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Am J Physiol Endocrinol Metab 292: E621-E628, 2007. First published October 17, 2006; doi:10.1152/ajpendo.00387.2006
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Chronic ethanol feeding to rats decreases adiponectin secretion by subcutaneous adipocytes

Xiaocong Chen,1,3 Becky M. Sebastian,3 and Laura E. Nagy1,2,3

1Department of Nutrition, Case Western Reserve University; and Departments of 2Gastroenterology and 3Pathobiology, Cleveland Clinic Foundation, Cleveland, Ohio

Submitted 2 August 2006 ; accepted in final form 11 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic ethanol feeding to mice and rats decreases serum adiponectin concentration and adiponectin treatment attenuates chronic ethanol-induced liver injury. Although it is clear that lowered adiponectin has pathophysiological importance, the mechanisms by which chronic ethanol decreases adiponectin are not known. Here, we have investigated the impact of chronic ethanol feeding on adiponectin expression and secretion by adipose tissue. Rats were fed a 36% Lieber-DeCarli ethanol-containing liquid diet or pair-fed control diet for 4 wk. Chronic ethanol feeding decreased adiponectin mRNA but had no effect on adiponectin protein in subcutaneous adipose tissue. Chronic ethanol feeding also reduced adiponectin secretion by isolated subcutaneous and retroperitoneal adipocytes despite the maintenance of equivalent intracellular concentrations of adiponectin between subcutaneous adipocytes from ethanol- and pair-fed rats. Treatment with brefeldin A suppressed adiponectin secretion by subcutaneous adipocytes from pair-fed rats but had little effect after ethanol feeding. In subcutaneous adipocytes from pair-fed rats, adiponectin was enriched in endoplasmic reticulum (ER)/Golgi relative to plasma membrane; however, after chronic ethanol feeding, adiponectin was equally distributed between plasma membrane and ER/Golgi fractions. In conclusion, chronic ethanol feeding impaired adiponectin secretion by subcutaneous and retroperitoneal adipocytes; impaired secretion likely contributes to decreased adiponectin concentrations after chronic ethanol feeding.

trafficking; adipose depots; Golgi

ADIPONECTIN [also known as adipocyte complement-related protein 30 kDa (Acrp30)] is an adipokine exclusively secreted by adipose tissue (28). Adiponectin is abundantly expressed by adipose tissues, composing ~0.01% of the total serum protein (3). Two forms of adiponectin, a low-molecular-weight (LMW) trimer and a high-molecular-weight (HMW) complex are primarily found in serum (24). Two isoforms of adiponectin receptors, AdipoR1 and AdipoR2, are predominantly expressed in skeletal muscle and liver, respectively (42). Adiponectin has important functions in the regulation of glucose and lipid metabolism in both liver and skeletal muscle; it improves insulin action in the liver by suppressing hepatic glucose production (4). Unlike other adipokines, such as leptin, expression of adiponectin is lower in obese humans (3). Serum adiponectin concentrations are also decreased in both humans and murine models of insulin resistance or type 2 diabetes (13, 14, 37). Targeted disruption of adiponectin expression in mice increases their sensitivity to diet-induced insulin resistance compared with wild-type mice (19). Adiponectin also has potent anti-inflammatory functions, decreasing the production of inflammatory cytokines such as TNF-{alpha} and IL-6 by macrophages and adipocytes (1, 39, 43).

Chronic alcohol consumption is characterized by an increase in the expression of a number of inflammatory mediators, including cytokines, reactive oxygen species, and nitrogen species. This proinflammatory state is due to an increased exposure to gut-derived endotoxin/lipopolysaccharide (LPS), as well as enhanced sensitivity of macrophages to activation by LPS (21, 33). Increased production of proinflammatory mediators, in particular TNF-{alpha}, is an important contributor to the progression of ethanol-induced liver injury (21, 33). Recent studies have shown that chronic ethanol consumption decreases serum adiponectin concentration in mice (41, 44) and rats (32). Importantly, treatment of mice with adiponectin during chronic ethanol exposure prevents the development of ethanol-induced liver injury (41) by increasing fatty acid oxidation in the liver, thus preventing ethanol-induced steatosis, as well as decreasing TNF-{alpha} expression in mice chronically exposed to ethanol (41). Furthermore, decreased serum adiponectin concentrations after chronic ethanol feeding are dependent on the type of fat in the diet (44). You et al. (44) reported that ethanol-containing diets high in unsaturated fats contribute to ethanol-induced decreases in adiponectin, whereas inclusion of saturated fats in the ethanol feeding protocol prevent decreased adiponectin. Importantly, the maintenance of serum adiponectin concentrations during ethanol feeding in the context of a high saturated fat diet are associated with a protective effect of saturated fats on the progression of ethanol-induced fatty liver injury (44).

Although these data demonstrate that decreased serum adiponectin concentration contributes to chronic ethanol-induced liver injury, the mechanisms by which chronic ethanol decreases serum adiponectin concentrations have not been studied. Here, we have investigated the effects of chronic ethanol consumption to rats on the production and secretion of adiponectin by adipocytes. We demonstrate that chronic ethanol feeding decreases adiponectin mRNA in subcutaneous adipose tissue. In addition, chronic ethanol feeding disrupts the intracellular distribution of adiponectin peptide and impaired adiponectin secretion by subcutaneous adipocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Adult male Wistar rats weighing 170–180 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). Lieber-DeCarli high-fat ethanol diet was purchased from Dyets (Bethlehem, PA). Cell culture reagents were purchased from GIBCO (Grand Island, NY). Lactate dehydrogenase (LDH) kit was from Promega (Madison, WI). PicoGreen dsDNA kit was from Invitrogen (Eugene, OR). Antibody against adiponectin was from Alpha Diagnostic (San Antonio, TX); anti-ERK1/2 antibody was from Upstate (Charlottesville, VA); anti-Na-K-ATPase was from the Developmental Hybridoma Studies Bank (University of Iowa); and antibodies against calreticulin and syntaxin 6 were from BD Bioscience (San Jose, CA).

