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Effect of leptin on liver alcohol dehydrogenase
BBRC Biochemical and Biophysical Research Communications 337 (2005) 1324–1329 www.elsevier.com/locate/ybbrc
Effect of leptin on liver alcohol dehydrogenase Esteban Mezey *, Lynda Rennie-Tankersley, James J. Potter Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2195, USA Received 21 September 2005 Available online 10 October 2005
Abstract The effect of leptin on liver alcohol dehydrogenase (ADH) was determined in male rats. Administration of one or three daily doses of leptin (1 lg/g of body weight intraperitoneally) increased ADH activity. Leptin enhanced ADH synthesis without an effect on ADH degradation. Leptin did not change ADH mRNA, indicating that the effect of leptin in enhancing ADH occurs at the post-transcriptional level. Leptin increased eukaryotic initiation factor (eIF) 2a, eIF2B activity, and the eIF4E–eIF4G complex, while it decreased the inhibitory complex of eIF4E with the eIF4E-binding protein-1 (4E-BP1). Leptin increased mammalian target of rapamycin (mTor) that phosphorylates 4E-BP1. In conclusion, leptin increases liver ADH activity and ADH protein due to an increase in synthesis which occurs at the post-transcriptional level. The effect of leptin in enhancing translational initiating factors may be of significance in the regulation not only of ADH but also of many other proteins. 2005 Elsevier Inc. All rights reserved. Keywords: Alcohol dehydrogenase; Leptin; Eukaryotic initiation factors; Mammalian target of rapamycin
Liver alcohol dehydrogenase (ADH:alcohol:NAD oxidoreductase, EC 1.1.1.1) is the principal enzyme responsible for ethanol oxidation. Liver alcohol dehydrogenase (ADH) is affected by a variety of hormones. Stress, which stimulates the hypothalamo–hypophyseal–adrenocortical axis and the sympathetic nervous system, increases the enzyme activity [1]. GH increases liver ADH due to increased synthesis that is initiated at the level of transcription [2,3]. The effects of stress and GH were associated with increases in the rate of elimination of ethanol [1–3]. Endotoxin originating from intestinal bacteria is an important mediator of hepatocellular inflammation in the intragastric feeding rat model of alcoholic liver disease [4,5]. We recently demonstrated that lipopolysaccharide (LPS), the endotoxin component of gram-negative bacteria, increased liver ADH [6]. An increase rate of formation of acetaldehyde (AC) caused by an enhanced ADH activity may contribute to worsening of alcoholic liver injury caused by endotoxin. *
Corresponding author. Fax: +1 410 955 9677. E-mail address: [email protected] (E. Mezey).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.003
Leptin is a cytokine that belongs structurally to the long-chain helical cytokine family, which includes IL-3, IL-11, and IL-12, and GH [7]. Leptin was shown to enhance liver fibrosis produced by a single dose of CCl4 or by chronic administration of thiocetamide [8,9]. Administration of LPS to rats increases plasma leptin [10]. Since GH and LPS both increase ADH, it is quite likely that leptin has a similar effect. An increase in ADH with increased formation of AC is a potential mechanism for enhancement of fibrogenesis by leptin in alcoholic liver disease. The purpose of this study was to determine the effects of leptin on liver ADH. This study shows that leptin enhances liver ADH and that this effect occurs at the post-transcriptional level. Methods Animals and materials. Male Sprague–Dawley rats were obtained from Charles River Laboratories, Wilmington, MA. All animals received humane care in compliance of the guidelines from the Animal Care and Use Committee of the Johns Hopkins University. Murine leptin was obtained from Biomol, Plymouth Meeting, PA. Sterile [4–5-3H]L-leucine and [14C]sodium bicarbonate (NaH14CO3) were obtained from ICN
E. Mezey et al. / Biochemical and Biophysical Research Communications 337 (2005) 1324–1329 Biomedicals, Irvine, CA. [8-3H]Guanosine 5 0 -diphosphate ([3H]GDP) was obtained from Amersham Biosciences, Piscataway, NJ. Animal treatment. Rats received intraperitoneal (i.p.) injections of leptin (1 lg/g of body weight) either once or for 3 consecutive days, while controls received isovolumetric amounts of saline. The animals were sacrificed at 6 and 24 h after one injection or 2 h after the third daily injection. Approximately 400–500 mg of the liver was homogenized in 4 vol of 0.25 M sucrose in 0.1 M Tris–HCl buffer, pH 7.4, centrifuged at 10,000g for 10 min, and the resulting supernatant was centrifuged at 100,000g. The 100,000g supernatant (cytosol) was used immediately for the determination of ADH activity. The remainder of the supernatant was frozen at 80 C for determinations of LDH activity and ADH protein. One section of 1.0–1.2 g of the liver was processed for RNA isolation. ADH activity. ADH activity was determined in the liver cytosol at 37 C by the method of Crow et al. [11]. The reaction mixture was 1.0 ml and consisted of 0.5 M Tris–HCl, pH 7.2, 18 mM ethanol, 2.8 mM NAD+, and 0.01 ml of the cytosol. Blank reactions were run without ethanol. One unit of enzyme activity is defined as the formation of 1 lmol of NADH per min. ADH activity was expressed per mg of protein. Cell protein was determined by the method of Lowry et al. [12]. LDH activity was determined by the method of Plagemann et al. [13]. Immunoreactive protein of ADH. Immunoreactive ADH protein was assayed by quantitative enzyme linked immunosorbent assay (ELISA) as described previously [14]. The antisera to the enzyme were produced in rabbits by subcutaneous injections of purified ADH in FreundÕs adjuvant. The antisera are purified with protein A–Sepharose CL-4B affinity chromatography prior to use in the assay. Determination of messenger RNA (mRNA) by quantitative real time PCR. Total cellular RNA was isolated using the guanidine isothiocyanate procedure of Chomcynski and Sacchi [15] as described previously [3]. The concentration of the isolated RNA was determined from the optical density at 260 nm and its purity from the 260/280 ratio. The isolated RNA was initially stored at 80 C. The cDNA template was synthesized with the Superscript III first-strand synthesis system (InVitrogen, Carlsbad, CA) using 5 lg of total RNA. ADH mRNA was determined by the TaqMan gene expression assay of Applied Biosystems, Foster City, CA. Glyceraldehyde 3 phosphate dehydrogenase (GADPH) mRNA was used as a reference control. The gene-specific oligonucleotide primers for rat ADH (#Rn01522111) and for rodent GADPH (#4308313) were obtained from Applied Biosystems. The reaction conditions were 50 C for 2 min, 95 for 10 min followed by 40 cycles of 95 C for 15 s and 60 C for 1 min. To quantify ADH gene expression, the DDCt method was used to calculate relative fold changes normalized against GADPH gene expression. ADH synthesis and degradation. To determine the effect of leptin on ADH synthesis, 16 male Sprague–Dawley rats were fasted overnight following which 8 were injected with leptin, 1 lg/g body weight i.p., while 8 were injected with isovolumetric amounts of saline. Six