Yoshihiro Nishi

Identification and Characterization of Acyl-Protein Thioesterase 1/Lysophospholipase I As a Ghrelin Deacylation/Lysophospholipid Hydrolyzing Enzyme in Fetal Bovine Serum and Conditioned Medium

Ghrelin contains an octanoic acid at the third residue serine, and the presence of octanoic acid on ghrelin is critical to its physiological functions. The precise mechanism for the deacylation of ghrelin in circulation remains to be clarified, although the level of deacylated ghrelin (des-acyl ghrelin) is higher than that of acylated ghrelin in serum. In this study, rapid identification of ghrelin deacylation activity was achieved by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and a ghrelin deacylation enzyme was purified 1515-fold from fetal bovine serum. Chromatographic separation showed a 24-kDa band on SDS-PAGE corresponding to ghrelin deacylation activity, and the protein band was identified as acyl-protein thioesterase 1 (APT1)/lysophospholipase I. A ghrelin deacylation enzyme in medium from HepG2 cells was also purified and identified as APT1. Although it lacks a secretion signal sequence, APT1 may be released by cells expressing APT1, mainly from liver in vivo. APT1 was originally purified as a cytosolic lysophospholipid hydrolyzing enzyme (lysophospholipase I), and recombinant APT1 exhibited deacylation activity as well as lysophospholipase activity in vitro. APT1 is released at high levels from RAW264.7 macrophage-like cells into the culture medium after stimulation with lipopolysaccharide (LPS), and LPS suppresses APT1 mRNA and protein expressions in these cells. More potent ghrelin deacylase activities were detected in sera from LPS-treated rats than in control sera. These results suggested that the serum activity of APT1 may play an important role in determination of the concentration of des-acyl ghrelin in circulation, especially under septic inflammation.

Structures and molecular forms of the ghrelin-family peptides.

Ghrelin is an acylated peptide hormone produced mainly from the stomach. The major active products of the ghrelin gene in the stomach of rats, mice and humans are 28-amino acid peptides acylated at the serine-3 position with an n-octanoyl group (C8:0), called simply ghrelin. However, recent studies have revealed that the ghrelin gene can generate a variety of bioactive molecules besides ghrelin. These include acyl forms of ghrelin other than C8:0-ghrelin (i.e., n-decanoyl ghrelin or n-decenoyl ghrelin), des-acyl ghrelin, obestatin and ghrelin-associated peptides originated from the ghrelin gene. This review surveys the structures of the ghrelin peptides and molecular forms of ghrelin gene-derived products, and summarizes the knowledge about the functions of these peptides, with an emphasis on the acyl forms of the ghrelin peptide.

Plasma levels of n-decanoyl ghrelin, another acyl- and active-form of ghrelin, in human subjects and the effect of glucose- or meal-ingestion on its dynamics.

Besides n-octanoyl ghrelin (O-ghrelin), there is another acyl-form of ghrelin; n-decanoyl ghrelin (D-ghrelin), which has a decanoic acid modification. In this study, we examined the kinetics of D-ghrelin immunoreactivity in human plasma in comparison to O-ghrelin or total ghrelin by using a D-ghrelin-specific radioimmunoassay. The dynamics of plasma D-ghrelin was assessed following glucose- or meal-ingestion in healthy, non-obese subjects (5 males and 5 females). Correlations were also analyzed between the levels of plasma D-ghrelin and anthropometric or metabolic indicators in healthy human subjects (n=111, BMI 17.4-34.3). The plasma levels of D-ghrelin, like O- or T-ghrelin, significantly declined (p<0.05 for male and p<0.01 for female) 60 min after the ingestion of glucose in non-obese subjects. However, in the same subjects, no significant decline was noted in the levels of D-ghrelin, unlike O- or T-ghrelin, upon the meal ingestion. A significant increase was observed in the proportion of plasma D-ghrelin levels to that of T-ghrelin (p<0.05) in the healthy human subjects as BMI increased, unlike the proportion of O-ghrelin to T-ghrelin, which did not change. Since D-ghrelin possesses almost the same potential as that of O-ghrelin with regard to the feeding-stimulation, these differences between the dynamics of D- and O-ghrelin in human plasma might influence appetite-control, especially in those with increased BMI.

Ghrelin Acylation by Ingestion of Medium-Chain Fatty Acids

We found in a primary study that ingestion of medium-chain fatty acids (MCFAs) or medium-chain triacylglycerols (MCTs) increased the stomach contents of acyl ghrelin, and we further showed that the carbon-chain length of the acyl groups that modified the nascent ghrelin peptides corresponded to that of the ingested MCFAs or MCTs. These findings clearly demonstrated that the ingested MCFAs are directly used for the acyl-modification of ghrelin. Before the discovery of ghrelin-O-acyltransferase (GOAT), our in vivo study suggested that the putative GOAT preferred MCTs (composed of C6:0 to C10:0 FFAs) to either short- or long-chain triglycerides. In another study, we suggested that MCFAs or MCTs might represent a potential therapeutic modality for the clinical manipulation of energy metabolism through the modulation of ghrelin activity. After the discovery of GOAT, many studies have been done on the acylation of ghrelin using MCFAs, MCTs, or their derivatives; however, results and interpretations have been inconsistent, largely due to the differences in experimental conditions. This chapter describes detailed methods for the analysis of ghrelin acylation in vivo to facilitate future research in this field.

