Amphetamine Anorexia Research Articles

Double immunofluorescent evidence that oxidative stress-associated activation of JNK/AP-1 signaling participates in neuropeptide-mediated appetite control

Amphetamine (AMPH), an appetite suppressant, alters expression levels of neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus. This study explored the potential role of cJun-N-terminal kinases (JNK) in appetite control, mediated by reactive oxygen species (ROS) and activator protein-1 (AP-1) in AMPH-treated rats. Rats were given AMPH daily for 4 days. Changes in feeding behavior and expression levels of hypothalamic NPY, CART, cFos, cJun, phosphorylated JNK (pJNK), as well as those of anti-oxidative enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione S-transferase (GST), were examined and compared. Following AMPH treatment, food intake and NPY expression decreased, whereas the other proteins expression and AP-1/DNA binding activity increased. Both cerebral cJun inhibition and ROS inhibition attenuated AMPH anorexia and modified detected protein, revealing a crucial role for AP-1 and ROS in regulating AMPH-induced appetite control. Moreover, both pJNK/CART and SOD/CART activities detected by double immunofluorescent staining increased in hypothalamic arcuate nucleus in AMPH-treated rats. The results suggested that pJNK/AP-1 signaling and endogenous anti-oxidants participated in regulating NPY/CART-mediated appetite control in rats treated with AMPH. These findings advance understanding of the molecular mechanism underlying the role of pJNK/AP-1 and oxidative stress in NPY/CART-mediated appetite suppression in AMPH-treated rats.

Amphetamine alters neural response to sucrose in healthy women

Amphetamine, likely via action on the brain's dopaminergic systems, induces anorectic eating behavior and blunts dopaminergic midbrain activation to rewards. Past work has hypothesized that this blunted reward responsivity is a result of increasing tonic over phasic DA activity. We sought to extend past findings to sweet taste during fMRI following single-blind administration of dextroamphetamine and placebo in 11 healthy women. We hypothesized that neural response in both limbic and cognitive sweet taste circuits would mirror past work with monetary rewards by effectively blunting sweet taste reward, and ‘equalizing’ it's rewarding taste with receipt of water. Behavioral results showed that amphetamine reduced self-reported hunger (supporting the existence of amphetamine anorexia) and increased self-report euphoria. In addition, region of Interest analysis revealed significant treatment by taste interactions in the middle insula and dorsal anterior cingulate confirming the ‘equalizing’ hypothesis in the cingulate, but unlike monetary reinforcers, the insula actually evinced enhanced separation between tastes on the amphetamine day. These results suggest a divergence from prior research using monetary reinforcers when extended to primary reinforcers, and may hint that altering dopaminergic signaling in the insula and anterior cingulate may be a target for pharmacological manipulation of appetite, and the treatment of obesity.

Involvement of oxidative stress in the regulation of NPY/CART-mediated appetite control in amphetamine-treated rats

Amphetamine (AMPH) treatment can suppress appetite and increase oxidative stress in the brain. AMPH-induced appetite suppression is associated with the regulation of neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus. The present study explored whether antioxidants, including glutathione S-transferase (GST) and glutathione peroxidase (GP), were involved in this NPY/CART-mediated appetite control. Rats were treated daily with AMPH for four days. Changes in food intake and expression levels of hypothalamic NPY, CART, GST, and GP were examined and compared. Results showed that, in AMPH-treated rats, (1) food intake and NPY expression decreased, while CART, GST, and GP expression increased; (2) NPY knockdown in the brain enhanced the decrease in NPY and the increases in CART, GST, and GP expression; and (3) central inhibition of reactive oxygen species production decreased GST and GP and modulated AMPH anorexia and the expression levels of NPY and CART. The present results suggest that oxidative stress in the brain participates in regulating NPY/CART-mediated appetite control in AMPH-treated rats. These results may advance the knowledge regarding the molecular mechanism of AMPH-evoked or NPY/CART-mediated appetite suppression.

