Everolimus therapy for progressive adrenocortical cancer.

Patients with advanced adrenocortical carcinoma (ACC) have limited treatment options after failure of chemotherapy. Tumor IGF2 expression has been shown to be amplified in the majority of cases of ACC and autocrine/paracrine activation of the IGF receptor (IGF-R) is thought to play a major role in the pathogenesis of ACC. It has been shown in vitro that inhibition of the IGF-R inhibits ACC cell proliferation. mTOR is a downstream effector of the IGFR signaling pathway; therefore, the rapamycin analog everolimus could prove to be useful for treatment of patients with ACC. Four women with ACC (ages 25-60 years) developed stage IV disease after surgery. All had progressive disease (PD) despite treatment with mitotane and other treatment modalities (etoposide, doxorubicin, cis-platinum in 3/4 patients, further streptozotocin + 5-FU in 1/4 patients, further thalidomide therapy in 2/4 patients; 1 patient progressed on an IGF-R antagonist). The patients were started on everolimus 10 mg/day orally and 2/4 patients also continued mitotane. Disease progression was monitored monthly by CT in 3/4 and after 3 months in 1/4. In all patients everolimus was well tolerated. In the three patients monitored monthly, PD was evident after 1, 3, and 4 months; in the patient evaluated after 3 months PD was also evident. In this small exploratory study, no clinically meaningful response was observed with everolimus in four patients with advanced ACC. The failure of efficacy could be related to an interaction with mitotane, multiple signaling pathways, and/or other downstream IGF-R effectors operative in the pathogenesis of ACC.

Repeated colonoscopic screening of patients with acromegaly: 15-year experience identifies those at risk of new colonic neoplasia and allows for effective screening guidelines.

OBJECTIVE: It is suggested that patients with acromegaly have an increased risk of colorectal cancer and pre-malignant adenomatous polyps. However, the optimum frequency with which colonoscopic screening should be offered remains unclear. DESIGN: To determine the optimum frequency for repeated colonoscopic surveillance of acromegalic patients. METHODS: We retrospectively reviewed the case records of all patients with acromegaly seen in our centre since 1992: 254 patients had at least one surveillance colonoscopy, 156 patients had a second surveillance colonoscopy, 60 patients had a third surveillance colonoscopy and 15 patients had a fourth surveillance colonoscopy. RESULTS: The presence of hyperplastic or adenomatous polyps was assessed in all patients, while one cancer was detected at the second surveillance. At the third surveillance, mean (+/-s.d.) serum IGF1 levels (ng/ml) in patients with hyperplastic polyps were significantly higher than those with normal colons (P<0.05). The presence of an adenoma rather than a normal colon at the first colonoscopy was associated with a significantly increased risk of adenoma at the second (odds ratio (OR) 4.4, 95% confidence interval (CI) 1.9-10.4) and at the third (OR 8.8, 95% CI 2.9-26.5) screens. Conversely, a normal colon at the first surveillance gave a high chance of normal findings at the second (78%) or third surveillance (78%), and a normal colon at the second colonoscopy was associated with normality at the third colonoscopy (81%). CONCLUSIONS: Repeated colonoscopic screening of patients with acromegaly demonstrated a high prevalence of new adenomatous and hyperplastic colonic polyps, dependent on both the occurrence of previous polyps and elevated IGF1 levels.

Activation of RAF/MEK/ERK and PI3K/AKT/mTOR pathways in pituitary adenomas and their effects on downstream effectors.

Raf/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascades are key signalling pathways interacting with each other to regulate cell growth and tumourigenesis. We have previously shown B-Raf and Akt overexpression and/or overactivation in pituitary adenomas. The aim of this study is to assess the expression of their downstream components (MEK1/2, ERK1/2, mTOR, TSC2, p70S6K) and effectors (c-MYC and CYCLIN D1). We studied tissue from 16 non-functioning pituitary adenomas (NFPAs), six GH-omas, six prolactinomas and six ACTH-omas, all collected at transsphenoidal surgery; 16 normal autopsy pituitaries were used as controls. The expression of phospho and total protein was assessed with western immunoblotting, and the mRNA expression with quantitative RT-PCR. The expression of pSer217/221 MEK1/2 and pThr183 ERK1/2 (but not total MEK1/2 or ERK1/2) was significantly higher in all tumour subtypes in comparison to normal pituitaries. There was no difference in the expression of phosphorylated/total mTOR, TSC2 or p70S6K between pituitary adenomas and controls. Neither c-MYC phosphorylation at Ser 62 nor total c-MYC was changed in the tumours. However, c-MYC phosphorylation at Thr58/Ser62 (a response target for Akt) was decreased in all tumour types. CYCLIN D1 expression was higher only in NFPAs. The mRNA expression of MEK1, MEK2, ERK1, ERK2, c-MYC and CCND1 was similar in all groups. Our data indicate that in pituitary adenomas both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways are upregulated in their initial cascade, implicating a pro-proliferative signal derangement upstream to their point of convergence. However, we speculate that other processes, such as senescence, attenuate the changes downstream in these benign tumours.