Chronic ethanol feeding. The chronic ethanol feeding model used in this study has been previously described (27). Randomly selected rats assigned to the ethanol-fed group were provided an ad libitum liquid diet containing ethanol as 36% of total caloric value for 4 wk. Control rats were pair fed a liquid diet with maltose dextrin isocalorically substituted for ethanol. The type of fat included in the Lieber-Decarli diet used in this study was 8.5 g/l corn oil, 28.4 g/l olive oil, and 2.7 g/l safflower oil. All animal protocols were approved by the Case Western Reserve University Institutional Animal Care and Use Committee. After the feeding period, ethanol-fed and their specific pair-fed controls were anesthetized, and adipose depots were removed and used immediately to isolate adipocytes or flash-frozen in liquid nitrogen and then stored at –80°C. Blood was collected and serum isolated and stored at –80°C. Experiments on ethanol-fed rats and their specific pair-fed controls were carried out in parallel.

Adipose tissue homogenization. Adipose tissue was homogenized (3 ml homogenate buffer/g tissue) in a glass homogenizer with a buffer containing 1% Triton-X100, 50 mM Tris·HCl, 6.4 mM NaCl, 1 mM EDTA, 1 mM sodium pyrophosphate, 1 mM activated sodium vanadate, 10 mM NaF, and protease inhibitor cocktail (Complete-EDTA Free; Roche Molecular Biochemicals, Indianapolis IN). The homogenate was gently rotated for 15 min at 4°C and then centrifuged at 16,000 g for 5 min. The infranatant was used to measure adiponectin by ELISA and then normalized to dsDNA by Picogreen dsDNA kit according to the manufacturer's instructions.

Adipocyte isolation, incubation, and lysate. Adipocytes were isolated as previously described (29), except that cells were washed and incubated in DMEM-F12 with 25 mM HEPES and 0.1% BSA (pH 7.4). After isolation, adipocytes were diluted to 0.5 x 106 cells/ml in wash buffer. Cells (0.1 x 106) were aliquoted to 5 ml of polypropylene round-bottom tubes and incubated at 37°C in a shaking water bath. At the end of the treatment periods, adipocyte suspensions were centrifuged briefly, and medium was removed for adiponectin ELISA or stored at –20°C. Adipocytes were washed once with ice-cold PBS and lysed with ice-cold RIPA buffer (50 mM Tris·HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail). Cell lysates were centrifuged at 7,000 g for 5 min. Infranatant was collected, flash-frozen in liquid nitrogen, and then used for adiponectin ELISA or stored at –80°C.

Real-time PCR for adiponectin and TNF-{alpha} mRNA expression. Total RNA was isolated from fat pads by using the RNeasy lipid tissue mini kit (Qiagen, Valencia, CA), and DNA digestion was performed by using an RNase-free DNase I set (Qiagen) according to manufacturer's instructions. One microgram of total RNA was reverse transcribed using the RETROscript kit (Ambion, Austin, TX) with random primers according to the manufacturer's protocol. Real-time polymerase chain reactions (real-time PCR) were performed by using the SYBR Green Core reagents (Applied Biosystems, Warrington, UK) and three sets of primers specific for adiponectin (forward: 5'-GAC ACG CAG GTG TTC TTG-3', reverse: 5'-CCT ACG CTG AAT GCT GAG-3'), TNF-{alpha} (forward: 5'-CAA GGA GGA GAA GTT CCC AA-3', reverse: 5'-CTC TGC TTG GTG GTT TGC TA-3'), and beta-actin (forward: 5'-CGG TCA GGT CAT CAC TAT CG-3', reverse: 5'-TTC CAT ACC CAG GAA GGA AG-3'). Primers were intron spanning to avoid interference with genomic DNA. Data were analyzed by the {Delta}CT method (18).

Adiponectin ELISA. Adiponectin concentrations in rat serum, incubation medium, adipocyte lysates, and adipose tissue homogenates were measured by ELISA (B-Bridge, San Diego CA) according to the manufacturer's protocol. Two different lots of this ELISA kit were used; the first lot was used for studies in GoFigs. 2A and 3C, and the second lot was used for all other experiments. The absolute values for adiponectin concentration varied slightly between these two different lots of the ELISA kit (data not shown).


Figure 1
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  		Fig. 1. Chronic ethanol (EtOH) feeding to rats decreased serum adiponectin concentration and adiponectin mRNA in subcutaneous adipose tissue. Rats were allowed free access to an ethanol-containing diet or pair-fed control diet for 4 wk. A: serum adiponectin concentrations were measured by ELISA. B: adiponectin and TNF-{alpha} mRNA level in subcutaneous adipose tissue were measured by real-time PCR and then normalized to beta-actin mRNA level. mRNA levels in ethanol-fed rats were then expressed relative to the mRNA levels in their specific pair-fed controls. C: adiponectin protein content in subcutaneous adipose tissue was measured by ELISA and normalized to dsDNA. Values represent means ± SE; n = 7 (A), n = 10 (B), n = 8 (C). *P < 0.05, chronic ethanol-fed vs. pair-fed controls.