Involvement of neuropeptide Y Y1 receptor in the regulation of amphetamine-mediated appetite suppression

Recently, we reported that an initial decrease followed by recovery of food intake was observed during four days of amphetamine (AMPH) treatment and suggested that these changes in response were mediated by changes in neuropeptide Y (NPY) and proopiomelanocortin (POMC). Here we investigated if Y1 receptor (Y1R) and/or Y5 receptor (Y5R) might be involved in this regulation. Rats were treated daily with AMPH for four days. Changes in the expression levels of Y1R, Y5R, melanocortin receptor 3 (MC3R), and NPY were assessed and compared. Results showed that Y1R and MC3R increased, with a maximal increase of about 210% on Day 2 but with a restoration to the normal level on Day 4. In contrast, NPY decreased with a biggest reduction of about 45% on Day 2 and the pattern of expression during AMPH treatment was opposite to those of Y1R and MC3R, while the expression of Y5R was not changed. Central inhibitions of NPY formation or Y1R activity modulated the anorectic response of AMPH and the reciprocal regulation of NPY and MC3R, revealing a crucial role of Y1R in this action. It is suggested that Y1R participates in the reciprocal regulation of NPY- and MC3R-containing neurons in the hypothalamus during the anorectic effect of AMPH. These results may further the understanding of Y1R in the control of eating.

NF-κB knockdown can modulate amphetamine-mediated feeding response

This study determined if transcription factor NF-κB is involved in the effect of amphetamine (AMPH)-mediated feeding response. Moreover, possible roles of hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) were also investigated. AMPH was administered daily to rats for four days. Changes in NF-κB, NPY and POMC expression were assessed and compared. The NPY gene was down-regulated with maximal response on Day 2 during AMPH treatment, which was consistent with the response to feeding behavior. In contrast, NF-κB and POMC genes were up-regulated, and their expression was increased by about 200% and 450%, respectively, with maximal response on Day 2. Moreover, NF-κB DNA binding ability and expression were increased similar to that of POMC. To examine further if NF-κB was involved, intracerebroventricular infusion of NF-κB antisense oligonucleotide was performed 1 h before the daily AMPH dosing in freely moving rats. Results showed that NF-κB knockdown could modify AMPH anorexia as well as NPY and POMC expression. The present findings prove that cerebral NF-κB participates in AMPH-mediated appetite suppression, possibly by modulating NPY and POMC expression. These results may aid in therapeutic research on AMPH and AMPH-like anti-obesity drugs.

Evidence for a difference in mechanism of action between fenfluramine- and amphetamine-induced anorexia

Abstract The influence of drugs, active on 5-hydroxytryptamine (5-HT) mechanisms, has been examined on the anorexigenic activity of fenfluramine and (+)-amphetamine in rats trained to consume their daily food ration during 6 h. Chlorimipramine, which inhibits the re-uptake mechanisms in central 5-HT neurons, and the 5-HT blocking drugs methergoline and methysergide were used. Fenfluramine, 7.5 mg kg−1, and amphetamine, 2.5 mg kg−1, given 1/2 h before feeding reduced the food intake during the following 2 h to approximately 40% compared with control days. Pretreatment with methergoline in the optimal dose (1 mg kg−1) produced only a weak but significant antagonism to amphetamine anorexia, whereas the fenfluramine anorexia was strongly antagonized by methergoline in all doses tested (0.3, 1 and 3 mg kg−1). Methysergide (0.1, 0.3, 1 and 3 mg kg−1) showed no significant antagonism against amphetamine or fenfluramine anorexia. Chlorimipramine produced a strong antagonistic effect to the fenfluramine anorexia, but showed no antagonism against amphetamine. In contrast the highest dose of chlorimipramine (20 mg kg−1) potentiated amphetamine anorexia. The present results together with other evidence discussed support the conclusion that 5-HT mechanisms are involved in fenfluramine anorexia, whereas amphetamine anorexia seems mainly correlated with catecholamine dependent mechanisms.

