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The Journal of Nutrition Vol. 128 No. 2 February 1998,
pp. 158-165
Nutritional Sciences Program, University of Missouri-Columbia, Columbia, MO 65211
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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Epidemiologic data and animal models have demonstrated a correlation between dietary fat composition and colon cancer risk. We have previously found that dietary fat alters cell proliferation in rat colon, which may influence the risk of colon cancer. Growth factors, including insulin-like growth factor (IGF) I and II, regulate the cell cycle in most mammalian tissues. Hence, we measured IGF-I and IGF-II receptor expression in colonocytes from Sprague-Dawley rats fed diets containing either beef tallow (BT) or corn oil (CO) at 12, 30 or 37% of energy for 4 wk. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using an internal standard was used to examine the relative expression of both IGF-I and II receptor mRNA in three sections of the colon. The IGF-I receptor protein was also measured by Western immunoblot. In the distal colon, IGF-I receptor gene expression and protein increased significantly as the percentage of CO increased. In both proximal and middle colon, an increased percentage of BT resulted in significantly increased IGF-II receptor expression. In the proximal colon, IGF-II receptor expression decreased with increasing CO concentration, whereas in the middle colon, rats fed 37% CO had significantly higher IGF-II receptor expression than rats fed 12 or 30% CO. IGF-II receptor gene expression in proximal colon decreased with increased fat quantity, independently of fat source, whereas in the middle colon, increased fat quantity resulted in increased IGF-II receptor expression. Thus IGF-I and IGF-II receptor mRNA and IGF-I receptor protein level in colon mucosa were significantly altered by dietary fat source and quantity, thereby suggesting a potential influence of dietary fat on the endocrine regulation of colon cell mitogenesis.
KEY WORDS: dietary fat · insulin-like growth factor receptors · rats · colon cancer · gene expression
An increased risk of developing colon cancer in humans has been correlated with consumption of diets high in fat (Graham et al. 1988 Excessive cell proliferation is positively associated with colon tumor risk; reduction in proliferation reduces risk (Lipkin 1991 A potential mechanism through which dietary fat may affect proliferation of colon cells is via alteration of growth factor activation of intracellular signals. Growth factors, including the insulin-like growth factors (IGF)6 play essential roles in the regulation of cellular growth and differentiation (Froesh et al. 1985). Moreover, a role for IGF as growth-promoting factors in several normal and cancer cell lines has been proposed (Kaleko et al. 1990 Dietary fat composition affects the physicochemical properties and biochemical functions of cell membranes (Foot et al. 1983, Innis and Clandinin 1981 Evidence suggests that dietary fat composition affects membrane-associated receptor activity and cell proliferation. On the basis of these observations, we postulated that dietary fat type and quantity may affect the expression of IGF receptors in the colon, which thus influence colon cell proliferation and thereby colon cancer risk. In this study, we examined the effect of dietary fat (both type and quantity) on the expression of the IGF receptors of rat colonocytes. Because the IGF-I and II receptor gene expression has been difficult to quantify due to low copy number in the gastrointestinal tract, we developed a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) method using an internal standard to provide relative comparisons in gene expression. Because the same amount of target cDNA from each sample within a colon region was amplified with known amounts of competitor, changes in expression due to dietary treatment could be observed. The relative amount of IGF-I receptor protein in the colon sections was also determined by Western immunoblot.
Animals and diets.
Thirty-six weanling male Sprague-Dawley rats (Sasco, St. Louis, MO) were removed from dams and randomly assigned to one of six groups fed diets shown in Table 1. The diets contained 12, 30 or 37% of total energy from corn oil (CO) or beef tallow (BT) and were of equivalent nutrient density for protein, vitamins and minerals. The reason for choosing to examine these three levels of dietary fat was to compare the current western diet level (37%) with the recommended lower fat intake (30%) and a very low fat level (12%). The fatty acid profiles of the 12 and 37% fat experimental diets have been reported previously (MacDonald et al. 1996
Western immunoblot.