hours later [4–5-3H]L-leucine (100 lC/lmol per 100 g body weight in a 0.1 ml vol) was injected i.p. The animals were sacrificed 1 h after the isotope injection. The livers were removed and homogenized in 4 vol of 0.25 M sucrose in 0.1 M Tris–HCl buffer, pH 7.4, containing a proteinase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), 0.1 mM dithiothreitol (DDT), and 1 mM phenylmethanesulfonyl fluoride (PMSF). The homogenate was centrifuged and the liver cytosol was prepared as described above. A portion of the cytosol was used for the assay of ADH activity and the remainder was frozen at 80 C for determination of the incorporation of radioactive leucine into ADH. For the determination of ADH turnover, 40 rats received a single i.p. injection of 1.0 mCi of NaH14CO3 (55 mCi/mmol) per 100 g of body weight as described previously [16]. Starting immediately after the injection of the isotope, leptin (1 lg/g body weight) was given i.p. each day to one-half the rats, while the other one-half were given isovolumetric amounts of saline. Eight animals, 4 injected with leptin and 4 controls, were sacrificed on days 1, 2, 3, 4, and 5 after the administration of the isotope. The livers were processed to isolate the liver cytosol as described above. To measure the incorporation of the isotopes into ADH protein, 400 lL of cytosol was precipitated with antibody to ADH. The antigen–
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antibody complex was allowed to form for 2 h at 25 C. The antigen– antibody complexes were precipitated from the incubation mixture with protein A bearing Staphylococcus aureus cells (Pansorbin, Calbiochem, San Diego, CA). The precipitate was centrifuged at 12,000g for 3 min and the pellet was resuspended in 0.05 M Tris–HCl buffer, pH 7.4, containing 300 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 1% Na deoxycholate, and 0.025 M sucrose [17]. The pellet was precipitated and washed 3 times in the same buffer, and then resuspended in 0.125 M Tris– HCl buffer, pH 6.2, containing 6 M urea, 3% sodium dodecyl sulfate, and 5% mercaptoethanol and heated for 5 min at 95 C. After cooling, the suspension was centrifuged and the supernatant was subjected to SDS– polyacrylamide electrophoresis [16]. The gels were fixed in a solution of 28% trichloroacetic acid and stained with Coomassie brilliant blue R-250. The protein bands corresponding to the 40,000 molecular weight subunit of ADH were identified from the electrophoresis of the purified enzyme [17]. The gels were sliced, incubated into Protosol, and counted in Ecolite (ICN, Biochemicals, Aurora, OH). The radioactivity incorporated into ADH was corrected for background radioactivity in adjacent slices not containing proteins. In the case of the ADH turnover study with NaH14CO3, the fractional rates of degradation (Kd) and synthesis (Ks) were obtained from the slopes of the regression lines of the decreases in total and specific radioactivity, respectively [18]. The absolute rate of synthesis (V) was obtained by multiplying Ks by the total liver ADH activity. Western blot analysis of eukaryotic initiation factors (eIFs). Liver was homogenized in 7 vol of buffer composed of 20 mM Tris–HCl, pH 7.4, 250 mM sucrose, 100 mM KCl, 1 mM DDT, 50 mM NaF, 1 mM PMSF, 1 mM benzamidine, and 0.5 mM vanadate. The samples were centrifuged to 10,000g for 10 min and the determinations were done with the resulting supernatant as described by Lang et al. [19]. The samples were mixed with 2· Laemmli SDS buffer boiled, centrifuged, subjected to electrophoresis at 60 mA in a 12.5% polyacrylamide gel, and transferred to nitrocellulose membranes. The membranes were washed and subsequently blocked with (5% wt/vol) non-fat milk in 25 mM Tris–HCl, pH 7.4, and the membranes were incubated with goat polyclonal antibodies to either eIF2a, phosphorylated eIF2a or eIFB2e (Cell signaling Technology, Beverly, MA) at 4 C overnight. After repeated washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution, Amersham Life Science) at room temperature for 1 h. The membranes were washed and visualized by enhanced chemiluminescence reaction (ECL-plus, Amersham Biosciences). Phosphorylation of mTOR. The phosphorylation of mTor was determined in the 10,000g liver supernatants as described by Kimball et al. [20]. The Western blots were carried out with rabbit polyclonal mTor and phospho-mTOr (Ser 2448) antibodies (Cell Signaling Technology). Quantitation of eIF4E–eIF4G and eIF4E–4E-BP1complexes. The associations of eIF4E with eIF4G or 4E-BP1 were determined as described by Kimball et al. [20]. Briefly, eIF4E was immunoprecipitated from the 10,000g liver supernatant with rabbit polyclonal eIF4E antibody (Santa Cruz), the precipitate resolved by SDS–PAGE, and transferred to nitrocellulose membranes. One set of membranes was re-probed with rabbit polyclonal anti-eIF4G antibody, and another set of membranes re-probed with rabbit 4E-BP1 (both from Santa Cruz). The membranes were visualized by the enhanced chemiluminescence reaction. The blots were later inactivated and re-probed with anti-eIF4E antibody. The values obtained with the anti-eIF4G and anti-4E-BP1 antibodies were normalized to eIF4E present in the samples. Determination of eIF2B activity. Liver eIF2B activity was determined in 10,000g liver supernatants from the rate of exchange of [3H]GDP in the eIF2 [3H]GDP complex for non-radioactively labeled GDP as described by Kimball et al. [21,22]. A binary complex of eIF2 and [3H]GDP was formed by incubation for 10 min at 30 C in a mixture containing 50 mM MOPS, pH 7.4, 100 mM KCl, 1 mM DDT, 200 lg/ml bovine albumin, 1.3 lM [3H]GDP (10.7 Ci/mmol), and recombinant human liver eIF2a (Cell Sciences, Canton, MA). Two millimolar magnesium acetate was added and the binary complex was placed on ice. A 35 lL aliquot of the liver supernatant was mixed with 140 lL of a reaction buffer containing 50 mM Mops, pH 7.4, 209 lM GDP, 2 mM magnesium acetate, 100 mM
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KCl, 1 mM DDT, and 200 mg/ml bovine serum albumin plus and 87.5 lL water. The reaction was initiated by the addition of 35 lL of the eIF-2 [3H]GDP complex (1–2 pmol) and the tubes were maintained in a water bath at 30 C. Aliquots (75 lL) of the reaction mixture were removed at 1, 2, and 3 min, and placed in tubes containing 2.5 ml ice-cold reaction mixture without bovine albumin. The contents were mixed, filtered through a nitrocellulose filter disk, the filters were dissolved in Filtron X (National Diagnostic, Atlanta, GA), and the radioactivity was counted. eIF2B activity was determined from the rate of decrease in the eIF2 [3H]GDP complex bound to nitrocellulose filters. Statistical analysis. The data were analyzed with StudentÕs t test when appropriate or by analysis of variance when comparing means of more than two groups. The Duncan new multiple range test was used to estimate differences between means [23]. Equality of variances around regression lines in the ADH turnover study was tested using the F test. The significance of the difference in slopes was determined by StudentÕs t distribution as described by Bailey [24].