A genetic study of the ghrelin and growth hormone secretagogue receptor (GHSR) genes and stature.

Growth and nutrition are interrelated and influenced by multiple genetic and environmental factors. We studied whether common variants in ghrelin and ghrelin receptor (GHSR) genes could play a role in stature variation in the general population and in families ascertained for obesity. Selected tagging SNPs in the ghrelin and GHSR genes were genotyped in 263 Caucasian families recruited for childhood obesity (1,275 subjects), and in 287 families from a general population (1,072 subjects). We performed familial testing for associations in the entire population and in a sub-set of the samples selected for a case-control study. In the case-control study for height (cases were selected from the obese cohort with mean ZH = 3.17 +/- 0.15 confidence interval (CI) versus controls with mean ZH 0.14 +/- 0.09), we found an association with a 2 base-pair intronic deletion in the GHSR gene (rs10618418) (p = 0.006, odds ratio (OR) 1.86, 95% CI [1.26;2.74] under additive model), although when adjusting for BMI, the association disappeared (p = 0.06). Individuals carrying no deletion or who were heterozygous were significantly more frequent among the tall obese population (52% vs. 36% in controls, p = 0.007, OR 1.97, 95%CI [1.22;3.18]). However, the association was not maintained after correcting for multiple testing. Familial association testing of the ghrelin and GHSR genes and their interaction testing failed to show that any combination of SNPs had any significant effect. Thus, our results suggest that common variants of the ghrelin and GHSR genes are not major contributors to height variation in a French population.

A new variation in the promoter region, the -604 C>T, and the Leu72Met polymorphism of the ghrelin gene are associated with protection to insulin resistance.

OBJECTIVE: Previous studies suggested that polymorphisms in the coding region of the preproghrelin were involved in the etiology of obesity and might modulate glucose-induced insulin secretion. We evaluated the association of a new variation, -604C>T, in the promoter region of the ghrelin gene, of Leu72Met (247C>A) and of Gln90Leu (265A>T), all haplotype-tagging single nucleotide polymorphisms (SNPs), with measures of insulin sensitivity in 1420 adult individuals. RESEARCH METHODS: The three SNPs were genotyped using ABI PRISM 7900 HT Sequence Detection System. We used multiple linear regression analysis for quantitative traits and THESIAS software for haplotype analysis. RESULTS: We observed a protective effect exerted by Met72 variant of Leu72Met SNP on insulin resistance parameters; a significant decreasing trend from Leu/Leu to Leu/Met and to Met/Met homozygous subjects in triglycerides, fasting insulin levels and HOMA-IR index (P=0.02, 0.01 and 0.003, respectively), and, consistently, an increase in ghrelin levels (P=0.003) was found. A significant decrease from CC to TC and to TT genotypes in insulin levels and HOMA-IR index was also detected (P=0.00l for both), but only in subjects homozygous for Leu72, where the protective effect of Met72 was not present. The haplotype analysis results supported the data obtained by the evaluation of each single SNP, showing the highest value of insulin levels and HOMA-IR index in the -604(c)247(c) haplotype intermediate value in -604(T)247(C) and lowest value in -604(C)247(A). CONCLUSION: Our observations suggest a protective role of the Met72 variant and of -604 T allele in modulating insulin resistance. These SNPs or an unknown functional variant in linkage disequilibrium could increase ghrelin levels and probably insulin sensitivity.

Single nucleotide polymorphisms in the neuropeptide Y2 receptor (NPY2R) gene and association with severe obesity in French white subjects.