Cells from proximal and distal colon were lysed in buffer containing 0.09% sodium dodecyl sulfate, 1 mmol/L sodium acetate and 100 mmol/L dithiothreitol, heated at 100°C for 5 min and sonicated. Aliquots of the cell lysates containing similar protein concentrations were electrophoresed on 10% acrylamide gels and then electroblotted onto nitrocellulose membranes (TransBlot, BioRad, Hercules, CA). As an internal control, a lysate of murine Swiss 3T3 cells and a high range biotinylated SDS-PAGE molecular weight marker (BioRad) were used on each gel. Membranes were incubated in a blocking buffer containing 5% nonfat dry milk, with 0.05% Tween-80 in Tris base/saline buffer overnight at 4°C. The primary antibody was chicken polyclonal anti-human IGF-I receptor (Upstate Biotechnology, Lake Placid, NY), and the secondary antibodies were biotinylated rabbit anti-chicken IgG (Pierce, Rockford, IL) and streptavidin-biotinylated horseradish peroxidase complex (Amersham, Arlington Heights, IL). Reactive bands were visualized using enhanced chemiluminescent substrate, SuperSignal Substrate (Pierce), and autoradiography. The band corresponding to the IGF-I receptor was quantified by densitometric scanning with an LKB UltraScan XL Enhanced Laser Densitometer (LKB Producter AB, Sweden). The resulting densities were corrected for the 3T3 control sample on the same blot and expressed as a ratio.
RT-PCR.
A competitive RT-PCR method, based on the mimic strategy (Clontech Laboratories, Palo Alto, CA), was used to measure IGF-I and II receptor mRNA levels in rat colon cells. A competitive internal standard with identical primer binding sites used to amplify the IGF-I cDNA was generated by amplifying a BamH1/EcoR1 fragment of v-erbB with two composite primers (40-mer) 5 Statistical methods.
Each sample was analyzed by PCR in triplicate in separate experiments. Each sample was analyzed by Western immunoblot in duplicate in separate experiments. The data are expressed as means ± SD. Statistical evaluation of the results was conducted by ANOVA (SAS/STAT Version 6.08, SAS Institute, Cary, NC) using a 2 × 3 factorial design in which the linear model contained fat type, fat quantity and fat type × fat quantity interaction. For variables for which significant interaction effects occurred, differences were determined for the interactive means using the least significant difference test (Snedecor and Cochran 1980 Animal growth and feed intake.
No differences in final mean body weight were observed in rats fed the defined diets (215 ± 20 and 210 ± 15 g overall mean ± pooled SD for CO- and BT-fed rats, respectively). Similar feed intake was also observed in the rats (range of 506-619 kJ/d at termination).
Gene expression of IGF-I receptor.
When CO was the fat source, increased dietary fat quantity resulted in increased IGF-I receptor gene expression in distal colon (Fig. 1). In rats fed the BT diets, however, 37% energy as fat resulted in the lowest IGF-I receptor gene expression. No differences in IGF-I receptor expression due to diet were observed in proximal or middle colon (data not shown).
Protein levels of IGF-I receptor.
In the distal colon, rats fed the 37% CO diet had significantly higher IGF-I receptor protein than rats fed 12 or 30% CO, but no difference in protein expression was observed in rats fed 12 or 37% BT (Fig. 2). In the proximal colon, dietary fat type affected IGF-I receptor expression, with a 20% higher expression in rats fed CO than BT, but no effect of fat quantity or interaction between fat type and quantity was present (data not shown).
Gene expression of IGF-II receptor.
In the proximal colon, increased IGF-II receptor gene expression with increased fat quantity was found when BT was the fat source, but increased CO quantity resulted in decreased IGF-II receptor gene expression (Fig. 3). The IGF-II receptor expression response of the middle colon to the BT diets was similar to that of the proximal colon; however, the response to CO was opposite (Fig. 4). IGF-II receptor expression in the proximal colon was lower when 30 or 37% fat was fed compared with 12% fat, whereas in the middle colon, increased percentage of fat resulted in increased IGF-II receptor expression (Fig. 5). No differences due to fat quantity were observed on IGF-II receptor expression in the distal colon.
