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Amylin Modulates the Mesolimbic Dopamine System to Control Energy Balance

July 2014
Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 40(2)

July 2014
40(2)

DOI:10.1038/npp.2014.180

Source
PubMed

Authors:

Elizabeth Mietlicki-Baase

University at Buffalo, The State University of New York

David Reiner

National Institute on Drug Abuse

Jackson J Cone

University of Chicago

Diana Olivos

The Children's Hospital of Philadelphia

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Amylin acts in the CNS to reduce feeding and body weight. Recently, the ventral tegmental area (VTA), a mesolimbic nucleus important for food intake and reward, was identified as a site-of-action mediating the anorectic effects of amylin. However, the long-term physiological relevance and mechanisms mediating the intake-suppressive effects of VTA amylin receptor activation are unknown. Data show that the core component of the amylin receptor, the calcitonin receptor (CTR), is expressed on VTA dopamine neurons and that activation of VTA amylin receptors reduces phasic dopamine in the nucleus accumbens core (NAcC). Suppression in NAcC dopamine mediates VTA amylin-induced hypophagia, as combined NAcC D1/D2 receptor agonists block the intake-suppressive effects of VTA amylin receptor activation. Knockdown of VTA CTR via AAV-shRNA resulted in hyperphagia and exacerbated body weight gain in rats maintained on high-fat diet. Collectively, findings show that VTA amylin receptor signaling controls energy balance by modulating mesolimbic dopamine signaling.Neuropsychopharmacology accepted article preview online, 18 July 2014; doi:10.1038/npp.2014.180.

Amylin receptors are expressed on VTA DA neurons. Representative

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VTA AmyR activation suppresses intake of a palatable HFD primarily by decreasing meal size. Intra-VTA injection of sCT (n=10) reduced cumulative HFD consumption (a) and meal size (b), but only the highest dose of sCT decreased meal number (c). Body weight gain over the 24 h test was also reduced by sCT (d). *Main effect of sCT (p<0.05); within a time bin, bars with different letters are significantly different from each other (p<0.05). The key next to d applies to all panels. All data are shown as mean±SEM.Download Power Point slide (974 KB)

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Intra-NAcC administration of a combination of D1 and D2 receptor agonists blocks the energy balance effects of VTA AmyR activation in rats maintained on palatable HFD. Administration of SKF-81297 (D1 receptor agonist) plus quinpirole (D2 receptor agonist) directly to the NAcC blocks the suppression of HFD intake (a) and 24-h body weight gain (b) after intra-VTA sCT administration (n=12). *Different from vehicle/vehicle (p<0.05); Ŧdifferent from vehicle/sCT (p<0.05). The key next to b applies to both panels. All data are shown as mean±SEM. See also Supplementary Figure S2.Download Power Point slide (562 KB)

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A novel AAV-shRNA targeting the calcitonin receptor (CTR) reduces

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Endogenous amylin activates the VTA to provide long-term control of energy balance. Rats maintained on HFD or chow received intra-VTA injection of the AAV to knock down CTR expression (CTR-KD) or the control virus (CTR-Ctrl), and subsequent energy intake and body weight were monitored for 1-month post-AAV injection (n=6–7 per AAV/dietary condition). (a) HFD-fed VTA CTR-KD rats gained more body weight than any other treatment group; total body weight gain over the course of the experiment is shown in c. The enhanced body weight gain of HFD-fed CTR-KD rats is explained by hyperphagia, as rats in this condition consume more kilocalories than animals in any other treatment group both in 48-h measurements (b) and over the course of the study (d). VTA knockdown of CTR-A expression was confirmed via qPCR (e). For (a, b), *different from CTR-Ctrl/Chow (p<0.05); ^different from CTR-Ctrl/HFD (p<0.05). The key in a also applies to b. For (c–e), bars with different letters are significantly different from each other (p<0.05); the key below e also applies to c and d. All data are shown as mean±SEM. See also Supplementary Figure S3.Download Power Point slide (1,176 KB)

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Accepted Article Preview: Published ahead of advance online publication

Amylin Modulates the Mesolimbic Dopamine System to

Control Energy Balance

Elizabeth G Mietlicki-Baase, David J Reiner, Jackson J Cone,

Diana R Olivos, Lauren E McGrath, Derek J Zimmer,

Mitchell F Roitman, Matthew R Hayes

Cite this article as: Elizabeth G Mietlicki-Baase, David J Reiner, Jackson J Cone,

Diana R Olivos, Lauren E McGrath, Derek J Zimmer, Mitchell F Roitman,

Matthew R Hayes, Amylin Modulates the Mesolimbic Dopamine System to

Control Energy Balance, Neuropsychopharmacology accepted article preview 18

July 2014; doi: 10.1038/npp.2014.180.

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Received 12 May 2014; revised 18 June 2014; accepted 7 July 2014; Accepted

article preview online 18 July 2014

Amylin modulates the mesolimbic dopamine system to control energy

balance

Elizabeth G. Mietlicki-Baase, Ph.D.1*, David J. Reiner, B.S.1, Jackson J. Cone,

Ph.D.2, Diana R. Olivos, B.A.1, Lauren E. McGrath, B.S.1, Derek J. Zimmer,

M.S.1, Mitchell F. Roitman, Ph.D.2, & Matthew R. Hayes, Ph.D.1*

1Translational Neuroscience Program, Department of Psychiatry, Perelman

School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA;

2Department of Psychology, University of Illinois at Chicago, Chicago, IL, USA

Running Title: VTA amylin modulates accumbens dopamine

*Address correspondence to:

Dr. Elizabeth G. Mietlicki-Baase: 125 S. 31st St., Philadelphia, PA 19104; Phone:

215-746-3664; Fax: 215-573-2041; Email: ebaase@mail.med.upenn.edu

Dr. Matthew R. Hayes: 125 S. 31st St., Philadelphia, PA 19104; Phone: 215-573-

6070; Fax: 215-573-2041; Email: hayesmr@mail.med.upenn.edu

Abstract

Amylin acts in the CNS to reduce feeding and body weight. Recently, the

ventral tegmental area (VTA), a mesolimbic nucleus important for food

intake and reward, was identified as a site-of-action mediating the anorectic

effects of amylin. However, the long-term physiological relevance and

mechanisms mediating the intake-suppressive effects of VTA amylin

receptor activation are unknown. Data show that the core component of the

amylin receptor, the calcitonin receptor (CTR), is expressed on VTA

dopamine neurons and that activation of VTA amylin receptors reduces

phasic dopamine in the nucleus accumbens core (NAcC). Suppression in

NAcC dopamine mediates VTA amylin-induced hypophagia, as combined

NAcC D1/D2 receptor agonists block the intake-suppressive effects of VTA

amylin receptor activation. Knockdown of VTA CTR via AAV-shRNA

resulted in hyperphagia and exacerbated body weight gain in rats

maintained on high-fat diet. Collectively, findings show that VTA amylin

receptor signaling controls energy balance by modulating mesolimbic

dopamine signaling.

Keywords: IAPP, obesity, reward, diabetes, feeding

Introduction

Amylin is a neuropeptide co-secreted with insulin from pancreatic β-cells

(Johnson et al, 1988; Kahn et al, 1990) that is involved in the regulation of blood

glucose (Scherbaum, 1998) and energy balance [see (Lutz, 2005; Mietlicki-

Baase and Hayes, 2014) for review]. Systemic administration of amylin and

amylin agonists such as salmon calcitonin (sCT) and pramlintide reduce food

intake and body weight in humans and animal models (Bello et al, 2008;

Chapman et al, 2005; Lutz et al, 1994; Lutz et al, 2000; Smith et al, 2007), effects

mediated by the CNS (Lutz et al, 1995a; Rushing et al, 2000). Previous research

has focused heavily on the notion that amylin receptor (AmyR) activation in the

area postrema (AP), a hindbrain circumventricular nucleus, is responsible for

amylin-induced hypophagia (Lutz et al, 1998; Mollet et al, 2004). Importantly,

however, amylin readily crosses the blood-brain barrier (Banks and Kastin, 1998;

Banks et al, 1995), and amylin binding sites are found not only in the AP, but also

in other feeding-relevant CNS nuclei (Christopoulos et al, 1995; Paxinos et al,

2004; Sexton et al, 1994). Together with the increasingly recognized distributed

nature of energy balance control (Grill, 2006; Grill and Kaplan, 2002), this

suggests that amylin may act at multiple sites throughout the brain to regulate

feeding and body weight. Recently, the ventral tegmental area (VTA), a

mesolimbic nucleus important in the control of food intake and reward [see

(Narayanan et al, 2010; Vucetic and Reyes, 2010) for review], was identified as a

novel site-of-action for the intake- and body weight-suppressive effects of amylin

(Mietlicki-Baase et al, 2013b). However, the long-term physiological relevance

and neuronal mechanisms by which VTA AmyR signaling controls energy

balance are unknown.

The VTA contains a large population of dopamine (DA) neurons that

project to sites throughout the brain that regulate feeding, food reinforcement,

and motivated behavior [see (Baik, 2013; Narayanan et al, 2010; Vucetic and

Reyes, 2010) for review]. In particular, phasic “spikes” in DA release in the

nucleus accumbens (NAc) evoked by aspects of food reward (Brown et al, 2011;

Roitman et al, 2004) have been shown to be modulated by feeding-related

hormones (Cone et al, 2014) and are sufficient for food reinforcement (Domingos

et al, 2011). Given that VTA AmyR activation reduces food intake as well as the

motivation to work for a palatable food (Mietlicki-Baase et al, 2013b), these data

collectively support the idea that a decrease in NAc DA signaling may be

involved in VTA AmyR-mediated hypophagia. However, the ability of VTA AmyR

activation to modulate NAc DA signaling to control feeding has not been tested.

Furthermore, although previous research showed that VTA AmyR signaling

acutely reduces food intake, it remains to be determined whether endogenous

VTA AmyR signaling is required for the day-to-day control of energy balance.