AIMS/HYPOTHESIS: Genetic variants of genes for peptide YY (PYY), neuropeptide Y2 receptor (NPY2R) and pancreatic polypeptide (PPY) were investigated for association with severe obesity. SUBJECTS AND METHODS: The initial screening of the genes for variants was performed by sequencing in a group of severely obese subjects (n=161). Case-control analysis of the common variants was then carried out in 557 severely obese adults, 515 severely obese children and 1,163 non-obese/non-diabetic control subjects. Rare variants were genotyped in 700 obese children and the non-obese/non-diabetic control subjects (n=1,163). RESULTS: Significant association was found for a 5' variant (rs6857715) in the NPY2R gene with both severe adult obesity (p=0.002) and childhood obesity (p=0.02). This significant association was further supported by a pooled allelic analysis of all obese cases (adults and children, n=928) vs the control subjects (n=938) (p=0.0004, odds ratio=1.3, 95% CI 1.1-1.5). Quantitative trait analysis of BMI and WHR was performed and significant association was observed for SNP rs1047214 in NPY2R with an increase in WHR in the severely obese children (co-dominant model p=0.005, recessive model p=0.001). Association was also observed for an intron 3 variant (rs162430) in the PYY gene with childhood obesity (p=0.04). No significant associations were observed for PPY variants. Only one rare variant in the NPY2R gene (C-5641T) was not found in lean individuals and this was found to co-segregate with obesity in one family. CONCLUSIONS/INTERPRETATION: These results provide evidence of association for NPY2R and PYY gene variants with obesity and none for PPY variants. A rare variant of the NPY2R gene showed evidence of co-segregation with obesity and its contribution to obesity should be investigated further.

Enhanced protein kinase B/Akt signalling in pituitary tumours.

Pituitary tumours have previously been shown to harbour several abnormalities that cause deregulation of the cell cycle, particularly down-regulation of expression of the cyclin-dependent kinase inhibitor p27. However, it has been unclear whether these are the primary initiating events, or are secondary to other more proximate alterations in signalling pathways. In other cellular systems the Akt signalling pathway has been associated with downstream modulation of cell-cycle control. The aim of the present study was to test the hypothesis that Akt signalling is enhanced in pituitary tumours, and to see if changes in Akt expression are related to previous findings on low expression levels of the nuclear cell-cycle inhibitor p27 in pituitary tumours. We examined normal and adenomatous human pituitary tissue for mRNA and protein expression of Akt1, Akt2 and p27, and the activation of Akt, as well the phosphatase involved in the inactivation of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). In pituitary adenomas Akt1 and Akt2 mRNA were found to be over-expressed compared with normal pituitary, while PTEN transcripts showed similar levels between the two tissue types. Immunohistochemical expression of phospho-Akt was found to be higher in the tumours than normal pituitaries, while the protein expression of nuclear p27 and PTEN was lower in the adenomas. However, the expression of p27 and Akt were not directly correlated. PTEN sequencing revealed no mutation in the coding region of the gene in pituitary adenomas, and thus we did not locate a cause for the increased phosphorylation of Akt. In summary, we have shown over-expression and activation of the Akt pathway in pituitary tumours, and we speculate that cell-cycle changes observed in such tumours are secondary to these more proximate alterations. Since Akt is a major downstream signalling molecule of growth factor-liganded tyrosine kinase receptors, our data are most compatible with an abnormality at this level as the primary driver of pituitary tumorigenesis.

The Krüppel-like transcription factor 6 gene in sporadic pituitary tumours.

