Glucose-dependent insulinotropic polypeptide monoclonal antibodies prevent and treat obesity in wild-type and hyperphagic mice.

OBJECTIVE: This study aimed to investigate whether a glucose-dependent insulinotropic polypeptide (GIP) monoclonal antibody (mAb) will promote weight loss in wild-type mice and to determine effects of this mAb in preventing weight gain in ob/ob mice. METHODS: Phosphate-buffered saline (PBS) or GIP mAb was injected intraperitoneally to wild-type mice fed a 60% high-fat diet (HFD). After 12 weeks, mice that received PBS were divided into two groups and were fed a 37% HFD for 5 weeks; one group received PBS, and one group received GIP mAb. In a separate study, PBS or GIP mAb was injected intraperitoneally to ob/ob mice fed normal mouse chow for 8 weeks. RESULTS: PBS-treated mice gained significantly more than those treated with GIP mAb, with no difference in food consumption detected. Obese mice fed a 37% HFD and PBS continued to gain weight (+2.1% ± 0.9%), whereas mice administered GIP mAb lost 4.1% ± 1.4% body weight (p < 0.01). Leptin-deficient mice consumed similar amounts of chow, and, after 8 weeks, the PBS- and GIP mAb-treated mice gained 250.4% ± 9.1% and 192.4% ± 7.3%, respectively (p < 0.01). CONCLUSIONS: These studies support the hypothesis that a reduction in GIP signaling appears to affect body weight without suppressing food intake and might provide a novel, useful method for the treatment and prevention of obesity.

Effects of gastric inhibitory polypeptide (GIP) immunoneutralization on mouse motor coordination and memory.

Gastric inhibitory polypeptide (GIP) is a regulatory peptide expressed in the mammalian upper small intestine, and both GIP and its receptor (GIPR) are expressed in the cortex and hippocampus regions of the brain as well. While learning and memory deficits have been observed in GIPR-/- mice, the effects of peripheral GIP immunoneutralization on motor-coordination, learning, and memory have not been examined. In the present study, adult GIPR-/- mice (KO) and age-matched wild-type C57BL/6 J mice (WT) received weekly vehicle PBS injections for 12 weeks, while a third group of wild-type mice were injected weekly for 12 weeks with 30 mg/kg body weight humanized GIP-mAb (AB) to assess the possibility of long-term effects of peripheral GIP antagonism on rodent memory and behavior. All mice groups then underwent a battery of tests that evaluated motor behavior, body coordination, and memory. Performance deficits in several memory studies after 12 weeks of treatment were demonstrated in KO, but not in AB or WT mice. Body coordination performance showed no significant differences among the 3 groups. A similar short-term study (3 injections over 9 days) was also conducted and the results were similar to those from the long-term study. Thus, short-term and long-term peripheral GIP antagonism by GIP-mAb did not appear to affect learning and memory in mice, consistent with the notion that the GIP-mAb does not cross the blood brain barrier. Furthermore, our studies indicate that GIP signaling in the brain appears to involve local neurocrine pathways.

Gastric inhibitory polypeptide immunoneutralization attenuates development of obesity in mice.

Previous reports have suggested that the abrogation of gastric inhibitory polypeptide (GIP) signaling could be exploited to prevent and treat obesity and obesity-related disorders in humans. This study was designed to determine whether immunoneutralization of GIP, using a newly developed specific monoclonal antibody (mAb), would prevent the development of obesity. Specific mAb directed against the carboxy terminus of mouse GIP was identified, and its effects on the insulin response to oral and to intraperitoneal (ip) glucose and on weight gain were evaluated. Administration of mAb (30 mg/kg body wt, BW) to mice attenuated the insulin response to oral glucose by 70% and completely eliminated the response to ip glucose coadministered with human GIP. Nine-week-old C57BL/6 mice injected with GIP mAbs (60 mg·kg BW(-1)·wk(-1)) for 17 wk gained 46.5% less weight than control mice fed an identical high-fat diet (P < 0.001). No significant differences in the quantity of food consumed were detected between the two treatment groups. Furthermore, magnetic resonance imaging demonstrated that subcutaneous, omental, and hepatic fat were 1.97-, 3.46-, and 2.15-fold, respectively, lower in mAb-treated animals than in controls. Moreover, serum insulin, leptin, total cholesterol (TC), low-density lipoprotein (LDL), and triglycerides were significantly reduced, whereas the high-density lipoprotein (HDL)/TC ratio was 1.25-fold higher in treated animals than in controls. These studies support the hypothesis that a reduction in GIP signaling using a GIP-neutralizing mAb might provide a useful method for the treatment and prevention of obesity and related disorders.

Obesity and the gastrointestinal tract: you are what you eat.

