Efficacy and safety of bariatric surgery for craniopharyngioma-related hypothalamic obesity: a matched case-control study with 2 years of follow-up.

BACKGROUND: Hypothalamic obesity is a devastating consequence of craniopharyngioma. Bariatric surgery could be a promising therapeutic option. However, its efficacy and safety in patients with craniopharyngioma-related hypothalamic obesity remain largely unknown. OBJECTIVES: We investigated the efficacy of bariatric surgery for inducing weight loss in patients with craniopharyngioma-related hypothalamic obesity. In addition, we studied the safety of bariatric surgery regarding its effects on hormone replacement therapy for pituitary insufficiency. METHODS: In this retrospective matched case-control study, we compared weight loss after bariatric surgery (that is, Roux-en-Y gastric bypass and sleeve gastrectomy) between eight patients with craniopharyngioma-related hypothalamic obesity and 75 controls with 'common' obesity during 2 years of follow-up. We validated our results at 1 year of follow-up in a meta-analysis. In addition, we studied alterations in hormone replacement therapy after bariatric surgery in patients with craniopharyngioma. RESULTS: Mean weight loss after bariatric surgery was 19% vs 25% (difference -6%, 95% confidence of interval (CI) -14.1 to 4.6; P=0.091) at 2 years of follow-up in patients with craniopharyngioma-related hypothalamic obesity compared with control subjects with 'common' obesity. Mean weight loss was 25% vs 29% (difference -4%, 95% CI -11.6 to 8.1; P=0.419) after Roux-en-Y gastric bypass and 10% vs 20% (difference -10%, 95% CI -14.1 to -6.2; P=0.003) after sleeve gastrectomy at 2 years of follow-up in patients with craniopharyngioma-related hypothalamic obesity vs control subjects with 'common' obesity. Our meta-analysis demonstrated significant weight loss 1 year after Roux-en-Y gastric bypass, but not after sleeve gastrectomy. Seven patients with craniopharyngioma suffered from pituitary insufficiency; three of them required minor adjustments in hormone replacement therapy after bariatric surgery. CONCLUSIONS: Weight loss after Roux-en-Y gastric bypass, but not sleeve gastrectomy, was comparable between patients with craniopharyngioma-related hypothalamic obesity and control subjects with 'common' obesity at 2 years of follow-up. Bariatric surgery seems safe regarding its effects on hormone replacement therapy.

Acylated and unacylated ghrelin during OGTT in Prader-Willi syndrome: support for normal response to food intake.

BACKGROUND: Prader-Willi syndrome (PWS) is characterized by hyperphagia with impaired satiety. PWS patients have very high acylated ghrelin (AG) with normal unacylated ghrelin (UAG) levels, resulting in an elevated AG/UAG ratio, suggesting an intrinsic defect in the ghrelin regulation. Normally, food intake induces satiety and a drop in AG and UAG levels, but it is unknown if these levels also decline in PWS. OBJECTIVE: To evaluate whether the high AG levels in PWS decline in response to glucose intake during an oral glucose tolerance test (OGTT), and to investigate the effects of growth hormone (GH) treatment on this response. METHOD: Serum levels of AG, UAG and AG/UAG ratio during an OGTT were determined in 24 GH-treated patients with PWS (median age 19·0, range 14·2-25·9 years) and in 10 GH-stop patients (of whom five were in GH-treated group; 18·5, 14·5-20·3 years). RESULTS: In GH-treated and GH-stop young adults with PWS, there was a sharp decline of AG levels and a decrease of UAG levels in the first 30 min after the glucose load, which resulted in a lower AG/UAG ratio. GH-treated patients had significantly lower AG levels than GH-stop patients at baseline and during the OGTT. All UAG levels and AG/UAG ratios were lower in the GH-treated patients, although not significantly. CONCLUSIONS: In young adults with PWS, an oral glucose load significantly reduces AG and UAG levels, suggesting normal regulation of the ghrelin axis by food intake. GH treatment results in lower AG levels at baseline and during OGTT, suggesting a more favourable metabolic profile. Our findings might suggest that the impaired satiety is not the result of an abnormal response of the orexigenic ghrelin to food intake.

Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-R1a.

Recent studies demonstrate widespread expression of ghrelin among tissues and have uncovered its pleiotropic nature. We have examined gene expression of ghrelin and its two receptor splice variants, growth hormone secretagogue receptors (GHS-R) 1a and 1b, in human bone biopsies and in the human pre-osteoblastic SV-HFO cell line during differentiation. Additionally, we examined proliferative effects of ghrelin and unacylated ghrelin (UAG) in differentiating and non-differentiating cells. We detected GHS-R1b mRNA in human bone and osteoblasts but not ghrelin's cognate receptor GHS-R1a, using two different real-time PCR assays and both total RNA and mRNA. In osteoblasts GHS-R1b mRNA expression remained low during the first 14 days of culture, but increased 300% in differentiating cells by day 21. Both human bone biopsies and osteoblasts expressed ghrelin mRNA, and osteoblasts were found to secrete ghrelin. Overall, ghrelin gene expression was greater in differentiating than non-differentiating osteoblasts, but was not increased during culture in either group. Ghrelin and UAG induced thymidine uptake dose-dependently, peaking at 1 and 10 nM respectively, at day 6 of culture in both non-differentiating and differentiating osteoblasts. The proliferative response to ghrelin and UAG declined with culture time and state of differentiation. The proliferative effects of ghrelin and UAG were suppressed by inhibitors of extracellular-signal-regulated kinase (ERK) and phosphoinositide-3 kinase, and both peptides rapidly induced ERK phosphorylation. Overall, our data suggest new roles for ghrelin and UAG in modulating human osteoblast proliferation via a novel signal transduction pathway.

What is the efficacy of switching to weekly pegvisomant in acromegaly patients well controlled on combination therapy?

CONTEXT: Although combination therapy of acromegaly with long-acting somatostatin analogs (LA-SSAs) and pegvisomant (PEGV) normalizes insulin-like growth factor-1 (IGF1) levels in the majority of patients, it requires long-term adherence. Switching from combination therapy to monotherapy with weekly PEGV could improve patients' comfort, but the efficacy is unknown. OBJECTIVE: To assess the efficacy of switching to PEGV monotherapy in patients well controlled on combination therapy of LA-SSAs and PEGV. DESIGN: Single-center, open-label observational pilot study. LA-SSA therapy was discontinued at baseline and all patients were switched to PEGV monotherapy for 12 months. If IGF1 levels exceeded 1.0 times upper limit of normal (ULN), PEGV dose was increased by 20 mg weekly. SUBJECTS AND METHODS: The study included 15 subjects (eight males), with a median age of 58 years (range 35-80) on combination therapy of high-dose LA-SSAs and weekly PEGV for >6 months, and IGF1 levels within the normal range. Treatment efficacy was assessed by measuring serum IGF1 levels. RESULTS: After 12 months of weekly PEGV monotherapy, serum IGF1 levels of 73% of the subjects remained controlled. In one patient, LA-SSA had to be restarted due to recurrence of headache. IGF1 levels increased from a baseline level of 0.62 × ULN (range 0.30-0.84) to 0.83 × ULN (0.30-1.75) after 12 months, while the median weekly PEGV dose increased from 60 (30-80) mg to 80 (50-120) mg. CONCLUSION: Our results suggest that switching from combination therapy of LA-SSAs and PEGV to PEGV monotherapy can be a viable treatment option for acromegaly patients without compromising efficacy.

Pegvisomant in combination with long-acting somatostatin analogues in acromegaly: the role of the GH receptor deletion of exon 3.

