Michael O. Boylan

Glucose-Dependent Insulinotropic Polypeptide Modulates Adipocyte Lipolysis and Reesterification

Objective: Glucose‐dependent insulinotropic polypeptide (GIP) is an incretin released from intestinal K‐cells during the postprandial period. Previous studies have suggested that GIP may play an etiologic role in obesity; thus, the GIP receptor may represent a target for anti‐obesity drugs. The present studies were conducted to elucidate mechanisms by which GIP might promote obesity by examining the effect of GIP on both glycerol release (indicative of lipolysis) and free fatty acid (FFA) release (indicative of both lipolysis and reesterification), as well as the ability of a GIP‐specific receptor antagonist (ANTGIP) to attenuate these effects.

Glucose‐dependent Insulinotropic Polypeptide (GIP) Stimulates Transepithelial Glucose Transport

The purpose of this study was to characterize the effects of glucose‐dependent insulinotropic peptide (GIP) on small intestinal glucose transport in vitro. Stripped proximal jejunum from fasted mice was mounted in Ussing chambers. The serosal side was bathed in Regular Ringer solution containing 5 mmol/l glucose, and the mucosal side, with solution containing 10 mmol/l 3‐O‐methyl glucose (3OMG). Intercellular cyclic adenosine monophosphate (cAMP), mucosa‐to‐serosa fluxes of 3OMG (Jms3OMG), and short‐circuit current (ISC) were measured in the presence and absence of GIP. GIP increased cAMP by 2.5‐fold in isolated enterocytes, consistent with a direct effect of GIP on these epithelial cells. GIP also increased ISC and Jms3OMG by 68 and 53%, respectively, indicating that the increase in Jms3OMG was primarily electrogenic, with a small electroneutral component. The stimulatory effect of GIP on Jms3OMG was concentration dependent. In addition, 1,000 nmol/l and 10 nmol/l GIP increased Jms3OMG by 70 and 30% over control, respectively, consistent with receptor activation. Phlorizin (20 μmol/l), an inhibitor of Na+‐glucose cotransporter (SGLT‐1), abolished the increase in ISC and decreased Jms3OMG by ∼65%. These results indicate that stimulation of SGLT‐1 activity by GIP partially accounts for the increase in Jms30MG. These studies are the first to demonstrate direct stimulation of intestinal glucose transport by GIP independent of its insulinotropic properties. GIP stimulates cellular accumulation of cAMP and thereby upregulates glucose transport. The GIP‐induced increase in glucose transport appears to be mediated, at least in part, by SGLT‐1.

Cell-specific expression of the glucose-dependent insulinotropic polypeptide gene functions through a GATA and an ISL-1 motif in a mouse neuroendocrine tumor cell line

BACKGROUND/AIMS Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that, in the presence of glucose, stimulates insulin secretion from beta-cells. GIP is expressed in gastrointestinal K-cells. Prior analysis of the GIP promoter demonstrated that 193 bases of the promoter are required to direct cell specific expression. Here we sought to identify and characterize the transcription factors involved. RESULTS By mutational analysis of the GIP promoter in a neuroendocrine cell line (STC-1), we identified two regions located between bases -193 and -182 and bases -156 and -151 that, when independently altered, were responsible for a 90% and 85% reduction in transcription, respectively. When we compared these two regions with known motifs from transcription factor databases, we identified the cis elements as potential GATA and ISL-1 binding sites. With subsequent electrophoretic mobility shift analysis (EMSA) using STC-1 nuclear extracts, we demonstrated the ability of these regions to form specific DNA protein complexes. Furthermore, we utilized antisera to confirm the specific binding of GATA-4 to the upstream site and ISL-1 to the downstream element. CONCLUSION These findings provide evidence for the involvement of the transcription factors GATA-4 and ISL-1 in the cell-specific expression of the GIP gene.

