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Molecular Endocrinology, doi:10.1210/me.2007-0036
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*ETHANOL
Molecular Endocrinology 21 (10): 2541-2550
Copyright © 2007 by The Endocrine Society

Dose-Dependent Effects of Alcohol on Insulin Signaling: Partial Explanation for Biphasic Alcohol Impact on Human Health

Ling He, John C. Marecki, Ginette Serrero, Frank A. Simmen, Martin J. J. Ronis and Thomas M. Badger

Departments of Physiology and Biophysics (L.H., F.A.S., T.M.B.), Pediatrics (J.C.M., M.J.J.R., T.M.B.), and Pharmacology and Toxicology (M.J.J.R.), University of Arkansas for Medical Sciences, and Arkansas Children’s Nutrition Center (L.H., J.C.M., F.A.S., M.J.J.R., T.M.B.), Little Rock, Arkansas 72202; and A&G Pharmaceutical Inc. (G.S.), Columbia, Maryland 21045

Address all correspondence and requests for reprints to: Thomas M. Badger, Arkansas Children’s Nutrition Center, 1120 Marshall St., Little Rock, Arkansas 72202. E-mail: badgerthomasm{at}uams.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Routine consumption of alcohol at low doses is associated with decreased risk of acquiring type 2 diabetes, whereas chronic and excessive alcohol consumption increases the risk. Although there is good epidemiologic evidence for these biphasic effects, careful validation of these effects on insulin signaling has not been reported, nor have biological mechanisms underlying these biphasic effects been proposed. In this study, we provide evidence in rats that low-dose alcohol intake (4 g/kg·d) enhances hepatic insulin signaling by suppressing p55{gamma} (a phosphatidylinositol 3-kinase regulatory subunit isoform) at the posttranscriptional level, leading to the increased association of the phosphatidylinositol 3-kinase catalytic subunit (p110) with insulin receptor substrate-1 (P < 0.05) and subsequent activation of downstream effectors such as Akt, glycogen synthase kinase 3ß, and nuclear sterol regulatory element binding protein (SREBP)-1. These results, combined with our previous data (confirmed in the present study) demonstrating that ethanol intake at high doses (13 g/kg·d) disrupts hepatic insulin signaling by inducing TRB3, a mammalian homolog of Drosophila (tribbles-related protein 3) that prevented activation of downstream effectors (such as Akt, GSK3ß, and nSREBP-1), provide clear mechanistic validation of the biphasic effects of ethanol on insulin signaling. We also report that ethanol induction of TRB3 can be partially blocked (P < 0.01) by compounds (4-phenyl butyric acid and taurine-ursodeoxycholic acid) known to reduce endoplasmic reticulum stress. Thus, alcohol exerts biphasic actions on hepatic insulin signaling, such that low doses activate insulin signaling pathways associated with reduced p55{gamma} to increase nSREBP-1, whereas high doses of ethanol elevate TRB3 and suppress insulin signaling to decrease SREBP-1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EIGHTEEN MILLION AMERICANS (more than 7%) suffer from alcohol abuse or dependence, and the total annual societal cost is estimated at greater than $185 billion (1). Interestingly, however, epidemiological data suggest that excessive and chronic alcohol consumption is associated with increased risk of type 2 diabetes as compared with abstainers, whereas the risk of type 2 diabetes is lower with light to moderate alcohol consumption (2, 3, 4, 5). Similarly, low to moderate alcohol consumption (one daily drink for women or two daily drinks for men) is associated with a lower prevalence of coronary heart disease (fewer myocardial infarctions), and an improved survival after myocardial infarction (6, 7, 8). In contrast, chronic and excessive alcohol intake increases the likelihood of developing congestive heart disease (9). The biological mechanisms underlying these biphasic effects of alcohol are far from clear.

Studies by our group focusing on the mechanisms underlying induction of the hepatic class I alcohol dehydrogenase (ADH) expression in response to chronic high-dose alcohol have pointed to a disruption in the insulin-dependent membrane to nucleus signaling machinery culminating in significant loss of mature sterol regulatory element binding protein-1c (SREBP-1c) (10, 11). SREBP-1c is the predominately expressed form of the transcription factor SREBP-1 in the liver, which mediates the transcription of genes responsible for lipogenesis, such as acetyl coenzyme A carboxylase, and fatty acid synthase (12, 13). SREBP-1c also acts as a repressor for the class I ADH gene in rats (11, 14). With chronic high-dose alcohol intake, the posttranscriptional reduction of nuclear SREPP-1 (nSREPP-1) and consequent induction of ADH expression was coincident with the induction of TRB3, a mammalian homolog of Drosophila (tribbles-related protein 3) thought to be induced in response to DNA damage and endoplasmic reticulum (ER) stress (15). TRB3 has also been shown to trigger the degradation of acetyl-coenzyme A in adipose tissue and protect against diet-induced obesity (16). Importantly, we have shown that the induction of TRB3 also suppresses insulin signal transduction by negatively modulating the phosphorylation of Akt at Thr308 in the liver (10). Our studies in rats exposed to high-dose ethanol demonstrated that alcohol-induced TRB3 efficiently bound the pleckstrin homology domain of Akt sequestering the complex in the cytosol, thus preventing the translocation to the membrane and subsequent Akt-mediated signaling (10). A similar induction of TRB3 in livers of rats fed high-ethanol containing diets (13 g/kg·d) has recently been reported by other investigators (17).