BACKGROUND: Doses of the GH receptor (GHR) antagonist pegvisomant (PEGV) that normalize insulin-like growth factor 1 (IGF1) levels vary widely among acromegaly patients. Predictors for PEGV response are baseline IGF1 levels, sex, body weight and previous radiotherapy. A GHR polymorphism lacking exon 3 (d3-GHR) is frequent in the general population. The influence of d3-GHR on PEGV responsiveness in acromegaly is unclear. OBJECTIVE: To assess the influence of d3-GHR on IGF1 levels and PEGV responsiveness in acromegaly patients using combined PEGV and long-acting somatostatin receptor ligand (LA-SRIF) treatment. DESIGN: Data were collected at the Rotterdam Pituitary Centre between 2004 and 2013. Patients with elevated IGF1 levels (>1.2 upper limit of normal; n=112) and over 6 months of high-dose LA-SRIF treatment were co-treated with PEGV. GHR genotype was assessed using genomic DNA in 104 patients. RESULTS: D3-GHR was observed in 51 (49.0%) of the patients (7.7% homozygous, 41.3% heterozygous) and was in Hardy-Weinberg equilibrium (P=0.859). Baseline characteristics were similar in d3-GHR and full-length (fl)-GHR genotypes. During PEGV/LA-SRIF treatment IGF1 levels were not different between d3-carriers and non-carriers. Similarly, no difference in PEGV dose required to normalize IGF1 (P=0.337) or PEGV serum levels (P=0.433) was observed between the two groups. However, adenoma size decreased significantly (>20% of largest diameter) in 25.6% of the fl-GHR genotype but only in 7.5% of d3-carriers (P=0.034, OR: 4.6 (CI: 1.1-18.9)). CONCLUSIONS: GHR genotype does not predict the IGF1 normalizing dose of PEGV in acromegaly patients using combination PEGV/LA-SRIF treatment. However, fewer d3-carriers showed significant reductions in adenoma size.

Elevated ratio of acylated to unacylated ghrelin in children and young adults with Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is characterized by a switch from failure to thrive to excessive weight gain and hyperphagia in early childhood. Hyperghrelinemia may be involved in the underlying mechanisms of the switch. The purpose of this study is to evaluate acylated ghrelin (AG) and unacylated ghrelin (UAG) levels in PWS and investigate their associations with hyperphagia. This is a cross-sectional clinical study conducted in three PWS expert centers in the Netherlands and France. Levels of AG and UAG and the AG/UAG ratio were determined in 138 patients with PWS (0.2-29.4 years) and compared with 50 age-matched obese subjects (4.3-16.9 years) and 39 healthy controls (0.8-28.6 years). AEBSF was used to inhibit deacylation of AG. As a group, PWS patients had higher AG but similar UAG levels as healthy controls (AG 129.1 vs 82.4 pg/ml, p = 0.016; UAG 135.3 vs 157.3 pg/ml, resp.), resulting in a significantly higher AG/UAG ratio (1.00 vs 0.61, p = 0.001, resp.). Obese subjects had significantly lower AG and UAG levels than PWS and controls (40.3 and 35.3 pg/ml, resp.), but also a high AG/UAG ratio (1.16). The reason for the higher AG/UAG ratio in PWS and obese was, however, completely different, as PWS had a high AG and obese a very low UAG. PWS patients without weight gain or hyperphagia had a similar AG/UAG ratio as age-matched controls, in contrast to those with weight gain and/or hyperphagia who had an elevated AG/UAG ratio. The switch to excessive weight gain in PWS seems to coincide with an increase in the AG/UAG ratio, even prior to the start of hyperphagia.

The expression of insulin-like growth factor (IGF)-binding protein-2 and IGF-II genes in the tissues of the developing ovine fetus.