 

Figure 2
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  		Fig. 2. Chronic ethanol feeding decreased adiponectin secretion by isolated subcutaneous adipocytes without affecting intracellular adiponectin content. Extracellular (medium; A) and intracellular (B) adiponectin in isolated subcutaneous adipocytes was measured by ELISA. C: subcutaneous adipocytes were incubated at 37°C or on ice for 60 min. After incubation, extracellular adiponectin concentration was measured by ELISA. Quantity of adiponectin was normalized to cell number. Values represent means ± SE; n = 8 (A), n = 6 (B), n = 4 (C). *P < 0.05, chronic ethanol-fed vs. pair-fed controls.

 

Figure 3
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  		Fig. 3. Chronic ethanol feeding had no effect on adiponectin protein content in epididymal or retroperitoneal adipose tissue but decreased adiponectin secretion by retroperitoneal adipocytes. A: adiponectin and TNF-{alpha} mRNA level in epididymal adipose tissue were measured by real-time PCR and then normalized to beta-actin mRNA level. mRNA levels in ethanol-fed rats were then expressed relative to the mRNA levels in their specific pair-fed controls. Adiponectin protein content in subcutaneous (B) and retroperitoneal (D) adipose tissue was measured by ELISA and then normalized to dsDNA. Extracellular adiponectin concentration after incubation of epididymal (C) or retroperitoneal (E) adipocytes was measured by ELISA and then normalized to cell number. Values represent means ± SE; n = 10 (A and B), n = 8 (C), n = 4 (D), n = 8 (E). *P < 0.05, chronic ethanol-fed vs. pair-fed controls.

 
Western blotting. Each sample with 1–4 µg of total protein was boiled 5 min and separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel before being transferred to a polyvinylidene difluoride membrane by using a semidry transfer technique. Blots were blocked with 3% BSA in Tris-buffered saline, pH 7.6, containing 0.1% Tween-20 before overnight incubation at 4°C with primary antibody. Dilutions of primary antibodies used were as follows: 1:1,000 for adiponectin, 1 µg/ml for Na-K-ATPase, 1:2,500 for calreticulin, 1:5,000 for syntaxin 6, and 1:10,000 for ERK1/2. After a 1-h incubation with secondary antibody and subsequent washes, bound antibodies were visualized using enhanced chemiluminescence reagent.

Subcellular fractionation. Subcellular fractions were prepared from isolated adipocytes as previously described, with minor modifications (26, 31). After isolation, subcutaneous adipocytes were homogenized 10 strokes in a Potter-Elvehjem tissue grinder with TSE buffer containing 20 mM Tris·HCl plus 1 mM EDTA plus 255 mM sucrose plus protease inhibitor cocktail (6 million cells/ml TSE). All procedures were performed at 4°C. In brief, homogenates were centrifuged at 16,000 g for 15 min. The pellet (P1) was resuspended in 500 µl of TSE and layered over a 750-µl sucrose pad (20 mM Tris·HCl-1 mM EDTA-1.12 M sucrose, pH 7.4) and centrifuged at 100,000 g for 70 min. The resulting interface, enriched in plasma membrane (PM), was collected and pelleted at 16,000 g for 15 min and then resuspended in 50 µl of RIPA buffer. The supernatant (S1) was centrifuged at 48,000 g for 20 min, and the pellet, containing high-density microsomes (HDM) enriched in endoplasmic reticulum (ER) and Golgi, was resuspended in 40 µl of RIPA. That supernatant (S2) was centrifuged at 200,000 g for 70 min and the pellet, containing low-density microsomes (LDM) enriched in Golgi, was resuspended in 40 µl of RIPA. The remaining supernatant, enriched in cytosol, was collected. Protein content was measured in all fractions and then used for Western blotting. Yield and enrichment for each subcellular fraction were monitored using specific marker proteins: Na-K ATPase for PM, calreticulin for endoplasmic reticulum, syntaxin 6 for Golgi, and ERK1/2 for cytosol.

Data analysis. Values are expressed as means ± SE. Data were analyzed by Student's t-test using InStat (GraphPad Software) or general linear models procedure followed by least square means analysis of differences between groups (SAS, Carey, IN). Data were log transformed, if needed, to obtain a normal distribution.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Chronic ethanol consumption decreases serum adiponectin concentration in mice (41, 44) and rats (32). Consistent with previous studies, serum adiponectin concentration was reduced by 30% in rats after chronic ethanol feeding (Fig. 1A). Because adiponectin is secreted mainly by adipose tissue, we hypothesized that decreased serum adiponectin concentration was due to the decreased adiponectin expression by adipose tissue after chronic ethanol feeding. To test this hypothesis, we first investigated the effect of chronic ethanol feeding on adiponectin mRNA and protein level in subcutaneous adipose tissue. Real-time PCR analysis showed that adiponectin mRNA was decreased by 40% in subcutaneous adipose tissue from ethanol-fed compared with pair-fed rats (Fig. 1B), whereas TNF-{alpha} mRNA was increased two- to threefold by chronic ethanol feeding. In contrast to the decrease in adiponectin mRNA, adiponectin protein content in subcutaneous adipose tissue did not differ between pair- and ethanol-fed rats (Fig. 1C).

