Effects of aging and caloric restriction on IGF-I, IGF-I receptor, IGFBP-3 and IGFBP-4 gene expression in the rat stomach and colon

Regulatory Peptides 89 (2000) 37–44 www.elsevier.com / locate / regpep

Effects of aging and caloric restriction on IGF-I, IGF-I receptor, IGFBP-3 and IGFBP-4 gene expression in the rat stomach and colon a

b

a,c

a,c ,

Lance M. Hallberg , Yuji Ikeno , Ella Englander , George H. Greeley Jr. a

*

Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555 -0725, USA b Department of Physiology, University of Texas Health Science Center, San Antonio, TX, USA c Shriners Hospitals for Children, Galveston, TX, USA Received 6 September 1999; received in revised form 31 December 1999; accepted 5 January 2000

Abstract The purpose of this study was to determine the effects of aging and caloric restriction (CR) on insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), IGF-binding protein-3 (IGFBP-3) and IGFBP-4 expression in the stomach and colon of male Fischer 344 rats. Stomach and colonic RNA were prepared from ad libitum (AL) fed or long-term CR rats. Stomach IGF-I, IGFBP-3 and IGFBP-4 mRNA levels increased significantly (P # 0.05), while colonic IGF-I mRNA levels were unchanged in aged AL rats. In aged CR rats, stomach IGFBP-3 mRNA levels decreased. Stomach and colonic IGF-IR mRNA levels declined with aging in AL and CR rats (P # 0.05). Colonic IGFBP-3 mRNA levels decreased significantly with aging in AL rats. There were no changes in colonic IGFBP-4 mRNA levels in aged AL or CR rats. Increased expression of stomach IGF-I, IGFBP-3 and IGFBP-4 in aged AL rats suggests that the stomach attempts to preserve IGF activity by increasing local expression of IGF-I and IGFBPs. Because the aging colon has a propensity to develop cancer, it may adapt to increased colonic IGF-I expression by reducing IGF-IR and IGFBP-3 expression. Additionally, CR lowers colonic IGF-I expression in aged rats (24 months) which may also be a protective adaptive mechanism.  2000 Elsevier Science B.V. All rights reserved. Keywords: Gastrointestinal; Insulin-like Growth Factor; Insulin-like Growth Factor Binding Proteins

1. Introduction Abundant data suggest that the insulin-like growth factor system (IGF) plays a major role in the regulation of mucosal epithelial proliferation in the intestine [1–6]. The IGF system is composed, in part, of IGF-I, the IGF-I receptor (IGF-IR) and IGF binding proteins (IGFBPs). IGF-I and the IGF-I receptor (IGF-IR) are expressed in the gastrointestinal tract [7] and treatment of rats or mice with IGF-I stimulates intestinal growth in a potent fashion [1–6]. Additionally, in the rat, intestinal expression of IGF-I is increased after small bowel resection, implying *Corresponding author. Tel.: 1 1-409-772-2094; fax: 1 1-409-7726368. E-mail address: [email protected] (G.H. Greeley Jr.).

that the IGF system participates in the regeneration of the intestinal mucosa [3,8]. A family of at least six different IGF binding proteins (IGFBPs) regulates IGF-I activity [9]. IGFBPs apparently modulate IGF-I action partly by affecting its half-life and tissue distribution [9,10]. IGFBPs are expressed in the gastrointestinal tract [11]. Earlier reports indicate that gastrointestinal function changes in aging laboratory animals and humans. Some examples are: reduced basal and gastrin-induced gastric acid secretion in aging rats [12]; decreased basal pepsin secretion and mucosal pepsinogen levels in aging humans [13]; diminished gastric mucosa integrity and gastric atrophy in humans with aging [13]; and decreased absorption of carbohydrate loads, zinc, calcium, vitamin B 12 and iron with aging in humans [13–15]. Caloric restriction (CR) has been shown to prolong

