Activation of Nuclear PPARγ Receptors by the Antidiabetic Agent Pioglitazone Suppresses Alcohol Drinking and Relapse to Alcohol Seeking

Activation of Nuclear PPAR␥ Receptors by the Antidiabetic Agent Pioglitazone Suppresses Alcohol Drinking and Relapse to Alcohol Seeking Serena Stopponi, Lorenzo Somaini, Andrea Cippitelli, Nazzareno Cannella, Simone Braconi, Marsida Kallupi, Barbara Ruggeri, Markus Heilig, Gregory Demopulos, George Gaitanaris, Maurizio Massi, and Roberto Ciccocioppo Background: Pioglitazone and rosiglitazone belong to the class of thiazolidinediones (TZDs). They were first developed as antioxidants and then approved for the clinical treatment of insulin resistance and Type 2 diabetes. TZDs bind with high affinity and activate peroxisome proliferator-activated receptor-gamma (PPAR␥) receptors, which in the brain are expressed both in neurons and in glia. Methods: We evaluated the effect of PPAR␥ activation by TZDs on alcohol drinking, relapse-like behavior, and withdrawal in the rat. We also tested the effect of TZDs on alcohol and saccharin self-administration. Results: We showed that activation of PPAR␥ receptors by pioglitazone (0, 10, and 30 mg/kg) and rosiglitazone (0, 10 and 30 mg/kg) given orally selectively reduced alcohol drinking. The effect was blocked by pretreatment with the selective PPAR␥ antagonist GW9662 (5 ␮g/rat) given into the lateral cerebroventricle, suggesting that this TZD’s effect is mediated by PPAR␥ receptors in the central nervous system. Pioglitazone abolished reinstatement of alcohol seeking, a relapse-like behavior, induced by yohimbine, a pharmacologic stressor, but did not affect cue-induced relapse. In the self-administration experiments, pioglitazone reduced lever pressing for alcohol but not for saccharin. Finally, pioglitazone prevented the expression of somatic signs of alcohol withdrawal. Conclusions: These findings provide new information about the role of brain PPAR␥ receptors and identify pioglitazone as candidate treatments for alcoholism and possibly other addictions.

Key Words: Addiction, alcohol drinking, pioglitazone, PPAR␥ receptors, relapse, thiazolidinediones eroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. At present, three distinct PPAR isoforms—PPAR␣, PPAR␤/␦, and PPAR␥— have been identified (1– 4). The PPAR␣ receptor isoform is highly expressed in the liver and kidney and regulates fatty acid catabolism; the PPAR␤/␦ is ubiquitously expressed and is involved in the regulation of various cellular processes, including adipocyte, keratinocyte, and oligodendrocyte differentiation, whereas the PPAR␥ receptors are predominantly expressed in adipose tissues and macrophages, where they are involved in adipocyte differentiation, regulation of sugar and lipid homeostasis, and control of inflammatory responses (4 – 6). Recent studies have shown that PPAR receptors are also expressed in neurons, oligodendrocytes, and astrocytes in the central nervous system (7–10). In particular, it has been demonstrated that the PPAR␥ receptor is highly expressed in the lateral hypothalamus (LH), the paraventricular nucleus of the hypothalamus (PVN), the arcuate nucleus (ARC), and the ventral tegmental area (VTA). In LH and ARC, the PPAR␥ receptors are expressed in ␣ melanocyte-

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From the School of Pharmacy (SS, AC, NC, SB, MK, BR, MM, RC), Pharmacology Unit, University of Camerino, Camerino; Addiction Treatment Centre (LS), Health Local Unit, Biella, Italy; Laboratory of Clinical and Translational Studies (AC, MH), National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland; and Omeros Corporation (GD, GG), Seattle, Washington. Address correspondence to Roberto Ciccocioppo, Ph.D., School of Pharmacy, Pharmacology Unit, University of Camerino, 62032 Camerino, Italy; E-mail: [email protected]. Received May 4, 2010; revised Dec 1, 2010; accepted Dec 1, 2010.

