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Keywords:

  • Galanin;
  • Paraventricular Nucleus;
  • Dietary Fat;
  • Lipids;
  • Feeding Behavior

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Background: Recent evidence has demonstrated that ethanol intake can stimulate the expression and production of the feeding-stimulatory peptide, galanin (GAL), in the hypothalamic paraventricular nucleus (PVN), and that PVN injection of this peptide, in turn, can increase the consumption of ethanol. To test the hypothesis that other feeding-related systems are involved in ethanol intake, this study examined the effect of ethanol on the hypothalamic opioid peptides, enkephalin (ENK), and dynorphin (DYN).

Method: Adult, male Sprague–Dawley rats were trained to voluntarily drink increasing concentrations of ethanol, up to 9% v/v, on a 12-hour access schedule or were given a single injection of ethanol (10% v/v) versus saline vehicle. The effect of ethanol on GAL, ENK, and DYN mRNA was measured using real-time quantitative polymerase chain reaction and radiolabeled in situ hybridization, while radioimmunoassay was used to measure peptide levels. In addition to blood alcohol, circulating levels of triglycerides (TG), leptin, and insulin were also measured.

Results: The data demonstrated that: (1) rats voluntarily drinking 9% v/v ethanol (approximately 2.0 g/kg/d) show a significant increase in GAL, ENK, and DYN mRNA in the PVN compared with water-drinking rats; (2) voluntary consumption of ethanol also increases peptide levels of ENK and DYN in the PVN; (3) acute injection of 10% ethanol (1.0 g/kg of 10% v/v) similarly increases the expression of GAL, ENK, and DYN in the PVN; and (4) ethanol consumption and injection, while having little effect on leptin and insulin, consistently increase circulating levels of TG as well as alcohol, both of which are strongly, positively correlated with peptide expression in the PVN.

Conclusions: These findings, together with published studies, suggest a possible role for hypothalamic opioid peptides in the drinking of ethanol. Based on evidence that dietary fat and lipid injections stimulate the PVN peptides and injection of the opiates and GAL increase ethanol intake, it is proposed that both TG and alcohol in the circulation, which are elevated by the ingestion or injection of ethanol, are involved in stimulating these peptides in the PVN, which in turn promote further consumption of ethanol.

THE STUDY OF neurochemicals involved in ingestive behavior may provide important insights into the mechanisms underlying the consumption of ethanol. Hypothalamic peptides are powerful controllers of the urge to eat and keep eating (Leibowitz and Hoebel, 2004b; Schwartz et al., 2000). Moreover, the expression of these peptides is strongly affected by the substances being ingested (Leibowitz and Hoebel, 2004b, 2004c). This is clearly reflected in the studies of galanin (GAL) in the hypothalamic paraventricular nucleus (PVN). Local injection of GAL into the PVN stimulates feeding behavior, particularly the intake of a fat-rich diet (Corwin et al., 1993; Kyrkouli et al., 1986; Yun et al., 2005), and the expression of this peptide, in turn, is stimulated in the PVN by ingestion of a high-fat diet (Akabayashi et al., 1994; Leibowitz et al., 2004a), suggesting a positive-feedback loop. Recent studies indicate a similar relationship between hypothalamic GAL and the consumption of ethanol. Injection of GAL into the PVN or ventricles increases the amount of ethanol consumed (Lewis et al., 2004; Rada et al., 2004), and voluntary ethanol intake and systemic injection of ethanol stimulate the expression of GAL mRNA in the PVN (Leibowitz et al., 2003). These results suggest that peptides in the hypothalamus that modulate food intake may also have a role in ethanol intake.

The focus of this report is on the opioid peptides, enkephalin (ENK) and dynorphin (DYN), in the hypothalamus and specifically the PVN. Like GAL, injection of these peptides into the PVN has a stimulatory effect on food intake, and this effect is strongest in rats fed a fat-rich diet (Arjune et al., 1991; Baile et al., 1986; Leibowitz, 2000; Mclean and Hoebel, 1983; Zhang et al., 1998). Further, a recent report demonstrates that ENK mRNA in the PVN increases with acute consumption of a high-fat compared with a low-fat diet (Chang et al., 2004), while other studies show DYN mRNA in this nucleus to be stimulated by a chronic high-fat diet (Archer et al., 2005; Levin and Dunn-Meynell, 2002; Welch et al., 1996). These similarities between GAL and the opioid peptides in their relationship with dietary fat suggest that the opioid peptides in the PVN may also be similar to GAL in their relation to ethanol intake (Leibowitz, 2005). Although studies with hypothalamic opiate injections have yet to be conducted, peripheral injections of morphine or leu-ENK are found to increase the drinking of ethanol, while opioid receptor antagonists reduce ethanol intake (Hubbell et al., 1986; Messiha, 1989; Oswald and Wand, 2004). A direct link between the hypothalamic opioid and GAL systems receives support from evidence that GAL-induced feeding is blocked by an opioid antagonist (Barton et al., 1996; Dube et al., 1994), GAL coexists with opioids in hypothalamic neurons (Kalra and Kalra, 1996; Merchenthaler et al., 1993), and both GAL and the opioids stimulate the release of dopamine in the nucleus accumbens (NAc) (Pothos et al., 1991; Rada et al., 1998).