0167-0115 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 00 )00095-1

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lifespan in rodents [16–19]. The effects of CR and aging on the IGF system in the liver and other tissues have been examined. Hepatic IGF-I, IGF-IR and IGFBP mRNA levels change with aging in rats [20,21]. CR causes a reduction in hepatic and kidney IGF-I and IGFBP mRNA levels in rats [20,22]. In humans, skeletal IGF-I and IGFBP-3 mRNA expression decrease with aging [23,24]. IGF-I is an important anabolic factor that regulates expression of multiple proteins with diverse functions [25]. IGF-I may influence effector systems that control motility, digestion absorption and other functions in the gastrointestinal tract. Age-related changes in the IGF system may affect these systems. Whether the gastrointestinal IGF system is influenced by aging or CR is not known. The purpose of the present study, therefore, was to determine the effects of aging and CR on expression of IGF-I, IGF-IR, IGFBP-3 and IGFBP4 in the stomach and colon in Fischer 344 rats.

2. Materials and methods

2.1. Animals Fischer 344 rats were used in this study since Fischer 344 rats are the preferred rat for aging studies. Male Fischer 344 rats were purchased as weanlings (26–30 days of age) from the Kingston, NY, plant of the Charles River Laboratories. Rats were maintained according to the guidelines of the University of Texas Health Science Center, San Antonio, TX. Rats were maintained in a barrier facility and were housed singly in plastic cages with wire-mesh floors suspended on the Hazleton-Enviro Rack System (Hazleton System, Aberdeen, MD) in order to maintain specific pathogen-free conditions. The barrier facilities have been described in detail previously [16]. A 12:12 h light / dark cycle was used. Rats were fed ad libitum until 6 weeks of age. To prevent the occurrence of chronic nephropathy, which is a major cause of fatality in Fischer 344 rats, soy protein was used as the protein source during the entire course of the experiment [26]. At 6 weeks of age, 72 rats were randomly assigned to two groups: Rats were either fed ad libitum (AL) or were calorie restricted (CR) by reducing the food intake to 60% of the food intake of control rats. Food intake of each AL rat was measured as described previously [16]. Each CR rat received a daily allotment of food approximately 1 h before the start of the dark phase of the light cycle (3:00 pm). Rats were sacrificed by decapitation at 6, 12, 18 and 24 months (N 5 9 rats / group). Sacrifice of rats and tissue collection were done in the morning and lasted approximately 2–3 h. The gastrointestinal tract was immediately removed from both ad libitum (AL) fed and caloricrestricted (CR) rats, frozen in liquid nitrogen, and stored in a 2 808C freezer. Tissues were shipped on dry ice to The University of Texas Medical Branch at Galveston and

processed for this study. In this study, the stomach fundus and the combined segments of the descending, mid- and ascending colon for each rat were used for preparation of stomach and colonic RNA extracts, respectively.