Although it has been established that diets high in fat and low in fiber increase the risk of colon cancer, the cellular mechanisms through which dietary fat affects cancer risk remain unknown. To determine the influence of dietary fat type and quantity on colon cells, we developed a dietary model that provides similar protein and micronutrient intake on an energy basis. With the use of this model, we previously found that cell proliferation in colon cells was higher in rats fed 37% compared with 12% CO, and lower in rats fed 37% compared with 12% BT (Thornton and MacDonald 1994
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Jain et al. 1980). Numerous animal studies have demonstrated that the induction of colon cancer in rats by chemical agents is exacerbated by increased levels of fat in the diet (Bull et al. 1979, Nigro et al. 1975, Reddy and Maruyama 1986, Reddy et al. 1991). However, the mechanism through which dietary fat affects colon cancer development remains undefined.
). Mucosa cells near tumor sites had higher rates of proliferation than those in more distant sections (Risio 1992). Dietary fat has been found to alter colon cell proliferation rates. Both rats and mice fed diets containing 40% of energy as corn oil had expanded proliferative regions in the sigmoid colon compared with those fed 12% corn oil (Reddy et al. 1976). In the proximal colon, hyperplasia and an increase in labeled cells occurred in rats fed 25% compared with 5% energy as fat (Steinbach et al. 1993). We have found that diets high in saturated fat reduced colon cell proliferation in rats compared with diets high in unsaturated fat (Thornton and MacDonald 1994).
). IGF-I and IGF-II are homologous to insulin in regard to amino acid sequence, structure and biological activity (Rechler and Nissley 1990). Biological response to IGF-I and IGF-II is mediated by IGF-I and IGF-II/mannose-6-phosphate (M-6-P) receptors, respectively, within the plasma membrane (LeRoith et al. 1995). IGF-I and IGF-II receptors are present on normal rat intestinal epithelium (Laburthe et al. 1988, Young et al. 1990), and both IGF-I and IGF-II mRNA has been reported in normal rat intestine (Lund et al. 1986). The presence of IGF and their receptors throughout the gastrointestinal tract suggests that IGF play an important role in the growth and function of this tissue.
). Changes in membrane lipid composition are associated with altered activity of membrane-associated receptors. In rat liver, an increase in dietary oleic acid resulted in increased 18:1 and total monounsaturated fatty acids in plasma membrane phospholipid, which correlated with increased glucagon-stimulated adenylate cyclase activity (Clandinin et al. 1985). Rats fed diets high in polyunsaturated fatty acids demonstrated increased linoleic acid [18:2(n-6)] content in adipocyte membrane and higher insulin binding compared with rats fed low fat diets (Field et al. 1990). However, a negative correlation between erythrocyte membrane linoleic or arachidonic acid composition and insulin receptor binding was reported in healthy humans (Pelikanova et al. 1989). We have observed that rats fed 12% energy as beef tallow or 38% energy as corn oil had lower insulin binding in jejunum compared with those fed 12% corn oil (MacDonald and Thornton 1993).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Rats were housed individually in wire-bottom stainless steel cages in an environmentally controlled room with a 12-h diurnal light cycle and allowed free access to food and water. Body weight and energy intake were periodically monitored during the study period. All animal protocols were approved by the University of Missouri Animal Care and Use Committee. After 4 wk, the rats were deprived of food for 20 h and then killed by injection of sodium pentobarbital (Abbott Laboratories, North Chicago, IL) and pneumothorax. The entire colon from the cecum to just proximal to the rectum was removed from each rat and divided into three equal sections: proximal, middle and distal. The colon segments were flushed with cold saline, inverted, filled with 10 mmol/L EDTA buffer (pH 7.4) and tied at both ends. The filled colon segments were suspended in 10 mmol/L EDTA buffer (pH 7.4) containing hyaluronidase (200 IU/L) and shaken in a 37°C water bath. The mucosal suspension was pelleted by centrifugation at 1300 × g for 10 min, then resuspended in 10 mmol/L EDTA (pH 7.3) and frozen at 
80°C for subsequent Western immunoblot analysis. For RNA extraction, mucosal cells were scraped into guanidinium thiocyanate buffer, immediately homogenized and stored at 80°C.
