RESUMO
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.
Assuntos
Anticorpos Monoclonais , Obesidade , Camundongos , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Obesidade/tratamento farmacológico , Obesidade/prevenção & controle , Polipeptídeo Inibidor Gástrico , Hiperfagia , Glucose , InsulinaRESUMO
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/6â¯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 30â¯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.
Assuntos
Anticorpos Monoclonais/farmacologia , Polipeptídeo Inibidor Gástrico/antagonistas & inibidores , Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Receptores dos Hormônios Gastrointestinais/fisiologia , Animais , Modelos Animais de Doenças , Polipeptídeo Inibidor Gástrico/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
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.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Polipeptídeo Inibidor Gástrico/imunologia , Obesidade/imunologia , Obesidade/prevenção & controle , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Polipeptídeo Inibidor Gástrico/antagonistas & inibidores , Imunoterapia/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular/métodos , Obesidade/diagnóstico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Resultado do TratamentoRESUMO
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.
Assuntos
Tecido Adiposo/metabolismo , Dieta/efeitos adversos , Metabolismo Energético , Polipeptídeo Inibidor Gástrico/metabolismo , Gastroenteropatias/etiologia , Trato Gastrointestinal/metabolismo , Insulina/metabolismo , Obesidade/etiologia , Adaptação Fisiológica , Tecido Adiposo/fisiopatologia , Animais , Gastroenteropatias/metabolismo , Gastroenteropatias/fisiopatologia , Trato Gastrointestinal/fisiopatologia , Humanos , Obesidade/metabolismo , Obesidade/fisiopatologia , Fatores de Risco , Transdução de SinaisRESUMO
Glucose-dependent insulinotropic polypeptide (GIP) is a mammalian incretin hormone released into the circulation following nutrient ingestion. We examined the functional evolution of GIP and its relationship with insulin to delineate their respective roles in promoting nutrient efficiency. Expression patterns were examined in the sea lamprey (Petromyzon marinus), a basal vertebrate lacking a distinct pancreas, and in the zebrafish, Xenopus laevis, chicken, and mouse, organisms possessing extraintestinal pancreata. Although sea lamprey genomic analysis predicted a potential GIP-like gene, transcripts were not detected, and insulin expression was confined to the caudal pancreatic bud. GIP was detected in both the intestine and pancreas of the zebrafish and X. laevis. In contrast, GIP and insulin expression were limited to the intestine and pancreas, respectively, in chicken and mouse. Phylogenetic analysis of the glucagon-like ligands suggested proglucagon as the common ancestor, supporting the theory that GIP arose as a gene duplication of proglucagon. Insulin-secreting cells in the sea lamprey intestine may have obviated the need for an enteroinsular axis, and zebrafish may represent an evolutionary transition where GIP does not yet function as an incretin hormone. These observations are consistent with the hypothesis that GIP and insulin influence survival advantage by enhancing the efficiency of nutrient absorption and energy storage.
Assuntos
Evolução Molecular , Polipeptídeo Inibidor Gástrico/genética , Insulina/genética , Petromyzon/genética , Proglucagon/genética , Peixe-Zebra/genética , Animais , Galinhas/genética , Polipeptídeo Inibidor Gástrico/metabolismo , Expressão Gênica , Insulina/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , Pâncreas/metabolismo , Petromyzon/metabolismo , Filogenia , Xenopus laevis/genética , Xenopus laevis/metabolismo , Peixe-Zebra/metabolismoRESUMO
Glucose-dependent insulinotropic polypeptide (GIP), an important component of the enteroinsular axis, is a potent stimulator of insulin secretion, functioning to maintain nutrient efficiency. Although well-characterized in mammals, little is known regarding GIP transcriptional regulation in Danio rerio (Dr). We previously demonstrated that DrGIP is expressed in the intestine and the pancreas, and we therefore cloned the Dr promoter to compare GIP transcriptional regulation in Dr and mammals. Although no significant homology was indentified between the highly conserved mammalian promoter and the DrGIP promoter, 1072-bp of the DrGIP promoter conferred tissue-specific expression in mammalian cell lines. Deletional analysis of the DrGIP promoter identified two regions that, when deleted, reduced transcription by 75% and 95%, respectively. Mutational analysis of the upstream region suggested involvement of an Nkx binding site, although we were unable to identify the factor binding to this site. The cis element in the downstream region was found to be a GATA binding site. Lastly, overexpression and shRNA experiments identified PAX4 as a potential repressor of DrGIP expression. These findings provide evidence that despite the identification of species-specific transcriptional regulators and differences in GIP expression patterns between D. rerio and mammals, a moderate degree of regulatory conservation appears to exist.
