The Journal of Nutrition Vol. 128 No. 8 August 1998, pp. 1262-1269

Energy Metabolism of Rat Colonocytes Changes during the Tumorigenic Process and Is Dependent on Diet and Carcinogen^1,2

Jianhu Zhang, Guoyao Wu, Robert S. Chapkin, and Joanne R. Lupton³

Faculty of Nutrition, Texas A&M University, College Station, TX 77843-2471

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Alterations in ATP production, intracellular energy levels and mitochondrial function have been shown to trigger cytokinetic events in vitro, including inhibition of cell division, abnormal or blocked differentiation and inhibition of apoptosis. Changes in colonic cytokinetics are directly related to colon tumorigenesis but alterations in energy metabolism during the tumorigenic process have never been reported. We conducted a 2 × 2 × 3 factorial design study in 120 male Sprague-Dawley rats with two diets (pectin or cellulose-supplemented), two injected subgroups (with or without the carcinogen azoxymethane, AOM) and three termination time points (6, 16 and 36 wk post-second injection). Colonocytes were isolated and incubated with their primary energy substrates (radiolabeled butyrate, glucose, glutamine and -hydroxybutyrate) for 60 min. Production of lactate, ketone bodies and CO₂ were determined. At 6 wk, there were no significant differences in metabolism among treatments. In contrast, at 16 wk, AOM-injected rats had dramatically lower rates of CO₂ production (P < 0.001) from both glucose and butyrate and lower rates of lactate and ketone body production than their saline counterparts. At 36 wk, when tumors developed, the depressed production of lactate and ketone bodies seen in AOM-injected rats at 16 wk returned to control values. However, in AOM-injected rats, CO₂ production from glucose and butyrate remained depressed. Cellulose feeding resulted in decreased oxidation of glucose, butyrate and glutamine and an increased production of ketone bodies from butyrate by colonocytes compared with pectin feeding at 36 wk. We conclude that colonocyte energy metabolism differs in AOM-injected rats vs. saline controls and changes during tumorigenesis, and suggest a relationship between intracellular energy status and changes in cell kinetics. This is the first report that such a relationship may exist in vivo.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Colon tumorigenesis is characterized by increased colonocyte proliferation, decreased differentiation and decreased apoptosis (Chang et al. 1997). The mechanism(s) by which these cytokinetic changes occur is not known but may be related to alterations in genetic events, i.e., deletion of tumor suppressor genes and/or activation of oncogenes as a result of carcinogen exposure. The connection between genetic alterations and changes in cell kinetics is the focus of considerable current attention (Kern 1996, Kern and Kinzler 1995).

Alternatively, or perhaps as a result of genetic changes, new studies suggest that alterations in ATP production, intracellular energy levels and mitochondrial function may also trigger cytokinetic events including inhibition of cell division, abnormal or blocked differentiation, and inhibition of apoptosis (Eguchi et al. 1997, Heerdt et al., 1997, Leist et al. 1997, Stefanelli et al. 1997). These elegant, mechanistic studies, have all been performed in vitro using inhibitors of ATP production or utilization, or effectors of mitochondrial function. Because normal colonocytes have proven refractory to culture, all of the mechanistic in vitro studies have been conducted in transformed cells. There are no reports in the literature as to whether the energy metabolism of colonocytes changes during the tumorigenic process. This is important because butyrate, the primary energy source for colonocytes, induces growth arrest, differentiation and apoptosis of transformed colonic epithelial cells (Barnard and Warwick 1993, Heerdt et al. 1994), but may actually stimulate cell division in normal colonocytes (Lupton 1995). It is reasonable to hypothesize that butyrate may be metabolized differently in normal vs. transformed cells. We provide, for the first time, data on the oxidative fates of the major substrates for colonocyte energy production (butyrate, glucose, -hydroxybutyrate and glutamine) and how their metabolism changes during experimental carcinogenesis in the well-established rat azoxymethane model. Metabolic measurements were determined in colonocytes from rats provided with the same diets and terminated at the same time points as our previously reported study (Chang et al. 1997), in an effort to relate changes in metabolism to changes in proliferation and apoptosis.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and study design.  An animal use protocol that included the procedures to be described was approved by the University Laboratory Animal Care Committee of Texas A&M University and conformed to NIH guidelines. One hundred twenty male weanling (21 d old) Sprague-Dawley rats (Harlan Sprague Dawley, Houston, TX) were housed individually in cages and maintained in a temperature- and humidity-controlled animal facility with a daily photoperiod of 12 h light and 12 h dark. This study used a 2 × 2 × 3 factorial design with two diets (pectin or cellulose), two injection groups (with or without carcinogen) and three termination time points (6, 16 and 36 wk post-second injection) for a total of 12 groups (10 rats/group). An outline of the experimental design is shown in Figure 1. Rats were stratified by body weight so that mean initial body weights of the 12 groups did not differ. Body weights were recorded weekly throughout the study.

