Ralf Nass

Novel ghrelin assays provide evidence for independent regulation of ghrelin acylation and secretion in healthy young men.

CONTEXT Ghrelin, an acylated peptide hormone secreted from the gut, regulates appetite and metabolism. Elucidating its pattern of secretion in the fed and fasted states is important in the face of the obesity epidemic. OBJECTIVE Our objective was to examine changes in circulating ghrelin and des-acyl ghrelin in response to meals and fasting using newly developed two-site sandwich assays and sample preservation protocols to allow specific detection of full-length forms. DESIGN Ten-minute sampling was done for 26.5 h during a fed admission with standardized meals and on a separate admission during the final 24 h of a 61.5-h fast and continuing for 2.5 h after terminating the fast. SETTING The study was conducted at the University Hospital General Clinical Research Center. PARTICIPANTS Eight male volunteers participated, mean +/- sd age 24.5 +/- 3.7 yr and body mass index 24 +/- 2.1 kg/m(2). MAIN OUTCOME MEASURES Ten-minute sampling profiles were assessed for ghrelin and des-acyl ghrelin, fed and fasting. RESULTS In the fed state, ghrelin and des-acyl ghrelin showed similar dynamics; both were sharply inhibited by meals and increased at night. During fasting, ghrelin decreased to nadir levels seen postprandially, and des-acyl ghrelin remained near peak levels seen preprandially. Total full-length ghrelin (acyl plus des-acyl) levels remained unchanged. CONCLUSIONS Meals inhibited secretion of both ghrelin and des-acyl ghrelin, yet long-term fasting inhibited acylation but not total secretion. Acylation may be regulated independently of secretion by nutrient availability in the gut or by esterases that cleave the acyl group. These studies highlight the importance of stringent conditions for sample collection and evaluation of full-length ghrelin and des-acyl ghrelin using specific two-site assays.

Effects of an oral ghrelin mimetic on body composition and clinical outcomes in healthy older adults: a randomized trial.