0006-3223/$36.00 doi:10.1016/j.biopsych.2010.12.010

stimulating hormone (␣-MSH), agouti-related protein (AgRP) and pro-opiomelanocortin-producing cells. Double immunostaining of VTA neurons showed colocalization of PPAR␥ and tyrosine hydroxylase, suggesting the expression of this receptor in dopaminergic cells (10). In the brain, it has been also shown that activation of PPAR␥ mediates neuroprotection against N-methyl-D-aspartate-mediated excitotoxic processes and inflammatory damage (2, 4 – 6). Activation of these receptors has been also associated to reduction in the expression of methamphetamine-induced locomotor sensitization in mice (11), an effect attributed to the ability of PPAR␥ to reduce glia-mediated inflammatory response in the brain (11). Considering these properties of PPAR␥ receptors and noting that they are localized in brain areas that are key for the regulation of reward processing and addiction, we investigated the role of this system in the regulation of alcohol-related behaviors. To this end, we used pioglitazone (Actos, Takeda, Japan) and rosiglitazone (Avandia GlaxoSmithKline, Brentford, Middlesex, United Kingdom), drugs belonging to the class of thiazolidinediones (TZDs) that are approved for clinical treatment of insulin resistance and Type 2 diabetes (1– 4).

Methods and Materials Animals Male genetically selected alcohol-preferring Marchigian Sardinian (msP) rats were used (12). All the procedures were conducted in adherence with the European Community Council Directive for Care and Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (for details, see Supplement 1). BIOL PSYCHIATRY 2011;69:642– 649 © 2011 Society of Biological Psychiatry

S. Stopponi et al. Drugs Pioglitazone and rosiglitazone were prepared from Actos (30 mg) and Avandia (8 mg) tablets, respectively. Yohimbine and GW9662 were purchased from Sigma-Aldrich (Milano, Italy) and Tocris (Bristol, United Kingdom) respectively. For drug preparation, see Supplement 1. Intracranial Surgery A 22-gauge guide cannula for intracerebroventricular (ICV) drug injections was stereotaxically implanted into the lateral cerebroventricle with the following coordinates: anteroposterior, ⫺1.0; lateral (L), ⫺1.8; ventral (V), 2.0 with reference to bregma (13). Experiment 1: Effect of Chronic Pioglitazone and Rosiglitazone on Alcohol Intake in msP Rats In a two-bottle choice test, msP rats were trained to drink 10% alcohol (v/v) for 24 hours per day (free choice between water and alcohol) until a stable baseline of alcohol intake was reached (12,14). At this point, rats were divided into three groups (n ⫽ 9/group) to be treated with pioglitazone (0, 10, or 30 mg/kg). The study was replicated using rosiglitazone (0, 10, or 30 mg/kg) under identical experimental condition in a different population of msP rats (n ⫽ 9 –10/group). Treatments were continued for 7 consecutive days, and drug or vehicle were administered twice daily at 12 hours and at 1 hour before the beginning of the dark period of the light– dark cycle. Alcohol, water, and food consumption were monitored daily. Experiment 2: Effect of Central PPAR␥ Antagonist Administration on Pioglitazone-Induced Reduction of Alcohol Intake To examine whether the effect of pioglitazone on alcohol intake is mediated by brain PPAR␥ receptors, another group of msP rats (n ⫽ 22) was treated ICV with 5 ␮g/rat of the PPAR␥ antagonist GW9662 (15) and then injected with pioglitazone (30 mg/kg) or its vehicle. In a between-subject design each group of animals (n ⫽ 5– 6/ group) received a different drug dose for 3 consecutive days. Pioglitazone or its vehicle was administered twice daily at 12 hours and 1 hour before the beginning of the dark period of the light– dark cycle. GW9662 was given immediately after pioglitazone and 15 min before access to alcohol. Alcohol, water, and food intake were recorded at 24 hours from the moment that alcohol was made available. Experiment 3: Effect of Pioglitazone on Alcohol, Saccharin, and Food Self-Administration Three groups of msP rats (n ⫽ 8/group) were separately trained to self-administer 10% alcohol, .2% saccharin, or 45 mg food pellets in 30-min daily sessions under a fixed-ratio 1 schedule of reinforcement in which each response resulted in delivery of .1 mL of fluids or one food pellet. Following acquisition of a stable self-administration baseline pioglitazone treatment began. In counterbalance order, 12 hours and 1 hour before the beginning of the operant sessions, animals were treated with pioglitazone (10 and 30 mg/kg) or its vehicle. Drug treatment was performed every fourth day. The first day after drug test, rats remained in their home cages, and on Day 2 and 3 after the test, baseline self-administration was reestablished. Experiment 4: Effect of Pioglitazone on Yohimbine-Induced Reinstatement of Alcohol Seeking The experiment consisted of three phases. In the operant selfadministration training phase, a group of msP rats (n ⫽ 10) was trained to self-administer 10% alcohol in 30-min daily sessions on

