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Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111
3To whom correspondence should be addressed. E-mail: xiang-dong.wang{at}tufts.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • retinoic acid • alcohol • IGF-I
Chronic and excessive alcohol intake is associated with a number of impairments in biochemical metabolism and nutritional status as well as a variety of chronic disorders, including liver disease, heart disease, obesity, cancer, and brain malfunction. The insulin-like growth factor-I (IGF-I)4 system plays a critical role in the regulation of cellular proliferation, differentiation, and apoptosis in a wide variety of cells and tissues (1). Liver has the highest expression of IGF-I, and circulating IGF-I in plasma is produced mainly in the liver (2). Decreased IGF-I levels in plasma and liver were reported in alcoholics (3,4) and in acute ingestion studies (5), as well as in chronic ethanol-feeding studies in animal models (6,7). Low IGF-I levels were attributed to both liver damage and metabolic dysfunction due to prolonged and excessive alcohol consumption (3,8–10).
The mechanisms by which alcohol consumption affects the IGF-I level in plasma and the expression of IGF in the liver remains unclear. Chronic alcohol intake interferes with retinoid [retinyl esters, retinol and retinoic acid (RA)] metabolism (11). In a previous study, we demonstrated that high doses of ethanol significantly reduced the RA concentration in both plasma and liver, compared with rats pair-fed an isoenergetic control diet containing the same amount of vitamin A (12). We also demonstrated that the restoration of liver RA levels by all-trans-RA supplementation suppresses alcohol-induced hepatocyte proliferation and the activation of the Jun N-terminal kinase pathway (13,14). In addition, it was reported that feeding a vitamin A–deficient diet decreased plasma IGF-I levels as well as mRNA transcription levels in liver, lung, testis, and heart in Japanese quail and rats (15). Although these studies offer indirect evidence regarding underlying biochemical mechanism(s) for the interference of RA signaling by alcohol and alcohol-induced hepatic injury, it is not known whether RA supplementation in alcohol-fed rats can restore normal hepatic IGF-I expression and plasma IGF-I concentrations. In the present study, we examined both plasma IGF-I levels and hepatic IGF-I mRNA expression in alcohol-fed rats that were or were not supplemented with RA for 6 mo.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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130–150 g; Charles River Breeding Laboratory) were distributed, by weight matching, into 1) control group (C); 2) ethanol-fed group (E); and 3) ethanol-fed plus dietary all-trans-RA supplementation group (ERA). All ethanol-fed groups were fed the Lieber-DeCarli liquid diet containing 36% of total energy as ethanol (6.2% v/v). In the control diet, ethanol was replaced by an isoenergetic amount of maltodextrin (Purina Test diets, Richmond, IN). Both diets contained 16.4% of total energy as protein and 35% as fat; 48.6% of total energy was provided from carbohydrates in the control diet, whereas 12.6% of total energy was from carbohydrates in the ethanol diet. The Lieber-DeCarli liquid diet contained vitamin A (0.9 mg/4.184 MJ) in an amount sufficient for both the control and experimental groups. There was no RA in this base liquid diet, as analyzed by HPLC (14). For dietary RA supplementation, all-trans-RA [100 µg/kg body weight (BW) (Sigma Chemical) dissolved in 95% ethanol] was added directly into the liquid diet every day. This dosage of RA supplementation was shown to restore alcohol-reduced RA levels to normal levels in our previous study (14). Because the liquid diet provides physiologic amounts of fluid, extra water was not given. Rats in the C and E groups were pair-fed the mean intake of the ERA group for 6 mo. Body weights of the rats were recorded weekly. During the 6-mo experiment, 3 rats in the E group and 1 rat in the ERA group died. At the end of the experimental period, all rats were terminally exsanguinated under AErrane (Fort Dodge Animal Health) anesthesia. Liver tissue and plasma were collected, frozen under liquid nitrogen, and stored at –80°C for further analysis. HPLC analysis. A gradient reverse phase HPLC was used for the analysis of retinoids as described previously (14).
Plasma IGF-I concentration. Rat plasma levels of IGF-I were measured in triplicate by enzyme immunoassays (EIA) using a Rat IGF-I EIA kit from Diagnostic Systems Laboratory as described in the manual. The concentration of plasma IGF-I was calculated on the basis of a standard curve measured with recombinant rat IGF-I at the same time. The sensitivity of the assay was 30 µg/L, and the CV of the assay ranged from 5 to 10%.