Results Leptin enhances ADH The administration of leptin (1 lg/g body weight) increased liver ADH activity. Fig. 1 shows the increases in ADH activity at 6 and 24 h after the administration of one dose of leptin. Furthermore, Fig. 1 also shows the increase in ADH activity after daily administration of leptin for 3 days. Leptin did not result in any significant changes in LDH activity (data not shown). ADH immunoprotein determined after 3 days of leptin administration was increased from 8.80 ± 0.69 in the controls to 14.58 ± 0.16 lmol/mg of cytosol protein in the leptin-treated rats (p < 0.001). Leptin had no significant effect on ADH mRNA at any time point. The relative values of ADH mRNA were 110 ± 2.9 and 101 ± 3.4 at 6 and 24 h after leptin administration as compared to respective saline-controls of 100 ± 2.8 and 100 ± 4.3. After administration of 3
Fig. 1. Effect of leptin on liver alcohol dehydrogenase (ADH) activity. Either one dose of leptin (1 lg/g of body weight) was injected intraperitoneally (i.p.) and the animals were sacrificed 6 and 24 h later, or 3 daily doses of leptin were given and the animals were sacrificed 2 h after the third injection (3 days). Control animals were given isovolumetric amounts of saline. ADH activity was determined in the liver cytosol. The values are expressed as means ± SE of 8 determinations. *p < 0.01 versus respective control value.
daily doses of leptin, the ADH mRNA values were 98 ± 10 and 100 ± 8 for the leptin-treated and saline-controls. Effect of leptin on ADH synthesis The effect of leptin on the synthesis of ADH was initially determined with radioactive leucine in 24 h-fasted rats. The rats were injected with [4–5-3H]L-leucine 6 h after the administration of one dose of leptin or saline and the incorporation of the isotope into ADH was determined 1 h later. Leptin resulted in a 22.3% increase in the incorporation of the labeled leucine into ADH. The values were 23.0 ± 3.1 · 103 dpm/mg protein for the leptin-treated as compared with 18.8 ± 2.6 · 103 dpm/mg protein for the saline-controls (p > 0.05). The effect of daily administration of leptin for 5 days on turnover of total radioactivity was subsequently determined with NaH14CO3 and found to be similar in leptin-treated and control rats. The fractional rate of degradation (Kd) was 0.10 for the leptin-treated and 0.11 per day for the controls rats. This corresponds to halflife (T1/2) values of 6.9 and 6.3 days for the leptin-treated and control rats, respectively. By contrast, the fractional rate of synthesis (Ks) of ADH was enhanced by the administration of leptin to 0.15 ± 0.011 for the leptin-treated as compared to 0.11 ± 0.012 for the control rats (p < 0.05). The absolute rate of synthesis (V) was increased 36% from 0.55 total liver units/day in the control rats to 0. 75 total liver units/day in the leptin-treated rats. Effects of leptin on translation Initiation of translation involves the binding of initiator methionyl RNA to the 40 S ribosomal subunit as a ternary complex with eIF2 and GTP. Leptin increased the a subunit of eIF2 (eIF2a) (p < 0.05), while it did not change significantly phospho-eIF2a, which inhibits eIF2B activity (Fig. 2). Leptin did not result in significant changes in eIFB2e, which is the catalytic subunit of eIF2B (data not shown). Leptin increased eIF2B activity, which stimulates the exchange of GTP to GDP that is required for formation of the ternary complex. The activities were 1.48 ± 0.27 and 0.82 ± 0.14 pmol of GDP exchanged per min per mg protein for the leptin-treated and saline-controls (p < 0.05). The subsequent step in translation is the binding of mRNA to the 43 S preinitiation complex which is regulated by a group of proteins forming a complex called eIF4F. The active eIF4F complex in the regulation of translation, which consists of the association between eIF4E with eIF4G, was increased by leptin (p < 0.05) (Fig. 3A). By contrast, the inactive 4E-BP-1 to eIF4E complex was decreased by leptin (p < 0.01) (Fig. 3B). The binding of 4E-BP1 to eIF4E is decreased by phosphorylation of 4EBP1, and in this study mTOR, which phosphorylates 4E-BP1, was increased by leptin (p < 0.05) (Fig. 4A), while phospho-mTOR was decreased by leptin (p < 0.05) (Fig. 4B).
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Fig. 2. Effect of leptin: on (A) eIF2a and on (B) phosphorylated eIF2a. Leptin (1 lg/g of body weight) was injected i.p. and the animals were sacrificed 6 h later. Control animals were given isovolumetric amounts of saline. The determinations were done by Western blot and quantitated by densitometry. The values are expressed as means ± SE of 8 determinations. *p < 0.01 versus control value.
Fig. 3. Effect of leptin on: (A) association of eIF4E with eIF4G assessed by immunoprecipitation (IP) with eIF4E followed by Western blot (WB) analysis with eIF4G. (B) Association of eIF4E with the inhibitory 4E-BP1 assessed by IP with eIF4E followed by WB analysis with 4E-BP1. Leptin (1 lg/g of body weight) was injected i.p. and the animals were sacrificed 6 h later. Control animals were given isovolumetric amounts of saline. The determinations were done by WB and quantitated by densitometry. The values of the association between eIF4E with either eIF4G or 4E-BP1 were normalized to the amount of eIF4E present in the samples. Relative values are expressed as means ± SE of 8 determinations. *p < 0.05 versus control value. **p < 0.01 versus control value.