Assuntos
Evolução Molecular , Polipeptídeo Inibidor Gástrico/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Camundongos , Células NIH 3T3 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Ratos , Peixe-ZebraRESUMO
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.
Assuntos
Neoplasias Colorretais/patologia , Polipeptídeo Inibidor Gástrico/farmacologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Polipeptídeo Inibidor Gástrico/genética , Humanos , Ligantes , Camundongos , Obesidade/genética , Obesidade/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , SuínosRESUMO
In mammals, glucose-dependent insulinotropic polypeptide (GIP) is synthesized predominately in the small intestine and functions in conjunction with insulin to promote nutrient deposition. However, little is known regarding GIP expression and function in early vertebrates like the zebrafish, a model organism representing an early stage in the evolutionary development of the compound vertebrate pancreas. Analysis of GIP and insulin (insa) expression in zebrafish larvae by RT-PCR demonstrated that although insa was detected as early as 24 h postfertilization (hpf), GIP expression was not demonstrated until 72 hpf, shortly after the completion of endocrine pancreatic development but prior to the commencement of independent feeding. Furthermore, whole mount in situ hybridization of zebrafish larvae showed expression of GIP and insa in the same tissues, and in adult zebrafish, RT-PCR and immunohistochemistry demonstrated GIP expression in both the intestine and the pancreas. Receptor activation studies showed that zebrafish GIP was capable of activating the rat GIP receptor. Although previous studies have identified four receptors with glucagon receptor-like sequences in the zebrafish, one of which possesses the capacity to bind GIP, a functional analysis of these receptors has not been performed. This study demonstrates interactions between the latter receptor and zebrafish GIP, identifying it as a potential in vivo target for the ligand. Finally, food deprivation studies in larvae demonstrated an increase in GIP and proglucagon II mRNA levels in response to fasting. In conclusion, the results of these studies suggest that although the zebrafish appears to be a model of an early stage of evolutionary development of GIP expression, the peptide may not possess incretin properties in this species.
Assuntos
Polipeptídeo Inibidor Gástrico/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Fatores Etários , Envelhecimento/genética , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Privação de Alimentos , Polipeptídeo Inibidor Gástrico/genética , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Insulina/genética , Insulina/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Larva/genética , Larva/metabolismo , Ligantes , Masculino , Dados de Sequência Molecular , Pâncreas/embriologia , Pâncreas/metabolismo , Proglucagon/genética , RNA Mensageiro/metabolismo , Ratos , Receptores dos Hormônios Gastrointestinais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
We report the case of a 22-year-old woman who was referred for evaluation of possible Zollinger-Ellison syndrome because of hypergastrinemia and a positive secretin stimulation test. She was being treated with proton pump inhibitors (PPIs) for severe gastroesophageal reflux disease during her initial evaluation. At cessation of PPI therapy, her fasting serum gastrin levels normalized, as did her response to secretin injection. Previous reports describing false-positive secretin tests have been limited to cases of hypergastrinemia in the setting of chronic atrophic gastritis, presumably a result of achlorhydria. This case represents a clearly documented instance of PPI-related hypergastrinemia with a false-positive secretin test, with subsequent normalization of serum gastrin and a negative secretin test after withdrawal of PPI therapy. The current case emphasizes the need to assess the acid secretory status of individuals with hypergastrinemia and to discontinue the use of potent antisecretory agents, principally PPIs, to avoid the erroneous diagnosis of gastrinoma and before embarking on expensive and potentially invasive evaluations for the purpose of tumor localization.
Assuntos
Erros de Diagnóstico , Gastrinoma/diagnóstico , Inibidores da Bomba de Prótons/uso terapêutico , Secretina , Adulto , Feminino , Gastrinas/sangue , Humanos , Adulto JovemRESUMO
BACKGROUND & AIMS: In addition to its role as the primary mediator of the enteroinsular axis, glucose-dependent insulinotropic polypeptide (GIP) may play a critical role in the development of obesity. The purpose of these studies was to characterize the effects of GIP and its receptor (GIPR) in adipocyte development and signaling. METHODS: Effects of GIP and GIPR on differentiated 3T3-L1 cells were analyzed using Western blot analysis, Oil-Red-O staining, cyclic adenosine monophosphate radioimmunoassay, immunofluorescence microscopy, and glucose uptake measurements. RESULTS: To determine whether GIP and GIPR are important components in adipocyte development, the expression profile of GIPR during differentiation was examined. GIPR protein expression was enhanced during the differentiation process, and coincubation with its ligand GIP augmented the expression of aP2, a fat cell marker. Conversely, the suppression of GIPR expression by a specific short hairpin RNA attenuated Oil-Red-O staining and aP2 expression, suggesting that the GIPR may play a critical role in adipocyte development. To investigate specific signaling components that may mediate the effects of GIP, we analyzed Akt, glucose transporter-4, and glucose uptake, all of which are modulated by insulin in fat cells. Like insulin, GIP induced the activation of Akt in a concentration-dependent manner, promoted membrane glucose transporter-4 accumulation, and enhanced [(3)H]-2-deoxyglucose uptake. CONCLUSIONS: These studies provide further evidence for an important physiologic role for GIP in lipid homeostasis and possibly in the pathogenesis of obesity. Furthermore, our data indicate that the GIPR might represent a suitable target for the treatment of obesity.