View larger version (8K): [in this window] [in a new window]   Fig 1. Overall time line for the study. Rats were 3 wk of age on arrival in the facility and began experimental diets at 4 wk of age. This time line shows the age of the rats (in weeks) at each killing time and the number of weeks the rats had received experimental diets at each time point, together with the number of weeks after the two injections with either azoxymethane (AOM) or saline.

Diets.  After a 1-wk acclimation period of consuming nonpurified diet, rats were assigned to one of two experimental diets (Table 1), which differed only in the type of dietary fiber (pectin or cellulose). This diet has been described in detail in a previous report (Maciorowski et al. 1997). The fibers were chosen because of their different degrees of fermentability. Pectin is more highly fermented in the colon, whereas cellulose is poorly fermented, resulting in different in vivo luminal concentrations of short-chain fatty acids (Zhang and Lupton 1994, Zoran et al. 1997). Because the short-chain fatty acid butyrate is the major oxidative fuel for colonocytes (Roediger 1982), in vivo exposure levels to this important oxidative substrate should be very different between the two fiber groups. The diet contained 6% fiber by weight, which corresponds to a daily intake of 30 g of fiber in humans and is well tolerated by rats. Food and water were freely available at all times. Food intake and fecal output over a 48-h period were measured 1 wk after injections and again 1 wk before each killing time.

Carcinogen treatment.  After 1 wk of receiving their experimental diets, half of the rats were injected with saline (controls) and the other half received azoxymethane. Azoxymethane (AOM,⁴ Sigma Chemical, St. Louis, MO) was injected subcutaneously (15 mg/kg body weight) on two occasions, separated by 1 wk, according to the standard protocol established by Reddy and Maruyama (1986). Control rats received an equal volume of saline. Forty rats (10/group) were terminated at each of the three different time points, one rat from each treatment for each day, in a random order. Thus rats were received into the facility, provided with diets and injected with AOM or saline in a staggered protocol so that at the time of killing there were no differences in age or time of exposure to carcinogen between diet groups. All rats were killed by carbon dioxide, followed by cervical dislocation. They were killed within a half-hour window starting at 1200 h to minimize the effects of diurnal variation. The entire colon from cecum to rectal ampulla was immediately resected.

View larger version (116K): [in this window] [in a new window]   Fig 2. Micrograph of an isolated intact crypt and free cells from rat colon (×10 magnification).

View larger version (131K): [in this window] [in a new window]   View larger version (129K): [in this window] [in a new window]   Fig 3. Micrographs of the colon mucosa of a rat (A) before and (B) after the cell isolation procedure (×10 magnification). (A) A section from the colon showing intact crypts. (B) All of the epithelial cells are removed, the lamina propria and muscle layers remain intact.

Isolation of colonocytes.  Colonocytes were isolated using a modification of the procedure originally described by Roediger and Truelove (1979). Briefly, the colon was flushed clean of luminal contents with calcium-free Krebs-Ringer bicarbonate (Ca²⁺ free-KRB) buffer. Calcium was omitted from the KRB buffer and 5 mmol/L EDTA, 5 mmol/L dithiothreitol (DTT) and 5 mmol/L HEPES (pH 7.4) were added. The buffer was saturated with O₂/CO₂ (95%:5%). Subsequently, the lumen was filled with the calcium-free KRB buffer and both ends were ligated. The ligated colon was placed in a flask with 100 mL of calcium-free KRB buffer for 30 min at 37°C in a waterbath with shaking at 50 cycles/min; then, the luminal fluid was drained into a polystyrene tube containing KRB buffer (119 mmol/L NaCl, 4.8 mmol/L KCl, 2.5 mmol/L CaCl₂, 1.2 mmol/L MgSO₄, 1.2 mmol/L KH₂PO₄ and 25 mmol/L NaHCO₃) supplemented with 5 mmol/L DTT and 5 mmol/L HEPES (pH 7.4). Next, the colon was everted onto a glass rod. Additional cells were isolated from the mucosa into the tube with the luminal fluid by gently stirring for a period of 30 s. The isolated cells were centrifuged at 100 × g for 5 min and washed twice in KRB buffer.

