PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Regul Pept. Author manuscript; available in PMC 2011 August 9.
Published in final edited form as:
PMCID: PMC2902700
NIHMSID: NIHMS207750

GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE STIMULATES THE PROLIFERATION OF COLORECTAL CANCER CELLS

Abstract

Although numerous epidemiological studies have provided convincing evidence for an increase in the prevalence of colorectal cancer (CRC) in obese individuals, the precise mechanisms involved have not been elucidated. Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal regulatory peptide whose primary physiologic role is to stimulate postprandial pancreatic insulin secretion. Like insulin, GIP has been linked to enhanced nutrient efficiency, which occurred during the course of evolution. Its expression is increased in obesity, and we thus initiated studies to examine whether GIP might contribute to the pathogenesis of obesity-related CRC. RT-PCR and Western analysis demonstrated the presence of the GIP receptor (GIPR) in several human CRC cell lines. GIP stimulated the proliferation of MC-26 cells, a mouse CRC cell line, in a concentration-dependent manner. Western analysis showed that GIP induced the activity of several downstream signaling molecules known to be involved in cellular proliferation in a concentration- and time-dependent manner. These studies indicate that the presence of GIP receptors in CRC may enable ligand binding and, in so doing, stimulate CRC cell proliferation. The overexpression of GIP, which occurs in obesity, might thereby be contributing to the enhanced rate of carcinogenesis observed in obesity.

Keywords: glucose-dependent insulinotropic polypeptide, GIP, obesity, colorectal cancer, Akt, mTOR

INTRODUCTION

Despite significant advances in radiotherapy, chemotherapy, and surgery during the past two decades, colorectal cancer (CRC) remains the third most common cause of cancer-related death in the United States, accounting for 10% of all deaths due to malignancy (1). The American Cancer Society estimates that in 2009, approximately 147,000 Americans will be diagnosed with CRC, of whom nearly 50,000 will die. The magnitude of this burden on the American health system emphasizes the importance of elucidating the basic mechanisms underlying the disease to permit the design of optimal treatment modalities.

The development of CRC appears to involve a multi-step process of genetic mutations combined with environmental factors, whereby normal epithelial cells undergo dysplastic transformation, followed by proliferation and histological progression to neoplasia (2,3). One such factor is obesity, which represents a global epidemic and is a leading cause of illness and death worldwide (4). An estimated 100 million adults in the United States are considered obese (5), which substantially raises their risk of morbidity. In addition to cosmetic effects, obesity is a known risk factor in the pathogenesis of type 2 diabetes mellitus, hypertension, pulmonary dysfunction, vascular diseases, gastrointestinal (GI) and biliary disorders, osteoarthritis, and various malignancies, including CRC. Although the precise relationship between CRC and obesity has not been elucidated, the American Cancer Society has estimated that obese individuals possess approximately a 1.5-2.0 relative risk for the development of CRC compared with those of normal weight, and that 20.8% and 35.4% of CRC cases may be attributable to weight in women and men, respectively.

Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid peptide hormone synthesized in, and released from, K-cells of the duodenum and jejunum after the ingestion of a meal. Upon its release, GIP induces insulin release from pancreatic islet β-cells, thus acting as the principal mediator of the enteroinsular axis and as a hormone that contributes to optimization of nutrient deposition (7). GIP is overexpressed in obese animal models and humans, and an etiologic role for GIP in the development of obesity has accordingly been suggested (8-12).

The present studies were conducted to assess the possibility that enhanced GIP expression, which occurs in obesity, might contribute to the pathogenesis of obesity-related carcinogenesis, and to investigate the intracellular mechanisms that might mediate GIP-induced CRC tumor growth. After first demonstrating the presence of the GIP receptor (GIPR) in CRC cells, we showed that GIP stimulated the proliferation of MC-26 cells, a mouse CRC cell line. Western analysis demonstrated that GIP induced several downstream signaling molecules known to be involved in cellular proliferation. These studies indicate that the presence of the GIPR in CRC may enable ligand binding and, in so doing, stimulate CRC cell proliferation. GIP overexpression might thereby be contributing to the enhanced rate of carcinogenesis observed in obesity.

