In this paper, we describe for the first time marked expression within gastric ghrelin cells of the secreted proteins RBP4 and TTR. For RBP4, this marked expression was first suggested at the mRNA level in FACS-sorted hrGFP-positive cells from ghrelin-hrGFP reporter mice, using gene chip technology. Such was confirmed by qPCR studies using not only the hrGFP/ghrelin cell-enriched samples but also two different mouse ghrelinoma cell lines. Similar to the observed enrichment of RBP4 noted in FACS-sorted hrGFP-positive cells, mRNA for TTR, which serves as a transport protein for both RBP4 and thyroid hormone, also was expressed within the stomach, where it was highly enriched within the ghrelin cell population. Although RBP4 was found in lysates and the media of cultured ghrelinoma cells, we did not observe octanoic acid or catecholamine stimulation of RBP4 secretion, as had been observed previously when the same conditions were used to assess acyl-ghrelin secretion 
. Nor did we observe changes in RBP4 mRNA levels in whole stomach upon caloric restriction. We also did not observe any statistically significant increase in plasma RBP4 in mice harboring ghrelinomas. Caloric restriction did, however, decrease TTR mRNA in whole stomach.
The significance of these findings stems not only from the known biological functions of RBP4 and TTR, but also from the mostly uncharacterized physiology of the ghrelin cell. RBP4 is a 21-kDa protein synthesized by and secreted mainly from the liver 
. Its best characterized function is to deliver retinol (vitamin A) from hepatic retinoid stores to tissues throughout the body, where it is metabolized to retinaldehyde and retinoic acid 
. More specifically, RBP4 is the only specific serum transport protein for retinol and thus serves an important function for a multitude of retinol-dependent functions, including vision, growth and development and immune regulation 
More recently, plasma RBP4 has been identified as an important signal of insulin resistance 
. This first became evident in insulin resistant mice lacking GLUT4 (glucose transporter 4) specifically in white adipose tissue, in which serum RBP4 was markedly elevated as was the level of white adipose tissue RBP4 mRNA 
. Previously in rodents, adipose tissue had been reported to contain relatively high levels of RPB4 mRNA, averaging between 6–37% those in liver 
. Circulating RBP4 is also elevated in several other mouse models of insulin resistance as well as in insulin-resistant humans 
. Interestingly, pharmacologic administration of RBP4 and transgenic overexpression of RBP4 both induce insulin resistance in mice, while genetic deletion of RBP4 enhances insulin sensitivity 
. These findings and others suggest that elevated serum RBP4 may play a causative role in the development of insulin resistance, although it is as yet unclear if this involves retinol-dependent mechanisms, retinol-independent mechanisms, or both 
Retinol-RBP4 circulates in the bloodstream in a 1
1 molar complex with TTR 
. Like RBP4, TTR also is synthesized mainly in the liver 
. Not only does TTR transport the retinol-RBP4 complex in the bloodstream, but also it prevents glomerular filtration of RBP4 at the kidney, thus helping to prevent loss of retinol and RBP4 in the urine 
. Regarding its role in thyroid hormone transport in the bloodstream, in humans, TTR carries about 10–15% of T4
(as opposed to thyroxine binding globulin, which carries about 70%, albumin, which carries about 15–20%, and lipoproteins, which carry a fraction), although its relatively low affinity for T4 and T3 leads to a more immediate delivery of thyroid hormone to target tissues 
. In mice, TTR serves as the major thyroid hormone transport protein 
. TTR also is synthesized in the choroid plexus, which likely accounts for its presence in cerebrospinal fluid 
. Within the CSF of both humans and mice, TTR functions as the major carrier of thyroid hormone; in this role, TTR is thought to facilitate the passage of thyroid hormone across the choroid plexus and then distribute it to the central nervous system 
Of interest, RBP4 and TTR expression have been localized to more sites than just the liver plus white adipose tissue (RBP4) and the liver plus choroid plexus (TTR). Using Northern Blot analysis on various rat tissues, RBP4 mRNA has been detected in the kidney at 5–10% of hepatic levels, in lung, spleen, brain, heart and skeletal muscle at 1–3% of hepatic levels, and in the intestine, testis and pancreas at <1% of hepatic levels 
. Other studies have shown RBP mRNA in rat eye, where it is localized to the retinal pigment epithelium, and in the visceral yolk sac 
. As it relates to the current study, Northern blot analysis also has revealed RBP4 mRNA levels in stomach at 1–3% of hepatic levels 
. Here, using qPCR, RBP4 mRNA levels within whole stomach reached only 0.4% of those in the liver while RBP4 mRNA levels within the hrGFP/ghrelin cell-enriched pools reached 31% of those in the liver. Thus, the current data suggests that the RBP4 expression within ghrelin cells likely accounts for the majority of its stomach expression. Like RBP4, TTR is also synthesized in the rat pigment epithelium 
. Additionally, it is produced in the placenta and in the islets of Langerhans 
. TTR expression within the gastrointestinal tract also has been described previously, although never before co-localized with ghrelin as described in the current study. In particular, transgenic mice in which Cre recombinase or red fluorescence protein expression are directed by TTR promoter elements manifest reporter activity in the gastric mucosa of E12.5–E16.5 embryonic animals 
. Here, in adult mice, TTR expression in the stomach approached 0.4% of its levels in liver, while that in hrGFP/ghrelin cell-enriched pools approached 62% of its levels in liver.
