There is controversy in the literature about the role of ghrelin in adipogenesis: in vitro studies on primary cultures of rat adipocytes show that ghrelin stimulates glycerol-3-phosphate dehydrogenase (GPDH) activity and increases the expression of PPAR-2 in adipocytes. Ghrelin promotes differentiation of preadipocytes, decreases the rate of lipolysis and facilitates adipogenesis via GHS-R (Choi, Roh et al. 2003).
Other studies demonstrate that ghrelin inhibits adipogenesis: transfection of 3T3-L1 cells with the ghrelin plasmid inhibits differentiation of preadipocytes into adipocytes compared to controls. Cells overexpressing ghrelin or stimulated with ghrelin exhibit increase in proliferation accompanied with a decrease in the expression of peroxisome proliferative activated receptor gamma 2 (PPAR- mRNA and protein levels. The mechanisms by which ghrelin is inhibiting adipogenesis appear to involve the mitogen-activated protein kinase (MAPK) pathways (Zhang, Zhao et al. 2004).
2.10.2 Ghrelin effects on the endocrine pancreas
Although initial studies indicated that ghrelin stimulates insulin secretion, a substantial number of studies, many of them in vivo, have now shown that ghrelin plays an inhibitory role on insulin secretion (Pusztai, Sarman et al. 2008).
The first study to appear on the effects of ghrelin on islet function demonstated the presence of both ghrelin and ghrelin receptor expression in rat islets. In this study, ghrelin was found to colocalize with glucagon, indicating that ghrelin is produced in the alpha cells of the endocrine pancreas. As discussed above, this conclusion is not uncontested. It was however found that insulin secretion by isolated islets incubated with ghrelin at a
dose of 1 pM in the presence of 8.3 mM glucose was slightly increased. An increase in the amplitude of [Ca2+]i oscillations was also seen at stimulatory glucose concentrations (8.3 mM) in the presence of ghrelin, while no effect was seen at non-stimulatory (2.8 mM) glucose concentrations. (Date, Nakazato et al. 2002). While many studies have been using a single concentration of ghrelin, one report gives a full ghrelin dose-response curve for the effects on isolated mice islets, measuring insulin and glucagon secretion (Salehi, Dornonville de la Cour et al. 2004). It was observed that ghrelin has an inhibitory effect on glucose-stimulated insulin release when used at low concentrations, whereas a stimulatory effect was found at high concentrations. On the contrary, glucagon secretion was stimulated at all ghrelin concentrations tested. In another study, the rat perfused pancreas preparation was used and the responses of islet hormones to various stimuli (glucose, arginine and carbachol) were measured in the presence and the absence of 10 nM ghrelin in the perfusate It was found that ghrelin caused a marked inhibition of both insulin and somatostatin secretion, whereas glucagon secretion was unaffected by ghrelin (Egido, Rodriguez-Gallardo et al. 2002).
When tested in vivo, high doses of ghrelin inhibit basal and glucose-stimulated insulin secretion, whereas low doses have no effect. Furthermore, ghrelin at a dose of 3.3 nmol/kg-1 promoted an impaired glucose tolerance. The authors conclude by questioning the physiological relevance of the ghrelin effects on insulin secretion and pointing out, in particular, that at the levels found in plasma either pre-or post-prandially, ghrelin has no effect. This conclusion is, however, complicated by the finding that ghrelin may be produced within the islets themselves, possibly implying that local concentrations in the islet interstitium may be very different from those found in the circulation (Salehi, Dornonville de la Cour et al. 2004). (Fig 13).