Most studies of endogenous opioids in the hypothalamus as they relate to ethanol consumption have analyzed the whole structure rather than discrete nuclei. Thus, mixed results have been obtained, with acute or chronic ethanol having no effect or causing either an increase or a decrease in levels, release, or expression of ENK, DYN, and β-endorphin in the hypothalamus (Cowen and Lawrence, 1999, 2001; de Gortari et al., 2000; Oswald and Wand, 2004; Patel and Pohorecky, 1989; Przewlocka and Lason, 1991; Schulz et al., 1980; Seizinger et al., 1983). To our knowledge, there are only 2 reports that have examined a specific hypothalamic nucleus, namely, the PVN. One study demonstrated that ENK mRNA in this nucleus is unaffected by acute, intragastric infusion of ethanol (2.5 g/kg of 30% ethanol) at 1 and 4 hours after injection but is significantly stimulated at 24 hours (de Gortari et al., 2000). The other showed no change after chronic consumption of a lower concentration of 5% ethanol (Cowen and Lawrence, 2001).

Thus, more systematic studies of endogenous opioid peptides in specific hypothalamic nuclei are clearly needed to understand their role in controlling ethanol consumption. The focus of this report is on the PVN, where GAL functions in close relation to ethanol intake as well as the ingestion of a fat-rich diet (Carrillo et al., 2004; Leibowitz, 2005). The objectives are to measure both the expression and peptide levels of ENK and DYN as well as GAL in the PVN, in relation to both chronic consumption and acute injection of ethanol. In addition, there is evidence that ethanol intake, like fat intake, can increase circulating levels of triglycerides (TG) (Contaldo et al., 1989; Goude et al., 2002) and that these lipids are closely, positively related to PVN expression of GAL and ENK (Chang et al., 2004; Leibowitz et al., 2004a; Leibowitz, 2005). Thus, this study also measured these lipids in addition to blood alcohol levels, to determine whether they shift in close relation to the ethanol-induced changes in endogenous peptides.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Subjects

Adult, male Sprague–Dawley rats (300–350 g, Taconic Farms, Germantown, NY) were housed individually, on a 12-hour reversed light/dark cycle. The rats in each set of water-drinking and ethanol-drinking groups were approximately matched for body weight, with an overall range of 300 to 350 g at the start of the experiment and 400 to 475 g at the end. All animals were allowed 1 week to acclimate to their individual housing conditions, during which time they received ad libitum access to standard rodent chow (LabDiet Rodent Chow 5001, St. Louis, MO; 12% fat, 60% carbohydrate, and 28% protein) and water, which was delivered via an automatic watering system from a spout in the back of the cage. Animals were well cared for in accordance with the Institutional Animal Care and Use Committee regulations at Princeton University.

Test Procedures

In Experiments 1 to 3, rats were given ad libitum access to lab chow, water, and in some cases ethanol. The water-drinking control rats (n=5–10/experiment) were maintained on chow and water, and the ethanol-drinking experimental groups (n=10–15/experiment) were additionally given access to ethanol (95% ethanol, David Sherman Corp., St. Louis, MO) diluted with tap water. This was presented at the front of the home cage in a 100-mL graduated glass cylinder, which was fitted with a sipper tube containing a steel ball as a tip valve to prevent spillage. Access to the ethanol-containing cylinder was provided for 12 hours each day for 28 to 30 days, with ethanol presented after a 4-hour delay following dark onset to enhance initial consumption. The concentration of ethanol was increased stepwise, every 4 days, from 1 to 2, 4, 7, and then 9% v/v, with the rats maintained for an additional 8 to 10 days on 9% ethanol. In each experiment, the ethanol-drinking rats were rank ordered and subdivided into 2 groups based on the average amount of 9% ethanol consumed in g/kg in 12 hours. They were designated either “low-ethanol” or “high-ethanol” drinkers, which, respectively, were the lowest and highest 50% (Experiment 1) or 33% (Experiments 2 and 3) of the group. On the final day of 9% ethanol drinking in these experiments, chow was removed 1 hour after dark onset (2 hours before access to ethanol), and either water or ethanol was presented 3 hours later at 4 hours into the dark cycle. Body weight and daily food intake were measured every 4 days. The groups were similar in body weight, and the measures of 24-hour food intake showed that the ethanol-drinking rats consumed slightly, but not significantly, fewer calories from chow compared with the water-drinking rats (96 vs 103 Kcals/d).

After being allowed to drink for 1 hour, the water-drinking and ethanol-drinking rats were killed by rapid decapitation, and their brains were removed at 5 hours after dark onset. In Experiment 1, the PVN and also the nucleus accumbens (NAc, core, and shell) and dorsal striatum were microdissected for measurements of GAL, ENK, and DYN mRNA using real-time quantitative polymerase chain reaction (PCR). In Experiment 3, the PVN was microdissected for measurements of ENK and DYN peptide levels via radioimmunoassay, while the whole brain in Experiment 2 was placed in a 4% paraformaldehyde solution for peptide measurements in the PVN using radiolabeled in situ hybridization histochemistry. In specific experiments, trunk blood was additionally collected and analyzed for blood alcohol concentration, TG levels, and, in Experiment 1, the hormones leptin and insulin. All analyses were performed blind to the treatment condition.

In Experiment 4, 2 sets of rats (n=12–16/set) were used to test the effect of ethanol injection on the peptides in the PVN as well as circulating levels of alcohol and TG. Rats were given an intraperitoneal (i.p.) injection of saline vehicle or 1.0 g/kg of a 10% v/v solution of ethanol in saline 1 hour after dark onset, with food removed at the time of the injection. In the first set, the rats (n=8/group) on separate days received 3 tests each with saline or ethanol injection (given in counterbalanced order), with blood collections at 30, 60, and 90 minutes after injection performed via tail vein puncture, as previously described (Chang et al., 2004). The saline values for the 3 tests were similar and thus averaged for the baseline score. The rats in the second set (n=6/group) received only 1 test with saline or ethanol injection. They were killed by rapid decapitation at 3 hours after injection, and their brains were removed (blood was not collected) and prepared for measurements of peptide expression in the PVN via real-time quantitative PCR.