Cell viability was routinely in the range of 80-90% using trypan blue exclusion. The isolation procedure was designed to maintain the crypt architecture as much as possible, because viability is higher with intact crypts than with single cells. A mixture of whole and fragmented crypts with individual free cells was obtained (Fig. 2). The completeness of the isolation procedure was confirmed by histological examination of the tissue remaining (Fig. 3). At 36 wk after the last AOM injection, macroscopic tumors had appeared. Tumors were not disaggregated by the cell isolation procedure, but rather remained attached to the colon tissue with the lamina propria and muscle layers (Figs. 4 and 5).

View larger version (123K): [in this window] [in a new window]   View larger version (136K): [in this window] [in a new window]   Fig 4. Micrographs of colon tumor from an azoxymethane (AOM)-injected rat before the cell isolation procedure. (A) Abnormal tumor crypts and "normal appearing" crypts contiguous to the tumor (×10 magnification). (B) Higher magnification (×40) of the boxed area in panel A.

View larger version (135K): [in this window] [in a new window]   View larger version (147K): [in this window] [in a new window]   Fig 5. Micrographs of colon tumor from an azoxymethane (AOM)-injected rat after the cell isolation procedure. (A) Tumor cells remain; the adjacent "normal appearing" epithelial cells have been removed by the cell isolation procedure (×10 magnification). (B) Higher magnification (×40) of the boxed area in panel A.

Incubation for cell metabolism experiments.  Cell suspensions (representing ~2.5 g protein/L) were placed in siliconized 25-mL Erlenmeyer flasks. Each flask contained the cell suspension, one or two metabolic substrates, a ¹⁴C-labeled substrate and KRB buffer (pH 7.4) with 5 g/L bovine serum albumin (fraction V, essentially fatty acid free, Sigma) in a volume of 2 mL. All incubation media contained 5.6 mmol/L glucose, which represents physiologic blood levels. Butyrate and glutamine were also supplied within the physiologic range but at two different levels (1 or 5 mmol/L) to test for a concentration effect. -Hydroxybutyrate was used at 1 mmol/L. The ¹⁴C-labeled substrates (DuPont NEN, Boston, MA) were D-[6-¹⁴C]glucose, n-[1-¹⁴C]butyrate, D-()-[3-¹⁴C] hydroxybutyrate and L-[U-¹⁴C]glutamine. The flasks were gassed with O₂/ CO₂ (95%:5%) and sealed by a rubber double-seal stopper with a center well. The flasks were incubated at 37^oC in a waterbath with shaking at 50 cycles/min for 60 min. The incubation was terminated by injection of 1.5 mol/L HClO₄ into the incubation flask. Then organic bases NCSII (Amersham Life Science, Arlington Heights, IL) were injected into the hanging center well to trap CO₂. After another 60 min of incubation, the center well with the alkali solution was transferred into a scintillation vial with 15 mL of a modified Bray's solution (5.0 g 2,5-diphenyloxazole, and 0.2 g 1,4-bis-2-(5-phenyloxazolyl)benzene in 0.5 L of toluene plus 0.5 L of methyl glycol monomethyl ether) (Wu and Thompon 1988). The medium in the incubation flask was neutralized with K₂CO₃, and centrifuged at 1000 × g for 20 min. The supernatant was stored at 80^o C. Bacterial contribution to metabolite formation was minimized by isolating and incubating cells with penicillin 1.8 × 10⁵ U/L (Sigma) and streptomycin 180 g/L (Sigma).

