Differential GH-releasing hormone regulation of GHRH receptor mRNA expression in the rat pituitary.

To understand the capacity of growth hormone-releasing hormone (GHRH) to regulate expression of the GHRH receptor, we studied the effects of GHRH on GHRH receptor mRNA expression in immature and adult rats by use of pituitary cell culture and immunoneutralization approaches. Pituitary cell cultures from neonatal (2-day-old) and adult (70-day-old) rats were treated with GHRH for 4, 24, or 72 h. The effect of GHRH on GHRH receptor mRNA expression depended on the duration of GHRH exposure in both age groups; short-term (4 h) GHRH treatment significantly reduced GHRH receptor mRNA expression (P < 0.05), whereas intermediate treatment (24 h) restored GHRH receptor mRNA to basal levels, and long-term treatment (72 h) stimulated GHRH receptor mRNA expression (P < 0.02). The long-term stimulatory effect of GHRH on GHRH receptor mRNA expression required the presence of serum in the culture medium, and, in the absence of serum, the stimulatory effect was completely abolished. Moreover, the capacity of the pituitary to increase GHRH receptor mRNA expression in response to 72-h GHRH treatment was age dependent, with neonatal pituitaries exhibiting a much greater stimulatory effect than adult pituitaries (P < 0.025). Immunoneutralization of endogenous GHRH significantly reduced GHRH receptor mRNA expression in neonatal (P < 0.004), juvenile (P < 0.003), and mature (P < 0.004) pituitaries compared with age-matched controls. Taken together, these results indicate that GHRH is a potent regulator of GHRH receptor gene expression in immature and mature pituitaries; however, the nature and direction of GHRH regulation of its receptor depend significantly on several variables, including the duration of GHRH exposure, the presence of permissive components in serum, and the developmental stage of the pituitary.

Energy restriction modulates the development of advanced preneoplastic lesions depending on the level of fat in the diet.

Our objective was to investigate the ability of preneoplastic colonic lesions at different stages of development to respond to the growth-retarding effects of energy restriction (ER). Male Fischer 344 rats were given three injections of azoxymethane (15 mg/kg s.c.) and fed a high-fat corn oil diet for 16 weeks. This duration allowed aberrant crypt foci (ACF) to develop and acquire different growth states. ACF growth was assessed by enumerating the number of crypts per focus. At Week 16, 10 animals were killed and their colons were enumerated for ACF (baseline). The remaining animals were then allocated to four dietary groups: high-fat (23% wt/wt), low-fat (5% wt/wt), high-fat energy-restricted (HFER), or low-fat energy-restricted (LFER). After the animals were fed the experimental diets for six weeks, ER decreased the total number of ACF regardless of the level of fat. At Week 12, the LFER diet retarded the appearance of advanced ACF, but this was not the case for the HFER diet. Consequently, the level of fat was identified as the significant variable in affecting the number of ACF with different crypt multiplicity. The animals fed the LFER diet had the fewest tumors and microadenomas per rat. The HFER diet was ineffective in modulating tumor outcome. To our knowledge, these findings are the first to suggest that ER modulated the development of advanced ACF and colonic tumors depending on the level of fat in the diet.

Diverse effect of fish oil on the growth of aberrant crypt foci and tumor multiplicity in F344 rats.

The main objective of the present study was to investigate the amenability of preneoplastic lesions at different developmental stages to the growth-regulatory effects of two types of dietary lipids. F344 male rats were given three injections of azoxymethane (15 mg/kg) and fed a low-fat corn oil diet for 12 weeks to allow preneoplastic lesions or aberrant crypt foci (ACF) to develop. At this time, the colons of rats had a large number of ACF exhibiting various crypt multiplicities (number of crypts/focus). These rats were then randomly allocated to three dietary groups: high-fat corn oil (HFC), high-fat fish oil (HFF), and low-fat corn oil (LFC). The number and crypt multiplicity of ACF and adenomatous lesions were determined after 6 and 12 weeks of dietary intervention. After six weeks, the HFF group had the highest number of ACF of all crypt multiplicities and microadenomas among the dietary groups. After 12 weeks of feeding, the HFC diet increased the number of tumors without significantly changing the number of ACF. In contrast, the HFF diet increased significantly (p < 0.05) the number of ACF with higher crypt multiplicity without affecting the number of tumors. Consequently, the total number of tumors per group in decreasing order was as follows: HFC > LFC > HFF. These findings suggest that dietary lipids varying in fatty acid composition, namely corn oil and fish oil, exerted a growth-enhancing and -inhibiting effect, respectively, on different preneoplastic stages in a selective and differential manner. Most notably, transition of microscopic preneoplastic lesions to macroscopic lesions (microadenomas or adenomas) appears to be retarded by an HFF diet.