Discussion This study shows that leptin enhances liver ADH protein and activity, and that this effect is due to increased enzyme synthesis. Leptin did not change ADH mRNA, indicating that the effect of leptin in enhancing ADH occurs at the post-transcriptional level. Although LPS has been shown to increase both leptin [10] and liver ADH [6], this study indicates that the effect of LPS is not mediated by leptin, since the effect of leptin, unlike that of LPS, was not associated with an increase in ADH mRNA. Leptin had no effect on ADH degradation. By contrast, castration was previously shown to enhance liver ADH due to a decrease in the rate of degradation of the enzyme [16] and, in turn, the suppressant effect of dihydrotestoster-
one on ADH was demonstrated in hepatocytes in culture to be due to increased enzyme degradation [17]. Fasting decreases ADH due to a decrease in ADH synthesis, which is associated with an increased ADH degradation [25]. The known rapid decrease in leptin levels with fasting [26] may contribute to the decrease in ADH with fasting by limiting optimal stimulation of ADH synthesis. The leptin-induced enhancement in ADH activity leading to increased formation of AC, which increases collagen production [27], is a potential mechanism for the effect of leptin on enhancing fibrogenesis in alcoholic liver disease. The effect of leptin on increasing ADH is most likely at the translational level. Previously, higher liver ADH activity in C57BL/6J (B6) mice than in B6.S congenic mice was found in the absence of differences in transcriptional rate
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Fig. 4. Effect of leptin on: (A) mTOR and on (B) phosphorylated mTOR. Leptin (1 lg/g of body weight) was injected i.p. and the animals were sacrificed 6 h later. Control animals were given isovolumetric amounts of saline. The determinations were done by Western blot using anti-mTOR and anti-phospho Ser2448 antibodies and quantitated by densitometry. The values are expressed as means ± SE of 8 determinations. *p < 0.01 versus respective control value.
indicating a post-transcriptional mechanism for regulation of ADH [28]. Leptin, in the present study, increased eIF2a and eIF2B activity, and complex formation of eIF4E with eIF4G. These changes were associated with a decrease in the inhibitory eiF4E–4E-BP1 complex. The increase in the active eIF4E–eIF4G complex in association with the decrease in the inhibitory eIF4E–4E-BP1 complex may be regulated by increases in phosphorylation of 4E-BP1 [29]. Leptin, in this study, increased mTor that phosphorylates 4E-BP1 and inactivates it [29] leading to decreased complex formation of 4E-BP1 with eIF4E. The effect of leptin on decreasing the phosphorylation of mTOR at Ser2448 may have contributed to increased availability of mTOR, however, the significance of mTOR phosphorylation remains unknown [30]. The Ser2448 phosphorylation appears to be mediated by protein kinase B rather than by autophosphorylation. In one study, acute alcohol intoxication in rats, which decreased myocardial protein synthesis, decreased myocardial mTOR phosphorylation in association with decreased 4E-BP1 phosphorylation on Ser2448 and SER2481, and increased formation of the inhibitory eIF4E–4E-BP1 complex [31]. The effect of leptin on increasing protein synthesis by mechanisms of translation could possibly be mediated by insulin which is known to increase eIF4E–eIF4G binding by increasing phosphorylation of 4E-BP1 [32]. Studies of the effects of leptin on plasma insulin have shown varied effects. In one study, administration of 2 doses of 300 lg leptin 12 h apart increased plasma glucose, and plasma insulin in fasted but not in fed mice [33]. On the other hand, infusion of leptin (10 lg/h for 48 h) decreased plasma insulin levels and insulin sensitivity determined from glucose utilization during hyperinsulinemic glucose clamp in fasted rats [34]. Both leptin and insulin which reduce food intake and body weight have in common a number of signaling pathways and influence each other. Insulin and leptin activate phosphoinositide-3-kinase through IRS-1 (insulin receptor-1) and IRS-2, respectively, and pretreatment of each reduces the expression of the receptor of the other [35].
Insulin administration increased liver ADH activity in rats [36]. In liver cell culture, although insulin has no direct effect on liver ADH [37], it had a permissive action on the activating effect of IGF-I on ADH, an effect that is due to the ability of insulin to maintain a high number of IGF-I-binding receptors in hepatocyte culture. In conclusion, this study shows that leptin increases liver ADH activity due to an increase in synthesis which occurs at the post-transcriptional level. The effect of leptin in enhancing translational initiating factors may be of significance in the regulation not only of ADH but of many other proteins. Acknowledgment This work was supported by Grant AA000626 from the United States Public Health Service. References [1] E. Mezey, J.J. Potter, R. Kvetnansky, Effect of stress by repeated immobilization on hepatic alcohol dehydrogenase activity and ethanol metabolism, Biochem. Pharmacol. 28 (1979) 657–663. [2] E. Mezey, J.J. Potter, Rat liver alcohol dehydrogenase activity. Effects of growth hormone and hypophysectomy, Endocrinology 104 (1979) 1667–1673. [3] J.J. Potter, V.W. Yang, E. Mezey, Influence of growth hormone on the synthesis of rat liver alcohol dehydrogenase in primary hepatocyte culture, Arch. Biochem. Biophys. 274 (1989) 548–555. [4] R.G. Thurman, Mechanisms of hepatic toxicity. II. Alcoholic liver injury involves activation of Kupffer cells by endotoxin, Am. J. Physiol. 275 (1998) G605–G611. [5] M. Yin, E. Gabele, M.D. Wheeler, H. Connor, B.U. Bradford, A. Dikalova, I. Rusyn, R. Mason, R.G. Thurman, Alcohol-induced free radicals in mice: direct toxicants or signaling molecules? Hepatology 34 (2001) 935–942. [6] J.J. Potter, L. Rennie-Tankersley, E. Mezey, Endotoxin enhances liver alcohol dehydrogenase by action through upstream stimulatory factor but not by nuclear factor-jB, J. Biol. Chem. 278 (2003) 4353– 4357. [7] F. Zhang, M.B. Basinski, J.M. Beals, S.L. Briggs, L.M. Churgay, D.K. Clawson, R.D. DiMarchi, T.C. Furman, J.E. Hale, H.M. Hsiung, B.E. Schoner, D.P. Smith, X.Y. Zhang, J.P. Wery, R.W.
