Michael O. Thorner

Role of the GH/IGF-1 axis in lifespan and healthspan: lessons from animal models

Animal models are fundamentally important in our quest to understand the genetic, epigenetic, and environmental factors that contribute to human aging. In comparison to humans, relatively short-lived mammals are useful models as they allow for rapid assessment of both genetic manipulation and environmental intervention as related to longevity. These models also allow for the study of clinically relevant pathologies as a function of aging. Data associated with more distant species offers additional insight and critical consideration of the basic physiological processes and molecular mechanisms that influence lifespan. Consistently, two interventions, caloric restriction and repression of the growth hormone (GH)/insulin-like growth factor-1/insulin axis, have been shown to increase lifespan in both invertebrates and vertebrate animal model systems. Caloric restriction (CR) is a nutrition intervention that robustly extends lifespan whether it is started early or later in life. Likewise, genes involved in the GH/IGF-1 signaling pathways can lengthen lifespan in vertebrates and invertebrates, implying evolutionary conservation of the molecular mechanisms. Specifically, insulin and insulin-like growth factor-1 (IGF-1)-like signaling and its downstream intracellular signaling molecules have been shown to be associated with lifespan in fruit flies and nematodes. More recently, mammalian models with reduced growth hormone (GH) and/or IGF-1 signaling have also been shown to have extended lifespans as compared to control siblings. Importantly, this research has also shown that these genetic alterations can keep the animals healthy and disease-free for longer periods and can alleviate specific age-related pathologies similar to what is observed for CR individuals. Thus, these mutations may not only extend lifespan but may also improve healthspan, the general health and quality of life of an organism as it ages. In this review, we will provide an overview of how the manipulation of the GH/IGF axis influences lifespan, highlight the invertebrate and vertebrate animal models with altered lifespan due to modifications to the GH/IGF-1 signaling cascade or homologous pathways, and discuss the basic phenotypic characteristics and healthspan of these models.

Endoscopic Transsphenoidal Surgery for Acromegaly: Remission Using Modern Criteria, Complications, and Predictors of Outcome

CONTEXTnDespite the growing application of endoscopic transsphenoidal surgery (ETSS), outcomes for GH adenomas are not clearly defined.nnnOBJECTIVEnWe reviewed our experience with ETSS with specific interest in remission rates using the 2010 consensus criteria, predictors of remission, and associated complications.nnnDESIGN AND SETTINGnThis was a retrospective single institution study. PATIENTS, INTERVENTIONS, AND OUTCOME MEASURES: Sixty acromegalic patients who underwent ETSS were identified. Remission was defined as a normal IGF-I and either a suppressed GH less than 0.4 ng/ml during an oral glucose tolerance test or a random GH less than 1.0 ng/ml.nnnRESULTSnRemission was achieved in all 14 microadenomas and 28 of 46 macroadenomas (61%). Tumor size, age, gender, and history of prior surgery were not predictive on multivariant analysis. In hospital postoperative morning GH levels less than 2.5 ng/ml provided the best prediction of remission (P < 0.001). Preoperative variables predictive of remission included Knosp score (P = 0.017), IGF-I (P = 0.030), and GH (P = 0.042) levels. New endocrinopathy consisted of diabetes insipidus in 5%, adrenal insufficiency in 5.4%, and new hypogonadism in 29% of men and 17% of women. However, 41% of hypogonadal men had normal postoperative testosterone levels and 83% of amenorrheic women regained menses. The most common complaints after surgery were sinonasal (36 of 60, 60%) resolving in all but two.nnnCONCLUSIONSnETSS for GH adenomas is associated with high rates of remission and a low incidence of new endocrinopathy. Despite the panoramic views offered by the endoscope, invasive tumors continue to have lower rates of remission.

