Christian G. Ziegler

Agonist of growth hormone-releasing hormone as a potential effector for survival and proliferation of pancreatic islets

Therapeutic strategies for transplantation of pancreatic islet cells are urgently needed to expand β-cell mass by stimulating islet cell proliferation and/or prolonging islet cell survival. Control of the islets by different growth factors provides a potential venue for augmenting β-cell mass. In the present study, we show the expression of the biologically active splice variant-1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor in rat insulinoma (INS-1) cells as well as in rat and human pancreatic islets. In studies in vitro of INS-1 cells, the GHRH agonist JI-36 caused a significant increase in cell proliferation and a reduction of cell apoptosis. JI-36 increased islet size and glucose-stimulated insulin secretion in isolated rat islets after 48–72 h. At the ultrastructural level, INS-1 cells treated with agonist JI-36 revealed a metabolic active stimulation state with increased cytoplasm. Coincubation with the GHRH antagonist MIA-602 reversed the actions of the agonist JI-36, indicating the specificity of this agonist. In vivo, the function of pancreatic islets was assessed by transplantation of rat islets under the kidney capsule of streptozotocin-induced diabetic non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice. Islets treated with GHRH agonist JI-36 were able to achieve normoglycemia earlier and more consistently than untreated islets. Furthermore, in contrast to diabetic animals transplanted with untreated islets, insulin response to an i.p. glucose tolerance test (IPGTT) in animals receiving islets treated with agonist Jl-36 was comparable to that of normal healthy mice. In conclusion, our study provides evidence that agonists of GHRH represent a promising pharmacological therapy aimed at promoting islet graft growth and proliferation in diabetic patients.

Human adipocytes induce an ERK1/2 MAP kinases-mediated upregulation of steroidogenic acute regulatory protein (StAR) and an angiotensin II — sensitization in human adrenocortical cells

Objectives:Hypertension is a major complication of overweight with frequently elevated aldosterone levels in obese patients. Our previous work suggests a direct stimulation of adrenal aldosterone secretion by adipocytes. Owing to aldosterones important role in maintaining blood pressure homeostasis, its regulation in obesity is of major importance. One objective was to determine the signaling mechanisms involved in adipocyte-induced aldosterone secretion. In addition to a direct stimulation, a sensitization toward angiotensin II (AngII) might be involved. The second objective was to determine a possible adipokines-induced sensitization of human adrenocortical cells to AngII.Design:Human subcutaneous adipocytes and adrenocortical cells, and the adrenocortical cell line NCI-H295R were used. Adrenocortical cells were screened for signal transduction protein expression and phosphorylation. Subsequently, steroidogenic acute regulatory protein (StAR), cAMP response element-binding protein (CREB), cAMP and phosphorylated extracellular regulated kinase were analyzed by Western blot, enzyme-linked immunosorbent assay, quantitative PCR, reporter gene assay and confocal microscopy to investigate their role in adipocyte-mediated aldosterone secretion.Results:AngII-mediated aldosterone secretion was largely increased by preincubating H295R cells with adipocyte secretory products. StAR mRNA and StAR protein were upregulated in a time-dependent way. This steroidogenic effect was independent of the cAMP-protein kinase A (PKA) pathway as cellular cAMP was unaltered and inhibition of PKA by H89 failed to reduce aldosterone secretion. However, CREB reporter gene activity was moderately elevated. Upregulation of StAR was accompanied by ERK1/2 MAP kinase activation and nuclear translocation of the kinases. Inhibition of MAP kinase by UO126 abolished adipokine-stimulated aldosterone secretion from primary human adrenocortical and H295R cells, and inhibited StAR gene activity. Adipokines stimulated steroidogenesis also in primary human adrenocortical cells, supporting a role in human physiology and/or pathology.Conclusions:Adipokines induce aldosterone secretion from human adrenocortical cells and sensitization of the cells to stimulation by AngII, possibly mediated via ERK1/2-dependent upregulation of StAR activity. This stimulation of aldosterone secretion could be one link between overweight and inappropriately elevated aldosterone levels.