Measurement of metabolic products.  On the day after the incubation experiments, ¹⁴CO₂ was quantitated using a liquid scintillation counter (model LS-3801, Beckman Instruments, Irvine, CA). The background ¹⁴CO₂ in flasks containing no cells represented the action of HClO₄ on ¹⁴C-labeled substrate. Generation of ¹⁴CO₂ (nmol) from ¹⁴C-labeled substrate was calculated by specific radioactivity (dpm/nmol) of ¹⁴C-labeled substrate in the incubation medium after the background had been subtracted. Data are expressed as nanomoles of ¹⁴CO₂ produced per milligram protein per hour. The concentration of protein in the cell suspensions was assayed by the method of Bradford (1976) using dye reagent (Bio-Rad, Hercules, CA) with bovine plasma albumin as a standard.

The supernatant of the incubation medium was used for measuring lactate and ketone bodies by enzymatic methods. L-Lactate was measured with lactate dehydrogenase (Boehringer Mannheim, Indianapolis, IN) as described by Gutmann and Wahlefeld (1974). Acetoacetate (Mellanby and Williamson 1974) and -hydroxybutyrate (Williamson and Mellanby 1974) were measured using -hydroxybutyrate dehydrogenase (Boehringer Mannheim).

Statistical analysis.  Data were analyzed by two-way ANOVA at each time point to determine the effect of dietary fiber, carcinogen and fiber × carcinogen. The effect of time after the injection was determined in each treatment group by one-way ANOVA. When the P-value for the interaction was <0.05, group means were separated using Duncan's multiple range test. Differences between means were considered significant at P < 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

Body weight, food and energy intake, fecal output and colon length.  Rat body weights throughout the 39-wk study are shown in Figure 6. There were no differences in weight or rate of weight gain. Food and energy intakes for the week before killings are shown in Table 2. There was no effect of diet or carcinogen on food intake at any time point, but at wk 16, rats receiving the cellulose diet ate more food (P < 0.05) if they had been injected with AOM than if they had been injected with saline. There was a 26-33% decrease (P < 0.05) in energy intake between wk 6 and 16 for all groups, when energy intake was normalized to body weight (Table 2).

View larger version (26K): [in this window] [in a new window]   Fig 6. Body weights of rats fed diets containing either cellulose or pectin, injected with azoxymethane (AOM) or saline and killed at one of three post-injection time points. Values are means, n = 10. Rats were weighed weekly until killed (dotted vertical lines). There were no differences in weight at any time during the study.

At 6 and 16 wk, rats receiving cellulose diets produced dry fecal weights that were approximately double those of rats receiving pectin diets (Table 2); however, the differences were reduced in saline-injected rats at 36 wk. There was no effect of fiber or carcinogen on colon length. As expected, colon length increased with the age of the rats (Table 2).

Histological evaluation of tissue and presence of tumors.  Each rat colon was carefully examined for the presence of visible tumors. No evidence of macroscopic tumors was found in the colon or other tissues of rats killed at 6 or 16 wk post-carcinogen injection. Histological evaluation of the tissue from other rats (part of a parent study) killed at the same time points, revealed no evidence of severe atypia, carcinoma in situ or microscopic adenocarcinomas at either of the first two time points (Chang et al. 1997). In contrast, at the final time point (36 wk post-injection), macroscopic tumors were detected in the AOM-injected rats. There was no effect of fiber on tumor incidence (7 of 10 rats in each diet group for this study, and 51 of 77 in the cellulose diet group vs. 38 out of 66 in the pectin diet group in the parent study).

Oxidation of glucose and butyrate as a function of diet, carcinogen and time.  At the first time point (6 wk post-injections), there were no treatment differences with respect to the oxidation of 5.6 mmol/L glucose, or 1 or 5 mmol/L butyrate (Table 3), nor was there any difference in the production of ¹⁴CO₂ from 1 mmol/L butyrate compared with 5 mmol/L butyrate at this or at any time point. In marked contrast to the rats at 6 wk, there were consistent differences in glucose and butyrate utilization in the AOM-injected and their saline-injected counterparts at the mid- and final points of tumor development (16 and 36 wk). In all saline-injected rats, the rate of ¹⁴CO₂ production from [6-¹⁴C] glucose was decreased at wk 16 and 36 compared with wk 6. AOM-injected rats had lower rates of CO₂ production from both glucose and butyrate than did the saline controls at wk 16 and 36 (Table 3).