Leptin Does Not Mediate Short-Term Fasting-Induced Changes in Growth Hormone Pulsatility but Increases IGF-I in Leptin Deficiency States

CONTEXTnStates of acute and chronic energy deficit are characterized by increased GH secretion and decreased IGF-I levels.nnnOBJECTIVEnThe objective of the study was to determine whether changes in levels of leptin, a key mediator of the adaptation to starvation, regulate the GH-IGF system during energy deficit.nnnDESIGN, SETTING, PATIENTS, AND INTERVENTIONnWe studied 14 healthy normal-weight men and women during three conditions: baseline fed and 72-h fasting (to induce hypoleptinemia) with administration of placebo or recombinant methionyl human leptin (r-metHuLeptin) (to reverse the fasting associated hypoleptinemia). We also studied eight normal-weight women with exercise-induced chronic energy deficit and hypothalamic amenorrhea at baseline and during 2-3 months of r-metHuLeptin treatment.nnnMAIN OUTCOME MEASURESnGH pulsatility, IGF levels, IGF and GH binding protein (GHBP) levels were measured.nnnRESULTSnDuring short-term energy deficit, measures of GH pulsatility and disorderliness and levels of IGF binding protein (IGFBP)-1 increased, whereas leptin, insulin, IGF-I (total and free), IGFBP-4, IGFBP-6, and GHBP decreased; r-metHuLeptin administration blunted the starvation-associated decrease of IGF-I. In chronic energy deficit, total and free IGF-I, IGFBP-6, and GHBP levels were lower, compared with euleptinemic controls; r-metHuLeptin administration had no major effect on GH pulsatility after 2 wk but increased total IGF-I levels and tended to increase free IGF-I and IGFBP-3 after 1 month.nnnCONCLUSIONSnThe GH/IGF system changes associated with energy deficit are largely independent of leptin deficiency. During acute energy deficit, r-metHuLeptin administration in replacement doses blunts the starvation-induced decrease of IGF-I, but during chronic energy deficit, r-metHuLeptin administration increases IGF-I and tends to increase free IGF-I and IGFBP-3.

Impact of the GH-cortisol ratio on the age-dependent changes in body composition

Aging is associated with a decrease in GH levels and this is paralleled by changes in body composition, i.e., increased visceral fat, and decreased lean body mass and bone mineral density. Similar changes in body composition are seen in the state of hypercortisolism. Increasing age has been shown to be associated with elevated evening cortisol levels in men. An increased exposure of several tissues to glucocorticoids with aging, i.e., visceral fat cells, in combination with the reduction of the lipolytic effects of declining GH levels, may contribute to the age-dependent increase of visceral fat accumulation. We hypothesize that the age-dependent changes in body fat are the result of an age-dependent decrease of the GH/cortisol ratio at the level of the adipocyte. This is caused by the decline in GH concentrations and the increase in cortisol levels and/or metabolism at the adipocyte.

AutoDecon: a robust numerical method for the quantification of pulsatile events.

This work presents a new approach to the analysis of aperiodic pulsatile heteroscedastic time-series data, specifically hormone pulsatility. We have utilized growth hormone (GH) concentration time-series data as an example for the utilization of this new algorithm. While many previously published approaches used for the analysis of GH pulsatility are both subjective and cumbersome to use, AutoDecon is a nonsubjective, standardized, and completely automated algorithm. We have employed computer simulations to evaluate the true-positive, the false-positive, the false-negative, and the sensitivity percentages of several of the routinely employed algorithms when applied to GH concentration time-series data. Based on these simulations, it was concluded that this new algorithm provides a substantial improvement over the previous methods. This novel method has many direct applications in addition to hormone pulsatility, for example, to time-domain fluorescence lifetime measurements, as the mathematical forms that describe these experimental systems are both convolution integrals.

Statement by the Growth Hormone Research Society on the GH/IGF-I Axis in Extending Health Span

Despite the fact that growth hormone (GH) has not been approved for antiaging purposes, its use for this indication is widespread and increasing. The Growth Hormone Research Society (GRS) convened an international workshop to critically review and debate the available evidence related to the use of GH in the older adults and the relationship between the GH/insulin-like growth factor I (IGF-I) axis and the aging process. This statement presents the conclusions reached and gives recommendations for future studies in this research field regarding the use of GH and growth hormone secretagogues (GHS) for promoting health span. The participants concluded that, until future clinical research in this area is conducted, in particular carefully designed, long-term studies, using validated outcome parameters, the clinical use of GH or GHS in older adults, alone or in combination with testosterone, cannot be recommended. In addition, future basic studies in model systems, to continue to unravel GH/IGF-I effects related to human life span and health span, were advocated.