Context The age-related decline of growth hormone secretion may play a role in sarcopenia and frailty. Content In this randomized trial, 65 healthy older adults were assigned to receive placebo or MK-677, an oral ghrelin mimetic that increased pulsatile growth hormone secretion to young-adult levels. Over 1 year, lean fat-free mass increased 1.1 kg with MK-677 and decreased 0.5 kg with placebo. MK-677 did not affect strength and function, but insulin sensitivity declined and mean serum glucose levels increased 0.28 mmol/L (5 mg/dL). Caution This short-term trial was underpowered to detect functional changes and adverse events. Implication An oral ghrelin mimetic increases pulsatile growth hormone secretion and alters body composition in healthy older adults. The Editors Aging is an inevitable process across all species. In humans, muscle mass declines after reaching its peak in the third decade of life. Muscle mass is important for physical fitness and metabolic regulation; sarcopenia is a major risk factor for frailty, loss of independence, and physical disability in elderly persons (1) and is associated with shortened survival in critically ill patients (2). As lifespans increase, more adults are becoming frail and dependent on others, which creates challenges for them, their families, and society. The decrease in fat-free mass correlates with the aging-associated decrease in growth hormone secretion (3, 4). Aging adults show decreases in fat-free mass and growth hormone secretion similar to those seen in growth hormonedeficient young adults (5). By the eighth decade, men and women lose approximately 7 and 3.8 kg of muscle mass, respectively (3), and gain intra-abdominal fat (6, 7). Previous trials in which growth hormone was administered to elderly persons were small, poorly controlled, or too short (8); in addition, growth hormone replacement does not restore pulsatile growth hormone secretion. MK-677, the first orally active ghrelin mimetic (a growth hormone secretagogue and growth hormone secretagoguereceptor agonist), increases pulsatile growth hormone secretion in older adults to levels observed in young adults (9, 10). Our primary objectives were to determine whether 25 mg of oral MK-677 daily would increase growth hormone and insulin-like growth factor I (IGF-I) levels in healthy older adults, prevent the decline in fat-free mass, and decrease abdominal visceral fat, with acceptable tolerability. Methods Design The General Clinical Research Center (GCRC) and the University of Virginia institutional review boards approved this study. All participants gave written informed consent. We performed a 2-year, randomized, double-blind, modified crossover trial in which healthy older men, women receiving hormone replacement therapy, and women not receiving hormone replacement therapy received oral MK-677, 25 mg, or placebo (in a 2:1 ratio) daily. After 1 year, participants receiving MK-677 were randomly assigned to continue receiving MK-677 (group 1) or change to placebo (group 2); participants receiving placebo were given MK-677 during year 2 (group 3). Appendix Figure 1 shows the study design. Supplement. Appendix Materials Setting and Participants We recruited healthy volunteers older than 60 years of age from the general population by advertisement and screened them by medical history, physical examination, and laboratory testing to rule out underlying disease. Exclusion criteria were body mass index greater than 35 kg/m2, strenuous exercise for more than 60 minutes per day, smoking, diabetes, history of cancer (other than some types of skin cancer), untreated hypertension or thyroid disease, or medications known to affect growth hormone secretion. We asked participants to maintain their typical diet and exercise throughout the study and to report any illnesses, medical procedures, or adverse effects. All participants were white, with the exception of 1 Hispanic and 1 African-American man. At baseline and every 6 months for 2 years, we admitted participants to the GCRC for measurement of body composition, body water, lipids, and bone mineral density; frequent blood sampling; and completion of quality-of-life questionnaires. We also performed tests of strength and function. During GCRC admissions, we standardized meals for caloric and nutrient content. Blood samples for growth hormone were drawn through an indwelling venous cannula every 10 minutes for 24 hours; participants were allowed to sleep after 9 p.m. Randomization and Intervention MK-677 and placebo tablets were provided by Merck Research Laboratories (Rahway, New Jersey) in a blinded manner and stored by a research pharmacist and dispensed in a blinded manner according to a randomization table with stratification for sex and hormone replacement therapy. Ten-mg tablets were provided for blinded back-titration. Participants were instructed to take the placebo or MK-677 tablets once daily between 7:00 and 9:00 a.m. (or at 9:00 a.m. during admissions). All research staff and volunteers remained blinded throughout the study and during data verification. We monitored adherence by pill counts. Outcome Measures We measured serum growth hormone and IGF-I levels in duplicate in the GCRC Core Laboratory. We assessed 24-hour mean growth hormone and endogenous growth hormone secretory dynamics by using the cluster method (11) and an automated multiple-parameter deconvolution method (9, 12). The Appendix provides details of all assay methods. We evaluated fat-free mass and total body fat by using a 4-compartment model (13) and dual x-ray absorptiometry (DXA) on a Hologic QDR-2000 (Hologic, Bedford, Massachusetts) in pencil-beam mode (14). Dual x-ray absorptiometry measurements included appendicular lean soft tissue of the arms and legs as an estimate of total appendicular skeletal muscle mass (TASM) (15); appendicular fat; and bone mineral density of the femoral neck, spine (L2L4), and total hip. We divided the DXA TASM estimates by height in square meters (TASM [kg]/m2) (15). We used this index of relative limb muscle mass to compute a T-score for each individual, relating the TASM/m2 to that of sex-concordant young adults (16). We defined sarcopenia as values more than 2 SD below values in young, sex-specific reference populations (17, 18). We used cross-sectional computed tomographic images to measure the areas of abdominal visceral and subcutaneous fat and midthigh skeletal muscle at predefined anatomical locations (19); we excluded data if the subsequent scan location differed or we had technical difficulties (4 placebo group recipients and 3 MK-677 recipients). One blinded observer analyzed the dual x-ray absorptiometry and computed tomographic scans. We measured total body water by using the deuterium oxide dilution technique (20) and extracellular water by using bromide dilution (21). We assessed intracellular water as the difference between total body water and extracellular water. To determine the relative relationships among total, extracellular, and intracellular water, we expressed each component in terms of kilograms of fat-free mass at each point. We chose the scale of measure for the analysis a priori. We also report the raw data in typical units for comparison. We determined concentric force during flexion and extension of the knee and shoulder every 6 months by using a Cybex II isokinetic dynamometer (CSM, Boston, Massachusetts). Participants performed 6 repetitions of maximal effort over 90 degrees at 60 degrees/s, and the mean of the last 5 repetitions was computed by using proprietary software (22). We calculated total work by multiplying the mean per repetition by 5. Function tests performed every 6 months included walking 30 meters as quickly as possible (best of 2 trials), walking as far as possible in 6 minutes on an indoor track, descending and ascending 4 flights of stairs, and rising and sitting 5 times from an armless chair with an 18-inch seat height. To compensate for differences in muscle mass between men and women, we analyzed all strength and function measurements in terms of kilograms of baseline appendicular skeletal muscle (lean) from DXA. We used arm lean and leg lean for shoulder and knee strength, respectively, and baseline TASM (sum of arms and legs) for the function tests. We chose the scale of measure used in this analysis a priori; the raw data are also reported. Participants completed 4 questionnaires every 6 months to assess quality of life and general well-being: the 20-item Short Form Health Survey, the Beck Depression Inventory, the Pittsburgh Sleep Quality Index, and the Body Cathexis Scale. The Appendix provides additional details of quality of life, muscle strength, and function assessments. We measured cholesterol, cortisol, and insulin sensitivity (estimated by the Quicki Index method [23] from fasting insulin and glucose) every 6 months. To determine whether the effects of MK-677 treatment were sustained for 2 years or reversed when changed to placebo, we analyzed several end points in a subgroup of participants who completed 24 months in each of the 3 treatment groups (Figure 1). Figure 1. Study flow diagram. FBG= fasting blood glucose; HRT= hormone replacement therapy; MI= myocardial infarction. Monitoring for Adverse Effects Each year, volunteers were seen monthly for the first 3 months and every 3 months thereafter for a physical examination, documentation of medications and vital signs, and questioning about side effects and overall well-being. We performed a complete blood count and chemistry panel and monitored levels of hemoglobin A1c (HbA1c) and fasting blood glucose in all participants and prostate-specific antigen and testosterone in men. Women received annual Papanicolaou smears and mammography. Statistical Analysis The 2 primary end points were fat-free mass and abdominal visceral fat. The study was powered for the pivotal first 12 months; the Appendix describes the power analysis in detail.

