Kanako Koike

Gastric leptin, but not estrogen and somatostatin, contributes to the elevation of ghrelin mRNA expression level in fasted rats

Ghrelin, an endogenous ligand for the GH secretagog receptor, is predominantly produced in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased after refeeding. It has also been reported that estrogen upregulates ghrelin expression and production and that somatostatin inhibits ghrelin secretion, whereas leptin has a paradoxical effect. Recently, several studies have shown that estrogen, somatostatin, and leptin are produced in the stomach, but the direct effects of these gastric hormones on ghrelin expression in a fasting state remain obscure. In this study, we examined the mRNA expression levels of gastric ghrelin, aromatase (estrogen synthetase), leptin and somatostatin, and concentrations of stomach leptin and portal vein 17beta-estradiol in fasted male rats. After 48 h of fasting, although gastric ghrelin mRNA level was significantly increased, both gastric leptin mRNA level and leptin content were decreased. Further, refeeding of fasted rats resulted in a decrease in ghrelin expression level and an increase in leptin expression level. On the other hand, gastric estrogen and somatostatin levels did not change after fasting. In vitro studies revealed that leptin dose-dependently inhibited ghrelin expression and also inhibited estrogen-stimulated ghrelin expression. Moreover, ghrelin cells were found to be tightly surrounded by leptin cells. RT-PCR analysis clearly showed that long and short forms of the leptin receptor are expressed in the rat stomach. These results strongly suggest that an elevated gastric ghrelin expression level in a fasting state is regulated by attenuated restraint from decreased gastric leptin level.

Physiological characteristics of gastric contractions and circadian gastric motility in the free-moving conscious house musk shrew (Suncus murinus)

Although many studies have demonstrated the physiological action of motilin on the migrating motor complex, the precise mechanisms remain obscure. To obtain new insights into the mechanisms, we focused on the house musk shrew (Suncus murinus, suncus used as a laboratory name) as a small model animal for in vivo motilin study, and we studied the physiological characteristics of suncus gastrointestinal motility. Strain gauge transducers were implanted on the serosa of the gastric body and duodenum, and we recorded gastrointestinal contractions in the free-moving conscious suncus and also examined the effects of intravenous infusion of various agents on gastrointestinal motility. During the fasted state, the suncus stomach and duodenum showed clear migrating phase III contractions (intervals of 80-150 min) as found in humans and dogs. Motilin (bolus injection, 100-300 ng/kg; continuous infusion, 10-100 ng·kg(-1)·min(-1)) and erythromycin (80 μg·kg(-1)·min(-1)) induced gastric phase III contractions, and motilin injection also increased the gastric motility index in a dose-dependent manner (P < 0.05, vs. saline). Pretreatment with atropine completely abolished the motilin-induced gastric phase III contractions. On the other hand, in the free-feeding condition, the suncus showed a relatively long fasting period in the light phase followed by spontaneous gastric phase III contractions. The results suggest that the suncus has almost the same gastrointestinal motility and motilin response as those found in humans and dogs, and we propose the suncus as a new small model animal for studying gastrointestinal motility and motilin in vivo.

DNA Introduction into Living Cells by Water Droplet Impact with an Electrospray Process