MATERIALS AND METHODS

Antibodies and cells

Rabbit anti-GIPR primary antibody was kindly provided by Dr. Joel Habener (Massachusetts General Hospital, Boston, MA). Other primary antibodies were purchased from Cell Signaling Technologies (Danvers, MA): rabbit anti-Phospho-AKT (Ser 473), rabbit anti-AKT antibody, rabbit anti-phospho-p70 S6 kinase (Thr 389), and rabbit anti-Phospho-p44/42 MAP Kinase antibody (Thr 202/204). Monoclonal β-actin primary antibody was purchased from Sigma (St. Louis, MO). The mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD98059 and PI3 kinase inhibitor Wortmannin were purchased from Calbiochem (San Diego, CA) and Roche (Indianapolis, IN), respectively.

MC-26 cells, a transplantable mouse CRC cell line, was obtained from Dr. K.K. Tanabe (Massachusetts General Hospital, Boston, MA), and HT29 cells, human CRC cell lines, were purchased from American Type Culture Collection (Manassas, VA). Cells were grown in Dulbecco's modified Eagle's Medium (DMEM; Cellgro Mediatech, Manassas, VA) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT), 100 U/ml penicillin G, and 100 μg/ml streptomycin at 37° C in a humidified atmosphere of 5% CO2.

Immunohistochemistry

Paraffin embedded blocks containing clinical colon biopsy specimens from four patients with stage 2-4 CRC were obtained from the Biospecimen Archive Research Core at Boston University School of Medicine and cut into 7-μm sections. Deparaffinized and rehydrated slides were subjected to antigen retrieval by steaming in a 10-mM citric acid buffer (pH 6.0) at 100 °C for 2 0 min and then were allowed to cool for 20 min at room temperature (RT). Slides were washed in phosphate-buffered saline (PBS) and then blocked with 5% normal donkey serum for 60 min at RT. The primary antibody, rabbit anti-GIPR (Abcam, Cambridge MA) was diluted 1:200 in PBS containing 1% bovine serum albumin (BSA) and incubated with the sections overnight at 4 °C. Slides were washed in PBS and incubated wi th Alexa Fluor 594 goat anti-rabbit secondary antibodies (Invitrogen, Carlsbad, CA) diluted 1:250 in PBS containing 1% BSA for 1 h at RT. Slides were washed in PBS and the nuclei were then counterstained with 4',6-diamidino-2-phenylindole (DAPI) and the sections mounted using UltraCruz mounting medium (Santa Cruz Biotechnology, Santa Cruz, CA). Sections were examined using a Nikon Deconvolution Wide-field Epiflourescence microscope. TIFF images were processed in Adobe Photoshop.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis

RT-PCR was used to determine the relative expression levels of GIPR. Total RNA was extracted from MC-26 and HT29 cells using the Qiagen RNeasy kit (Qiagen Inc., Valencia, CA). For amplification, starting with 2 μg of RNA as template, single-stranded cDNA was synthesized from total RNA using an oligo (dT) primer (50 pmol) and ThermoScript RT-PCR system (Invitrogen). The single-stranded cDNA was then amplified using published human gene-specific primer sequences for GIPR or for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The GIPR 5′ forward primer was (5′-CTG CCT GCC GCA CGG CCC AGA T-3′), and the 3′ reverse primer (5′-GCG AGC CAG CCT CAG CCG GTA A-3′). For amplification of GAPDH cDNA, the forward primer was (5’-GGG GAG CCA AAA GGG TCA TCA TCT-3’), and the 3’ reverse primer was (5’- GAC GCC TGC TTC ACC ACC TTC TTG-3’).

The PCR reaction was carried out in a total volume of 50 μl and consisted of PCR mix (50 mM KCl, 10 mM/Tris–HCl, 1.5 mM MgCl2, 0.2 mM dNTP's and 25 U Taq DNA polymerase, final pH 8.3; Roche, Mannheim, Germany). The amplification reaction involved denaturation at 94 °C for 2 min, followed by cycling as follows: 94 °C for 1 min, primer annealing at 53 °C for 1 min and extension at 72 °C for 2 min. After cycling, terminal elongation of 5 min at 72 °C was performed. Amplification required 30 cycles for GAPDH and 35 cycles for GIPR. PCR products were analyzed by electrophoresis on a 1% agarose gel with ethidium bromide staining and photographed under UV transillumination.