BIOL PSYCHIATRY 2011;69:642– 649 643 an FR-1 schedule of reinforcement, in which each response resulted in delivery of .1 mL of fluid. Training continued until stable baseline of alcohol responding was achieved. In the extinction phase, after the last alcohol self-administration session, animals were subjected to 30-min extinction sessions for 16 consecutive days. Responses at the lever activated the delivery mechanism but did not result in the delivery of alcohol. In the yohimbine-induced reinstatement phase, the day after the last extinction session in each cycle (see below), msP rats were subjected to a reinstatement test, during which yohimbine (1.25 mg/kg) was given intraperitoneally 30 min after the last pioglitazone administration. To evaluate whether pioglitazone was able to prevent the effect of the yohimbine, rats were orally treated with the PPAR␥ agonist (10 or 30 mg/kg) or vehicle 12 hours and 1 hour before the respective reinstatement test. Animals received vehicle or the two pioglitazone doses in a counterbalanced Latin square design. A 4-day interval, during which animals were subjected to daily extinction sessions, was allowed between drug tests (16). Experiment 5: Effect of Pioglitazone on Cue-Induced Reinstatement of Alcohol Seeking The experiments consisted of three phases (16 –18). In the conditioning phase, msP rats were trained to discriminate between 10% alcohol and water. The discriminative stimulus for alcohol consisted of the odor of an orange extract (S⫹), whereas water availability (i.e., no reward) was signaled by an anise extract (S⫺). In addition, each lever-press resulting in delivery of alcohol or water was paired with 5-sec illumination of the chamber’s house light or with 5-sec activation of the white noise tone, respectively. In the extinction phase, after the last conditioning day, rats were subjected to 30-min extinction sessions for 12 consecutive days. The reinstatement testing phase began the day after the last extinction session. This test lasted 30-min under conditions identical to those during the conditioning phase, except that alcohol and water were not made available. To evaluate whether pioglitazone was able to prevent cue-induced reinstatement of alcohol seeking, in a withinsubject (Latin square) design, rats (n ⫽ 14) were treated with the PPAR␥ agonist (0, 10, 30 mg/kg) 12 hours and 1 hour before the reinstatement test. A 4-day interval, during which animals remained in their home cages, was allowed between drug tests (16 –18). Experiment 6: Effect of Pioglitazone on the Expression of Alcohol Withdrawal Signs in Wistar Rats Chronic intoxication and physical dependence were induced by 6-day repeated daily intragastric alcohol administrations as recently described (19). Pioglitazone (0, 10, and 30 mg/kg) was administered twice, 12 hours and 1 hour before rating withdrawal symptoms. Behavioral signs of withdrawal consisted of 1) presence of the ventromedial distal flexion response, 2) tail stiffness/rigidity, and 3) tremors (20). Each sign was rated during 3 to 5 min of observation time on a scale of 0 to 2 (0 ⫽ absence of withdrawal signs, 1 ⫽ moderate withdrawal, 2 ⫽ severe withdrawal) by an experienced observer blind to treatment condition (20,21). Behavioral observations were carried out 12 hours after the last alcohol dose. Experiment 7: Measurement of Blood Alcohol and Blood Glucose Levels Following Administration of Pioglitazone msP rats (n ⫽ 36) were divided into six groups (n ⫽ 6/group); three groups received .7 g/kg of 10% alcohol by gavage, and the other three were treated with 2.5 g/kg of 20% alcohol. Pioglitazone (10 and 30 mg/kg) or its vehicle were given 12 hours and 1 hour before alcohol in a between subjects design for each alcohol dose. www.sobp.org/journal

644 BIOL PSYCHIATRY 2011;69:642– 649 Tail blood (approximately 200 ␮L) was collected 30, 60, and 120 min after alcohol administration. Samples were kept on ice and then immediately centrifuged (10 min, 1400 g). Alcohol content was then assayed from 5-␮L plasma aliquots using an oxygenate alcohol analyzed (Analox Instruments, Lunenburg, Massachusetts). Additional msP rats (n ⫽ 12) were divided into three groups (n ⫽ 4/groups) and were tested for the effect of pioglitazone (10 and 30 mg/kg) or its vehicle on blood glucose levels. Drug treatments were given 12 hours and 1 hour before measuring blood glucose levels at 30, 60, and 120 min. Glucose values were obtained immediately after sampling using a glucose reagent strip and a glucometer (Accu-Chek, Roche, Milan, Italy). To parallel the study of pioglitazone on alcohol drinking, rats used in this experiment were fed ad libitum and had free choice between water and 10% alcohol. Statistical Analysis The analysis of variance (ANOVA) followed by post hoc Newman–Keuls test was used (Supplement 1).