The only time dietary fiber affected oxidation of glucose and butyrate by colonocytes was when rats were killed at 36 wk (Table 3). Production of ¹⁴CO₂ was higher (P < 0.05) in colonocytes from rats consuming pectin than from rats receiving cellulose diets.

Oxidation of -hydroxybutyrate and glutamine.  The rate of ¹⁴CO₂ production from [3-¹⁴C] -hydroxybutyrate was measured only at the first and second time points. At the final time point (36 wk) when rats had developed tumors, we chose to evaluate glutamine metabolism instead of -hydroxybutyrate because glutamine is a major energy source in certain cancer cells (Kovacevic and Morris 1972, Medina et al. 1992, Souba 1993), and we were limited in the number of cells per rat that we could use. At wk 6, there were no treatment differences in CO₂ production from 1 mmol/L -hydroxybutyrate, but at wk 16, the carcinogen-treated rats produced less CO₂ than their saline counterparts (Table 3). Rates of CO₂ production from -hydroxybutyrate declined (P < 0.05) from wk 6 to 16 for AOM-injected rats.

Production of ¹⁴CO₂ from [U-¹⁴C] glutamine (1 mmol/L) was greater (P < 0.05) in cells isolated from saline-injected rats than in cells from rats injected with the carcinogen (Table 3). Production of ¹⁴CO₂ was significantly higher in rats consuming pectin than in rats receiving cellulose diets. Increasing the concentration of glutamine from 1 to 5 mmol/L increased the production of ¹⁴CO₂ (P < 0.05).

Production of lactate and ketone bodies as a function of diet, carcinogen and time.  There were no treatment effects on lactate production from glucose at 6 and 36 wk, and means within a group did not differ at these time points (Table 4). However, at wk 16, AOM-injected rats produced less lactate than did the saline controls. Ketone bodies were measured as the sum of acetoacetate and -hydroxybutyrate. The production of acetoacetate from butyrate was about two to three times that of -hydroxybutyrate in all groups (data not shown). There were no treatment differences with respect to ketone body production from 1 or 5 mmol/L butyrate at 6 wk (Table 4). At wk 16, there was a drop (P < 0.001) in ketone body production in the AOM-injected rats. At wk 36, the rate of ketone body production was lower in rats consuming pectin than in those consuming cellulose (Table 4). Increasing butyrate from 1 to 5 mmol/L in the medium had no effect on the amounts of ketone bodies produced.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

This is the first report on the metabolism of colonocytes during the tumorigenic process. The major finding of this study is that colonocyte metabolism differs in AOM-injected rats vs. saline controls and the rates of substrate utilization change during tumorigenesis.

Overall energy metabolism is depressed when rats are developing tumors.  Energy metabolism in AOM-injected rats vs. their saline counterparts was similar at 6 wk post-carcinogen injection. The reason why metabolism did not change at 6 wk after carcinogen treatment is unknown, although precancerous lesions were observed at this time in a companion study (Hong et al. 1997). At 16 wk, there was an overall reduction in the metabolism of energy substrates in AOM-injected rats. This is illustrated by a lower flux of glucose through the tricarboxylic acid cycle (less CO₂ production) in AOM-injected rats than in saline controls. There was also a lower production of lactate from glucose in the AOM-treated rats, suggesting lower utilization of anaerobic as well as aerobic glycolysis. Oxidation of butyrate, the primary energy source for colonocytes (Roediger 1982), was also depressed in AOM-treated rats compared with controls by wk 16, as was ketone body production from butyrate. Interestingly, once tumors had developed, rates of lactate and ketone body production returned to those of saline-injected rats. Rats injected with AOM continued to have a lower rate of glucose and butyrate oxidation, and a lower rate of glutamine oxidation.

