A Somatostatin Receptor Subtype-3 (SST3) Peptide Agonist Shows Antitumor Effects in Experimental Models of Nonfunctioning Pituitary Tumors.

PURPOSE: Somatostatin analogues (SSA) are efficacious and safe treatments for a variety of neuroendocrine tumors, especially pituitary neuroendocrine tumors (PitNET). Their therapeutic effects are mainly mediated by somatostatin receptors SST2 and SST5. Most SSAs, such as octreotide/lanreotide/pasireotide, are either nonselective or activate mainly SST2. However, nonfunctioning pituitary tumors (NFPTs), the most common PitNET type, mainly express SST3 and finding peptides that activate this particular somatostatin receptor has been very challenging. Therefore, the main objective of this study was to identify SST3-agonists and characterize their effects on experimental NFPT models. EXPERIMENTAL DESIGN: Binding to SSTs and cAMP level determinations were used to screen a peptide library and identify SST3-agonists. Key functional parameters (cell viability/caspase activity/chromogranin-A secretion/mRNA expression/intracellular signaling pathways) were assessed on NFPT primary cell cultures in response to SST3-agonists. Tumor growth was assessed in a preclinical PitNET mouse model treated with a SST3-agonist. RESULTS: We successfully identified the first SST3-agonist peptides. SST3-agonists lowered cell viability and chromogranin-A secretion, increased apoptosis in vitro, and reduced tumor growth in a preclinical PitNET model. As expected, inhibition of cell viability in response to SST3-agonists defined two NFPT populations: responsive and unresponsive, wherein responsive NFPTs expressed more SST3 than unresponsive NFPTs and exhibited a profound reduction of MAPK, PI3K-AKT/mTOR, and JAK/STAT signaling pathways upon SST3-agonist treatments. Concurrently, SSTR3 silencing increased cell viability in a subset of NFPTs. CONCLUSIONS: This study demonstrates that SST3-agonists activate signaling mechanisms that reduce NFPT cell viability and inhibit pituitary tumor growth in experimental models that expresses SST3, suggesting that targeting this receptor could be an efficacious treatment for NFPTs.

Insulin resistance determines a differential response to changes in dietary fat modification on metabolic syndrome risk factors: the LIPGENE study.

BACKGROUND: Previous data support the benefits of reducing dietary saturated fatty acids (SFAs) on insulin resistance (IR) and other metabolic risk factors. However, whether the IR status of those suffering from metabolic syndrome (MetS) affects this response is not established. OBJECTIVE: Our objective was to determine whether the degree of IR influences the effect of substituting high-saturated fatty acid (HSFA) diets by isoenergetic alterations in the quality and quantity of dietary fat on MetS risk factors. DESIGN: In this single-blind, parallel, controlled, dietary intervention study, MetS subjects (n = 472) from 8 European countries classified by different IR levels according to homeostasis model assessment of insulin resistance (HOMA-IR) were randomly assigned to 4 diets: an HSFA diet; a high-monounsaturated fatty acid (HMUFA) diet; a low-fat, high-complex carbohydrate (LFHCC) diet supplemented with long-chain n-3 polyunsaturated fatty acids (1.2 g/d); or an LFHCC diet supplemented with placebo for 12 wk (control). Anthropometric, lipid, inflammatory, and IR markers were determined. RESULTS: Insulin-resistant MetS subjects with the highest HOMA-IR improved IR, with reduced insulin and HOMA-IR concentrations after consumption of the HMUFA and LFHCC n-3 diets (P < 0.05). In contrast, subjects with lower HOMA-IR showed reduced body mass index and waist circumference after consumption of the LFHCC control and LFHCC n-3 diets and increased HDL cholesterol concentrations after consumption of the HMUFA and HSFA diets (P < 0.05). MetS subjects with a low to medium HOMA-IR exhibited reduced blood pressure, triglyceride, and LDL cholesterol levels after the LFHCC n-3 diet and increased apolipoprotein A-I concentrations after consumption of the HMUFA and HSFA diets (all P < 0.05). CONCLUSIONS: Insulin-resistant MetS subjects with more metabolic complications responded differently to dietary fat modification, being more susceptible to a health effect from the substitution of SFAs in the HMUFA and LFHCC n-3 diets. Conversely, MetS subjects without IR may be more sensitive to the detrimental effects of HSFA intake. The metabolic phenotype of subjects clearly determines response to the quantity and quality of dietary fat on MetS risk factors, which suggests that targeted and personalized dietary therapies may be of value for its different metabolic features. This study was registered at clinicaltrials.gov as NCT00429195.