Gastrin stimulates receptor-mediated proliferation of human esophageal adenocarcinoma cells

The prevalence of esophageal adenocarcinoma in the setting of Barretts metaplasia continues to increase in Western nations at a rate greater than any other cancer. The trophic properties of gastrin have been documented in gastric, pancreatic and colon cancer cell lines, suggesting a potential role for this regulatory peptide in the growth of these malignancies. The aims of these studies were to identify and characterize the presence of functional cholecystokinin type-2 (gastrin) receptors on the membranes of human esophageal adenocarcinoma cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated the presence of cholecystokinin type-2 receptor transcripts in human esophageal adenocarcinoma cell lines. Competitive binding assays revealed specific binding of gastrin in SEG-1 cells (IC50 of 2.4 x 10(-8) M). This finding was confirmed by laser scanning confocal microscopy through internalization of rhodamine green labeled gastrin heptapeptide in SEG-1 cells. Gastrin caused a dose-dependent increase in proliferation of SEG-1 cells when compared to controls. This effect was abolished by co-incubation with L365,260, a CCK-2-specific receptor antagonist. Gastrin-induced phosphorylation of the p44 and p42 mitogen-activated protein kinases was demonstrated by Western blot analysis. In conclusion, the studied human esophageal adenocarcinoma cell lines possess cholecystokinin type-2 (gastrin) receptors. Receptors bind gastrin, resulting in increased proliferation in SEG-1 cells.

GLUCOSE-DEPENDENT INSULINOTROPIC PEPTIDE (GIP) GENE EXPRESSION IN THE RAT SALIVARY GLAND

Previous studies have indicated that following nutrient ingestion, GIP is released principally from the upper small intestine. In addition to its presence in the rat small intestine, GIP transcripts have also been localized to the submandibular salivary gland (SSG). The present studies were directed to further characterize expression of the GIP gene in the SSG. Pregnant rats were sacrificed at gestational days 18 and 20, followed by the removal of rat fetuses. The duodenum pancreas, and SSG were then excised from the fetuses, as well as from neonatal pups at ages 1, 3, 7, 10, 14, and 21 days. RNA was extracted and measured by Northern blot analysis using specific rat GIP probes. GIP transcripts were first detected in the duodenum in the 18-day fetus and reached maximum levels at birth. In contrast, GIP mRNA was not observed in the SSG until 10 days postnatally and was not detected at all in either the fetal or neonatal pancreas. In situ hybridization of the SSG using an 35S-labelled antisense GIP RNA probe demonstrated expression of the GIP gene to be limited to ductal cells, with no transcripts present in acini. In separate experiments, rats fasted overnight were given water or 10% glucose. While no changes were detected in water-fed rats following oral glucose ingestion, small, but significant increases in SSG GIP gene expression were detected at 60 and 240 min. The results of these initial studies suggest the possibility of a functional role for GIP in the rat salivary gland by the demonstration of GIP mRNA in the SSG by Northern analysis and in situ hybridization, as well as by an increase in SSG GIP gene expression following a glucose meal.

GATA-4 upregulates glucose-dependent insulinotropic polypeptide expression in cells of pancreatic and intestinal lineage

A thorough examination of glucose-dependent insulinotropic polypeptide (GIP) expression has been hampered by difficulty in isolating widely dispersed, GIP-producing enteroendocrine K-cells. To elucidate the molecular mechanisms governing the regulation of GIP expression, 14 intestinal and pancreatic cell lines were assessed for their suitability for studies examining GIP expression. Both STC-1 cells and the pancreatic cell line betaTC-3 were found to express GIP mRNA and secrete biologically active GIP. However, levels of GIP mRNA and bioactive peptide and the activity of transfected GIP reporter constructs were significantly lower in betaTC-3 than STC-1 cells. When betaTC-3 cells were analyzed for transcription factors known to be important for GIP expression, PDX-1 and ISL-1, but not GATA-4, were detected. Double staining for GIP-1 and GATA-4 in mouse duodenum demonstrated GATA-4 expression in intestinal K-cells. Exogenous expression of GATA-4 in betaTC-3 cells led to marked increases in both GIP transcription and secretion. Lastly suppression of GATA-4 via RNA interference, in GTC-1 cells, a subpopulation of STC-1 cells with high endogenous GIP expression resulted in a marked an attenuation of GIP promoter activity. Our data support the hypothesis that GATA-4 may function to augment or enhance GIP expression rather than act as an initiator of GIP transcription.

Microscale sample preparation for PCR of C. difficile infected stool.

In this paper, we describe the design of a microfluidic sample preparation chip for human stool samples infected with Clostridium difficile. We established a polymerase chain reaction able to distinguish C. difficile in the presence of several other organisms found in the normal intestinal flora. A protocol for on-chip extraction of nucleic acids from clinical samples is described that can detect target DNA down to 5.0x10(-3) ng of template. The assay and sample preparation chip were then validated using known positive and known negative clinical samples. The work presented has potential applications in both the developed and developing world.