Assuntos
Adipócitos/fisiologia , Polipeptídeo Inibidor Gástrico/fisiologia , Glucose/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Diferenciação Celular , Células Cultivadas , Transportador de Glucose Tipo 4/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Receptores dos Hormônios Gastrointestinais/biossíntese , Transdução de SinaisRESUMO
PURPOSE OF REVIEW: This review is intended to provide an overview of this section on 'Gastrointestinal regulatory peptides' and to emphasize both similarities and differences between 'classic' hormones and those peptides synthesized within, and released from, the gastrointestinal tract. It will also discuss recent investigation involving these peptides and their physiological properties and pathologic potential. RECENT FINDINGS: More recent investigation, much of which is discussed in this section, has looked at the central role of the gastrointestinal tract, and specifically gastrointestinal regulatory peptides, in nutrient homeostasis and in the pathogenesis of obesity and other nutritional disorders. SUMMARY: Regulatory peptides are chemical messengers that provide a means of communication between two cells which are commonly located in different organ systems. The peptides interact via a shared aqueous environment; whereas this environment is endocrine in nature for classic hormones and gastrointestinal peptides, the latter also include those peptides that communicate more directly with their target via paracrine, neurocrine, and autocrine routes. The field of gastrointestinal regulatory peptides is in its infancy, and the coming decades will witness the development of these peptides, as well as analogues and antagonists, as potential new forms of therapy of obesity and other nutrition-related disorders, as well as other maladies.
Assuntos
Hormônios Gastrointestinais/fisiologia , Trato Gastrointestinal/fisiologia , Hormônios Peptídicos/fisiologia , Animais , Homeostase , Humanos , Distúrbios Nutricionais/etiologia , Fenômenos Fisiológicos da Nutrição , Obesidade/etiologiaRESUMO
PURPOSE OF REVIEW: As we strive to improve our understanding of the factors that contribute to the pathogenesis of obesity, it is certainly logical to speculate that a nutrition-dependent component emanating from the gastrointestinal tract, which has the capacity to regulate insulin expression, could represent an etiologic factor in this serious health issue. One such regulatory peptide that possesses these properties is glucose-dependent insulinotropic polypeptide. Release of this peptide is stimulated by the ingestion of glucose and fat from the upper portion of the small intestine, which in turn enhances the release of insulin from the pancreas. RECENT FINDINGS: In addition to its indirect effects in inducing obesity through the stimulation of insulin, a potent efficiency hormone, glucose-dependent insulinotropic polypeptide, like insulin, also directly modulates a variety of factors that are important in adipocyte homeostasis. Such factors include lipoprotein lipase, Akt, and GLUT-4, all of which promote the storage of fat. SUMMARY: In this review, various mechanisms by which glucose-dependent insulinotropic polypeptide may serve as a link that indirectly and directly contributes to the development of obesity will be discussed. Understanding the peptide's role will potentially help provide novel ways to prevent and treat obesity.
Assuntos
Polipeptídeo Inibidor Gástrico/metabolismo , Obesidade/etiologia , Adipócitos/metabolismo , Animais , Transportador de Glucose Tipo 4/metabolismo , Homeostase , Humanos , Insulina/metabolismo , Secreção de Insulina , Metabolismo dos Lipídeos , Lipase Lipoproteica/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
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.