Hormone and Metabolite Assays

Blood alcohol levels were measured using the Analox GM7 Fast Enzymatic Metabolic Analyser (Lunenburg, MA), and serum TG levels were assayed using a Triglyceride Assay Kit (Sigma, St Louis, MO) and E-Max Microplate Reader. Serum from trunk blood was also assayed for insulin and leptin, using radioimmunoassay kits from Linco Research Inc. (St. Charles, MO).

Brain Dissection

Immediately after killing, the brain was placed in a matrix slicing guide with the ventral surface facing up. For dissections of the PVN, three 1.0 mm coronal sections were made, with the middle of the optic chiasm as the anterior boundary. These sections were placed on a glass slide, and the PVN at Bregma −1.3 to −2.1 mm, according to the atlas of Paxinos and Watson (1986), was rapidly microdissected under a microscope, as a reversed isosceles triangle, 1.0 mm bilateral to the third ventricle and between the fornix structures (Chang et al., 2004). For microdissections of the NAc core and shell and the dorsal striatum in Experiment 1, two 0.5 mm coronal sections were made starting 1.0 mm anterior to the anterior border of optic chiasm, with the first section at Bregma 0.7 to 1.2 mm and the second section at Bregma 1.2 to 1.7 mm. The lateral ventricle (LV) and anterior commissure anterior (ACA) were used as landmarks. For the core, the area microdissected from the first section included the bottom one-sixth of the LV, starting from 0.1 mm up to 1.0 mm lateral to LV, extending along the LV to the ACA, and including the area 1.5 mm dorsal and lateral and 0.5 mm medioventral to the ACA. For the shell in this first section, an area 1.0 mm medial to the same level of the LV and medially parallel to the core was microdissected. In the second section, the area microdissected for the core started from the bottom of LV, like a pear with its apex pointed to the bottom of the LV, and it extended to the ACA and included 0.5 to 1.0 mm surrounding the ACA. The area for the shell was 0.5 to 1.0 mm medially, running parallel to the core with the septum as a medial border. In these same 2 sections, the dorsal striatum was easily dissected, with the corpus callosum and LV as landmarks. These microdissections were immediately frozen in liquid nitrogen and stored at −80° C until processed.

Real-Time Quantitative PCR

As previously described (Chang et al., 2004), total RNA from pooled microdissected samples was extracted with TRIzol reagent. RNA was treated with RNase-free DNase I before RT. For quantitative PCR, cDNA and minus RT were synthesized using an oligo-dT primer with or without SuperScript II Reverse Transcriptase. The SYBR Green PCR core reagents kit (Applied Biosystems, Foster City, CA) was used, with β-actin as an endogenous control. PCR was performed in MicroAmp Optic 96-well Reaction Plates (Applied Biosystems) on an ABI PRISM 7700 Sequence Detection system (Applied Biosystems), under the condition of 2 minutes at 50°C, 10 minutes at 95°C, and then 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Each study consisted of 4 independent runs of PCR in triplicate, and each run included a standard curve, a nontemplate control, and a negative RT control. The levels of target gene expression were quantified relative to the level of β-actin, using the standard curve method. The primers, designed with ABI Primer Express V.1.5a software based on published sequences, were: (1) β-actin: 5′-GGCCAACCGTGAAAAGATGA-3′ (forward) and 5′-CACAGCCTGGATGGCTACGT-3′ (reverse); (2) GAL: 5′-TTCCCACCACTGCTCAAGATG-3′ (forward) and 5′-TGGCTGACAGGGTTGCAA-3′ (reverse); and (3) ENK: 5′-GGACTGCGCTAAATGCAGCTA-3′ (forward) and 5′-GTGTGCATGCCAGGAAGTTG-3′ (reverse); and (4) DYN: 5′-CAGCGGACTGCCTGTCCTT-3′ (forward) and 5′-TCAGGGTGAGAAAAGACCAAAAG-3′ (reverse). The concentrations of primers were 100 to 200 nM. All reagents, unless indicated, were from Invitrogen (Carlsbad, CA).

Radiolabeled in Situ Hybridization Histochemistry

Besides real-time quantitative PCR, mRNA levels of GAL, ENK, and DYN were measured with radiolabeled in situ hybridization histochemistry in rats drinking water or 9% ethanol. Antisense RNA probes and sense probes were labeled with 35S-UTP (Amersham Biosciences, Piscataway, NJ), as described (Leibowitz et al., 1998; Lucas et al., 1998). Alternative free-floating coronal sections were consecutively processed as follows: 10 minutes in 0.001% proteinase K, 5 minutes in 4% paraformaldehyde, and 10 minutes each in 0.2 N HCl and acetylation solution, with a 10-minute wash in PB between each step. After wash, the sections were hybridized with a 35S-labeled probe (103 cpm/μL) at 55° C for 18 hours. Following hybridization, the sections were washed in 4 × sodium chloride and sodium citrate (SSC), and the nonspecifically bound probe was removed by RNase (Sigma) treatment for 30 minutes at 37°C. Then, sections were run through further stringency washes with 0.1 M dithiothreitol (Sigma) in 2 × SSC and 1 × SSC and 0.1 × SSC at 55°C. Finally, sections were mounted, air-dried, and exposed to a Kodak BioMax MR film for 8 to 18 hours at −80°C, when films were developed and macroscopically analyzed. The sense probe control was performed in the same tissue, and no signal was found.

Computer-assisted microdensitometry of autoradiographic images was determined as described (Lucas et al., 1998; Reagan et al., 2004) on the MCID image analysis system (Image Research Inc., St. Catherines, ON, Canada). Microscale 14C standards (Amersham Biosciences) were exposed on the same Kodak film with the sections and digitized. Gray-level/optical density calibrations were performed by using a calibrated film-strip ladder (Imaging Research Inc.) for optical density. Optical density was plotted as a function of microscale calibration values. It was determined that all subsequent optical density values of digitized autoradiographic images fell within the linear range of the function. The values obtained represent the average of measurements taken from 10 to 12 sections per animal. In each section, the optical density for the PVN was recorded, from which the background optical density from a same-size area in the thalamus was subtracted. The mean value of the ethanol-drinking group in each experiment was reported as a percentage of the water-drinking control group.