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[22] S.R. Kimball, W.V. Everson, K.E. Flaim, L.S. Jefferson, Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes, Am. J. Physiol. 256 (1989) C28–C34. [23] R.G.D. Steel, J.H. Torrie, Principles and procedures of statistics with special reference to the biological sciences, McGraw-Hill, New York, 1960, pp. 106–112. [24] N.T.J. Bailey, Statistical methods in biology, third ed., Cambridge University Press, Cambridge, 1955, pp. 104–111. [25] W.F. Bosron, D.W. Crabb, T.A. Housinger, T.K. Li, Effect of fasting on the activity and turnover of rat liver alcohol dehydrogenase, Alcohol Clin. Exp. Res. 8 (1984) 196–200. [26] J.M. Friedman, J.L. Halaas, Leptin and the regulation of body weight in mammals, Nature 395 (1998) 763–770. [27] K. Holt, M. Bennett, M. Chojkier, Acetaldehyde stimulates collagen and noncollagen protein production by human fibroblasts, Hepatology 4 (1984) 843–848. [28] L. Tussey, M.R. Felder, Tissue-specific genetic variation in the level of mouse alcohol dehydrogenase is controlled transcriptionally in kidney and posttrascriptionally in liver, Proc. Natl. Acad. Sci. USA 86 (1989) 5903–5907. [29] A.C. Gingras, B. Raught, N. Sonneberg, Regulation of translation initiation by FRAP/mTOR, Genes Dev. 15 (2001) 807–826. [30] K. Yonezawa, K.I. Yoshino, C. Tokunaga, K. Hara, Kinase activities associated with mTOR, Curr. Top. Microbiol. Immunol. 279 (2004) 271–282. [31] T.C. Vary, G. Deiter, S.A. Goodman, Acute alcohol intoxication enhances myocardial eIF4G phosphorylation despite reducing mTOR signaling, Am. J. Physiol. Heart Circ. Physiol. 288 (2005) H121– H128. [32] W. Shen, D. Mallon, D.W. Boyle, E.A. Liechty, IGF-1 and insulin regulate eIF4F formation by different mechanisms in muscle and liver in the ovine fetus, Am. J. Physiol. Endocrinol. Metab. 283 (2002) E593–E603. [33] B. Ahren, P.J. Havel, Leptin increases circulating glucose, insulin and glucagon via sympathetic neural activation in fasted mice, Int. J. Obes. Relat. Metab. Disord. 23 (1999) 660–665. [34] W.I. Sivitz, S.A. Walsh, D.A. Morgan, M.J. Thomas, W.G. Haynes, Effects of leptin on insulin sensitivity in normal rats, Endocrinology 138 (1997) 3395–3401. [35] Y. Benomar, A.F. Roy, A. Aubourg, J. Djiane, M. Taouis, Cross down-regulation of leptin and insulin receptor expression signaling in a human neuronal cell line, Biochem J. 388 (2005) 929–939. [36] M.R. Lakshman, L.L. Chambers, S.J. Chirtel, N. Ekarohita, Rates of hormonal and nutritional factors in the regulation of rat liver alcohol dehydrogenase activity and ethanol elimination in vivo, Alcohol Clin. Exp. Res. 12 (1988) 407–411. [37] E. Mezey, J.J. Potter, L. Mishra, S. Sharma, M. Janicot, Effect of insulin-like growth factor I on rat alcohol dehydrogenase in primary hepatocyte culture, Arch. Biochem. Biophys. 280 (1990) 390–396.
Effect of leptin on liver alcohol dehydrogenase Esteban Mezey *, Lynda Rennie-Tankersley, James J. Potter Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2195, USA Received 21 September 2005 Available online 10 October 2005
Abstract The effect of leptin on liver alcohol dehydrogenase (ADH) was determined in male rats. Administration of one or three daily doses of leptin (1 lg/g of body weight intraperitoneally) increased ADH activity. Leptin enhanced ADH synthesis without an effect on ADH degradation. Leptin did not change ADH mRNA, indicating that the effect of leptin in enhancing ADH occurs at the post-transcriptional level. Leptin increased eukaryotic initiation factor (eIF) 2a, eIF2B activity, and the eIF4E–eIF4G complex, while it decreased the inhibitory complex of eIF4E with the eIF4E-binding protein-1 (4E-BP1). Leptin increased mammalian target of rapamycin (mTor) that phosphorylates 4E-BP1. In conclusion, leptin increases liver ADH activity and ADH protein due to an increase in synthesis which occurs at the post-transcriptional level. The effect of leptin in enhancing translational initiating factors may be of significance in the regulation not only of ADH but also of many other proteins. 2005 Elsevier Inc. All rights reserved. Keywords: Alcohol dehydrogenase; Leptin; Eukaryotic initiation factors; Mammalian target of rapamycin
Liver alcohol dehydrogenase (ADH:alcohol:NAD oxidoreductase, EC 1.1.1.1) is the principal enzyme responsible for ethanol oxidation. Liver alcohol dehydrogenase (ADH) is affected by a variety of hormones. Stress, which stimulates the hypothalamo–hypophyseal–adrenocortical axis and the sympathetic nervous system, increases the enzyme activity [1]. GH increases liver ADH due to increased synthesis that is initiated at the level of transcription [2,3]. The effects of stress and GH were associated with increases in the rate of elimination of ethanol [1–3]. Endotoxin originating from intestinal bacteria is an important mediator of hepatocellular inflammation in the intragastric feeding rat model of alcoholic liver disease [4,5]. We recently demonstrated that lipopolysaccharide (LPS), the endotoxin component of gram-negative bacteria, increased liver ADH [6]. An increase rate of formation of acetaldehyde (AC) caused by an enhanced ADH activity may contribute to worsening of alcoholic liver injury caused by endotoxin. *
Corresponding author. Fax: +1 410 955 9677. E-mail address: [email protected] (E. Mezey).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.003
Leptin is a cytokine that belongs structurally to the long-chain helical cytokine family, which includes IL-3, IL-11, and IL-12, and GH [7]. Leptin was shown to enhance liver fibrosis produced by a single dose of CCl4 or by chronic administration of thiocetamide [8,9]. Administration of LPS to rats increases plasma leptin [10]. Since GH and LPS both increase ADH, it is quite likely that leptin has a similar effect. An increase in ADH with increased formation of AC is a potential mechanism for enhancement of fibrogenesis by leptin in alcoholic liver disease. The purpose of this study was to determine the effects of leptin on liver ADH. This study shows that leptin enhances liver ADH and that this effect occurs at the post-transcriptional level. Methods Animals and materials. Male Sprague–Dawley rats were obtained from Charles River Laboratories, Wilmington, MA. All animals received humane care in compliance of the guidelines from the Animal Care and Use Committee of the Johns Hopkins University. Murine leptin was obtained from Biomol, Plymouth Meeting, PA. Sterile [4–5-3H]L-leucine and [14C]sodium bicarbonate (NaH14CO3) were obtained from ICN