Dietary fat alters the expression of cortistatin and ghrelin systems in the PBMCs of elderly subjects: putative implications in the postprandial inflammatory response.

SCOPE: Dietary fat influences systemic inflammatory status, which determines the progression of age-associated diseases. Since somatostatin (SST), cortistatin (CORT), and ghrelin systems modulate inflammatory response, we aim to comprehensively characterize the presence and regulation of the components of these systems in the peripheral blood mononuclear cells (PMBCs), a subset of white blood cells placed at the crossroad between diet and inflammation, in response to diets with different fat composition, and during the postprandial phase in elderly subjects. METHODS AND RESULTS: The applied nutrigenomic, inflammation-related PBMC-based approach revealed that the majority of components of SST/CORT and ghrelin systems are present in the human PBMCs. Particularly, CORT, SST/CORT receptors (sst2, sst3, sst5, and sst5TMD4), ghrelin, its acylating enzyme (GOAT), In1-ghrelin variant, and GHSR1b were detected in PBMCs. Their expression was altered in the long-term by diet composition, and in the short-term, during the postprandial phase. Of particular relevance is the postprandial elevation of CORT, sst2, and sst5 expression in PBMCs of subjects under n-3 PUFAs-enriched diet. CONCLUSION: Our results suggest a potential relevant role of CORT/ssts and ghrelin systems in regulating PBMCs response to nutrient intake, which could help to explain the positive effects of n-3 PUFAs-enriched diets in reducing the inflammatory response.

Obesity-induced hypogonadism in the male: premature reproductive neuroendocrine senescence and contribution of Kiss1-mediated mechanisms.

Reproduction is sensitive to insufficient body energy reserves, especially in females. Metabolic regulation of the male reproductive axis is less obvious, and the impact of conditions of persistent energy excess has received moderate attention. Yet, the escalating prevalence of obesity and the clinical evidence of its deleterious effects on male fertility have raised considerable concerns. We report here phenotypic and mechanistic studies of the reproductive impact of postnatal nutritional manipulations (mainly overnutrition) coupled to a high-fat diet (HFD) after weaning. Metabolic and hormonal analyses in young (4 months old) and middle-aged (10 months old) animals revealed that HFD caused profound metabolic perturbations, including glucose intolerance, which were worsened by precedent postnatal overfeeding; these were detectable already in young males but aggravated in 10-month-old rats. Impairment of reproductive parameters took place progressively, and HFD alone was sufficient to explain most of these alterations, regardless of postnatal under- or overnutrition. In young males, testosterone (T) levels and steroidogenic enzyme expression were suppressed by HFD, without compensatory increases of LH levels, which were in fact partially inhibited in heavier males. In addition, obese males displayed suppressed hypothalamic Kiss1 expression despite low T, and HFD inhibited LH responses to kisspeptin. Overweight anticipated some of the neuroendocrine effects of aging, such as the suppression of hypothalamic Kiss1 expression and the decline in serum T and LH levels. Nonetheless, HFD per se caused a detectable worsening of key reproductive indices in middle-aged males, such as basal LH and FSH levels as well as LH responses to kisspeptin. Our study demonstrates that nutritional stress, especially HFD, has a profound deleterious impact on metabolic and gonadotropic function as well as on the Kiss1 system and precipitates neuroendocrine reproductive senescence in the male.

Endogenous somatostatin is critical in regulating the acute effects of L-arginine on growth hormone and insulin release in mice.

l-arginine (l-Arg) rapidly stimulates GH and insulin release in vivo. It has been hypothesized that l-Arg stimulates GH release by lowering hypothalamic somatostatin (SST) tone. l-Arg may also act directly at the pituitary to stimulate GH release. Moreover, l-Arg has a direct stimulatory effect on ß-cells, which is thought to be blunted by the release of SST from pancreatic δ-cells. To confirm the role of endogenous SST on l-Arg-induced GH and insulin release, wild-type (WT) and SST-knockout (SST-KO) mice were injected with l-Arg (ip; 0.8 g/kg), and pre-/post-injection GH, insulin, and glucose levels were measured. In WT mice, l-Arg evoked a 6-fold increase in circulating GH. However, there was only a modest increase in GH levels in WT pituitary cell cultures treated with l-Arg. In contrast, l-Arg failed to increase GH in SST-KO beyond their already elevated levels. These results further support the hypothesis that the primary mechanism by which l-Arg acutely increases GH in vivo is by lowering hypothalamic SST input to the pituitary and not via direct pituitary effects. Additionally, l-Arg induced a clear first-phase insulin secretion in WT mice, but not in SST-KO. However, SST-KO, but not WT mice, displayed a robust and sustained second-phase insulin release. These results further support a role for endogenous SST in regulating l-Arg-mediated insulin release.