Results Experiment 1: Effect of Chronic Pioglitazone and Rosiglitazone on Alcohol Intake in msP Rats Pioglitazone administration significantly reduced voluntary alcohol intake in msP rats (Figure 1A). An ANOVA revealed a significant overall effect of treatment [F (2,24) ⫽ 11.3, p ⬍ .001]. At the highest drug dose (30 mg/kg), the effect appeared from the first day of treatment (Day 4) and remained significant throughout the treatment period (Days 4 –10) as shown by Newman–Keuls tests. At the lower dose (10 mg/kg), the effect of treatment increased over time,

S. Stopponi et al. and a statistical difference from controls was observed on Days 8 and 10 (fifth and seventh treatment days). Water consumption was not modified (data not shown), whereas food intake (Figure 1C) was significantly increased by pioglitazone [F (2,24) ⫽ 11.9, p ⬍ .001]. The effect was higher after administration of the lowest dose (10 mg/kg) of the drug. At the end of treatment, rats gradually recovered from drug effect and alcohol intake progressively returned to pretreatment levels. For rosiglitazone, ANOVA revealed a significant overall effect of treatment [F (2,25) ⫽ 33.8; p ⬍ .01]. Post hoc Newman–Keuls tests demonstrated that treatment with 10 mg/kg of rosiglitazone significantly reduced alcohol consumption on Days 4, 5, and 10 (p ⬍ .01). At 30 mg/kg, the drug reduced alcohol intake for the entire treatment period (p ⬍ .01) except on Day 4 when the difference was not significant (Figure 1B). Water (data not shown) and food consumption were not significantly affected by rosiglitazone treatment (Figure 1D). Experiment 2. Effect of a Centrally Administered PPAR␥ Antagonist on Pioglitazone-Induced Reduction of Alcohol Intake There was a main effect of pioglitazone treatment (30 mg/kg) to reduce alcohol drinking [F (3,16) ⫽ 7.67, p ⬍ .01; Figure 2A]. Newman–Keuls post hoc tests showed that pioglitazone treatment reduced alcohol consumption for the entire duration of treatment (p ⬍ .01 vs. vehicle / vehicle controls at each time point). There was no main effect of the PPAR␥ antagonist GW9662 on alcohol drinking [F (3,16) ⫽ 2.33, ns]. However, GW9662 blocked the effect of pioglitazone for whole treatment period (p ⬍ .05). Water intake was

Figure 1. Effect of chronic pioglitazone (Pio) and rosiglitazone (Rosi) on ethanol and food intake in alcohol-preferring Marchigian Sardinian rats at 24 hours. Voluntary 10% alcohol intake (g/kg) following treatment with (A) pioglitazone (0, 10, and 30 mg/kg) or (B) rosiglitazone (0, 10, and 30 mg/kg) administered 12 hours and 1 hour before access to ethanol. Voluntary food intake (g/kg) following treatment with (C) pioglitazone and (D) rosiglitazone. Data represent the mean ⫾ SEM. *p ⬍ .05, **p ⬍ .01 vs. vehicle (Veh). Where not indicated, difference from vehicles were not statistically significant.

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S. Stopponi et al.

BIOL PSYCHIATRY 2011;69:642– 649 645 (mean ⫾ SEM) the day before reinstatement tests were 11.2 ⫾ 2.1 (first test), 9.3 ⫾ 1.9 (second test), and 14.7 ⫾ 2.8 (third test). Furthermore, in yohimbine vehicle–treated rats the degree of reinstatement did not vary over repeated cycles of yohimbine challenge (number of responses, mean ⫾ SEM: 35.3 ⫾ 3.8 (first test), 28.7 ⫾ 5.2 (second test), and 45 ⫾ 9.7 (third test). These data demonstrate that rats can be repeatedly tested for yohimbineinduced reinstatement and that there are no carryover effects from prior relapse tests. Finally, inactive lever responses were unaffected by all treatments, indicating the selectivity of the effect of yohimbine in eliciting reinstatement of alcohol seeking. Experiment 5: Effect of Pioglitazone on Cue-Induced Reinstatement of Alcohol Seeking Throughout the conditioning phase, in which animals discriminated between alcohol and water, rats exhibited a strong preference for alcohol, and the ANOVA revealed a significant overall effect of conditioning [F (1,13) ⫽ 139.36, p ⬍ .001]. On the last day of the discrimination period, animals reached response rates for alcohol of

