Jyothis T. George

The kisspeptin-GnRH pathway in human reproductive health and disease

BACKGROUND The discovery of kisspeptin as key central regulator of GnRH secretion has led to a new level of understanding of the neuroendocrine regulation of human reproduction. The related discovery of the kisspeptin-neurokinin B-dynorphin (KNDy) pathway in the last decade has further strengthened our understanding of the modulation of GnRH secretion by endocrine, metabolic and environmental inputs. In this review, we summarize current understanding of the physiological roles of these novel neuropeptides, and discuss the clinical relevance of these discoveries and their potential translational applications. METHODS A systematic literature search was performed using PUBMED for all English language articles up to January 2014. In addition, the reference lists of all relevant original research articles and reviews were examined. This review focuses mainly on published human studies but also draws on relevant animal data. RESULTS Kisspeptin is a principal regulator of the secretion of gonadotrophins, and through this key role it is critical for the onset of puberty, the regulation of sex steroid-mediated feedback and the control of adult fertility. Although there is some sexual dimorphism, both neuroanatomically and functionally, these functions are apparent in both men and women. Kisspeptin acts upstream of GnRH and, following paracrine stimulatory and inhibitory inputs from neurokinin B and dynorphin (KNDy neuropeptides), signals directly to GnRH neurones to control pulsatile GnRH release. When administered to humans in different isoforms, routes and doses, kisspeptin robustly stimulates LH secretion and LH pulse frequency. Manipulation of the KNDy system is currently the focus of translational research with the possibility of future clinical application to regulate LH pulsatility, increasing gonadal sex steroid secretion in reproductive disorders characterized by decreased LH pulsatility, including hypothalamic amenorrhoea and hypogonadotropic hypogonadism. Conversely there may be scope to reduce the activity of the KNDy system to reduce LH secretion where hypersecretion of LH adds to the phenotype, such as in polycystic ovary syndrome. CONCLUSIONS Kisspeptin is a recently discovered neuromodulator that controls GnRH secretion mediating endocrine and metabolic inputs to the regulation of human reproduction. Manipulation of kisspeptin signalling has the potential for novel therapies in patients with pathologically low or high LH pulsatility.

Kisspeptin-10 Is a Potent Stimulator of LH and Increases Pulse Frequency in Men

CONTEXT Kisspeptins stimulate GnRH and thus gonadotropin secretion. Kisspeptin-10 is the minimal kisspeptin sequence with full intrinsic bioactivity, but it has not been studied in man. OBJECTIVE We investigated our hypothesis that kisspeptin-10 increases GnRH and thus LH pulse frequency. DESIGN AND PARTICIPANTS The dose response of kisspeptin-10 was investigated by administering iv bolus doses (0.01-3.0 μg/kg) and vehicle to healthy men. Effects on LH pulse frequency and size were determined by deconvolution analysis during infusion of kisspeptin-10 for up to 22.5 h. RESULTS Intravenous bolus kisspeptin-10 resulted in a rapid and dose-dependent rise in serum LH concentration, with maximal stimulation at 1 μg/kg (4.1 ± 0.4 to 12.4 ± 1.7 IU/liter at 30 min, P < 0.001, n = 6). Administration of 3 μg/kg elicited a reduced response vs. 1 μg/kg (P < 0.05). Infusion of kisspeptin-10 at 4 μg/kg · h for 22.5 h elicited an increase in LH from a mean of 5.4 ± 0.7 to 20.8 ± 4.9 IU/liter (n = 4; P < 0.05) and serum testosterone increased from 16.6 ± 2.4 to 24.0 ± 2.5 nmol/liter (P < 0.001). LH pulses were obscured at this high rate of secretion, but a lower dose infusion of kisspeptin-10 (1.5 μg/kg · h) increased mean LH from 5.2 ± 0.8 to 14.1 ± 1.7 IU/liter (n = 4; P < 0.01) and increased LH pulse frequency from 0.7 ± 0.1 to 1.0 ± 0.2 pulses/h (P < 0.05) and secretory burst mass from 3.9 ± 0.4 to 12.8 ± 2.6 IU/liter (P < 0.05). CONCLUSIONS Kisspeptin-10 boluses potently evoke LH secretion in men, and continuous infusion increases testosterone, LH pulse frequency, and pulse size. Kisspeptin analogues have therapeutic potential as regulators of LH and thus testosterone secretion.

Kisspeptin Restores Pulsatile LH Secretion in Patients with Neurokinin B Signaling Deficiencies: Physiological, Pathophysiological and Therapeutic Implications

Pulsatile gonadotropin-releasing hormone (GnRH) is crucial to normal reproductive function and abnormalities in pulse frequency give rise to reproductive dysfunction. Kisspeptin and neurokinin B (NKB), neuropeptides secreted by the same neuronal population in the ventral hypothalamus, have emerged recently as critical central regulators of GnRH and thus gonadotropin secretion. Patients with mutations resulting in loss of signaling by either of these neuroendocrine peptides fail to advance through puberty but the mechanisms mediating this remain unresolved. We report here that continuous kisspeptin infusion restores gonadotropin pulsatility in patients with loss-of-function mutations in NKB (TAC3) or its receptor (TAC3R), indicating that kisspeptin on its own is sufficient to stimulate pulsatile GnRH secretion. Moreover, our findings suggest that NKB action is proximal to kisspeptin in the reproductive neuroendocrine cascade regulating GnRH secretion, and may act as an autocrine modulator of kisspeptin secretion. The ability of continuous kisspeptin infusion to induce pulsatile gonadotropin secretion further indicates that GnRH neurons are able to set up pulsatile secretion in the absence of pulsatile exogenous kisspeptin.