2.2. Analysis of IGF-I, IGF-IR, IGFBP-3 and IGFBP-4 mRNA levels Total RNA from the rat stomach and colonic tissues was isolated by homogenizing the stomach or colon in 4 M guanidine thiocyanate (including 25 mM sodium citrate [pH 7.0], 0.5% sodium N-lauroylsarcosine, and 0.1 M 2-mercaptoethanol). Total RNA was purified by centrifugation through a CsCl density gradient (2 ml, 5.7 M, 18 h, 30 000 rpm) as previously described [27]. In some cases, poly(A)1 RNA was prepared using oligo (dT) (Stratagene). RNA concentrations were determined spectrophotometrically at 260 nm. We prepared mini-Northern blots in order to monitor washing stringency of the slot blots. Ten micrograms of poly(A)1 was separated by electrophoresis on a 1% denaturing agarose gel in 1X MOPS (20 mM 3-[N-morpholino] propanesulfonic acid [pH 7.0]) containing 8 mM sodium acetate and 1 mM EDTA (pH 8.0). Gel-separated poly(A)1 mRNA was transferred onto nitrocellulose filters (Schleicher and Schuell, Keene, NH) by capillary blotting in 20X standard saline citrate (SSC) (20X SSC 5 3 M sodium chloride, 0.3 M trisodium citrate, and dihydrate, pH 7). RNA was cross-linked to the nitrocellulose filter by baking in a vacuum at 808C for 2 h. RNA in gels and on filters were visualized with ethidium bromide staining under UV transillumination. For slot blot analysis, 5 mg of total RNA was applied onto nitrocellulose filters using a Minifold II slot blot apparatus (Schleicher and Schuell, Keene, NH). RNA was cross-linked to the filter by baking (2 h, 808C) in a vacuum oven. Slot blots and mini-Northern blots were processed simultaneously to monitor appropriate slot blot washing stringency. Membranes were prehybridized and hybridized in a buffer containing 5X SSPE (20X SSPE 5 2.98 mM sodium chloride, 0.2 M sodium phosphate, 0.02 M EDTA, pH 7.4), 5X Denhart’s solution (100X 5 2% solution of BSA, Ficoll-400, polyvinylpyrrolidone), 0.1% sodium dodecyl sulfate (SDS), 100 mg ml 21 sonicated salmon sperm DNA and 50% formamide. Membranes were prehybridized overnight at 658C and then hybridized with a 32 P-labeled IGF-I, IGF-IR, IGFBP-3 or IGFBP-4 cRNA probe (5 3 10 26 disintegrations min 21 ml 21 buffer) for 24 h. Antisense cRNA probes were produced by using SP6 polymerase-directed transcription of plasmids encoding IGF-I, IGF-IR, IGFBP-3 or IGFBP-4 sequences in the presence of [a- 32 P] CTP ( | 3000 Ci mmol 21 ) and the Riboprobe Gemini system (Promega, Madison, WI) according to the manufacturer’s directions. The rodent IGF-I cDNA was provided by C. Roberts [28] containing a 404-bp EcoRI restriction fragment. The IGF-IR cDNA was

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provided by D. LeRoith [29] containing a 310-bp EcoRI restriction fragment. The IGFBP-3 and IGFBP-4 cDNAs were provided by S. Shimasaki [30,31]. The IGFBP-3 cDNA contains a 699-bp ApaI restriction fragment and the IGFBP-4 probe contains a 444-bp SmaI restriction fragment. To correct for loading errors, slot blot membranes were rehybridized with a rat ribosomal 18S-cDNA probe. Autoradiographs were densitometrically scanned (BioImage Visage 60, Ann Arbor, MI) and quantified.

2.3. Statistical analysis Data were analyzed using analysis of variance for a two-factor factorial experiment. The two factors are age (6, 12, 18 and 24 months old) and diet (ad libitum and calorie restricted). Fisher’s least-significant difference procedure is used for multiple comparisons with Bonferroni adjustment for the number of comparisons. The 0.05 level of significance is used for an experiment-wise error rate.

3. Results

3.1. Effects of aging and caloric restriction on IGF-I mRNA levels in rat stomach and colon In AL rats (Fig. 1), stomach IGF-I mRNA levels increased by approximately 100% in 18- and 24-month-old rats when compared to 6-month-old AL rats (mean6S.E.: 0.3960.04 vs. 0.806 0.05, 0.8360.06, P # 0.05). Stomach IGF-I levels did not change significantly in CR rats with aging (0.6960.05 vs. 0.5660.05, 0.6260.09, 0.6960.15). Stomach IGF-I mRNA levels were higher by 74% in 6-month-old CR rats when compared to 6-month-old AL rats (0.6960.05 vs. 0.3960.04, P # 0.05). Stomach IGF-I mRNA levels were significantly lower in CR rats when compared to AL rats at 24 months of age (0.5660.07 vs. 0.8360.06, P # 0.05). Colonic IGF-I mRNA levels in AL fed rats did not vary significantly with age (0.5560.05 vs. 0.4160.05, 0.4560.04, 0.4960.06) (Fig. 2). Colonic IGF-I mRNA levels in 24-month-old CR rats decreased significantly ( | 70%) when compared to 6- and 12-month-old CR rats (0.7460.10, 0.6160.07 vs. 0.2160.03, P # 0.05). In 24month-old rats, colonic IGF-I mRNA levels in CR rats decreased significantly when compared to AL rats.