Here, we directly address these questions by examining whether VTA AmyR

activation modulates NAc phasic DA neurotransmission, as well as evaluating

the requirement of reduced D1/D2 receptor signaling in mediating the intake-

suppressive effects of more-palatable and less-palatable food intake following

VTA AmyR activation. Using a novel adeno-associated virus short hairpin RNA

(AAV-shRNA) construct for the calcitonin receptor (core component of the AmyR)

we also assess the requirement of endogenous VTA AmyR signaling in the long-

term control of food intake and body weight in rats maintained on standard chow

or high-fat diet (HFD).

Materials and Methods

Subjects. Adult male Sprague Dawley rats (Charles River) were housed

individually in a temperature- and humidity-controlled environment on a 12h/12h

light-dark cycle. Animals were housed in plastic bins for voltammetric testing and

in hanging wire cages for all other studies to allow for accurate measurement of

food spillage. Food and water were available ad libitum except where noted.

Animal care and use was in accordance with the National Institutes for Health

Guide for the Care and Use of Laboratory Animals, and all procedures received

approval from the Institutional Animal Care and Use Committee at the University

of Pennsylvania or the University of Illinois at Chicago. All behavioral studies

utilized a within-subjects, counterbalanced design except where noted.

Drugs. The amylin receptor agonist salmon calcitonin (sCT; Bachem) was

dissolved in sterile artificial cerebrospinal fluid (aCSF; Harvard Apparatus) for

central injections and in sterile 0.9% NaCl for peripheral injections. SKF-81297

(Sigma) and quinpirole (Sigma) were dissolved in aCSF. LiCl (Sigma) was

dissolved in 0.9% NaCl. Doses for drugs were selected from the literature

(Kanoski et al, 2012; Mietlicki-Baase et al, 2013b; Schmidt and Pierce, 2006;

Swanson et al, 1997).

Surgery. Chronic cannula implantation for feeding studies: Rats were

anesthetized by intramuscular (IM) injection of a mixture containing ketamine

(90mg/kg), xylazine (2.7mg/kg), and acepromazine (0.64mg/kg) (KAX) and then

placed into a stereotaxic apparatus. Analgesia was provided for all surgical

procedures (2mg/kg meloxicam). Each rat was stereotaxically implanted with a

bilateral guide cannula (26-ga; Plastics One) aimed at the VTA (guide cannula

coordinates: ±0.5mm lateral to midline, 6.8mm posterior to bregma, 6.6mm

ventral to skull; internal cannula aimed 8.6mm ventral to skull). For DA receptor

agonist experiments, rats were also stereotaxically implanted with a unilateral

guide cannula aimed at the NAcC (guide cannula coordinates: 2.5mm anterior to

bregma, 1.4mm left of midline, 4.5mm ventral to skull; internal cannula aimed

6.5mm ventral to skull). Cannula placements were verified post-mortem by

injection of pontamine sky blue (100nl). Adeno-associated virus (AAV)

parenchymal delivery: Rats were anesthetized by IM injection of KAX and then

placed into a stereotaxic apparatus. Analgesia was provided (2mg/kg

meloxicam). Each rat received a bilateral VTA infusion (250nl per hemisphere) of

an AAV designed to reduce expression of CTR-A/B (CTR-KD), or a control AAV

expressing eGFP (CTR-Ctrl); injection coordinates: ±0.5mm lateral to midline,

6.8mm posterior to bregma, 8.6mm ventral to skull. Surgical preparation for fast-

scan cyclic voltammetry (FSCV): Rats were anesthetized by IP injection of

ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (10 mg/kg). A

guide cannula (Bioanalytical Systems) was implanted dorsal to the right NAcC

(+1.3mm anterior and +1.5mm lateral to bregma, -2.5mm ventral to skull). An

infusion cannula (Plastics One) was also implanted aimed at the VTA: 26-ga

11mm cannula (C315), -5.8mm anterior and +2.9mm lateral to bregma, -6.5mm

ventral to skull, angled 15° away from midline. VTA coordinates were chosen to

maximize the likelihood of affecting VTA neurons that project to the NAcC

(Ikemoto, 2010). A chlorinated silver reference electrode was placed in left

forebrain. Stainless steel skull screws and dental cement secured implants to the

skull.

VTA colocalization of the CTR and tyrosine hydroxylase: Rats (n=6) maintained

on HFD were deeply anesthetized and transcardially perfused with 0.1M PBS

(pH 7.4) followed by 4% paraformaldehyde in 0.1M PBS. Brains were removed

from the crania and post-fixed in 4% paraformaldehyde for 6h, then stored in

20% sucrose in 0.1M PBS at 4°C overnight. Coronal VTA-containing sections

(30μm) were sliced and collected using a cryostat and stored in cryoprotectant at

-20°C until the start of immunohistochemistry (IHC). Briefly, tissue was washed

with 0.1M PBS followed by 50% ethanol to remove the cryoprotectant, followed

by washes in 1% sodium borohydride and 0.3% H2O2. Sections were incubated

in blocking solution (5% normal donkey serum, Jackson Immunoresearch)

followed by 16h incubation in rabbit anti-CTR primary antibody (1:1000; Abcam)

at room temperature. Tissue was then incubated in donkey anti-rabbit fluorescent

secondary antibody (AlexaFluor 488, 1:222; Jackson Immunoresearch) for 2h.

Next, sections were then incubated in rabbit anti-tyrosine hydroxylase (TH)

primary antibody (1:1000; Cell Signaling) at room temperature for 16h, followed

by 2h incubation in donkey anti-rabbit fluorescent secondary antibody

(AlexaFluor 594, 1:500; Jackson Immunoresearch) for 2h. Sections were

mounted and visualized using fluorescence microscopy (Nikon 80i; NIS-Elements

AR 3.0) at 20x magnification. CTR and/or TH immunoreactivity were

semiquantified from VTA-containing coronal sections between -5.2 to -6.8 from

bregma, using a rat brain atlas (Paxinos and Watson, 2006).

Modulation of NAcC phasic DA release by VTA AmyR activation: Rats (n=9) had

ad libitum access to water but were food (chow)-restricted to approximately 90%

of their free-feeding body weight during training and testing. All training and

experimental sessions took place during the light phase in standard operant

chambers (Med Associates) with a food receptacle and magazine for the delivery

of single 45mg sugar pellets (3.58kcal/g; BioServ). Rats were trained to retrieve

sugar pellets that were delivered with a random inter-trial interval (delivery

interval range: 30-90s; mean: 60 +/- 8.2s). Following 5d of training, rats were

surgically prepared for FSCV. After returning to pre-surgery body weight, rats

were retrained for 2d prior to the experimental session.

During an experimental session, rats were placed into operant chambers

as above. FSCV in awake and behaving rats and analyte identification and

quantification have been extensively described (Cone et al, 2013; Phillips et al,

2003). Briefly, a micromanipulator containing a glass-insulated carbon fiber

(~75µm; Goodfellow USA) (recording) electrode was inserted into the NAcC

guide cannula. The recording electrode was then lowered into NAcC and locked

into place. A FSCV headstage (University of Washington EME Shop) was used

to tether the rat, apply voltage changes and measure resultant current changes.

The electrode voltage was held at -0.4V and ramped in a triangular fashion (-0.4

to +1.3 to -0.4V; 400V/s) at 10Hz. In addition, a 33-ga injector connected to a

10µL Hamilton syringe was inserted into the VTA infusion cannula (2mm

projection). To verify that food reward reliably evoked phasic DA release, a single

sugar pellet was delivered. If this failed to evoke DA release, the electrode was

advanced 0.16mm and the process was repeated.

Once a stable release site was confirmed, the experimental session

began. Electrochemical data was synced with video and recorded during the

entire session. After 10 pellets (mid-session), an infusion pump was activated to

deliver intra-VTA sCT (0.04µg) or vehicle (100nl aCSF). After the recording

session, electrodes were removed; rats were disconnected from the headstage

and returned to their home cage. Ad libitum chow intake was monitored for

30min. Following experiments, all recording electrodes were calibrated in a flow-

cell (Sinkala et al, 2012) to convert detected current to concentration. The

average calibration factor for all electrodes used in these experiments was 44.58

+/- 4.0nM/nA.

All rats retrieved and ingested all pellets before and after pharmacological

manipulations. Individual trials were background-subtracted and DA

concentration during the 10s before to 10s after pellet retrieval was extracted

from voltammetric data using principal component analysis (Heien et al, 2004).

We calculated peak DA concentration evoked by pellet retrieval by finding the

maximum DA concentration during the 1s before to 1s after retrieval of each

individual pellet. These values were averaged and compared before (baseline)

and after VTA infusions to obtain a measure of percent change in peak DA.

Following completion of experiments, rats were deeply anesthetized with

sodium pentobarbital (100mg/kg) and a small electrolytic lesion was made at the

voltammetry recording site using a polyimide-insulated stainless steel electrode

(A-M Systems, Inc.). Rats were then transcardially perfused with cold 0.01M

phosphate-buffered saline followed by 10% buffered formalin solution (Sigma).

Brains were removed and stored in formalin for 24h and transferred to 30%

sucrose in 0.1M phosphate buffer (PB). All brains were sectioned at 40µm on a

cryostat. NAc sections were mounted and lesion locations were verified using

light microscopy (Paxinos and Watson, 2006). All recordings were confirmed to

be from the NAcC. Additionally, cannula tips from all animals were confirmed to

be in the VTA using light microscopy.

Feeding and body weight effects of NAcC DA receptor agonists: Separate groups

of chow-maintained rats with NAcC cannulae were given unilateral intra-NAcC

injections of either the D1 receptor agonist SKF-81297 (0, 0.1, 0.6, 1.2μg; n=6),

the D2 receptor agonist quinpirole (0, 1.5, 3, 6μg; n=6), or a combination of SKF-

81297 and quinpirole (1.2μg and 6μg, respectively; n=6), just before the onset of

the dark phase. For all experiments, vehicle was 200nl aCSF. Chow intake and

body weight change were measured over the 24h post-injection.

We also tested the effects of intra-NAcC delivery of SKF-81297 and

quinpirole on HFD intake by conducting similar studies in HFD-maintained rats.

