Glucagon-Like Peptide-1 Regulates Cholecystokinin Production in ß-Cells to Protect From Apoptosis.

Cholecystokinin (CCK) is a classic gut hormone that is also expressed in the pancreatic islet, where it is highly up-regulated with obesity. Loss of CCK results in increased ß-cell apoptosis in obese mice. Similarly, islet α-cells produce increased amounts of another gut peptide, glucagon-like peptide 1 (GLP-1), in response to cytokine and nutrient stimulation. GLP-1 also protects ß-cells from apoptosis via cAMP-mediated mechanisms. Therefore, we hypothesized that the activation of islet-derived CCK and GLP-1 may be linked. We show here that both human and mouse islets secrete active GLP-1 as a function of body mass index/obesity. Furthermore, GLP-1 can rapidly stimulate ß-cell CCK production and secretion through direct targeting by the cAMP-modulated transcription factor, cAMP response element binding protein (CREB). We find that cAMP-mediated signaling is required for Cck expression, but CCK regulation by cAMP does not require stimulatory levels of glucose or insulin secretion. We also show that CREB directly targets the Cck promoter in islets from obese (Leptin(ob/ob)) mice. Finally, we demonstrate that the ability of GLP-1 to protect ß-cells from cytokine-induced apoptosis is partially dependent on CCK receptor signaling. Taken together, our work suggests that in obesity, active GLP-1 produced in the islet stimulates CCK production and secretion in a paracrine manner via cAMP and CREB. This intraislet incretin loop may be one mechanism whereby GLP-1 protects ß-cells from apoptosis.

An important role for cholecystokinin, a CLOCK target gene, in the development and treatment of manic-like behaviors.

Mice with a mutation in the Clock gene (ClockΔ19) have been identified as a model of mania; however, the mechanisms that underlie this phenotype, and the changes in the brain that are necessary for lithium's effectiveness on these mice remain unclear. Here, we find that cholecystokinin (Cck) is a direct transcriptional target of CLOCK and levels of Cck are reduced in the ventral tegmental area (VTA) of ClockΔ19 mice. Selective knockdown of Cck expression via RNA interference in the VTA of wild-type mice produces a manic-like phenotype. Moreover, chronic treatment with lithium restores Cck expression to near wild-type and this increase is necessary for the therapeutic actions of lithium. The decrease in Cck expression in the ClockΔ19 mice appears to be due to a lack of interaction with the histone methyltransferase, MLL1, resulting in decreased histone H3K4me3 and gene transcription, an effect reversed by lithium. Human postmortem tissue from bipolar subjects reveals a similar increase in Cck expression in the VTA with mood stabilizer treatment. These studies identify a key role for Cck in the development and treatment of mania, and describe some of the molecular mechanisms by which lithium may act as an effective antimanic agent.

Roux-en-Y gastric bypass surgery increases number but not density of CCK-, GLP-1-, 5-HT-, and neurotensin-expressing enteroendocrine cells in rats.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) surgery is very effective in reducing excess body weight and improving glucose homeostasis in obese subjects. Changes in the pattern of gut hormone secretion are thought to play a major role, but the mechanisms leading to both changed hormone secretion and beneficial effects remain unclear. Specifically, it is not clear whether changes in the number of hormone-secreting enteroendocrine cells, or changes in the releasing stimuli, or both, are important. METHODS: We estimated numbers of enteroendocrine cells after immunohistochemical staining in fixed tissue samples from rats at 10-11 months after RYGB. KEY RESULTS: Numbers of glucagon-like peptide-1 (GLP-1) (L-cells, co-expressing peptide YY (PYY)), cholecystokinin (CCK), neurotensin, and 5-HT-immunoreactive cells were significantly increased in the Roux and common limbs, but not the biliopancreatic limb in RYGB rats compared with sham-operated, obese rats fed high-fat diet, and chow-fed controls. This increase was mostly accounted for by general hyperplasia of all intestinal wall layers of the nutrient-perfused Roux and common limbs, and less to increased density of expression. The number of ghrelin cells in the bypassed stomach was not different among the three groups. CONCLUSIONS & INFERENCES: The findings suggest that the number of enteroendocrine cells increases passively as the gut adapts, and that the increased total number of L- and I-cells is likely to contribute to the higher circulating levels of GLP-1, PYY, and CCK, potentially leading to suppression of food intake and stimulation of insulin secretion. Whether changes in releasing stimuli also contribute to altered circulating levels will have to be determined in future studies.