Because chronic ethanol feeding had no effect on total adiponectin protein content in subcutaneous adipose tissue despite a suppression in adiponectin mRNA, we hypothesized that chronic ethanol might decrease serum adiponectin level by impairing the secretion of adiponectin from adipocytes. To test this hypothesis, we utilized an ex vivo model of isolated adipocytes. Isolated adipocytes provide a homogeneous population of cells that retain many physiological activities of adipocytes in vivo (12, 38), where secretion of adiponectin can be monitored independently of potential changes in clearance from the circulation. Although adiponectin secretion increased over time in adipocytes from both pair-fed and ethanol-fed rats, chronic ethanol feeding decreased adiponectin secretion by 40% from subcutaneous adipocytes compared with pair-fed controls (Fig. 2A). However, intracellular adiponectin content was not changed by either time of incubation or chronic ethanol feeding (Fig. 2B). More than 60% of the total adiponectin content (the sum of intracellular adiponectin and adiponectin released into the medium) remained in the intracellular compartment, suggesting that impaired adiponectin secretion by chronic ethanol feeding was not due to reduced intracellular adiponectin peptide in adipocytes.

Several control experiments were conducted to ensure that the integrity of the isolated adipocytes was maintained over the 2-h incubation and did not differ between adipocytes isolated from pair-fed and ethanol-fed rats. If the release of adiponectin is a regulated process, then incubation of adipocytes on ice should decrease the rate of release. When subcutaneous adipocytes were incubated on ice for 60 min, no increase in extracellular adiponectin over baseline concentrations was observed in subcutaneous adipocytes isolated from either pair-fed or ethanol-fed rats (Fig. 2C). Furthermore, although some LDH, an intracellular/cytosolic enzyme, was detected in the extracellular media from adipocyte incubations, there were no differences in LDH concentration after incubation for 60 min compared with baseline or between adipocytes from pair- and ethanol-fed rats (pair-fed rats, 0.31 ± 0.1, ethanol-fed rats, 0.27 ± 0.09 units of activity/105 cells; n = 6) after 60 min of incubation.

We also measured the effect of chronic ethanol feeding on adiponectin expression and secretion by epididymal and retroperitoneal adipose tissue, as well as adipocytes isolated from epididymal or retroperitoneal adipose tissue. Chronic ethanol feeding had no effect on adiponectin mRNA or protein content in epididymal adipose tissue (Fig. 3, A and B). Epididymal adipose tissue was not completely insensitive to the chronic effects of ethanol, since TNF-{alpha} mRNA was increased by chronic ethanol feeding in epididymal adipose tissue (Fig. 3A). Adiponectin secreted by epididymal adipocytes from pair-fed rats increased over time; however, chronic ethanol feeding did not affect adiponectin secretion by epididymal adipocytes (Fig. 3C). Chronic ethanol feeding had no effect on the total intracellular adiponectin content in retroperitoneal adipose tissue (Fig. 3D) but decreased secretion of adiponectin over 60 min in isolated retroperitoneal adipocytes compared with adipocytes from pair-fed controls (Fig. 3E). Taken together, these data suggest that the inhibition of adiponectin secretion by chronic ethanol feeding was adipose tissue depot specific.

Brefeldin A (BFA), a drug that disrupts protein trafficking through the Golgi apparatus, severely inhibits adiponectin secretion by 3T3-L1 adipocytes (40). We hypothesized that chronic ethanol feeding would impair adiponectin secretion by affecting its trafficking in subcutaneous adipocytes. Treatment with BFA strongly inhibited adiponectin secretion by subcutaneous adipocytes isolated from pair-fed rats (Fig. 4) but had no effect on adipocytes from ethanol-fed rats. In contrast, treatment with cycloheximide (CHX), a drug that inhibits protein synthesis, had no effect on adiponectin secretion by subcutaneous adipocytes from either pair- or ethanol-fed rats, suggesting that secreted adiponectin was derived mainly from an already synthesized pool of adiponectin. Taken together, these data suggest that impaired adiponectin secretion after chronic ethanol feeding might be due, at least in part, to impaired adiponectin trafficking in subcutaneous adipocytes.


Figure 4
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  		Fig. 4. Effect of chronic ethanol feeding, brefeldin A (BFA), and cyclohexamide (CHX) on adiponectin secretion by subcutaneous adipocytes. Subcutaneous adipocytes were incubated with or without BFA or CHX at 37°C for 60 min. After incubation, extracellular adiponectin concentration was measured by ELISA. Quantity of adiponectin was normalized to cell number. Values represent means ± SE. *P < 0.05, chronic ethanol-fed vs. pair-fed controls; +P < 0.05 vs. cells not treated with BFA.

 
The lack of sensitivity of adiponectin secretion to BFA after chronic ethanol feeding suggests that chronic ethanol disrupts the trafficking of adiponectin through the Golgi itself and/or upstream of the Golgi in the ER. If chronic ethanol impaired trafficking of adiponectin through the ER and Golgi, we would expect to observe an abnormal intracellular distribution of adiponectin after chronic ethanol feeding. To test this hypothesis, adipocytes were fractionated by standard procedures into four compartments: PM-enriched, high-density microsomes (HDM; enriched in ER and Golgi), light-density microsomes (LDM; enriched in Golgi), and cytosol. In adipocytes from pair-fed rats, adiponectin was mainly detected in the PM and HDM fractions but barely detectable in LDM or cytosol fractions. Adiponectin was most abundant in the HDM fractions, exhibiting a fourfold enrichment over the PM in adipocytes from pair-fed rats. This enrichment in ER/Golgi is consistent with previous reports in 3T3-L1 adipocytes (40) and adipose tissue explants from mice (8). Chronic ethanol feeding had no effect on the distribution of either total proteins (data not shown) or marker proteins (Fig. 5A) to each subcellular fraction. However, after chronic ethanol feeding, the amount of adiponectin in the HDM fraction was decreased, resulting in a loss of enrichment of adiponectin in the HDM fraction (Fig. 5B). This decrease of adiponectin in the ER/Golgi compartment was consistent with the inability of BFA to suppress adiponectin secretion after chronic ethanol feeding.