Figure 2. Voluntary intake of (A) 10% alcohol and (B) food following chronic intracerebroventricular injection of 5 ␮g/rat of GW9662 (GW5) and of 30 mg/kg of pioglitazone (Pio) at 24 hours. Data represent the mean ⫾ SEM **p ⬍ .01 vs. vehicles (Veh). Where not indicated, difference from vehicle were not statistically significant.

low and not affected by drug treatments (data not shown). ANOVA also revealed no significant overall changes in food intake, but a significant treatment time interaction was observed [F (3,18) ⫽ 2.23, p ⬍ .05]. As revealed by post hoc tests (Figure 2C), food intake was increased by pioglitazone alone on treatment Day 3 (p ⬍ .05). Experiment 3: Effect of Pioglitazone on Alcohol, Saccharin, or Food Self-Administration Pioglitazone significantly reduced operant responding for alcohol [F (2,21) ⫽ 7.02; p ⬍ .01], but responses at the inactive control lever were not modified [F (2,21) ⫽ 2.59; p ⬎ .05]. As shown in Figure 3, post hoc analysis revealed a significant reduction of alcohol selfadministration following treatment with 30 mg/kg of pioglitazone (p ⬍ .05). Pioglitazone did not affect ether saccharin [F (2,21) ⫽ .39, ns; Figure 3B] or food self-administration [F (2,21) ⫽ .02, ns; Figure S1 in Supplement 1). Experiment 4: Effect of Pioglitazone on Yohimbine-Induced Reinstatement of Alcohol Seeking Administration of yohimbine significantly reinstated responding on the lever that previously had delivered alcohol [F (1,9) ⫽ 38.6 p ⬍ .001]. Pretreatment with pioglitazone significantly reduced the effect of yohimbine [F (2,9) ⫽ 6.0, p ⫽ .01]. As shown in Figure 4, post hoc analysis demonstrated a significant inhibition of reinstatement following administration of both doses (10 and 30 mg/kg) of pioglitazone tested (p ⬍ .05). The number of extinction responses

Figure 3. Effect of pioglitazone (Pio; 0, 10, and.30 mg/kg) treatment on alcohol and saccharin self-administration. Values represent the mean (⫾ SEM) number of responses at the (A) active or at (B) inactive lever. Differences from vehicle-treated rats (controls), **p ⬍ .01. Where not indicated, difference from vehicle were not statistically significant.

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646 BIOL PSYCHIATRY 2011;69:642– 649

Figure 4. Effect of pioglitazone (Pio) on yohimbine-induced reinstatement of alcohol seeking behavior in Marchigian Sardinian rats. During the training phase, animals reached a stable baseline of responding for 10% v/v ethanol that progressively decreased under extinction (Ext) condition. Compared with extinction, yohimbine (1.25 mg/kg intraperitoneal injection) elicited a significant reinstatement of responding that was significantly reduced following treatment with Pio (0, 10, and 30 mg/kg). Values represent the mean (⫾ SEM) number of responses at (A) alcohol active lever and (B) inactive lever. Significant difference from extinction, ## p ⬍ .01. Significant difference from vehicle (.0), *p ⬍ .05.

87.4 ⫾ 8.3 in 30 min, whereas the response for water was 13.6 ⫾ 1.9 in the same time period. During extinction, lever pressing progressively decreased, and in the reinstatement test, cues had a significant overall effect on alcohol seeking [F (4,13) ⫽ 6.99; p ⬍ .001]. A more detailed analysis showed a robust reinstatement of respond-

S. Stopponi et al.

Figure 6. Effect of pioglitazone (Pio; 0, 10, and .30 mg/kg) on the expression of alcohol withdrawal. Total withdrawal score was obtained from each animal by cumulating the score of the three withdrawal signs: 1) presence of the ventromedial distal flexion response, 2) tail stiffness/rigidity, and 3) tremors. Total withdrawal score ranged between 0 and 6. Significant difference from vehicle (.0), **p ⬍ .01.