Insulin-like growth factor-II (IGF-II) regulates the growth and differentiation of tissues during fetal life and is bound to a significant extent to IGF-binding protein-2 (IGFBP-2) in blood and tissue fluids during this period of development. We have compared the expression of IGFBP-2 and IGF-II genes during development by determining the levels of stable mRNAs in various tissues of fetal [50 days gestational age to term (145-147 days)] and postnatal sheep [lambs (2 days, 4 weeks and 9 weeks) and adults; n = 3-4 in each age group]. Both IGFBP-2 and IGF-II mRNAs were observed from day 50 of gestation onward. The level of IGF-II mRNAs was high in fetal tissues from early gestational ages and decreased with maturation. Seven IGF-II mRNA transcripts [1.2-6 kilobases (kb)] were expressed by most tissues except the adult liver, which expressed only a single 5.1-kb transcript, and the choroid plexus, which expressed only six transcripts. A single IGFBP-2 transcript of approximately 1.5 kb was observed. Expression of the IGFBP-2 gene was ubiquitous in tissues from fetuses younger than 80 days gestational age, but from 120 days, it was restricted mainly to the liver, kidney, and choroid plexus. In general, the IGFBP-2 mRNA level in tissues was high in early gestation and decreased with maturation, thus following the same pattern of expression as IGF-II. However, this pattern was reversed in the liver. The concurrent expression of both IGFBP-2 and IGF-II mRNAs in the same tissues in early pregnancy suggests that both proteins are synthesized together in these tissues and act by autocrine and/or paracrine mechanisms. In later gestational ages and early postnatal life, when IGF-II mRNAs were expressed in decreasing levels, IGFBP-2 mRNA was present only in selected tissues. Liver was the only tissue that continued to express abundant IGFBP-2 mRNA levels, indicating that it was the major source of this BP at later gestational ages and postnatal life, when the protein functions as an endocrine factor. Plasma IGFBP-2 levels were relatively low in fetuses of early gestational ages (< 0.5 gestation) when most tissues were expressing this gene. Instead, plasma IGFBP-2 levels appeared to mirror the level of IGFBP-2 mRNA in the liver, further supporting the hypothesis that liver IGFBP-2 gene expression is the principal determinant of plasma IGFBP-2 levels.(ABSTRACT TRUNCATED AT 400 WORDS)

The regulation of acid-labile subunit gene expression and secretion by cyclic adenosine 3',5'-monophosphate.

Circulating acid-labile subunit (ALS) is mainly hepatocyte derived and is GH dependent. ALS buffers the metabolic effects of the insulin-like growth factors by sequestering them in a ternary complex with insulin-like growth factor-binding protein-3. Nutritional regulation of ALS may be mediated by cAMP and changes in circulating GH levels or tissue GH sensitivity. Therefore, we examined the regulation by cAMP of ALS steady state messenger RNA (mRNA) levels and secretion in isolated hepatocytes under basal and GH-induced conditions. Increasing intracellular cAMP in primary hepatocytes produced a dose-dependent suppression of ALS mRNA levels and secretion. This effect was not related to a reduction in mRNA stability. In the presence of GH there was a parallel suppression of mRNA levels and secretion. However, under basal conditions cAMP had less effect on ALS mRNA levels than on secretion. Thus, in the absence of GH, expression of ALS may be predominantly posttranscriptionally regulated by cAMP. Our study suggests that cAMP affects ALS gene transcription, perhaps by interrupting the GH signaling pathway, and also inhibits posttranscriptional events in ALS expression.

Insulin-like growth factor-II (IGF-II) messenger ribonucleic acid is expressed in steroidogenic cells of the developing ovine adrenal gland: evidence of an autocrine/paracrine role for IGF-II.