Hepatic IGF-I mRNA expression. Total RNA was extracted from liver tissue by TriPure Isolation Reagent (Roche) as the protocol indicates. A 291-bp IGF-I cDNA fragment was obtained by RT-PCR, and cloned into a pCR II-TOPO vector (Invitrogen). A biotin-labeled IGF-I probe was generated by in vitro transcription using the pCR II-TOPO/IGF-I vector as a template with Biotin RNA labeling mix (Roche). A biotin-labeled 28S rRNA probe was synthesized using the same method with the template provided in the RPA III kits (Ambion). Rat liver IGF-I mRNA expression was determined with a ribonuclease protection assay using RPA III kits as indicated in the manual. The relative IGF-I density of IGF-I mRNA was normalized using a densitometric value of 28S rRNA. All of the measurements were repeated 3 times.
Statistical analysis. All group values are expressed as means ± SEM. Group means were compared using one-way ANOVA followed by a Tukey’s honestly significant different test to adjust for multiple comparisons. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Hepatic retinoid concentration. Hepatic retinol concentration in rats that consumed ethanol (E group) did not differ from the C group (Table 1). In the ERA group, the hepatic retinol concentration was greater than in the other groups (Table 1). Concentrations of retinyl palmitate, a retinyl ester stored in the liver, were significantly lower in ethanol-fed rats that were or were not supplemented with RA compared with the C group (Table 1). Hepatic RA tended to be lower (P = 0.06) in the E group than in controls and tended to be restored (P = 0.08) in the ERA group.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The important observation in the present study is that alcohol-impaired hepatic RA status may contribute to the decreased plasma IGF-I levels and hepatic IGF-I expression seen in ethanol-fed rats. In agreement with previous studies showing that chronic ethanol feeding for 4.5 mo decreased plasma IGF-I levels in rats (20), we showed that both plasma IGF-I concentration and hepatic mRNA expression of IGF-I were decreased in rats fed an ethanol-based diet for 6 mo. The correlation between hepatic IGF-I mRNA expression and plasma IGF-I concentration in this study indicated that the change in plasma IGF-I level was due in part to the decrease in hepatic IGF-I synthesis. We also showed, for the first time, that the alcohol-reduced plasma IGF-I levels and hepatic IGF-I expression can be partially restored by RA supplementation, whereas RA supplementation did not affect the plasma IGF-I levels in rats fed the control diet (Lian, F. and Wang, X. D., unpublished results). IGF-I is an anabolic hormone that regulates important biochemical processes of the body, including glucose metabolism, protein synthesis, as well as growth and differentiation (1). The impaired IGF-I synthesis after alcohol consumption was postulated to be at least partly responsible for some alcohol-induced disorders (6,21). Therefore, the partial restoration of the normal IGF-I system by RA may provide beneficial protection against certain alcohol-related injuries. This is supported by our previous studies showing that the restoration of normal hepatic RA status by RA supplementation plays a role in the maintenance of normal hepatocyte growth in alcohol-fed rats (13,14).
The exact mechanism(s) by which RA regulates hepatic IGF-I expression is unknown. Because no retinoic acid receptor response element was found in the promoter region of IGF-I and RA supplementation did not affect plasma IGF-I level in vitamin A–sufficient rats, it seems that the regulation of IGF-I levels by RA in this study was indirect and involved other factors. This hypothesis is supported by the observation that the restoration of hepatic RA did not completely restore plasma IGF-I concentration and liver mRNA expression in alcohol-fed rats. There are several possible factors that affect RA status and IGF-I level, including growth hormone, which is a major regulator of IGF-I expression and is regulated by RA. It was shown that RA stimulated both expression and secretion of growth hormone in human pituitary cells (22,23), whereas the synthesis of growth hormone was decreased by alcohol consumption (24). In addition, IGF binding protein-3, a major binding protein for circulating IGF-I that can also be induced by RA, may be involved in the regulation of plasma IGF-I level (1). It was shown that ethanol decreases plasma IGF binding protein-3 (25). Recently, we observed that chronic alcohol intake for 6 mo reduced hepatic IGF binding protein-3 mRNA level (Lian, F. and Wang, X. D., unpublished results). However, the exact mechanism(s) relating alcohol and RA homeostasis to circulating levels of IGF-I and regulation of IGF-I expression require further investigation.
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FOOTNOTES |
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2 Present address: Department of Food & Nutrition, Kyung Hee University, Seoul, Korea.
4 Abbreviations used: BW, body weight; C, control; E, ethanol; IGF-I, insulin-like growth factor-1; RA, retinoic acid.
Manuscript received 21 July 2004. Initial review completed 5 August 2004. Revision accepted 1 September 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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