Figure 5
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  		Fig. 5. Subcellular distribution of adiponectin in subcutaneous adipocytes. Subcutaneous adipocytes were homogenized and separated by differential centrifugation into 4 subcellular fractions: plasma membrane (PM), high-density microsomes (HDM), low-density microsomes (LDM), and cytosol. A: adiponectin protein content was detected by Western blotting. Marker proteins for PM, endoplasmic reticulum (ER), Golgi, and cytosol were Na-K-ATPase, calreticulin, syntaxin 6, and ERK1/2, respectively. Images are representative of 5–7 different subcellular fractionations per experimental group. P, pair fed; E, ethanol fed. B: adiponectin content in HDM and PM fractions from ethanol-fed rats was normalized to the quantity of adiponectin in HDM from adipocytes from their specific pair-fed rats. Values represent means ± SE; n = 5–7 rats per group. *P < 0.05, chronic ethanol-fed vs. pair-fed controls; +P < 0.05, HDM vs. PM within an experimental group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Chronic ethanol consumption decreases serum adiponectin concentration in mice (41, 44) and rats (32) (Fig. 1). This decrease in adiponectin contributes to the development of ethanol-induced liver injury, as delivery of exogenous adiponectin via osmotic pump (41) protects mice from ethanol-induced liver injury. Thus, although it is clear that decreased adiponectin concentrations are important to the pathophysiology of chronic ethanol-induced liver injury, the mechanisms by which chronic ethanol feeding decreases serum adiponectin concentration have not been investigated. As adipose tissue is the major source of adiponectin in the circulation, we hypothesized that chronic ethanol feeding decreased adiponectin production by adipose tissue. In this study, we identify two mechanisms by which chronic ethanol decreases adiponectin production: first, chronic ethanol decreases adiponectin mRNA in subcutaneous adipose tissue; and second, chronic ethanol decreased the secretion of adiponectin from subcutaneous and retroperitoneal adipocytes. Decreased secretion from subcutaneous adipocytes was associated with a disruption in the intracellular trafficking of adiponectin after chronic ethanol feeding.

Chronic ethanol feeding decreased adiponectin mRNA in subcutaneous adipose tissue without affecting the intracellular pool of adiponectin peptide. A number of DNA binding sites have been identified in the adiponectin promoter region, including sites for peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), CCAAT/enhancer-binding protein-beta (C/EBP-beta), nuclear factor-Y (NFY), and sterol regulatory element-binding protein 1c (SREBP1c) (15, 25, 30). Mutation analysis of the SREBP1c, C/EBP-beta, and PPAR{gamma} sites showed that these elements are required for basal promoter activity of adiponectin (15, 25, 30). Therefore, it is likely that decreased adiponectin mRNA in subcutaneous adipose tissue in response to chronic ethanol feeding is regulated by one or more of these elements. Data from HeLa cells transfected with the adiponectin promoter suggest that ethanol may interact with the specific fatty acids in the media to downregulate adiponectin promoter activity (44). Also of particular relevance may be the interaction between the inflammatory cytokine TNF-{alpha} and adiponectin, which suppress each other's expression both in vivo and in vitro (19, 41). Suppression of adiponectin expression by TNF-{alpha} has been associated with inactivation of C/EBP-beta (17), as well as regulation of PPAR{gamma} DNA binding activity (30). After chronic ethanol exposure, increased circulating TNF-{alpha} is observed in both rodent models and humans (32, 33). Thus it is likely that increased TNF-{alpha} in the circulation creates an "inflammatory environment" for adipocytes, contributing to the downregulation of adiponectin expression.

Adiponectin gene expression is also inhibited by beta-adrenergic stimulation via protein kinase A (PKA) both in vivo and in vitro (8, 11). However, chronic ethanol feeding decreases beta-adrenergic receptor-mediated activation of PKA in isolated adipocytes (16). Therefore, it is unlikely that the suppression of adiponectin mRNA in response to chronic ethanol feeding involves inhibition by PKA.

The second major mechanism for decreased adiponectin production by adipocytes identified here is a decrease in the secretion of adiponectin from adipocytes isolated from subcutaneous, as well as retroperitoneal, adipose tissue. Secretion was suppressed despite the maintenance of a large intracellular pool of adiponectin peptide. This intracellular pool of adiponectin was not reduced either by chronic ethanol feeding or by incubation for 2 h ex vivo. Furthermore, inhibition of adiponectin synthesis by treatment with CHX did not reduce adiponectin secretion, indicating that the presynthesized pool of adiponectin was sufficient to support secretion for at least 2 h. This observation is consistent with a previous report demonstrating that treatment with CHX does not affect adiponectin secretion by 3T3-L1 adipocytes for 6 h (40). On the basis of these results, it appears unlikely that impaired adiponectin secretion after chronic ethanol feeding is due to a decrease in the total intracellular pool of adiponectin. Because the intracellular concentration of adiponectin is determined by the rate of synthesis of adiponectin peptide and by its rate of secretion from the adipocyte, we hypothesize that the intracellular pool of adiponectin was maintained after chronic ethanol feeding due to the combined impact of decreased quantity of adiponectin mRNA in response to chronic ethanol feeding and the ethanol-induced decrease in adiponectin secretion.

