Micaela Iantorno, M.D. and Michael J. Quon, M.D., Ph.D., Diabetes Unit, National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland 20892, USA

Ghrelin is a 28 amino acid peptide hormone secreted from X/A-like cells in the oxyntic mucosa of the stomach that has central actions to stimulate growth hormone release and orexigenesis [1]. The cognate receptor for ghrelin, GHSR-1a, is a 7-transmembrane G-protein coupled receptor that mediates most of the biological actions of ghrelin [2]. The principal physiological function of GHSR-1a was previously thought to be stimulation of growth hormone release from pituitary. However, GHSR-1a is expressed in many cell types including cardiomyocytes, myocardium, vascular endothelium, and monocytes raising the possibility that ghrelin may also have physiological actions in peripheral tissues. Indeed, ghrelin has direct peripheral actions (independent of growth hormone) to regulate cardiovascular homeostasis, inflammation, adipocyte function, bone formation, and gastric motility. The cardiovascular phenotypes of ghrelin null mice, ghrelin receptor null mice, and humans with ghrelin receptor mutations have not been reported to date. However, ghrelin has direct vasodilator, cardiotropic, cardioprotective, and anti-inflammatory actions [3].
Intravenous infusion of ghrelin acutely lowers blood pressure in humans and rats. However, in rats, the hypotensive effects of ghrelin appear to be independent of vasodilator actions of nitric oxide (NO). Furthermore, studies in vivo [4] and ex vivo [5] suggest that ghrelin has acute NO-independent vasodilator actions. On the other hand, in patients with metabolic syndrome who have lower circulating ghrelin levels than healthy subjects, intra-arterial ghrelin infusion acutely improves endothelial dysfunction by increasing bioavailability of NO [6]. Moreover, a number of both central and ex vivo peripheral actions of ghrelin are NO-dependent. Therefore, mechanisms by which ghrelin exerts its peripheral and/or central hemodynamic actions have not been fully elucidated. In particular, the roles of vascular endothelium and NO in mediating cardiovascular actions of ghrelin remain unresolved. Intriguingly, ghrelin and insulin share some common actions to regulate energy homeostasis including stimulation of food intake and glucose metabolism [7]. The actions of insulin to regulate energy homeostasis are PI 3-kinase-dependent. Vasodilator actions of insulin to stimulate production of NO in vascular endothelium that help to couple metabolic and hemodynamic homeostasis are also PI 3-kinase-dependent [8]. Recently, it was found that ghrelin acutely activates endothelial nitric oxide synthase (eNOS) in vascular endothelium resulting in increased production of NO using PI 3-kinase-dependent signaling pathways shared in common with insulin [9].

Ghrelin-Stimulated Production of NO in Vascular Endothelial Cells Requires GHSR-1a and Involves Akt and e NOS, But Not MAP-Kinase

Treatment of aortic endothelial cells in primary culture with physiological concentrations of ghrelin increases NO production in a dose- and time-dependent manner that requires expression of GHSR-1a as well as activation of PI 3-kinase (but not MAP-kinase). Ghrelin-stimulated phosphorylation of Akt and eNOS at its Akt phosphorylation site also requires expression of GHSR-1a. Since PI 3-kinase-dependent phosphorylation of Akt and eNOS is known to increase production of NO, this strongly suggest that in vascular endothelial cells, ghrelin binds to its cognate receptor (GHSR-1a) resulting in activation of PI 3-kinase that then stimulates phosphorylation and activation of Akt that, in turn, phosphorylates and activates eNOS, resulting in increased production of NO. It is possible that ghrelin is also stimulating production of NO in endothelial cells through activation of other receptors known to bind ghrelin (e.g. CD36). However, this seems unlikely since reducing expression of GHSR-1a completely inhibits ghrelin-stimulated production of NO. These results are concordant with studies demonstrating that ghrelin stimulates phosphorylation of Akt in cardiomyocytes [10] and human endothelial cells [11]. Importantly, these data are fully consistent with human studies demonstrating NO-dependent vasodilator actions of ghrelin [6].
The post-receptor signaling pathway used by ghrelin to stimulate production of NO in vascular endothelium is partially overlapping with that of insulin (insulin receptor/IRS-1/PI 3-kinase/PDK-1/Akt/eNOS) [8]. Thus, ghrelin joins the growing list of hormones involved with regulation of metabolism including insulin [12], estrogen [13], leptin [14], adiponectin [15], HDL [16], and DHEA [17] that acutely activate eNOS in vascular endothelium by a PI 3-kinase-dependent signaling mechanism leading to phosphorylation of eNOS by Akt. Metabolic and vasodilator actions of insulin are both regulated by highly parallel signaling pathways requiring PI 3-kinase (but not MAP-kinase). This represents one potential mechanism underlying the ability of vascular actions of insulin to help couple metabolic and hemodynamic homeostasis. Thus, ghrelin may also have functions to couple metabolic and cardiovascular physiology.