The consequences of this depression in energy production and its relationship to tumorigenesis are not known, but several recent papers suggest a connection between ATP production and changes in cell proliferation and apoptosis (Eguchi et al. 1997, Heerdt et al. 1997, Leist et al. 1997, Stefanelli et al. 1997). In a companion study that used the same rats, diets and termination times, we reported a higher rate of cell proliferation and a lower rate of apoptosis at 16 wk in rats injected with AOM than in their saline counterparts (Chang et al. 1997). Rapidly proliferating cells have decreased numbers of mitochondria compared with more quiescent cells (Pedersen 1978) and thus may have decreased capacity for oxidative metabolism. Butler et al. (1992) studied the relationship of colonocyte metabolism to cell proliferation using a food-deprivation/refeeding intervention. Consistent with our findings of depressed oxidative metabolism and increased cell proliferation, they reported a decrease in CO₂ production from butyrate in distal colonocytes during refeeding (the time of active proliferation). Also supportive of a relationship between energy metabolism and cell proliferation is the finding of Roediger (1980) that in inflammatory disorders with actively proliferating colonocytes (such as ulcerative colitis), butyrate oxidation is suppressed.

The depression of energy metabolism, as observed in colonocytes from rats injected with AOM at 16 wk, may also inhibit energy-requiring processes such as DNA damage repair and targeted apoptosis. This would favor survival of DNA-damaged cells rather than their repair or removal and allow for their future clonal expansion. Maintenance of cellular ATP levels is required for cells to undergo programmed cell death (apoptosis) (Richter et al. 1996, Stefanelli et al. 1997), and apoptosis can be blocked by ATP depletion (Eguchi et al. 1997, Kass et al. 1996, Leist et al. 1997, Richter et al. 1996). Inhibition of apoptosis during carcinogenesis is now considered a key factor in colon tumor promotion (Bedi et al. 1995).

In addition to the importance of intracellular energy relative to apoptosis and chemoprevention, ATP levels may be important to cancer treatment. Chemotherapy and radiation treatments attempt to trigger apoptosis (Fisher 1994, Piazza et al. 1995). Drug resistance is one of the major problems of chemotherapy. Chou et al. (1995) and Liu et al. (1994) reported that intracellular ATP is required for anticancer drug-induced apoptotic cell death, which is blocked if intracellular ATP content is reduced.

The major effect of dietary fiber type on metabolism was at the final time point.  Type of dietary fiber had little effect on colonocyte metabolism until the final time point (36 wk) when higher rates of glucose, butyrate and glutamine oxidation and a lower rate of ketone body production were observed in pectin-fed rats than in those fed cellulose. Pectin supplementation results in chronic exposure to higher colonic luminal levels of all short-chain fatty acids, including butyrate (Zhang and Lupton 1994, Zoran et al. 1997); presumably, this could result in an adaptive response to upregulate oxidative metabolism.

The lack of effect of dietary fiber intake on colonocyte metabolism at the earliest time points (6 and 16 wk) is consistent with previously published studies. Clausen and Mortensen (1994) found little effect of prefeeding 10% pectin or 20% tributyrin for 14 d on colonocyte metabolism, including ketone body production, and SCFA or glucose oxidation. A study examining the effect of high versus low fiber diets on oxidative metabolism by isolated pig colonocytes also did not find differences in substrate oxidation when pigs were adapted to a high fiber diet (Darcy-Vrillon et al. 1993). Marsman and McBurney (1996) also did not observe an effect of fiber on oxidation of glucose and butyrate in colonocytes isolated from rats after 2 wk of consuming a high fiber diet.

In summary, this study shows that the type of fiber affects colonocyte metabolism after a long exposure time (36 wk). Most importantly, the metabolism of colonocytes changes during the tumorigenic process. Energy metabolism is depressed in rat colonocytes 16 wk after they have been injected with AOM. Oxidation of metabolic substrates in rat colonocytes exposed to a carcinogen remains depressed at 36 wk, but lactate and ketone production rise to values of control rats when tumors develop. This depression in energy metabolism, seen at 16 wk, is coincident with an increase in cell proliferation and a decrease in apoptosis observed in a companion study. It suggests a relationship between intracellular energy status and changes in cell kinetics. Although manipulation of intracellular energy levels has been documented to affect proliferation and apoptosis in vitro (Eguchi et al. 1997, Herwig and Strauss 1997, Kass et al. 1996, Leist et al. 1997, Richter et al. 1996), this is the first report that such a relationship may exist in vivo.