Whereas our data clearly demonstrate that chronic consumption of high levels of alcohol resulting in blood ethanol concentrations (BECs) greater than 200 mg/dl reduce hepatic nSREBP-1 abundance in rats and significantly induce ADH I gene expression, other investigators have documented an increased level of nSREBP-1 in mice fed alcohol-containing diets (18, 19). These differences may partially reflect dissimilarity in metabolism and endocrinology of rats and mice. For example, mice are well known to have greater ethanol metabolism than rats, which results in lower BECs at the same ethanol intake (20). We, therefore, considered the possibility that low BECs may result in increased nSREBP-1 through enhancement of insulin signaling. To address this issue and to elucidate mechanisms underlying possible biphasic effects of alcohol on hepatic insulin signaling and nSREBP-1, we studied rats fed isocaloric diets containing low or high doses of ethanol. Herein, we provide evidence that the hepatic protein level of P55{gamma} [an isoform of the phosphatidylinositol 3-kinase (PI3K) regulatory subunit] was suppressed concurrently with an increased association of the PI3K catalytic subunit (p110) with insulin receptor substrate (IRS)-1 (P < 0.05) in rats fed low-dose ethanol-containing diets (4 g/kg·d), resulting in enhanced insulin signaling. Also in this report, we have linked TRB3 induction and insulin resistance after high-dose ethanol consumption with the development of ER stress.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of Ethanol-Containing Diets on Rat Urine Ethanol Concentrations (UECs)
UECs of rats fed diets containing 13 g/kg·d ethanol fluctuated in the form of large regular pulses such that concentrations varied between a nadir of near zero to peak levels of greater than 500 mg/dl (Fig. 1Go) in a manner nearly identical with our previous observations (21). However, UECs ranged between 0 and 25 mg/dl in rats fed the diets containing low-dose ethanol (4 g/kg·d). UECs were measured because previous studies have demonstrated that UECs accurately track blood alcohol concentrations (22).


Figure 1
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  		Fig. 1. UECs

Data are daily alcohol concentrations of 24-h rat urines from one representative rat in each alcohol-fed group (5 rats/group). Rats fed high-dose ethanol (closed circle) were killed when the mean UECs high on the descending limb of the ethanol pulse (usually greater than 300 mg/dl).

 
Effects of Ethanol on Insulin Signaling in Rat Liver
We examined hepatic insulin signaling because ethanol is metabolized predominantly in the liver and previous studies have linked increases in alcohol metabolism at high blood ethanol concentrations with impaired insulin signaling (10, 11). Data shown in Fig. 2AGo reconfirmed our previous results (10, 11) showing decreased (P < 0.05) phosphorylation of GSK3ß and Akt-Thr308, increased (P < 0.05) phosphorylation of Akt-Ser473, and decreased (P < 0.05) nuclear form of SREBP-1 protein (Fig. 2BGo, right panel) in livers of rats fed high-dose ethanol (13 g/kg·d). However, phosphorylation of GSK3ß, Akt308 and Akt473 increased (P < 0.05) and nSREBP-1 levels were greater (P < 0.05) in livers of rats fed diets with the low ethanol dose (4 g/kg·d) (Fig. 2Go, A and B, left panels). These data demonstrate a dose-dependent biphasic effect of ethanol on hepatic insulin signaling, whereby low-dose intake increases insulin signaling and high-dose intake blocks this pathway. To study the effects of ethanol on proximal insulin signaling, we monitored the phosphorylation status of IRS. Low-dose alcohol did not significantly affect tyrosine phosphorylation of IRS-1 or -2 (Fig. 2CGo, left panel). In agreement with the results of a previous report (23), high-dose alcohol increased (P < 0.01) IRS tyrosine phosphorylation (Fig. 2CGo, right panel). The above results are graphically summarized in Fig. 2DGo. It is important to note that the low- and high-dose alcohol animal studies reported in the figures were designed with paired control animals infused at the same time, and the exposure time for each Western blot was varied to provide signals within the linear range of the x-ray film.


Figure 2
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  		Fig. 2. Effects of Ethanol on Hepatic Insulin Signaling

Adult male rats were fed diets containing either no ethanol (Control), 4 g/kg·d ethanol (low-dose ethanol, experiment 1), or 13 g/kg·d (high-dose ethanol, experiment 2). Each lane represents liver whole cell extract from an individual rat. Akt phosphorylated at Thr308 (p-Akt-Thr308); Akt phosphorylated at Ser473 (p-Akt-Ser473), Akt, phospho-GSK3ß (p-GSK3ß), and total GSK3 (GSK3) protein levels are shown (A). Western blot analyses of SREBP-1 are shown (B). Tyrosine phosphorylation of IRS was examined after immunoprecipitation with antiphosphotyrosine antibody (aPy IP, 4G10) followed by Western blot (WB) or by direct immunoblotting for IRS-1 and -2 input using liver whole cell lysates (C). Densitometric analysis (means ± SE) are shown (D). *, P < 0.05 as compared with control (5 rats/group). ND, Not detected; ADU, arbitrary densitometric units.