Western blot analysis

Western blot hybridization analysis was performed using previously published methods (7). Immunoreactive protein bands were identified by protein size using known molecular weight standards provided by Bio-Rad Kaleidoscope Prestained Standards (Hercules, CA, USA). Ponceau S (Sigma) staining, and immunoblots with monoclonal β-actin antibody were used to confirm equal loading of Western blot membranes. Audioradiographs were scanned with a Bio-Rad GS 700 Imaging Densitometer (Bio-Rad), and photodensitometric analysis was performed with the NIH ImageJ program.

In-vitro cell proliferation assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay]

Cell proliferation was assessed using a CellTiter 96 AQ One Solution Cell Proliferation Assay kit (Promega, Madison, WI). MC-26 cells at a density of 3×103 cells per well were plated onto a 96-well microtiter plate and incubated in DMEM with 10% FBS overnight. The FBS containing medium was replaced with serum free medium the following day and incubated for 24 h. To determine the effect of GIP on proliferation, cells were treated in triplicate with increasing concentrations of porcine GIP for 24, 48, 72, and 96 h. At the end of each experiment, 20 μL of CellTiter96 Aqueous solution Reagent were added to each well, and the plate was incubated for 1 h at 37 °C in a humidified 5% CO2 atmosphere. The absorbance at 490 nm was then recorded using a 96-well ELx800 universal microplate reader (BIO-TEK Instruments, Winooski, VT).

Cyclic Adenosine Monophosphate (cAMP) measurement

Intracellular cAMP was measured using a cAMP HTS Chemiluminescent Immunoassay Kit (Millipore, Billerica, MA). MC-26 cells were seeded at 80% confluency on a 48-well plate in DMEM containing 10% FBS for 24 h for cells to attach, after which they were incubated in serum free DMEM media for an additional 24 h. Cells were pretreated for I h with a 1 x isobutyl-1-methylxanthine (IBMX) and then treated with porcine GIP in DMEM for 10 min. At that time, media were removed, and 200 μL of cold lysis buffer were added to each well and incubated for 10 min at room temperature. Experiments were performed in triplicate on 96-well anti-rabbit coated assay plates, using 50 :l of each sample to assay. The assay was performed in accordance with the manufacturer's instructions, and the plates were read for 1 sec with a POLARstar OPTIMA luminometer (MG LABTECH, Durham, NC).

Akt, ERK1/2 pathways

To investigate the effects of GIP on Akt and ERK1/2 activation, MC-26 and HT29 cells were serum-starved overnight. Triplicate wells of cells were then incubated in the absence or presence of 100 nM GIP, with and without pretreatment with 20 nM rapamycin, 1 μM Wortmannin, or 40 μM PD98059 for 30 min. Whole cell lysates were extracted and Western analysis was performed using specific antibodies to total Akt, phosphorylated ERK1 and ERK2 (pERK1/2), or p70S6K, or a phospho-specific antibody that recognizes serine 473 phosphorylation of Akt (pAkt).

Statistical analysis

The two-way Student's T-test was performed for paired comparisons. Statistical significance was assigned if P < 0.05.

RESULTS

Presence of the GIPR in CRC and in CRC cell lines

An immunohistochemical analysis of all four paraffin-embedded CRC specimens demonstrated strongly positive staining for the GIPR; representative slides from one patient with advanced CRC are shown in Fig. 1. Total RNA was extracted from different CRC cell lines, after which cDNA was synthesized and amplified by PCR using GIPR-specific primers. As shown in Fig. 2A, GIPR transcripts were detected in both the control (HEK 293-T cells transfected with the GIPR) and two CRC cell lines. Cellular lysates were then extracted and assessed by Western analysis for the expression of the GIPR using rabbit anti-GIP receptor specific antibodies. As seen in Fig 2B, GIP receptor protein was present in both MC-26 cells and HT29 cells.