A major lineage of enteroendocrine cells coexpress CCK, secretin, GIP, GLP-1, PYY, and neurotensin but not somatostatin.

Enteroendocrine cells such as duodenal cholecystokinin (CCK cells) are generally thought to be confined to certain segments of the gastrointestinal (GI) tract and to store and release peptides derived from only a single peptide precursor. In the current study, however, transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the CCK promoter demonstrated a distribution pattern of CCK-eGFP positive cells that extended throughout the intestine. Quantitative PCR and liquid chromatography-mass spectrometry proteomic analyses of isolated, FACS-purified CCK-eGFP-positive cells demonstrated expression of not only CCK but also glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide (GIP), peptide YY (PYY), neurotensin, and secretin, but not somatostatin. Immunohistochemistry confirmed this expression pattern. The broad coexpression phenomenon was observed both in crypts and villi as demonstrated by immunohistochemistry and FACS analysis of separated cell populations. Single-cell quantitative PCR indicated that approximately half of the duodenal CCK-eGFP cells express one peptide precursor in addition to CCK, whereas an additional smaller fraction expresses two peptide precursors in addition to CCK. The coexpression pattern was further confirmed through a cell ablation study based on expression of the human diphtheria toxin receptor under the control of the proglucagon promoter, in which activation of the receptor resulted in a marked reduction not only in GLP-1 cells, but also PYY, neurotensin, GIP, CCK, and secretin cells, whereas somatostatin cells were spared. Key elements of the coexpression pattern were confirmed by immunohistochemical double staining in human small intestine. It is concluded that a lineage of mature enteroendocrine cells have the ability to coexpress members of a group of functionally related peptides: CCK, secretin, GIP, GLP-1, PYY, and neurotensin, suggesting a potential therapeutic target for the treatment and prevention of diabetes and obesity.

Effect of heat exposure on gene expression of feed intake regulatory peptides in laying hens.

The aim of this paper was to investigate the effect of heat stress on the regulation of appetite-associated genes in laying hens. Forty eight laying hens were randomly divided into two circumstances: high (31 ± 1.5°C; relative humidity, 82.0 ± 2.2%) or normal (20 ± 2°C, control; relative humidity, 60.1 ± 4.5%) ambient environment. Heat stress decreased body weight gain (P < 0.01), feed intake (P < 0.01), laying rate (P < 0.05), average egg mass (P < 0.01), egg production (P < 0.01), shell thickness (P < 0.01), and feed efficiency (P < 0.05). High ambient temperature decreased plasma uric acid (P < 0.05). Heat stress significantly increased mRNA levels of ghrelin and cocaine- and amphetamine-regulated transcript (P < 0.05) and decreased mRNA levels of cholecystokinin (P < 0.05) in the hypothalamus. Heat stress significantly increased (P < 0.05) mRNA levels of ghrelin in the glandular stomach and jejunum but significantly decreased (P < 0.05) mRNA levels of cholecystokinin in the duodenum and jejunum. In conclusion, heat stress plays a unique role in some special neuropeptides (e.g., ghrelin, cocaine- and amphetamine-regulated transcript, and cholecystokinin), which might participate in the regulation of feed intake in laying hens under high ambient temperature.

Food deprivation explains effects of mouthbrooding on ovaries and steroid hormones, but not brain neuropeptide and receptor mRNAs, in an African cichlid fish.

Feeding behavior and reproduction are coordinately regulated by the brain via neurotransmitters, circulating hormones, and neuropeptides. Reduced feeding allows animals to engage in other behaviors important for fitness, including mating and parental care. Some fishes cease feeding for weeks at a time in order to provide care to their young by brooding them inside the male or female parent's mouth. Maternal mouthbrooding is known to impact circulating hormones and subsequent reproductive cycles, but neither the full effects of food deprivation nor the neural mechanisms are known. Here we ask what effects mouthbrooding has on several physiological processes including gonad and body mass, brain neuropeptide and receptor gene expression, and circulating steroid hormones in a mouthbrooding cichlid species, Astatotilapia burtoni. We ask whether any observed changes can be explained by food deprivation, and show that during mouthbrooding, ovary size and circulating levels of androgens and estrogens match those seen during food deprivation. Levels of gonadotropin-releasing hormone 1 (GnRH1) mRNA in the brain were low in food-deprived females compared to controls and in mouthbrooding females compared to gravid females. Levels of mRNA encoding two peptides involved in regulating feeding, hypocretin and cholecystokinin, were increased in the brains of food-deprived females. Brain mRNA levels of two receptors, GnRH receptor 2 and NPY receptor Y8c, were elevated in mouthbrooding females compared to the fed condition, but NPY receptor Y8b mRNA was differently regulated by mouthbrooding. These results suggest that many, but not all, of the characteristic physiological changes that occur during mouthbrooding are consequences of food deprivation.