	FOOTNOTES

Manuscript received 12 December 1997. Initial reviews completed 22 January 1998. Revision accepted 27 March 1998.

	ACKNOWLEDGMENT

We thank Sid Tracy (Traco Labs, Seymour, IL) for kindly donating the corn oil.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Barnard J. A., Warwick G. Butyrate rapidly induces growth inhibition and differentiation in HT-29 cells. Cell Growth & Diff. 1993; 4:495-501[Abstract]
• Bedi A., Psricha P. J., Akhtar A. J., Barber J. P., Bedi G. C., Giardiello F. M., Zehnbauer B. A., Hammiton S. R., Jones R. J. Inhibition of apoptosis during development of colorectal cancer. Cancer Res. 1995; 55:1811-1816[Abstract/Free Full Text]
• Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976; 72:248-254[Medline]
• Butler, R. N., Goland, G., Jarrett, I. G., Fettman, M. J. & Roberts-Thomson, I. C. (1992) Glucose metabolism in proliferating epithelial cells from the rat colon. Comp. Biochem. Physiol. 101B: 573-576.
• Chang W.-C., Chapkin R. S., Lupton J. R. Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis. Carcinogenesis 1997; 18:721-730[Abstract/Free Full Text]
• Chou C. C., Lam C. Y., Yung B.Y.M. Intracellular ATP is required for actinomycin D-induced apoptotic cell death in HeLa cells. Cancer Lett. 1995; 96:181-187[Medline]
• Clausen M. R., Mortensen P. B. Kinetic studies on the metabolism of short-chain fatty acids and glucose by isolated rat colonocytes. Gastroenterology 1994; 106:423-432[Medline]
• Darcy-Vrillon B., Morel M.-T., Cherbuy C., Bernard F., Posho L., Blachier F., Meslin J. C., Duee P.-H. Metabolic characteristics of pig colonocytes after adaptation to a high fiber diet. J. Nutr. 1993; 123:234-243
• Eguchi Y., Shimizu S., Tsujimoto Y. Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res. 1997; 57:1835-1840[Abstract/Free Full Text]
• Gutmann, I. & Wahlefeld, A. W. (1974) Lactate determination with lactate dehydrogenase and NAD. In: Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), vol. 3, pp. 1464-1472. Academic Press, New York, NY.
• Heerdt B. G., Houston M. A., Augenlicht L. H. Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines. Cancer Res. 1994; 54:3288-3294[Abstract/Free Full Text]
• Heerdt B. G., Houston M. A., Augenlicht L. H. Short-chain fatty acid-initiated cell cycle arrest and apoptosis of colonic epithelial cells is linked to mitochondrial function. Cell Growth & Diff. 1997; 8:523-532[Abstract]
• Herwig S., Strauss M. The retinoblastoma protein: a master regulator of cell cycle, differentiation and apoptosis. Eur. J. Biochem. 1997; 246:581-601[Medline]
• Hong M. Y., Chang W.-C., Chapkin R. S., Lupton J. R. Relationship among colonocyte proliferation, differentiation, and apoptosis as a function of diet and carcinogen. Nutr. Cancer 1997; 28:20-29[Medline]
• Kass G.E.N., Eriksson J. E., Weis M., Orrenius S., Chow S. C. Chromatin condensation during apoptosis requires ATP. Biochem. J. 1996; 318:749-752
• Kern, S. E. (1996) Oncogenes/proto-oncogenes, tumor-suppressor genes, and DNA-repair genes in human neoplasia. In: Cellular and Molecular Pathogenesis (Sirica, A. E., ed.), pp. 321-340. Lippincott-Raven, Philadelphia, PA.
• Kern, S. E. & Kinzler, K. W. (1995) Molecular genetics of colorectal carcinoma. In: Gastrointestinal Cancers: Biology, Diagnosis, and Therapy (Rustgi, A. K., ed.), pp. 413-422. Lippincott-Raven, Philadelphia, PA.
• Kovacevic Z., Morris H. P. The role of glutamine in the oxidative metabolism of malignant cells. Cancer Res. 1972; 32:326-333[Abstract/Free Full Text]