3.2. Effects of aging and caloric restriction on IGF-I receptor mRNA levels rat in stomach and colon Stomach IGF-IR mRNA levels (Fig. 3) decreased (50%) in 24-month-old AL rats when compared to 12- and 18month-old AL rats (0.5660.08 vs. 1.0560.11, 1.0960.13,

Fig. 1. Top: Effects of aging and caloric restriction (CR) on stomach IGF-I mRNA levels. Slot blots were hybridized with specific 32 P-labeled rat IGF-I cRNA probe. Blots were stripped and rehybridized with an a- 32 P-labeled rat 18S ribosomal probe to monitor differences in loading. mRNA was visualized by autoradiography. Only slots of three rats are shown in each group (N 5 9 rats / group). Bottom: Stomach IGF-I mRNA levels in rats at various ages fed either ad libitum (AL) or caloricrestricted (CR) diets. CR rats received 60% of the daily caloric intake of AL rats. The data are shown as the ratio of stomach IGF-I mRNA over rat 18S ribosomal mRNA densitometric readings. (a) P # 0.05 vs. 6-monthold rats of the respective group; (b) P # 0.05 vs. 12-month-old rats of the respective group; (c) P # 0.05 ad libitum vs. caloric-restricted rats.

P # 0.05). IGF-IR mRNA levels also declined in 24month-old CR rats ( | 45%) when compared to 6- and 18-month-old CR rats (0.5560.08 vs. 1.1160.04, 0.9260.12, P # 0.05). Colonic IGF-IR mRNA levels in 12-, 18- and 24-monthold AL rats (Fig. 4) declined significantly when compared to 6-month-old AL rats (0.8960.13, 0.8060.04, 0.5260.05 vs. 1.5960.09, P # 0.05). Colonic IGF-IR also declined in 24-month-old CR rats when compared to younger (6-, 12-, 18-month-old) CR rats (0.34 60.07 vs. 0.9860.06, 0.8960.09, 1.0560.10, P # 0.05). Colonic IGF-IR mRNA levels in CR rats declined when compared to AL rats only in young (6-month-old) rats (0.9860.06 vs. 1.5960.09, P # 0.05).

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Fig. 2. Colonic IGF-I mRNA levels in rats at various ages fed either ad libitum (AL) or caloric-restricted (CR) diets. (a) P # 0.05 vs. 6-month-old rats of the respective group; (b) P # 0.05 vs. 12-month-old rats of the respective group; (c) P # 0.05 ad libitum vs. caloric-restricted rats.

Fig. 3. Stomach IGF-IR mRNA levels in rats at various ages fed either ad libitum (AL) or caloric-restricted (CR) diets. (a) P # 0.05 vs. 6-month-old rats of the respective group; (b) P # 0.05 vs. 12-month-old rats of the respective group; (c) P # 0.05 vs. 18-month-old rats of the respective group.

3.3. Effects of aging and caloric restriction on IGFBP-3 and IGFBP-4 RNA levels in rat stomach and colon In AL rats, stomach IGFBP-3 mRNA levels increased significantly in 18-month-old rats when compared to 6and 12-month-old rats (0.5160.06 vs. 0.2860.06, 0.2660.03, P # 0.05) (Fig. 5). In CR rats, stomach IGFBP-3 levels decreased significantly at 24 months when compared to 12-month-old CR rats. When compared to AL rats, CR did not affect stomach IGFBP-3 levels. Stomach IGFBP-4 mRNA levels increased at 18 months when compared to 6- and 12-month-old AL rats

Fig. 4. Top: Effects of aging and caloric restriction (CR) on colonic IGF-IR mRNA levels. Slot blots were hybridized with specific 32 Plabeled rat IGF-IR cRNA probe. Blots were stripped and rehybridized with an a- 32 P-labeled rat 18S ribosomal probe to monitor differences in loading. mRNA was visualized by autoradiography. Only slots of three rats are shown in each group (N 5 9 rats / group). Bottom: Colonic IGF-IR mRNA levels in rats at various ages fed either ad libitum (AL) or caloric-restricted (CR) diets. (a) P # 0.05 vs. 6-month-old rats of the respective group; (b) P # 0.05 vs. 12-month-old rats of the respective group; (c) P # 0.05 vs. 18-month-old rats of the respective group; (d) P # 0.05 ad libitum vs. caloric-restricted rats.