Biliopancreatic diversion induces villi elongation and cholecystokinin and ghrelin increase.

INTRODUCTION: Factors leading to weight loss and weight stabilization after bariatric surgery are not fully understood. Our aim was to evaluate, in Sprague-Dawley rats, the histological and gut hormonal changes after Larrad-biliopancreatic diversion (Larrad-BPD). MATERIALS AND METHODS: Rats randomly underwent the following protocols: Larrad-BPD (n=4) versus pair fed (PF) (n=4). Weight and food intake were measured every day. By immunohistochemistry ghrelin was examined in the stomach, while cholecystokinin (CCK), glucagon-like-peptide-1 (GLP-1), peptide YY (PYY) and serotonin (5-HT) expression were analyzed in alimentary limb and ileum following or not the Larrad-BPD. RESULTS: Larrad-BPD rats exhibited significant (P<0.05) weight loss compared to PF rats. Villi enlongation was observed in Larrad-BPD rats. In residual stomach, ghrelin was diminished. In the alimentary limb, ghrelin and CCK positive cells were detected more than in the ileum of PF rats. GLP-1 expression was decreased and PYY expression was absent after Larrad-BPD compared with PF rats. DISCUSSION: Larrad-BPD is followed by histological changes and a pleiotropic gut endocrine response aimed to compensate the reduction of intestinal area exposed to food. Until now, the hormones responsible for the intestinal hypertrophy have not been defined.

Effect of Salvia miltiorrhiza pretreatment on the CCK and VIP expression in hepatic ischemia-reperfusion-induced digestive tract congestion.

The inhibitory effect of different reperfusion periods 45 min following hepatic ischemia on the expression of cholecystokinin (CCK) and vasoactive intestinal peptide (VIP) in the jejunum and the effect of salvia miltiorrhiza pretreatment were investigated, and the possible mechanism and implications were explored. Eighty rats were randomly divided into four groups: normal control group (CO group), sham-operated group (SO group), ischemia/reperfusion (I/R) injury group (IR group) and salvia miltiorrhiza pretreatment group (SM group). The rat model of I/R was established by using a non-invasive artery clamp to clip (45 min) or relax the hepatic pedicle. In the SM group, saline (40 mL/kg) and salvia miltiorrhiza injection (6 g/kg) were injected via the tail vein 30 min before clipping the hepatic pedicle. In the SO group only the porta hepatis was dissected after laparotomy without clamping the hepatic pedicle. At 0, 3, 12, 24 and 72 h post-reperfusion, respectively, upper jejunum samples were taken for immunohistochemistry of CCK and VIP. It was found that 0 h after I/R, the expression of CCK and VIP in the upper jejunum was upregulated. With prolongation of the reperfusion period, the expression of CCK and VIP was also increased, reached the peak at the 24th h, and gradually returned to the normal level at the 72nd h after reperfusion. The levels of both CCK and VIP in the SM group were lower than those in the IR group. It is suggested that the digestive tract congestion injury caused by liver ischemia can upregulate the expression of CCK and VIP in the jejunum following reperfusion. Salviae pretreatment can partly reduce the increased expression of CCK and VIP in the jejunum in the same period, which might contribute to the early recovery of gastrointestinal motility.

[Overdose of iodine on expression of CCK gene in rat brains].