Recently, a mechanism for ghrelin inhibition of insulin secretion has been proposed by Dezaki et al. By studying ghrelin effect on insulin secretion both in vitro and in vivo they show that insulin secretion is attenuated by ghrelin and that the attenuation is abolished after pre-treatment with pertussis toxin (PTX), an inhibitor of the Gi/o subclass of trimeric G-proteins. By using single rat -cells, they also show that glucose-induced increases in cytosolic Ca2+ and electrical activity are reduced in the presence of ghrelin. The effect on cytosolic Ca2+ was abolished by PTX. Finally, using voltage clamp measurements of voltage-dependent currents, they demonstrate that the delayed rectifier K+ current (Kv
current) is enhanced by ghrelin, an effect which is also PTX sensitive. The Kv current is acting by reducing the electrical activity and blocking this current with tethra-ethyl ammonium (TEA) resulted in a doubling of insulin release from isolated islets. It may thus be concluded that ghrelin reduces insulin secretion by acting on trimeric G-proteins coupled to the Kv channel to enhance the current density, leading to a reduction of electrical activity, thereby decreasing insulin secretion (Dezaki, Hosoda et al. 2004).
2.10.3 Effects of ghrelin on hepatocytes
Modulation of glucose metabolism by insulin and ghrelin was studied in vitro using HepG2 cells, a human hepatocellular carcinoma cell line and H4-II-E cells, a rat hepatoma cellline. In HepG2 cells, it was shown that ghrelin dose-depently increased the amount of tyrosine-phosphorylated IRS-1 compared to controls. This action was cancelled by the antagonist of the GHS-R, (d-Lys-3) GHRP6. Additionally, when ghrelin and insulin were administrated together in Hep G2 cells, the amount of tyrosine-phosphorylated IRS-1 increased in an additive manner. Furthermore, stimulation with ghrelin and insulin increased the association between growth factor receptor bound protein 2 (GRB2) and IRS-1, as well as between PI3K and IRS-1 in the same cells. The activity of MAPK kinase was also increased by ghrelin and insulin and was additive when both hormones were added in HepG2 cells. On the contrary, the AKT kinase activity increased in response to insulin, but not following ghrelin. Ghrelin also induced cell proliferation of HepG2 cells via MAPK kinase (Murata, Okimura et al. 2002).
In H4-II-E cells, ghrelin stimulation also increased the amount of tyrosine- phosphorylation, specifically of IRS-1. The expression of phosphoenolpyruvate carboxykinase(PEPCK) in H4-II-E cells was also studied. Insulin treatment reduced the expression of PEPCK and pre-incubation with ghrelin for 1 to 2 h before insulin treatment partially reversed the down-regulating effect of insulinon PEPCK expression (Murata, Okimura et al. 2002).
In vivo, it has been shown by using the hyperinsulinemic-euglycemic clamp that ghrelin given intravenously inhibits insulin action on hepatic glucose production (Heijboer, van den Hoek et al. 2006).
From these studies, it appears that ghrelin is modulating insulin signalling and mitogenic processes inhepatocytes. Furthermore, the results on PEPCK activity would imply that ghrelin has a stimulatory effect on gluconeogenesis (Murata, Okimura et al. 2002) (Fig.13).
2.10.4 Ghrelin and gastrointestinal function
Like histamine, ghrelin administred i.v. leads to a dose-dependent increase in gastric acid secretion and motility in male Sprague-Dawley rats. These effects are abolished by cervical vagotomy and atropine but not by blockade of the histamine H(2)-receptor.
Similar results are observed in response to i.c.v. ghrelin. Thus, ghrelin exerts a positive effect on gastric acid secretion and motility which is mediated by the vagus nerve (Masuda, Tanaka et al. 2000). Moreover, c-fos expression in the medulla oblongata is increased in the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMNV) in ghrelin-treated rats. These loci are both known to be involved in the central control of gastric acid secretion (Date, Nakazato et al. 2001). Another study demonstrated a specific and dose-dependent effect of ghrelin on gastric emptying with no concomitant stimulation of gastric acid secretion. In keeping with these results, a new ghrelin receptor agonist, TZP-101, was shown to increase gastric emptying in a dose-dependent manner in male Sprague-Dawley rats with postoperative ileus (POI) treated or not with morphine. Such data indicate that TZP-101 could possibly be used in humans to prevent this postoperative complication (Venkova, Fraser et al. 2007) (Fig.13).