Knocking down the transcript of protein kinase C-lambda modulates hypothalamic glutathione peroxidase, melanocortin receptor and neuropeptide Y gene expression in amphetamine-treated rats

It has been reported that neuropeptide Y (NPY) contributes to the behavioral response of amphetamine (AMPH), a psychostimulant. The present study examined whether protein kinase C (PKC)-λ signaling was involved in this action. Moreover, possible roles of glutathione peroxidase (GP) and melanocortin receptor 4 (MC4R) were also examined. Rats were given AMPH daily for 4 days. Hypothalamic NPY, PKCλ, GP and MC4R were determined and compared. Pretreatment with α-methyl-para-tyrosine could block AMPH-induced anorexia, revealing that endogenous catecholamine was involved in regulating AMPH anorexia. PKCλ, GP and MC4R were increased with maximal response on Day 2 during AMPH treatment, which were concomitant with the decreases in NPY. cAMP response element binding protein (CREB) DNA binding activity was increased during AMPH treatment, revealing the involvement of CREB-dependent gene transcription. An interruption of cerebral PKCλ transcript could partly block AMPH-induced anorexia and partly reverse NPY, MC4R and GP mRNA levels to normal. These results suggest that PKCλ participates in regulating AMPH-induced anorexia via a modulation of hypothalamic NPY gene expression and that increases of GP and MC4R may contribute to this modulation. Our results provided molecular evidence for the regulation of AMPH-induced behavioral response.

Amphetamine, an appetite suppressant, decreases neuropeptide Y immunoreactivity in rat hypothalamic paraventriculum

Amphetamine (AMPH) is a well-known anorectic agent. The mechanism underlying the anorectic response of AMPH has been attributed to its inhibitory effect on hypothalamic neuropeptide Y (NPY), an orexigenic peptide in the brain. However, there is still lack of genomic or in situ immunohistochemical evidence to prove it. The present study was aimed to assess the molecular mechanism of AMPH anorexia by immunostaining of hypothalamic NPY protein in the area of paraventricular nucleus (PVN) and by detecting the change of hypothalamic NPY mRNA level using RT-PCR. Results revealed that an AMPH treatment might reduce the expression of NPY at both transcriptional and posttranslational levels. Comparatively, a treatment of clomipramine, a serotonin transporter inhibitor, was unable to reduce NPY mRNA level, revealing the noninvolvement of hypothalamic NPY gene in serotonin anorexia. Our results provided genomic and in situ immunohistochemical evidence to confirm the mediation of hypothalamic NPY neurons in the anorectic action of AMPH.

Further evidence for the mediation of both subtypes of dopamine D1/D2 receptors and cerebral neuropeptide Y (NPY) in amphetamine-induced appetite suppression

Amphetamine (AMPH) is known as an anorectic agent. Repeated treatments of AMPH for several days induced a marked anorexia on day 1 followed by a gradual return of food intake to normal level. Previously, using daily food intake (DFI) as an indicator, we found that both dopamine (DA) D1 and D2 receptors were involved in AMPH anorexia. In the present study, using DFI and body weight change (BWC) as indicators, we took a further step to examine whether repeated co-administrations of D1 and D2 agonists might produce an AMPH-like action. Results revealed that repeated co-administrations of D1 and D2 agonists, but not D1 or D2 agonist alone, produced an AMPH-like action (i.e., tolerant DFI and BWC), confirming the previous study. In addition, we examined whether cerebral neuropeptide Y (NPY), an orexigenic neurotransmitter reported to mediate AMPH anorexia, was involved in the anorectic action of D1/D2 co-administration. Our result revealed that injection of NPY antisense into brain could modify the anorectic action of repeated D1/D2 agonists, indicating the involvement of NPY. Taken together, the present results confirmed that both subtypes of D1 and D2 receptors and cerebral NPY were involved in the anorectic action of AMPH.