Dynamics of placental ghrelin production and its receptor expression in a Dahl salt-sensitive rat model of intrauterine growth restriction

BACKGROUND Ghrelin, a peptide hormone produced mainly in the stomach, is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). The existence of placental ghrelin and its receptor has been confirmed in normal pregnancy. However, few reports have so far referred to placental ghrelin and its receptor in intrauterine growth restriction (IUGR). OBJECTIVES The dynamics of ghrelin production and its receptor expression was investigated to clarify the role of placental ghrelin in an IUGR pregnancy using pregnant Dahl salt-sensitive (Dahl S) rats as a model for IUGR. METHODS Pregnant Dahl S rats were fed a high-salt diet to develop hypertensive pregnancy with IUGR (IUGR-preg). The levels of ghrelin peptide in the placenta, stomach and plasma of the dams, together with the expression levels of mRNAs for ghrelin and its functional receptor (GHS-R1a) in the placenta, were measured in the IUGR-preg rats at 2 and 3 weeks of gestation, and compared to those in the control pregnant Dahl S rats fed standard chow (Normal-preg). RESULTS The levels of placental ghrelin peptide at 2 weeks of gestation and placental ghrelin mRNA at each gestational week in IUGR-preg were significantly higher than those in Normal-preg. The level of GHS-R1a mRNA in the placenta of IUGR-preg, which was lower at 2 weeks of gestation in comparison to Normal-preg, significantly increased from 2 to 3 weeks of gestation. No significant difference was observed in the level of ghrelin peptide in the plasma or stomach of the dams between the two groups. CONCLUSION The profile of placental ghrelin production and the expression of its receptor using Dhal S rats in the IUGR-preg was different from that in the control. The placental ghrelin-ghrelin receptor system thus continues to work until the term of pregnancy in the IUGR-preg in contrast to Normal-preg, which might act as a compensational mechanism for fetal growth.

Ghrelin levels are reduced in Rett syndrome patients with eating difficulties.

Most patients with Rett syndrome (RTT) have both gastrointestinal problems and somatic growth failure, including microcephaly. Ghrelin is a peptide hormone involved in growth hormone secretion, interdigestive motility, and feeding behavior. Plasma ghrelin assays have previously been described for other neurodevelopmental disorders. To examine the pathophysiology of RTT, we measured plasma levels of ghrelin in patients with RTT. A case–control study examining plasma levels of ghrelin, serum growth hormone, and insulin‐like growth factor‐1 (IGF‐1) was performed on 27 patients with RTT and 53 controls. Plasma levels of total (T)‐ and octanoyl (O)‐ghrelin were significantly lower in patients with RTT than in controls. Plasma levels of T‐ghrelin correlated significantly with serum IGF‐1 levels and head circumference. Significantly lower levels of plasma T‐ghrelin and O‐ghrelin were observed in RTT patients with eating difficulties, while lower levels of plasma T‐ghrelin were observed in RTT patients with constipation, in comparison to patients without either of these symptoms. Alterations in plasma ghrelin levels may reflect various clinical symptoms and signs in RTT patients, including growth failure, acquired microcephalus, autonomic nerve dysfunction, and feeding difficulties. We describe the role of ghrelin in RTT and suggest this peptide as a novel biological marker in patients with RTT.

Increased production of active ghrelin is relevant to hyperphagia in nonobese spontaneously diabetic Torii rats

An abnormal eating behavior is often associated with diabetes mellitus in individuals. In the present study, we investigated the mechanisms underlying the relationship among uncontrolled diabetes, food intake, and the production of ghrelin, an orexigenic hormone, in spontaneous diabetic Torii (SDT) rats. Male SDT rats and age-matched control Sprague-Dawley (SD) rats were housed from 8 to 38 weeks of age. Body weight and daily food intake were measured weekly, whereas blood and whole stomach samples were obtained at the age of 8, 25, and 38 weeks in both SDT and SD rats. The SDT rats at both 25 and 38 weeks of age demonstrated significantly lower body weights despite almost doubled food consumption compared with the SD rats of the same age. The SDT rats showed overt hyperglycemia at 25 and 38 weeks of age with concomitant hypoinsulinemia. The plasma active ghrelin levels and the ratio to total ghrelin levels of SDT rats at 38 weeks of age were significantly higher than those of SD rats of the same age. Stomach ghrelin and ghrelin O-acyltransferase messenger RNA expression levels were higher in SDT rats than in SD rats after the induction of diabetes, with a concomitant decrease of stomach ghrelin-immunopositive cell numbers in SDT rats at 38 weeks of age. The SDT rats with uncontrolled hyperglycemia show hyperphagia with a concomitant increase of plasma active ghrelin concentration. This report is the first to clarify the relevance of ghrelin to hyperphagia in diabetic state over an extended period.