Figure 1
Immunohistochemical demonstration of GIP receptors in human CRC
Figure 2
Expression of GIP receptors in mouse and human CRC and effects of GIP on cell proliferation

GIP stimulates proliferation of CRC lines expressing the GIPR

We next examined the effects of GIP on cell proliferation. MC-26 cells were incubated with various concentrations of GIP for 24-96 h, and cell proliferation was measured using MTT assays. GIP induced a concentration-dependent increase in cell proliferation at all time points examined (Fig. 2C), with maximum levels measured using 100 nM GIP, during which proliferation increased by 29.8% over basal levels (P<0.001).

Activation of the GIPR increases intracellular cAMP

Previous studies using various cell lines have demonstrated that GIP signals through adenylyl cyclase, thereby inducing the generation and accumulation of intracellular cAMP (13). Thus, to determine whether GIP likewise induces signaling through this pathway in CRC cells, cAMP assays were performed using a specific ELISA to measure the intracellular cAMP concentration in MC-26 cells. As shown in Fig. 3, intracellular cAMP concentration increased in response to GIP stimulation at 10 min in comparison to untreated cells. These results indicate that GIP may exert its effects in CRC cells by activating adenylyl cyclase and, in turn, increasing the intracellular generation of cAMP.

Figure 3
Intracellular cAMP accumulation in MC-26 cells in response to GIP

GIP signals through PI3 Kinase/Akt pathway and its downstream components in CRC cells

Akt plays a central role in mediating critical cellular responses, including cell growth and survival, angiogenesis, and transcriptional regulation (7,14). GIP promoted Akt phosphorylation in a concentration-dependent manner. After 30 min of incubation, phospho-Akt levels increased by ~3.3-fold in cells treated with 100 nM GIP, when compared to control (P<0.01, Fig. 4 A,B). The effects of GIP on Akt phosphorylation were attenuated by pretreatment of cells for 30 min with 1 μM Wortmannin, and observed results were similar in HT29 cells (Data not shown).

Figure 4
Enhanced signaling of Akt and its downstream targets induced by GIP in MC-26 cells

As discussed above, past studies have demonstrated that Akt phosphorylation may activate the mTOR pathway, which is a protein kinase that promotes protein synthesis and cell growth (15,16). Because GIP promoted Akt activation in MC-26 cells, we next examined whether mTOR might also play a role in GIP-induced CRC cell proliferation. Cells were pretreated with 20 nM of rapamycin, a known mTOR inhibitor, for 30 min prior to adding 100 nM GIP for an additional 30 min. Cell lysates were then prepared, and Western analysis was performed to measure the phosphorylation of p70S6K, a principal component of the mTOR pathway. GIP promoted p70S6K phosphorylation (Fig. 4C), which was abolished in the presence of rapamycin in both MC-26 and HT29 cells (latter not shown).

We next examined whether Akt activated by GIP could, in turn, activate any of its many substrates. Western analysis was performed using a phospho-Akt substrate-specific antibody that recognizes proteins phosphorylated by Akt exclusively at a conserved serine/threonine motif, which includes an arginine residue at either the –5 or –3 position. GIP activated the substrate-specific for Akt in a concentration-dependent manner in MC-26 cells (Fig. 4D).

GIP activates MAPK pathway

Because previous studies have demonstrated that GIP induces a signaling cascade involving the MAPK pathway in pancreatic islet β-cells (17), we next performed studies to determine whether this pathway might likewise mediate the effects of GIP on CRC cells. Utilizing an antibody specific for the phosphorylated forms of MAPK extracellular signal-regulated protein kinases, ERK1 and ERK2, Western blot analysis was performed on protein extracted from MC-26 cells incubated in the presence of increasing concentrations of GIP for 30 min. A concentration-dependent increase in phosphorylated ERK1 and ERK2 was detected in M-26 cells treated with GIP for 30 min (Fig. 5), with a maximum relative increase of ~6.3-fold detected with 100 nM GIP (P<0.05). The effects of GIP on the levels of ERK1/2 phosphorylation were nearly abolished by the preincubation of cells for 30 min with 40 μM of the MEK inhibitor PD98059.