The oncogenes and/or tumour suppressor genes which may be involved in the transformation process for the vast majority of pituitary tumours remain unknown. There is substantial evidence for derangement of cell cycle control in such tumours, but cell cycle protein mutations identified in other human malignancies are restricted to only a very small subset of sporadic pituitary neoplasms. Krüppel-like factors are DNA-binding transcriptional regulators with diverse effects including the upregulation of the cell cycle protein p21(WAF1/CIP1). It has been reported that the Krüppel-like transcription factor 6 (KLF6) gene is mutated in a proportion (15-55%) of human prostate cancers, and more recent data are emerging regarding mutated KLF6 in nasopharyngeal carcinomas, astrocytoid gliomas and colorectal cancer. We therefore speculated that other tumours such as pituitary adenomas might also harbour such mutations that may be involved in the control of cell proliferation in the pituitary. The aim of the current study was thus to analyse the KLF6 gene for mutations in sporadic pituitary tumours. We analysed 60 pituitary adenomas (15 GH-, four ACTH-, two PRL-secreting and 39 non-functioning) with direct sequence analysis of exons 2 and 3 of the KLF6 gene, the region where most of the previously described mutations are located. Three non-functioning pituitary adenomas of the 60 pituitary tumours (5%) had two identical sequence changes in exon 2 (missense mutation Val165Met, 523G-->A and a silent substitution in Ser77Ser codon 261C-->T). Analysis of genomic DNA extracted from peripheral lymphocytes in one patient confirmed these changes to be present in the germline and they therefore probably represent polymorphisms, although we cannot exclude the possibility that these are predisposing germline mutations. We conclude that mutations of the KLF6 gene are unlikely to play an important role in sporadic pituitary tumorigenesis.

Activating point mutations in cyclin-dependent kinase 4 are not seen in sporadic pituitary adenomas, insulinomas or Leydig cell tumours.

Cell cycle dysregulation is one of the defining features of cancer. Cyclin-dependent kinase 4 (CDK4), together with its regulatory subunit cyclin D, governs cell cycle progression through the G1 phase. Cyclin-dependent kinase inhibitors, including p16(INK4A) (encoded by CDKN2A), in turn regulate CDK4. In particular, dysregulation of the p16/CDK4/cyclin D complex has been established in a variety of types of human tumours. Dominant activating mutations affecting codon 24 of the CDK4 gene (replacement of Arg24 by Cys or His) render CDK4 insensitive to p16(INK4) inhibition and are responsible for melanoma susceptibility in some kindreds. However, 'knock-in' mice homozygous for the CDK4(R24C) mutation were noted to develop multiple neoplasia, most commonly including endocrine tumours: pituitary adenomas, insulinomas and Leydig cell testicular tumours. We therefore speculated that sporadic human endocrine tumours might also harbour such mutations. The aim of the current study was to analyze the CDK4 gene for the two characterized activating mutations, R24C and R24H, in sporadic human pituitary adenomas, insulinomas and Leydig cell tumours. We used DNA extracted from 61 pituitary adenomas, and paired tumorous and neighboring normal genomic DNA extracted from 14 insulinoma and 6 Leydig cell tumour samples. Genomic DNA from patients with familial melanoma harbouring the R24C or the R24H mutations served as positive controls. All samples were subjected to PCR, mutation-specific restriction digests and/or sequencing. Both methodologies failed to detect mutations at these two sites in any of the sporadic endocrine tumours including pituitary adenomas, benign or malignant insulinomas or Leydig cell tumours, while the positive controls showed the expected heterozygote patterns. Protein expression of CDK4 was demonstrated by immunohistochemistry and Western blotting in pituitary and pancreatic samples. These data suggest that the changes in the regulatory 'hot-spot' on the CDK4 gene, causing various endocrine tumours in CDK4(R24C/R24C )mice, are not a major factor in sporadic pituitary, insulin beta-cell or Leydig cell tumorigenesis.

Macrophage migration inhibitory factor expression is increased in pituitary adenoma cell nuclei.

Macrophage migration inhibitory factor (MIF) is an essential regulator of the macrophage responses to endotoxin. MIF also has the ability to override the anti-inflammatory actions of glucocorticoids during an immune response, and is thus an important pro-inflammatory factor. The presence of MIF in cells of the anterior pituitary has been described, and high levels of MIF in other rapidly proliferating tIssues have also been demonstrated. It has been hypothesised that MIF release from these cells is influenced by the hypothalamo-pituitary-adrenal axis, and that ACTH and MIF are released simultaneously to exert counter-regulatory effects on cortisol. However, another intracellular role for MIF has also been suggested as it has been shown that MIF exerts an effect on the inhibitory cell cycle control protein p27 through an interaction with Jab1, a protein implicated in p27 degradation. We studied MIF expression in different normal and adenomatous human pituitary samples using immunohistochemistry and RT-PCR. There was evidence of co-immunoprecipitation of MIF with Jab1, suggesting an interaction of the two proteins. Our results showed that there is increased expression of MIF protein in the nuclei of all pituitary adenomas compared with normal tIssue (P=0.0067), but there was no statistically significant difference in nuclear MIF expression between the different adenoma types. Nuclear MIF expression correlated positively with p27 and its phosphorylated form in normal tIssue (P=0.0028 and P<0.0001); however, this relationship was not seen in the adenoma samples. Cytoplasmic expression of MIF was found to be variable both in normal and adenomatous samples, with no consistent pattern. MIF mRNA was demonstrated to be present in all tumour and normal samples studied. Somatotroph tumours showed higher MIF mRNA expression compared with normal pituitary or other types of adenomas. In conclusion, MIF is expressed in cell nuclei in pituitary adenomas to a greater extent than in normal pituitary tIssue. We speculate that it may play a role in the control of the cell cycle, but whether its higher level in adenomas is a cause or a consequence of the tumorigenic process remains to be clarified.