Obesity represents a complex multifactorial syndrome that develops from interactions among genetic and environmental factors and is a leading cause of illness and death. The prevalence of obesity in the United States has increased dramatically since 1975. Although often ignored, the gastrointestinal tract, and the gastrointestinal regulatory peptides in particular, constitutes an ideal starting point for defining and investigating obesity as it represents the route by which all nutrients are ingested, processed, and absorbed. Another important factor to consider when evaluating the etiology of obesity is the capacity for all animals to store nutrients. Insulin is the most potent anabolic hormone, and it appears to have evolved from the need to maximize energy efficiency, obviating the requirement to continuously forage for food. Organisms expressing this important peptide possessed a distinct survival advantage and flourished. During the course of evolution, insulin biosynthesis translocated from the intestine to pancreatic islets, which necessitated a messenger from the intestine to complete the "enteroinsular axis." The eventual development of glucose-dependent insulinotropic polypeptide (GIP) and other incretins fulfilled this requirement. GIP appears to offer an additional survival benefit by not only stimulating intestinal glucose transport and maximally releasing insulin to facilitate nutrient storage but also by its insulin-mimetic properties, including enhanced uptake of glucose by adipocytes. This physiological redundancy offered by insulin and GIP ensured the survival of organisms during times when food was scarce. As food is no longer scarce, at least in the West, this survival advantage appears to have contributed to the current obesity epidemic.

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) 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 (J(ms)(3OMG)), and short-circuit current (I(SC)) 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 I(SC) and J(ms)(3OMG) by 68 and 53%, respectively, indicating that the increase in J(ms)(3OMG) was primarily electrogenic, with a small electroneutral component. The stimulatory effect of GIP on J(ms)(3OMG) was concentration dependent. In addition, 1,000 nmol/l and 10 nmol/l GIP increased J(ms)(3OMG) by 70 and 30% over control, respectively, consistent with receptor activation. Phlorizin (20 mumol/l), an inhibitor of Na(+)-glucose cotransporter (SGLT-1), abolished the increase in I(SC) and decreased J(ms)(3OMG) by approximately 65%. These results indicate that stimulation of SGLT-1 activity by GIP partially accounts for the increase in J(ms)(30MG). 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.

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.

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. RESEARCH METHODS AND PROCEDURES: Isolated rat adipocytes were perifused on a column with 10 nM GIP alone or in combination with 10 microU/mL insulin, 1 microM isoproterenol, or 1 microM ANTGIP. Samples were collected every minute and assayed for FFA, glycerol, and lactate. RESULTS: GIP significantly increased FFA reesterification (decreased FFA release by 25%), stimulated lipolysis (increased glycerol release by 22%), and attenuated the lipolytic response to isoproterenol by 43%. These properties were similar to those of insulin in vitro, suggesting that GIP possesses insulin-like lipogenic effects on adipocytes. Finally, ANTGIP reversed the effects of GIP on both basal and stimulated adipocyte metabolism. DISCUSSION: These studies provide further evidence for an important physiological role for GIP in lipid homeostasis and possibly in the pathogenesis of obesity. They also suggest that the GIP receptor may represent an excellent target for the prevention and treatment of obesity and obesity-related type 2 diabetes.

Sp1/Sp3 binding is associated with cell-specific expression of the glucose-dependent insulinotropic polypeptide receptor gene.

The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor (GIPR) expression has been implicated in abnormal GIP responses associated with type 2 diabetes mellitus and food-induced Cushing's syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between -1 and -100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5'-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 bp. Subsequent serial deletion analyses showed that the region between -85 and -40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions -77, -60, and -50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at -50 and -60 led to 36 and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at -70 did not affect promoter activity. Cotransfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long form of Sp3 activate transcription through binding to the Sp1 sites located between -100 and -40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cell-specific expression of GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter.

Cell-specific expression of glucose-dependent-insulinotropic polypeptide is regulated by the transcription factor PDX-1.

Glucose-dependent insulinotropic polypeptide (GIP) is a potent stimulator of insulin secretion and comprises an important component of the enteroinsular axis. GIP is synthesized in enteroendocrine K-cells located principally in the upper small intestine. The homeobox-containing gene PDX-1 is also expressed in the small intestine and plays a critical role in pancreatic development and in the expression of pancreatic-specific genes. Previous studies determined that the transcription factors GATA-4 and ISL-1 are important for GIP expression. In this study, we demonstrate that PDX-1 is also involved in regulating GIP expression in K-cells. Using immunohistochemistry, we verified the expression of PDX-1 protein in the nucleus of GIP-expressing mouse K-cells and evaluated the expression of PDX-1, serotonin, and GIP in wild-type and PDX-1(-/-) mice at 18.5 d after conception. Although we demonstrated a 97.8% reduction in the number of GIP-expressing cells in PDX-1(-/-) mice; there was no statistical difference in the number of serotonin-positive cells. Additionally, PDX-1 transcripts and protein were detected in a GIP-expressing neuroendocrine cell line, STC-1. Electromobility shift assays using STC-1 nuclear extracts demonstrated the specific binding of PDX-1 protein to a specific regulatory region in the GIP promoter. Using chromatin immunoprecipitation analysis, we demonstrated binding of PDX-1 to this same region of the GIP promoter in intact cells. Lastly, overexpression of PDX-1 in transient transfection assays led to a specific increase in the activity of GIP/Luc reporter constructs. The results of these studies indicate that the transcription factor PDX-1 plays a critical role 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 Barrett's 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.

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.