Hypothesis: Kisspeptin Mediates Male Hypogonadism in Obesity and Type 2 Diabetes

Hypogonadism occurs commonly in men with type 2 diabetes (T2DM) and severe obesity. Current evidence points to a decreased secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus and thereby decreased secretion of gonadotropins from the pituitary gland as a central feature of the pathophysiology in these men. Hyperglycaemia, inflammation, leptin and oestrogen-related feedback have been proposed to make aetiological contributions to the hypogonadotropic hypogonadism of T2DM. However, the neuroendocrine signals that link these factors with modulation of GnRH neurons have yet to be identified. Kisspeptins play a central role in the modulation of GnRH secretion and, thus, downstream regulation of gonadotropins and testosterone secretion in men. Inactivating mutations of the kisspeptin receptor have been shown to cause hypogonadotropic hypogonadism in man, whilst an activating mutation is associated with precocious puberty. Data from studies in experimental animals link kisspeptin expression with individual factors known to regulate GnRH secretion, including hyperglycaemia, inflammation, leptin and oestrogen. We therefore hypothesise that decreased endogenous kisspeptin secretion is the common central pathway that links metabolic and endocrine factors in the pathology of testosterone deficiency seen in men with obesity and T2DM. We propose that the kisspeptin system plays a central role in integrating a range of metabolic inputs, thus constituting the link between energy status with the hypothalamic-pituitary-gonadal axis, and put forward potential clinical studies to test the hypothesis.

Exploring the pathophysiology of hypogonadism in men with type 2 diabetes: Kisspeptin‐10 stimulates serum testosterone and LH secretion in men with type 2 diabetes and mild biochemical hypogonadism

Low serum testosterone is commonly observed in men with type 2 diabetes (T2DM), but the neuroendocrine pathophysiology remains to be elucidated.

Molecular clocks, type 2 diabetes and cardiovascular disease

The westernised world is in the midst of an epidemic of type 2 diabetes and associated cardiovascular disease. These closely interlinked conditions have a common pathophysiological basis underpinned by insulin resistance and the metabolic syndrome. Contemporary changes in environmental factors on a background of genetic susceptibility are thought to account for the increases seen. Life on earth is governed by the 24-hour environment of light and darkness cycling with the rotation of the earth. Numerous metabolic and physiological pathways are coordinated to this 24-hour cycle by an endogenous clock. Recent epidemiological evidence and animal data suggest that disturbance of circadian rhythms through genetic and environmental influences on the molecular clock is pivotal in the pathogenesis of obesity, type 2 diabetes and cardiovascular disease. This review describes current knowledge on the topic.

Kisspeptin antagonists: unraveling the role of kisspeptin in reproductive physiology.

Kisspeptin has recently been identified as a key neuroendocrine gatekeeper of reproduction and is essential for the initiation of human puberty and maintenance of adult reproduction. Kisspeptin neurons appear to be integrative sensors, as they respond to changes in numerous internal and external factors including nutrient and fat status, stress and sex steroids, thus providing a link between these factors and reproduction. We have pioneered the development of kisspeptin antagonists as powerful tools for interrogating the role of kisspeptin in reproductive physiology and pathology, and as potential treatments for hormone-dependent disease. This article summarizes their development and key findings to date. These demonstrate an essential role for kisspeptin in GnRH neuron firing, GnRH pulsatile secretion, negative feedback by gonadal steroids, the onset of puberty, and the ovulatory LH surge. These studies establish that kisspeptin antagonists are powerful investigative tools and set the scene for more extensive physiological and pathophysiological studies as well as therapeutic intervention.

Rationale and design of the EXenatide study of cardiovascular event lowering (EXSCEL) trial