Evidence for Acyl-Ghrelin Modulation of Growth Hormone Release in the Fed State

CONTEXT The timing and frequency of GH secretory episodes is regulated by GHRH and somatostatin. This study provides evidence for amplification of these GH pulses by endogenous acyl-ghrelin. DESIGN Blood was sampled every 10 min for 26.5 h during a fed admission with standardized meals and also during the final 24 h of a 61.5-h fast. GH secretion profiles were derived from deconvolution of 10-min sampling data, and full-length acyl-ghrelin levels were measured using a newly developed two-site sandwich assay. SETTING The study was conducted at a university hospital general clinical research center. PARTICIPANTS Participants included eight men with mean (+/- sd) age 24.5 +/- 3.7 yr (body mass index 24 +/- 2.1 kg/m(2)). RESULTS Correlations were computed between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-min interval preceding each GH burst. In the fed state, the peak correlations were positive for all subjects and significantly higher than in the fasting state when acyl-ghrelin levels declined [mean (+/- sem): 0.7 (0.04) vs. 0.29 (0.08), P = 0.017]. In addition, long-term fasting was associated with an increase in the GH secretory pulse mass and amplitude but not frequency [fed vs. fasting pulse mass: 0.22 (0.05) vs. 0.44 (0.06) microg/liter, P = 0.002; amplitude: 5.2 (1.3) vs. 11.8 (1.9) microg/liter/min, P = 0.034; pulses per 24 h: 19.4 (0.5) vs. 22.0 (1.4), P = 0.1]. CONCLUSION Our data support the hypothesis that under normal conditions in subjects given regular meals endogenous acyl-ghrelin acts to increase the amplitude of GH pulses.