Radioimmunoassay

The PVN tissue was homogenized in 1 mL of 0.1 M acetic acid and centrifuged at 14,000 ×g for 15 minutes at 4°C. The entire supernatant was removed, boiled for 10 minutes, and frozen at −80°C until use. For radioimmunoassay, commercially available kits for met-ENK and DYN-A (Peninsula laboratories Inc., San Carlos, CA) were used.

Data Analysis

The data in the figures, for circulating alcohol and TG levels and PVN peptides, are expressed as mean±SEM. Statistical analyses of these data were performed using a 1-way ANOVA, followed by post hoc tests (Bonferroni or Fisher for the peptides and Holm–Sidak for blood alcohol and TG) for multiple comparisons between groups, or using an unpaired t-test where appropriate. The different within-group measures were related using Pearson's product–moment correlation. The probability values given in the text or legends to the figures and tables reflect the results of these tests.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Experiment 1: Effect of Voluntary Ethanol Consumption on Peptide mRNA Measured by Quantitative PCR

Building on previous work showing 9% ethanol intake to stimulate GAL expression in the PVN (Leibowitz et al., 2003), this experiment used the same paradigm to determine whether the opioid peptides, ENK and DYN, in this hypothalamic nucleus are similarly affected by the consumption of ethanol. Two groups of rats, a water-drinking control group (n=5) and an ethanol-drinking experimental group (n=10), with matched body weights and chow intake scores (see Methods), were maintained for 28 days with either water and chow available ad libitum or water and chow ad libitum plus ethanol available on a 12-hour schedule, respectively (see Methods). After 4 days on 9% ethanol, the rats consumed an average of 1.8 g/kg/d, with individual scores ranging from 0.5 to 2.8 g/kg/d. Because of this wide spread, these rats could be separated into 2 subgroups, referred to as low-ethanol or high-ethanol drinkers, which consumed an average of 1.0 or 2.5 g/kg/d, respectively. On the final test day, the rats were killed after 1 hour of water or 9% ethanol access, and their trunk blood was collected for measurements of alcohol and TG, as well as leptin and insulin.

The high-ethanol drinkers had significantly elevated levels of blood alcohol compared with the low-ethanol drinkers, which had higher levels than the water drinkers (Table 1). Across the entire group, these alcohol levels were positively correlated with the amount of ethanol consumed (r=+0.64, p<0.05). While the ethanol drinking had little impact on leptin (8.3±1.2 vs 6.3±1.4 ng/mL) or insulin (2.3±0.5 vs 1.8±0.4 ng/mL), it produced a significant increase in circulating levels of TG. These lipids were elevated by 90% (p<0.01) in the high-ethanol drinkers compared with the low-ethanol and water drinkers (Table 1). Their close relationship with ethanol is supported by the positive correlations between TG and both the amount of ethanol consumed (r=+0.62, p<0.05) and the blood alcohol levels (r=+0.66, p<0.05).

Table 1.   Alcohol and Triglyceride Levels After Chronic Consumption of 9% Ethanol
 Alcohol (mg/dL)Triglycerides (mg/dL)
  • Results presented as mean ± SEM with n=5/group, reveal, in ethanol-drinking versus water-drinking rats, a significant increase in circulating levels of alcohol in Experiment 1 [F(2, 13)=6.31, p<0.01] and Experiment 2 [F(2, 17)=5.06, p<0.02], and also of triglyceride in Experiment 1 [F(2, 14)=5.12, p<0.03] and Experiment 2 [F(2, 17)=4.21, p< 0.04]. nd; no data.

  • *

    p<0.05 for direct comparisons with water drinkers.

  • **

    p<0.05 for direct comparisons with low-ethanol and water drinkers.

Experiment 1
 Water drinkers0.1 ± 0.0176 ± 8
 Low-ethanol drinkers6.3 ± 20*77 ± 12
 High-ethanol drinkers17.3 ± 28**143 ± 31**
Experiment 2
 Water drinkers0.1 ± 0.0164 ± 8
 Low-ethanol drinkersnd65 ± 8
 High-ethanol drinkers17.5 ± 2.9*117 ± 21**

Further differences between these groups were seen in the measurements of peptide gene expression using real-time quantitative PCR, as revealed by statistically significant (p<0.01) 1-way ANOVAs. Direct comparisons between the groups showed that the high-ethanol drinkers had significantly higher GAL mRNA levels in the PVN compared with the low-ethanol and water drinkers (Fig. 1), confirming the results obtained previously using in situ hybridization (Leibowitz et al., 2003). Measurements of the opioids ENK and DYN mRNA in the PVN revealed a similar increase in gene expression in the high-ethanol drinkers (Fig. 1). These results demonstrate a close association between voluntary ethanol intake, circulating levels of TG and alcohol, and opioid peptides in the PVN, similar to GAL.

image

Figure 1.  Effects of voluntary intake of 9% ethanol on the expression of galanin (GAL), enkephalin (ENK), and dynorphin (DYN) in the paraventricular nucleus (PVN), as measured by real-time quantitative polymerase chain reaction (Experiment 1). Three groups of rats (n=5/group), water, low-ethanol, and high-ethanol drinkers, were examined. The data (mean±SEM) reveal a significant increase in expression of GAL [F(2, 14)=5.15, p<0.03], ENK [F(2, 14)=10.74, p<0.002], and DYN [F(2, 14)=18.12, p<0.001], with the high-ethanol drinkers showing greater expression when directly compared with both the low-ethanol and water drinkers (**p<0.05).