Acute Peripheral Metabolic Effects of Intraarterial Ghrelin Infusion in Healthy Young Men

CONTEXTnGhrelin is the endogenous agonist for the growth hormone secretagogue receptor (GHS-R). Intravenous administration of ghrelin induces insulin resistance and hyperglycemia and increases the levels of free fatty acids (FFA).nnnOBJECTIVEnTo investigate whether these effects are mediated directly by ghrelin in skeletal muscle tissue.nnnDESIGNnThis study was single blinded, randomized, and placebo controlled. Eight healthy men (25.5 ± 3.1 years) received 240 min of intraarterial ghrelin infusion (4.2 ng × kg(-1) × min(-1)) into one femoral artery and intraarterial placebo infusion into the contralateral artery. Simultaneous blood samples were drawn from both femoral veins and muscle biopsies were obtained from both legs during both a basal period and during a hyperinsulinemic and euglycemic clamp period.nnnRESULTSnGhrelin significantly elevated venous FFA levels and venous dilution of palmitate, suggestive of increased lipolysis. Glucose metabolism was unchanged, and there were no direct effects on pertinent enzymes in the insulin signaling cascade. The metabolic clearance rate of acyl ghrelin was 12.5 ± 3.3 ml × kg(-1) × min(-1). Acyl and desacyl ghrelin levels both increased.nnnCONCLUSIONSnThe results of this study suggest that ghrelin may stimulate lipolysis directly in skeletal muscle.

Growth Hormone Exerts Acute Vascular Effects Independent of Systemic or Muscle Insulin-like Growth Factor I

CONTEXTnEndothelial dysfunction is common in patients with GH deficiency who are at increased risk for premature cardiovascular death. GH regulates vascular tone and reactivity in humans.nnnOBJECTIVEnOur objective was to explore the mechanisms underlying the GHs acute vascular effects. DESIGN AND STUDY SETTING: There were 10 healthy, lean and young, volunteers studied after an overnight fast. GH was infused systemically for 6 h at 0.06 microg/kg.min. Biopsy of the vastus lateralis muscle was done in seven subjects before and after GH infusion. Human aortic endothelial cells (HAECs) were incubated with GH in vitro.nnnRESULTSnGH infusion increased plasma GH to 32.9 +/- 1.5 ng/ml and forearm blood flow by 66% (P < 0.001). GH infusion did not significantly change plasma IGF-I concentrations, muscle IGF-I mRNA expression, and muscle Akt phosphorylation, suggesting a lack of IGF-I action in muscle. Because it was reported that GH exerts an acute vascular effect via a nitric oxide (NO)-dependent mechanism, we performed additional in vitro experiments using HAECs. HAECs express abundant GH receptors. Incubating HAECs with GH at 30 ng/ml for 3 or 6 h did not alter endothelial NO synthase (eNOS) protein content but time dependently increased the phosphorylation and activity of eNOS, thus demonstrating a direct effect of GH on endothelial cells.nnnCONCLUSIONSnGH exerts an acute vascular effect independent of both systemic and local IGF-I production, and this effect is likely via direct action on GH receptors and eNOS in the vascular endothelium.

Effects of glucose and insulin on acyl ghrelin and desacyl ghrelin, leptin, and adiponectin in pregnant women with diabetes

The aim of the study was to compare the regulation of ghrelin, leptin, and adiponectin by insulin and glucose during the second and third trimesters of pregnancy in women with diabetes. We studied 9 pregnant women with diabetes. All women were treated with insulin and omitted the morning dose on the day of the test. After collection of baseline fasting samples, we performed 3 successive glucose clamps: 2 euglycemic clamps (glucose, 5 mmol/L; insulin infusion at 20 and 40 mU m(-2) min(-1)) and 1 hyperglycemic clamp (glucose, 10 mmol/L; insulin infusion at 40 mU m(-2) min(-1)). We determined concentrations of acyl and desacyl ghrelin (using a double-antibody sandwich assay that recognizes the full-length molecule), leptin, and adiponectin. Fasting desacyl ghrelin concentrations decreased, whereas insulin and leptin concentrations increased, between the second and third trimesters of pregnancy (P < or = .011). During the clamp studies, desacyl ghrelin concentrations decreased by 33% (second trimester, P = .004) and 27% (third trimester, P = .09) with increasing glucose and insulin concentrations, whereas acyl ghrelin, leptin, and adiponectin concentrations were unaffected. Glucose and insulin regulate desacyl ghrelin concentrations in pregnant women with diabetes. Impaired desacyl ghrelin regulation may affect energy metabolism in pregnant women with poorly controlled diabetes.

Ghrelin, not just another growth hormone secretagogue.