Enhancement of amphetamine anorexia after chronic administration of sulpiride in rats

Long-term administration of sulpiride induces hyperphagia and obesity in female rats. After sulpiride withdrawal, a significant hypophagia has been observed. The hyperphagia could be related to the blockade and the hypophagia to supersensitivity of dopamine D 2 receptors, in particular those D 2 receptors located in the perifornical hypothalamus. If this were the case, an enhancement of anorexia induced by amphetamine and dopamine should be observed after interruption of long-term supiride treatment. Two doses of systemic sulpiride (20 or 200 mg/kg) and one dose of intrahypothalamic sulpiride (15 μg) were tested. Amphetamine was administered by systemic or intrahypothalamic infusion. Dopamine was administered in the hypothalamus. After withdrawal of systemic administration of sulpiride (200 mg/kg), an enhancement of anorexia induced by systemic amphetamine was observed. However, the anorexia induced by intrahypothalamic injections of amphetamine or dopamine was not affected by the interruption of the sulpiride treatment. These results suggest that the hypophagia following chronic sulpiride treatment is not due to supersensitivity of D 2 dopamine receptors in the lateral hypothalamus. Moreover, the change in the response to amphetamine might be related to supersensitivity of extrahypothalamic D 2 receptors.

Serotonin may play a role in the anorexia induced by amphetamine injections into the lateral hypothalamus

Amphetamine (AMPH) injections into the lateral hypothalamus (LH) are known to inhibit feeding and this effect has been shown to be mediated by the release and the reuptake blockade of catecholamines. LH serotonin (5-HT) has been suggested to be involved in feeding inhibition and a recent study showed that LH amphetamine infusion increases extracellular dopamine (DA), norepinephrine (NE) and 5-HT, which suggests that 5-HT might also be involved in amphetamine anorexia. The present study investigated this possibility. A correlational study was performed between the anorectic effect of LH unilateral microinjections of each monoamine and the anorectic effect of AMPH. Six groups of male rats were used. The rats in each group were submitted to 2 series of 6 experimental sessions. Each session consisted of one microinjection in 24 h food-deprived rats, followed by the measurement of food intake 30 min later. The first series was similar for all groups and explored the AMPH effect (difference between the mean food intake after 3 AMPH injections (40 μg/0.5 μl) and the mean food intake after 3 saline injections (0.5 μl). The second series explored the effects of DA (40 μg), NE (25 μg), EPI (25 μg), 5-HT (25 μg) or AMPH again, in a similar way as described for AMPH in the first series. Linear regression analysis on the first series AMPH effect and the amine effect of the 2nd series showed a positive correlation between both series of AMPH, AMPH and DA, and AMPH and 5-HT. This last correlation was replicated in a different group. No correlation was found between AMPH and NE or AMPH and epinephrine (EPI). These results suggest that LH AMPH anorexia could be mediated through DA and 5-HT release.

Procedures affecting maintenance and loss of tolerance to amphetamine-induced anorexia and cross-tolerance to haloperidol-induced catalepsy

Procedures affecting the maintenance and loss of tolerance to amphetamine anorexia were investigated. Following a period in which rats acquired tolerance to amphetamine-induced anorexia via the contingent- tolerance procedure, a variety of manipulations were investigated to determine which effected a loss of tolerance. The procedures included: continued milk availability or its absence, saline injections or no injections and a switch to non-contingent amphetamine injections. These procedures were each investigated while animals continued to be food-deprived or were maintained at their ad libitum weights. The animals maintained at 100% ad lib. lost tolerance whereas tolerance was maintained by the 85% groups, regardless of what other manipulations were given. Subsequently all animals were tested for catalepsy induced by 1.25 mg/kg haloperidol. Animals that had maintained tolerance to amphetamine anorexia (85% groups) were significantly less cataleptic than the animals that had lost tolerance to amphetamine anorexia (100% groups). The finding that tolerance to amphetamine anorexia is lost in animals that have ad lib. access to food indicates that a homeostatic imbalance caused by food-deprivation is perhaps the most important factor in the maintenance of tolerance to amphetamine anorexia.