The ghrelin O-acyltransferase–ghrelin system: a novel regulator of glucose metabolism

Purpose of reviewGhrelin, an orexigenic hormone secreted from the stomach, exists in the circulation in two isoforms: des-acyl and acyl ghrelin. Acylation by the enzyme ghrelin O-acyl-transferase (GOAT) enables ghrelin to activate the ghrelin receptor. This review discusses recent findings illustrating the role of acyl ghrelin, des-acyl ghrelin and GOAT in regulating glucose homeostasis. Recent findingsRecent publications support a role of ghrelin in modulating glucose homeostasis. Novel cellular mechanisms have been proposed to explain these effects. Controversy on this topic continues to exist owing to inconsistent observations made in both rodents and humans. Many recent studies are uncovering a role of des-acyl ghrelin in glucose metabolism specifically in modulating insulin sensitivity and glucose uptake into adipocytes. A novel role of ghrelin acylation by the enzyme GOAT in regulating glucose metabolism during caloric deprivation has newly been discovered. SummaryGhrelin plays a role in regulating glucose homeostasis through the modulation of insulin secretion and insulin sensitivity. Acyl ghrelin and des-acyl ghrelin appear to have opposing glucoregulatory effects and regulation of acylation by the enzyme GOAT appears to play a role in mediating glucose metabolism. Modulation of GOAT or ghrelin signaling may be a clinically relevant strategy to treat metabolic diseases such as type II diabetes.

The role of ghrelin in GH secretion and GH disorders

In humans, growth hormone (GH) is secreted from the anterior pituitary in a pulsatile pattern. The traditional view is that this secretory pattern is driven by two counter regulatory neurohormones, GHRH and somatostatin. Ghrelin, the natural ligand for the growth hormone (GH)-secretagogue receptor (GHS-R), is produced in the stomach. Ghrelin is the strongest GH secretagogue known to date, but the role of endogenous ghrelin in the regulation of circulating GH levels remains controversial. The following review examines the evidence suggesting that endogenous ghrelin may be a key regulator of GH peak amplitude and discusses studies of diseases with altered GH levels, where it is found that in these states GH and ghrelin levels change in a similar way.

The Ghrelin Axis in Disease; Potential Therapeutic Indications

Ghrelin, the natural ligand for the growth hormone (GH)-secretagogue receptor (GHS-R), is produced predominantly in the stomach. It is present in the circulation in two major forms, an acylated and an unacylated form, both of which have reported activities. Some of the best understood actions of acylated ghrelin administration are its orexigenic effects, and the stimulation of GH secretion. Ghrelin also seems to play a role in glucose homeostasis, lipid metabolism and immune function. Based on its orexigenic and metabolic effects, ghrelin and ghrelin mimetics have potential benefit in antagonizing protein breakdown and weight loss in catabolic conditions such as cancer cachexia, renal, cardiac and pulmonary disease, and age-related frailty. Ghrelin also has potentially useful positive effects on cardiac function and gastric motility. Ghrelin antagonists may be of benefit to increase insulin sensitivity and potentiate weight loss. The following chapter presents some background on ghrelin and ghrelin assays and discusses some of the potential therapeutic approaches for the use of ghrelin, ghrelin mimetic compounds and ghrelin antagonists in clinical disease.

Ghrelin and growth hormone: story in reverse.

The current epidemic of obesity and diabetes in the face of a surfeit of calories contrasts the natural selection to survive famine that confronted our ancestors. The report in PNAS by Zhao et al. (1) shows that ghrelin is an important hormone in this process. By studying mice under severe caloric restriction, they show that knockout (KO) of the enzyme ghrelin O-acyltransferase (GOAT), necessary to convert ghrelin to its active form, manifests the first clear phenotype seen in KO of ghrelin activity; ghrelin is necessary for triggering the growth-hormone (GH) response to nutritional deprivation that prevents hypoglycemia and death.

Age-dependent decline in acyl-ghrelin concentrations and reduced association of acyl-ghrelin and growth hormone in healthy older adults.

