Rhonda D. Kineman

Enhanced Growth of Mice Lacking the Cyclin-Dependent Kinase Inhibitor Function of p27Kip1

SUMMARY Disruption of the cyclin-dependent kinase-inhibitory domain of p27 enhances growth of mice. Growth is attributed to an increase in cell number, due to increased cell proliferation, most obviously in tissues that ordinarily express p27 at the highest levels. Disruption of p27 function leads to nodular hyperplasia in the intermediate lobe of the pituitary. However, increased growth occurs without an increase in the amounts of either growth hormone or IGF-I. In addition, female mice were infertile. Luteal cell differentiation is impaired, and a disordered estrus cycle is detected. These results reflect a disturbance of the hypothalamic-pituitary-ovarian axis. The phenotypes of these mice suggest that loss of p27 causes an alteration in cell proliferation that can lead to specific endocrine dysfunction.

Quantitative analysis of somatostatin receptor subtypes (1-5) gene expression levels in somatotropinomas and correlation to in vivo hormonal and tumor volume responses to treatment with octreotide LAR

OBJECTIVE To determine whether the somatostatin receptor subtype (SSTR) expression profile correlates with hormonal and tumor volume responses to postsurgical octreotide long acting repeatable (OCT LAR) treatment. DESIGN AND METHODS Quantitative real-time RT-PCR was used to evaluate the absolute mRNA copy numbers for all five SSTR subtypes in 22 somatotropinomas. Response to OCT LAR was studied by hormone levels (GH and IGF-I) and tumor volume (sella turcica magnetic resonance imaging). RESULTS SSTR5 was present at the highest level followed by SSTR2, SSTR3, SSTR1, and SSTR4 (2327 (1046-5555), 2098 (194-23 954), 97 (0-460), 14 (0-29 480), and 0 (0-652) copies respectively). Positive correlations were found between SSTR2 levels and the percentage decrease of GH and IGF-I after 3 (r=0.49, P<0.027 and r=0.49, P<0.029 respectively) and 6 (r=0.59, P<0.006 and r=0.58, P<0.008 respectively) months of OCT LAR. A negative correlation was found between SSTR5 mRNA levels and the percentage decrease of GH after 3 months of OCT LAR (r=-0.52, P=0.016, n=21). A higher SSTR2/SSTR5 ratio was observed among patients who obtained hormonal control with OCT LAR, when compared with those uncontrolled (2.4 (0.7-10) vs 0.3 (0.1-7.7), P=0.001). A ROC curve analysis showed a SSTR2/SSTR5 ratio of 1.3 as the best predictor of disease control, with a sensitivity of 88% and a specificity of 92% - area under curve, 0.9. A positive correlation was also found between SSTR2 mRNA levels and the percentage decrease in tumor volume after 6 months of OCT LAR (r=0.79, P=0.002, n=12). CONCLUSIONS Somatostatin receptor subtype 2 mRNA expression levels in somatotropinomas correlate positively with in vivo hormonal and tumor volume responses to OCT LAR.

Expression Analysis of Dopamine Receptor Subtypes in Normal Human Pituitaries, Nonfunctioning Pituitary Adenomas and Somatotropinomas, and the Association between Dopamine and Somatostatin Receptors with Clinical Response to Octreotide-LAR in Acromegaly

CONTEXT Dopamine receptor (DR) and somatostatin receptor subtype expression in pituitary adenomas may predict the response to postsurgical therapies. OBJECTIVES Our objectives were to assess and compare the mRNA levels of DR1-5 and somatostatin receptors 1-5 in normal pituitaries (NPs), nonfunctioning pituitary adenomas (NFPAs), and somatotropinomas. In addition, we determined whether the level of DR expression correlates with the in vivo response to octreotide-LAR in acromegalic patients. DESIGN AND PATIENTS Eight NPs, 30 NFPAs, and 39 somatotropinomas were analyzed for receptor mRNA levels by real-time RT-PCR. The DR2 short variant was estimated as the DR2 long/DR2 total (DR2T). The relationship between DR expression and the postsurgical response to octreotide-LAR was assessed in 19 of the acromegalic patients. RESULTS DR3 was not detected. The relationship between expression levels of DR subtypes in NPs and somatotropinomas was DR2T>>>DR4>>DR5>DR1, whereas in NFPAs, DR2T>>>DR4>>DR1>DR5. The DR2 short variant was the predominant DR2 variant in the majority of samples. In acromegalics treated with octreotide-LAR, DR1 was negatively correlated with percent GH reduction (3 months: r = -0.67, P = 0.002; and 6 months: r = -0.58, P = 0.009), and DR5 was positively correlated with percent IGF-I reduction (3 months: r = 0.55, P = 0.01; and 6 months: r = 0.47, P = 0.04). CONCLUSIONS DR2 is the predominant DR subtype in NPs, NFPAs, and somatotropinomas. The fact that DR1, DR4, and DR5 are also expressed in many adenomas tested suggests that these receptors might also play a role in the therapeutic impact of postsurgical medical therapies in patients with NFPA and acromegaly. This was supported by the finding that the in vivo response to octreotide-LAR was negatively associated with DR1 and positively associated with DR5.