Electrospraying is performed by applying a high voltage to a capillary tip. A highly charged droplet at the capillary tip splits into microor nanoscale droplets, and a charged fineliquid aerosol is accelerated by a high-voltage electric field. This method has been used for soft ionization in the mass spectrometric analysis of macromolecules and biomaterials. Moreover, in desorption electrospray ionization (DESI) for mass spectrometry, the electrically charged droplets hit the surface molecules and are ionized under ambient conditions. Electrospraying is also used for the production of nanofibers and protein chips. Delivery of a foreign nucleic acid into a living cell is an important technique in molecular biology and the medical field. Previously developed transfection techniques, including methods that use a cationic polymer, lipofection, a gene gun, and electroporation, have been reported. Moreover, Pui et al. used electrospraying to increase the momentum of particles in the gene-gun method. However, these techniques sometimes cause severe damage to cells, especially mammalian cells, or require complex and expensive devices. We hypothesized that liquid droplet impact by electrospraying would transport a nucleic acid into a cell by making a transient channel through mild damage on the cell surface. Herein, we describe a technique for the introduction of a gene into eukaryotic (mammalian) and prokaryotic (bacterial) cells and avian embryonic tissue by spraying water droplets produced by an electrospray device without any cytotoxic reagents. Figure 1 shows a diagram of the electrospray equipment for gene delivery. Purified water is sprayed onto the cells as a charged aerosol generated by static electricity repulsion at the tip of the tube. Only water or phosphate-buffered saline (PBS) is used in this method, and the device is a simple structure basically consisting of a tube connected to a highvoltage power supply that is a constant potentiostat with the current limited to 100 mA. A stainless-steel capillary (caliber 0.1 mm, outside diameter 0.3 mm, length 40 mm) was impressed with a high voltage, and water was supplied at a flow rate of 100–200 mLmin . In this experiment, the stainless-steel tube was impressed from 7 to 18 kV. This voltage (over 7 kV) is higher than that used for mass spectrometry (1–3 kV), as an organic solvent, which is usually used in mass analysis for reduction of surface tension, cannot be applied to gene transfection because of its cytotoxicity. The sprayed dishes were placed on an electrically grounded square-plate electrode, and the inside of the culture dish was also grounded by attaching a small piece of metal foil to the plate. Adhesive Chinese hamster ovary (CHO) cells and HeLa cells were tested as models, and almost the same results were obtained. CHO cells were cultured in minimum essential medium (a-MEM, Gibco, USA) supplemented with 10% fetal bovine serum. Cells were plated in a 35-mm culture dish (Falcon, USA) at 2.0 ? 10 cells per dish and cultured at 37 8C under 5% CO2 in air. Three days later, the cells were used for electrospraying. The culture medium was removed from the dish and an aqueous solution (100 mL) of plasmid vector pEGFP-N1 (100 mgmL 1 in water, Clonetech, USA), as green fluorescence protein (GFP) encoding DNA, was added to the dish. Water was electrosprayed onto the cells from a height of 2 cm at 10 kV, and culture mediumwas directly added to the dish. After 24 h of cultivation, GFP-positive cells were counted with a hemocytometer under a fluorescence microscope (Olympus, Japan). We used nonmoving equipment in this experiment, which resulted in a limited spraying area on the dish. We evaluated the transfection rate of isolated GFP-positive cells to total cells, which included cells located in the external region of the sprayed zone, and obtained a transfection rate of 0.05 to 1.6%. The number of cells showing fluorescence increased with an increase in applied voltage, which indicates that the Figure 1. Diagram of the electrospray equipment for gene delivery. The plasmid DNAs located around the plasma membrane are introduced into cells by collision of water microdroplets.

Macrophage Colony-Stimulating Factor Induces Prolactin Expression in Rat Pituitary Gland

We investigated the role of macrophage colony-stimulating factor (M-CSF) in the pituitary gland to understand the effect of M-CSF on pituitary hormones and the relationship between the endocrine and immune systems. When we attempted to establish pituitary cell lines from a thyrotropic pituitary tumor (TtT), a macrophage cell line, TtT/M-87, was established. We evaluated M-CSF-like activity in conditioned media (CM) from seven pituitary cell lines using TtT/M-87 cells. TtT/M-87 proliferation significantly increased in the presence of CM from TtT/GF cells, a pituitary folliculostellate (FS) cell line. M-CSF mRNA was detected in TtT/GF and MtT/E cells by reverse transcriptase-polymerase chain reaction (RT-PCR), and its expression in TtT/GF cells was increased in a lipopolysaccharide (LPS) dose-dependent manner. M-CSF mRNA expression was also increased in rat anterior pituitary glands by LPS. M-CSF receptor (M-CSFR) mRNA was only detected in TtT/M-87 cells and increased in the LPS-stimulated rat pituitary glands. In rat pituitary glands, M-CSF and M-CSFR were found to be localized in FS cells and prolactin (PRL)-secreting cells, respectively, by immunohistochemistry. The PRL concentration in rat sera was significantly increased at 24 h after M-CSF administration, and mRNA levels significantly increased in primary culture cells of rat anterior pituitary glands. In addition, TNF-&agr; mRNA was increased in the primary culture cells by M-CSF. These results revealed that M-CSF was secreted from FS cells and M-CSF regulated PRL expression in rat pituitary glands.