OBJECTIVE: To observe the effect of overdose iodine on the expression of CCK gene in brains of rats and identify the possible mechanisms. METHODS: One-month weaning Wistar rats were randomly divided into five groups which were fed with normal feedstuff and water supplemented with different concentrations of potassium iodide, named A group (iodine ration was about 6.15 microg per day), B group (iodine ration was about 30.75 microg per day), C group (iodine ration was about 61.5 microg per day), D group (iodine ration was about 307.5 microg per day) and E group (iodine ration was about 615 microg per day). Rats were sacrificed after being fed for three or six months. Then serum thyroid hormones were measured by radioimmunoassay and the mRNA level of CCK gene was studied by using RT-PCR technique. RESULTS: At the end of three months, the values of thyroid hormones in E group [TT4 (45.2 +/- 13.7) nmol/L, TI'3 (0.65 +/- 0.20) nmol/L, FT3 (0.93 +/- 0.45) pmol/L, FT4 (7.07 +/- 2.43) pmol/L, rT3 (0.15 +/- 0.04) nmol/L] were all lower than those in A group [TT4 (76.0 +/- 18.8) nmol/L, TT3 (1.34 +/- 0.41) nmol/L, FT3 (2.45 +/- 0.62) pmol/L, FT4 (15.12 +/- 3.40) pmol/L, rT3 (0.24 +/- 0.04) nmol/L]. There were significant differences between E group and A group on the levels of serum TH (F values are 14.68, 16.03, 21.16, 20.25, 13.52 respectively, P < 0.01); FT3 levels in C and D groups were significantly decreased as compared to A and B groups (F = 21.16, P < 0.05). rT3 level in D group was significantly decreased compared with A,B and C groups (F = 13.52, P < 0.05). At the end of six months, the levels of serum TH in E group (TT4 (51.84 +/- 15.83) nmol/L, TT3 (0.77 +/- 0.22) nmol/L, FT4 (6.88 +/- 2.23) pmol/L, FT3 (0.74 +/- 0.28) pmol/L, rT3 (0.14 +/- 0.03) nmol/L) were lower than those in any other groups (F values were 6.05, 12.22, 11.25, 13.42, 5.89 respectively, P < 0.05). At the end of both three and six months, the mRNA levels of CCK gene in E group were lower than any other groups (F values were 4.04, 3.95 respectively, P < 0.01). The results of correlation analysis showed that serum FT4 had linear correlation with levels of CCK mRNA (r values were 0.990, 0.948 respectively; P < 0.05); However serum FT3 had no linear correlation with the levels of CCK mRNA (r values are 0.970, 0.932 respectively). CONCLUSIONS: Exposure to overdose of iodine (iodine ration was 100-fold higher than that of A group) could decrease the mRNA level of CCK gene. Compared with FT3, FT4 might have more important role on the regulation of CCK mRNA induced by excess of iodine.

Cholecystokinin (CCK) and CCK receptor expression by human gliomas: Evidence for an autocrine/paracrine stimulatory loop.

Cholecystokinin (CCK) is a gut-brain peptide has been described to be able to induce mitosis according to recent studies. Additionally, conflicting data has been published on whether tumours of the central and peripheral nervous system in general, and gliomas in particular, express CCK receptors. In the present in vitro study we employed reverse transcription followed by the polymerase chain reaction (RT-PCR) to investigate whether mRNA for CCK-A and CCK-B receptors as well as CCK peptide itself is present in primary human gliomas and the U-87 MG GBM cell line. The data show that 14/14 (100%) of the primary gliomas exhibited mRNA expression for the CCK peptide gene and the B receptor including the U-87 MG cells, whereas, only 2/14 (14%) showed presence of the CCK-A receptor. The presence of CCK receptors together with CCK peptide expression itself suggests presence of an autocrine loop controlling glioma cell growth. In support of this conclusion, a neutralizing antibody against the CCK peptide exhibited a dose dependent inhibition of cell growth whereas, antagonists to CCK caused a dose depend inhibition of exogenous stimulated glioma cell growth in vitro, via the CCK-B receptor which is PKC activated. Assessment of apoptosis and proteasome activity were undertaken and we report that treatment with CCK antagonists decreased proteasome and increased caspase-3 activity. These data indicate that CCK peptide and CCK-B are abundant in human gliomas and they act to stimulate cell growth in an autocrine manner, primarily via the high affinity CCK-B receptor, which was blocked by antagonists to CCK, perhaps via apoptosis.

The cell-specific pattern of cholecystokinin peptides in endocrine cells versus neurons is governed by the expression of prohormone convertases 1/3, 2, and 5/6.