View this table:
Table 1.
Composition of experimental diets
-ATG AGT ACA ACT ACC GCT GC GGC AAG TGA AAT CTC CTC CG-3 and 5-TAC ATG CTC TGG GTG CTG TT TTG AGT CCA TGG GGA GCT TT-3. The first 20 nucleotides of the primer were complementary to the IGF-I receptor, and the following 20 nucleotides were complementary to v-erbB. The internal standard was synthesized, purified and quantified by spectrophotometry following protocols described by the manufacturer. The primers used to amplify the IGF-I receptor were 5-ATG AGT ACA ACT ACC GCT GC (5 primer) and 5-TAC ATG CTC TGG GTG CTG TT (3 primer). A 364-bp stretch of the IGF-I receptor and a 596-bp stretch of the competitor were amplified. The primers used to create the IGF-II receptor internal standard were 5-GAC CAG GAC AGT GAG GAT GA GGC AAG TGA AAT CTC CTC CG-3 and 5-TTT GGT TGG AGG TGC TTG GC TTG AGT CCA TGG GGA GCT TT-3, and the primers used to amplify the IGF-II receptor and the internal standard were, 5-GAC CAG GAC AGT GAG GAT GA-3 (5 primer) and 5-TTT GGT TGG AGG TGC TTG GC-3 (3 primer). A 320-bp stretch of the IGF-II receptor and a 596-bp stretch of the competitor were amplified. The amplification efficiency of the mimic sequences showed no significant difference from the target cDNA sequences.
). After denaturation (90°C, 2 min), 1 µg of total RNA preparation was reverse transcribed using 1 µmol/L oligo(dT)18 primer, 0.5 µmol/L (each) deoxynucleoside triphosphates, 20 U of recombinant RNase inhibitor and 200 U of MMLV (Moloney-Murine Leukemia Virus) reverse transcriptase (Clontech) in a final volume of 20 µL containing 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl and 3 mmol/L MgCl2. The reaction was conducted at 42°C for 60 min. After the reaction was stopped by heating at 94°C for 5 min, the volume was brought to 100 µL. Equal amounts of target cDNA were amplified with different dilutions of known amounts of mimic DNA, and the ratio of mimic band intensity to target band intensity was determined. For each of the three colon regions, a 10-fold serial dilution (6.88 × 10
15-6.88 × 1022 or 7.3 × 1015-7.3 × 1022 mol/µL for IGF-I or IGF-II receptor, respectively) of mimic was used to estimate the concentration needed to obtain a 1:1 ratio of mimic to target. Then a more specific concentration range was determined using a two-fold dilution series around the equivalent concentration. A single concentration of mimic was then used in each of the PCR analyses of the rat colon samples (~1018 or 1020 mol/µL for IGF-I or IGF-II receptor, respectively). Target cDNA was used in PCR amplification in a final volume of 50 µL containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.0 mmol/L MgCl2, 0.2 mmol/L each dNTP, 1.0 µmol/L each primer, 2 U of Taq DNA polymerase (Fisher, St. Louis, MO), and 2 µL of competitor DNA. Amplification was performed in a MiniCycler thermal cycler (MJ Research, Watertown, MA) and 30 cycles conducted as follows: denaturation for 45 s at 95°C, annealing of primers at 63°C for 45 s and extension at 72°C for 2 min, followed by a final 7-min incubation at 72°C. Aliquots of the PCR product were resolved on 1.5% agarose gels, then stained with ethidium bromide. Band intensities of photographed ethidium bromide-stained gels were determined by densitometric scanning.
). The probability level at which the differences were considered significant was P < 0.05.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View larger version (49K):
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Fig 1.
Insulin-like growth factor I (IGF-I) receptor expression in distal colon of rats fed beef tallow or corn oil at 12, 30 or 37% of energy for 4 wk. mRNA from colon mucosa was reverse transcribed and IGF-I receptor mRNA (target) amplified by polymerase chain reaction (PCR). An internal competitor (mimic) was simultaneously amplified and the ratio of target/mimic determined. Values are means ± SD. Each mean represents triplicate replicates of colon samples from 5 rats. Differences among the means were determined by least significant difference test after 2 × 3 ANOVA. Bars with different letters are significantly different (P < 0.05).