Although the total intracellular content of adiponectin was not decreased, the intracellular distribution of adiponectin was disrupted by chronic ethanol feeding. The mechanisms by which adiponectin is trafficked through the adipocyte and secreted are not well understood. Several lines of evidence suggest that adiponectin localized in the Golgi is an important source of intracellular adiponectin available for secretion. Adiponectin is predominantly colocalized with Golgi marker proteins in 3T3-L1 adipocytes (5) and is found in HDM fractions, which are enriched in Golgi and ER, in mouse adipose explants (8) and isolated rat subcutaneous adipocytes (Fig. 5). When adipocytes are treated with BFA, adiponectin is shifted from the Golgi compartment, and adiponectin secretion is inhibited in 3T3-L1 adipocytes (40) as well as subcutaneous adipocytes from pair-fed rats (Fig. 4). Adiponectin secretion is also dependent on GGA proteins (for Golgi localizing {gamma}-adaptin ear homology domain ARF-binding protein) (40) and Vti1a, a v-Snare protein found in the trans-Golgi network (6) in 3T3-L1 adipocytes.

In contrast to the predominant localization of adiponectin to Golgi/Golgi-enriched compartments in control adipocytes, adiponectin was shifted from the HDM fraction to a PM-enriched fraction after chronic ethanol feeding (Fig. 5). Furthermore, adiponectin secretion was no longer sensitive to inhibition by BFA after chronic ethanol feeding (Fig. 4). These data suggest that chronic ethanol feeding acts to disrupt the normal trafficking of adiponectin through the Golgi apparatus. Ethanol exposure disrupts protein trafficking in a number of cell types, targeting multiple steps in protein trafficking, including receptor-mediated endocytosis and the flow of newly synthesized proteins to the PM (22, 35). In adipocytes, chronic ethanol consumption disrupts insulin-stimulated GLUT4 trafficking in rat adipocytes (26) as well as localization of membrane proteins to lipid rafts (29). In other cell types, trafficking of both endogenous and viral marker proteins through the Golgi apparatus is disrupted by chronic ethanol exposure (20, 34). This impairment may be related to the effect of chronic ethanol on required posttranslational modifications, including glycosylation, that occur within the ER and Golgi apparatus (7) and/or changes in the activity of small GTP-binding proteins required for Golgi function (20, 34), since posttranslational glycosylation, as well as formation of disulfide bonds within the adiponectin trimer, are required for the functional activity of adiponectin (24, 36). It is possible that impairment of either appropriate glycosylation and/or trimer formation contributes to the abnormal intracellular trafficking of adiponectin as well as the reduced rates of secretion observed after chronic ethanol feeding.

Chronic ethanol feeding differentially regulated adiponectin mRNA expression in subcutaneous compared with epididymal adipose tissue. In contrast, TNF-{alpha} mRNA was increased by chronic ethanol in both adipose depots. Individual adipose depots exhibit varied rates of lipolysis and respond differently to nutrient or insulin action; these depot-specific responses are thought to relate to the specific metabolic demands required by the individual organs in the body associated with each depot (9, 10, 23). Very few data are available regarding the depot-specific expression of adiponectin, although one study suggests that a shift in the depot-specific expression of adiponectin may be a contributing factor for the development of insulin resistance in Zucker fatty rats (2). In that study, Zucker fatty rats had lower adiponectin mRNA in both subcutaneous and visceral adipose than lean controls. Furthermore, the relative expression of adiponectin is shifted from higher relative expression in visceral fat in lean rats to a higher expression of adiponectin in subcutaneous fat in the fatty rats (2). In contrast to the suppression of adiponectin mRNA in several adipose depots in obesity (2), the effects of chronic ethanol on adiponectin mRNA were not global, in that adiponectin mRNA was decreased specifically in subcutaneous adipose tissue.

Chronic ethanol feeding specifically suppressed adiponectin secretion by adipocytes isolated from retroperitoneal adipose, considered a visceral fat depot, as well as subcutaneous adipocytes, but not epididymal adipocytes. Because chronic ethanol feeding decreased adiponectin secretion from the two major types of adipose tissue, subcutaneous and visceral, it is likely that these changes contribute to decreased serum adiponectin concentration after chronic ethanol feeding. It is also possible that chronic ethanol feeding increases the clearance of adiponectin by the kidneys or liver, which could also contribute to the decrease in circulating adiponectin observed after chronic ethanol feeding.

In summary, we have demonstrated here that chronic ethanol feeding to rats decreases adiponectin mRNA in subcutaneous adipocytes and disrupts the typical intracellular distribution of adiponectin from an ER/Golgi-enriched compartment to the PM. This impaired intracellular trafficking was associated with a suppression in the rate of adiponectin secretion by subcutaneous adipocytes. Because decreased adiponectin concentration contributes to the progression of chronic ethanol-induced liver injury (41), it is likely that the suppression of adiponectin secretion reported here contributes to the pathophysiological effects of chronic ethanol feeding.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institute on Alcohol Abuse and Alcoholism Grant RO1-AA-11876.