Hyperthyroidism differentially regulates neuropeptide S system in the rat brain.

Thyroid hormones play an important role in the regulation of energy balance, sleep and emotional behaviors. Neuropeptide S (NPS) is a recently discovered neuropeptide, regulating feeding, sleep and anxiety. Here, we examined the effect of hyperthyroidism on the gene and protein expression of neuropeptide S and its receptor (NPS-R) in the hypothalamus, brainstem and amygdala of rats. Our results showed that the expression of NPS and NPS-R was differentially modulated by hyperthyroidism in the rat brain. NPS and NPS-R mRNA and protein levels were decreased in the hypothalamus of hyperthyroid rats. Conversely NPS-R expression was highly increased in the brainstem and NPS and NPS-R expression were unchanged in the amygdala of these rats. These data suggest that changes in anxiety and food intake patterns observed in hyperthyroidism could be associated with changes in the expression of NPS and NPS-R. Thus, the NPS/NPS-R system may be involved in several hyperthyroidism-associated comorbidities.

Mediterranean diet supplemented with coenzyme Q10 induces postprandial changes in p53 in response to oxidative DNA damage in elderly subjects.

Coenzyme Q10 (CoQ) is a powerful antioxidant that reduces oxidative stress. We explored whether the quality of dietary fat alters postprandial oxidative DNA damage and whether supplementation with CoQ improves antioxidant capacity by modifying the activation/stabilization of p53 in elderly subjects. In this crossover study, 20 subjects were randomly assigned to receive three isocaloric diets during 4 weeks each: (1) Mediterranean diet (Med diet), (2) Mediterranean diet supplemented with CoQ (Med+CoQ diet), and (3) saturated fatty acid-rich diet (SFA diet). Levels of mRNAs were determined for p53, p21, p53R2, and mdm2. Protein levels of p53, phosphorylated p53 (Ser20), and monoubiquitinated p53 were also measured, both in cytoplasm and nucleus. The extent of DNA damage was measured as plasma 8-OHdG. SFA diet displayed higher postprandial 8-OHdG concentrations, p53 mRNA and monoubiquitinated p53, and lower postprandial Mdm2 mRNA levels compared with Med and Med+CoQ diets (p < 0.05). Moreover, Med+CoQ diet induced a postprandial decrease of cytoplasmatic p53, nuclear p-p53 (Ser20), and nuclear and cytoplasmatic monoubiquitinated p53 protein (p < 0.05). In conclusion, Med+CoQ diet improves oxidative DNA damage in elderly subjects and reduces processes of cellular oxidation. Our results suggest a starting point for the prevention of oxidative processes associated with aging.

Somatostatin and its receptors contribute in a tissue-specific manner to the sex-dependent metabolic (fed/fasting) control of growth hormone axis in mice.

Somatostatin (SST) inhibits growth hormone (GH) secretion and regulates multiple processes by signaling through its receptors sst1-5. Differential expression of SST/ssts may contribute to sex-specific GH pattern and fasting-induced GH rise. To further delineate the tissue-specific roles of SST and sst1-5 in these processes, their expression patterns were evaluated in hypothalamus, pituitary, and stomach of male and female mice under fed/fasted conditions in the presence (wild type) or absence (SST-knockout) of endogenous SST. Under fed conditions, hypothalamic/stomach SST/ssts expression did not differ between sexes, whereas male pituitary expressed more SST and sst2A/2B/3/5A/5TMD2/5TMD1 and less sst1, and male pituitary cell cultures were more responsive to SST inhibitory actions on GH release compared with females. This suggests that local pituitary SST/ssts can contribute to the sexually dimorphic pattern of GH release. Fasting (48 h) reduced stomach sst2A/B and hypothalamic SST/sst2A expression in both sexes, whereas it caused a generalized downregulation of pituitary sst subtypes in male and of sst2A only in females. Thus, fasting can reduce SST sensitivity across tissues and SST input to the pituitary, thereby jointly contributing to enhance GH release. In SST-knockout mice, lack of SST differentially altered sst subtype expression levels in both sexes, supporting an important role for SST in sex-dependent control of GH axis. Evaluation of SST, IGF-I, and glucocorticoid effects on hypothalamic and pituitary cell cultures revealed that these hormones could directly account for alterations in sst2/5 expression in the physiological states examined. Taken together, these results indicate that changes in SST output and sensitivity can contribute critically to precisely define, in a tissue-dependent manner, the sex-specific metabolic regulation of the GH axis.