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Fig 2.
Insulin-like growth factor I (IGF-I) receptor protein quantity in distal colon of rats fed beef tallow or corn oil at 12, 30 or 37% of energy for 4 wk. Colon mucosa was analyzed by Western immunoblot using anti-IGF-I receptor antibody. The density of the band corresponding to the IGF-I receptor was determined by densitometry, corrected for an internal control and expressed as a ratio in arbitrary units (AU). Values are means ± SD. Each mean represents triplicate replicates of colon samples from 5 rats. ND, not determined. Differences among the means were determined by least significant difference test after 2 × 3 ANOVA. Bars with different letters are significantly different (P < 0.05).
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Fig 3.
Insulin-like growth factor II (IGF-II) receptor expression in proximal colon of rats fed beef tallow or corn oil at 12, 30 or 37% of energy for 4 wk. mRNA from colon mucosa was reverse transcribed and IGF-II receptor mRNA (target) amplified by PCR. An internal competitor (mimic) was simultaneously amplified and the ratio of target to mimic determined. Values are means ± SD. Each mean represents triplicate replicates of colon samples from 5 rats. Differences among the means were determined by least significant difference test after 2 × 3 ANOVA. Bars with different letters are significantly different (P < 0.05).
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Fig 4.
Insulin-like growth factor II (IGF-II) receptor expression in middle colon of rats fed beef tallow or corn oil at 12, 30 or 37% of energy for 4 wk. mRNA from colon mucosa was reverse transcribed and IGF-II receptor mRNA (target) amplified by PCR. An internal competitor (mimic) was simultaneously amplified and the ratio of target to mimic determined. Values are means ± SD. Each mean represents triplicate replicates of colon samples from 5 rats. Differences among the means were determined by least significant difference test after 2 × 3 ANOVA. Bars with different letters are significantly different (P < 0.05).
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Fig 5.
Insulin-like growth factor II (IGF-II) receptor expression in proximal, middle and distal colon of rats fed 12, 30 or 37% of energy from either beef tallow or corn oil for 4 wk. mRNA from colon mucosa was reverse transcribed and IGF-II receptor mRNA (target) amplified by PCR. An internal competitor (mimic) was simultaneously amplified and the ratio of target to mimic determined. Values are least squares means ± SEM for the combined beef tallow- and corn oil-fed rats at each of the three concentrations of dietary fat. Each mean represents triplicate replicates of colon samples from 10 rats. Differences among the means were determined by least significant difference test after 2 × 3 ANOVA. Bars for a region with different letters are significantly different (P < 0.05).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). This suggested that CO fed at levels consumed by Americans increased colon cell proliferation, whereas BT at the same level decreased cell proliferation. The IGF system, which includes IGF-I, IGF-II, IGF-I and IGF-II membrane-associated receptors and numerous binding proteins, is critical for normal cellular growth and development, and a role for this system in malignancy has been suggested (LeRoith et al. 1995). Therefore, alteration of the IGF system by dietary fat may provide a mechanism to explain the influence of dietary fat on cell proliferation, and perhaps colon cancer risk. To examine this hypothesis, we measured expression of the IGF receptors in colon from rats fed defined diets containing two types and three levels of fat.