Exenatide once-weekly is an extended release formulation of exenatide, a glucagon-like peptide-1 receptor agonist, which can improve glycemic control, body weight, blood pressure, and lipid levels in patients with type 2 diabetes mellitus (T2DM). The EXenatide Study of Cardiovascular Event Lowering (EXSCEL) will compare the impact of adding exenatide once-weekly to usual care with usual care alone on major cardiovascular outcomes. EXSCEL is an academically led, phase III/IV, double-blind, pragmatic placebo-controlled, global trial conducted in 35 countries aiming to enrol 14,000 patients with T2DM and a broad range of cardiovascular risk over approximately 5 years. Participants will be randomized (1:1) to receive exenatide once-weekly 2 mg or matching placebo by subcutaneous injections. The trial will continue until 1,360 confirmed primary composite cardiovascular end points, defined as cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke, have occurred. The primary efficacy hypothesis is that exenatide once-weekly is superior to usual care with respect to the primary composite cardiovascular end point. EXSCEL is powered to detect a 15% relative risk reduction in the exenatide once-weekly group, with 85% power and a 2-sided 5% alpha. The primary safety hypothesis is that exenatide once-weekly is noninferior to usual care with respect to the primary cardiovascular composite end point. Noninferiority will be concluded if the upper limit of the CI is <1.30. EXSCEL will assess whether exenatide once-weekly can reduce cardiovascular events in patients with T2DM with a broad range of cardiovascular risk. It will also provide long-term safety information on exenatide once-weekly in people with T2DM. ClinicalTrials.gov Identifier: NCT01144338.

Kisspeptin and the Hypothalamic Control of Reproduction: Lessons from the Human

The hypothalamic hormone GnRH is a central driver of pituitary gonadotropin secretion, controlling pulsatile gonadotropin secretion, modulating gonadal steroid feedback, and bringing about full fertility in the adult. Thus, understanding GnRH neuronal regulation is essential to understanding the neurohumoral control of human reproduction. Genetic tools were used in patients with GnRH deficiency (i.e. idiopathic hypogonadotropic hypogonadism), a clinical syndrome that results from the failure of a normal pattern of pulsatile GnRH, to discover upstream modulators of GnRH secretion (1). In 2003, homozygosity mapping of two consanguineous pedigrees led to the identification of loss of function mutations in KISS1R (a G protein coupled receptor) by two groups (2, 3). In parallel, the Kiss1r(-/-) mouse was shown to be a phenocopy of the human GnRH-deficient state, demonstrating that the function of KISS1R/Kiss1r is conserved across mammalian species (4). Just before these human genetic discoveries, the ligand for kisspeptin-1 receptor [KISS1R; also known as G protein coupled receptor 54 (GPR54)], was discovered to be kisspeptin. Soon thereafter a large array of experimental studies began assembling genetic, expression, physiologic, transgenic, knockdown, and electrophysiological data to characterize the physiology of kisspeptin and its seminal role in modulating GnRH release.

Empagliflozin and Clinical Outcomes in Patients With Type 2 Diabetes Mellitus, Established Cardiovascular Disease, and Chronic Kidney Disease

Background: Empagliflozin, a sodium-glucose cotransporter 2 inhibitor, reduced cardiovascular morbidity and mortality in patients with type 2 diabetes mellitus and established cardiovascular disease in the EMPA-REG OUTCOME trial (Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients). Urinary glucose excretion with empagliflozin decreases with declining renal function, resulting in less potency for glucose lowering in patients with kidney disease. We investigated the effects of empagliflozin on clinical outcomes in patients with type 2 diabetes mellitus, established cardiovascular disease, and chronic kidney disease. Methods: Patients with type 2 diabetes mellitus, established cardiovascular disease, and estimated glomerular filtration rate (eGFR) ≥30 mL·min−1·1.73 m−2 at screening were randomized to receive empagliflozin 10 mg, empagliflozin 25 mg, or placebo once daily in addition to standard of care. We analyzed cardiovascular death, hospitalization for heart failure, all-cause hospitalization, and all-cause mortality in patients with prevalent kidney disease (defined as eGFR <60 mL·min−1·1.73 m−2 and/or urine albumin-creatinine ratio >300 mg/g) at baseline. Additional analyses were performed in subgroups by baseline eGFR (<45, 45–<60, 60–<90, ≥90 mL·min−1·1.73 m−2) and baseline urine albumin-creatinine ratio (>300, 30–⩽300, <30 mg/g). Results: Of 7020 patients treated, 2250 patients had prevalent kidney disease at baseline, of whom 67% had a diagnosis of type 2 diabetes mellitus for >10 years, 58% were receiving insulin, and 84% were taking angiotensin-converting enzyme inhibitors or angiotensin receptor blockers. In patients with prevalent kidney disease at baseline, empagliflozin reduced the risk of cardiovascular death by 29% compared with placebo (hazard ratio [HR], 0.71; 95% confidence interval [CI], 0.52–0.98), the risk of all-cause mortality by 24% (HR, 0.76; 95% CI, 0.59–0.99), the risk of hospitalization for heart failure by 39% (HR, 0.61; 95% CI, 0.42–0.87), and the risk of all-cause hospitalization by 19% (HR, 0.81; 95% CI, 0.72–0.92). Effects of empagliflozin on these outcomes were consistent across categories of eGFR and urine albumin-creatinine ratio at baseline and across the 2 doses studied. The adverse event profile of empagliflozin in patients with eGFR <60 mL·min−1·1.73 m−2 was consistent with the overall trial population. Conclusions: Empagliflozin improved clinical outcomes and reduced mortality in vulnerable patients with type 2 diabetes mellitus, established cardiovascular disease, and chronic kidney disease. Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01131676.