Glucose-Dependent Insulinotropic Polypeptide (GIP)

The term “enterogastrone” was proposed by Kosaka and Lim (1) in 1930 to describe a putative hormone that was secreted in response to fat or its digestive products in the intestinal lumen and inhibited gastric acid secretion. Two candidate intestinal hormones apparently satisfying these requirements were secretin, which was isolated because of its ability to stimulate pancreatic secretion (2) and cholecystokinin (CCK) (3), a potent stimulant of gallbladder contraction. Impure preparations of both were found to have acid inhibitory activity in the vagally denervated (Heidenhain) canine gastric pouch (4). However, in contrast to the acid inhibitory effects of impure CCK, Magee and Nakamura (5) observed that intravenously administered CCK preparations stimulated acid secretion under fasting conditions. Brown and Pederson (6) tested the hypothesis that the gastric effects were caused by factors other than CCK present in the impure preparations of hog upper small intestinal mucosa. The acid secretory effects of two different preparations of CCK (designated 10 and 40% pure, respectively, on the basis of gallbladder stimulating potency) were compared in dogs prepared with Heidenhain pouches. The 40% pure preparation produced a greater stimulatory effect on acid secretion than the 10% pure preparation at doses that yielded comparable gallbladder activity. It was proposed that either a gastric stimulant had been concentrated or an inhibitor of acid secretion had been removed during the purification process. The latter hypothesis was further pursued.

Engineering of Murine Embryonic Stem Cells to Express and Release Human Growth Hormone in a Murine Model

Background: Growth factors including EGF have emerged as potential therapeutic options for patients with surgical short gut syndrome. The potential augmentation of the adaptive response by EGF makes it an appealing therapy to promote intestinal autonomy. Crypt epithelium responds to small bowel (SB) resection with increased proliferation and fissioning. EGF augments the intestinal epithelial cell (IEC) responses to intestinal resection although its role in regulating β-catenin is unclear. To examine the potential that EGF-induced PI3K mediates the epithelial response to β-catenin activation in ICR, we utilized an inducible genetic model of intestinal epithelial specific deletion of Class IA PI3K p85α AhCre/pi3kr1 ). Our laboratory demonstrated cross-talk between PI3K and the Wnt/β-catenin signaling pathways in inflammatory bowel disease tissue. Previous data from Clevers and colleagues showed that the proliferative responses originate in intestinal stem cells (ISC) and are dependent on Wnt/β-catenin signaling. Here we test the notion that EGF stimulates IEC via β-catenin activation after ICR through PI3K pathway. Design/Methods: C57BL/6 wild type (WT) mice and AhCre/pi3kr1 animals underwent sham surgery, ICR, or no surgery. All animals were given 50 ug/kg/day of rEGF in osmotic pumps immediately after the surgery. Five days later, SB morphometrics, immunohistochemistry (IHC) for BrdU incorporation (2hr), phospho(p) AKT(Thr308), and pBcatenin(ser552) and western blots of purified IEC proteins including cleaved caspase-3 and nuclear total β-catenin were performed. Results: Compared to EGF-treated sham, EGF-treated ICR mice have increased crypt length (63.3 ± 7 vs 118.3 ±2 8.9 μm, p< 0.05), BrdU incorporation, pAKT(Thr308), pβ-catenin(ser552) by IHC, and nuclear total β-catenin (WB). Data on AhCre/pi3kr1 ICR mice revealed reduced crypt length (118.3±28.9 vs. 67.1 ± 15.5 μm, p< 0.05 ), incorporation of pBcatenin(ser552) (1.43 ± 0.58 vs. 0.85 ± 0.19 counts/crypt, p< 0.05), and a 90% reduction of nuclear total β-catenin by WB. Interestingly, BrdU incorporation in AhCre/pi3kr1 ICR mice was similar to EGF-treated ICR alone, whereas cleaved caspase-3 (WB) doubled, suggesting enhanced apoptosis attenuated the observed induction of IEC proliferation. Conclusions: These data indicate that the epithelial response to ICR in EGF-treated mice enhanced crypt length and IEC proliferation along with PI3K and β-catenin signaling. Furthermore, data indicate that PI3K enhanced IEC crypt lengths, β-catenin activation, and cell survival. Thus, we propose that enhanced IEC PI3K signaling in short bowel patients may facilitate the recovery of absorptive surface through epithelial restitution.