(1.1360.12 vs. 0.7360.08, 0.6760.04, P # 0.05) (Fig. 6), and increased at 24 months when compared to 12-monthold AL rats (1.0560.112 vs. 0.6760.04). Aging did not affect IGFBP-4 expression in CR rats. Additionally, CR did not affect IGFBP-4 levels when compared to AL rats over age. In AL rats, colonic IGFBP-3 mRNA levels significantly declined in 12-, 18- and 24-month-old AL rats when compared to 6-month-old AL rats (0.6460.16 vs. 0.2860.04, 0.2960.05, 0.1860.02, P # 0.05) (Fig. 7). Aging showed no significant effect on colonic IGFBP-3 levels of CR rats. Additionally, CR did not significantly

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Fig. 5. Stomach IGFBP-3 mRNA levels in rats at various ages fed either ad libitum (AL) or caloric-restricted (CR) diets. (a) P # 0.05 vs. 6-monthold rats of the respective group; (b) P # 0.05 vs. 12-month-old rats of the respective group.

Fig. 6. Stomach IGFBP-4 mRNA levels in rats at various ages fed either ad libitum (AL) or caloric-restricted (CR) diets. (a) P # 0.05 vs. 6-monthold rats of the respective group; (b) P # 0.05 vs. 12-month-old rats of the respective group.

Fig. 7. Top: Effects of aging and caloric restriction (CR) on colonic IGFBP-3 mRNA levels. Slot blots were hybridized with specific 32 Plabeled rat IGFBP-3 cRNA probe. Blots were stripped and rehybridized with an a- 32 P-labeled rat 18S ribosomal probe to monitor differences in loading. mRNA was visualized by autoradiography. Only slots of three rats are shown in each group (N 5 9 rats / group). Bottom: Colonic IGFBP-3 mRNA levels in colon in rats at various ages fed either ad libitum (AL) or caloric-restricted (CR) diets. (a) P # 0.05 vs. 6-month-old rats of the respective group.

affect IGFBP-3 expression when compared to AL rats. Colonic IGFBP-4 mRNA levels in AL and CR rats showed no significant changes with aging (Fig. 8).

4. Discussion The purpose of the present study was to determine the effects of aging and caloric restriction (CR) on the IGF system in the stomach and colon of Fischer-344 rats. The IGF system in the gastrointestinal tract appears to play a major role in the stimulation of intestinal growth and maintenance of mucosal proliferation [2–5].

Fig. 8. Colonic IGFBP-4 mRNA levels in rats at various ages fed either ad libitum (AL) or caloric-restricted (CR) diets.