Effects of haloperidol on anorexia induced by 1-norephedrine and d-amphetamine in adult rats

Although amphetamine anorexia has been linked to activation of dopaminergic receptors within the lateral aspects of the hypothalamus, the receptor type by which phenylpropanolamine (PPA: the racemic mixture of d- and l-norephedrine) induces anorexia has not been identified. In the present experiment, separate groups of adult male rats were pretreated (IP) with either 0.9% saline or haloperidol (either 0.4 or 0.8 mg/kg) 45 minutes prior to treatment (IP) with either saline or 20 mg/kg l-NEP (the active enantiomer of PPA) and were then allowed 180 minutes access to food and water. Treatment with 20 mg/kg l-NEP induced comparable reductions in food intake of approximately 30% in rats pretreated with either dose of haloperidol or saline. In a sub-experiment, it was demonstrated that 1.0 mg/kg d-amphetamine sulfate reduced food intake by 25%, but this anorexic action was completely attenuated by 0.8 mg/kg haloperidol given 45 minutes prior to feeding. These results add to a growing body of literature that documents important differences between the mechanisms by which amphetamine and PPA produce their anorexic actions.

Sulpiride injections in the lateral hypothalamus induce feeding and drinking in rats

Amphetamine injections into the lateral hypothalamus inhibit feeding. This effect is blocked by local administration of neuroleptics, suggesting a role for dopamine in feeding inhibition. However, the type of dopamine receptor involved in satiety is not known. Therefore, we tested the effect of intrahypothalamic injections of sulpiride, a specific D 2 receptor blocker, on amphetamine anorexia in food-deprived rats, and on spontaneous feeding and drinking in satiated rats. Sulpiride attenuated by 36% the anorexia produced by intrahypothalamic injections of amphetamine. In satiated rats, sulpiride (8 μg/0.5 μl) elicited feeding (mean food intake after sulpiride: 5.4 g, and after vehicle 1.6 g, p<0.001), and drinking (mean water intake after sulpiride: 12.3 ml, and after vehicle: 0.9 ml, p<0.001). A dose response relationship was found between sulpiride dose and feeding or drinking. Sulpiride-induced drinking was observed in the absence of food, showing that it is not a postprandial phenomenon. These results suggest that hypothalamic D 2 receptors might be involved in feeding and drinking regulation.

Behavioural microanalysis of the role of dopamine in amphetamine anorexia

A microstructural analysis paradigm was used to study amphetamine anorexia. Doses above 0.40 mg/kg significantly reduced food intake by reducing eating time; in contrast, eating rate was increased at these doses. Examination of the frequency distribution of interresponse times (IRTs) revealed a significant shift to shorter IRTs at doses as low as 0.125 mg/kg. Pimozide blocked amphetamine anorexia at 0.5 and 1.0 mg/kg, suggesting that at both doses amphetamine anorexia has a dopaminergic substrate. However, the atypical neuroleptic thioridazine did not antagonize amphetamine. Furthermore, effects of amphetamine were additive with those of apomorphine, administered at a dose known to suppress feeding by inhibiting mesolimbic DA neurons. These results provide evidence against an involvement of the mesolimbic DA system in amphetamine anorexia.

Receptor blocking drugs and amphetamine anorexia in human subjects.

The interaction between the receptor antagonist thymoxamine (THYM), propranolol (PPL) and metergoline (MTG) with dexamphetamine (d-Amp)-induced anorexia was examined in a series of studies in normal female volunteers. Visual analogue scale (VAS) ratings of hunger were made and food intake was measured using an automated solid food dispenser (AFD). d-Amp (10 mg) significantly depressed hunger ratings compared to placebo in two of the three studies and its effect was countered by the addition of MTG (4 mg). d-Amp significantly reduced food intake compared to placebo in two studies. In all trials the reduction in food intake following d-Amp was significantly greater than would have been predicted from its effect on hunger. THYM (160 mg) and PPL (40 mg) were associated with no changes in food intake when given alone or with d-Amp, MTG increased food intake (but not significantly) and the combined effects of MTG and d-Amp was the algebraic sum of the effect of each; but there appeared to be no true pharmacological interaction between blocker and anorectic. The results indicated that there may be some dissociation between the effect of d-Amp on hunger and food intake but have failed to produce evidence that noradrenergic pathways are involved. The results are consistent with the theories that d-Amp anorexia does not involve the release of 5-hydroxytryptamine (5-HT) but that 5-HT pathways are involved in the feeding process.