 
Role of PI3K-P55{gamma} in Activation of Insulin Signaling in Rats Fed Low-Dose Alcohol
The phosphorylation of Akt-Thr308, phosphorylation of GSK3ß, and levels of nSREBP-1 were increased in livers of rats fed low-dose ethanol. However, tyrosine phosphorylation of IRS-1 and -2 did not differ significantly from controls. To gain an understanding of the mechanism resulting in increased insulin signaling, we examined the levels of PI3K catalytic subunit (PI3K-p110), PTEN, and phospho-PTEN protein levels and found no effect of low-dose ethanol on the abundance of these proteins in liver (data not shown). The lack of effects of ethanol treatment on PTEN expression and phosphorylation status suggests that PTEN does not play an important role in ethanol effects on hepatic insulin signaling in this model, but the possibility remains that PTEN activity could be altered without changes in expression or phosphorylation status. However, using an antibody against PI3K-p85 (Cell Signaling, Beverly, MA), we found that expression of one protein subunit was suppressed in rats fed low-dose alcohol (Fig. 3AGo). This antibody recognizes p85{alpha}, p85ß, and p55{gamma}; therefore, the approximately 60-kDa molecular mass band that this ethanol-suppressed protein is likely to be is p55{gamma}. To confirm this, we performed Western blot analysis using an antibody against the N-SH2 domain of PI3K-p85, which recognizes all the p85{alpha}, p55{alpha}, and p50{alpha} isoforms of the Pik3r1 gene (Fig. 3BGo), and we found no significant changes in these isoforms between the control and alcohol-fed groups. The data also suggest that the ethanol-sensitive protein is most likely to be p55{gamma}. To substantiate this, we examined p55{gamma} protein abundance using antiserum raised against p55{gamma} (24). The abundance of p55{gamma} protein was decreased in a pool made from whole liver lysates of rats fed low-dose ethanol (Fig. 3DGo). These data were further corroborated by using small interfering RNA (siRNA) specific for rat p55{gamma} to knock down the expression of p55{gamma}. The siRNA-suppressed protein migrated in the same position as the protein suppressed by ethanol (Fig. 3DGo). Using RT-PCR, we determined that the mRNA levels of p55{gamma} were not affected by feeding low alcohol containing diets (data not shown). Thus, the effect of low-dose alcohol on p55{gamma} expression occurs at a posttranscriptional level.


Figure 3
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  		Fig. 3. Effects of Low-Dose Ethanol on Hepatic PI3K-p55{gamma} Protein Levels

Western blot analysis with antibody (Cell Signaling) recognizing p85{alpha}, p85ß, and p55{gamma} in liver whole cell lysates (panel A). Western blot analysis with antibody (Upstate Biotechnology) recognizing all isoforms of p85{alpha} (p85{alpha}, p55{alpha}, and p50{alpha}) (panel B). Each lane in panels A and B represents a sample from an individual rat (5 rats/group). Panel C, Densitometric analysis of p55{gamma} in panel A. The bars are means ± SEM and represent densitometric analysis of the five lanes per group. p55{gamma} protein levels in liver whole cell lysates are presented in right side of panel D [each lane represents pooled sample (five rats); C, control; E, ethanol] were examined with antiserum raised against p55{gamma} (19 ). On the left side of panel D is the p55{gamma} protein levels in FGC-4 cells transfected with negative control siRNA duplex (C) or with either 25 nM or 100 nM p55{gamma} siRNA oligonucleotides. Rats were fed a diet containing either 4 g/kg·d ethanol [ethanol (low dose)] or no ethanol [control]. *, P < 0.05 as compared with control. ADU, Arbitrary densitometric units.

 
Because p55{gamma} is not thought to be a major PI3K regulatory subunit in the liver or in the FGC-4 cells, we wanted to confirm that p55{gamma} reductions would result in the enhancement of insulin signaling consistent with low-dose ethanol effects. Therefore, we treated FGC-4 cells with insulin (100 nM for 5 min) and/or siRNA (negative control or p55{gamma} siRNA as described above) and assessed p-Akt-Thr308 and p-GSK3ß (Fig. 4Go). Insulin treatment induced p-Akt-Thr308 and p-GSK3ß (P < 0.05) and siRNA knockdown cells had elevated (P < 0.05) phosphorylation of Akt and GSK3ß and enhanced (P < 0.05) the effects of insulin. An important role for PI3K-regulatory subunits (p85{alpha}, p85ß, p55{alpha}, p55{gamma}, p50{alpha}) is to affect recruitment of PI3K-catalytic subunits (PI3K-p110s) to the plasma membrane by binding to IRS, and allowing PI3K-P110s to make contact with membrane lipid substrates. We therefore examined low-dose ethanol effects on the association of IRS with PI3K-p110 and found an increased association of p110 with IRS-1 (P < 0.05) but no effect on the association of PI3K-p110s with IRS-2 (Fig. 5Go, A and B). This suggests that the increased association of IRS-1 with p110 resulting from loss of p55{gamma} may be responsible for the activation of downstream effectors.