Age-dependent regulation of chromaffin cell proliferation by growth factors, dehydroepiandrosterone (DHEA), and DHEA sulfate

The adrenal gland comprises two endocrine tissues of distinct origin, the catecholamine-producing medulla and the steroid-producing cortex. The inner adrenocortical zone, which is in direct contact with the adrenomedullary chromaffin cells, produces dehydroepiandrostendione (DHEA) and DHEA sulfate (DHEAS). These two androgens exhibit potential effects on neurogenesis, neuronal survival, and neuronal stem cell proliferation. Unlike the closely related sympathetic neurons, chromaffin cells are able to proliferate throughout life. The aim of this study was to investigate the effect of DHEA and DHEAS on proliferation of bovine chromaffin cells from young and adult animals. We demonstrated that graded concentrations of leukemia inhibitory factor induced proliferation of chromaffin cells from young animals, whereas EGF had no effect. On the contrary, EGF increased the cell proliferation in cells from adult animals, whereas leukemia inhibitory factor was inactive. In both cases, DHEA decreased the proliferative effect induced by the growth factors. Surprisingly, DHEAS enhanced, in a dose-dependent-manner, the effect of growth factors on proliferation in cells from adult animals but not from young animals. Flutamide, ICI 182,780, and RU 486 had no effect on the action of DHEA or DHEAS on chromaffin cell proliferation. These data show that DHEA and its sulfated form, DHEAS, differentially regulate growth-factor-induced proliferation of bovine chromaffin cells. In addition, the sensitivity of chromaffin cells to different growth factors is age-dependent. Furthermore, these two androgens may act through a receptor other than the classical steroid receptors.

Modulation of pancreatic islets-stress axis by hypothalamic releasing hormones and 11β-hydroxysteroid dehydrogenase

Corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone (GHRH), primarily characterized as neuroregulators of the hypothalamic-pituitary-adrenal axis, directly influence tissue-specific receptor-systems for CRH and GHRH in the endocrine pancreas. Here, we demonstrate the expression of mRNA for CRH and CRH-receptor type 1 (CRHR1) and of protein for CRHR1 in rat and human pancreatic islets and rat insulinoma cells. Activation of CRHR1 and GHRH-receptor significantly increased cell proliferation and reduced cell apoptosis. CRH stimulated both cellular content and release of insulin in rat islet and insulinoma cells. At the ultrastructural level, CRHR1 stimulation revealed a more active metabolic state with enlarged mitochondria. Moreover, glucocorticoids that promote glucose production are balanced by both 11b-hydroxysteroid dehydrogenase (11β-HSD) isoforms; 11β-HSD–type-1 and 11β-HSD–type-2. We demonstrated expression of mRNA for 11β-HSD-1 and 11β-HSD-2 and protein for 11β-HSD-1 in rat and human pancreatic islets and insulinoma cells. Quantitative real-time PCR revealed that stimulation of CRHR1 and GHRH-receptor affects the metabolism of insulinoma cells by down-regulating 11β-HSD-1 and up-regulating 11β-HSD-2. The 11β-HSD enzyme activity was analyzed by measuring the production of cortisol from cortisone. Similarly, activation of CRHR1 resulted in reduced cortisol levels, indicating either decreased 11β-HSD-1 enzyme activity or increased 11β-HSD-2 enzyme activity; thus, activation of CRHR1 alters the glucocorticoid balance toward the inactive form. These data indicate that functional receptor systems for hypothalamic-releasing hormone agonists exist within the endocrine pancreas and influence synthesis of insulin and the pancreatic glucocorticoid shuttle. Agonists of CRHR1 and GHRH-receptor, therefore, may play an important role as novel therapeutic tools in the treatment of diabetes mellitus.