Response of serum macrophage migration inhibitory factor levels to stimulation or suppression of the hypothalamo-pituitary-adrenal axis in normal subjects and patients with Cushing's disease.

Macrophage migration inhibitory factor (MIF) is a proinflammatory pituitary and immune cell cytokine and a critical mediator of septic shock. It has been reported that MIF is secreted in parallel with ACTH from the pituitary in response to stress or inflammatory stimuli. MIF release from immune cells is also induced rather than inhibited by glucocorticoids. It has therefore been suggested that MIF may be a novel counterregulatory hormone of glucocorticoid action that acts both as a paracrine and endocrine modulator of host responses. We have measured circulating MIF levels, using a human MIF ELISA, in normal subjects and patients under numerous pathophysiological conditions. Serum MIF was measured in normal subjects who underwent stimulation of the hypothalamo-pituitary-adrenal axis with an insulin tolerance test (n = 8), a CRH-stimulation test (n = 5), a short synacthen test (n = 5), and following a low-dose dexamethasone suppression test (n = 6). We also sampled from a peripheral vein and both inferior petrosal sinuses before and after CRH stimulation in four patients with a histologically proven diagnosis of Cushing's disease. Immunostaining of the pituitary tumors for MIF was also performed. In normal subjects serum MIF levels did not rise in parallel with cortisol during the insulin tolerance or CRH test or after administration of synthetic ACTH. In all subjects cortisol levels became undetectable after the low-dose dexamethasone suppression test, and no consistent change was observed in serum MIF levels during the test. In patients with Cushing's disease, there was no basal central-to-peripheral gradient in MIF, and no consistent changes occurred in serum MIF levels in either the left or right inferior petrosal sinus after CRH stimulation; however, immunostaining of the surgically removed pituitary tumors from the same patients showed strong staining for both ACTH and MIF. These results show that in humans acute modulation of the hypothalamo-pituitary-adrenal axis does not significantly alter circulating MIF levels. In addition, ACTH-secreting pituitary tumors that express MIF do not release MIF either spontaneously or in response to CRH stimulation, and there is no gradient for MIF in the venous drainage of the pituitary. Our study suggests that the pituitary gland is not the major contributor to circulating MIF; an autocrine or paracrine role for pituitary-derived MIF is more likely.

The release of leptin and its effect on hormone release from human pituitary adenomas.