Most peptide hormone genes are, in addition to endocrine cells, also expressed in neurons. The peptide hormone cholecystokinin (CCK) is expressed in different molecular forms in cerebral neurons and intestinal endocrine cells. To understand this difference, we examined the roles of the neuroendocrine prohormone convertases (PC) 1/3, PC2, and PC5/6 by measurement of proCCK, processing intermediates and bioactive, alpha-amidated, and O-sulfated CCK peptides in cerebral and jejunal extracts of null mice, controls, and in the PC5/6-expressing SK-N-MC cell-line. In PC1/3 null mice, the synthesis of bioactive CCK peptide in the gut was reduced to 3% of the translational product, all of which was in the form of alpha-amidated and tyrosine O-sulfated CCK-22, whereas the neuronal synthesis in the brain was largely unaffected. This is opposite to the PC2 null mice in which only the cerebral synthesis was affected. SK-N-MC cells, which express neither PC1/3 nor PC2, synthesized alone the processing intermediate, glycine-extended CCK-22. Immunocytochemistry confirmed that intestinal endocrine CCK cells in wild-type mice express PC1/3 but not PC2. In contrast, cerebral CCK neurons contain PC2 and only little, if any, PC1/3. Taken together, the data indicate that PC1/3 governs the endocrine and PC2 the neuronal processing of proCCK, whereas PC5/6 contributes only to a modest endocrine synthesis of CCK-22. The results suggest that the different peptide patterns in the brain and the gut are due to different expression of PCs.

Central expressions of ghrelin and cholecystokinin in rats with gastric electrical stimulation.

BACKGROUND: Gastric electrical stimulation (GES) has recently been proposed for the treatment of obesity. The aim of this study was to explore the possible central mechanisms involved in GES by investigating the expression of orexigenic and anorexigenic peptides in the rodent hypothalamus and hippocampus. METHODS: The experiment was designed in two parts: an acute experiment with 2 h GES and a chronic experiment with 14-day continuous GES. After stimulation, the expressions of an orexigenic hormone, ghrelin, in the hypothalamus and an anorexigenic hormone, cholecystokinin (CCK), in the hippocampus were detected by the immunohistochemical method. GES was performed using parameters similar to those used in clinical studies for treating obesity. RESULTS: Compared with the control group, 2 h GES resulted in a decrease in the number of ghrelin-immunoreactive (ghrelin-IR) neurons in the hypothalamic paraventricular nucleus (PVN, 34.8 +/- 1.86 vs 57.2 +/- 2.95, P = 0.02) and the supraoptic nucleus (SON, 51.2 +/- 3.21 vs 82.8 +/- 3.08, P = 0.01); the CCK-immunoreactive (CCK-IR) neurons in the hippocampus were of no changes (7.4 +/- 0.87 vs 6.2 +/- 0.58, P = 0.29). After the 14-day GES, the number of CCK-IR neurons in the hippocampus was increased compared with that of the control group (4.0 +/- 0.32 vs 2.4 +/- 0.51, P = 0.03). However, there were no changes in the number of ghrelin-IR neurons either in the PVN or in the SON. CONCLUSIONS: These results indicate that the expression of ghrelin and CCK can be altered by GES. GES may be able to alter energy homeostasis by modulating the expressions of food intake-related hormones in the central nervous system: reducing the level of orexigenic ghrelin acutely and increasing the level of anorexigenic CCK chronically.

Cholecystokinin (CCK) receptor and CCK gene expression in human pituitary adenomas and in vitro effects of CCK peptides on GH and gonadotrophin secretion.

There is growing evidence that cholecystokinin (CCK) affects growth and differentiation of anterior pituitary cells, via the CCK-B receptor. The possibility of an autocrine / paracrine role for CCK to modulate hormone secretion in human pituitary tumour cells is demonstrated here by RT-PCR and direct sequencing. In support of this conclusion, a neutralising antibody against the CCK peptide exhibited a dose dependent inhibition of hormone secretion by functionless pituitary adenomas. Total RNA was extracted from human pituitary adenomas, reverse transcribed into cDNA and subjected to PCR using primers specific for the gene for CCK, CCK-A and CCK-B receptors. PCR bands of the predicted length were observed in all tumours using human CCK gene and CCK-B receptor primers. Restriction digestion and direct sequence analysis provided further evidence that they represented both the human CCK peptide along with the CCK-A and/B receptor mRNA. CCK-33 and CCK octapeptide sulphate (CCK-8s) both powerfully stimulated phosphatidylinositol hydrolysis, providing evidence for functional activity of the CCK-A and/B receptors. A direct stimulatory effect of CCK peptides on both LH and FSH secretion is reported for the first time, whereas stimulatory effects on GH were blocked by antagonists to CCK. These results may indicate an autocrine role for CCK in the functioning and perhaps development of human pituitary tumours.