Figure 4
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  		Fig. 4. PI3K-p55{gamma} Affects Insulin Signaling

Total Akt, p55{gamma}, p-Akt-Thr308, p-GSK3ß, and GSK3 protein levels in FGC-4 cells transfected with negative control (Con) siRNA duplex or with p55{gamma} siRNA oligonucleotides and/or treated with 100 nM insulin for 5 min. Bars represent means ± SEM for n = 3/treatment. ADU, Arbitrary densitometric units.

 

Figure 5
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  		Fig. 5. Effects of Low-Dose Ethanol on the Association of PI3K-p110 with Insulin Receptor Substances

A, PI3K-p110 in IRS-1 and -2 immunoprecipitates (IP). B, IRS-1 and -2 in PI3K-p110 immunoprecipitates. Each lane represents an individual rat liver. Bars represent means ± SEM for n = 3/treatment. *, P < 0.05 as compared with controls. ADU, Arbitrary densitometric units.

 
Identification of the Pathway for Induction of TRB3 in Rats Fed High-Dose Ethanol
We previously demonstrated that up-regulation of TRB3 expression by high-dose ethanol treatment is responsible for impaired signaling through the Akt-GSK3ß-SREBP-1c pathway in rat liver (10). However, we did not examine the mechanism for TRB3 induction in those studies. Because ethanol has been shown to induce ER stress (25), and ER stress has been suggested to induce TRB3 via CCAAT/enhancer binding protein homologous protein (CHOP-10) (26), we have examined this possible pathway in the current study using in vivo and in vitro approaches. The induction of TRB3 was observed only in rats fed high-dose alcohol-containing diets (Fig. 6Go). We examined hepatic CHOP-10 protein levels in rats fed diets containing low or high levels of ethanol and found that this protein was induced approximately 8-fold (P < 0.01) only by high-dose ethanol (Fig. 6Go). We further monitored the ER stress protein, p-PERK, and found elevated phosphorylation of PERK at Thr981 (3-fold, P < 0.01) in livers of rats fed high-dose alcohol (Fig. 6Go). Feeding low-dose ethanol did not increase ER stress effectors (Fig. 6Go). These data demonstrate that high-dose ethanol consumption caused ER stress and suggests that this led to the induction of TRB3. If this were indeed the case, one might expect a blunted TRB3 induction by alcohol when alcohol-induced ER stress was blocked. To study this, FGC-4 cells were treated with ethanol in the presence or absence of two compounds known to block ER stress; 4-phenyl butyric acid (PBA) and taurine-ursodeoxycholic acid (TUDCA) (27). Induction of TRB3, p-PERK, and CHOP-10 by ethanol was reduced (P < 0.01) in FGC-4 cells pretreated with PBA or TUDCA (Fig. 7Go).


Figure 6
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  		Fig. 6. Effects of Ethanol on ER Stress Proteins in Whole Cell Lysates of Rat Liver

Western immunoblot analyses of TRB3, CHOP-10, p-PERK, and PERK. Each lane represents an individual rat liver. Asterisks indicate significant differences from the respective control at P < 0.05. Bars represent means ± SEM for n = 5/group. ADU, Arbitrary densitometric units.

 

Figure 7
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  		Fig. 7. Chemical Blockade of Ethanol-Induced ER Stress and TRB3 in FGC-4 Cells

Cells were pretreated with 500 µg/ml TUDCA for 16 h (A) or 10 mM PBA for 16 h (B), followed ethanol (200 mM) treatment for the indicated time points. *, P < 0.05 as compared with corresponding control for TRB3, CHOP-10, and p-PERK at each timing point. Bars represent means ± SEM for n = 3 wells/group. ADU, Arbitrary densitometric units.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Individuals who regularly consume less than two drinks a day (20 g/d) have significantly lower risk of death from all health-related causes than do abstainers (8, 28). The risk of total mortality is increased in individuals who consume higher amounts of alcohol (>70 g/d) (28). These and other studies suggest that the health effects of alcohol are dose dependent and biphasic, with low regular alcohol intake being beneficial and chronic, excessive alcohol intake increasing the risk of adverse health outcomes. For example, when compared with abstainers, the incidence of type 2 diabetes was lower in alcohol drinkers who consumed low levels of alcohol, whereas the risk was greater in heavy drinkers (29). The impairment of insulin signaling by excessive alcohol intake in animal models and potential mechanisms underlying these effects have been previously reported (10, 11, 23). However, experimental animal data to confirm the existence of biphasic effects of alcohol consumption on insulin signaling (especially the effects of low-dose ethanol intake) was heretofore lacking. One interesting and potentially relevant finding is that chronic intake of alcohol induces hepatic nSREBP-1 in mice (18, 19), but decreases nSREBP-1 in rats (11, 30). These reports of two completely opposite effects of alcohol on SREBP-1, an important downstream mediator of insulin signaling, could be indications of the biphasic nature of alcohol-health effects given that mice have much greater rate of ethanol clearance and attain much lower blood ethanol concentrations than rats at the same dose.