Expression of neuropeptide hormone receptors in human adrenal tumors and cell lines: Antiproliferative effects of peptide analogues

Peptide analogues targeting various neuropeptide receptors have been used effectively in cancer therapy. A hallmark of adrenocortical tumor formation is the aberrant expression of peptide receptors relating to uncontrolled cell proliferation and hormone overproduction. Our microarray results have also demonstrated a differential expression of neuropeptide hormone receptors in tumor subtypes of human pheochromocytoma. In light of these findings, we performed a comprehensive analysis of relevant receptors in both human adrenomedullary and adrenocortical tumors and tested the antiproliferative effects of peptide analogues targeting these receptors. Specifically, we examined the receptor expression of somatostatin-type-2 receptor, growth hormone-releasing hormone (GHRH) receptor or GHRH receptor splice variant-1 (SV-1) and luteinizing hormone-releasing hormone (LHRH) receptor at the mRNA and protein levels in normal human adrenal tissues, adrenocortical and adrenomedullary tumors, and cell lines. Cytotoxic derivatives of somatostatin AN-238 and, to a lesser extent, AN-162, reduced cell numbers of uninduced and NGF-induced adrenomedullary pheochromocytoma cells and adrenocortical cancer cells. Both the splice variant of GHRH receptor SV-1 and the LHRH receptor were also expressed in adrenocortical cancer cell lines but not in the pheochromocytoma cell line. The GHRH receptor antagonist MZ-4–71 and LHRH antagonist Cetrorelix both significantly reduced cell growth in the adrenocortical cancer cell line. In conclusion, the expression of receptors for somatostatin, GHRH, and LHRH in the normal human adrenal and in adrenal tumors, combined with the growth-inhibitory effects of the antitumor peptide analogues, may make possible improved treatment approaches to adrenal tumors.

Expression and Function of Endocannabinoid Receptors in the Human Adrenal Cortex

Endogenous cannabinoids are important signaling molecules in neuroendocrine control of homeostatic and reproductive functions including stress response and energy metabolism. The hypothalamic paraventricular and supraoptic nuclei have been shown to release endocannabinoids, which act as retrograde messengers to modulate the synaptic release of glutamate during stress response. This study endeavors to elucidate possible interaction of the endocannabinoid system with the regulation of adrenocortical function at the adrenal level. Human adrenocortical NCI-H295R cells and normal human adrenal glands were used to study the possible effects of anandamide and cannabinoid receptor 1 (CB1) antagonist SR141716A on aldosterone and cortisol secretion. Our data indicate the expression of CB1 in human adrenal cortex and adrenocortical NCI-H295R cells; CB2 was not expressed. Furthermore, anandamide inhibited basal release and stimulated release of adrenocortical steroids (corticosterone and aldosterone); this effect was reversed by CB1 antagonist (SR141716A). Therefore, the endocannabinoid system at the level of the adrenal, can directly influence adrenocortical steroidogenesis.

DHEA reduces NGF-mediated cell survival in serum-deprived PC12 cells.

Abstract:  Adrenocortical androgens, including dehydroepiandrosterone (DHEA), are produced in the inner zone of the adrenal cortex that is in direct contact with the neural crest–derived catecholamine‐producing chromaffin cells. DHEA has recently been identified as a crucial regulator of neuronal stem cell proliferation. Thus, DHEA might play a hitherto unknown role in intra‐adrenal tissue formation. In the present study, we examined the influence of DHEA on nerve growth factor (NGF)–mediated survival in serum‐deprived PC12 cells and analyzed the influence of DHEA on NGF‐induced ERK1/2 mitogen‐activated protein (MAP) kinase activation by enzyme‐linked immunosorbent assay (ELISA). Cell survival promoted by NGF in serum‐deprived PC12 cells and neurite outgrowth was reduced by DHEA, pointing toward a role of DHEA in the differentiation process of chromaffin cells. Furthermore, NGF‐induced ERK 1/2 activation was significantly inhibited by DHEA. Hence, we speculate that DHEA might influence NGF‐mediated chromaffin differentiation processes using the ERK1/2 MAP kinase pathway during adrenal tissue development.

Aldosterone Rapidly Induces Leukocyte Adhesion to Endothelial Cells: A New Link Between Aldosterone and Arteriosclerosis?