ing under the S⫹ (p ⬍ .001) but not under the S⫺ compared with the last day of extinction. As shown in Figure 5, conditioned reinstatement of alcohol-seeking was not significantly modified by pretreatment with pioglitazone [F (2,13) ⫽ .16; p ⬎ .05]. Responses at the inactive lever were not influenced by the treatment (data not shown). Experiment 6: Effect of Pioglitazone on the Expression of Alcohol Withdrawal Signs in Wistar Rats Following intoxication, marked withdrawal symptoms were observed after 12 hours from the last alcohol administration. Kruskal– Wallis ANOVA showed a significant overall effect of pioglitazone treatment on total withdrawal scores [H(2,19) ⫽ 11.064; p ⬍ .01]. As shown in Figure 6, total withdrawal signs were significantly reduced after administration of both 10 mg/kg and 30 mg/kg of pioglitazone (p ⬍ .01). Experiment 7: Measurement of Blood Alcohol and Blood Glucose Levels Following Administration of Pioglitazone Pioglitazone treatment did not modify blood alcohol levels following administration of either low or high alcohol doses [F (2,15) ⫽ 2.9, NS; F (2,14) ⫽ .1, NS, respectively]. There was also no effect of pioglitazone treatment on blood glucose levels [F (2,9) ⫽ 2.19, ns; Table 1].

Discussion

Figure 5. Effect of pioglitazone (Pio) on cue-induced reinstatement of alcohol seeking-behavior in Marchigian Sardinian rats. During the training phase animals learned to discriminate between ethanol and water availability. Lever responding then decreased progressively under extinction (Ext) conditions. Compared with extinction, cues predictive of alcohol availability (S⫹) elicited a significant reinstatement of responding that was not affected by treatment with pioglitazone (0, 10, and 30 mg/kg). Following presentation of stimuli predictive of water availability (S⫺) responding remained at extinction levels. Values represent the mean (⫾ SEM) number of responses at (A) alcohol active lever and (B) inactive lever. Significant difference from extinction, ##p ⬍ .01. Significant difference from vehicle (.0).

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We show here that activation of PPAR␥ by pioglitazone and rosiglitazone significantly reduces alcohol intake in rats genetically selected for high alcohol consumption. The effect appeared from the first day after drug administration and was maintained for the entire treatment period. Alcohol consumption progressively returned to baseline levels after drug treatment was stopped. At the lowest dose, pioglitazone showed increased efficacy over repeated administrations, suggesting that chronic treatment may lead to more pronounced suppression of alcohol drinking. A modest increase in daily food consumption was observed following a low dose of pioglitazone, whereas rosiglitazone did not modify food consumption. Contrary to the effects on alcohol intake, the effect on food consumption decreased over time. Water intake was not affected by administration of the two PPAR␥ agonists. Together these findings suggest that the effect of TZD on alcohol intake is specific and is independent of any general inhibition of feeding behavior.

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S. Stopponi et al. Table 1. Effect of Pioglitazone on Blood Alcohol and Blood Glucose Levels Time (min) BALs (mg/dL) Following .7 g/kg of Ethanol 30 60 120 30 BALs (mg/dL) Following 2.5 g/kg of Ethanol 30 60 120 Blood Glucose Levels (mg/dL) 30 60 120

Vehicle

Pio 10 mg/kg

Pio 30 mg/kg

29.5 ⫾ 3.4 28.7 ⫾ 5.5 17.0 ⫾ 1.0 29.5 ⫾ 3.4

39.8 ⫾ 7.8 27.2 ⫾ 5.0 14.18 ⫾ .9 39.8 ⫾ 7.8

29.9 ⫾ 2.2 14.9 ⫾ .8 7.2 ⫾ 2.4 29.9 ⫾ 2.2

125.5 ⫾ 28.8 105.1 ⫾ 18.3 89.7 ⫾ 16.8

105.6 ⫾ 17.4 100.1 ⫾ 12.1 85.0 ⫾ 16.1

106.0 ⫾ 24.1 98.0 ⫾ 17.2 80.0 ⫾ 15.9

125.0 ⫾ 1.5 124.8 ⫾ 3.2 127.0 ⫾ 3.0

124.5 ⫾ 4.1 115.5 ⫾ 5.5 115.8 ⫾ 3.5

116.0 ⫾ 4.3 116.0 ⫾ 2.5 116.5 ⫾ 2.0

BAL, blood alcohol level; Pio, pioglitazone.