To address the potential biphasic actions of alcohol on insulin signaling, we assessed the effects of low- and high-dose ethanol consumption on insulin signaling in the TEN rat model. We found that tyrosine phosphorylation of IRS-1 and -2 was not affected by low-dose alcohol but was increased by high doses of ethanol. However, although there were clear and significant increases in phosphorylation of downstream insulin signaling via Akt and GSK3ß, and in the abundance of active SREBP-1 protein (nSREBP) in whole liver lysates of rats fed diets with low-dose ethanol, the opposite effects were observed with high-dose ethanol despite increased IRS phosphorylation. The latter results in livers of rats fed high-dose ethanol-containing diets replicated our previously published data and appear to be mediated via impaired Akt signaling by ethanol-induced TRB3 (10).

An examination of livers from rats fed low-dose ethanol found significantly decreased abundance of the PI3K regulatory subunit P55{gamma} (p55PIK), whereas the p85, p55{alpha}, p50{alpha}, and p110 isoforms were unaffected and the association of PI3K-p110s with IRS1 was increased. PI3K regulatory subunit (p85{alpha}, p85ß, p55{alpha}, and p50{alpha})-deficient mice have been reported to have enhanced insulin sensitivity and improved glucose tolerance (31, 32, 33). This suggested to us the possibility that the increased phosphorylation of Akt at Thr308 and Ser473 and GSK3ß in rats consuming low doses of ethanol involved enhanced P13K signaling mediated by reductions in p55{gamma}. To test this hypothesis, we used siRNA to knock down the p55{gamma} in FGC-4 cells and found enhanced activation of downstream effectors upon insulin treatment. PI3K regulatory subunits exist in excess to catalytic p110 subunits and IRS proteins, and the free monomeric PI3K regulatory subunit serves as a competitor of the dimeric PI3K kinase by competing for binding to the IRS protein (34). Thus, our data suggest that consumption of low-dose alcohol suppressed p55{gamma} protein expression and improved the stoichiometry between the regulatory and catalytic subunits, resulting in increased association of PI3K-p110 with IRS1 and enhanced the insulin signaling. Whereas our data are consistent with a PI3K-dependent phosphorylation pathway for Thr308 on Akt and ethanol activation of this pathway via effects on p55{gamma} expression, this may not be the only explanation for the current data. Other pathways have also been implicated in Akt phosphorylation at this site. Recent reports suggest that the TORC2 complex can also catalyze this activity (35) and that a protein called Sin1 with a similar function to Rictor is essential for this TORC2 activity (36). These results provide a mechanism for epidemiological studies that suggest that regular consumption of low levels of alcohol enhances insulin sensitivity.

Our results also offer a potential explanation for the discrepancy observed between mice (18, 19) and rats (11, 18, 30) with regard to nSREBP-1 levels. In good part, this may relate to the much greater alcohol metabolism of mice as compared with rats (20). Ethanol metabolism is much greater in mice with nearly twice as much alcohol is required in mice to produce the same hepatic damage as in rats. This is presumably in large part related to the requirement for a threshold level of alcohol in the target tissue. Although the level of alcohol intake (24–34 g/kg·d) was reported in these mice studies (18, 19), the actual blood alcohol levels were not presented. One possible explanation of why those mice had elevated hepatic nSREBP-1 levels is a low tissue ethanol concentration in the hours before tissue collection.

We previously reported that chronic high alcohol consumption led to the induction of TRB3 (10), a negative regulator of Akt (15). TRB3 impaired insulin signaling by binding to the pleckstrin homology domain of Akt sequestering Akt in the cytosol, and preventing Akt phosphorylation (10). However, the mechanism underlying the induction of TRB3 by high-dose ethanol consumption is unknown. Recent reports demonstrate that TRB3 is a novel ER stress-related protein and is induced via CHOP-10 pathway (26). In this report, we found high-dose ethanol consumption significantly increased CHOP-10 protein levels and phosphorylation of PERK at Thr981. These data imply that high-dose ethanol consumption caused ER stress with subsequent induction of TRB3. To test this hypothesis, we used the same in vitro model system (FGC-4 cells) in which we previously demonstrated ethanol induction of TRB3 and impairment of Akt phosphorylation (10). In the current study, we further treated the cells with PBA and TUDCA, each of which has been reported to alleviate ER stress (27). We found that the ethanol-induced increases in TRB3 levels were delayed and decreased significantly. These latter results suggest a crucial role of ER stress on TRB3 induction by high-dose ethanol consumption. It is possible that high tissue ethanol concentrations could result in ER stress in other tissues such as muscle, fat, and heart. This could provide an explanation for biphasic effects of ethanol on disease states such as diabetes and cardiovascular disease. The data also raise a very interesting and important question concerning how high alcohol consumption results in ER stress. Whereas these data are consistent with ER stress in ethanol-exposed cells producing the induction of TRB3 via CHOP, as suggested by Ohoka et al. (26), the data do not preclude the possibility that TRB3 may also be induced through alternate pathways. Future studies in our laboratory will examine these mechanisms.