Briefly, one group of rats (n=12) received unilateral intra-NAcC injections of SKF-

81297 (1.2μg), quinpirole (6μg), or vehicle (200nl aCSF); a second group of

HFD-maintained animals (n=8) received NAcC injections of the combination of

SKF-81297 and quinpirole (1.2μg and 6μg, respectively) or vehicle (200nl aCSF)

just before the onset of the dark phase. HFD intake and body weight were

measured for 24h post-injection.

Energy balance effects of VTA AmyR activation in combination with NAcC DA

receptor agonism: Just before the onset of the dark phase, chow-maintained rats

(n=10) with VTA and NAcC cannulae were given a unilateral intra-NAcC injection

containing a mixture of SKF-81297 and quinpirole (1.2μg and 6μg, respectively)

or vehicle (200nl aCSF), followed by a unilateral VTA injection of sCT (0.04μg) or

its vehicle (100nl aCSF). Subsequent chow intake was measured at 1, 3, 6, and

24h, and 24h body weight change was calculated. A separate group of animals

(n=12) maintained on HFD (60% kcal from fat) were given the same NAcC and

VTA drug treatments, and HFD intake (1, 3, 6, and 24h) and 24h body weight

change were assessed.

Effect of VTA AmyR activation on conditioned taste avoidance (CTA): A two-

bottle CTA test was performed as described previously (Garcia et al, 1955;

Kanoski et al, 2012). Briefly, rats were habituated to a water deprivation schedule

for 7d in which water access was given once daily for 90min, 2h after the onset of

the light phase. Food was available ad libitum. After habituation, rats were

assigned to treatment groups: 1) 0.15M LiCl, IP (n=8); 2) 0.04μg sCT, intra-VTA

(n=9); 3) 0.4μg sCT, intra-VTA (n=6). The vehicle for IP injection was 1ml/kg

0.9% NaCl, while the vehicle for VTA injections was 100nl aCSF. CTA training

consisted of two training days. On each training day during the normal 90min

fluid access period, rats were given access to two burettes containing the same

flavor of Kool-Aid [either cherry or grape, flavors equally preferred (Lucas and

Sclafani, 1996); 0.14oz Kool-Aid mix, 10g saccharin, 3L water]. Immediately after

flavor exposure, each rat received IP or intra-VTA injection as described above.

Drug treatments were counterbalanced across training days and for paired flavor.

Each training day was succeeded by a non-injection day where water was

available during the 90min fluid access period. Two days after the second

training day, animals were presented with both flavors (one flavor per burette)

during the 90min fluid access period. Fluid intake was recorded after 45min, at

which point the side of flavor presentation was switched to avoid side preference.

Fluid intake was recorded again at 90min.

Effect of VTA AmyR activation on glycemic control: Rats (n=7) were deprived of

food overnight (16h) to ensure an empty gastrointestinal tract. Oral glucose

tolerance testing was conducted mid-light phase; food remained unavailable for

the duration of the test. First, a small sample of tail tip blood was obtained from

each rat and a standard glucometer (Accucheck, Roche Diagnostics) was used

to measure baseline blood glucose (BG) values (time = -20min). Immediately

after baseline BG was assessed, each rat received a unilateral intra-VTA

injection of sCT (0, 0.004, 0.04, or 0.4μg in 100nl aCSF). Twenty minutes after

VTA injection (time = 0min), BG was measured again, and each rat received a

glucose load via oral gavage (25%; 2g/kg). Subsequent BG readings were taken

at 20, 40, 60, and 120min post-glucose load. A minimum of 72h separated test

sessions.

Meal pattern effects of VTA AmyR activation on high-fat diet (HFD) intake: Rats

(n=10) were housed in a custom-built automated feedometer system. Each

feedometer consisted of a hanging wire mesh cage with a small access hole

leading to a food cup resting on an electronic scale. Food cup weights were

recorded by computer software (LabView) every 10s for 24h. Rats were

maintained on HFD (60% kcal from fat; Research Diets) prior to and throughout

testing. Just before dark phase onset, each rat received a unilateral VTA injection

of sCT (0.01, 0.04, 0.4μg) or vehicle (100nl aCSF) before receiving access to

preweighed HFD. Food intake and body weight change were monitored at 24h

post-injection. For meal pattern analyses, a meal was defined as at least 0.25g of

food intake and a minimum of 10min between feeding bouts (Mietlicki-Baase et

al, 2013a; Mietlicki-Baase et al, 2013b).

Development of AAV for CTR knockdown: To create a short hairpin RNA

(shRNA)-AAV targeting CTR-A/B, we screened commercially available potential

sequences for reduced CTR expression (TG712173; Origene) in an in vitro

model. Using a commercially available rat immortalized hypothalamic neuronal

cell line (R-19; Cedarland Labs), a plasmid designed to over-express CTR

(NM_053816; Origene) was transiently transfected into R-19 cells either alone or

in combination with a plasmid sequence to reduce CTR expression. The most

robust knockdown of overexpressed CTR was obtained using the following

sequence: [GAT CGT CCA GTT CTT CAG GCT CCT ACC AAT CTC ATC AAG

AGT GAG ATT GGT AGG AGC CTG AAG AAC TGG ATT TTT T] targeting CTR-

A/B. This sequence was packaged into an AAV vector (serotype I; titer = 5.3e12)

designed to express shRNA-CTR-A/B driven by the cytomegalovirus (CMV)

promoter, as well as enhanced green fluorescent protein (eGFP) for visualization.

An eGFP-expressing empty vector AAV (titer = 5.0e11) was used as a control.

To confirm the ability of the AAV to reduce CTR expression in vivo, chow-

maintained rats received bilateral, intra-VTA AAV infusion as described above

(CTR-KD, n=5; CTR-Ctrl, n=4). Three weeks after viral injection, rats were killed,

and brains were rapidly removed from the crania and flash-frozen in -70°C

isopentane. To evaluate AAV knockdown efficacy via quantitative real-time PCR

(qPCR), 1mm micropunches of VTA tissue were collected bilaterally from each

brain. qPCR was performed as described previously (Mietlicki-Baase et al,

2013b). CTR-A and -B levels were quantified, with rat GapDH (VIC-MGB) serving

as an internal control. PCR reactions for CTR were completed with Taqman gene

expression kits (CTR-A: Rn01526770_m1; CTR-B: Rn01526768_m1; GapDH:

Rn01775763_g1) and PCR reagents (Applied Biosystems). Analysis of samples

was conducted using an Eppendorf Mastercycler ep realplex2. The comparative

threshold cycle method (Bence et al, 2006) was used for relative mRNA

expression calculations. Correct placement of AAV injections was verified by

collecting a VTA-containing coronal section (30μm) from each brain and

visualizing eGFP (Fig. 6c).

Effect of VTA CTR knockdown on acute response to IP sCT: Chow-maintained

rats were given bilateral intra-VTA AAV infusion (CTR-KD, n=9; CTR-Ctrl, n=7).

After surgery, approximately 3 weeks elapsed before initiating behavioral testing

to ensure full viral expression. Each rat received an IP injection of sCT (2μg/kg)

or vehicle (1ml/kg sterile 0.9% NaCl) just before the onset of the dark phase.

Chow intake (1, 3, 6, 24h) and 24h body weight gain were measured.

Long-term effect of VTA CTR knockdown on energy balance: Separate rats were

maintained on chow or on HFD for 1 week prior to surgical intra-VTA injection of

AAV as described above. Within each dietary condition, half of the rats received

VTA injection of AAV to knock down expression of CTR (CTR-KD; chow, n=7;

HFD, n=7) and the remaining animals were injected with the AAV control vector

(CTR-Ctrl; chow, n=7; HFD, n=7). Food intake (accounting for spillage) and body

weight were measured prior to surgery to make baseline comparisons between

treatment groups. Beginning post-surgery Day 1, measurements continued every

48h for a total of 30 days (Days 1-31 post-viral injection). To make accurate

intake comparisons between dietary conditions, all food intake measurements

were converted to kilocalories for analyses (chow = 3.36kcal/g; HFD =

5.24kcal/g). Two rats were excluded from behavioral analyses due to technical

issues with daily measurements (1 CTR-KD/HFD, 1 CTR-Ctrl/Chow). After the

conclusion of the behavioral study, rats were killed, and brains were rapidly

removed and flash-frozen in -70°C isopentane. VTA CTR-A knockdown was

confirmed via qPCR, with rat GapDH (VIC-MGB) as the internal control.

Statistical analyses: The α level was set at p<0.05 for all studies. Statistical

analyses were performed using Statistica (StatSoft), GraphPad 5.0 (Prism Inc.),

and Microsoft Excel. For FSCV studies, percent change in peak DA and post-

session chow intake were compared using unpaired t-tests. For oral glucose

tolerance testing and for all feeding studies, binned data were analyzed using

separate mixed-design ANOVAs to account for the within-subjects experimental

design while assessing between-subjects effects (e.g., drug treatment, AAV

condition, dietary condition). Statistically significant effects were probed using

Student-Newman-Keuls post hoc analyses, with the exception of the experiment

testing the ability of IP sCT to promote negative energy balance in CTR-KD rats,

in which Bonferroni post hoc comparisons were used to be conservative in the

interpretation of the acute behavioral effects of the novel AAV. Finally,

knockdown of CTR expression achieved with the novel AAV was analyzed by t-

test for in vitro and preliminary in vivo testing, and by ANOVA with Student-

Newman-Keuls post hoc analyses for the chronic dietary experiment.

Results

The calcitonin receptor (CTR), the core signaling component of the AmyR

complex, is predominantly expressed on VTA DA neurons

Given the potential involvement of DA signaling in mediating the energy

balance effects of VTA AmyR signaling, we hypothesized that AmyR may be

expressed on VTA DA neurons. The AmyR is a complex consisting of a dimer of

the CTR and a receptor activity modifying protein (Christopoulos et al, 1999;

Young, 2005). Therefore, to analyze expression and colocalization of the AmyR

on VTA DA neurons, we performed double-label immunohistochemistry for CTR

and the DA marker tyrosine hydroxylase (TH) in the VTA. As shown in Fig. 1,

62.6 ± 6.1% of VTA CTR-expressing neurons co-express TH; of the TH-positive

neurons within the VTA, 11.8 ± 3.6% co-express the CTR. This suggests that

VTA AmyR activation may directly influence DA neurotransmission, pointing

toward a potential mechanism by which VTA amylin could reduce food intake.