In addition to its pharmacologic reinforcing properties, alcohol is also a caloric substance. An alternative account of the data that must be considered is that alcohol consumption was decreased to compensate for the increase of food intake produced by pioglitazone. However, we consider this hypothesis to be unlikely for several reasons. First, there was no correlation between the effect of pioglitazone on ingestion of food and that on alcohol drinking. In fact, over repeated dosing, the effect on food ingestion decreased, whereas suppression of alcohol drinking became progressively greater in magnitude. Further support for a specificity of action on alcohol intake by TZDs is that rosiglitazone also reduced alcohol intake and did so in the absence of feeding effects. Lastly, the daily calories that msP rats obtain from alcohol and food are about 40 to 50 and 150 kcal/kg body weight, respectively. Alcohol is therefore only a modest source of energy for msP rats compared with food. In fact, these animals largely drink alcohol for its pharmacologic effects. Alcohol drinking reduction was more pronounced for pioglitazone than for rosiglitazone. This was somewhat surprising because the affinity for PPAR␥ receptors is between 30 and 100 times higher for rosiglitazone than for pioglitazone (22–24). Alcohol drinking is largely controlled by central nervous system mechanisms. It is therefore possible that the lack of correlation between the pharmacologic potency and the efficacy of these two TZDs reflects their different brain penetrant properties. To our knowledge direct comparisons for brain permeability between pioglitazone and rosiglitazone have never been conducted. However, rodent studies showed that after oral administration of .5 mg/kg, approximately 10% of plasma pioglitazone reaches the brain, leading to an exposure of about .06 ␮g of drug per gram of brain tissue (25). In contrast, the ability of rosiglitazone to cross the blood– brain barrier appears to be lower, and after intravenous administration, only .045% of an injected dose per gram of tissue is taken up by the brain (26,27). Alternatively, the higher efficacy of pioglitazone could be attributed to off-target effects associated to its lower selectivity for PPAR␥ receptors. For instance, pioglitazone also binds to PPAR␣ receptors, but rosiglitazone does not (6). Notably, recent studies have shown that activation of central nervous system PPAR␣ receptors by oleoylethanolamide reduces nicotine self-administration and lowers nicotine-induced firing in dopaminergic VTA cells (28,29). In our view, however, off-target mediation of pioglitazone effects on alcohol drinking is unlikely, because we found that this effect was completely blocked by intracerebroventricular pretreatment with the selective PPAR␥ receptor antagonist GW9662 (15). Alcoholism is a chronic relapsing disorder characterized by high relapse rates (30,31). Stress is a major relapse trigger in abstinent

alcoholics. Yohimbine, an ␣-2 adrenoreceptor antagonist, increases cell firing and release of brain noradrenalin and acts as a pharmacologic stressor (32–36). Yohimbine is known to increase alcohol craving in humans (37) and to reinstate alcohol seeking following extinction in rats trained to self-administer alcohol (35,38). This stressor was therefore used to investigate the effect of pioglitazone on stress-induced reinstatement of drug seeking. Results demonstrated that pretreatment with pioglitazone significantly reduced reinstatement of alcohol seeking induced by yohimbine. In fact, at the highest pioglitazone dose tested, responding at the previously alcohol-paired lever returned to extinction levels. Neither yohimbine nor pioglitazone had effects on inactive lever responding, providing some evidence against nonspecific actions. Environmental conditioning factors are also known to play a pivotal role in alcohol relapse (31, 39 – 41). To evaluate the effect of pioglitazone on cue-induced relapse, we trained rats on an extinction reinstatement procedure in which reexposure to environmental stimuli predictive of alcohol availability elicited a robust resumption of lever responding (17,42). Pioglitazone did not show any activity in this model, indicating a high degree of specificity to inhibit stress-induce relapse-like behavior. Reinstatement induced by stress and by conditioned cues, respectively, has partly dissociable neural substrates, and effects of these two stimulus categories are additive (43). In patients, both types of stimuli contribute as relapse triggers (44). The opioid antagonist naltrexone, a medication that blocks cue-induced relapse-like behavior in preclinical models, is approved for human use. In contrast, pioglitazone and rosiglitazone are, to our knowledge, the first medications with activity in stress-induced relapse models approved for use in humans. In the brain, PPAR␥ receptors have been described in LH and PVN neurons, where they colocalize with ␣-MSH, AgRP, and pro-opiomelanocortin-immunoreactivity cells (10). Of note, stimulation of PPAR␥ receptors leads to a downregulation of the transcript for corticotropinreleasing factor in the PVN, suggesting a potential mechanism for the antirelapse effects of pioglitazone in the presence of stress (27). PPAR␥ receptors are also present in the VTA, where they colocalize with tyrosine hydroxylase, suggesting the expression of this receptor in dopaminergic cells (10). The VTA dopamine system has a well-established role in drug dependence (45,46), and a growing body of evidence indicates that the ␣-MSH and the AgRP systems regulate functions associated with positive reinforcement and drug reward (47–50). One possibility is, therefore, that PPAR␥ receptor agonists regulate the response of these neurotransmitter systems to alcohol, leading to a reduction of its motivational properties and consumption. However, this possibility is perhaps made less likely, given the important role of www.sobp.org/journal