Immunoelectron study of pancreatic carcinomas using antibodies to gastrointestinal hormones.

The aim of this study was to investigate the ultrastructural appearance of pancreatic adenocarcinoma combined with glucagon and gastrin/cholecystokinin (CCK) expression. The authors investigated the ultrastructure and the immunocytochemistry of 12 human pancreatic cancer specimens and used 3 chronic pancreatitis samples and 6 adjacent histological normal pancreatic tissues (away from the tumor) as controls. The ultrastructural study revealed that chronic pancreatitis tissues were characterized by alterations of the secretory cells. The enzymic and secretory changes were confirmed by electron immunogold results. Glucagon appeared to be located not only in islet alpha cells but also in intermediate alpha acinar cells. The changes were more significant in adenocarcinoma cases. Abnormality in the immunoreaction of the peptides was indicated not only in the tumor area but also in the islets near the cancer. Cells immunoreactive with antibodies were found in all 12 adenocarcinoma cases. Abnormal co-location of both hormones in the same type of endocrine cell was also found. Moderately to poorly differentiated adenocarcinomas were poorly granulated compared with differentiated tumors. Increased and ectopic gastrin/CCK expression was correlated with pancreatic adenocarcinomas exhibiting poor histological grade and neoplastic endocrine cells, providing a potential marker for pancreatic adenocarcinomas with aggressive behavior.

GPR93 activation by protein hydrolysate induces CCK transcription and secretion in STC-1 cells.

In the intestinal lumen, protein hydrolysate increases the transcription and release of cholecystokinin (CCK) from enteroendocrine cells of the duodenal-jejunal mucosa. Our recent discovery that a G protein-coupled receptor, GPR93, is activated by dietary protein hydrolysate causing induced intracellular calcium-mediated signaling events in intestinal epithelial cells raises a possibility that GPR93 might be involved in the protein hydrolysate induction of CCK expression and/or secretion. Using the enteroendocrine STC-1 cells as a model, the present study demonstrates that increasing expression of GPR93 amplifies the peptone induction of endogenous CCK mRNA levels. A similar increase in CCK transcription, indicated by the luciferase reporter activity driven by an 820-bp CCK promoter, is also observed in response to peptone at a dose as little as 6.25 mg/ml, but not to lysophosphatidic acid (LPA), an agonist of GPR93. We discovered that the upregulation of CCK transcription involves ERK1/2, PKA, and calmodulin-dependent protein kinase-mediated pathways. Additionally, GPR93 activation by peptone induces a response in CCK release at 15 min, which continues over a 2-h period. The cAMP level in STC-1 cells overexpressing GPR93 is induced at a greater extent by peptone than by LPA, suggesting a possible explanation of the different effects of peptone and LPA on CCK transcription and secretion. Our data indicate that GPR93 can contribute to the observed induction of CCK expression and secretion by peptone and provide evidence that G protein-coupled receptors can transduce dietary luminal signals.

[Construction of recombinant plasmid pIRES2-EGFP/CCK and its expression in vivo and in vitro].

OBJECTIVE: To construct eukaryotic expression plasmid of porcine CCK gene pIRES2-EGFP/CCK and express it in COS-7 cells and hamsters. Methods The aimed segments were obtained from intermediate vector pMD18-T/CCK and were inserted into an eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid pIRES2-EGFP/CCK. The recombinant expression plasmid was transfected into COS-7 cells by liposome-mediated gene transfer method and was observed through fluorescence microscope. The plasmid was injected into the skeletal muscle of hamsters directly to detect the expression of the recombinant plasmid in vivo. RESULTS: A recombinant eukaryotic expression plasmid pIRES2-EGFP/CCK was successfully constructed. Green fluorescent protein could be detected in the transfected COS-7 cells 24, 48, and 72 hours after the transfection. On the 4th day postinjection into the skeletal muscle of hamsters, the protein could be detected at the injection site and the fluorescence intensity became much stronger on the 14th day than that on the 4th day. On the 42nd day the protein level increased. The green fluorescence protein was never expressed in the untransfected cells. CONCLUSION: The porcine CCK gene eukaryotic expression plasmid pIRES2-EGFP/CCK is constructed successfully, and is expressed in mammal COS-7 cells and hamsters in vivo. The research paves the way for the cross immunity therapy of hamster pancreatic carcinoma.