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A very different pattern was evident in the forebrain, where comparisons between the high-ethanol and water drinkers revealed no differences in the ratio of peptide mRNA/β-actin mRNA, in the NAc shell (GAL: 0.062 vs 0.073; ENK: 0.096 vs 0.088; DYN: 0.061 vs 0.066), NAc core (GAL: 0.072 vs 0.066; ENK: 0.065 vs 0.059; DYN: 0.050 vs 0.056), or dorsal striatum (GAL: 0.094 vs 0.162; ENK: 0.060 vs 0.063; DYN: 0.042 vs 0.048). The only significant effect observed in these areas was seen for the 3 peptides in the low-ethanol drinkers, which, compared with the water drinkers, were actually reduced by 20 to 35% (p<0.01) in the NAc shell and core but not the dorsal striatum. These results demonstrate clear region specificity for the effects of chronic ethanol consumption on these peptides.

Experiment 2: Effect of Voluntary Ethanol Consumption on Peptide mRNA Measured by in Situ Hybridization

This experiment was conducted to confirm the results of Experiment 1 using radiolabeled in situ hybridization histochemistry. Two groups of rats, a water-drinking control (n=5) and a 9% ethanol-drinking group (n=13), with similar body weights and daily caloric intake, were examined using the same test paradigm as Experiment 1. The ethanol-drinking rats drank an average of 1.4 g/kg/d of ethanol, with the high drinkers consuming 1.9 g/kg/d and low drinkers consuming 0.8 g/kg/d. When killed on the final day after 1 hour of ethanol access, the high-ethanol drinking rats had significantly elevated levels of blood alcohol compared with the water drinkers (no data for low-ethanol rats) and also higher levels of TG compared with the low-ethanol and water drinkers (Table 1). Across the entire group, significant positive correlations were obtained between the amount of ethanol consumed and circulating levels of TG (r=+0.61, p<0.05).

The radiolabeled in situ hybridization technique revealed a distribution pattern of GAL-, ENK-, and DYN-expressing neurons similar to that previously described (Chou et al., 2001; Harlan et al., 1987; Leibowitz et al., 1998), with a dense signal seen in the PVN and PFH and a relatively weak signal in the ARC. Group comparisons showed significantly increased mRNA levels (measures expressed as percentage of control) for all 3 peptides in the PVN of high-ethanol drinkers compared with water or low-ethanol drinkers (Fig. 2). This comparison between high-ethanol and water drinking rats is illustrated in the photomicrographs of Fig. 3. These PVN peptides were also significantly higher in the low-ethanol compared with water-drinking rats (Fig. 2). A close relationship between these PVN peptides, ethanol intake, and circulating levels of TG was revealed by the strong, positive correlations consistently detected between these measures, ranging from r=+0.64 to r=+0.80 (p<0.01). These findings, like those obtained in Experiment 1, support the idea that the opioid peptides in the PVN are similar to GAL in their close relation to circulating lipids as well as to ethanol.

image

Figure 2.  Effects of voluntary intake of 9% ethanol on the expression of galanin (GAL), enkephalin (ENK), and dynorphin (DYN) in the hypothalamic paraventricular nucleus (PVN), as measured by radiolabeled in situ hybridization (Experiment 2). The data (mean±SEM) for the 3 groups (n=5/group), water, low-ethanol, and high-ethanol drinkers, are presented as percent of water control. They show a significant increase in expression of GAL [F(2, 14)=44.56, p<0.001], ENK [F(2, 13)=109.3, p<0.002], and DYN [F(2, 13)=56.3, p<0.01], with greater expression seen in the high-ethanol compared with low-ethanol and water drinkers (**p<0.05) and in the low-ethanol drinkers compared with water drinkers (*p<0.05).

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image

Figure 3.  Photomicrographs illustrating the stimulatory effect (high-ethanol vs water drinker) of voluntary 9% ethanol intake on galanin (GAL), enkephalin (ENK), and dynorphin (DYN) mRNA in the paraventricular nucleus (PVN), as measured using radiolabeled in situ hybridization and graphed in Fig. 2 (Experiment 2).

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Experiment 3: Voluntary Ethanol Consumption and Peptide Levels Measured by Radioimmunoassay

To determine whether this increase in opioid gene expression produced significant changes in the levels of the opioid peptides in the PVN, similar to the results obtained with GAL (Leibowitz et al., 2003), this experiment tested the effect of ethanol drinking on PVN levels of ENK and DYN as measured via radioimmunoassay. Once again, 2 groups of rats, water control (n=9) and ethanol-drinking (n=14), with similar daily caloric intake and body weight measures were maintained for 28 days with chow and water available ad libitum or chow and water ad libitum plus ethanol available on a 12-hour schedule, respectively. At 9% ethanol, the rats consumed an average of 1.8 g/kg/d, with the high-ethanol drinkers ingesting 2.7 g/kg/d compared with 0.7 g/kg/d for the low-ethanol drinkers.

Analysis of peptide levels in the PVN revealed an increase in the opioids in response to voluntary ethanol consumption. Both ENK and DYN levels were significantly elevated in the high-ethanol rats compared with the low-ethanol or water-drinking rats, respectively, while there was no difference detected between the low-ethanol and water drinkers (Fig. 4). Significant positive correlations were obtained between the amount of ethanol consumed and PVN peptide levels of ENK (r=+0.66, p<0.05) and DYN (r=+0.63, p<0.05). This experiment indicates that a change in peptide levels in the PVN accompanies the ethanol-induced increase in gene expression shown in Experiments 1 and 2.

image

Figure 4.  Effects of voluntary intake of 9% ethanol on peptide levels of enkephalin (ENK) and dynorphin (DYN) in the paraventricular nucleus (PVN), as measured via radioimmunoassay (Experiment 3). The data (mean±SEM) for the 3 groups (n=7–9/group), water, low-ethanol, and high-ethanol drinkers show a significant increase in peptide levels of ENK [F(2, 17)=5.10, p<0.02] and DYN [F(2, 13)=6.20, p<0.01], with higher levels of ENK and DYN seen in the high-ethanol compared, respectively, with low-ethanol or water drinkers (**p<0.05).