2.10.5 Ghrelin and cardiovascular functions
In vitro studies show that ghrelin and des-acyl ghrelin inhibit apoptosis in primary adult cardiomyocytes and H9c2 cardiomyocytes via tyrosine phosphorylation, activation of extracellular signal-regulated kinase (ERK)-1/2 and Akt serine kinase. As H9c2 do not express the GHSR1a, ghrelin and des-acyl ghrelin may bind to a receptor-type that has not yet been identified and protect them from apoptosis (Baldanzi, Filigheddu et al.
2002). Moreover, incubation of H9c2 cardiomyocytes with hexarelin and ghrelin increases thymidine incorporation into DNA in a dose-dependent and specific manner,
indicating that GHSs increase cell proliferation in the H9c2 cell line (Pettersson, Muccioli et al. 2002) (Fig. 13).
In vivo experimental findings suggest that GH has beneficial effects on the cardiovascular system. In one study (Tivesten, Bollano et al. 2000), the effects of hexarelin (a potent GH secretagogue) and GH treatment after experimentally induced myocardial infarction were compared. Both hexarelin and GH lead to a decrease in total peripheral resistance, accompanied by an increase in stroke volume, cardiac output and cardiac index.
Due to these observations, the effects of subcutaneous ghrelin administration on left ventricular (LV) dysfunction and cachexia were tested in rats with chronic heart failure (CHF). It was observed that rats with CHF receiving ghrelin have a reduced weight loss compared to vehicle-injected animals. In addition, measurement of various hemodynamic parameters allowed to conclude that ghrelin decreases mean arterial blood pressure and significantly decreases systemic vascular resistance in CHF rats. Moreover, right ventricle (RV) systolic pressure and right atrial pressure tended to be lower. Thus, ghrelin may have a positive role in the treatment of cardiac infarction (Nagaya, Uematsu et al. 2001).
With regard to ghrelins effect on blood pressure in rats, it was observed that unilateral microinjection of ghrelin in the nucleus of the solitary tract decreases the mean arterial pressure, heart rate and the renal sympathetic nerve activity up to 30% (Lin, Matsumura et al. 2004; Garcia and Korbonits 2006).
Ghrelin may also have beneficial effects on cardiovascular function independently from its effect on GH secretion. In one study, ghrelin was subcutaneously injected for 3 weeks in GH-deficient rats and the relaxation of thoracic aorta in response to norepinephrine and acetylcholine was measured. It was found that GH-deficient rats have impaired acetylcholine-induced relaxation compared to normal rats, a defect that was improved by the ghrelin treatment. Additional results showed that ghrelin may improve endothelial function by causing an increase in the biovailability of nitric oxide, via an increase in eNOS expression in the aorta (Shimizu, Nagaya et al. 2003)
(Fig. 13).
2.10.6 Ghrelin and bone repair
In primary osteoblastic cells obtained from rat calvarie, ghrelin or hexarelin induced enhanced osteoblast proliferation and [3H]thymidine DNA-incorporation, together with increased alkaline phosphatase (ALP) and osteocalcin (OC) production compared to controls. Moreover, the presence of GHS-R1a mRNA and protein was seen in calvarial osteoblasts, suggesting that ghrelin and hexarelin may exert their proliferative actions via the GHSR1a receptor (Maccarinelli, Sibilia et al. 2005). In vivo studies in rats receiving ghrelin show a significant increase in new bone formation compared to controls at 6 and 12 weeks. Moreover, ALP, OC, and type I collagen were also higher compared to saline controls (Deng, Ling et al. 2008) (Fig. 13).
Peripheral effects of ghrelin
Figure 13. Peripheral effects of ghrelin in various tissues. A question mark indicates points of controversy.
gastrointestinal secretion and motility
ghrelin adipogenesis?
rate of lipolysis
insulin secretion ? glucagon secretion somatostatin secretion
proliferation of hepatocytes gluconeogenesis
apoptosis of cardiomyocytes blood preasure
cardiac output osteoblastic proliferation
bone formation
adipose tissue
pancreas
stomach
heart
bone liver
2.11 Animal models of ghrelin knockout mice and GHSR-/- mice