Figure 5
Increased phosphorylation of MAPK species ERK1 and ERK2 in MC-26 cells treated with GIP

Proposed Model of GIP action on CRC

As depicted in Fig. 6, the results of our studies indicate that GIP appears to function through multiple interrelated pathways that lead to cell growth. GIP appears to bind to its G-protein coupled receptor, which then activates multiple intracellular proteins, such as the cAMP and MAPK pathways. In addition, GIP activates PI3K/Akt, a major growth stimulating pathway, as well as its downstream signals, as evident by its effects on mTOR components and proteins phosphorylated by Akt exclusively at a conserved serine/threonine motif.

Figure 6
Proposed model for GIP signaling in CRC

DISCUSSION

Despite its original description as an inhibitor of gastric acid secretion (18), the primary biological property of GIP appears to be as a mediator of the enteroinsular axis, whereby the peptide stimulates the release of insulin from pancreatic islet β-cells following the ingestion of glucose and fat. The existence of a chemical stimulant of the endocrine pancreas had been suggested by Moore et al. in 1906 (19), who coined the term “incretin” as an insulinotropic substance emanating from small intestine released into the circulation following glucose-containing meals. Of the several GI peptides that have been proposed as candidates, only glucagon-like peptide-1 (GLP-1) and GIP have been shown to function as physiological incretins (20,21).

As mentioned above, several previous reports have suggested an etiologic role for GIP in the development of obesity. Postprandial GIP secretion has been shown to be increased in obese subjects compared with age-matched healthy controls (22). Animal models of obesity, represented by leptin-deficient (ob/ob) mice, have also demonstrated that the concentrations of plasma GIP are enhanced when chronically fed a high-fat diet. In addition, diets high in fat content induced K-cell hyperplasia in these mice (23). Miyawaki et al. (12) demonstrated that while normal mice became obese and developed insulin resistance and T2DM in response to a high-fat diet, GIPR-deficient (GIPR-/-) mice were protected and remained normal while consuming the identical diet. More recently, Althage et al. (24) used regulatory elements for the rat GIP promoter to express an attenuated Diphtheria toxin in transgenic mice. K-cell number, GIP transcripts, and plasma GIP levels were all profoundly diminished, and body weight was reduced by 25% in the transgenic mice.

Although a relationship between obesity and cancer had long been suspected (25), Calle et al. (6) conducted a prospective analysis of 900,000 American adults, who were free of cancer at the outset of the study. During the ensuing 16 years, 6.3% (57,145) died from cancer-related illness. From their extensive analysis, they estimated that cancer-related deaths in 14% and 20% of men and women, respectively, were associated with excess weight. The relative risk of mortality due to CRC increased directly with the body-mass index (BMI): 1.20 for overweight male individuals (BMI of 25.0-29.9), 1.47 for men with functional class I obesity (BMI of 30.0-34.9), and 1.84 for men with a BMI of 35.0-39.9. The risk of CRC-related death was somewhat diminished in women compared with men. The relative risk of mortality due to CRC was 1.36 and 1.46 for women with a BMI of 35.0-39.9 and >40, respectively.

The cause of obesity-related CRC is likely multifactorial, and previous studies have suggested that insulin-like peptides likely represent one of the principal biochemical mediators (26-29). The precise mechanisms by which insulin and insulin-like growth factors might contribute to the development of CRC have not been elucidated. Upon binding to the receptor, insulin activates its receptor's intrinsic kinase, leading to autophosphorylation and tyrosine phosphorylation of several substrates, including members of the insulin receptor substrate (IRS) family (30). IRS phosphorylation, in turn, recruits other signaling molecules, including PI3K (30). One of the downstream targets of PI3K is protein kinase B (Akt), and its activation causes a cascade of cellular responses (31). Akt appears to contribute to the development of carcinogenesis via several downstream pathways, including attenuation of apoptosis and stimulation of cell proliferation (32).