Identification and characterization of new functional truncated variants of somatostatin receptor subtype 5 in rodents.

Somatostatin and cortistatin exert multiple biological actions through five receptors (sst1-5); however, not all their effects can be explained by activation of sst1-5. Indeed, we recently identified novel truncated but functional human sst5-variants, present in normal and tumoral tissues. In this study, we identified and characterized three novel truncated sst5 variants in mice and one in rats displaying different numbers of transmembrane-domains [TMD; sst5TMD4, sst5TMD2, sst5TMD1 (mouse-variants) and sst5TMD1 (rat-variant)]. These sst5 variants: (1) are functional to mediate ligand-selective-induced variations in [Ca(2+)]i and cAMP despite being truncated; (2) display preferential intracellular distribution; (3) mostly share full-length sst5 tissue distribution, but exhibit unique differences; (4) are differentially regulated by changes in hormonal/metabolic environment in a tissue- (e.g., central vs. systemic) and ligand-dependent manner. Altogether, our results demonstrate the existence of new truncated sst5-variants with unique ligand-selective signaling properties, which could contribute to further understanding the complex, distinct pathophysiological roles of somatostatin and cortistatin.

Metabolic regulation of ghrelin O-acyl transferase (GOAT) expression in the mouse hypothalamus, pituitary, and stomach.

Ghrelin acts as an endocrine link connecting physiological processes regulating food intake, body composition, growth, and energy balance. Ghrelin is the only peptide known to undergo octanoylation. The enzyme mediating this process, ghrelin O-acyltransferase (GOAT), is expressed in the gastrointestinal tract (GI; primary source of circulating ghrelin) as well as other tissues. The present study demonstrates that stomach GOAT mRNA levels correlate with circulating acylated-ghrelin levels in fasted and diet-induced obese mice. In addition, GOAT was found to be expressed in both the pituitary and hypothalamus (two target tissues of ghrelin's actions), and regulated in response to metabolic status. Using primary pituitary cell cultures as a model system to study the regulation of GOAT expression, we found that acylated-ghrelin, but not desacyl-ghrelin, increased GOAT expression. In addition, growth-hormone-releasing hormone (GHRH) and leptin increased, while somatostatin (SST) decreased GOAT expression. The physiologic relevance of these later results is supported by the observation that pituitary GOAT expression in mice lacking GHRH, SST and leptin showed opposite changes to those observed after in vitro treatment with the corresponding peptides. Therefore, it seems plausible that these hormones directly contribute to the regulation of pituitary GOAT. Interestingly, in all the models studied, pituitary GOAT expression paralleled changes in the expression of a dominant spliced-variant of ghrelin (In2-ghrelin) and therefore this transcript may be a primary substrate for pituitary GOAT. Collectively, these observations support the notion that the GI tract is not the only source of acylated-ghrelin, but in fact locally produced des-acylated-ghrelin could be converted to acylated-ghrelin within target tissues by locally active GOAT, to mediate its tissue-specific effects.

Melanotrope secretory cycle is regulated by physiological inputs via the hypothalamus.

Previously, it has been shown that background color conditions regulate the overall activity of the frog intermediate lobe by varying the proportions of the two subtypes of melanotropes existing in the gland, the highly active or secretory melanotropes and hormone storage melanotropes, depending on melanocyte-stimulating hormone requirements. However, the factors and mechanisms underlying these background-induced changes are still unknown. In the present study, we investigated whether hypothalamic factors known to regulate melanotrope cell function can induce changes in vitro similar to those caused by background adaptation in vivo. We found that the inhibitors apomorphine (a dopamine receptor agonist) and neuropeptide Y decreased the number of active melanotropes and increased simultaneously that of storage melanotropes. On the other hand, the stimulator TRH increased the number of active cells and concomitantly reduced that of storage cells. Inasmuch as none of these treatments modified the apoptotic and proliferation rates in melanotrope cells, it appears that these hypothalamic factors caused actual interconversions of cells from a subpopulation to its counterpart. Taken together, these findings suggest that the hypothalamus would control melanotrope activity not only through short-term regulation of hormone synthesis and release, but also through a long-term regulation of the secretory phenotype of these cells whereby the activity of the intermediate lobe would be adjusted to fulfill the hormonal requirements imposed by background conditions.