Detailed morphogenetic analysis of the embryonic chicken pars tuberalis as glycoprotein alpha subunit positive region

The pars tuberalis (PT) is a part of the anterior pituitary gland that is located as a thin cell layer surrounding the median eminence. The characteristics of PT, including cell shape and cell composition, differ from those of the pars distalis (PD), suggesting that PT has unique physiological functions and different morphogenesis compared to PD. In this study, we used chicken embryos and showed for the first time that most hormone-producing cells in PT at embryonic day (E) 20.0 were only glycoprotein α subunit (αGSU)-positive staining cells. Then, using serial frontal and sagittal sections, we examined the detailed distribution of the αGSU mRNA-expressing region, as a marker of PT in the chicken embryonic pituitary gland during the E3.0–20.0 period. This three-dimensional expression pattern analysis clarified that αGSU mRNA expression initially appeared only in the bilateral regions of the Rathke’s recess (RR) at E3.5, and this region expanded and showed a ring-like structure on RR. Subsequently, this αGSU mRNA-expressing region gradually expanded upward and reached the diencephalon at E8.0. This region became thinner as it surrounded the base of the diencephalon from E12.0 to E20.0. In this study, we demonstrated the detailed morphological changes of the chicken PT primordium by detecting αGSU mRNA, and we also showed that PT is a unique region in the early developmental stage.

Measurement of Contractile Activity in Small Animal's Digestive Organ by Carbon Nanotube-Based Force Transducer

A carbon nanotube (CNT)-based force transducer designed to be embedded in the body of a live animal was fabricated and implanted into the stomach of a rat omit to measure contractile movement. The transducer comprised dispersed poly(ethylene glycol)-grafted multiwalled CNTs applied to a comb-like Au-electrode formed on a poly(dimethylsiloxane) sheet. The implanted rat was injected with acetylcholine to induce muscular contractions and changes in the resistance of the transducer were measured. Such changes arise owing to strain in the CNT network upon distortion. The measured resistance change was found to be proportional to the concentration of injected acetylcholine.

M2024 Ghrelin Stimulates Gastric Contraction in a Specific Physiological Condition In Vivo and In Vitro in the House Musk Shrew (Suncus Murinus), a Ghrelin-And Motilin-Producing Laboratory Animal

Background: While gastroparesis usually presents with nausea or vomiting, abdominal pain also may be part of the presenting illness. The importance of pain in gastroparesis and its associated factors are largely unexplored. Aims: Define abdominal pain prevalence in gastroparesis and relate to demography, etiology, severity, quality of life, gastric retention, and analgesic vs. prokinetic/antiemetic drug use. Methods: 339 gastroparesis patients were enrolled from 6 centers of the NIDDK Gastroparesis Clinical Research Consortium from 1/ 07-11/09. Survey, exam, and gastric emptying data were compared in those with pain vs. no pain and pain as the predominant symptom vs. predominant nausea or vomiting. Results: 243 patients (72%) noted abdominal pain; pain was the predominant symptom in 65 (19%) vs. nausea or vomiting in 194 (57%). On Patient Assessment of GI Symptoms surveys, upper abdominal pain scores were higher in those with pain (3.5±1.4) vs. no pain (1.9±1.8, P<0.0001). Lower abdominal pain scores also were higher with (2.3±1.7) vs. no pain (1.4±1.5, P<0.0001). Higher percentages of those with pain were women (86 vs. 76%, P= 0.04) and had idiopathic etiology (70 vs. 54%, P=0.007). Infection-like prodromes were similar with (17%) vs. no pain (19%, P=0.75). Investigator-rated gastroparesis severity was similar with vs. no pain (P=0.25). Patient Assessment of GI Quality of Life (PAGI-QOL) scores were lower with pain (2.3±1.1 vs. 2.8±1.1, P<0.0007). SF-36 physical function (P<0.0001), energy/fatigue (P=0.004), emotional wellbeing (P=0.02), social function (P= 0.0004), pain (P<0.0001), and general health (P=0.02) scores were lower with pain. 4 hr gastric retention (normal <10%) was similar with (32±24%) vs. no pain (33±22%, P=0.91). Opiate use was higher with pain (P<0.0001), while antidepressant (P=0.22), neuropathic pain modulator (P=0.10), prokinetic (P=0.40), and antiemetic (P=0.13) use did not significantly differ. Compared to those with predominant nausea or vomiting, patients with predominant pain had higher upper abdominal pain scores (P<0.0001), but similar severity (P= 0.91), lower abdominal pain scores (P=0.21), 4 hr gastric retention (P=0.96), PAGI-QOL and SF-36 scores (P=NS). Conclusions: Abdominal pain is prevalent in gastroparesis and is predominant in 1 in 5 patients. Pain, like nausea and vomiting, impairs quality of life. Pain is associated with female gender and idiopathic etiology but not gastric retention. Increased opiate use reflects its health care impact and underscores a need to investigate new treatment options for pain. Our findings provide insight into this important symptom and emphasize its negative influence in gastroparesis.