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Experiment 4: Effect of Acute Ethanol Injection on Peptide Expression Measured by Real-Time Quantitative PCR

Building on the results of Experiments 1 to 3 showing that PVN peptides and circulating TG levels as well as blood alcohol increase with chronic ethanol consumption, this experiment tested whether a similar pattern of results can be obtained in response to an acute ethanol treatment that bypasses the ingestion process. This experiment tested 2 sets of rats, each with 2 groups injected i.p. with saline or 10% ethanol 1 hour before dark onset, as reported in a prior publication (Leibowitz et al., 2003). In the first set of rats (n=8/group), blood was collected via tail vein puncture at 30, 60, and 90 minutes after saline or ethanol injection, to examine postinjection changes in levels of alcohol and TG that precede a change in the PVN peptides. In the second set (n=6/group), the rats were killed 3 hours after injection, and their brains were dissected for analysis of peptide expression in the PVN using real-time quantitative PCR.

As shown by the first set of rats, circulating levels of alcohol and TG were significantly elevated at 30 minutes after ethanol compared with saline injection, and they remained higher at subsequent time periods up to 90 minutes after injection (Table 2). In the second set of rats, measurements of GAL, ENK and DYN mRNA in the PVN at 3 hours postinjection also revealed clear differences between the ethanol-injected and saline-injected groups, with the expression of each of these peptides significantly elevated by ethanol (Fig. 5). Together, these results showed a single injection of 10% ethanol to produce changes in circulating alcohol and TG levels and also in PVN peptides that are very similar to those seen 1 hour after the drinking of 9% ethanol (Experiments 1–3) in rats given daily access for 12 hours.

Table 2.   Circulating Alcohol and Triglyceride Levels After Injection of 10% Ethanol
 SalineEthanol injection (min)
306090
  • Results presented as mean ± SEM with n=8/group, reveal a significant increase in circulating levels of alcohol [F(2, 18)=157.8, p<0.001] and triglyceride [F(3, 28)=5.97, p<0.003] in ethanol-injected compared with saline-injected rats. nd, no data.

  • *

    p<0.05 after ethanol injection compared with saline baseline.

Alcohol (mg/dL)0.1 ± 0.01116 ± 11*nd79 ± 4.4*
Triglycerides (mg/dL)63 ± 8175 ± 34*120 ± 13*127 ± 13*
image

Figure 5.  Effects of 10% ethanol injection (1.0 g/kg) on the expression of galanin (GAL), enkephalin (ENK), and dynorphin (DYN) in the paraventricular nucleus (PVN), as measured by real-time quantitative polymerase chain reaction (Experiment 4). Two groups of rats (n=6/group), saline-injected and ethanol-injected, were tested 3 hours after injection. The data (mean±SEM) show a significant increase in GAL, ENK, and DYN expression after ethanol versus saline injection, as compared by an unpaired t-test (*p<0.05).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The new findings of this study demonstrate that the opioid peptides ENK and DYN, like GAL, are significantly stimulated in the PVN by the consumption or injection of ethanol. In addition, this effect is consistently associated with an increase in circulating levels of TG, which, together with blood alcohol, may contribute to the ethanol-induced stimulation of PVN peptides.

Effect of Ethanol on GAL in the PVN

One goal of this paper was to confirm, using additional techniques, our original finding that ethanol stimulates the expression of GAL in the PVN (Leibowitz et al., 2003). Using both real-time quantitative PCR and radiolabeled in situ hybridization, the results demonstrate that GAL mRNA in the PVN is stimulated by chronic consumption of ethanol. This effect was observed when comparing high-ethanol drinkers consuming 2.0 g/kg/d with either water-drinking rats or low-ethanol drinkers consuming 0.8 g/kg/d. This increase in GAL was also seen after acute injection of 10% ethanol (1.0 g/kg) compared with saline. Further, the levels of GAL mRNA in the PVN were positively correlated with the amount of ethanol consumed. These findings confirm our earlier report on GAL (Leibowitz et al., 2003) and are substantiated by a recent report demonstrating an association of GAL haplotypes with alcoholism in distinct populations (Belfer et al., 2006).