In summary, we provide evidence that alcohol has biphasic effects on hepatic insulin signaling through different mechanisms. High, chronic doses of alcohol increase ER stress and TRB3 levels to impair insulin signaling, whereas low doses decrease what appears to be a negative regulatory subunit of the PI3K, p55{gamma}, to increase insulin signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
All chemicals, unless otherwise specified, were purchased from Sigma. Antibodies were purchased from commercial suppliers: SREBP-1, CHOP-10, PI3K-p110 (pan) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); GSK3, PI3K-p85 (N-SH2 domain), IRS2, antiphosphotyrosine (4G10), and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were from Upstate Biotechnology (Lake Placid, NY); phospho-GSK3ß, Akt, phospho-Akt473, phospho-Akt308, phospho-PTEN, PI3K-p85, and IRS1 were from Cell Signaling, Inc. Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA). The TRB3 antibody was kindly provided by Dr. Marc Montminy (15) and antiserum to p55{gamma} (P55PIK) was generated as reported previously (24). Tauroursodeoxycholic acid was purchased from Calbiochem (San Diego, CA); 4-phenylbutyrate was from Acros Organics (Morris Plains, NJ).

Animal Experimental Protocols
All animal experiments conformed to ethical guidelines for animal research and received prior approval by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. Adult male Sprague Dawley rats were purchased from Harlan Industries (Indianapolis, IN). An intragastric cannula was surgically implanted into each animal and during the 10 d of recovery, rats were infused 25 ml/d water and allowed ad libitum access to standard rodent diet and bottled water as previously described (37). Thereafter, rats were fed by total enteral nutrition using either an ethanol-containing diet (13 g/kg·d or 4 g/kg·d) or the same diet except that ethanol was isocalorically replaced with carbohydrate. Using a computer-driven programmable pump, diets were supplied continuously, except for 1 h each day needed to refill the syringes, as described previously (37). Rats were killed after 21–30 d of continuous diet infusion. Rats fed the low-dose ethanol-containing diets had UECs below 25 mg/dl, whereas those fed the high-ethanol dose had pulsatile UECs (ranging from near zero to >500 mg/dl). The high-ethanol dose group was killed at a time when the UECs were high on the descending limb of the UEC pulse as described previously (10, 11, 21). Animals were killed during diet infusion to avoid any fasting-induced metabolic or hormonal changes. Livers were collected and stored at –70 C.

Western Immunoblot Analysis and Immunoprecipitation
Samples containing equal amounts of protein were resolved on polyacrylamide gels. Gels were transferred to Hybond-P membrane (Amersham Biosciences, Piscataway, NJ), or stained with Coomassie Blue to confirm equal sample loading. Membranes were blocked overnight at 4 C in Tris-buffered saline with Tween (0.1%) plus 5% (wt/vol) milk powder with gentle shaking and were then incubated with primary antibodies diluted in Tris-buffered saline with Tween (0.1%). Proteins were visualized using the enhanced chemiluminescence plus system (ECL Plus; Amersham Biosciences, Piscataway, NJ) after incubation with secondary antibodies. Detection by autoradiography was described previously (14). For immunoprecipitation, hepatic lysates were incubated with antibodies and protein A agarose beads with gentle rotation at 4 C overnight or 48 h (for precipitating the tyrosine phosphorylated IRS in livers of rats fed low-dose ethanol). Immunoprecipitates were collected, washed, and separated by SDS-PAGE followed by Western blotting.

Cell Culture and RNA Interference
Rat FGC-4 cells were cultured as previously described (10). At approximately 60% confluence, PBA (10 mM) and TUDCA (500 µg/ml) were added for 16 h in glucose-free DMEM supplemented with 1 mM pyruvate and 5% fetal bovine serum. Because insulin resistance has been shown to develop in cultured hepatic cells exposed to superphysiological levels of glucose required for the maintenance and propagation of the FGC-4 cells in vitro, we performed the short-term PBA and TUDCA experiments in glucose-free DMEM supplemented with pyruvate as previously described (10) to avoid this issue. After this 16-h incubation period, ethanol (200 mM or 0.92 g/dl) was added and caps were tightly closed for the remainder of the experiments to prevent evaporative loss of alcohol. For siRNA knockdown experiment, double-stranded stealth RNA duplexes (Invitrogen) corresponding to rat PI3K-p55{gamma} (p55PIK) coding region (5'-CCAAGAACAACACAGUAAAGACUAU-3') were transfected into FGC-4 cells using Lipofectamine 2000. The effect of RNA interference was measured after 48 h of incubation. The negative control siRNA was used in control transfections (Ambion, Austin, TX). Total cell lysates were obtained and separated by SDS-PAGE followed by Western blotting.

Statistics
Student’s t test was used to determine whether group means differed at a significance level of P < 0.05.


   FOOTNOTES
 
This work was supported by the National Institutes on Alcohol Abuse and Alcoholism Grant AA008645 (to T.M.B.).

Disclosure Statement: Authors have nothing to disclose.