E. Mezey et al. / Biochemical and Biophysical Research Communications 337 (2005) 1324–1329 Biomedicals, Irvine, CA. [8-3H]Guanosine 5 0 -diphosphate ([3H]GDP) was obtained from Amersham Biosciences, Piscataway, NJ. Animal treatment. Rats received intraperitoneal (i.p.) injections of leptin (1 lg/g of body weight) either once or for 3 consecutive days, while controls received isovolumetric amounts of saline. The animals were sacrificed at 6 and 24 h after one injection or 2 h after the third daily injection. Approximately 400–500 mg of the liver was homogenized in 4 vol of 0.25 M sucrose in 0.1 M Tris–HCl buffer, pH 7.4, centrifuged at 10,000g for 10 min, and the resulting supernatant was centrifuged at 100,000g. The 100,000g supernatant (cytosol) was used immediately for the determination of ADH activity. The remainder of the supernatant was frozen at 80 C for determinations of LDH activity and ADH protein. One section of 1.0–1.2 g of the liver was processed for RNA isolation. ADH activity. ADH activity was determined in the liver cytosol at 37 C by the method of Crow et al. [11]. The reaction mixture was 1.0 ml and consisted of 0.5 M Tris–HCl, pH 7.2, 18 mM ethanol, 2.8 mM NAD+, and 0.01 ml of the cytosol. Blank reactions were run without ethanol. One unit of enzyme activity is defined as the formation of 1 lmol of NADH per min. ADH activity was expressed per mg of protein. Cell protein was determined by the method of Lowry et al. [12]. LDH activity was determined by the method of Plagemann et al. [13]. Immunoreactive protein of ADH. Immunoreactive ADH protein was assayed by quantitative enzyme linked immunosorbent assay (ELISA) as described previously [14]. The antisera to the enzyme were produced in rabbits by subcutaneous injections of purified ADH in FreundÕs adjuvant. The antisera are purified with protein A–Sepharose CL-4B affinity chromatography prior to use in the assay. Determination of messenger RNA (mRNA) by quantitative real time PCR. Total cellular RNA was isolated using the guanidine isothiocyanate procedure of Chomcynski and Sacchi [15] as described previously [3]. The concentration of the isolated RNA was determined from the optical density at 260 nm and its purity from the 260/280 ratio. The isolated RNA was initially stored at 80 C. The cDNA template was synthesized with the Superscript III first-strand synthesis system (InVitrogen, Carlsbad, CA) using 5 lg of total RNA. ADH mRNA was determined by the TaqMan gene expression assay of Applied Biosystems, Foster City, CA. Glyceraldehyde 3 phosphate dehydrogenase (GADPH) mRNA was used as a reference control. The gene-specific oligonucleotide primers for rat ADH (#Rn01522111) and for rodent GADPH (#4308313) were obtained from Applied Biosystems. The reaction conditions were 50 C for 2 min, 95 for 10 min followed by 40 cycles of 95 C for 15 s and 60 C for 1 min. To quantify ADH gene expression, the DDCt method was used to calculate relative fold changes normalized against GADPH gene expression. ADH synthesis and degradation. To determine the effect of leptin on ADH synthesis, 16 male Sprague–Dawley rats were fasted overnight following which 8 were injected with leptin, 1 lg/g body weight i.p., while 8 were injected with isovolumetric amounts of saline. Six hours later [4–5-3H]L-leucine (100 lC/lmol per 100 g body weight in a 0.1 ml vol) was injected i.p. The animals were sacrificed 1 h after the isotope injection. The livers were removed and homogenized in 4 vol of 0.25 M sucrose in 0.1 M Tris–HCl buffer, pH 7.4, containing a proteinase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), 0.1 mM dithiothreitol (DDT), and 1 mM phenylmethanesulfonyl fluoride (PMSF). The homogenate was centrifuged and the liver cytosol was prepared as described above. A portion of the cytosol was used for the assay of ADH activity and the remainder was frozen at 80 C for determination of the incorporation of radioactive leucine into ADH. For the determination of ADH turnover, 40 rats received a single i.p. injection of 1.0 mCi of NaH14CO3 (55 mCi/mmol) per 100 g of body weight as described previously [16]. Starting immediately after the injection of the isotope, leptin (1 lg/g body weight) was given i.p. each day to one-half the rats, while the other one-half were given isovolumetric amounts of saline. Eight animals, 4 injected with leptin and 4 controls, were sacrificed on days 1, 2, 3, 4, and 5 after the administration of the isotope. The livers were processed to isolate the liver cytosol as described above. To measure the incorporation of the isotopes into ADH protein, 400 lL of cytosol was precipitated with antibody to ADH. The antigen–
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antibody complex was allowed to form for 2 h at 25 C. The antigen– antibody complexes were precipitated from the incubation mixture with protein A bearing Staphylococcus aureus cells (Pansorbin, Calbiochem, San Diego, CA). The precipitate was centrifuged at 12,000g for 3 min and the pellet was resuspended in 0.05 M Tris–HCl buffer, pH 7.4, containing 300 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 1% Na deoxycholate, and 0.025 M sucrose [17]. The pellet was precipitated and washed 3 times in the same buffer, and then resuspended in 0.125 M Tris– HCl buffer, pH 6.2, containing 6 M urea, 3% sodium dodecyl sulfate, and 5% mercaptoethanol and heated for 5 min at 95 C. After cooling, the suspension was centrifuged and the supernatant was subjected to SDS– polyacrylamide electrophoresis [16]. The gels were fixed in a solution of 28% trichloroacetic acid and stained with Coomassie brilliant blue R-250. The protein bands corresponding to the 40,000 molecular weight subunit of ADH