Effect of Ethanol on Opioid Peptides in the PVN

Building on the theory that PVN peptides stimulated by dietary fat are similarly affected by ethanol in the diet, the second goal of this study was to determine whether ENK and DYN in the PVN are similar to GAL in their response to ethanol consumption. The present results demonstrate that the gene expression of both ENK and DYN in this hypothalamic nucleus is increased by chronic consumption of 9% ethanol and also by acute injection of 10% ethanol. This effect is also seen with measurements of ENK and DYN peptide levels, indicating that the ethanol-induced change in gene expression may result in an increase in peptide production. The possibility that these elevated peptides have functional consequences needs to be confirmed with opioid injection studies, similar to those performed with GAL (Lewis et al., 2004; Rada et al., 2004). In a prior report with measurements of ENK mRNA in the PVN, intragastric administration of ethanol (2.5 g/kg) was found to stimulate gene expression at 24 hours but not at 1 or 4 hours after the infusion (de Gortari et al., 2000). The failure to observe a change at these earlier times, in contrast to the present study, may be attributed to the difference in the mode of administration, with ethanol introduced intragastrically rather than consumed voluntarily. The finding that PVN ENK mRNA is unaffected by chronic consumption of a lower concentration (5%) of ethanol (Cowen and Lawrence, 2001) suggests that the stimulatory effect is dose dependent. In this regard, it is interesting that, whereas all high-ethanol drinkers in the different experiments consuming 2.5 g/kg/d showed a significant increase in ENK mRNA in the PVN compared with water drinkers, the low-ethanol drinkers consuming 1.0 mg/kg/d showed this effect only in Experiment 2, in which the tissue was analyzed using radiolabeled in situ hybridization. This finding argues for the anatomical specificity of the effect, apparently localized to the PVN. In contrast to the in situ hybridization, which is more anatomically precise, the microdissected samples examined in Experiments 1 and 3 very likely included some tissue outside the borders of the PVN, which diluted any effect at the lower levels of ethanol consumption. The present findings are the first indication that DYN in the PVN is similar to ENK in its response to the consumption or injection of ethanol, with a significant increase in DYN mRNA also observed with the drinking of 1.0 g/kg/d of ethanol.

It is noteworthy that the NAc, a structure known to be involved in motivated behavior and drug taking (Cowen and Lawrence, 2001, Di Chiara et al., 2004; Lindholm et al., 2000), shows clear differences from this hypothalamic nucleus in its response to ethanol. Although acute injection of ethanol can increase ENK mRNA or extracellular ENK levels in this forebrain nucleus (Marinelli et al., 2005; de Gortari et al., 2000), ENK mRNA is unaffected or decreased by chronic ethanol exposure (Cowen and Lawrence, 1999, 2001). These latter results are consistent with the present findings that chronic ethanol consumption at low levels can reduce ENK mRNA in the NAc, both the shell and core, although it has no effect in the dorsal striatum. The measures of GAL and DYN mRNA in the NAc revealed a pattern similar to ENK: a significant decline in low-ethanol drinkers with no change in the high-ethanol drinkers. While this result with DYN does not agree with evidence showing acute and chronic ethanol to stimulate DYN expression in the NAc (de Gortari et al., 2000; Li et al., 1998; Lindholm et al., 2000; Oswald and Wand, 2004; Seizinger et al., 1983; Schulz et al., 1980), this phenomenon in the NAc was very consistent, observed with each of the 3 peptides, in both the shell and core of the NAc, and only with the lower amount (1.0 g/kg/d) of ethanol consumed. Thus, in future investigations, it would be informative not only to examine possible differences between the forebrain and hypothalamic structures but also to vary the amounts of ethanol exposure.

Ethanol Stimulation of GAL and Opioids in Relation to Blood and Brain Alcohol Levels

Consistent with the close relationship of PVN GAL, ENK, and DYN with ethanol consumption is the finding that these peptides are also correlated with blood alcohol levels, which in turn reflect the amount consumed. This relationship was observed in Experiments 1 to 3 with voluntary intake of 9% ethanol, which increased blood levels to 17 to 18 mg/dL after 60 minutes of ethanol access, at the same time when PVN peptide expression was increased. It was similarly seen in Experiment 4 with acute injection of 10% ethanol, which raised blood alcohol to peak levels of 116 mg/dL at 30 minutes after injection, consistent with published results (Marinelli et al., 2005), and somewhat lower levels of 79 mg/dL at 90 minutes, after which PVN peptide mRNA was enhanced. This close association between ethanol and the opioids or GAL in the PVN is further supported by the strong, positive correlations detected between these peptides and the measures of both ethanol intake and blood alcohol levels recorded in the animals of Experiments 2 and 3. A similar relationship between ENK and blood alcohol is suggested by the results obtained in the NAc, where met-ENK release after acute peripheral ethanol injection (1.6–3.2 g/kg) is stimulated in proportion to blood alcohol levels (Marinelli et al., 2005) and ENK mRNA after intragastric infusion of ethanol (2.5 g/kg) is increased at a time when blood alcohol levels reach their peak (Li et al., 1998). The present results, showing a significant change in peptide expression that is consistently positively correlated with blood alcohol, indicate that the relatively low levels obtained (17–18 mg/dL) after only 60 minutes of access to the ethanol in voluntarily drinking rats have some impact on the brain and also on the periphery, where circulating TG levels were significantly elevated.

Ethanol in the brain, which peaks before circulating blood alcohol (Lumeng et al., 1982; Nurmi et al., 1994), may also be a factor in producing the increased expression of the peptides in the PVN. Although there are no studies with intracerebroventricular (i.c.v.) injection of ethanol combined with opioid peptide measurements, there is evidence that i.c.v. ethanol significantly increases the expression in the PVN of pro-opiomelanocortin and corticotrophin-releasing factor 1 (Lee et al., 2004). It also stimulates c-Fos-IR specifically in the PVN and NAc but not in other areas examined (Crankshaw et al., 2003). Thus, whether consumed or injected, ethanol may have direct effects in the brain, including the stimulation of opioid-expressing and GAL-expressing neurons in the PVN.