Assuntos
Adipócitos/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Fármacos Gastrointestinais/farmacologia , Metabolismo dos Lipídeos , Lipólise/efeitos dos fármacos , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevenção & controle , Esterificação/efeitos dos fármacos , Ácidos Graxos não Esterificados/metabolismo , Polipeptídeo Inibidor Gástrico/antagonistas & inibidores , Fármacos Gastrointestinais/antagonistas & inibidores , Humanos , Masculino , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/prevenção & controle , Ratos , Ratos Sprague-DawleyRESUMO
Peroxisome proliferators-activated receptor gamma (PPARgamma) has been shown to suppress cell proliferation and tumorigenesis, whereas the gastrointestinal regulatory peptide gastrin stimulates the growth of neoplastic cells. The present studies were directed to determine whether changes in PPARgamma expression might mediate the effects of gastrin on the proliferation of colorectal cancer (CRC). Initially, using growth assays, we determined that the human CRC cell line DLD-1 expressed both functional PPARgamma and gastrin receptors. Amidated gastrin (G-17) attenuated the growth suppressing effects of PPARgamma by decreasing PPARgamma activity and total protein expression, in part through an increase in the rate of proteasomal degradation. G-17-induced degradation of PPARgamma appeared to be mediated through phosphorylation of PPARgamma at serine 84 by a process involving the biphasic phosphorylation of ERK1/2 and activation of the epidermal growth factor receptor (EGFR). These results were confirmed through the use of EGFR antagonist AG1478 and MEK1 inhibitor PD98059. Furthermore, mutation of PPARgamma at serine 84 reduced the effects of G-17, as evident by inability of G-17 to attenuate PPARgamma promoter activity, degrade PPARgamma, or inhibit the growth suppressing effects of PPARgamma. The results of these studies demonstrate that the trophic properties of gastrin in CRC may be mediated in part by transactivation of the EGFR and phosphorylation of ERK1/2, leading to degradation of PPARgamma protein and a decrease in PPARgamma activation.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Gastrinas/metabolismo , PPAR gama/metabolismo , Animais , Proliferação de Células , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptor de Colecistocinina B/metabolismo , Serina/química , Ativação TranscricionalRESUMO
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.
Assuntos
Polipeptídeo Inibidor Gástrico/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Polipeptídeo Inibidor Gástrico/metabolismo , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Ligação Proteica , Ratos , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/metabolismoRESUMO
Vitamin D deficiency has been associated with increased risk of colon cancer in epidemiologic and prospective clinical studies. In vitro and in vivo studies demonstrated that 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and its analogs inhibit colon cancer cell proliferation. Few studies have evaluated the effect of vitamin D deficiency on the development and growth of colon cancer. To assess the antiproliferative effects of 25-hydroxyvitamin D [25(OH)D] and 1,25(OH)2D3 in vitro, we cultured MC-26 (a colon cancer cell line) in the presence of 25(OH)D3 and 1,25(OH)2D3 and performed [3H]thymidine incorporation. The proliferation of MC-26 was significantly inhibited by both 25(OH)D3 and 1,25(OH)2D3. To determine the effect of vitamin D deficiency on colon cancer proliferation, Balb/c mice were rendered vitamin D deficient by feeding them a vitamin D-deficient diet for 3 mo. A group of vitamin D-sufficient mice was given the same diet with supplemental vitamin D. The mice were injected with MC-26 colon cancer cells and the tumors were measured daily for 20 d. Vitamin D-sufficient mice had 40% smaller tumors than vitamin D-deficient mice. The tumors were evaluated for mRNA expression of the vitamin D receptor (VDR) and 25-hydroxvitamin D-1alpha-hydroxylase (1alpha-OHase) by quantitative RT-PCR. The expression of the mRNA for the VDR and the 1alpha-OHase was 37- and 6-fold higher, respectively, in the vitamin D-sufficient mice compared with the vitamin D-deficient mice. We conclude that vitamin D deficiency enhances the growth of colon cancer in mice. The tumor expression of VDR and 1alpha-OHase indicates possible autocrine/paracrine cell growth regulation by vitamin D.
Assuntos
Calcitriol/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/fisiopatologia , Deficiência de Vitamina D/fisiopatologia , Vitaminas/farmacologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Animais , Calcitriol/sangue , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Receptores de Calcitriol/genética , Transplante Heterólogo , Vitaminas/sangueRESUMO
Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits growth and survival of hepatocellular carcinoma (HCC) cell lines. To further investigate the function of PPARgamma in HCC, PPARgamma expression patterns in primary tumors were examined, and the responses of two HCC cell lines to PPARgamma activation and inhibition were compared. PPARgamma expression was increased in HCC and benign-appearing peritumoral hepatocytes compared with remote benign hepatocytes. Both compound PPARgamma inhibitors and PPARgamma small interfering RNAs prevented HCC cell lines from adhering to the extracellular matrix. Loss of adhesion was followed by caspase-dependent apoptosis (anoikis). PPARgamma inhibitors had no effect on initial beta1 integrin-mediated adhesion, or on total focal adhesion kinase levels but did reduce focal adhesion kinase phosphorylation. The PPARgamma inhibitor T0070907 was significantly more efficient at causing cancer cell death than the activators troglitazone and rosiglitazone. T0070907 caused cell death by reducing adhesion and inducing anoikis, whereas the activators had no direct effect on adhesion and caused cell death at much higher concentrations. In conclusion, PPARgamma overexpression is present in HCC. Inhibition of PPARgamma function causes HCC cell death by preventing adhesion and inducing anoikis-mediated apoptosis. PPARgamma inhibitors represent a potential novel treatment approach to HCC.