). The proliferative compartment of normal colorectal mucosa of individuals at low risk for colorectal cancer, and of normal rodents, is located in the lower part of the colonic crypts. However, patients at high risk of colon cancer showed a shift of the proliferative compartment toward the top of the crypts where cells were more likely to be exposed to carcinogens and become mutated while undergoing division (Scalmati and Lipkin 1993). Dietary fat alters the lipid composition and function of mucosal membranes (Garg et al. 1988, Thomson et al. 1988). An increase in the arachidonic acid content of rat colonic mucosal phospholipid was associated with increased rates of cell proliferation (Lee et al. 1993b). Because membrane phospholipid fatty acids are used in the production of intracellular signals such as diacylglycerol and prostaglandins, it has been suggested that modifications in the fatty acid profile may affect the composition and function of these systems and thereby influence cell proliferation. The expression and quantity of IGF-I receptors in distal colon of rats (Figs. 1 and 2) were significantly increased with increasing concentrations of CO in the diet, but only moderately affected by BT. This may reflect a specific response of the IGF-I receptor to the availability of polyunsaturated fatty acids, or an indirect response of the receptor to an increase in cell proliferation. We have previously found lower insulin binding capacity in colon of rats fed a high fat diet compared with a high carbohydrate diet (MacDonald and Thornton 1993). Field et al. (1989) reported that insulin binding to rat epidiymal adipocytes was positively correlated with the polyunsaturated fatty acid content in membrane phosphatidylethanolamine. Hence, the polyunsaturated fatty acid content of membrane phospholipids may specifically affect membrane-associated receptors and thereby influence cellular responses to growth factors and cell proliferation.
) because IGF-I administration improved recovery in rats following gut resection (Lemmey et al. 1991) and small bowel transplantation (Zhang et al. 1995). High affinity IGF-I receptors were present in cultured colon cancer cells and in human colon tumor biopsies, and IGF-I stimulated proliferation of the tumor cells in vitro, thus suggesting a role for IGF-I in regulation of colon tumor growth (Guo et al. 1992). However, the presence or absence of IGF-I receptors on colon tumors was not correlated with clinical outcome in colon cancer patients (Bhatavdekar et al. 1995). In a comparison of two patients, Zenilman and Graham (1997) found no differences in IGF-I receptor expression, measured by PCR, in colon tumor and normal tissue. Because no systematic analysis of IGF receptor expression in colon tumors has been done, it remains unclear if IGF-I receptor expression varies in colon cancer of different stages, or with cellular origin. We found no differences in IGF-I receptor expression in the proximal or middle colon due to dietary fat intake, although differences in the distal colon due to CO were evident. This suggests that the IGF-I receptor may have distinct roles in these regions of the rat colon and may be differentially influenced by environmental factors.
). Although the rat colon differs from that of the human in anatomical characteristics, it is of interest that we found the IGF-I receptor to be influenced by dietary fat only in the distal colon of rats (Figs. 1 and 2). Lee et al. (1993a) observed the dietary fat composition to affect primarily the distal colon, with increased dietary BT correlated with increased cell proliferation. Recently, changes in apoptosis, or programed cell death, in colon cells have been proposed to contribute to cancer risk (Payne et al. 1995). Merritt et al. (1995) compared the incidence of both spontaneous and damage-induced apoptosis of epithelial cells in longitudinal sections of the crypts of the small intestine and colon of mice with the expression of bcl-2, a suppressor of apoptosis. The higher incidence of apoptosis in colon compared with small intestine, and stem cells compared with crypts of both small intestine and colon led to the suggestion that lower apoptosis might contribute to the higher incidence of tumors in these regions. Resistance to butyrate-induced apoptosis was observed in nontumor mucosa from patients with colon cancer, suggesting potential survival of DNA-damaged cells. The recent observation that IGF-I inhibits apoptosis suggests that increased expression of the IGF-I receptor might increase tumor risk (Parrizas et al. 1997). Cells with targeted disruption of the IGF-I receptor gene (R-cells) are resistant to transformation by the SV40 large T antigen (Sell 1995). And cells transfected with IGF-I receptors in which carboxy-terminal tyrosines were mutated to prevent receptor phosphorylation induced fewer tumors in mice than wild-type cells (Blakesley et al. 1996). Lower IGF-I receptor expression was associated with a suppression of the malignant phenotype in human rhabdomysarcoma (Shapiro et al. 1992) and growth inhibition in breast cancer cells (Neuenshwander et al. 1995). Hence, higher IGF-I receptor expression and protein quantity in colon cells as observed in rats fed the higher concentrations of CO may inhibit apoptosis and increase colon cancer risk.