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 ghrelins 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.

Cdk4 Is Indispensable for Postnatal Proliferation of the Anterior Pituitary

For proper development and tissue homeostasis, cell cycle progression is controlled by multilayered mechanisms. Recent studies using knock-out mice have shown that animals can develop relatively normally with deficiency for each of the G1/S-regulatory proteins, D-type and E-type cyclins, cyclin-dependent kinase 4 (Cdk4), and Cdk2. Although Cdk4-null mice show no embryonic lethality, they exhibit specific endocrine phenotypes, i.e. dwarfism, infertility, and diabetes. Here we have demonstrated that Cdk4 plays an essential non-redundant role in postnatal proliferation of the anterior pituitary. Pituitaries from wild-type and Cdk4-null embryos at embryonic day 17.5 are morphologically indistinguishable with similar numbers of cells expressing a proliferating marker, Ki67, and cells expressing a differentiation marker, growth hormone. In contrast, anterior pituitaries of Cdk4-null mice at postnatal 8 weeks are extremely hypoplastic with markedly decreased numbers of Ki67+ cells, suggesting impaired cell proliferation. Pituitary hyperplasia induced by transgenic expression of human growth hormone-releasing hormone (GHRH) is significantly diminished in the Cdk4+/– genetic background and completely abrogated in the Cdk4–/– background. Small interfering RNA (siRNA)-mediated knockdown of Cdk4 inhibits GHRH-induced proliferation of GH3 somato/lactotroph cells with restored expression of GHRH receptors. Cdk4 siRNA also inhibits estrogen-dependent cell proliferation in GH3 cells and closely related GH4 cells. In contrast, Cdk6 siRNA does not diminish proliferation of these cells. Furthermore, Cdk4 siRNA does not affect GHRH-induced proliferation of mouse embryonic fibroblasts or estrogen-dependent proliferation of mammary carcinoma MCF-7 cells. Taken together, Cdk4 is dispensable for prenatal development of the pituitary or proliferation of other non-endocrine tissues but indispensable specifically for postnatal proliferation of somato/lactotrophs.

Liver-Derived IGF-I Regulates GH Secretion at the Pituitary Level in Mice

We have reported that liver-specific deletion of IGF-I in mice (LI-IGF-I−/−) results in decreased circulating IGF-I and increased GH levels. In the present study, we determined how elimination of hepatic IGF-I modifies the hypothalamic-pituitary GH axis to enhance GH secretion. The pituitary mRNA levels of GH releasing factor (GHRF) receptor and GH secretagogue (GHS) receptor were increased in LI-IGF-I−/− mice, and in line with this, their GH response to ip injections of GHRF and GHS was increased. Expression of mRNA for pituitary somatostatin receptors, hypothalamic GHRF, somatostatin, and neuropeptide Y was not altered in LI-IGF-I−/− mice, whereas hypothalamic IGF-I expression was increased. Changes in hepatic expression of major urinary protein and the PRL receptor in male LI-IGF-I−/− mice indicated an altered GH release pattern most consistent with enhanced GH trough levels. Liver weight was enhanced in LI-IGF-I−/− mice of both genders. In conclusion, loss of liver-derived IGF-I enhances GH release by...

Kisspeptin Regulates Gonadotroph and Somatotroph Function in Nonhuman Primate Pituitary via Common and Distinct Signaling Mechanisms

Kisspeptins (Kps) have emerged as key players in the control of reproductive-axis function, in which they operate as primary regulators of hypothalamic GnRH release. In addition, recent data indicate that Kps can also directly act on the pituitary to stimulate LH and GH release in primary pituitary cell culture prepared from rats, cows, and sheep. We present herein evidence that Kps (specifically Kp-10) can also stimulate LH and GH release in primary pituitary cell cultures prepared from female baboons (Papio anubis), a species that more closely models human physiology. The stimulatory effect of Kp-10 on LH and GH release was dose and time dependent and enhanced the hormonal responses to their major regulators (GnRH for LH; GHRH/ghrelin for GH) without affecting the release of other pituitary hormones (TSH, FSH, ACTH, prolactin). Use of pharmacological intracellular signaling blockers indicated Kp-10 signals through phospholipase C, protein kinase C, MAPK, and intracellular Ca(2+) mobilization, but not adenylyl cyclase, protein kinase A, extracellular Ca(2+) influx (through L-type channels), or nitric oxide synthase, to stimulate both LH and GH release. Interestingly, blockade of mammalian target of rapamycin or phosphoinositol 3-kinase activity fully abolished the stimulatory effect of Kp-10 on LH but not GH release. Of note, estradiol enhanced the relative LH response to Kp-10, alone or in combination with GnRH. In sum, our data are the first to provide evidence that, in a primate model, there is a functional Kp-signaling system within the pituitary, which is dynamically regulated and may contribute to the direct control of gonadotropic and somatotropic axes.