[Construction of recombinant plasmid pIRES2-EGFP/CCK and its expression in vivo and in vitro].

OBJECTIVE: To construct eukaryotic expression plasmid of porcine CCK gene pIRES2-EGFP/ CCK and express it in COS-7 cells and hamsters. METHODS: The aimed segments were obtained from intermediate vector pMD18-T/CCK by the method of restricted enzymatic resection and were inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid pIRES2-EGFP/CCK. The recombinant expression plasmid was transfected into COS-7 cells by liposome-mediated gene transfer method and observed through Fluorescence microscopy. The plasmid was injected into the skeletal muscle of hamsters directly to detect the expression of the recombinant plasmid in vivo. RESULTS: A recombinant eukaryotic expression plasmid pIRES2-EGFP/CCK was successfully constructed. Green fluorescent protein could be detected in the transfected COS-7 cells 24, 48, and 72 hours post transfection and the expression of green fluorescent protein reached its peak 72 h post transfection. The green fluorescent protein could be detected at the injection site on the 4th day post injection and the fluorescence intensity became stronger on the 14th day. The level of fluorescence became ever stronger on the 42nd day. No expression of green fluorescence was detected in the control group. CONCLUSION: Porcine CCK cDNA eukaryotic expression plasmid pIRES2-EGFP/CCK has been successfully constructed and expressed in mammal cells COS-7 and hamster in vivo. The research paved the way for cross immunity therapy of hamster pancreatic carcinoma.

Clinical endocrinology and metabolism. Cholecystokinin.

Cholecystokinin (CCK) is a peptide hormone discovered in the small intestine. Together with secretin and gastrin, CCK constitutes the classical gut hormone triad. In addition to gallbladder contraction, CCK also regulates pancreatic enzyme secretion and growth, intestinal motility, satiety signalling and the inhibition of gastric acid secretion. CCK is, however, also a transmitter in central and intestinal neurons. Notably, CCK is the most abundant neuropeptide in the human brain. Owing to difficulties in developing accurate assays, knowledge about CCK secretion in disease is limited. Available data indicate, however, that proCCK is expressed in certain neuroendocrine tumours and sarcomas, whereas the secretion of CCK is impaired in celiac disease and bulimia nervosa. Stimulation with exogenous CCK has proved useful in diagnostic tests of gallbladder and pancreatic diseases, as well as medullary thyroid carcinomas.

An immunohistochemical study of endocrine cells in the alimentary tract of the grass lizard, Takydromus wolteri Fischer (Laceridae).

Distribution patterns and the relative frequency of different types of endocrine cells were demonstrated in the alimentary tract of the grass lizard, Takydromus wolteri, using nine specific antibodies raised against mammalian regulatory peptides. The alimentary tract of the lizard was divided into six portions from the esophagus to the rectum. Most endocrine cells were found in the epithelial lining and were generally spindle shaped with long cytoplasmic processes ending in the lumen (open cell type), whereas cells that were spherical in shape (closed cell type) were occasionally found in gastric, esophageal and intestinal glands. Endocrine cells were stained for the following regulatory peptides: bovine Sp-1/chromogranin (BCG), serotonin, somatostatin, gastrin, cholecystokinin (CCK)-8, glucagon, insulin, human pancreatic polypeptide (HPP) and secretin. Cells stained for BCG and serotonin were present throughout the entire gastrointestinal tract and they occurred with the highest frequency in stomach and pylorus, respectively. Somatostatin-positive cells were detected throughout the entire gastrointestinal tract except for the esophagus and large intestine, and were most predominant in pylorus and duodenum. Cells stained for gastrin were restricted to the pylorus and duodenum and occurred with a relatively low frequency. CCK-8-positive cells were observed from pylorus to small intestine and showed the highest frequency in the pylorus. Glucagon- and insulin-containing cells were located in duodenum and small intestine but were found only rarely. HPP-stained cells were detected in duodenum and small intestine with the highest frequency in duodenum. Cells stained for secretin were restricted to duodenum and were found only rarely. In conclusion, distribution patterns and the relative frequency of these endocrine cells correspond well with previous reports on distribution patterns of endocrine cells in reptile species but some deviating patterns were also observed.