 175 ± 12% increase in arginine vasopressin (AVP) release (p < 0.001) compared to basal. Agrp(83–132) (100 nM) administered to hypothalamic explants resulted in a 161 ± 20% increase in CRH (p < 0.01) and 174 ± 13% increase in AVP release (p < 0.001) compared to basal. Hypothalamic explants treated with the combination of α-MSH and Agrp(83–132) (100 nM) resulted in a 179 ± 31% increase in CRH release (p < 0.01) and 130 ± 9% increase in AVP release (p < 0.01) compared to basal, but there was no additive effect. This is the first report that both α-MSH and Agrp(83–132) stimulate the HPA axis. The combination of α-MSH and Agrp(83–132) has no additive effect in vitro and in vivo in male rats. These results suggest that there may be another receptor independent of the known melanocortin receptors at which Agrp is acting.

The Effects of Kisspeptin-10 on Reproductive Hormone Release Show Sexual Dimorphism in Humans

BACKGROUND Kisspeptin peptides are critical in human reproductive physiology and are potential therapies for infertility. Kisspeptin-10 stimulates gonadotropin release in both male and female rodents. However, few studies have investigated the effects of kisspeptin-10 on gonadotropin release in humans, and none have investigated the effect in women. If kisspeptin is to be useful for treating reproductive disease, its effects in both men and women must be established. AIM To compare the effects of kisspeptin-10 administration on reproductive hormone release in healthy men and women. METHODS Intravenous bolus kisspeptin-10 was administered to men and women (n = 4-5 per group). Subcutaneous bolus and i.v. infusion of kisspeptin-10 was also administered to female women (n = 4-5 per group). Circulating reproductive hormones were measured. RESULTS In healthy men, serum LH and FSH were elevated after i.v. bolus kisspeptin-10, at doses as low as 0.3 and 1.0 nmol/kg, respectively. In healthy women during the follicular phase of the menstrual cycle, no alterations in serum gonadotropins were observed after i.v. bolus, s.c. bolus, or i.v. infusion of kisspeptin-10 at maximal doses of 10 nmol/kg, 32 nmol/kg, and 720 pmol/kg/min, respectively. In women during the preovulatory phase, serum LH and FSH were elevated after i.v. bolus kisspeptin-10 (10 nmol/kg). CONCLUSION Kisspeptin-10 stimulates gonadotropin release in men as well as women during the preovulatory phase of menstrual cycle but fails to stimulate gonadotropin release in women during the follicular phase. The sexual dimorphism of the responsiveness of healthy men and women to kisspeptin-10 administration has important clinical implications for the potential of kisspeptin-10 to treat disorders of reproduction.