Glucocorticoids Regulate Pituitary Growth Hormone Secretagogue Receptor Gene Expression

Glucocorticoids regulate growth hormone (GH) secretion by modulating both hypothalamic and pituitary function. At the level of the pituitary, glucocorticoids increase GH and GH‐releasing hormone receptor (GHRH‐R) gene expression. To test if glucocorticoids might also regulate the pituitary expression of the recently identified GH secretagogue (GHS) receptor, GHS‐R; adult male rats were adrenalectomized or sham operated, and treated with the synthetic glucocorticoid (dexamethasone, 200 µg/day) or vehicle for 8 days. Pituitary GHS‐R mRNA levels were assessed by reverse transcriptase polymerase chain reaction (RT‐PCR). Adrenalectomy decreased pituitary GHS‐R mRNA to 45% of vehicle‐treated, sham‐operated rats (P < 0.05). Administration of dexamethasone increased GHS‐R mRNA levels in sham‐operated as well as in adrenalectomized rats (199 ± 24% (P < 0.05) and 369 ± 48% (P < 0.01) of vehicle‐treated controls). Addition of dexamethasone to primary rat pituitary cell cultures increased GHS‐R mRNA levels in a dose‐ and time‐dependent manner while the transcriptional inhibitor, actinomycin D, completely blocked the stimulatory action of dexamethasone. Taken together, these results suggest glucocorticoids directly increase pituitary GHS‐R mRNA levels by stimulating GHS‐R gene transcription.

Role of ghrelin system in neuroprotection and cognitive functions: implications in Alzheimer’s disease

Alzheimers disease (AD) is a multifactorial progressive neurodegenerative disorder characterized by loss of memory and cognitive deficits, strongly influenced by the metabolic status, in which the impairment of neuropeptides/neurotransmitters systems has been previously observed. Ghrelin is a multifunctional hormone produced in a wide variety of tissues, which has been associated with the progression of obesity and metabolic syndrome, but has been also linked to neuromodulation, neuroprotection and memory and learning processes. In addition, ghrelin system also acts in an autocrine/paracrine fashion where the majority of its components [ghrelin variants (native ghrelin, In1-ghrelin), acylation enzyme (GOAT) and receptors (GHS-Rs)] are expressed in the different regions of central nervous system. In spite of all these pieces of information strongly suggesting a close association between ghrelin system and AD, which could be of pathophysiological relevance, few studies have been addressed to clarify this relationship. In this work, the role of ghrelin system in neuroprotection, memory consolidation and learning is reviewed, and its influence in AD, as well as the regulation of its expression in the brain of AD patients, is discussed.

Expression of the Ghrelin and Neurotensin Systems is Altered in the Temporal Lobe of Alzheimer's Disease Patients

Ghrelin and neurotensin (NTS) are neuroendocrine peptides that exert opposite effects on food intake and energy homeostasis, but share comparable actions in improving memory and learning. Ghrelin and NTS mediate their effects via receptors with high evolutionary identity: two ghrelin G-protein coupled receptors (GPCRs; GHS-R1a/1b) and three NTS-receptors, two GPCRs (NTSR1/2) and one non-GPCR (NTSR3). Because ghrelin and NTS systems are tightly linked to energy balance regulation and cognitive processes, they have been proposed to be altered in Alzheimers disease (AD), a dementia syndrome markedly influenced by the metabolic status. Although it has been demonstrated that ghrelin and NTS can attenuate AD-related cognitive impairment, a comprehensive analysis of these systems in AD has not been conducted. Here, we used quantitative real time-RT-PCR to analyze expression of the ghrelin/NTS axis in one of the cortical regions most affected in AD, the temporal gyrus. Results unveiled a striking reduction of mRNA levels for ghrelin, and its newly discovered In2-ghrelin variant, as well as for the enzyme responsible for ghrelin acylation, ghrelin-O-acyltransferase and GHS-R1a, while expression of GHS-R1b was markedly increased. In addition, expression levels of NTSR1 and NTSR2 were profoundly decreased in AD, whereas mRNA levels of NTS only declined slightly, and those of NTSR3 (which is involved in neuronal apoptosis) did not vary. Taken together, our results provide the first quantitative evidence showing that ghrelin/NTS systems are markedly altered in the brain of AD patients, thereby suggesting that these systems may contribute to the severe cognitive deficit observed in this pathology.