Insulin-like growth factors (IGFs) are potent mitogenic and differentiation-promoting factors that regulate the growth and development of many fetal tissues. Their role in the development of the adrenal gland and activation of its function is not known. The latter is crucial in providing the stimulus for the maturation of various fetal organs and determines the onset of parturition in sheep. To examine the hypothesis that IGFs are important autocrine/paracrine regulators of fetal adrenal development in vivo, we localized IGF-I and IGF-II mRNAs and peptides in the adrenal glands of developing sheep fetuses and correlated the cellular distribution with localization of 3 beta-hydroxysteroid dehydrogenase, tyrosine hydroxylase, and phenylethanolamine-N-methyltransferase enzymes by immunohistochemistry. Adrenal glands from 60- to 75-day-old (n = 4), 100- to 110-day-old (n = 4), 120- to 130-day-old (n = 4), and 145- to 147-day-old (term; n = 4) fetal sheep and 1- to 4-day-old newborn lambs (n = 4) were dissected and either snap-frozen or fixed. Total RNAs were subjected to Northern analysis using ovine IGF-I and IGF-II cDNA probes. Seven IGF-II transcripts of 1.2-6.0 kilobases (kb) were identified in the adrenal glands of fetuses at all gestational ages, and in the newborn. By densitometry, the abundance of IGF-II mRNA was highest in the fetal adrenal gland at 60 days, decreased slightly between 60 and 100 days, remained relatively constant until term, and decreased significantly after birth. At all gestational ages, IGF-II mRNA was detectable in significantly greater abundance than IGF-I mRNA. IGF-I and IGF-II mRNAs were localized by in situ hybridization using 35S-labeled anti-sense cRNA probes, and the peptides by immunohistochemistry using specific antisera. Low levels of IGF-I mRNA were detected in the zona fasciculata, but not in the zona glomerulosa. There was strong hybridization of the IGF-II cRNA to the zona glomerulosa and fasciculata and to the capsule. The hybridization signal was greater in the zona fasciculata than in the zona glomerulosa. IGF-II mRNA was also detected in groups of cells within the medulla. Localization of IGF-II mRNA by in situ hybridization correlated well with the distribution of IGF-II immunoreactivity and with 3 beta-hydroxysteroid dehydrogenase-positive cells in the cortex and in groups of cells within the medulla.(ABSTRACT TRUNCATED AT 400 WORDS)

Prolonged hypoxia induced by the reduction of maternal uterine blood flow alters insulin-like growth factor-binding protein-1 (IGFBP-1) and IGFBP-2 gene expression in the ovine fetus.

Insulin-like growth factors (IGF-I and IGF-II) are potent mitogenic and differentiating peptides which are synthesized by many fetal tissues. In the circulation and tissue fluids, IGFs are bound to binding proteins (BPs) which not only function as carrier proteins, but also inhibit or modulate the biological actions of IGFs. We have previously shown that prolonged hypoxia in the ovine fetus induced by the reduction of maternal uterine blood flow for 24 h causes a reduction in the DNA synthesis rate in selected fetal tissues. To determine if this effect is due to alterations in the local synthesis of tissue IGFs and their binding proteins or to changes in systemic concentrations of IGFs and IGFBPs, we have investigated the abundance of mRNAs encoding IGFs and IGFBPs in selected tissues and changes in plasma IGFs and IGFBPs. Ovine fetuses (115-120 days gestation; n = 6) underwent 24 h of hypoxia by the reduction of maternal uterine blood flow (RUBF). Controls (n = 6) underwent the same surgical procedure without RUBF. Serial plasma samples were collected before, during, and after the experiment, and tissues were collected at the end of 24 h. Mean plasma IGF-I and IGF-II concentrations tended to be lower in hypoxic fetuses than in controls during the course of hypoxia, but these differences were not statistically significant. Tissue mRNA levels for IGF-I and IGF-II in lung, muscle, thymus, and kidney were similar in control and hypoxic fetuses after 24 h of hypoxia. The relative abundance of liver IGF-I and IGF-II mRNAs was lower in hypoxic fetuses, but only IGF-I mRNA levels were significantly different from the control values (P < 0.05). Compared to control fetuses, IGFBP-1 mRNA levels in the liver of hypoxic fetuses were increased 3- to 7-fold, and IGFBP-1 mRNA expression was induced in kidneys of some hypoxic fetuses (two of six). In addition, IGFBP-2 mRNA levels were decreased in the liver (50%) and kidney (30%) of hypoxic fetuses. The increase in liver IGFBP-1 mRNA abundance and the decrease in liver and kidney IGFBP-2 mRNA abundance were accompanied by an increase in IGFBP-1 levels and a decrease in IGFBP-2 levels in fetal plasma. No changes were observed in either plasma levels or tissue mRNA abundance for IGFBP-3. Analysis of the time course of changes in plasma revealed that the changes in IGFBP-1 and IGFBP-2 occurred within 4 h of hypoxia.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute and short-term effects of growth hormone on insulin-like growth factors and their binding proteins: serum levels and hepatic messenger ribonucleic acid responses in humans.

