Context: The lack of sensitive and robust analytical methods has hindered the reliable quantification of estrogen metabolites in subjects with low concentrations. Objective: To establish sex-specific reference ranges for estrone (E1) and estradiol (E2) throughout life and to evaluate sex-differences using the state-of-the-art liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of E1, E2, and estriol (E3). Design: LC-MS/MS method development and construction of estrogen reference ranges. Settings: Population-based cross-sectional cohorts from the greater Copenhagen and Aarhus areas. Participants: Healthy participants aged 3 months to 61 years (n = 1838). Results: An isotope diluted LC-MS/MS method was developed and validated for measurements of serum E1, E2, and E3. Limits of detections (LODs) were 3 pmol/L (E1), 4 pmol/L (E2), and 12 pmol/L (E3), respectively. This sensitive method made it possible to differentiate between male and female concentration levels of E1 and E2 in children. In girls, E2 levels ranged from <LOD to 100 pmol/L during mini-puberty, whereas it was ≤20 pmol/L during childhood. E1 and E2 increased with age and pubertal breast stage and varied during the menstrual cycle; E1 was lower than E2 in girls and premenopausal women, and higher than E2 in postmenopausal women. In boys, E1 and E2 increased with age and pubertal stage, whereas little changes with age were observed in men. High E3 concentrations were confirmed in pregnant women. Conclusion: Reference ranges of simultaneous quantification of E1 and E2 by this novel specific and highly sensitive LC-MS/MS method provide an invaluable tool in clinical practice and in future research studies.

OBJECTIVE—Ghrelin is a gut-derived peptide and an endogenous ligand for the growth hormone (GH) secretagogue receptor. Exogenous ghrelin stimulates the release of GH (potently) and adrenocorticotropic hormone (ACTH) (moderately). Ghrelin is also orexigenic, but its impact on substrate metabolism is controversial. We aimed to study direct effects of ghrelin on substrate metabolism and insulin sensitivity in human subjects. RESEARCH DESIGN AND METHODS—Six healthy men underwent ghrelin (5 pmol · kg⁻¹ · min⁻¹) and saline infusions in a double-blind, cross-over study to study GH signaling proteins in muscle. To circumvent effects of endogenous GH and ACTH, we performed a similar study in eight hypopituitary adults but replaced with GH and hydrocortisone. The methods included a hyperinsulinemic-euglycemic clamp, muscle biopsies, microdialysis, and indirect calorimetry. RESULTS—In healthy subjects, ghrelin-induced GH secretion translated into acute GH receptor signaling in muscle. In the absence of GH and cortisol secretion, ghrelin acutely decreased peripheral, but not hepatic, insulin sensitivity together with stimulation of lipolysis. These effects occurred without detectable suppression of AMP-activated protein kinase phosphorylation (an alleged second messenger for ghrelin) in skeletal muscle. CONCLUSIONS—Ghrelin infusion acutely induces lipolysis and insulin resistance independently of GH and cortisol. We hypothesize that the metabolic effects of ghrelin provide a means to partition glucose to glucose-dependent tissues during conditions of energy shortage.

Summary: Background  TZP‐101 is a synthetic, selective ghrelin agonist in development for gastroparesis.Aim  To assess safety and effects of TZP‐101 in diabetes patients with symptomatic gastroparesis.Methods  Adults with type 1 or type 2 diabetes mellitus received placebo and TZP‐101 (80, 160, 320 or 600 μg/kg) infusions in a cross‐over manner following a radiolabelled meal. Blood glucose levels were stabilized using a hyperinsulinemic‐euglycemic clamp. Primary endpoints were gastric half emptying and latency times. Secondary measures included assessment of gastroparesis symptoms and endocrine responses.Results  Ten patients with type 1 (n = 7) or 2 (n = 3) diabetes, moderate‐to‐severe gastroparesis symptoms and ≥29% retention 4 h after a radiolabelled solid meal were enrolled. TZP‐101 produced significant reductions in solid meal half‐emptying (20%, P = 0.043) and latency (34%, P = 0.037) times vs. placebo. Reductions in overall postmeal symptom intensity (24%) and postprandial fullness (37%) following TZP‐101 infusion were not statistically significant. Most adverse events were mild and self‐limiting and there were no identifiable differences in numbers or types of adverse events between TZP‐101 and placebo.Conclusions  This proof‐of‐concept study demonstrates that the ghrelin agonist TZP‐101 is well‐tolerated in diabetes patients with moderate‐to‐severe chronic gastroparesis and shows statistically significant improvements in gastric emptying.