Intra-VTA AmyR activation attenuates food-evoked phasic DA signaling in

the NAc core

One of the major afferent targets of VTA DA neurons is the NAc (Fallon

and Moore, 1978), and DA signaling within the core subregion (NAcC) is

important for food intake and reward (Brown et al, 2011; Vucetic and Reyes,

2010). To test whether VTA AmyR activation reduces NAcC DA, we performed

fast-scan cyclic voltammetry (FSCV) to assess changes in food-evoked phasic

DA signaling in the NAcC associated with VTA AmyR activation. Indeed, food

reward stimulates phasic increases in NAcC DA (Cone et al, 2014). Rats that

were food-restricted to 90% of their free-feeding body weight (McCutcheon et al,

2012) received random deliveries of a sucrose pellet, an event that reliably

evoked a phasic increase in NAcC DA (see Fig. 2a-b). The magnitude of DA

evoked during retrieval of sucrose pellets was compared within-session, before

and after unilateral intra-VTA infusion of the AmyR agonist salmon calcitonin

[sCT, 0.04µg; dose chosen from (Mietlicki-Baase et al, 2013b)] or vehicle (100nl

aCSF). Intra-VTA sCT significantly reduced peak NAcC DA evoked during pellet

retrieval compared to vehicle infusion [Fig. 2c; % change from pre-infusion

values, aCSF: 100.3 +/- 5.3%, sCT 73.8 +/- 4.0%; t(7)=4.11, p<0.01]. Intra-VTA

sCT also suppressed 30min post-session home cage chow intake (Fig. 2d;

t(7)=3.25, p<0.05).

NAcC DA receptor activation attenuates the chow intake- and body weight-

suppressive effects of VTA AmyR activation

The reduction in NAcC DA signaling observed in the FSCV studies likely

corresponds with decreased DA receptor activation within the core. This led us to

hypothesize that reduced NAcC DA receptor activation mediates VTA sCT-

induced hypophagia. As both D1 and D2 receptors are present in the NAcC (Lu

et al, 1998), we first established doses of the D1 agonist SKF-81297 (0.1, 0.6,

1.2μg; vehicle, 200nl aCSF) and separately, the D2 agonist quinpirole (1.5, 3,

6μg; vehicle, 200nl aCSF) that are subthreshold for effects on chow intake when

delivered unilaterally to the NAcC (SKF-81297, Fig. S1a-b, F3,15=0.31, p=0.82;

quinpirole, Fig. S1c-d, F3,15=0.19, p=0.90) and 24h body weight gain (SKF-

81297, F3,15=1.00, p=0.42; quinpirole, F3,15=0.71, p=0.56). We also ensured that

the combined NAcC administration of the D1 and D2 agonists (1.2μg SKF-81297

plus 6μg quinpirole) was subthreshold for feeding or body weight effects (Fig.

S1e-f; 24h food: F1,5=0.002, p=0.96; 24h body weight: F1,5=0.16, p=0.71). This

dose combination was then used to determine whether activating NAcC D1/D2

receptors could attenuate the reduction in food intake following VTA AmyR

activation.

Separate chow-maintained rats received an intra-NAcC injection of the

combined D1 and D2 receptor agonists (1.2µg SKF-81297 and 6µg quinpirole;

D1+D2) or vehicle (200nl), followed by an intra-VTA injection of sCT (0.04µg) or

vehicle (100nl). As shown in Fig. 3a, activation of NAcC D1 and D2 receptors

attenuated the reduction of chow intake produced by VTA AmyR activation (main

effect of sCT 1, 3, 6, and 24h, all ANOVAs F1,9≥5.81, p≤0.04; interaction between

D1+D2 and sCT at 24h, F1,9=7.99, p<0.02; planned comparisons between

vehicle/sCT and D1+D2/sCT at 24h, p<0.05). NAcC DA receptor activation also

inhibited the suppression of body weight produced by intra-VTA sCT (Fig. 3b;

interaction between D1+D2 and sCT; F1,9=13.01, p<0.01). These data indicate

that decreased NAcC D1/D2 receptor signaling mediates the intake- and body

weight-suppressive effects of VTA AmyR activation.

VTA AmyR activation does not produce conditioned taste avoidance or

alter blood glucose levels

Previous research from our laboratory showed that AmyR activation in the

VTA did not induce pica (Mietlicki-Baase et al, 2013b), a measure of

nausea/malaise in rodents (Andrews and Horn, 2006), suggesting that the intake-

suppressive effects of VTA amylin are not caused by malaise. To rule out the

possibility that VTA AmyR activation suppresses feeding by producing a

conditioned avoidance response to food, rats underwent a two-bottle conditioned

taste avoidance (CTA) experiment (Garcia et al, 1955; Kanoski et al, 2012). Two

groups of rats received intra-VTA injection of aCSF (CS-; 100nl) or sCT (CS+),

with one group receiving sCT at a dose effective at reducing food intake in the

VTA but subthreshold for effect when given ICV (0.04µg), and the other group

receiving a higher dose of sCT that reduces food intake when delivered ICV

(0.4µg). A third group of rats received IP injection of saline (CS-; 1ml/kg) or LiCl

(CS+; 0.15M), a substance known to elicit nausea/malaise in rodents (Kanoski et

al, 2012; Spector et al, 1988), serving as a positive control for the CTA

procedure. As shown in Fig. 3c, LiCl-treated rats consumed significantly less of

the (CS+)-paired flavor than the (CS-)-paired flavor during the test, indicating the

presence of a CTA (interaction between drug treatment and flavor, F2,22=10.22,

p<0.001; within LiCl rats, (CS-)-paired flavor versus (CS+)-paired flavor, p<0.05).

However, VTA AmyR activation did not produce a CTA, as consumption of (CS-)-

paired and (CS+)-paired flavors did not differ (p>0.05 for both sCT doses). These

findings provide further evidence that the anorexia produced by VTA AmyR

activation is independent of nausea/malaise and/or a conditioned avoidance to

food.

As systemic administration of AmyR agonists lowers blood glucose levels

(Scherbaum, 1998) via action within the CNS [see (Hayes et al, 2014; Lutz,

2005) for review], it was important to determine whether VTA AmyR activation

influences glycemia, an effect that could indirectly influence feeding. Accordingly,

the effects of intra-VTA injections of sCT (0.004, 0.04, or 0.4µg; vehicle, 100nl)

on blood glucose were assessed in an oral glucose tolerance test. Intra-VTA sCT

did not alter blood glucose concentrations at any time (Fig. 3d; all ANOVAs

F3,18≤2.27, p≥0.12), suggesting that the intake-suppressive effects of VTA AmyR

signaling are separable from glycemic changes, and that other AmyR-expressing

CNS nuclei mediate the glycemic effects of amylin and amylin receptor agonists.

VTA AmyR activation decreases intake of a palatable high-fat food by

reducing meal size

Previous data show that intra-VTA sCT injection reduces intake of a

palatable sucrose solution (Mietlicki-Baase et al, 2013b), but the effects of VTA

AmyR activation on palatable high-fat food intake are unknown. To test whether

acute VTA AmyR activation reduces intake of a palatable HFD (60% kcal from

fat), rats were given intra-VTA injections of sCT (0.01, 0.04, 0.4µg) or vehicle

(100nl) and HFD intake, meal patterns, and body weight were monitored for 24h

post-injection. Fig. 4a shows a dose-dependent suppression of cumulative HFD

intake by sCT (significant effects at 0.5, 1, 1.5, 2, 4, 12, 24h; ANOVAs F3,27≥3.00,

p<0.05). Meal pattern analyses reveal main effects of VTA sCT on meal size at

all times (Fig. 4b; all ANOVAs F3,27≥3.02, p<0.05), with fewer effects on meal

number (Fig. 4c; significant effects at 1, 1.5, 2, 12, and 24h; ANOVAs F3,27≥3.71,

p≤0.02). The most robust reductions in meal size were observed with 0.4µg sCT,

although all doses significantly reduced meal size compared to vehicle treatment

at 12h and 24h (p<0.05). Consistent with this finding, average meal duration over

the 24h test period was also reduced by all doses of sCT (in minutes, mean ±

SEM: vehicle, 15.88 ± 1.87; 0.01µg sCT, 8.79 ± 1.70; 0.04µg sCT, 11.59 ± 1.31;

0.4µg sCT, 8.05 ± 1.62; F3,27=7.34, p<0.001). However, only the highest dose of

sCT (0.4µg) reliably suppressed meal number (0.4µg versus vehicle, p<0.05 at

all listed times) or latency to consume the first meal (in minutes, mean ± SEM:

vehicle, 5.2 ± 1.79; 0.01µg sCT, 58.53 ± 45.26; 0.04µg sCT, 32.28 ± 16.69;

0.4µg sCT, 418.17 ± 192.95; F3,27=4.03, p<0.02; vehicle versus 0.4µg sCT,

p<0.05). In addition, the highest dose of sCT reduced 24h BW gain (Fig. 4d;

F3,27=8.11, p<0.001). Together, these data indicate that the primary behavioral

mechanism driving the reduction in HFD intake by VTA AmyR activation is

suppression of meal size, rather than decreased meal number.