   ACKNOWLEDGMENTS
 
We thank Dr. Michele T. Pritchard for help with the real-time PCR setup and primer design. We are also grateful to Megan R. McMullen and Brian T. Pratt for technical support. The monoclonal antibody against subunit 1 of Na-K-ATPase (6F), developed by D. M. Fambrough, was obtained from the Developmental Studies Hybridoma Bank and maintained by the University of Iowa, Department of Biological Sciences, under Contract NO1-HD-7-3263.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. E. Nagy, Cleveland Clinic Foundation, Lerner Research Institute/NE40, 9500 Euclid Ave, Cleveland, OH 44195 (e-mail: nagyl3{at}ccf.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ajuwon KM, Spurlock ME. Adiponectin inhibits LPS-induced NF-{kappa}B activation and IL-6 production and increases PPAR{gamma}2 expression in adipocytes. Am J Physiol Regul Integr Comp Physiol 288: R1220–R1225, 2005.[Abstract/Free Full Text]
  2. Altomonte J, Harbaran S, Richter A, Dong H. Fat depot-specific expression of adiponectin is impaired in Zucker fatty rats. Metabolism 52: 958–963, 2003.[CrossRef][ISI][Medline]
  3. Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K, Shimomura I, Nakamura T, Miyaoka K, Kuriyama H, Nishida M, Yamashita S, Okubo K, Matsubara K, Muraguchi M, Ohmoto Y, Funahashi T, Matsuzawa Y. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 257: 79–83, 1999.[CrossRef][ISI][Medline]
  4. Berg AH, Combs TP, Du X, Brownlee M, Scherer PE. The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat Med 7: 947–953, 2001.[CrossRef][ISI][Medline]
  5. Bogan JS, Lodish HF. Two compartments for insulin-stimulated exocytosis in 3T3-L1 adipocytes defined by endogenous ACRP30 and GLUT4. J Cell Biol 146: 609–620, 1999.[Abstract/Free Full Text]
  6. Bose A, Guilherme A, Huang S, Hubbard AC, Lane CR, Soriano NA, Czech MP. The v-SNARE Vti1a regulates insulin-stimulated glucose transport and Acrp30 secretion in 3T3-L1 adipocytes. J Biol Chem 280: 36946–36951, 2005.[Abstract/Free Full Text]
  7. Cottalasso D, Domenicotti C, Traverso N, Pronzato M, Nanni G. Influence of chronic ethanol consumption on toxic effects of 1,2-dichloroethane:glycolipoprotein retention and impairment of dolichol concentration in rat liver microsomes and Golgi apparatus. Toxicology 178: 229–240, 2002.[CrossRef][ISI][Medline]
  8. Delporte ML, Funahashi T, Takahashi M, Matsuzawa Y, Brichard SM. Pre- and post-translational negative effect of beta-adrenoceptor agonists on adiponectin secretion: in vitro and in vivo studies. Biochem J 367: 677–685, 2002.[CrossRef][ISI][Medline]
  9. Einstein FH, Atzmon G, Yang XM, Ma XH, Rincon M, Rudin E, Muzumdar R, Barzilai N. Differential responses of visceral and subcutaneous fat depots to nutrients. Diabetes 54: 672–678, 2005.[Abstract/Free Full Text]
  10. Engfeldt P, Arner P. Lipolysis in human adipocytes, effects of cell size, age and of regional differences. Horm Metab Res Suppl 19: 26–29, 1988.[Medline]
  11. Fasshauer M, Klein J, Neumann S, Eszlinger M, Paschke R. Adiponectin gene expression is inhibited by beta-adrenergic stimulation via protein kinase A in 3T3-L1 adipocytes. FEBS Lett 507: 142–146, 2001.[CrossRef][ISI][Medline]
  12. Honnor RC, Dhillon GS, Londos C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior. J Biol Chem 260: 15122–15129, 1985.[Abstract/Free Full Text]
  13. Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, Iwahashi H, Kuriyama H, Ouchi N, Maeda K, Nishida M, Kihara S, Sakai N, Nakajima T, Hasegawa K, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Hanafusa T, Matsuzawa Y. Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol 20: 1595–1599, 2000.[Abstract/Free Full Text]
  14. Hu E, Liang P, Spiegelman BM. AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271: 10697–10703, 1996.[Abstract/Free Full Text]
  15. Iwaki M, Matsuda M, Maeda N, Funahashi T, Matsuzawa Y, Makishima M, Shimomura I. Induction of adiponectin, a fat-derived antidiabetic and antiatherogenic factor, by nuclear receptors. Diabetes 52: 1655–1663, 2003.[Abstract/Free Full Text]
  16. Kang L, Nagy LE. Chronic ethanol feeding suppresses (beta)-adrenergic receptor-stimulated lipolysis in adipocytes isolated from epididymal fat. Endocrinology 147: 4330–4338, 2006.[Abstract/Free Full Text]
  17. Kita A, Yamasaki H, Kuwahara H, Moriuchi A, Fukushima K, Kobayashi M, Fukushima T, Takahashi R, Abiru N, Uotani S, Kawasaki E, Eguchi K. Identification of the promoter region required for human adiponectin gene transcription: Association with CCAAT/enhancer binding protein-beta and tumor necrosis factor-alpha. Biochem Biophys Res Commun 331: 484–490, 2005.[CrossRef][ISI][Medline]
  18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2[–Delta Delta C(T)] method. Methods 25: 402–408, 2001.[CrossRef][ISI][Medline]
  19. Maeda N, Shimomura I, Kishida K, Nishizawa H, Matsuda M, Nagaretani H, Furuyama N, Kondo H, Takahashi M, Arita Y, Komuro R, Ouchi N, Kihara S, Tochino Y, Okutomi K, Horie M, Takeda S, Aoyama T, Funahashi T, Matsuzawa Y. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nat Med 8: 731–737, 2002.[CrossRef][ISI][Medline]
  20. Marmillot P, Rao MN, Lakshman MR. Chronic ethanol exposure in rats affects rabs-dependent hepatic trafficking of apolipoprotein E and transferrin. Alcohol 25: 195–200, 2001.[CrossRef][ISI][Medline]