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One of the primary findings of this study is that IGF-I expression increases in the stomach with aging in AL rats. In this study, colonic IGF-I mRNA levels did not change with aging in AL rats. However, in another study from our laboratory, we have found that colonic IGF-I mRNA levels increased significantly with aging. In this parallel study, colonic IGF-I expression was analyzed using poly(A)1 RNA and Northern blotting analysis. Northern blotting analysis of poly(A)1 may represent a more specific method than slot blotting of total RNA and may be a more representative measure of IGF-I expression since IGF-I mRNA consists of multiple-sized transcripts [8,20,21,32]. Body and tissue growth have been attributed to the stimulatory effects of growth hormone on the liver, which is mediated in an endocrine fashion, through secretion of IGF-I [33,34]. However, recent studies indicate that local production of IGF-I is more significant to growth than circulating IGF-I [35,36]. Therefore, it is reasonable to propose that stomach and colonic IGF-I mRNA levels increase with aging, in order to compensate for the agingassociated reduction in serum IGF levels [20,21]. Another finding of the present study is that stomach and colonic levels of IGF-IR decrease with aging in AL rats. Since the IGF system is composed of multiple interdependent components, suggesting a mechanism for the age-associated changes in the individual components necessitates consideration of all components of the IGF system. Presumably, changes in IGF-IR and IGFBP expression with aging occur in order to modulate IGF-I activity. In other words, age-related changes in IGF-IR and IGFBPs may be secondary to changes in tissue IGF-I expression and occur in order to either increase or decrease IGF-I activity in the gastrointestinal tract. Therefore, the age-related drop in stomach IGF-IR expression may be linked to the rise in stomach IGF-I expression, in order to regulate stomach IGF-I activity. We also suggest that IGFBP-3 and IGFBP-4 expression increase in the stomach, in response to decreasing serum IGF-I levels with aging, in order to preserve stomach IGF-I activity. IGFBPs can function to either enhance or reduce IGF-I activity [37,38]. For example, low levels of IGFBP-3 enhance the stimulatory effects of IGF-I whereas high levels of IGFBP-3 appear to block IGF-I action [37,39]. Because IGFBPs and IGF-IR are part of the IGF-I pathway, IGF-IR may decrease with aging as stomach IGFBP-3 and IGFBP-4 expression increases. The decrease of colonic IGF-IR with aging may be an age-related adaptation to the increased colonic IGF-I expression that occurs with aging and an attempt to decrease colonic IGF-I activity. The colon has a propensity for development of cancer with aging and IGF-I is exceptionally mitogenic and anti-apoptotic [2,4,8,32,40– 45]. In contrast to the stomach, colonic IGFBP-3 expression decreased with aging whereas IGFBP-4 expression was unchanged. As mentioned earlier, since the colon is prone to develop cancer with aging, the colon may adapt to

increased colonic IGF-I expression by reducing local IGFIR and IGFBP-3 expression with aging and thereby attempt to decrease IGF-I activity. Additionally, the lack of change in IGFBP-4 expression with aging suggests that IGFBP-3 is more important in regulation of IGF-I activity in the colon, in contrast to the stomach. An important finding of the present study is that caloric restriction reduced colonic IGF-I expression at 24 months. This age-related reduction in colonic IGF-I expression with caloric restriction may be an adaptive mechanism to decrease cancer since the colon has a high incidence of colon cancer with aging [40–45]. In contrast, the stomach IGF system was not affected in a major way. This finding is a surprise in view of the earlier reports that the gastrointestinal IGF system is sensitive to metabolic changes [46]. Our observations in the stomach also contradict the effects of CR on other organ systems. For instance, in rats, CR results in decreased liver IGF-I mRNA levels [20]. Short-term fasting (48 h) has also been shown to decrease liver IGF-I mRNA levels [21,47]. In the intestine, short-term fasting (3 days) can decrease jejunal IGFBP-3 mRNA levels but did not affect IGF-IR mRNA levels [48]. Although speculative, the absence of any major change in the IGF system with CR in the stomach suggests that the gastric IGF system is not affected by long-term metabolic changes. Alternatively, the gastric IGF system may be affected initially by CR but then reestablishes normal expression levels soon after CR initiation. In our study, stomach IGF-I mRNA levels were higher in 6-month-old CR rats when compared to AL rats; however, in 24-month-old rats, stomach IGF-I levels were higher in AL rats rather than in CR rats. These findings indicate that the effect of CR on the stomach IGF system is independent of age. In addition to its role as a trophic agent for the gastrointestinal tract, IGF-I is an important anabolic factor that regulates expression of multiple proteins with diverse functions [25]. Hence, alterations in the IGF-I system might affect effector systems in the gastrointestinal tract that control motility, digestion, absorption and other functions which change with aging.

Acknowledgements Supported by grants from the National Institutes of Health (PO1 DK35608, RO1 DK15241, AG 01188).

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