A novel methodological approach to study the level of specific protein and gene expression in aberrant crypt foci putative preneoplastic colonic lesions by Western blotting and RT-PCR.

Aberrant crypt foci (ACF) represent microscopic preneoplastic lesions, present in the carcinogen-treated rodent colons. The cellular and molecular changes occurring within these lesions may provide important clues to the sequence of events leading to advanced preneoplastic or neoplastic lesions. The main objective of this investigation was to determine whether intact mRNA and protein can be isolated from fixed tissue and studied. A pure population of ACF was harvested from colonic tissue that had been preserved in 70% ethanol or 10% buffered formalin, by using a dissecting microscope and plucking the ACF out using fine forceps. The standard procedure of isolating RNA was performed successfully on ACF and normal tissue preserved in 70% ethanol. The expression of a housekeeping gene, beta-actin was demonstrated. Analysis of ethanol-preserved ACF for phosphorylated proteins was carried out by immunoblotting. The present study demonstrated that ACF are easily harvested from fixed tissues and that intact RNA and protein can be isolated from ACF or normal epithelium which can be used in techniques such as immunoblotting and RT-PCR.

Inability of low- or high-fat diet to modulate late stages of colon carcinogenesis in Sprague-Dawley rats.

The main objective of the present proposal was to investigate the effect of feeding a low- or high-fat diet in the early and late stages of colon carcinogenesis. Sprague-Dawley male rats were injected with azoxymethane (20 mg/kg/week) for 2 weeks. One week later they were randomly allocated to eat a low-fat (4% beef tallow + 1% corn oil) or a high-fat (18.6% beef tallow + 4.7% corn oil) diet (LF or HF). After 10 weeks of feeding, 10 animals per group were killed, and their colons were evaluated for tumors. The remaining animals in each group were divided further into LF and HF groups. The four experimental groups consisted of groups receiving LF or HF diet throughout the study (LF-LF or HF-HF) and the groups fed LF or HF diet for the first 10 weeks, then assigned the alternate diet for the remainder of the duration (LF-HF or HF-LF). By week 26, the remaining animals were killed, and their colons were evaluated for the number, location and size of tumors. The tumor incidence in the HF-HF and HF-LF groups were higher than the LF-LF and LF-HF groups (81.6 and 84.8% versus 71.4 and 60.0%). Tumor multiplicity ranged from 1.86 +/- 0.26 to 2.54 +/- 0.33 in all groups. The average size of tumors and total tumor area/rat were affected significantly by the time at which the diet was fed. Average size and total tumor area in the animals fed HF diet during early stages (HF-HF and HF-LF) were significantly higher than those fed the LF diet during the early stages. Late intervention by specific diets did not affect tumor outcome. Sequential enumeration of aberrant crypt foci of different growth features representing early preneoplastic stages corroborated the findings of the tumor outcome. It was concluded that early preneoplastic stages were more sensitive than their advanced counterparts to the dietary interventions of the present study.

Modulation of aberrant crypt foci by dietary fat and caloric restriction: the effects of delayed intervention.

Recent investigations have established that caloric restriction (CR) reduces end tumor incidence in the rat colon. The present study was conducted to determine whether CR at the level of 20% of the ad libitum (AL) intake and dietary fat would alter the growth of intermediate preneoplastic colonic aberrant crypt foci (ACF). F344 rats were given injections of 15mg/kg azoxymethane, fed AL for 11 weeks, and then allocated to 1 of 4 dietary groups (n-20/group): high fat (23% w/w) or low fat (5% w/w) AL (HFAL, LFAL), or high fat or low fat CR (HFCR, LFCR). After 4 weeks only the HFAL and HFCR groups had identifiable adenomas with incidences of 50 and 30%, respectively. There was a significant positive correlation between total fat consumed/day (grams) and the number of ACF with 4-6 crypts focus. After 12 weeks of feeding, the total number of ACF was lower (P < or = 0.05) in the CR groups relative to the AL groups in both the high and low fat diets. The number of ACF with 4-6 crypts/focus and > 6 crypts/focus were lower in the LFCR group compared to the LFAL group, whereas the number of ACF with 1-3 crypts/focus was lower in the HFCR group compared to the HFAL group. CR was the main variable affecting the number and growth of ACF at week 12. Positive correlations were demonstrated between increased mean daily intake of energy and the number of total ACF/colon, ACF with 4-6 crypts/focus, and ACF with > 7 crypts/focus at week 12. Cell proliferation indices did not correlate with ACF or tumor incidence data. These findings demonstrated that (a) dietary fat affects tissue growth characteristics more rapidly than CR, (b) CR alters the development of ACF depending on the level of fat and experimental duration, and (c) ACF with varying growth features respond differently to CR and dietary fat. These findings also suggest that subtle dietary manipulations in fat and caloric content used at the later stages of colon carcinogenesis can modulate tumor development.