NAcC DA receptor agonism attenuates the intake- and body weight-

suppressive effects of VTA AmyR activation in HFD-fed rats

Current data show that the reductions in chow intake produced by intra-

VTA sCT administration are attenuated by NAcC pretreatment with DA receptor

agonists. To assess whether a similar DAergic mechanism mediates the

suppression in HFD intake produced by VTA AmyR activation, we first confirmed

that intra-NAcC administration of the highest doses of SKF-81297 (1.2µg) and

quinpirole (6µg) tested in the chow studies did not alter intake or body weight

gain in HFD-fed rats when administered either separately (Fig. S2a-b; food:

F2,22=0.89, p=0.42; body weight: F2,22=1.29, p=0.29) or in combination (Fig. S2c-

d; food: F1,7=0.15, p=0.71; body weight: F1,7=0.06, p=0.81). Next, we tested

whether intra-NAcC injection of a combination of SKF-81297 plus quinpirole

(D1+D2; 1.2µg and 6µg, respectively; vehicle, 200nl) would attenuate the

hypophagia and body weight loss produced by intra-VTA sCT (0.04µg; vehicle,

100nl) in HFD-fed rats. Fig. 5a shows that VTA sCT potently suppressed HFD

intake (main effect of sCT at 3, 6, 24h; all ANOVAs F1,11≥5.78, p≤0.04); however,

intra-NAcC pretreatment with the D1+D2 agonists significantly attenuated these

effects (interaction between D1+D2/sCT at all times; all ANOVAs F1,11≥4.96,

p<0.05; vehicle/sCT versus D1+D2/sCT, p<0.05 at 3, 6, 24h). Similar results

were observed for 24h body weight gain, as intra-NAcC D1+D2 treatment

attenuated the weight loss produced by intra-VTA sCT (Fig. 5b; main effect of

sCT; F1,11=6.33, p=0.03; interaction between D1+D2/sCT, F1,11=7.18, p=0.02;

vehicle/sCT versus D1+D2/sCT, p<0.05). These results indicate that the novel

NAcC DA mechanism identified in the earlier chow study also mediates

suppression of palatable food intake after VTA AmyR activation. NAcC D1/D2

activation attenuated the suppression of HFD intake by VTA sCT delivery at

earlier times than observed in the chow study, suggesting the intriguing

possibility that the changes in DA signaling induced by VTA AmyR signaling may

have more impact on the intake of palatable compared to bland foods.

A novel AAV decreases CTR expression in vitro and in vivo

To evaluate the effects of chronically reduced VTA AmyR signaling on

energy balance, we developed an AAV to site-specifically knockdown expression

of CTR-A/B. We selected a potential sequence for shRNA interference-mediated

CTR knockdown and tested its ability to reduce CTR expression in vitro in an

immortalized rat neuronal cell line. Transient transfection of neuronal R-19 cells

overexpressing CTR-A, the more highly expressed CTR subtype within the CNS

(Young, 2005) and VTA (Mietlicki-Baase et al, 2013b), with the shRNA-CTR

sequence produced a dramatic reduction (>95%) in CTR-A mRNA expression

(Fig. 6a; t(2)=14.63, p<0.01), indicating the potential utility of this sequence for in

vivo CTR knockdown. After packaging the sequence into an AAV (serotype I) co-

expressing eGFP, we tested the efficacy of the AAV-shRNA in vivo by

administering the AAV-CTR knockdown (CTR-KD) or a control AAV expressing

eGFP (CTR-Ctrl) bilaterally into the VTA of rats. Consistent with the in vitro

results, VTA CTR-KD produced robust (>90%) knockdown of CTR-A mRNA in

the VTA compared to CTR-Ctrl treatment (Fig. 6b; t(7)=24.30, p<0.0001). VTA

CTR-B expression was below threshold for significant detection in both CTR-Ctrl

and CTR-KD rats, consistent with previous data showing higher expression of

CTR-A compared to CTR-B within the VTA (Mietlicki-Baase et al, 2013b).

Knockdown of VTA AmyR by AAV-CTR-KD blunts the chow intake- and

body weight-suppressive effects of a peripheral AmyR agonist

Peripheral sCT injection activates VTA AmyR to reduce chow intake and

body weight (Mietlicki-Baase et al, 2013b). To confirm the behavioral relevance

of VTA CTR-KD, chow-maintained CTR-KD and CTR-Ctrl rats received IP

injection of sCT (2μg/kg) or vehicle (1ml/kg 0.9% NaCl) and subsequent intake

and body weight change were measured. In CTR-Ctrl rats, IP sCT potently

suppressed food intake (Fig. 6d; main effect of sCT at all times, all ANOVAs

F1,14≥9.58, p≤0.01, planned comparisons between CTR-Ctrl/vehicle and CTR-

Ctrl/sCT, p<0.05 at 3, 6, 24h) and 24h body weight gain (Fig. 6e; main effect of

sCT, F1,14=8.09, p=0.01; planned comparison between CTR-Ctrl/vehicle and

CTR-Ctrl/sCT, p<0.05). In contrast, systemic sCT was ineffective at reducing

food intake or body weight gain in VTA CTR-KD rats (all CTR-KD/vehicle versus

CTR-KD/sCT planned comparisons, p>0.05). AAV condition alone had no effect

on feeding or body weight (CTR-Ctrl/vehicle versus CTR-KD/vehicle, all p>0.05)

in this acute test. These data provide critical evidence supporting the VTA AmyR

as a site-of-action for peripheral amylin agonists and show that reduction of CTR,

and thus functional AmyR, in the VTA has important acute behavioral

consequences.

Endogenous VTA AmyR signaling is required for the long-term control of

energy balance

To assess the relevance of chronic endogenous VTA amylin signaling for

energy balance control, rats were maintained on chow or on HFD; within each

dietary condition, rats received either bilateral VTA CTR-KD or CTR-Ctrl AAV

injections, resulting in a total of 4 experimental groups. Body weight was

measured 24h prior to viral injection to confirm that baseline values did not differ

between groups (Fig. S3a; no effect of diet or planned AAV condition, no

interaction; all F1,22≤3.38, p>0.05). Food intake was also monitored for 48h prior

to viral injection (-72h to -24h); although HFD-maintained rats consumed more

kilocalories than chow-fed rats (Fig. S3b; main effect of diet, F1,22=29.21,

p<0.0001), no differences were observed within dietary condition in rats that were

to be assigned to each AAV condition (planned comparisons between CTR-

Ctrl/Chow versus CTR-KD/Chow and CTR-Ctrl/HFD versus CTR-KD/HFD,

p>0.05).

Beginning the day after viral injection, food intake and body weight were

recorded every 48h to evaluate the long-term energy balance effects of

chronically reduced VTA AmyR signaling in each dietary condition. In chow-fed

rats, no differences in total body weight gain (Fig. 7c; main effects of diet and of

AAV, all F1,22≥4.93, p≤0.04; planned comparison between CTR-KD/Chow versus

CTR-Ctrl/Chow, p>0.05) or total energy intake (Fig. 7d; main effects of diet and

of AAV, all F1,22≥8.31, p≤0.01; planned comparison between CTR-KD/Chow

versus CTR-Ctrl/Chow, p>0.05) were observed. Regardless of viral condition,

HFD-maintained rats gained more body weight and consumed more kilocalories

over the course of the study than did chow-fed rats. However, HFD-fed CTR-KD

rats showed an exacerbated hyperphagia and gained more total body weight

than did HFD-fed CTR-Ctrl rats (planned comparisons of CTR-KD/HFD versus

CTR-Ctrl/HFD for total energy intake and total body weight gain, all p<0.05). The

increased total kilocalorie intake and body weight gain results of HFD CTR-KD

rats were recapitulated in 48h measurements for the duration of the study (Fig.

7a-b; kcal: main effect of AAV, main effect of diet, and/or diet x AAV interaction

for all bins except Days 1-3, all significant ANOVAs F1,22≥4.44, p<0.05; body

weight: main effect of AAV, main effect of diet, and/or diet x AAV interaction for

all days, all significant ANOVAs F1,22≥4.73, p<0.05). Effective AAV-mediated

knockdown of VTA CTR-A expression was confirmed by qPCR at the end of the

study (Fig. 7e; main effect of AAV; F1,24=16.93, p<0.001; planned comparisons

between CTR-Ctrl/Chow versus CTR-KD/Chow and CTR-Ctrl/HFD versus CTR-

KD/HFD, p<0.05; no main effect of diet or diet x AAV interaction, all F1,24≤2.38,

p>0.05). These data demonstrate a role for endogenous VTA amylin signaling in

the long-term control of energy balance, particularly in rats fed a palatable HFD.

Discussion

As amylin-based pharmacotherapies gain attention as potential treatments

for obesity (Lutz, 2013; Roth et al, 2009; Sadry and Drucker, 2013), it is clear that

a deeper comprehension of the distributed CNS effects of AmyR signaling is

urgently needed. The existence of non-AP CNS nuclei that respond to amylin

and amylin analogs, and the potential role of these sites in the regulation of

feeding, has been particularly under-investigated. Only recently has the VTA

been recognized as an important nucleus mediating the energy balance effects

of amylin (Mietlicki-Baase et al, 2013b). The current data provide novel insight

into a neuroanatomical mechanism mediating the energy balance effects of VTA

AmyR activation, demonstrating that acute VTA AmyR activation produces

hypophagia and weight loss by reducing DA signaling from the VTA to the NAcC.

Additionally, we provide the first evidence for site-specific, long-term control of

energy balance by endogenous amylin, as AAV-mediated knockdown of VTA

AmyR resulted in hyperphagia and enhanced body weight gain in HFD-fed rats.

The fact that VTA AmyR activation reduces palatable food intake and

motivation to obtain a palatable food (Mietlicki-Baase et al, 2013b), together with

the established role of VTA DA signaling in the regulation of feeding and reward,

highlighted the possible involvement of DA in the energy balance effects of VTA

amylin. We focused our attention on DA signaling from the VTA to the NAcC, a

pathway known to influence food intake and motivated behavior (Cone et al,

2014; Roitman et al, 2004; Vucetic and Reyes, 2010; Wanat et al, 2010).

Several feeding-related peptides modulate VTA to NAc DA neurotransmission,

including leptin (Krugel et al, 2003) and ghrelin (Cone et al, 2014; Skibicka et al,

2013). Here, we found that VTA AmyR activation reduces NAcC D1/D2 receptor

activation to decrease feeding. Our FSCV data suggest that this is likely due to

VTA amylin-induced suppression of DA release in the NAcC. Interestingly, the

ability of NAcC D1/D2 receptor activation to attenuate the energy balance effects

of VTA sCT was observed both in rats that were fed chow or a palatable HFD,

highlighting a common DA-mediated mechanism by which VTA AmyR activation

reduces intake of both bland and palatable foods.