We investigated the acute (4-5 h) and short-term (5 days) effects of GH treatment on hepatic messenger RNA (mRNA) levels of the genes for the insulin-like growth factors (IGFs), insulin-like growth factor binding protein-1, -2, and -3 (IGFBPs), and the acid labile subunit (ALS), as well as serum levels of these proteins in humans. At the mRNA level, we observed an increase in IGF-1 transcription (+173%) following GH treatment in the acute group, which remained elevated in the short-term treatment group. IGFBP-2 mRNA decreased after short-term GH treatment, without changes in IGFBP-1 or -3 expression. The ALS transcript level increased after 5 days. In serum, we found increased levels of IGF-I and insulin, and decreased levels of IGF-II, in the short-term treatment group. IGFBP-1 decreased in both treatment groups, whereas IGFBP-2 was reduced after 5 days treatment. ALS increased in the short-term group. We observed increased IGFBP-3 serum levels after 5 days of GH treatment, likely due to increased formation of the ternary complex. Our results show that the metabolic effects by GH on the IGF axis are complex. In addition to a direct stimulation of IGF-I and ALS expression, GH inhibits IGFBP-1 serum levels and IGFBP-2 expression in an indirect manner, possibly facilitating enhanced IGF bioavailability to target tissues.

Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity.

We investigated the metabolic actions of ghrelin in humans by examining the effects of acute administration of acylated ghrelin, unacylated ghrelin, and the combination in eight adult-onset GH-deficient patients. We followed glucose, insulin, and free fatty acid concentrations before and after lunch and with or without the presence of GH in the circulation. We found that acylated ghrelin, which is rapidly cleared from the circulation, induced a rapid rise in glucose and insulin levels. Unacylated ghrelin, however, prevented the acylated ghrelin-induced rise in insulin and glucose when it was coadministered with acylated ghrelin. Surprisingly, the injection of acylated ghrelin induced an acute increase in unacylated ghrelin and therefore total ghrelin levels. Finally, acylated ghrelin decreased insulin sensitivity up to the end of a period of 6 h after administration. This decrease in insulin sensitivity was prevented by coinjection of unacylated ghrelin. This combined administration of acylated and unacylated ghrelin even significantly improved insulin sensitivity, compared with placebo, for at least 6 h, which warrants studies to investigate the long-term efficacy of this combination in the treatment of disorders with disturbed insulin sensitivity.

Sp1 and Sp3 transactivate the RET proto-oncogene promoter.

The RET proto-oncogene plays an important role in the initiation and progression of tumors derived from the neural crest. The cis-regulatory elements responsible for RET basal promoter activity have not been identified. To characterize these elements, a RET promoter DNA fragment (-453 to +227bp) was fused to a luciferase reporter and introduced into TT, a neural crest-derived cell line. Sequential 5' deletions of the promoter revealed that optimal expression of the RET promoter in TT cells required only 70bp of sequence upstream of the transcription start site, and contains two Sp1 binding sites. DNase I footprinting, electrophoretic mobility shift analysis (EMSA), and supershift assays revealed that this region binds both Sp1 and its related protein, Sp3. Additionally, RET basal promoter activity was abrogated by removal of these Sp1/Sp3 binding sites. The proximal two GC boxes were sufficient to allow transactivation of the RET promoter in Drosophila SL2 cells. Sp3 expression in these cells caused an additional activation of the promoter. These results demonstrate that the transactivation of the RET promoter within a neural crest-derived cell line is dependent on Sp1 and Sp3.