We have previously reported that, like insulin, GIP induces the activation of the Akt in adipocytes via a wortmannin-sensitive pathway, which, in turn, promoted fat cell membrane GLUT-4 accumulation and enhanced [3H]-2-deoxyglucose uptake (7). The results of the present studies provide further evidence for the insulin mimetic properties of GIP. After demonstrating the presence of the GIPR in human and mouse CRC cell lines, we found that GIP stimulated the proliferation of the cells in a concentration-dependent manner. We next showed that GIP promoted Akt phosphorylation in a concentration-dependent manner (Figs. 4A and 4B) and activated the substrate-specific for Akt in a concentration-dependent manner (Fig. 4D).

In addition to its effects on Akt activation, GIP treatment of CRC cells also promoted the phosphorylation of p70S6K, a principal component of the mTOR pathway, which was abolished in the presence of rapamycin (Fig. 4C). Emerging evidence suggests that mTOR plays a key role in several pathways that are involved in human cancer (15), as well as obesity and its sequelae (33,34). Two mTOR complexes, mTORC1 and mTORC2, have been described, each possessing distinct features, including different combinations of interacting components and variable sensitivity to rapamycin; mTORC1, but not mTORC2, is rapamycin sensitive (35). One of the principal downstream targets of mTORC1 is p70S6K, and the p70S6K through phosphorylation represent important events involved in the regulation of protein synthesis.

The notion that GIP might possess proliferative properties has been reported in studies employing β-cells and surrogate insulin-secreting cell lines. GIP has been shown to activate mitogenic signaling, such as the mitogen-activated protein kinase (MAPK) and PI3K/Akt pathways, in β-cells (15-17). Akt is a major mediator of GIP action on pancreatic islets, increasing β-cell mass and function and promoting β-cell survival (36). Trδmper et al. (37) reported that GIP and glucose acted synergistically as anti-apoptotic factors in the well-differentiated β-cell line INS-1 and involved the activation of several pathways, including adenylyl cyclase, MAPK, and PI3K/Akt. Similarly, Kim et al. (13) reported that GIP stimulated the anti-apoptotic Bcl-2 gene through the adenylyl cyclase pathway in INS-1 cells. Using this same cell line, they later showed that GIP regulated β-cell apoptosis by down-regulating the pro-apoptotic bax gene and up-regulating Bcl-2 via the activation of PI3K (38). Down-regulation of the bax gene via PI3K appeared to be mediated by GIP-induced phosphorylation of the Forkhead/winged helix family member Foxo1 (38).

As stated above, Akt activation has been shown enhance cell growth both through the attenuation of apoptosis and the stimulation of cell proliferation (32). Although the effects of GIP apoptosis in CRC were not examined in the present study, we have demonstrated that the proliferation of CRC cells appears to involve several known mitogenic pathways, including the downstream target target p70S6K, as well as adenylyl cyclase and MAPK. Moreover, because GIP is overexpressed in obesity, these studies provide another potential link between obesity and CRC and possibly other malignancies. Additional studies will be necessary to determine the effects of GIP on apoptosis, as well as to elucidate more completely the cellular mechanisms involved in mediating the mitogenic properties of GIP in CRC. Finally, in vivo experiments and epidemiological studies in obese humans will be required to provide further evidence linking obesity and enhanced GIP expression with CRC.

ACKNOWLEDGMENTS

This study was supported by grants R01 DK53158 (M.M. Wolfe) and R01 CA118992 (M.M. Wolfe) from the National Institutes of Health.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