648 BIOL PSYCHIATRY 2011;69:642– 649 dopamine in the regulation of conditioned reinstatement of drug seeking and the lack of pioglitazone activity on cue-induced relapse in our study. An alternative hypothesis is that pioglitazone reduces excessive alcohol use and related behaviors by acting on glial cells that are also rich in PPAR␥ receptors. It is known that increases in antiinflammatory mediators or glia-derived neurotrophic factor reduce cocaine abuse vulnerability (51) and lower alcohol consumption (52), whereas partial deletion of the glial cell derived neurotrophic factor transcript increases sensitivity to morphine and methamphetamine reward (53,54). Moreover, activation of glia-derived proinflammatory mediators such as interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha increases locomotor and neurotoxic effects of psychostimulants (55,56) and at least in parts mediates the development of opiate tolerance (57,58). Of note, in a series of recent studies, it has also been shown that ibudilast, a brain penetrant phosphodiesterase inhibitor (59) that decreases glia-derived proinflammatory cytokine production, lowers morphine reward (60), prevents withdrawal (61), and reduces development of tolerance (62,63). These effects of ibudilast, in part, appear to be linked to its ability to prevent opioids from activating toll-like receptor 4, a key sensor of endogenous damage signal (62– 64). Like ibudilast, TZDs inhibit proinflammatory IL-1beta, IL-6, and TNF-alpha production and can block the increase of toll-like 4 receptor expression after lipopolysaccharide toxin insult (65). These findings suggest a possible role for glial PPAR␥ receptors in the effects of pioglitazone on alcohol addiction. Recently, the sensitizing role of lipopolysaccharide, IL-1beta, and TNF-alpha on the expression of somatic and emotional (i.e., anxiety) signs of alcohol withdrawal has been also established (66). In light of this observation, one could explain the protective effects of pioglitazone on alcohol withdrawal and on stress-induced reinstatement with its ability to modulate glial PPAR␥ functions. In conclusion, the results of this study demonstrate that activation of PPAR␥ receptors mediates inhibition of alcohol consumption, reduces relapse vulnerability, and prevents the expression of alcohol withdrawal. Together these findings point to the possibility that PPAR␥ agonists such as pioglitazone may be useful in the treatment of alcoholism, and possibly other forms of addiction. In addition, as shown is several recent studies, pioglitazone has important hepatoprotective, neuroprotective, and anti-neuroinflammatory actions that may offer additional benefit for the treatment of alcoholism (4 – 6, 67). Pioglitazone has been in clinical use for several years in the treatment of Type 2 diabetes. Its tolerability and favorable safety profile have been largely demonstrated. Its ability to lower alcohol drinking, relapse behavior, and alcohol withdrawal therefore opens the possibility for immediate clinical investigation to determine its efficacy in alcoholism.

This study was supported by the University of Camerino (Grant FAR to RC). We thank Alfredo Fiorelli, Rina Righi, and Marino Cucculelli for expert technical assistance. Dr. Demopulos is president and Dr. Gaitanaris is vice-president of OMEROS Corporation. The company owns exclusive intellectual property rights on the use of PPAR␥ receptors and drugs targeting PPAR␥ receptors as a treatment target for addiction. Dr. Ciccocioppo is applicant on a patent application relating to the therapeutic use of PPAR␥ agonist in addiction. He is entitled to receive patent royalties from Omeros Corporation. All other authors report no biomedical financial interests or potential conflicts of interest. Supplementary material cited in this article is available online. www.sobp.org/journal

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