BACKGROUND Acyl-ghrelin is thought to have both orexigenic effects and to stimulate GH release. A possible cause of the anorexia of aging is an age-dependent decrease in circulating acyl-ghrelin levels. OBJECTIVES The purpose of the study was to compare acyl-ghrelin and GH concentrations between healthy old and young adults and to examine the relationship of acyl-ghrelin and GH secretion in both age groups. METHODS Six healthy older adults (age 62-74 y, body mass index range 20.9-29 kg/m(2)) and eight healthy young men (aged 18-28 y, body mass index range 20.6-26.2 kg/m(2)) had frequent blood samples drawn for hormone measurements every 10 minutes for 24 hours. Ghrelin was measured in an in-house, two-site sandwich ELISA specific for full-length acyl-ghrelin. GH was measured in a sensitive assay (Immulite 2000), and GH peaks were determined by deconvolution analysis. The acyl-ghrelin/GH association was estimated from correlations between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-minute interval preceding each GH burst. RESULTS Twenty-four-hour mean (±SEM) GH (0.48 ± 0.14 vs 2.2 ± 0.3 μg/L, P < .005) and acyl-ghrelin (14.7 ± 2.3 vs 27.8 ± 3.9 pg/mL, P < .05) levels were significantly lower in older adults compared with young adults. Twenty-four-hour cortisol concentrations were higher in the old than the young adults (15.1 ± 1.0 vs 10.6 ± 0.9 μg/dL, respectively, P < .01). The ghrelin/GH association was more than 3-fold lower in the older group compared with the young adults (0.16 ± 0.12 vs 0.69 ± 0.04, P < .001). CONCLUSIONS These results provide further evidence of an age-dependent decline in circulating acyl-ghrelin levels, which might play a role both in the decline of GH and in the anorexia of aging. Our data also suggest that with normal aging, endogenous acyl-ghrelin levels are less tightly linked to GH regulation.

Growth Hormone Axis and Aging

Growth hormone (GH) and/or ghrelin mimetics represent potential treatment and/or prevention options for musculoskeletal impairment associated with aging. Use of improvement in muscle function as an outcome in studies of GH and ghrelin mimetics is complicated by the lack of a standardized definition for clinically meaningful efficacy of this end point. Based on preliminary study results, the use of ghrelin mimetics may be more suitable for use in this age group than GH itself. There are still several unanswered questions related to the use of ghrelin mimetics in the elderly, which prevents recommendation for its use at the current time.

Hexarelin Modulates the Expression of Growth Hormone Secretagogue Receptor Type 1a mRNA at Hypothalamic and Pituitary Sites

Ghrelin and the synthetic growth hormone secretagogues (GHSs) activate a G-protein-coupled receptor (GHS-R) originally cloned from the pituitary, but which is also expressed in the hypothalamus, in other areas of the brain and in numerous peripheral tissues. Several studies have shown that growth hormone (GH)-releasing hormone (GHRH) is necessary for GHSs to exert maximal GH release in vivo. The exact mechanism of this synergism is not clear. Previous data suggest that GHSs can affect pituitary GHS-R mRNA expression; however, it is unknown whether this effect is age dependent and whether hypothalamic GHS-Rs are also affected. In this study, we tested whether (a) the synthetic GHS hexarelin regulates mRNA expression of its own receptor at the pituitary and/or hypothalamus and whether this effect is age dependent, and (b) whether short-term treatment with GHRH or, conversely, passive immunization against GHRH affects pituitary GHS-R1a mRNA expression in infant (10 days old) and young adult rats. GHS-R1a mRNA expression was measured with competitive reverse transcriptase-polymerase chain reaction. Hexarelin treatment significantly increased pituitary and hypothalamic GHS-R1a mRNA levels in normal infant rats, but not in normal young adult rats. In addition, hexarelin administration also stimulated pituitary GHS-R1a mRNA in infant as well as in young adult rats passively immunized against GHRH. GHRH treatment significantly enhanced pituitary GHS-R1a mRNA expression in GHRH-deprived young adult rats, though it did not affect the basal levels of GHS-R1a mRNA in normal infant and adult rats. These data further support the hypothesis that GHRH can affect GHS-R1a expression and that hexarelin upregulates the expression of its own receptor at the pituitary as well as the hypothalamus in an age-dependent fashion.