were identified from the electrophoresis of the purified enzyme [17]. The gels were sliced, incubated into Protosol, and counted in Ecolite (ICN, Biochemicals, Aurora, OH). The radioactivity incorporated into ADH was corrected for background radioactivity in adjacent slices not containing proteins. In the case of the ADH turnover study with NaH14CO3, the fractional rates of degradation (Kd) and synthesis (Ks) were obtained from the slopes of the regression lines of the decreases in total and specific radioactivity, respectively [18]. The absolute rate of synthesis (V) was obtained by multiplying Ks by the total liver ADH activity. Western blot analysis of eukaryotic initiation factors (eIFs). Liver was homogenized in 7 vol of buffer composed of 20 mM Tris–HCl, pH 7.4, 250 mM sucrose, 100 mM KCl, 1 mM DDT, 50 mM NaF, 1 mM PMSF, 1 mM benzamidine, and 0.5 mM vanadate. The samples were centrifuged to 10,000g for 10 min and the determinations were done with the resulting supernatant as described by Lang et al. [19]. The samples were mixed with 2· Laemmli SDS buffer boiled, centrifuged, subjected to electrophoresis at 60 mA in a 12.5% polyacrylamide gel, and transferred to nitrocellulose membranes. The membranes were washed and subsequently blocked with (5% wt/vol) non-fat milk in 25 mM Tris–HCl, pH 7.4, and the membranes were incubated with goat polyclonal antibodies to either eIF2a, phosphorylated eIF2a or eIFB2e (Cell signaling Technology, Beverly, MA) at 4 C overnight. After repeated washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution, Amersham Life Science) at room temperature for 1 h. The membranes were washed and visualized by enhanced chemiluminescence reaction (ECL-plus, Amersham Biosciences). Phosphorylation of mTOR. The phosphorylation of mTor was determined in the 10,000g liver supernatants as described by Kimball et al. [20]. The Western blots were carried out with rabbit polyclonal mTor and phospho-mTOr (Ser 2448) antibodies (Cell Signaling Technology). Quantitation of eIF4E–eIF4G and eIF4E–4E-BP1complexes. The associations of eIF4E with eIF4G or 4E-BP1 were determined as described by Kimball et al. [20]. Briefly, eIF4E was immunoprecipitated from the 10,000g liver supernatant with rabbit polyclonal eIF4E antibody (Santa Cruz), the precipitate resolved by SDS–PAGE, and transferred to nitrocellulose membranes. One set of membranes was re-probed with rabbit polyclonal anti-eIF4G antibody, and another set of membranes re-probed with rabbit 4E-BP1 (both from Santa Cruz). The membranes were visualized by the enhanced chemiluminescence reaction. The blots were later inactivated and re-probed with anti-eIF4E antibody. The values obtained with the anti-eIF4G and anti-4E-BP1 antibodies were normalized to eIF4E present in the samples. Determination of eIF2B activity. Liver eIF2B activity was determined in 10,000g liver supernatants from the rate of exchange of [3H]GDP in the eIF2 [3H]GDP complex for non-radioactively labeled GDP as described by Kimball et al. [21,22]. A binary complex of eIF2 and [3H]GDP was formed by incubation for 10 min at 30 C in a mixture containing 50 mM MOPS, pH 7.4, 100 mM KCl, 1 mM DDT, 200 lg/ml bovine albumin, 1.3 lM [3H]GDP (10.7 Ci/mmol), and recombinant human liver eIF2a (Cell Sciences, Canton, MA). Two millimolar magnesium acetate was added and the binary complex was placed on ice. A 35 lL aliquot of the liver supernatant was mixed with 140 lL of a reaction buffer containing 50 mM Mops, pH 7.4, 209 lM GDP, 2 mM magnesium acetate, 100 mM
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KCl, 1 mM DDT, and 200 mg/ml bovine serum albumin plus and 87.5 lL water. The reaction was initiated by the addition of 35 lL of the eIF-2 [3H]GDP complex (1–2 pmol) and the tubes were maintained in a water bath at 30 C. Aliquots (75 lL) of the reaction mixture were removed at 1, 2, and 3 min, and placed in tubes containing 2.5 ml ice-cold reaction mixture without bovine albumin. The contents were mixed, filtered through a nitrocellulose filter disk, the filters were dissolved in Filtron X (National Diagnostic, Atlanta, GA), and the radioactivity was counted. eIF2B activity was determined from the rate of decrease in the eIF2 [3H]GDP complex bound to nitrocellulose filters. Statistical analysis. The data were analyzed with StudentÕs t test when appropriate or by analysis of variance when comparing means of more than two groups. The Duncan new multiple range test was used to estimate differences between means [23]. Equality of variances around regression lines in the ADH turnover study was tested using the F test. The significance of the difference in slopes was determined by StudentÕs t distribution as described by Bailey [24].
Results Leptin enhances ADH The administration of leptin (1 lg/g body weight) increased liver ADH activity. Fig. 1 shows the increases in ADH activity at 6 and 24 h after the administration of one dose of leptin. Furthermore, Fig. 1 also shows the increase in ADH activity after daily administration of leptin for 3 days. Leptin did not result in any significant changes in LDH activity (data not shown). ADH immunoprotein determined after 3 days of leptin administration was increased from 8.80 ± 0.69 in the controls to 14.58 ± 0.16 lmol/mg of cytosol protein in the leptin-treated rats (p < 0.001). Leptin had no significant effect on ADH mRNA at any time point. The relative values of ADH mRNA were 110 ± 2.9 and 101 ± 3.4 at 6 and 24 h after leptin administration as compared to respective saline-controls of 100 ± 2.8 and 100 ± 4.3. After administration of 3
Fig. 1. Effect of leptin on liver alcohol dehydrogenase (ADH) activity. Either one dose of leptin (1 lg/g of body weight) was injected intraperitoneally (i.p.) and the animals were sacrificed 6 and 24 h later, or 3 daily doses of leptin were given and the animals were sacrificed 2 h after the third injection (3 days). Control animals were given isovolumetric amounts of saline. ADH activity was determined in the liver cytosol. The values are expressed as means ± SE of 8 determinations. *p < 0.01 versus respective control value.