The characterization and expression of ovine insulin-like growth factor-binding protein-2.

We have isolated an ovine insulin-like growth factor-binding protein-2 (IGFBP-2) cDNA from an adult sheep cDNA library, to determine the structure of ovine IGFBP-2 and to examine the pattern of IGFBP-2 gene expression in adult sheep tissues. This cDNA had 81, 96 and 87% identity with the rat, bovine and human sequences respectively. The deduced amino acid sequence of the ovine IGFBP-2 showed 86, 95 and 85% homology with the rat, bovine and human peptide sequences respectively. The ovine IGFBP-2 cDNA encoded a precursor protein of 317 amino acids which comprised a 33 residue hydrophobic leader sequence and a 284 residue, 30.9 kDa, mature peptide. The 18 cysteine residues, which are a characteristic feature of IGFBPs, were conserved. Also, an Arg-Gly-Asp (RGD) sequence near the C terminus was present. A single transcript of approximately 1.5 kb was expressed in abundance in selected tissues of an adult sheep, namely liver, kidney, adrenal, pituitary and choroid plexus. Southern blot analysis of ovine genomic DNA with the cDNA probe demonstrated that IGFBP-2 is encoded by a single gene. These findings indicate that the ovine IGFBP-2 protein is similar to that in other species and that, in the adult, the mRNA is expressed only in selected tissues.

Cloning and characterization of the rat gene for the acid-labile subunit of the insulin-like growth factor binding protein complex.

The acid-labile subunit (ALS) of the ternary insulin-like growth factor-binding protein complex has a central role in controlling the bioavailability of circulating insulin-like growth factors. We have shown that gene expression of ALS is regulated by a number of factors, particularly growth hormone. Our aim was to characterize the ALS gene in order to define the mechanism of its regulation. Southern analysis suggests a single copy of the ALS gene in the rat genome. The protein-coding and 3'-untranslated regions span approximately 3.5 kilobases of rat genome and are divided into two exons. The 5' flanking region of the gene lacks a consensus TATA-box or Inr sequence, and primer extension and reverse transcriptase PCR experiments locate multiple transcriptional initiation sites between -505 and -385 nucleotides relative to the translational initiation codon. This putative promoter region, when inserted upstream of the luciferase reporter gene, directs luciferase expression when transfected into H4-II-E cells. Our data demonstrate the uncomplicated structure of the rat ALS gene, and the promoter function and presence of potential regulatory elements in the region upstream of the protein-coding sequence.

Desacyl ghrelin is influenced by changes in insulin concentration during an insulin tolerance test.

OBJECTIVE: Ghrelin, a gut-brain peptide, regulates energy homeostasis and glucose metabolism and is present in acylated and nonacylated form in the circulation. Although desacyl ghrelin (DAG), the predominant form of ghrelin, is associated with insulin sensitivity and improved metabolic state, not much is known about its direct regulation by insulin. We aimed to assess changes in DAG in response to the rapid increase in insulin concentration during an insulin tolerance test (ITT) in normal weight and obese subjects. DESIGN: We performed an observational single center study. An ITT was assessed in eight subjects (four males), median age of 29.9 years (range 19.6-42.0). DAG concentrations were measured at 20, 40, 60 and 90 min after insulin infusion. Homeostatic Model Assessment (HOMA) was calculated from fasting insulin and glucose. Body mass index (BMI) and waist circumference were assessed. RESULTS: Three subjects were obese (BMI ≥ 30 kg/m(2)), one subject was overweight (BMI = 25-30 kg/m(2)) and four subjects had normal weight (BMI = 18.5-25 kg/m(2)). Median DAG decreased after insulin infusion (90 pg/mL, p = 0.028), especially in normal weight subjects. Baseline DAG was lower in subjects with higher BMI (ρ = -0.76, p = 0.028) and higher fasting insulin (ρ = -0.76, p = 0.030). DAG changes correlated with fasting insulin levels (ρ = -0.85, p = 0.007), HOMA (ρ = -0.86, p = 0.007), BMI (ρ = -0.83, p = 0.010) and waist circumference (ρ = -0.93, p < 0.001). CONCLUSION: DAG levels rapidly decreased in response to insulin administration in normal subjects, but not in insulin-resistant obese who are in a state of relative DAG deficiency.