Assuntos
Anoikis/efeitos dos fármacos , Anoikis/fisiologia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , PPAR gama/antagonistas & inibidores , Anilidas/farmacologia , Benzamidas/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Adesão Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Cromanos/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Integrina beta1/biossíntese , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , PPAR gama/biossíntese , Proteínas Tirosina Quinases/metabolismo , Piridinas/farmacologia , Rosiglitazona , Tiazolidinedionas/farmacologia , TroglitazonaRESUMO
PURPOSE: To determine whether the nonselective and relatively inexpensive nonsteroidal anti-inflammatory drug ibuprofen would be effective in inhibiting colorectal cancer and might improve mortality in a mouse model. EXPERIMENTAL DESIGN: The effects of ibuprofen on tumor growth inhibition and animal survival have been examined in both mouse and human colorectal cancer tumor models. Angiogenesis was measured by in vitro endothelial cell tube formation and immunohistochemistry. RESULTS: Ibuprofen significantly inhibited cell proliferation in mouse (MC-26) and human (HT-29) colorectal cancer cell lines. In vitro angiogenesis assays also indicated that ibuprofen decreased both cell proliferation and tube formation. The administration of chow containing 1,360 ppm ibuprofen, which achieved an average plasma concentration of ibuprofen lower than the peak level achieved in humans at therapeutic doses, inhibited tumor growth by 40% to 82%. Fewer liver metastases were found in the ibuprofen group compared with the control group. In combination therapy with the standard antineoplastic agents, 5-fluorouracil, or irinotecan (CPT-11), tumor volumes in the groups with ibuprofen +/- CPT-11 or 5-fluorouracil were smaller than in the control group. Ibuprofen was similar to the cyclooxygenase-2 selective inhibitor rofecoxib in its ability to suppress tumor growth and improve overall survival. CONCLUSIONS: Ibuprofen, in part by modulating tumor angiogenesis, decreases both tumor growth and metastatic potential in mice. The ibuprofen doses were in the low range of therapeutic human plasma concentrations. Ibuprofen potentiates the antitumor properties of CPT-11 and improves survival of mice without increasing gastrointestinal toxicity.
Assuntos
Camptotecina/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Inibidores de Ciclo-Oxigenase/farmacologia , Ibuprofeno/farmacologia , Neovascularização Patológica/prevenção & controle , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Western Blotting , Camptotecina/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/metabolismo , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/administração & dosagem , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Fluoruracila/administração & dosagem , Células HT29 , Humanos , Ibuprofeno/administração & dosagem , Irinotecano , Lactonas/administração & dosagem , Lactonas/farmacologia , Neoplasias Hepáticas/prevenção & controle , Neoplasias Hepáticas/secundário , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/patologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Sulfonas/administração & dosagem , Sulfonas/farmacologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
As gastrin may play a role in the pathophysiology of gastrointestinal (GI) malignancies, the elucidation of the mechanisms governing gastrin-induced proliferation has recently gained considerable interest. Several studies have reported that a large percentage of colorectal tumours overexpress or stabilise the beta-catenin oncoprotein. We thus sought to determine whether gastrin might regulate beta-catenin expression in colorectal tumour cells. Amidated gastrin-17 (G-17), one of the major circulating forms of gastrin, not only enhanced beta-catenin protein expression, but also one of its target genes, cyclin D1. Furthermore, activation of beta-catenin-dependent transcription by gastrin was confirmed by an increase in LEF-1 reporter activity, as well as enhanced cyclin D1 promoter activity. Finally, G-17 prolonged the tau(1/2) of beta-catenin protein, demonstrating that gastrin appears to exert its mitogenic effects on colorectal tumour cells, at least in part, by stabilising beta-catenin.
Assuntos
Neoplasias Colorretais/metabolismo , Proteínas do Citoesqueleto/metabolismo , Gastrinas/farmacologia , Transativadores/metabolismo , Animais , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Ciclina D1/efeitos dos fármacos , Ciclina D1/metabolismo , Proteínas do Citoesqueleto/efeitos dos fármacos , Camundongos , Regiões Promotoras Genéticas/efeitos dos fármacos , Transativadores/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , beta CateninaRESUMO
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.