First Published Online July 10, 2007

Abbreviations: ADH, Class I alcohol dehydrogenase; BEC, blood ethanol concentrations; CHOP, CCAAT/enhancer binding protein homologous protein; ER, endoplasmic reticulum; IRS, insulin receptor substrate; nSREBP, nuclear SREBP; PBA, 4-phenyl butyric acid; PI3K, phosphatidylinositol 3-kinase; p-PERK, ER stress protein; siRNA, small interfering RNA; SREBP, sterol regulatory element binding protein-1c; TRB3, a mammalian homolog of Drosophila (tribbles-related protein 3); TUDCA, taurine-ursodeoxycholic acid; UEC, urine ethanol concentrations.

Received for publication January 19, 2007. Accepted for publication July 3, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Li TK, Hewitt BG, Grant BF 2004 Alcohol use disorders and mood disorders: a National Institute on Alcohol Abuse and Alcoholism perspective. Biol Psychiatry 56:718–720[Medline]
  2. Wannamethee SG, Camargo Jr CA, Manson JE, Willett WC, Rimm EB 2003 Alcohol drinking patterns and risk of type 2 diabetes mellitus among younger women. Arch Intern Med 163:1329–1336[Abstract/Free Full Text]
  3. Beulens JW, Stolk RP, van der Schouw YT, Grobbee DE, Hendriks HF, Bots ML 2005 Alcohol consumption and risk of type 2 diabetes among older women. Diabetes Care 28:2933–2938[Abstract/Free Full Text]
  4. Koppes LL, Dekker JM, Hendriks HF, Bouter LM, Heine RJ 2005 Moderate alcohol consumption lowers the risk of type 2 diabetes: a meta-analysis of prospective observational studies. Diabetes Care 28:719–725[Abstract/Free Full Text]
  5. Hodge AM, English DR, O’Dea K, Giles GG 2006 Alcohol intake, consumption pattern and beverage type, and the risk of Type 2 diabetes. Diabet Med 23:690–697[CrossRef][Medline]
  6. Gigleux I, Gagnon J, St Pierre A, Cantin B, Dagenais GR, Meyer F, Despres JP, Lamarche B 2006 Moderate alcohol consumption is more cardioprotective in men with the metabolic syndrome. J Nutr 136:3027–3032[Abstract/Free Full Text]
  7. McElduff P, Dobson AJ 1997 How much alcohol and how often? Population based case-control study of alcohol consumption and risk of a major coronary event. BMJ 314:1159–1164[Abstract/Free Full Text]
  8. Agarwal DP 2002 Cardioprotective effects of light-moderate consumption of alcohol: a review of putative mechanisms. Alcohol Alcohol 37:409–415[Abstract/Free Full Text]
  9. Piano MR 2002 Alcoholic cardiomyopathy: incidence, clinical characteristics, and pathophysiology. Chest 121:1638–1650[CrossRef][Medline]
  10. He L, Simmen FA, Mehendale HM, Ronis MJ, Badger TM 2006 Chronic ethanol intake impairs insulin signaling in rats by disrupting Akt association with the cell membrane. Role of TRB3 in inhibition of Akt/protein kinase B activation. J Biol Chem 281:11126–11134[Abstract/Free Full Text]
  11. He L, Simmen FA, Ronis MJ, Badger TM 2004 Post-transcriptional regulation of sterol regulatory element-binding protein-1 by ethanol induces class I alcohol dehydrogenase in rat liver. J Biol Chem 279:28113–28121[Abstract/Free Full Text]
  12. Horton JD, Goldstein JL, Brown MS 2002 SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 109:1125–1131[CrossRef][Medline]
  13. Brown MS, Goldstein JL 1997 The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340[CrossRef][Medline]
  14. He L, Ronis MJ, Badger TM 2002 Ethanol induction of class I alcohol dehydrogenase expression in the rat occurs through alterations in CCAAT/enhancer binding proteins ß and {gamma}. J Biol Chem 277:43572–43577[Abstract/Free Full Text]
  15. Du K, Herzig S, Kulkarni RN, Montminy M 2003 TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver. Science 300:1574–1577[Abstract/Free Full Text]
  16. Qi L, Heredia JE, Altarejos JY, Screaton R, Goebel N, Niessen S, Macleod IX, Liew CW, Kulkarni RN, Bain J, Newgard C, Nelson M, Evans RM, Yates J, Montminy M 2006 TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism. Science 312:1763–1766[Abstract/Free Full Text]
  17. Bardag-Gorce F, French BA, Dedes J, Li J, French SW 2006 Gene expression patterns of the liver in response to alcohol: in vivo and in vitro models compared. Exp Mol Pathol 80:241–251[CrossRef][Medline]
  18. You M, Fischer M, Deeg MA, Crabb DW 2002 Ethanol induces fatty acid synthesis pathways by activation of sterol regulatory element-binding protein (SREBP). J Biol Chem 277:29342–29347[Abstract/Free Full Text]