daily doses of leptin, the ADH mRNA values were 98 ± 10 and 100 ± 8 for the leptin-treated and saline-controls. Effect of leptin on ADH synthesis The effect of leptin on the synthesis of ADH was initially determined with radioactive leucine in 24 h-fasted rats. The rats were injected with [4–5-3H]L-leucine 6 h after the administration of one dose of leptin or saline and the incorporation of the isotope into ADH was determined 1 h later. Leptin resulted in a 22.3% increase in the incorporation of the labeled leucine into ADH. The values were 23.0 ± 3.1 · 103 dpm/mg protein for the leptin-treated as compared with 18.8 ± 2.6 · 103 dpm/mg protein for the saline-controls (p > 0.05). The effect of daily administration of leptin for 5 days on turnover of total radioactivity was subsequently determined with NaH14CO3 and found to be similar in leptin-treated and control rats. The fractional rate of degradation (Kd) was 0.10 for the leptin-treated and 0.11 per day for the controls rats. This corresponds to halflife (T1/2) values of 6.9 and 6.3 days for the leptin-treated and control rats, respectively. By contrast, the fractional rate of synthesis (Ks) of ADH was enhanced by the administration of leptin to 0.15 ± 0.011 for the leptin-treated as compared to 0.11 ± 0.012 for the control rats (p < 0.05). The absolute rate of synthesis (V) was increased 36% from 0.55 total liver units/day in the control rats to 0. 75 total liver units/day in the leptin-treated rats. Effects of leptin on translation Initiation of translation involves the binding of initiator methionyl RNA to the 40 S ribosomal subunit as a ternary complex with eIF2 and GTP. Leptin increased the a subunit of eIF2 (eIF2a) (p < 0.05), while it did not change significantly phospho-eIF2a, which inhibits eIF2B activity (Fig. 2). Leptin did not result in significant changes in eIFB2e, which is the catalytic subunit of eIF2B (data not shown). Leptin increased eIF2B activity, which stimulates the exchange of GTP to GDP that is required for formation of the ternary complex. The activities were 1.48 ± 0.27 and 0.82 ± 0.14 pmol of GDP exchanged per min per mg protein for the leptin-treated and saline-controls (p < 0.05). The subsequent step in translation is the binding of mRNA to the 43 S preinitiation complex which is regulated by a group of proteins forming a complex called eIF4F. The active eIF4F complex in the regulation of translation, which consists of the association between eIF4E with eIF4G, was increased by leptin (p < 0.05) (Fig. 3A). By contrast, the inactive 4E-BP-1 to eIF4E complex was decreased by leptin (p < 0.01) (Fig. 3B). The binding of 4E-BP1 to eIF4E is decreased by phosphorylation of 4EBP1, and in this study mTOR, which phosphorylates 4E-BP1, was increased by leptin (p < 0.05) (Fig. 4A), while phospho-mTOR was decreased by leptin (p < 0.05) (Fig. 4B).
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Fig. 2. Effect of leptin: on (A) eIF2a and on (B) phosphorylated eIF2a. Leptin (1 lg/g of body weight) was injected i.p. and the animals were sacrificed 6 h later. Control animals were given isovolumetric amounts of saline. The determinations were done by Western blot and quantitated by densitometry. The values are expressed as means ± SE of 8 determinations. *p < 0.01 versus control value.
Fig. 3. Effect of leptin on: (A) association of eIF4E with eIF4G assessed by immunoprecipitation (IP) with eIF4E followed by Western blot (WB) analysis with eIF4G. (B) Association of eIF4E with the inhibitory 4E-BP1 assessed by IP with eIF4E followed by WB analysis with 4E-BP1. Leptin (1 lg/g of body weight) was injected i.p. and the animals were sacrificed 6 h later. Control animals were given isovolumetric amounts of saline. The determinations were done by WB and quantitated by densitometry. The values of the association between eIF4E with either eIF4G or 4E-BP1 were normalized to the amount of eIF4E present in the samples. Relative values are expressed as means ± SE of 8 determinations. *p < 0.05 versus control value. **p < 0.01 versus control value.
Discussion This study shows that leptin enhances liver ADH protein and activity, and that this effect is due to increased enzyme synthesis. Leptin did not change ADH mRNA, indicating that the effect of leptin in enhancing ADH occurs at the post-transcriptional level. Although LPS has been shown to increase both leptin [10] and liver ADH [6], this study indicates that the effect of LPS is not mediated by leptin, since the effect of leptin, unlike that of LPS, was not associated with an increase in ADH mRNA. Leptin had no effect on ADH degradation. By contrast, castration was previously shown to enhance liver ADH due to a decrease in the rate of degradation of the enzyme [16] and, in turn, the suppressant effect of dihydrotestoster-
one on ADH was demonstrated in hepatocytes in culture to be due to increased enzyme degradation [17]. Fasting decreases ADH due to a decrease in ADH synthesis, which is associated with an increased ADH degradation [25]. The known rapid decrease in leptin levels with fasting [26] may contribute to the decrease in ADH with fasting by limiting optimal stimulation of ADH synthesis. The leptin-induced enhancement in ADH activity leading to increased formation of AC, which increases collagen production [27], is a potential mechanism for the effect of leptin on enhancing fibrogenesis in alcoholic liver disease. The effect of leptin on increasing ADH is most likely at the translational level. Previously, higher liver ADH activity in C57BL/6J (B6) mice than in B6.S congenic mice was found in the absence of differences in transcriptional rate
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Fig. 4. Effect of leptin on: (A) mTOR and on (B) phosphorylated mTOR. Leptin (1 lg/g of body weight) was injected i.p. and the animals were sacrificed 6 h later. Control animals were given isovolumetric amounts of saline. The determinations were done by Western blot using anti-mTOR and anti-phospho Ser2448 antibodies and quantitated by densitometry. The values are expressed as means ± SE of 8 determinations. *p < 0.01 versus respective control value.
indicating a post-transcriptional mechanism for regulation of ADH [28]. Leptin, in the present study, increased eIF2a and eIF2B activity, and complex formation of eIF4E with eIF4G. These changes were associated with a decrease in the inhibitory eiF4E–4E-BP1 complex. The increase in the active eIF4E–eIF4G complex in association with the decrease in the inhibitory eIF4E–4E-BP1 complex may be regulated by increases in phosphorylation of 4E-BP1 [29]. Leptin, in this study, increased mTor that phosphorylates 4E-BP1 and inactivates it [29] leading to decreased complex formation of 4E-BP1 with eIF4E. The effect of leptin on decreasing the phosphorylation of mTOR at Ser2448 may have contributed to increased availability of mTOR, however, the significance of mTOR phosphorylation remains unknown [30]. The Ser2448 phosphorylation appears to be mediated by protein kinase B rather than by autophosphorylation. In one study, acute alcohol intoxication in rats, which decreased myocardial protein synthesis, decreased myocardial mTOR phosphorylation in association with decreased 4E-BP1 phosphorylation on Ser2448 and SER2481, and increased formation of the inhibitory eIF4E–4E-BP1 complex [31]. The effect of leptin on increasing protein synthesis by mechanisms of translation could possibly be mediated by insulin which is known to increase eIF4E–eIF4G binding by increasing phosphorylation of 4E-BP1 [32]. Studies of the effects of leptin on plasma insulin have shown varied effects. In one study, administration of 2 doses of 300 lg leptin 12 h apart increased plasma glucose, and plasma insulin in fasted but not in fed mice [33]. On the other hand, infusion of leptin (10 lg/h for 48 h) decreased plasma insulin levels and insulin sensitivity determined from glucose utilization during hyperinsulinemic glucose clamp in fasted rats [34]. Both leptin and insulin which reduce food intake and body weight have in common a number of signaling pathways and influence each other. Insulin and leptin activate phosphoinositide-3-kinase through IRS-1 (insulin receptor-1) and IRS-2, respectively, and pretreatment of each reduces the expression of the receptor of the other [35].
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