It has been proposed that the sites of RBP4 and TTR biosynthesis may help dictate their functions. In a study specifically designed to investigate the physiological role of circulating RBP4 produced outside the liver, handling of retinol was examined in mice in which endogenous RBP4 expression was genetically deleted and replaced with RBP4 overexpressed in muscle. Unlike liver-derived RBP4, the extrahepatically synthesized RBP4 failed to mobilize liver retinol stores in this mouse model 
. This led the authors of that study to postulate that RBP4 “has a function specific to its tissue of origin” 
. Supporting this statement, it has been suggested that RBP4 and TTR made in the visceral yolk sac may be involved in transport of retinol and thyroid hormone from the maternal circulation to the developing fetus 
. As mentioned, choroid plexus-derived TTR is thought to mediate the distribution of thyroid hormone to the central nervous system, and furthermore, also may help protect against ischemia-induced brain injury 
. In the case of islet-derived TTR, it has been theorized that the protein could affect the processing of glucagon, and furthermore that it may influence deposition of islet amyloid, which is found in at least 95% of subjects with Type II diabetes mellitus 
An as-of-yet unanswered question raised by the current study is why RBP4 and TTR are expressed within ghrelin cells, especially at such high relative levels. One might postulate that their expression is related to the emerging role of ghrelin cell-derived products, and thus, the ghrelin cell as a whole, in metabolism. As mentioned, acyl-ghrelin, desacyl-ghrelin, obestatin, nesfatin-1 and RBP4 all have been shown (albeit in certain cases, inconsistently) to affect food intake, body weight and/or blood glucose handling. Relationships of RBP4 to some of these other ghrelin cell products has been examined in at least one human study, in which 6 mo following gastric band surgery, which resulted in a reduction of BMI of on average 6 kg/m2
, circulating levels of total ghrelin increased, obestatin increased and RBP4 decreased 
. Whereas low circulating ghrelin has been associated with elevated fasting insulin and insulin resistance in humans 
, the opposite is true for RBP4 
. Here, regulation of TTR mRNA expression in the stomach by caloric restriction also suggests that ghrelin-cell derived TTR may be involved in metabolism.
Despite the high level of RBP4 mRNA within ghrelin cells, especially as compared to liver and white adipose tissue, its overall contribution to the complement of RBP4 circulating in the bloodstream is seemingly low. The stomach is a relatively small organ, especially as compared to the liver and white adipose tissue, and furthermore, the ghrelin cell population is limited to approximately 0.3–1% cells comprising the entire gastric mucosa 
. Even with the marked expansion of ghrelin cells in the mice harboring ghrelinomas, total circulating RBP4 level remained unchanged. This perhaps suggests that ghrelin cell-derived RBP4 and TTR may not impact whole body insulin resistance and/or thyroid hormone transport. Rather, ghrelin cell-derived RBP4 and TTR instead may have effects that are more localized to the stomach, just as has been suggested for other tissues. Although co-expression of RBP4 with TTR within the ghrelin cell does suggest that there exists a shared function, the roles of ghrelin cell-derived RBP4 and TTR could be unrelated.
In conclusion, we have found relatively high levels RBP4 and TTR expression by gastric ghrelin cells. Thus, RBP4 and TTR join a short list of other proteins known to be secreted by ghrelin cells, including acyl-ghrelin, desacyl-ghrelin, obestatin and nesfatin-1. At this time, the etiology of their expression within ghrelin cells can only be speculated, although it is assumed that this could be related to an emerging general role for the ghrelin cell in blood glucose handling and metabolism. Alternatively, RBP4 and TTR may have a more regional function unique to ghrelin cells and their neighboring cells. Future studies in which RBP4 and/or TTR expression can be deleted specifically from ghrelin cells may be helpful in pinpointing the actions of ghrelin cell-derived RBP4 and TTR.