VTA AmyR activation with sCT produces acute reductions in ad libitum

intake of a palatable 15% sucrose solution (Mietlicki-Baase et al, 2013b). Here,

we show that intra-VTA sCT also suppresses HFD intake, indicating a broader

role for VTA amylin signaling in the control of palatable food intake. Interestingly,

VTA AmyR-mediated suppression of sucrose intake and of HFD intake occurs at

a dose of sCT (0.01μg) that is ineffective at significantly reducing intake of chow

(Mietlicki-Baase et al, 2013b). Although VTA AmyR activation may more potently

suppress intake of palatable foods compared to less palatable chow, intra-VTA

sCT-induced reductions in chow and in HFD intake are both mediated primarily

by reductions in meal size rather than meal number, consistent with systemic

amylin’s satiating properties (Bello et al, 2008; Lutz, 2005; Lutz et al, 1995b).

Along with the current findings indicating a role for NAcC DA signaling in VTA

amylin-mediated control of feeding, these data suggest that VTA AmyR activation

may engage common physiological (DA) and behavioral (meal size) mechanisms

to produce the suppression of intake of both palatable and bland foods by VTA

AmyR activation. These data also raise the point that VTA amylin signaling may

exert more robust anorectic effects on palatable ingesta. A similar possibility is

that amylin may have stronger anorectic effects with regard to non-

homeostatic/hedonic feeding. A previous report from our laboratory showed that

VTA amylin receptor activation can reduce the motivation to obtain a palatable

food, as measured by operant responding for sucrose pellets in a progressive

ratio task (Mietlicki-Baase et al, 2013b). Although operant responding for HFD

has not been evaluated, it is possible that issues of food reward/motivation and

not simply palatability may also impact the ability of VTA amylin to suppress HFD

intake.

VTA DA neurons project to several other feeding-relevant CNS sites

besides the NAcC, including the NAc shell (Fallon and Moore, 1978), the

prefrontal cortex (Berger et al, 1976), the central nucleus of the amygdala (Fallon

et al, 1978; Mogenson and Wu, 1982), and the hippocampus (Scatton et al,

1980). The ability of VTA AmyR signaling to modulate DA signaling to these

other sites remains untested, but represents an extremely important extension of

the current results. For example, dopamine signaling in the NAc shell is relevant

for many aspects of feeding and food reward (Baldo and Kelley, 2007; Bassareo

and Di Chiara, 1999; Beyene et al, 2010). The dopaminergic projection from the

VTA to the shell may also contribute to the intake-suppressive effects of VTA

amylin receptor activation. Indeed, elucidating the impact of VTA amylin receptor

activation on dopamine release in the shell would be an intriguing first step

toward addressing a possible role of the VTA-NAc shell connection in the ability

of VTA amylin to promote negative energy balance. Additionally, as amylin

binding is observed in many CNS nuclei (Christopoulos et al, 1995; Paxinos et al,

2004; Sexton et al, 1994), it is likely that sites other than the VTA contribute

directly or indirectly to the effects of amylin not only for feeding but for other

physiological effects such as glycemic control. Our current data support this

notion as VTA amylin receptor activation with sCT did not change blood glucose

levels in an oral glucose tolerance test, suggesting that other CNS site(s)

mediate the ability of amylin to regulate glycemia (Hayes et al, 2014).

In addition, although VTA CTR are highly expressed on DA neurons, it is

clear that a percentage of VTA CTR-expressing cells do not co-localize with TH-

positive neurons. The VTA contains sizable non-DA neuronal populations (e.g.,

glutamate, GABA) (Dobi et al, 2010), and testing the potential co-expression of

the AmyR with different neurotransmitter phenotypes may provide clues as to

other mechanisms involved in the acute energy balance effects of VTA amylin

signaling. Although the energy balance effects of VTA sCT are almost completely

reversed by NAc core administration of dopamine receptor agonists in HFD-

maintained rats, sCT-induced hypophagia and body weight loss are attenuated

but not reversed by intra-core D1+D2 receptor agonism in chow-fed rats. This

suggests that the ability of VTA amylin receptor activation to promote negative

energy balance in chow-fed animals may also involve dopamine-independent

mechanisms or processing of dopamine signaling in other CNS nuclei. In

contrast, in HFD-maintained animals the hypophagic effects of VTA amylin

receptor signaling appear to rely more heavily on dopamine signaling in the NAc

core. Thus, it is certainly possible that VTA amylin receptor activation may

engage additional neurotransmitter systems other than dopamine to partially

mediate the anorectic effects of VTA amylin receptor activation. At present, with

the absence of widely accepted and validated antibodies for these other

neurotransmitter systems, future studies using transgenic fluorescent-reporter

mice and neuropharmacological analyses will be needed to further such

explorations.

There is a paucity of data in the literature examining the role of the amylin

system in the chronic regulation of feeding and body weight. The limited number

of energy balance studies in amylin knockout mice demonstrate only minimal

phenotypic alterations, with no effect on food intake and minimal or no increase

in body weight (Dacquin et al, 2004; Gebre-Medhin et al, 1998; Lutz, 2005;

Olsson et al, 2012). In lean chow-fed rats, chronic systemic administration of the

amylin receptor antagonist AC187 has no effect on feeding or body weight

(Grabler and Lutz, 2004), although in contrast, chronic central AC187

administration increased food intake and adiposity (Rushing et al, 2001).

Together, these previous reports indicate that a centrally-targeted manipulation

of the amylin system induces more potent energy balance effects. Here, we

chose a novel and deductive approach to examine the long-term role of

endogenous central amylin signaling in control of energy balance by site-

specifically and chronically knocking down AmyR signaling in the VTA. Data

show that VTA AmyR can be directly activated by systemic amylin administration,

and furthermore, that endogenous amylin signaling in the VTA is involved in the

control of energy balance. Interestingly, VTA CTR-KD-mediated increases in

feeding and weight gain were observed only in rats maintained on HFD; chow-

fed CTR-KD animals were nearly identical to chow-fed AAV controls in their

energy intake and body weight. The earlier finding that chow intake was

increased by chronic central AmyR blockade (Rushing et al, 2001) contrasts with

these VTA CTR-KD data. However, the previous study used icv administration of

the AmyR antagonist (Rushing et al, 2001) as opposed to the site-specific

knockdown of AmyR signaling in the current experiment. This emphasizes the

intriguing possibility that other CNS sites, likely including the AP, contribute more

strongly to the chow intake-suppressive effects of amylin, whereas the VTA has

more robust effects on more palatable food. Importantly, the newly developed

CTR-AAV will allow for the investigation of long-term energy balance control by

amylin signaling in other isolated CNS sites, such as the AP, providing the

opportunity to gain a clearer understanding of the site- and diet-specific effects of

AmyR signaling.

The discovery that VTA amylin-induced hypophagia and body weight loss

is mediated by decreased NAcC DA signaling is a critical first step in

understanding the downstream neuroanatomical targets and mechanisms

engaged by VTA AmyR activation. Furthermore, the long-term control of

palatable food intake by VTA amylin, identified in our CTR-KD studies,

represents a new technology for CNS amylin research. Collectively, these results

provide evidence for novel behavioral and neuroanatomical mechanisms

mediating the regulation of energy balance by VTA AmyR signaling. As VTA

amylin signaling exerts both acute and chronic control over body weight and

palatable food intake, the current findings add support to the notion that amylin-

based compounds may be effective pharmacotherapies for the treatment of

obesity.

Funding and Disclosure

The authors declare no conflict of interest.

Acknowledgments

We thank Thomas Lutz for guidance in CTR IHC, Heath Schmidt for technical

advice on D1/D2 agonist studies, and the University of Pennsylvania Viral Core

for their expertise in developing the CTR shRNA-AAV. Valuable technical

assistance was provided by Marco Guevara, Kieran Koch-Laskowski, Daniela

Mendez, Orianne Montaubin, Chan Nguyen, Amit Pujari, and Christopher Turner.

This research was supported by NIH-DK097954 (E.G.M.-B.), DA025634

(M.F.R.), and DK096139 (M.R.H.).

Supplementary information is available at the Neuropsychopharmacology

website.

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Figure Legends

Figure 1. Amylin receptors are expressed on VTA DA neurons. Representative

high-magnification (20x) images from a coronal section containing the VTA show

TH alone in red (a), CTR alone in green (b), and colocalized neurons expressing

both TH and CTR (c). The dotted box in (c) is further magnified in (d).

Semiquantification of VTA neurons (n=6 rats) demonstrated that approximately

63% of VTA CTR-expressing neurons also express TH, whereas approximately

12% of TH-positive neurons express the CTR (e). Data are expressed as mean ±

SEM.

Figure 2. VTA AmyR activation decreases phasic DA in the NAcC evoked by

food reward. (a) Top: Average colorplot (10 trials/rat) depicting current changes

(color) as a function of electrode potential (y-axis) in the 10 s before and after (x-

axis) pellet retrieval. DA [identified by its oxidation (~+0.6 V; green) and reduction

(~-0.2 V; light yellow) features] was transiently evoked during pellet retrieval,

prior to vehicle infusion. Middle: Average colorplot after VTA vehicle infusion.

Bottom: Average DA concentration aligned to retrieval before and after VTA

aCSF extracted from individual colorplots using chemometric analysis (Heien et

al, 2004). (b) Average colorplots prior to (top) and after VTA sCT (middle; 10

trials/rat). Average DA concentration aligned to pellet retrieval before and after

VTA administration of the AmyR agonist sCT (bottom). (c) Percent change in

NAcC peak DA evoked during pellet retrieval, following VTA vehicle or sCT. (d)

Thirty min post-session chow intake following intra-VTA vehicle or sCT. Bar data

are mean ± SEM; n=9; *, p<0.05; **, p<0.01.