• Leist M., Single B., Castoldi A. F., Kuhnle S., Nicotera P. Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J. Exp. Med. 1997; 8:1481-1486
• Liu Y., Bhalla K., Hill C., Priest D. G. Evidence for involvement of tyrosine phosphorylation in taxol-induced apoptosis in a human ovarian tumor cell line. Biochem. Pharmacol. 1994; 48:1265-1272[Medline]
• Lupton J. R. Butyrate and colonic cytokinetics: differences between in vitro and in vivo studies. Eur. J. Cancer Prev. 1995; 4:373-378[Medline]
• Maciorowski K. G., Turner N. D., Lupton J. R., Chapkin R. S., Shermer C. L., Ha S. D., Ricke S. C. The effect of diet and carcinogen on colonic microbial populations in rats. J. Nutr. 1997; 127:449-457[Abstract/Free Full Text]
• Marsman K. E., McBurney M. I. Influence of dietary fiber consumption on oxidative metabolism and anapleurotic flux in isolated rat colonocytes. Comp. Biochem. Physiol. 1996; 126:1429-1437
• Medina M. A., Sanchez-Jimenez F., Marquez J., Quesada A. R., de Castro I. N. Relevance of glutamine metabolism to tumor cell growth. Mol. Cell. Biochem. 1992; 113:1-15[Medline]
• Mellanby, J. & Williamson D. H. (1974) Acetoacetate. In: Methods of Enzymatic Analysis (Bergmeyer H. U., ed.), vol. 4, pp. 1840-1843. Academic Press, New York, NY.
• Pedersen P. L. Tumor mitochondria and the bioenergetics of cancer cells. Prog. Exp. Tumor Res. 1978; 22:190-274[Medline]
• Piazza G. A., Rahm K.A.L., Krutzsch M., Sperl G., Paranka S. N., Gross P. H., Brendel K., Burt R. W., Alberts D. S., Pamukcu R., Ahnen D. J. Antineoplastic drugs sulindac sulfide and sulfone inhibit cell growth by inducing apoptosis. Cancer Res. 1995; 55:3110-3116[Abstract/Free Full Text]
• Reddy B. S., Maruyama H. Effect of dietary fish oil on azoxymethane-induced colon carcinogenesis in male F344 rats. Cancer Res. 1986; 46:3367-3370[Abstract/Free Full Text]
• Richter C., Schweizer M., Cossarizza A., Franceschi C. Control of apoptosis by the cellular ATP level. FEBS Lett. 1996; 378:107-110[Medline]
• Roediger, W.E.W. (1980) The colonic epithelium in ulcerative colitis: an energy deficiency disease? Lancet I: 712-715.
• Roediger W.E.W. Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 1982; 83:424-429[Medline]
• Roediger W.E.W., Truelove S. C. Method of preparing isolated colonic epithelial cells (colonocytes) for metabolic studies. Gut 1979; 20:484-488[Abstract/Free Full Text]
• Souba W. W. Glutamine and cancer. Ann. Surg. 1993; 218:715-728[Medline]
• Stefanelli C., Bonavita F., Stanic I., Farruggia G., Falcieri E., Robuffo I., Pignatti C., Muscari C., Rossoni C., Guarnieri C., Caldarera C. M. ATP depletion inhibits glucocorticoid-induced thymocyte apoptosis. Biochem. J. 1997; 322:909-917
• Williamson, D. H. & Mellanby, J. (1974) D-(-)-3-Hydroxybutyrate. In: Methods of Enzymatic Analysis (Bergmeyer H. U., ed.), vol. 4, pp. 1836-1839. Academic Press, New York, NY.
• Wu G., Thompson J. R. The effect of ketone bodies on alanine and glutamine metabolism in isolated skeletal muscle from the fasted chick. Biochem. J. 1988; 255:139-134[Medline]
• Zhang J., Lupton J. R. Dietary fiber stimulates colonic cell proliferation by different mechanisms at different sites. Nutr. Cancer 1994; 22:267-276[Medline]
• Zoran D., Barhoumi R., Burghardt R., Chapkin R. S., Lupton J. R. Diet and carcinogen alter luminal butyrate concentration and intracellular pH in isolated rat colonocytes. Nutr. Cancer 1997; 27:222-230[Medline]

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