1. Chang AJ, Song DH, Wolfe MM. Gastrin stimulation of colorectal cancer cell proliferation is mediated in part by attenuation of peroxisome proliferator activated receptor gamma (PPAR) activity and proteasomal degradation of PPARγ protein. J. Biol. Chem. 2006;281:14700–14710. [PubMed]
2. Nakata H, Wang S-L, Chung DC, Westwick JK, Tillotson LG. Oncogenic Ras induces gastrin gene expression in colon cancer. Gastroenterology. 1998;115:1144–1153. [PubMed]
3. Fearnon RR, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61:759–767. [PubMed]
4. Cummings DE, Weigle DS, Frayo S, Breen PA, Ma MK, Dellinger EP, Purnell JQ. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N. Engl. J. Med. 2002;346:1623–1630. [PubMed]
5. Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of overweight and obesity in the United States, 1999-2004. JAMA. 2006;295:1549–1555. [PubMed]
6. Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N. Engl. J. Med. 2003;348:1625–1638. [PubMed]
7. Song DH, Getty-Kaushik L, Tseng E, Simon J, Corkey BE, Wolfe MM. Glucose-dependent insulinotropic polypeptide enhances adipocyte development and glucose uptake in part through Akt activation. Gastroenterology. 2007;123:1796–1805. [PMC free article] [PubMed]
8. Service FJ, Rizza RA, Westland RE, Hall LD, Gerich JE, Go VL. Gastric inhibitory polypeptide in obesity and diabetes mellitus. J. Clin. Endocrinol. Metab. 1984;58:1133–1140. [PubMed]
9. Ebert R, Frerichs H, Creutzfeldt W. Impaired feedback control of fat induced gastric inhibitory polypeptide (GIP) secretion by insulin in obesity and glucose intolerance. Eur. J. Clin. Inves.t. 1979;9:129–135. [PubMed]
10. Groop PH. The influence of body weight, age and glucose tolerance on the relationship between GIP secretion and beta-cell function in man. Scand. J. Clin. Lab. Invest. 1989;49:367–379. [PubMed]
11. Pederson RA, Kieffer TJ, Pauly R, Kofod H, Kwong J, McIntosh CH. The enteroinsular axis in dipeptidyl peptidase IV-negative rats. Metabolism: Clin. Exp. 1996;45:1335–1341. [PubMed]
12. Miyawaki K, Yamada Y, Ban N, Ihara Y, Tsukiyama K, Zhou H, Fujimoto S, Oku A, Tsuda K, Toyokuni S, Hiai H, Mizunoya W, Fushiki T, Holst JJ, Makino M, Tashita A, Kobara Y, Tsubamoto Y, Jinnouchi T, Jomori T, Seino Y. Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Nat. Med. 2002;8:738–742. [PubMed]
13. Kim S-J, Nian C, Widenmaier S, McIntosh CHS. Glucose-dependent insulinotropic polypeptide-mediated up-regulation of [exists]-cell antiapoptotic Bcl-2 gene expression is coordinated by cyclic AMP (cAMP) response element binding protein (CREB) and cAMP-responsive CREB coactivator 2. Mol. Cell Biol. 2008;28:1644–1656. [PMC free article] [PubMed]
14. Kodach LL, Bos CL, Durán N, Peppelenbosch MP, Ferreira CV, Hardwick JCH. Violacein synergistically increases 5-fluorouracil cytotoxicity, induces apoptosis and inhibits Akt-mediated signal transduction in human colorectal cancer cells. Carcinogenesis. 2006;27:508–516. [PubMed]
15. Sabatini DM. mTOR and cancer: insights into a complex relationship. Nature Rev. 2006;6:729–734. [PubMed]
16. Sarbassov DD, Ali SM, Sabatini DM. Growing roles for the mTOR pathway. Curr. Opin. Cell Biol. 2005;17:596–603. [PubMed]
17. Kubota A, Yamada Y, Yasuda K, Someya Y, Ihara Y, Kagimoto S, Watanabe R, Kuroe A, Ishida H, Seino Y. Gastric inhibitory polypeptide activates MAP kinase through the wortmannin-sensitive and -insensitive pathways. Biochem. Biophys. Res. Commun. 1997;235:171–175. [PubMed]
18. Wolfe MM, Hocking MP, Maico DG, McGuigan JE. Effects of antibodies to gastric inhibitory peptide on gastric acid secretion and gastrin release in the dog. Gastroenterology. 1983;84:941–948. [PubMed]