). Normal human colon also demonstrated IGF-II receptors in a distribution similar to that of rat colon (Pillion et al. 1993). The IGF-II receptor is a bifunctional binding protein with distinct binding domains for IGF-II and M-6-P-containing ligands. The IGF-II/M-6-P receptor targets lysosomal enzymes and plays a role in protein turnover (Johnson and Kornfeld 1992). Recent evidence suggests that another critical role for the IGF-II receptor is to regulate the concentration of IGF-II within the extracellular environment and that IGF-II, via association with the IGF-I receptor, stimulates growth in utero. Mice with an inherited mutation in the IGF-II receptor gene had elevated circulating and tissue concentrations of IGF-II and increased growth in utero (Ludwig et al. 1996). When the IGF-II or IGF-I receptor gene was simultaneously mutated in the mice, growth was normal, suggesting IGF-II mediated cell proliferation via the IGF-I receptor. IGF-II overexpression by tumor cells increased the tumorigenic phenotype of the cells (Lamonerie et al. 1995) and inhibited cell differentiation (Zarrilli et al. 1996), thus further suggesting stimulation of cell proliferation by IGF-II. In our study, the IGF-II receptor expression in the proximal colon was increased with increased percentage of BT and decreased with decreased percentage of CO in the diet (Fig. 3). This pattern is the opposite of that of our previous report concerning the response of cell proliferation in the colon to these diets (Thornton and MacDonald 1994), suggesting that IGF-II receptor expression is inversely correlated with cell proliferation. It will be necessary to demonstrate increased IGF-II availability in colon cells in which IGF-II receptor expression is decreased and to correlate these changes with cell proliferation to determine if this mechanism of regulation is present in the colon. The response of the IGF-II receptor in the middle colon to increased percentage of BT was similar to that of the proximal colon, but a different pattern was observed when CO was fed (Fig. 4), and no differences due to dietary fat were observed in the distal colon. The middle colon responded dramatically to the percentage of fat in the diet, independently of the fat source, but only minor changes in the proximal and no changes in the distal colon were observed (Fig. 5). Regional differences in colon tumor incidence have been well documented in humans (Cappell and Forde 1989), and cellular responses to dietary fiber and fat in rodent models have been found to be differentially expressed along the colon, thus suggesting regional patterning in this tissue. Such regional variation in response to diet composition may be explained in part by differences in the membrane characteristics of cells within the colon. Brasitus and Dudeja (1985) found that the brush border membrane of colon cells in the distal colon have lower lipid fluidity than in the proximal colon, primarily as a result of increasing cholesterol and saturation index along the proximal to distal axis. This fluidity gradient may explain the regional differences in sodium and water absorption in the colon. Brush border membranes of proximal colon cells had a higher concentration of linoleic acid than distal cells, presumably because of the availability of this fatty acid from the diet (Brasitus and Dudeja 1985). However, when rats were fed diets enriched in linoleic acid, all regions of the colon had increased linoleic acid content and the regional differences were eliminated. In rats, cell proliferation in the distal colon responded to the type of dietary fiber fed, but no changes were observed in the proximal colon (Galloway et al. 1987). In contrast, Lee et al. (1993b) found cell proliferation to be affected by dietary fiber only in the proximal colon. In the same rats, the concentration of arachidonic acid was positively correlated with cell proliferation in the distal colon, but no correlation was observed in the proximal colon. We have found that the strongest correlation between dietary fat and IGF-I receptor expression occurred in the distal colon, whereas IGF-II receptor expression was most affected by dietary fat in the proximal colon. Hence, the IGF receptors in colonocytes appear to be independently affected by dietary fat and to display distinct functions within regions of the colon. This suggests that both receptors influence cellular proliferation and metabolic processes in the colon, and are important factors in mediating the carcinogenic process. Further studies are required to define the cellular location of the IGF receptors in the colon and the regulation of their expression in situ, particularly during the carcinogenic process. The observed changes in IGF receptor expression in colon cells in response to dietary fat demonstrate that these receptors are affected by diet composition, and their correlation with altered rates of cellular proliferation suggests that they may provide a cellular mechanism through which dietary fat affects colon cancer risk.
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FOOTNOTES |
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Manuscript received 27 May 1997. Initial reviews completed 25 June 1997. Revision accepted 25 September 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-kinase and mitogen-activated protein kinase pathways.
J. Biol. Chem.
1997;
272:154-161
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