In 1977, Bowers and colleagues published their seminal work describing the growth hormone releasing properties of peptides derived from met-enkephalin that lacked any opiate properties (Bowers et al., 1977). The first compound, growth hormone releasing peptide (GHRP), produced a weak GH response in vitro and was not effective in vivo. Further development produced GHRP-6, a hexapeptide with two D-amino acids; further methylation of the D-tryptophan produced hexarelin. Both compounds were stable, had improved potency and were active in vitro and in vivo. Both compounds were potent GH secretagogues when administered intravenously or subcutaneously; however, the bioavailability of both compounds orally was poor. These observations, together with the simple structure of the GHRPs, made them attractive molecules to be developed for enhancing GH secretion. The pharmaceutical industry became involved and committed major resources to the development of peptide and nonpeptide compounds. Perhaps the most significant of these agents was the spiroindolone MK0677, developed by Merck. The primary action of the GHRPs was the stimulation of GH release from the somatotroph acting via a mechanism distinct from that of GH releasing-hormone (GHRH). GHRP-6 activated the phospolipase-C pathway, resulting in a rise in inositol triphosphate and intracellular calcium, a pathway distinct from the cAMP-phosphokinase A pathway of the GHRH receptor. In 1996, the Merck group combined expression cloning and their compound MK0677, looking for changes in intracellular calcium to indicate a positive response, to identify and clone the GH secretagogue (GHS) receptor (Howard et al., 1996). The GHS receptor is a 7 transmembrane, G-protein linked receptor of 366 amino acids belonging to the rhodopsin family. The receptor was detected in the hypothalamus and the pituitary, adding weight to the speculation that GHRP was a third factor involved in GH regulation. Subsequent studies have demonstrated binding of radiolabelled GHRP in many tissues, some of which do not express the GHS receptor, suggesting that GHRP might have other physiological roles and that its effects may be exerted via other, as yet unidentified, receptors. The cloning of the GH secretagogue receptor provided the tool necessary to identify its natural ligand. In December 1999, the group led by Kojima and Kangawa published the structure of the natural ligand for the GHS receptor, designated ghrelin (derived from ghre, the proto Indo-European root of the word `grow) (Kojima et al., 1999). They used the GHS-receptor expressed in a cell line as a bioassay to screen tissue extracts from rats, a positive response causing a rise in intracellular calcium. The group did not assume that the ligand would be present in the hypothalamus, but assayed extracts from multiple organs; surprisingly, the strongest signal was obtained using extracts from the stomach, not the hypothalamus. Sequencing the purified ligand revealed that ghrelin was a 28 amino acid peptide, although the third residue was indeterminate. The mRNA encoding ghrelin indicated that this residue was a serine; however, the synthetic peptide did not activate the GHS-receptor, had a shorter retention time in reversed-phase high-performance liquid chromatography (RP-HPLC) and had a lower molecular mass than purified ghrelin. Futher characterization using mass spectrophotometry revealed that ghrelin was subjected to a unique post-translational modification: the hydroxyl group of the third residue serine was esterified by octanoic acid. The addition of the octanoyl residue to the synthetic peptide increased the hydrophobicity of the ghrelin molecule, produced the same RP-HPLC profile, had the same molecular weight and activity at the GHS-receptor as purified ghrelin. In both rats and humans, the ghrelin gene encodes a 117 amino acid peptide, preproghrelin, that shares 82% homology indicating that ghrelin is conserved between the species. Ghrelin expression is found in the intestine, from the stomach to the colon, with the greatest concentration being in the fundus of the stomach. Expression is restricted to the X/A-like cells of the oxyntic gland, a group of cells whose endocrine function was hitherto undefined (Date et al., 2000a). Ghrelin was also isolated in the arcuate nucleus of the hypothalamus an area rich in GHRH secreting neurones (Kojima et al., 1999); however, it is not clear at present whether ghrelin is synthesized there or transported there from the periphery. Many of the effects of ghrelin could be predicted from the known effects of the GHRPs and synthetic GH secretagogues. Ghrelin is a potent GH secretagogue in vitro and in vivo, in rats and in humans. A rapid, dose dependent GH response was observed following intravenous (i.v.) and intracerebroventricular (i.c.v.) injection of ghrelin (Date et al., 2000b; Seoane et al., 2000). The GH response to i.v. ghrelin in healthy human