19. Moore B, Edie ES, Abram JH. On the treatment of diabetes mellitus by acid extract of duodenal mucous membrane. Biochem. J. 1906;1:26–38. [PubMed]
20. Wang Z, Wang RM, Owji AA, Smith DM, Ghatei MA, Bloom SR. Glucagon-like peptide-1 is a physiological incretin in rat. J. Clin. Invest. 1995;95:417–421. [PMC free article] [PubMed]
21. Tseng CC, Kieffer TJ, Jarboe LA, Usdin TB, Wolfe MM. Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat. J. Clin. Invest. 1996;98:2440–2445. [PMC free article] [PubMed]
22. Eckel RH, Fujimoto WY, Brunzell JD. Gastric inhibitory polypeptide enhanced lipoprotein lipase activity in cultured preadipocytes. Diabetes. 1979;28:1141–1142. [PubMed]
23. Knapper JM, Puddicombe SM, Morgan LM, Fletcher JM. Investigations into the actions of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 (7-36) amide on lipoprotein lipase activity in explants of rat adipose tissue. J. Nutrition. 1995;125:183–188. [PubMed]
24. Althage MC, Ford EL, Wang S, Tso P, Polonsky KS, Wice BM. Targeted ablation of GIP-producing cells in transgenic mice reduces obesity and insulin resistance induced by high fat diet. J. Biol. Chem. 2008;283:18365–18376. [PubMed]
25. Lew EA, Garfinkel L. Variations in mortality by weight among 750,000 men and women. J. Chronic Dis. 1979;32:563–576. [PubMed]
26. Frezza EE, Wachtel MS, Chiriva-Internati M. Influence of obesity on the risk of developing colon cancer. Gut. 2006;55:285–291. [PMC free article] [PubMed]
27. Kaaks R, Toniola P, Akhmedkhanov A, Lukanova A, Biessy C, Dechaud H, Rinaldi S, Zeleniuch-Jacquotte A, Shore RE, Riboli E. Serum c-peptide, insulin-like growth factor (IGF)-1, IGF binding proteins, and colorectal cancer risk in women. J. Natl. Cancer Inst. 2000;92:1592–1600. [PubMed]
28. Giovannucci E. Insulin, insulin-like growth factors and colon cancer: a review of the evidence. J. Nutr. 2001;131:3109S–3120S. [PubMed]
29. Higginbotham S, Zhang ZF, Lee IM, Cook NR, Giovannucci E, Buring JE, Liu S. Dietary glycemic load and risk of colorectal cancer in the Women's Health Study. J. Natl. Cancer Inst. 2004;96:229–233. [PubMed]
30. Sridhar SS, Goodwin PJ. Insulin-insulin-like growth factor axis and colon cancer. J. Clin. Oncol. 2009;27:165–167. [PubMed]
31. Zhao L. Class I PI3K in oncogenic cellular transformation. Oncogene. 2008;27:5486–5496. [PMC free article] [PubMed]
32. Huang X-F, Chen J-Z. Obesity, the PI3/Akt signal pathway and colon cancer. Obes. Rev. 2009 Jun 12; [Epub ahead of print] [PubMed]
33. Fearnon RR, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61:759–767. [PubMed]
34. Hursting SD, Nunez NP, Varticovski L, Vinson C. The obesity-cancer link: lessons learned from a fatless mouse. Cancer Res. 2007;67:2391–2393. [PubMed]
35. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell. 2006;124:471–484. [PubMed]
36. Widenmaier SB, Sampaio AV, Underhill TM, McIntosh CHS. Noncanonical activation of Akt/protein kinase B in [exists]cells by the incretin hormone glucose-dependent insulinotropic polypeptide. J. Biol. Chem. 2009;284:10764–10773. [PubMed]
37. Trhħmper A, Trhħmper K, H[r with grave]sch D. Mechanisms of mitogenic and anti-apoptotic signaling by glucose-dependent insulinotropic polypeptide in ((INS-1)-cells. J. Endocrinol. 2002;174:233–246. [PubMed]
38. Kim S-J, Winter K, Nian C, Tsuneoka M, Koda Y, McIntosh CHS. Glucose-dependent insulinotropic polypeptide stimulation of pancreatic [exists]-cell survival is dependent upon phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling, inactivation of the forkhead transcription factor Foxo1, and down-regulation of bax expression. J. Biol. Chem. 2005;280:22297–22307. [PubMed]