Amphetamine-induced anorexia: Analysis with hypothalamic lesions and knife cuts

The present study examined the hypotheses that the midlateral perifornical region of the hypothalamus (PFH), at the level of the ventromedial nucleus, plays a crucial role in amphetamine (AMPH)-induced anorexia and that mediating fibers ascending to this brain region follow a midlateral course through the caudal hypothalamus. Electrolytic lesions that destroyed the PFH region attenuated the feeding suppression induced by intraperitoneal administration of AMPH. Lesions placed anterior, dorsal, or medial to this region, in contrast, did not decrease AMPH's effect. The medially-placed paraventricular nucleus lesion, in fact, enhanced drug response. Midlateral coronal wire-knife cuts in the caudal hypothalamus also attenuated AMPH anorexia. The crucial midlateral caudal hypothalamic cut also disrupted anorexia induced by direct injection of AMPH into the PFH area. The results obtained from the lesion data support the hypothesis that the PFH region is essential to AMPH's suppressive effect upon feeding, and the KC data suggest that crucial catecholamine fibers mediating this drug response ascend specifically through the midlateral portion of the hypothalamus.

Brain serotonin and eating behavior

Studies indicate that hypothalamic monoamine systems involved in the control of food intake have specific effects on temporal feeding patterns and on appetite for specific macronutrients. Based on the evidence obtained in rats, it is proposed that serotonin acts, in part, through a satiety mechanism of the medial hypothalamus, to reduce ingestion of carbohydrate while sparing protein intake. In controlling the ratio of carbohydrate to protein intake, this serotonergic system, which is responsive to the anorectic agent fenfluramine, is believed to function in direct opposition to the alpha 2-noradrenergic system of the paraventricular nucleus, which inhibits satiety for carbohydrate and thereby potentiates the size of carbohydrate meals. This serotonergic system may also indirectly oppose the catecholaminergic systems of the lateral hypothalamus, which mediate amphetamine anorexia and which inhibit a hunger-stimulating system for protein intake, thereby delaying the initiation of protein meals. Examination of the rats' normal eating patterns, in conjunction with particular biochemical analyses, has indicated specific points in the circadian eating cycle where these hypothalamic monoamine systems, in association with changes in circulating hormones and nutrients, may be physiologically activated.

Effects of guanethidine sympathectomy on feeding, drinking, weight gain and amphetamine anorexia in the rat

Adult female rats that underwent sympathectomy induced by guanethidine treatment (10, 20 or 40 mg/kg) exhibited markedly increased water intake, but did not display significant alterations of either food intake, body weight, or the Lee Index of obesity. Guanethidine treatment did not attenuate amphetamine anorexia as evidenced by comparable dose-dependent reductions in food intake to d-amphetamine sulfate (0.25, 0.50, 1.0, and 2.0 mg/kg) in sympathectomized and control rats. These data are not consistent with the hypothesis that amphetamine anorexia is partially mediated via enhanced BAT thermogenesis.

Tolerance pattern to amphetamine anorexia after selective lesions in the hypothalamic dopaminergic projection

Tolerance to the anorexic effect of d-amphetamine was studied in rats with selective dopamine lesions in the forebrain by means of 6-hydroxy dopamine, and measuring the food intake during two consecutive 2 h periods. Lesions placed in the perifornical hypothalamus (PFH) strongly antagonised the anorexic effect, whereas, lesions produced via intraventricular injections affected the anorexia only marginally. Amphetamine anorexia observed in the first 2 h in control and lesioned groups remained persistently, without any evidence of tolerance, upto 2 weeks of treatment. The second 2 h food intake exhibited a progressive increase which contributed to the apparent tolerance seen in total 4 h food intake in the control and lesioned animals. The onset and completion of this apparent tolerance was markedly delayed in the dopamine depleted group; lesions placed in the relatively medial areas delayed the tolerance development more effectively than that of PFH lesions. The stimulant effect of amphetamine on locomotion was abolished in lesioned animals. The results indicate that an apparent tolerance to amphetamine anorexia still developed in animals with forebrain dopamine loss. Although both the beta adrenergic and dopaminergic systems act together in mediating AMPH anorexia, the onset and the rate of completion of tolerance appear to be under the influence of hypothalamic dopaminergic system.