  21. Nagy LE. New insights into the role of the innate immune response in the development of alcoholic liver disease. Expt Biol Med 228: 882–890, 2003.[Abstract/Free Full Text]
  22. Nagy LE, Lakshman MR, Casey CA, Bearer CF. Ethanol and membrane protein trafficking: diverse mechanisms of ethanol action. Alcohol Clin Exp Res 26: 287–293, 2002.[CrossRef][ISI][Medline]
  23. Ostman J, Arner P, Engfeldt P, Kager L. Regional differences in the control of lipolysis in human adipose tissue. Metabolism 28: 1198–1205, 1979.[CrossRef][ISI][Medline]
  24. Pajvani UB, Du X, Combs TP, Berg AH, Rajala MW, Schulthess T, Engel J, Brownlee M, Scherer PE. Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. Implications for metabolic regulation and bioactivity. J Biol Chem 278: 9073–9085, 2003.[Abstract/Free Full Text]
  25. Park SK, Oh SY, Lee MY, Yoon S, Kim KS, Kim JW. CCAAT/enhancer binding protein and nuclear factor-Y regulate adiponectin gene expression in adipose tissue. Diabetes 53: 2757–2766, 2004.[Abstract/Free Full Text]
  26. Poirier LA, Rachdaoui N, Nagy LE. GLUT4 vesicle trafficking in rat adipocytes after ethanol feeding: regulation by heterotrimeric G-proteins. Biochem J 354: 323–330, 2001.[CrossRef][ISI][Medline]
  27. Rachdaoui N, Sebastian BM, Nagy LE. Chronic ethanol feeding impairs endothelin-1-stimulated glucose uptake via decreased G{alpha}11 expression in rat adipocytes. Am J Physiol Endocrinol Metab 285: E303–E310, 2003.[Abstract/Free Full Text]
  28. Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF. A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 270: 26746–26749, 1995.[Abstract/Free Full Text]
  29. Sebastian BM, Nagy LE. Decreased insulin-dependent glucose transport by chronic ethanol feeding is associated with dysregulation of the Cbl/TC10 pathway in rat adipocytes. Am J Physiol Endocrinol Metab 289: E1077–E1084, 2005.[Abstract/Free Full Text]
  30. Seo JB, Moon HM, Noh MJ, Lee YS, Jeong HW, Yoo EJ, Kim WS, Park J, Youn BS, Kim JW, Park SD, Kim JB. Adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element-binding protein 1c regulates mouse adiponectin expression. J Biol Chem 279: 22108–22117, 2004.[Abstract/Free Full Text]
  31. Simpson EA, Yver DR, Hissin PJ, Wardzala LJ, Darnieli E, Salans LB, Cushman SW. Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cell: characterization of subcellular fractions. Biochim Biophys Acta 763: 393–407, 1983.[Medline]
  32. Thakur V, Pritchard MT, McMullen MR, Nagy LE. Adiponectin normalizes LPS-stimulated TNF-{alpha} production by rat Kupffer cells after chronic ethanol feeding. Am J Physiol Gastrointest Liver Physiol 290: G998–G1007, 2006.[Abstract/Free Full Text]
  33. Thurman RG. Mechanisms of Hepatic Toxicity II. Alcoholic liver injury involves activation of Kupffer cells by endotoxin. Am J Physiol Gastrointest Liver Physiol 275: G605–G611, 1998.[Abstract/Free Full Text]
  34. Tomas M, Marin P, Megias L, Egea G, Renau-Piqueras J. Ethanol perturbs the secretory pathway in astrocytes. Neurobiol Dis 20: 773–784, 2005.[CrossRef][ISI][Medline]
  35. Tuma DJ, Casey CA, Sorrell MF. Effects of ethanol on hepatic protein trafficking: impairment of receptor-mediated endocytosis. Alcohol Alcohol 25: 117–125, 1990.[Abstract/Free Full Text]
  36. Wang Y, Xu A, Knight C, Xu LY, Cooper GJ. Hydroxylation and glycosylation of the four conserved lysine residues in the collagenous domain of adiponectin. Potential role in the modulation of its insulin-sensitizing activity. J Biol Chem 277: 19521–19529, 2002.[Abstract/Free Full Text]
  37. Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Pratley RE, Tataranni PA. Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 86: 1930–1935, 2001.[Abstract/Free Full Text]
  38. Wilkes JJ, DeForrest LL, Nagy LE. Chronic ethanol feeding in a high-fat diet decreases insulin-stimulated glucose transport in rat adipocytes. Am J Physiol Endocrinol Metab 271: E477–E484, 1996.[Abstract/Free Full Text]
  39. Wulster-Radcliffe MC, Ajuwon KM, Wang J, Christian JA, Spurlock ME. Adiponectin differentially regulates cytokines in porcine macrophages. Biochem Biophys Res Commun 316: 924–929, 2004.[CrossRef][ISI][Medline]
  40. Xie L, Boyle D, Sanford D, Scherer PE, Pessin JE, Mora S. Intracellular trafficking and secretion of adiponectin is dependent on GGA-coated vesicles. J Biol Chem 281: 7253–7259, 2006.[Abstract/Free Full Text]
  41. Xu A, Wang Y, Keshaw H, Xu LY, Lam KS, Cooper GJ. The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice. J Clin Invest 112: 91–100, 2003.[CrossRef][ISI][Medline]
  42. Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423: 762–769, 2003.[CrossRef][Medline]
  43. Yokota T, Oritani K, Takahashi I, Ishikawa J, Matsuyama A, Ouchi N, Kihara S, Funahashi T, Tenner AJ, Tomiyama Y, Matsuzawa Y. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood 96: 1723–1732, 2000.[Abstract/Free Full Text]
  44. You M, Considine RV, Leone TC, Kelly DP, Crabb DW. Role of adiponectin in the protective action of dietary saturated fat against alcoholic fatty liver in mice. Hepatology 42: 568–577, 2005.[CrossRef][ISI][Medline]



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