Figure 3. Activation of NAcC DA receptors with a combination of D1 and D2

receptor agonists attenuates the energy balance effects of VTA amylin receptor

activation in chow-fed rats. Direct intra-NAcC injection of a combination of SKF-

81297 (D1 agonist) and quinpirole (D2 agonist) attenuates the reduction in chow

intake (a) and 24h body weight gain (b) produced by intra-VTA injection of sCT

(n=10). *, different from vehicle/vehicle (p<0.05); Ŧ, different from vehicle/sCT

(p<0.05). The key next to (b) also applies to (a). (c) Although IP injection of the

known nausea/malaise-inducing agent LiCl produces conditioned taste

avoidance (CTA) in rats, intra-VTA administration of sCT does not cause rats to

exhibit CTA to the drug-paired flavor (n=6-9 per stimulus condition). ^, different

from vehicle-paired flavor (p<0.05). (d) VTA AmyR activation does not alter blood

glucose in an oral glucose tolerance test (n=7). All data shown as mean ± SEM.

See also Figure S1.

Figure 4. VTA AmyR activation suppresses intake of a palatable HFD primarily

by decreasing meal size. Intra-VTA injection of sCT (n=10) reduced cumulative

HFD consumption (a) and meal size (b), but only the highest dose of sCT

decreased meal number (c). Body weight gain over the 24h test was also

reduced by sCT (d). *, main effect of sCT (p<0.05); within a time bin, bars with

different letters are significantly different from each other (p<0.05). The key next

to (d) applies to all panels. All data shown as mean ± SEM.

Figure 5. Intra-NAcC administration of a combination of D1 and D2 receptor

agonists blocks the energy balance effects of VTA amylin receptor activation in

rats maintained on palatable high-fat diet. Administration of SKF-81297 (D1

receptor agonist) plus quinpirole (D2 receptor agonist) directly to the NAcC

blocks the suppression of high-fat diet intake (a) and 24h body weight gain (b)

after intra-VTA sCT administration (n=12). *, different from vehicle/vehicle

(p<0.05); Ŧ, different from vehicle/sCT (p<0.05). The key next to (b) applies to

both panels. All data shown as mean ± SEM. See also Figure S2.

Figure 6. A novel AAV-shRNA targeting the calcitonin receptor (CTR) reduces

CTR expression in vitro and in vivo. (a) Transient transfection of immortalized

neuronal R-19 cells overexpressing CTR-A with a plasmid expressing the

sequence targeting CTR mRNA decreased CTR expression (n=4). (b) Injection of

the CTR knockdown AAV (CTR-KD) into the VTA of rats robustly reduced VTA

CTR mRNA levels compared to rats given intra-VTA injection of an empty vector

control (CTR-Ctrl; n=4-5 per AAV condition). Representative VTA AAV-induced

eGFP expression for each viral condition is shown in (c). Although an IP injection

of sCT reliably decreases chow intake and 24h body weight gain in VTA CTR-

Ctrl rats, the same dose of sCT has no significant effect on feeding or weight

change in VTA CTR-KD animals (d, e; n=7-9 per AAV condition). ^, different from

CTR-Ctrl (p<0.05); *, different from vehicle control within dietary condition

(p<0.05). All data shown as mean ± SEM.

Figure 7. Endogenous amylin activates the VTA to provide long-term control of

energy balance. Rats maintained on high-fat diet (HFD) or chow received intra-

VTA injection of the AAV to knock down CTR expression (CTR-KD) or the control

virus (CTR-Ctrl), and subsequent energy intake and body weight were monitored

for 1 month post-AAV injection (n=6-7 per AAV/dietary condition). (a) HFD-fed

VTA CTR-KD rats gained more body weight than any other treatment group; total

body weight gain over the course of the experiment is shown in (c). The

enhanced body weight gain of HFD-fed CTR-KD rats is explained by

hyperphagia, as rats in this condition consume more kilocalories than animals in

any other treatment group both in 48h measurements (b) and over the course of

the study (d). VTA knockdown of CTR-A expression was confirmed via qPCR (e).

For (a) and (b), *, different from CTR-Ctrl/Chow (p<0.05); ^, different from CTR-

Ctrl/HFD (p<0.05). The key in (a) also applies to (b). For (c), (d), and (e), bars

with different letters are significantly different from each other (p<0.05); the key

below (e) also applies to (c) and (d). All data shown as mean ± SEM. See also

Figure S3.

Supplemental Figures and Legends

Figure S1. Twenty-four hour chow intake and body weight gain are not

significantly altered by intra-NAcC administration of the D1 receptor agonist SKF-

81297 (a, b; n=6), the D2 receptor agonist quinpirole (c, d; n=6), or a combination

of SKF-81297 plus quinpirole (e, f; n=6). All data shown as mean ± SEM.

Figure S2. Twenty-four hour high-fat diet consumption and body weight gain are

not changed by NAcC injection of the D1 receptor agonist SKF-81297, the D2

receptor agonist quinpirole (a, b), or a combination of SKF-81297 plus quinpirole

(c, d). All data shown as mean ± SEM; n=12.

Figure S3. Baseline body weight (a) and 48h energy intake (b) of chow-

maintained and high-fat diet-maintained rats prior to intra-VTA AAV treatment

(n=6-7 per AAV/dietary condition). AAV conditions for each group refer to the

eventual treatment assigned. *, p<0.05. Key applies to both panels. All data

shown as mean ± SEM.

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Full-text available

Jul 2023

The N -acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) catalyzes the production of N -acylethanolamines (NAEs), a family of endogenous bioactive lipids, which are involved in various biological processes ranging from neuronal functions to energy homeostasis and feeding behaviors. Reward-dependent behaviors depend on the dopamine (DA) transmission between the ventral tegmental area (VTA) and the nucleus accumbens (NAc) which conveys reward-values and scales reinforced behaviors. However, whether and how NAPE-PLD may contribute to the regulation of feeding and reward-dependent behaviors has not been investigated yet. This biological question is of paramount importance since NAEs are altered in obesity and metabolic disorders. Here, we show that transcriptomic meta-analysis highlights a potential role for NAPE-PLD within the VTA→NAc circuit. Using brain-specific invalidation approaches, we report that the integrity of NAPE-PLD is required for the proper homeostasis of NAEs within the midbrain VTA and it affects food-reward behaviors. Moreover, region-specific knock-down of NAPE-PLD in the VTA resulted in enhanced food-reward seeking and reinforced behaviors which were associated with increased in vivo DA release dynamics in response to both food and non-food-related rewards together with heightened tropism towards food consumption. Furthermore, midbrain knock-down of NAPE-PLD, which led to increased energy expenditure and adapted nutrients partitioning, elicited a relative protection against high-fat diet-mediated body fat gain and obesity-associated metabolic features. In conclusion, these findings unravel a new key role of VTA NAPE-PLD in shaping DA-dependent events, feeding behaviors and energy homeostasis, thus providing new insights on the regulation of body metabolism. Highlights - NAPE-PLD and NAEs are enriched in the VTA and regulate food-reinforced behaviors and reward processes. - NAPE-PLD scales in vivo VTA→NAc dopamine dynamics. - NAPE-PLD in the VTA contributes to whole-body energy balance and metabolic efficiency. - Downregulation of VTA NAPE-PLD ameliorates obesity-associated metabolic features.

Calcitonin receptor signaling in nucleus accumbens D1R- and D2R-expressing medium spiny neurons bidirectionally alters opioid taking in male rats

Article

Jun 2023

The high rates of relapse associated with current medications used to treat opioid use disorder (OUD) necessitate research that expands our understanding of the neural mechanisms regulating opioid taking to identify molecular substrates that could be targeted by novel pharmacotherapies to treat OUD. Recent studies show that activation of calcitonin receptors (CTRs) is sufficient to reduce the rewarding effects of addictive drugs in rodents. However, the role of central CTR signaling in opioid-mediated behaviors has not been studied. Here, we used single nuclei RNA sequencing (snRNA-seq), fluorescent in situ hybridization (FISH), and immunohistochemistry (IHC) to characterize cell type-specific patterns of CTR expression in the nucleus accumbens (NAc), a brain region that plays a critical role in voluntary drug taking. Using these approaches, we identified CTRs expressed on D1R- and D2R-expressing medium spiny neurons (MSNs) in the medial shell subregion of the NAc. Interestingly, Calcr transcripts were expressed at higher levels in D2R- versus D1R-expressing MSNs. Cre-dependent viral-mediated miRNA knockdown of CTRs in transgenic male rats was then used to determine the functional significance of endogenous CTR signaling in opioid taking. We discovered that reduced CTR expression specifically in D1R-expressing MSNs potentiated/augmented opioid self-administration. In contrast, reduced CTR expression specifically in D2R-expressing MSNs attenuated opioid self-administration. These findings highlight a novel cell type-specific mechanism by which CTR signaling in the ventral striatum bidirectionally modulates voluntary opioid taking and support future studies aimed at targeting central CTR-expressing circuits to treat OUD.

Cone JJ, McCutcheon JE, Roitman MF. Ghrelin acts as an interface between physiological state and phasic dopamine signaling. J Neurosci 34: 4905-4913

Article

Full-text available

Apr 2014
J NEUROSCI

Brief, high-concentration (phasic) spikes in nucleus accumbens dopamine critically participate in aspects of food reward. Although physiological state (e.g., hunger, satiety) and associated hormones are known to affect dopamine tone in general, whether they modulate food-evoked, phasic dopamine specifically is unknown. Here, we used fast-scan cyclic voltammetry in awake, behaving rats to record dopamine spikes evoked by delivery of sugar pellets while pharmacologically manipulating central receptors for the gut "hunger" hormone ghrelin. Lateral ventricular (LV) ghrelin increased, while LV ghrelin receptor antagonism suppressed the magnitude of dopamine spikes evoked by food. Ghrelin was effective when infused directly into the lateral hypothalamus (LH), but not the ventral tegmental area (VTA). LH infusions were made in close proximity to orexin neurons, which are regulated by ghrelin and project to the VTA. Thus, we also investigated and found potentiation of food-evoked dopamine spikes by intra-VTA orexin-A. Importantly, intra-VTA blockade of orexin receptors attenuated food intake induced by LV ghrelin, thus establishing a behaviorally relevant connection between central ghrelin and VTA orexin. Further analysis revealed that food restriction increased the magnitude of dopamine spikes evoked by food independent of any pharmacological manipulations. The results support the regulation of food-evoked dopamine spikes by physiological state with endogenous fluctuations in ghrelin as a key contributor. Our data highlight a novel mechanism by which signals relating physiological state could influence food reinforcement and food-directed behavior.