Ethanol-Induced Stimulation of GAL and Opioids in Relation to Circulating TG Levels

In addition to blood and brain ethanol, the results suggest that circulating lipids may also be involved, possibly causally related, in the effect of ethanol on opioid peptides and GAL in the PVN. A close relationship between TG and PVN peptides has previously been demonstrated in studies with a high-fat diet. These reports show that PVN GAL is stimulated by dietary fat or injection of Intralipid and is positively correlated with TG levels (Chang et al., 2004; Leibowitz et al., 2004a). Similarly, ENK in the PVN is increased by injection of Intralipid, which elevates TG levels (Chang et al. 2004). The present findings demonstrate that ethanol, like fat, consistently increases circulating TG. This increase in lipids is seen in chronic ethanol-drinking rats as well as after acute injection of ethanol, with the TG levels positively correlated with blood alcohol levels. This finding is consistent with earlier studies showing a positive relation between ethanol intake and circulating TG in rats and humans (Contaldo et al., 1989; Goude et al., 2002). This relationship may be attributed, in part, to the fact that ethanol reduces the clearance of very low-density lipoproteins-TG and chylomicrons-TG from the blood (Baraona and Lieber, 1998; Siler et al., 1998) and decreases fat oxidation in the body (Siler et al., 1998). The finding that TG levels are strongly, positively correlated with the PVN peptides as shown here and in studies with dietary fat (Chang et al., 2004; Leibowitz et al., 2004a, Leibowitz, 2005) supports their causal role in the ethanol-induced increase in peptide expression.

These circulating nutrients, alcohol and TG, may function together through a common mechanism, specifically in the PVN, which underlies their similar effects on the peptides. This is supported by evidence indicating that the peptide effect induced by ethanol or dietary fat is anatomically specific, seen in the PVN but generally weak in the ARC (Chang et al., 2004; Leibowitz et al., 2003, 2004a). It is possible that ethanol and TG act indirectly on PVN neurons through some common effects on peripheral fat metabolism. This does not appear, however, to involve a change in fat oxidation, which is stimulated by dietary fat (Leibowitz et al., 2004a) while suppressed by ethanol (Siler et al., 1998). As ethanol and fatty acids are known to have direct effects on central neural processes, producing changes in enzyme activity, neural activity, and gene expression (Ammouche et al., 1993; DeWille and Farmer, 1993; Merkel et al., 2002; Oomura et al., 1975), studies with central administration of these nutrients should help to elucidate their specific roles in the peptide-induced changes observed here.

In contrast to alcohol and TG, the present results revealed little effect of ethanol on levels of leptin and insulin. These hormones were measured based on evidence showing leptin to be associated with craving for alcohol and lifetime alcohol intake (Kiefer et al., 2001) and fasting insulin levels to be inversely related to ethanol intake (Konrat et al., 2002). The finding here, showing these hormones to remain stable in the ethanol-drinking rats, suggests that they are not involved in producing the increase in opioid peptides and thus underscores the specificity of the change in TG.

Functional Consequences of Increased Peptide Expression

With both ethanol and dietary fat increasing the gene expression and peptide levels of GAL, ENK, and DYN in the PVN, one is led to consider the functional consequences of this effect. The opioids and GAL have long been known to stimulate feeding behavior when injected into the PVN (Baile et al., 1986; Kyrkouli et al., 1986; Yun et al., 2005), and they are found to produce a stronger effect in rats on a high-fat diet and in fat-preferring rats (Yun et al., 2005; Zhang et al., 1998). It is remarkable that the exogenous peptides stimulate ingestion of a diet that further promotes endogenous production of the peptides. This suggests that each may function within a positive feedback loop. A similar relationship may also exist with the drinking of ethanol. There is evidence that ethanol intake is stimulated by peripheral injection of opioid agonists, and it is decreased by an opioid receptor antagonist (Hubbell et al., 1986; Messiha, 1989; Oswald and Wand, 2004) or by deletion of the κ-opioid receptor gene (Kovacs et al., 2005). Also, 2 recent studies from our laboratories show that PVN or i.c.v. injection of GAL can increase ethanol consumption, and a GAL receptor antagonist can produce the opposite effect: a marked decrease in ethanol intake (Lewis et al., 2004; Rada et al., 2004). This suggests a possible cause-and-effect relationship, in the form of a positive feedback circuit, between the peptides and ethanol, similar to that seen for fat. In this neural circuit, GAL and the opioids may work closely together in stimulating consumption of ethanol, as well as in increasing intake of a fat-rich diet.

Role of PVN Peptides in the Relationship Between Fat and Ethanol Intake

There is accumulating evidence to suggest a positive relationship between eating fat and drinking ethanol. In animal studies, rats maintained on a high-fat diet or exhibiting a preference for fat are found to consume more ethanol (Carrillo et al., 2004; Krahn and Gosnell, 1991; Pekkanen et al., 1978). Also, clinical studies show that fat intake is elevated in ethanol drinkers, with bingeing on fat-rich foods associated with high rates of alcoholism (Herbeth et al., 1988; Swinburn et al., 1998), and drinkers maintained on a fat-rich diet compared with a carbohydrate-rich diet exhibit shorter periods of ethanol abstinence (Forsander, 1998; Yung et al., 1983). A direct and possibly causal relationship between dietary fat and ethanol intake is demonstrated by the finding that a high-fat meal, compared with a low-fat/high-carbohydrate meal, can significantly increase ethanol intake right after the meal (Carrillo et al., 2004). Moreover, injection of intralipid compared with glucose or saline produces a similar avidity for ethanol, revealing the effect of fat in the absence of taste (Carrillo et al., 2004). While the palatability of a high-fat meal may potentiate ethanol intake by activating orosensory-reward systems, in particular the opioids (Glass et al., 1999; Kelley et al., 2002; Vaccarino and Kastin, 2001), the findings with Intralipid injection, which bypasses the orosensory system, indicate that the fat-induced increase in ethanol consumption cannot be attributed solely to taste. It very likely involves postingestive factors, presumably related to circulating lipids or fat metabolism, which potentiate the endogenous opioids.

Thus, the variables studied herein may provide an animal model for exploring some of the metabolic and neural antecedents of alcohol dependence. The results obtained with this model lead us to propose that the opioid peptides ENK and DYN, similar to GAL in the PVN, are responsive to circulating levels of TG and alcohol, and together, they may be involved in promoting further consumption of ethanol, particularly on a fat-rich diet.

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  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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