Ghrelin Stimulates Phosphorylation of MAP-Kinase but Not Secretion of ET-1 from Vascular Endothelial Cells

Ghrelin, like insulin, acutely stimulates phosphorylation of MAP-kinase in vascular endothelial cells [9,18,19]. Interestingly, by contrast with insulin, ghrelin does not stimulate secretion of the vasoconstrictor ET-1 from endothelial cells (a MAP kinase-dependent function of insulin). Thus, ghrelin mimics the vasodilator but not the vasoconstrictor actions of insulin in endothelial cells.
Insulin resistance in diabetes and obesity is characterized by pathway-selective impairment in PI 3-kinase-dependent insulin signaling, but not MAP-kinase-dependent insulin signaling. Thus, in insulin-resistant states, compensatory hyperinsulinemia that serves to maintain euglycemia results in an imbalance between opposing PI 3-kinase-dependent vasodilator actions of insulin and opposing MAP-kinase-dependent vasoconstrictor actions of insulin that predisposes to endothelial dysfunction and hypertension. Ghrelin only mimics the PI 3-kinase-dependent vasodilator actions of insulin but not the opposing MAP-kinase-dependent vasoconstrictor actions of insulin. Therefore, ghrelin may have novel therapeutic potential for both metabolic and cardiovascular diseases characterized by reciprocal relationships between endothelial dysfunction and insulin resistance. Indeed, circulating ghrelin levels are abnormally low in insulin-resistant conditions including diabetes, obesity, metabolic syndrome, hypertension, coronary heart disease, and atherosclerosis. Moreover, polymorphisms in the human ghrelin gene are associated with diabetes, impaired glucose tolerance, and hypertension [20]. Interestingly, lifestyle interventions including exercise and/or therapeutic interventions that result in weight loss increase plasma ghrelin levels [21]. Thus, ghrelin-stimulated production of NO from vascular endothelium may contribute to the beneficial metabolic and cardiovascular outcomes resulting from the successful implementation of these strategies.

Conclusions

Novel vascular actions of ghrelin directly stimulate production of NO from vascular endothelial cells using PI 3-kinase-dependent signaling pathways that mimic those of insulin. These findings may explain, in part, molecular mechanisms underlying some beneficial cardiovascular actions of ghrelin. This raises the possibility that novel or existing therapeutic strategies to increase circulating ghrelin levels or ghrelin action may be beneficial for metabolic and cardiovascular diseases characterized by reciprocal relationships between insulin resistance and endothelial dysfunction.

References

1. Kojima M, et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402(6762):656-60.
2. Kojima M, Kangawa K. Drug insight: The functions of ghrelin and its potential as a multitherapeutic hormone. Nat Clin Pract Endocrinol Metab 2006;2(2):80-88.
3. Cao JM, Ong H, Chen C. Effects of ghrelin and synthetic GH secretagogues on the cardiovascular system. Trends Endocrinol Metab 2006;17(1):13-18.
4. Okumura H, et al. Vasodilatory effect of ghrelin, an endogenous peptide from the stomach. J Cardiovasc Pharmacol 2002;39(6):779-83.
5. Wiley KE, Davenport AP. Comparison of vasodilators in human internal mammary artery: ghrelin is a potent physiological antagonist of endothelin-1. Br J Pharmacol 2002;136(8):1146-52.
6. Tesauro M, et al. Ghrelin improves endothelial function in patients with metabolic syndrome. Circulation 2005;112(19):2986-92.
7. Schwartz MW, Porte D Jr. Diabetes, obesity, and the brain. Science 2005;307(5708):375-79.
8. Kim JA, et al. Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms. Circulation 2006;113(15):1888-904.
9. Iantorno M, et al. Ghrelin has novel vascular actions that mimic only PI 3-kinase-dependent insulin-stimulated production of nitric oxide (NO) but not MAP-kinase-dependent insulin-stimulated secretion of ET-1 from endothelial cells. Am J Physiol Endocrinol Metab 2007: in press.
10. Baldanzi G, et al. Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT. J Cell Biol 2002;159(6):1029-37.
11. Rossi F, Bertone C, Petricca S, Santiemma V. Ghrelin inhibits angiotensin II-induced migration of human aortic endothelial cells. Atherosclerosis 2006 Aug 29; [Epub ahead of print].
12. Montagnani, M. et al. Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells. Mol Endocrinol 2002;16(8):1931-42.
13. Simoncini T, et al. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 2000;407(6803):538-41.
14. Vecchione C, et al. Leptin effect on endothelial nitric oxide is mediated through Akt-endothelial nitric oxide synthase phosphorylation pathway. Diabetes 2002;51(1):168-73.
15. Chen H, et al. Adiponectin stimulates production of nitric oxide in vascular endothelial cells. J Biol Chem 2003;278(45):45021-26.
16. Mineo C, et al. High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J Biol Chem 2003;278(11):9142-49.
17. Formoso G, et al. Dehydroepiandrosterone mimics acute actions of insulin to stimulate production of both nitric oxide and endothelin 1 via distinct phosphatidylinositol 3-kinase- and mitogen-activated protein kinase-dependent pathways in vascular endothelium. Mol Endocrinol 2006;20(5):1153-63.
18. Delhanty PJ, et al. 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. J Endocrinol 2006;188(1):37-47.
19. Yeh AH, et al. Ghrelin and a novel preproghrelin isoform are highly expressed in prostate cancer and ghrelin activates mitogen-activated protein kinase in prostate cancer. Clin Cancer Res 2005;11(23):8295-303.
20. Poykko S, et al. Ghrelin Arg51Gln mutation is a risk factor for Type 2 diabetes and hypertension in a random sample of middle-aged subjects. Diabetologia 2003;46(4):455-58.
21. Cummings DE, et al. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med 2002;346(21):1623-30.

Please address correspondence to:
Michael J. Quon, M.D., Ph.D.
Chief, Diabetes Unit
NCCAM, NIH
10 Center Drive
Building 10, Room 6C-205
Bethesda, MD 20892-1632
Tel: (301) 496-6269
Fax: (301) 402-1679
Email: quonm[at]nih.gov