Dopamine Signaling in reward-related behaviors

Article

Full-text available

Oct 2013

Ja-Hyun Baik

Dopamine (DA) regulates emotional and motivational behavior through the mesolimbic dopaminergic pathway. Changes in DA mesolimbic neurotransmission have been found to modify behavioral responses to various environmental stimuli associated with reward behaviors. Psychostimulants, drugs of abuse, and natural reward such as food can cause substantial synaptic modifications to the mesolimbic DA system. Recent studies using optogenetics and DREADDs, together with neuron-specific or circuit-specific genetic manipulations have improved our understanding of DA signaling in the reward circuit, and provided a means to identify the neural substrates of complex behaviors such as drug addiction and eating disorders. This review focuses on the role of the DA system in drug addiction and food motivation, with an overview of the role of D1 and D2 receptors in the control of reward-associated behaviors.

The food intake-suppressive effects of glucagon-like peptide-1 receptor signaling in the ventral tegmental area are mediated by AMPA/kainate receptors

Article

Full-text available

Oct 2013
AM J PHYSIOL-ENDOC M

Glucagon-like peptide-1 receptor (GLP-1R) activation in the ventral tegmental area (VTA) is physiologically relevant for the control of palatable food intake. Here, we tested whether the food intake-suppressive effects of VTA GLP-1R activation are mediated by glutamatergic signaling within the VTA. Intra-VTA injections of the GLP-1R agonist exendin-4 (Ex-4) reduced palatable high-fat food intake in rats primarily by reducing meal size; these effects were mediated in part via glutamatergic AMPA/kainate, but not NMDA, receptor signaling. Additional behavioral data indicated that GLP-1R expressed specifically within the VTA can partially mediate the intake- and body-weight-suppressive effects of systemically administered Ex-4, offering the intriguing possibility that this receptor population may be clinically relevant for food intake control. Intra-VTA Ex-4 rapidly increased tyrosine hydroxylase levels within the VTA, suggesting that GLP-1R activation modulates VTA dopaminergic signaling. Further evidence for this hypothesis was provided by electrophysiological data showing that Ex-4 increased the frequency of AMPA-mediated currents and reduced paired-pulse ratio in VTA dopamine neurons. Together, these data provide novel mechanisms by which GLP-1R agonists in the mesolimbic reward system control for palatable food intake.

Divergent circuitry underlying food reward and intake effects of ghrelin: Dopaminergic VTA-accumbens projection mediates ghrelin's effect on food reward but not food intake

Article

Full-text available

Jun 2013

Obesity has reached global epidemic proportions and creating an urgent need to understand mechanisms underlying excessive and uncontrolled food intake. Ghrelin, the only known circulating orexigenic hormone, potently increases food reward behavior. The neurochemical circuitry that links ghrelin to the mesolimbic reward system and to the increased food reward behavior remains unclear. Here we examine whether VTA-NAc dopaminergic signalling is required for the effects of ghrelin on food reward and intake. In addition, we examine the possibility of endogenous ghrelin acting on the VTA-NAc dopamine neurons. A D1-like or a D2 receptor antagonist was injected into the NAc in combination with ghrelin microinjection into the VTA to investigate whether this blockade attenuates ghrelin-induced food reward behavior. VTA injections of ghrelin produced a significant increase in food motivation/reward behavior, as measured by sucrose-induced progressive ratio operant conditioning, and chow intake. Pretreatment with either a D1-like or D2 receptor antagonist into the NAc, completely blocked the reward effect of ghrelin, leaving chow intake intact. We also found that this circuit is potentially relevant for the effects of endogenously released ghrelin as both antagonists reduced fasting (a state of high circulating levels of ghrelin) elevated sucrose-motivated behavior but not chow hyperphagia. Taken together our data identify the VTA to NAc dopaminergic projections, along with D1-like and D2 receptors in the NAc, as essential elements of the ghrelin responsive circuits controlling food reward behavior. Interestingly results also suggest that food reward behavior and simple intake of chow are controlled by divergent circuitry, where NAc dopamine plays an important role in food reward but not in food intake.

Catecholamine innervation of the basal forebrain: II. Amygdala, suprahinal cortex and entorhinal cortex

Article

Jan 1978

The rat brain in stereotaxic coordinates, Compact

Article

Jan 1997

Amylin: A Novel Action in the Brain to Reduce Body Weight

Article

Feb 2000

P. A. Rushing

Amylin is a 37-amino acid peptide hormone that is co-secreted with insulin by pancreatic B cells in response to a nutrient stimulus (e.g., during meals). To test the hypothesis that amylin acts within the brain to reduce long-term food intake and body weight, we examined the effects of acute and chronic 3rd-ventricular (i3vt) infusion of low doses of amylin on food intake and body weight in rats. In one experiment, separate groups of ad lib-fed male Long Evans rats were given one i3vt infusion (3 μl over 30 s) of synthetic cerebrospinal fluid vehicle or 1 to 100 pmol amylin, and food intake and body weight were monitored for 7 days. Amylin potently and dose-dependently reduced 1-h food intake, with all doses producing significant reductions. The largest dose (100 pmol) significantly reduced 24-h intake by over 30%. The effect was persistent in that both 7-day cumulative food intake and body weight change were significantly decreased over the 7 days following a single injection of 100 pmol of amylin. Other groups of rats received continuous i3vt infusion (0.5 μl/h volume) of saline or 2.0 pmol/h amylin via osmotic minipumps over 10 days. Food intake over the 10-day infusion was significantly suppressed in amylin-treated rats as compared to that of controls. Consequently, by the 4th day of infusion, amylin rats weighed significantly less than baseline relative to saline controls, and this difference persisted throughout the remainder of the infusion period. At sacrifice (Day 10), the percent of body weight from retroperitoneal fat depots was significantly lower in the amylin-treated rats, indicative of a reduction of total body adiposity. In summary, the results support the hypothesis that amylin acts as a signal to the brain contributing to the maintenance of long-term energy balance.

Inhibition of Central Amylin Signaling Increases Food Intake and Body Adiposity in Rats

Article

Nov 2001

Paul A. Rushing

Amylin is a 37-amino acid peptide hormone that is co-secreted with insulin by pancreatic β cells in response to feeding. We recently reported that amylin potently reduces food intake, body weight, and adiposity when delivered into the 3rd cerebral ventricle (i3vt) of rats. We have now infused i3vt a specific antagonist (AC187) to ascertain the physiological relevance of central amylin in the control of energy balance. After establishing the ability of i3vt AC187 to block the anorexic effect of i3vt amylin, we performed an experiment to examine the impact of acute inhibition of central amylin signaling on feeding. Separate groups (n = 7/group) of ad lib-fed male Long Evans rats were given one bolus i3vt infusion of synthetic cerebrospinal fluid vehicle (CSF) or AC187 (250 or 1000 pmol). Acute infusion of AC187 tended to increase 1-h food intake and significantly elevated 4-h intake. Both the 250 and 1000 pmol doses produced significant increases as compared to CSF. In another experiment designed to tonically inhibit central amylin signaling over an extended period, two other groups of rats (n = 6/group) received continuous i3vt infusion of CSF or 100 pmol/h AC187 over 14 days via implantable osmotic pumps. Rats receiving AC187 ate significantly more food over the 14-day infusion period relative to controls (CSF = 322 ± 6 g, AC187 = 360 ± 12 g). Although body weight was not significantly affected, body fat was increased by about 30% in the AC187 rats, with no difference in lean tissue between the groups. Additionally, although fasting plasma glucose did not differ between the CSF and AC187 groups after 14 days of infusion, plasma insulin was significantly elevated in the AC187 rats. In summary, the present results document significant increases of food intake and body adiposity resulting from inhibition of central amylin signaling. They are consistent with our hypothesis that CNS actions of endogenous amylin contribute to the long-term regulation of energy balance.

Incretins and Amylin: Neuroendocrine Communication Between the Gut, Pancreas, and Brain in Control of Food Intake and Blood Glucose

Article

Apr 2014
ANNU REV NUTR

Arguably the most fundamental physiological systems for all eukaryotic life are those governing energy balance. Without sufficient energy, an individual is unable to survive and reproduce. Thus, an ever-growing appreciation is that mammalian physiology developed a redundant set of neuroendocrine signals that regulate energy intake and expenditure, which maintains sufficient circulating energy, predominantly in the form of glucose, to ensure that energy needs are met throughout the body. This orchestrated control requires cross talk between the gastrointestinal tract, which senses the incoming meal; the pancreas, which produces glycemic counterregulatory hormones; and the brain, which controls autonomic and behavioral processes regulating energy balance. Therefore, this review highlights the physiological, pharmacological, and pathophysiological effects of the incretin hormones glucagon-like peptide-1 and gastric inhibitory polypeptide, as well as the pancreatic hormone amylin, on energy balance and glycemic control. Expected final online publication date for the Annual Review of Nutrition Volume 34 is July 17, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Amylin activates distributed CNS nuclei to control energy balance

Article

Jan 2014

Amylin is a pancreas-derived neuropeptide that acts in the central nervous system (CNS) to reduce food intake. Much of the literature describing the anorectic effects of amylin are focused on amylin's actions in the area postrema, a hindbrain circumventricular structure. Although the area postrema is certainly an important site that mediates the intake-suppressive effects of amylin, several pieces of evidence indicate that amylin may also promote negative energy balance through action in additional CNS nuclei, including hypothalamic and mesolimbic structures. Therefore, this review highlights the distributed neural network mediating the feeding effects of amylin signaling with special attention being devoted to the recent discovery that the ventral tegmental area is physiologically relevant for amylin-mediated control of feeding. The production of amylin by alternative, extra-pancreatic sources and its potential relevance to food intake regulation is also considered. Finally, the utility of amylin and amylin-like compounds as a component of combination pharmacotherapies for the treatment of obesity is discussed.

Amylin Modulates the Mesolimbic Dopamine System to Control Energy Balance

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