  19. Ji C, Kaplowitz N 2003 Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice. Gastroenterology 124:1488–1499[CrossRef]
  20. Kono H, Bradford BU, Yin M, Sulik KK, Koop DR, Peters JM, Gonzalez FJ, McDonald T, Dikalova A, Kadiiska MB, Mason RP, Thurman RG 1999 CYP2E1 is not involved in early alcohol-induced liver injury. Am J Physiol 277:G1259–G1267
  21. Badger TM, Hoog JO, Svensson S, McGehee Jr RE, Fang C, Ronis MJ, Ingelman-Sundberg M 2000 Cyclic expression of class I alcohol dehydrogenase in male rats treated with ethanol. Biochem Biophys Res Commun 274:684–688[CrossRef][Medline]
  22. Badger TM, Crouch J, Irby D, Hakkak R, Shahare M 1993 Episodic excretion of ethanol during chronic intragastric ethanol infusion in the male rat: continuous vs. cyclic ethanol and nutrient infusions. J Pharmacol Exp Ther 264:938–943[Abstract/Free Full Text]
  23. Onishi Y, Honda M, Ogihara T, Sakoda H, Anai M, Fujishiro M, Ono H, Shojima N, Fukushima Y, Inukai K, Katagiri H, Kikuchi M, Oka Y, Asano T 2003 Ethanol feeding induces insulin resistance with enhanced PI 3-kinase activation. Biochem Biophys Res Commun 303:788–794[CrossRef][Medline]
  24. Xia X, Serrero G 1999 Multiple forms of p55PIK, a regulatory subunit of phosphoinositide 3-kinase, are generated by alternative initiation of translation. Biochem J 341(Pt 3):831–837
  25. Kaplowitz N, Ji C 2006 Unfolding new mechanisms of alcoholic liver disease in the endoplasmic reticulum. J Gastroenterol Hepatol 21(Suppl 3):S7–S9
  26. Ohoka N, Yoshii S, Hattori T, Onozaki K, Hayashi H 2005 TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death. EMBO J 24:1243–1255[CrossRef][Medline]
  27. Ozcan U, Yilmaz E, Ozcan L, Furuhashi M, Vaillancourt E, Smith RO, Gorgun CZ, Hotamisligil GS 2006 Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 313:1137–1140[Abstract/Free Full Text]
  28. Rehm J, Gmel G, Sempos CT, Trevisan M 2003 Alcohol-related morbidity and mortality. Alcohol Res Health 27:39–51[Medline]
  29. Conigrave KM, Rimm EB 2003 Alcohol for the prevention of type 2 diabetes mellitus? Treat Endocrinol 2:145–152[CrossRef][Medline]
  30. French BA, Dedes J, Bardag-Gorce F, Li J, Wilson L, Fu P, Nan L, French SW 2005 Microarray analysis of gene expression in the liver during the urinary ethanol cycle in rats fed ethanol intragastrically at a constant rate. Exp Mol Pathol 79:87–94[CrossRef][Medline]
  31. Chen D, Mauvais-Jarvis F, Bluher M, Fisher SJ, Jozsi A, Goodyear LJ, Ueki K, Kahn CR 2004 p50{alpha}/p55{alpha} phosphoinositide 3-kinase knockout mice exhibit enhanced insulin sensitivity. Mol Cell Biol 24:320–329[Abstract/Free Full Text]
  32. Terauchi Y, Tsuji Y, Satoh S, Minoura H, Murakami K, Okuno A, Inukai K, Asano T, Kaburagi Y, Ueki K, Nakajima H, Hanafusa T, Matsuzawa Y, Sekihara H, Yin Y, Barrett JC, Oda H, Ishikawa T, Akanuma Y, Komuro I, Suzuki M, Yamamura K, Kodama T, Suzuki H, Yamamura K, Kodama T, Suzuki H, Koyasu S, Aizawa S, Tobe K, Fukui Y, Yazaki Y, Kadowaki T 1999 Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 {alpha} subunit of phosphoinositide 3-kinase. Nat Genet 21:230–235[CrossRef][Medline]
  33. Ueki K, Fruman DA, Yballe CM, Fasshauer M, Klein J, Asano T, Cantley LC, Kahn CR 2003 Positive and negative roles of p85 alpha and p85 beta regulatory subunits of phosphoinositide 3-kinase in insulin signaling. J Biol Chem 278:48453–48466[Abstract/Free Full Text]
  34. Saltiel AR, Pessin JE 2002 Insulin signaling pathways in time and space. Trends Cell Biol 12:65–71[CrossRef][Medline]
  35. Hietakangas V, Cohen SM 2007 Re-evaluating AKT regulation: role of TOR complex 2 in tissue growth. Genes Dev 21:632–637[Abstract/Free Full Text]
  36. Yang Q, Inoki K, Ikenoue T, Guan KL 2006 Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. Genes Dev 20:2820–2832[Abstract/Free Full Text]
  37. Badger TM, Ronis MJ, Lumpkin CK, Valentine CR, Shahare M, Irby D, Huang J, Mercado C, Thomas P, Ingelman-Sundberg M 1993 Effects of chronic ethanol on growth hormone secretion and hepatic cytochrome P450 isozymes of the rat. J Pharmacol Exp Ther 264:438–447[Abstract/Free Full Text]



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