Ghrelin and glucose homeostasis.

Ghrelin plays an important physiological role in modulating GH secretion, insulin secretion and glucose metabolism. Ghrelin has direct effects on pancreatic islet function. Also, ghrelin is part of a mechanism that integrates the physiological response to fasting. However, pharmacologic studies indicate the important obesogenic/diabetogenic properties of ghrelin. This is very likely of physiological relevance, deriving from a requirement to protect against seasonal periods of food scarcity by building energy reserves, predominantly in the form of fat. Available data indicate the potential of ghrelin blockade as a means to prevent its diabetogenic effects. Several studies indicate a negative correlation between ghrelin levels and the incidence of type 2 diabetes and insulin resistance. However, it is unclear if low ghrelin levels are a risk factor or a compensatory response. Direct antagonism of the receptor does not always have the desired effects, however, since it can cause increased body weight gain. Pharmacological suppression of the ghrelin/des-acyl ghrelin ratio by treatment with des-acyl ghrelin may also be a viable alternative approach which appears to improve insulin sensitivity. A promising recently developed approach appears to be through the blockade of GOAT activity, although the longer term effects of this treatment remain to be investigated.

Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway.

Ghrelin is expressed in normal human adrenocortical cells and induces their proliferation through growth hormone secretagogue receptor 1a (GHS-R1a). Consequently, it was of interest to us to determine whether acylated ghrelin and its predominant serum isoform, unacylated ghrelin, also act as factors for adrenocortical carcinoma cell growth. To examine a potential ghrelin-regulated system in adrenocortical tumors, we measured proliferative effects of acylated and unacylated ghrelin in the adrenocortical carcinoma cell lines SW-13 and NCI-H295R. We also examined the expression of ghrelin, GHS-R1a, and corticotrophin-releasing factor receptor 2 (CRF-R2). Acylated and unacylated ghrelin in the nanomolar range dose-dependently induced adrenocortical cell growth up to 200% of untreated controls, as measured by thymidine uptake and WST1 assay. The proliferative effects of acylated and unacylated ghrelin in SW-13 cells was blocked by [D-Lys(3)]growth hormone-releasing peptide 6 (GHRP6), but a CRF-R2 antagonist had no effect on unacylated ghrelin growth stimulation. Cell cycle analysis suggests that acylated and unacylated ghrelin suppress the sub-G(0)/apoptotic fraction by up to 50%. Measurement of DNA fragmentation and caspase-3 and -7 activity in SW-13 cells confirmed that acylated and unacylated ghrelin suppress apoptotic rate. SW-13 cells express preproghrelin mRNA and secrete ghrelin, and [D-Lys(3)]GHRP6 suppresses their basal proliferation rate, strongly suggesting that ghrelin could act as an auto/paracrine growth factor. Acylated and unacylated ghrelin are potential auto/paracrine factors acting through an antiapoptotic pathway to stimulate adrenocortical tumor cell growth. Unacylated ghrelin-stimulated growth is suppressed by an antagonist of GHS-R1a, suggesting either that unacylated ghrelin is acylated before its action or that ghrelin, unacylated ghrelin, and [D-Lys(3)]GHRP-6 bind to a novel receptor in these cells.