BACKGROUND: Leptin is the protein product of the obese gene, known to play an important role in body energy balance. The leptin receptor exists in numerous isoforms, the long isoform being the major form involved in signal transduction. Leptin expression has recently been demonstrated in the human pituitary, both in normal tissue and in pituitary adenomas. The long isoform of the leptin receptor has also been shown to be present in pituitary adenomas; however, contrasting results have been obtained regarding its expression in the normal human pituitary. AIM: The aim of this study was (i) to investigate the presence and pattern of distribution of leptin mRNA and the long isoform of its receptor mRNA in the normal pituitary and in different types of pituitary adenomas with RT-PCR; (ii) to study leptin secretion from human pituitary tumours in culture and (iii) to assess in vitro pituitary hormone release following stimulation with human leptin. RESULTS: Leptin receptor long isoform expression was detected in 2/4 GH-secreting adenomas, 12/17 non-functioning adenomas, 5/9 ACTH-secreting adenomas, 1/2 prolactinomas, 2/2 FSH-secreting adenomas and 5/5 normal pituitaries. The receptor long isoform did not segregate with any particular tumour type, and varying levels of expression were detected between the tissues studied. Leptin mRNA was detected at a low level of expression in 2/7 GH-secreting adenomas, 9/14 non-functioning adenomas, 2/3 ACTH-secreting adenomas, 1/3 prolactinomas and 1/3 FSH-secreting adenomas. We were unable to detect leptin mRNA in any of the five normal pituitaries removed at autopsy; however, immunostaining of a non-tumorous pituitary adjacent to an adenoma removed at transsphenoidal surgery showed scattered leptin positive cells. Culture of pituitary adenomas showed that 16/47 released leptin into the incubation media. Leptin release did not correlate with tumour type or with any of the other pituitary hormones released. In vitro leptin stimulation of pituitary tumours caused stimulation of FSH and alpha-subunit secretion from a non-functioning adenoma and TSH secretion from a somatotroph adenoma. CONCLUSION: We conclude that not only is leptin stored within the pituitary, but it may also be released from pituitary cells and modulate other pituitary hormone secretion. Pituitary leptin may therefore be a novel paracrine regulator of pituitary function.

Leptin in pituitary adenomas--a novel paracrine regulatory system.

A growing number of physiological and pathophysiological processes have been shown to be influenced by leptin apart from its first recognised role as a modulator of hypothalamic appetite and weight control centers. We investigated the presence and pattern of distribution of leptin mRNA and the mRNA of the long isoform of the leptin receptor in the normal pituitary and in different types of pituitary adenomas. We also studied leptin secretion from human pituitary tumors in culture, and the in vitro pituitary hormone release following stimulation with human leptin. Leptin mRNA expression was detected at a low level of expression in 50% of tumors but in none of the normal pituitaries. By immunohistochemistry, leptin was present in occasional scattered cells in the normal pituitary and in pituitary tumors. The leptin receptor long isoform was detected in the majority (65%) of pituitary tumors and in all normal pituitaries. It did not segregate with any particular tumor type, and varying levels of expression were detected between the tissues studied. 34% of pituitary adenomas showed leptin release into the incubation media during in vitro culture. Leptin mRNA, the mRNA of the long isoform of the receptor, or in vitro leptin release, did not correlate with tumor type or with any of the other pituitary hormones released. In vitro leptin stimulation of pituitary tumors caused stimulation of FSH and a-subunit secretion from a non-functioning adenoma and TSH secretion from a somatotroph adenoma. As the co-localisation of ACTH and leptin in corticotroph cells was previously suggested, we investigated whether in vivo ACTH release is accompanied by a simultaneous plasma leptin level rise (i) in peripheral plasma samples after food intake-induced ACTH rise in healthy obese and nonobese individuals and (ii) in petrosal sinus samples after CRH injection in Cushing's disease patients. Our data suggest that a rise in ACTH levels is not accompanied by detectable rise in leptin levels in peripheral and in petrosal sinus blood samples. In summary, leptin is synthesized and stored within the pituitary and may modulate other pituitary hormone secretion, although probably it does not contribute to plasma leptin level changes. Pituitary leptin may therefore be a novel paracrine regulator of pituitary function.

Leptin and puberty: a review.

Over the last few years, many studies have focused on leptin, the product of the LEP (ob) gene, searching for a possible link between energy balance and reproduction. The involvement of this peptide in the regulation of the hypothalamo-pituitary-adrenal, gonadal, thyroid and somatotroph axes suggests that leptin might play a pivotal role in coordinating the activity of these axes and their relationship with the body's energy balance. The effects of leptin on hypothalamic, pituitary and peripheral hormone levels, as well as the presence of the leptin receptor in a variety of tissues, suggest both an endocrine and a paracrine mode of action. Particular attention was paid to the effect of leptin on the gonadal axis as infertility, a characteristic feature of both the leptin deficient ob/ob mice and the leptin receptor mutant db/db mice, could be corrected in ob/ob mice by leptin administration. Considerable leptin level changes were observed during puberty both in animal and human studies. A matter of controversy is the precise role of leptin in the onset of puberty: is leptin the signal that initiates puberty and the accompanying hormonal changes, or has leptin only a permissive but key role for the onset of puberty, as likely seems to be the case for the maintenance of a functional gonadotroph axis? On current evidence, a mainly permissive role seems most probable.