Context: Ghrelin infusion increases plasma glucose and nonesterified fatty acids, but it is uncertain whether this is secondary to the concomitant release of GH. Objective: Our objective was to study direct effects of ghrelin on substrate metabolism. Design: This was a randomized, single-blind, placebo-controlled two-period crossover study. Setting: The study was performed in a university clinical research laboratory. Participants: Eight healthy men aged 27.2 ± 0.9 yr with a body mass index of 23.4 ± 0.5 kg/m² were included in the study. Intervention: Subjects received infusion of ghrelin (5 pmol·kg⁻¹·min⁻¹) or placebo for 5 h together with a pancreatic clamp (somatostatin 330 μg·h⁻¹, insulin 0.1 mU·kg⁻¹·min⁻¹, GH 2 ng·kg⁻¹·min⁻¹, and glucagon 0.5 ng·kg⁻¹·min⁻¹). A hyperinsulinemic (0.6 mU·kg⁻¹·min⁻¹) euglycemic clamp was performed during the final 2 h of each infusion. Results: Basal and insulin-stimulated glucose disposal decreased with ghrelin [basal: 1.9 ± 0.1 (ghrelin) vs. 2.3 ± 0.1 mg·kg⁻¹·min⁻¹, P = 0.03; clamp: 3.9 ± 0.6 (ghrelin) vs. 6.1 ± 0.5 mg·kg⁻¹·min⁻¹, P = 0.02], whereas endogenous glucose production was similar. Glucose infusion rate during the clamp was reduced by ghrelin [4.0 ± 0.7 (ghrelin) vs. 6.9 ± 0.9 mg·kg⁻¹·min⁻¹; P = 0.007], whereas nonesterified fatty acid flux increased [131 ± 26 (ghrelin) vs. 69 ± 5 μmol/min; P = 0.048] in the basal period. Regional lipolysis (skeletal muscle, sc fat) increased insignificantly with ghrelin infusion. Energy expenditure during the clamp decreased after ghrelin infusion [1539 ± 28 (ghrelin) vs. 1608 ± 32 kcal/24 h; P = 0.048], but the respiratory quotient did not differ. Minor but significant elevations in serum levels of GH and cortisol were observed after ghrelin infusion. Conclusions: Administration of exogenous ghrelin causes insulin resistance in muscle and stimulates lipolysis; these effects are likely to be direct, although a small contribution of GH and cortisol cannot be excluded.

Ghrelin levels fluctuate rapidly and dynamically with surges before meal times and postprandial troughs, and ghrelin increases appetite and food intake. Circulating ghrelin correlates negatively with body mass index (BMI), but obese individuals have a reduced postprandial decrease in ghrelin levels. Whether this reflects changes in secretion or clearance of ghrelin is uncertain. We therefore studied the pharmacokinetics of ghrelin in relation to anthropometric and biochemical measures. We also studied the effects of ghrelin on hormones and metabolites. In fasting humans, we used a constant infusion rate of ghrelin lasting 180 min at 5 pmol·kg body wt⁻¹·min⁻¹ in a randomized, double-blind, placebo-controlled crossover study. Serum ghrelin (s-ghrelin; total levels) was distributed and eliminated according to a two-compartment model. s-Ghrelin initial half-life was 24 ± 2 min and terminal half-life 146 ± 36 min, respectively. Mean residence time (MRT) of ghrelin was 93 ± 16 min. MRT correlated positively with both BMI ( r = 0.51, P < 0.001) and high-density cholesterol (HDL) levels ( r = 0.75, P < 0.001). Serum insulin levels remained constant during ghrelin infusion, whereas plasma glucose increased 0.3 ± 0.1 mmol/l ( P < 0.01) and free fatty acid levels more than doubled (to 1.03 ± 0.08 mmol/l, P < 0.001), translating into a significant reduction of insulin sensitivity ( P < 0.001). In conclusion, 1) we describe novel pharmacokinetics of ghrelin that are useful when tailoring ghrelin infusion rates in clinical experiments, 2) BMI and HDL correlate positively with MRT of infused ghrelin, and 3) supraphysiological ghrelin levels impair insulin sensitivity.

Patients with active acromegaly are insulin-resistant and glucose-intolerant, whereas children with growth hormone (GH) deficiency (GHD) are insulin-sensitive and may develop fasting hypoglycaemia. Surprisingly, however, hypopituitary adults with unsubstituted GHD tend to be insulin-resistant, which may worsen during GH substitution. During fasting, which may be considered the natural domain for the metabolic effects of GH, the induction of insulin resistance by GH is associated with enhanced lipid oxidation and protein conservation. In this particular context, insulin resistance appears to constitute a favourable metabolic adaptation. The problem is that GH substitution results in elevated circadian GH levels in non-fasting patients. The best way to address this challenge is to employ evening administration of GH and to tailor the dose. Insulin therapy may cause hypoglycaemia and GH substitution may cause hyperglycaemia. Such untoward effects should be minimized by carefully monitoring the individual patient.