To the Editor: Aldosterone is known to induce cardiovascular dysfunction, including fibrosis, inflammation, and endothelial dysfunction, as well as thrombosis formation.1 Clinical trials have shown aldosterone to be an independent predictor of increased mortality in patients with chronic heart failure,2 and high circulating plasma aldosterone levels predict the clinical outcome in patients after myocardial infarction.3 Mineralocorticoid receptor blockade proved to exert beneficial effects in clinical trials, such as the Randomised Aldactone Evaluation Study and the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study.4 Recent studies provided evidence for a role of aldosterone in the pathogenesis of arteriosclerosis.5 However, the exact mechanisms of adverse aldosterone actions in the cardiovascular system are largely unknown. Here, we aimed at elucidating rapid (60 minutes) aldosterone effects on interactions between endothelial cells and leukocytes. …

Multimodal somatostatin receptor theranostics using [64Cu]Cu-/[177Lu]Lu-DOTA-(Tyr3)octreotate and AN-238 in a mouse pheochromocytoma model

Pheochromocytomas and extra-adrenal paragangliomas (PHEO/PGLs) are rare catecholamine-producing chromaffin cell tumors. For metastatic disease, no effective therapy is available. Overexpression of somatostatin type 2 receptors (SSTR2) in PHEO/PGLs promotes interest in applying therapies using somatostatin analogs linked to radionuclides and/or cytotoxic compounds, such as [177Lu]Lu-DOTA-(Tyr3)octreotate (DOTATATE) and AN-238. Systematic evaluation of such therapies for the treatment of PHEO/PGLs requires sophisticated animal models. In this study, the mouse pheochromocytoma (MPC)-mCherry allograft model showed high tumor densities of murine SSTR2 (mSSTR2) and high tumor uptake of [64Cu]Cu-DOTATATE. Using tumor sections, we assessed mSSTR2-specific binding of DOTATATE, AN-238, and somatostatin-14. Therapeutic studies showed substantial reduction of tumor growth and tumor-related renal monoamine excretion in tumor-bearing mice after treatment with [177Lu]Lu-DOTATATE compared to AN-238 and doxorubicin. Analyses did not show agonist-dependent receptor downregulation after single mSSTR2-targeting therapies. This study demonstrates that the MPC-mCherry model is a uniquely powerful tool for the preclinical evaluation of SSTR2-targeting theranostic applications in vivo. Our findings highlight the therapeutic potential of somatostatin analogs, especially of [177Lu]Lu-DOTATATE, for the treatment of metastatic PHEO/PGLs. Repeated treatment cycles, fractionated combinations of SSTR2-targeting radionuclide and cytotoxic therapies, and other adjuvant compounds addressing additional mechanisms may further enhance therapeutic outcome.

Dehydroepiandrosterone-sulphate (DHEA-S) promotes neuroendocrine differentiation of chromaffin pheochromocytoma PC12 cells

The major source for dehydroepiandrosterone (DHEA) and its sulphate compound DHEA-S is the inner zone of the adrenal cortex, which is in direct contact to adrenomedullary chromaffin cells. Due to their close proximity, direct interactions of DHEA and DHEA-S with chromaffin cells during adrenal gland development and throughout the whole life span are hypothesized. A possible direct effect of DHEA-S and the cellular and molecular mechanisms of DHEA-S action on chromaffin cells remain unresolved. Therefore, in this study, we aimed at clarifying DHEA-S effects and mechanisms of action on rat chromaffin PC12 cells. DHEA-S (10(-6)mol/l) inhibited nerve growth factor (NGF, 20ng/ml)-induced cell proliferation by 66% (n=4, p<0.001). In NGF-stimulated cells, neuronal differentiation was inhibited by DHEA-S, as demonstrated by a 22% reduction (n=3; p<0.05) of neuronal differentiation marker expression, synaptosome-associated protein of 25kDa (SNAP-25), and a 59% (n=6; p<0.001) decrease in neurite outgrowth. Moreover, DHEA-S stimulated expression of endocrine marker chromogranin A (CgA) by 31% (n=4; p<0.05 vs. control) and catecholamine release from NGF-treated PC12 cells by 229% (n=3-5; p<0.001), indicating a DHEA-S-induced shift towards neuroendocrine differentiation. On a molecular level, DHEA-S diminished NGF-induced ERK1/2 phosphorylation. Taken together, DHEA-S inhibited NGF-induced proliferation and neuronal differentiation and shifted cells towards a more endocrine phenotype. Interference of DHEA-S with NGF-stimulated ERK1/2 activation might be involved in this effect. Our study provides support for the notion that adrenocortical-derived DHEA-S impacts adrenomedullary chromaffin cells during development and differentiation.