UNIVERSITY OF CINCINNATI
Date:______
I, ______, hereby submit this work as part of the requirements for the degree of: in:
It is entitled:
This work and its defense approved by:
Chair: ______
Novel fuel sensing mechanisms in the regulation of food intake
A dissertation submitted to the
Division of Research and Advanced Studies of the University of Cincinnati
in partial fulfillment of the requirements for the degree of
DOCTORATE OF PHILOSOPHY (Ph.D.)
in the Graduate Program in Neuroscience of the College of Medicine
May 22, 2006
by Karine Proulx
B.S., Université Laval, 2000 M.S., McGill University, 2002
Committee Chair: Randy J. Seeley, Ph.D. ABSTRACT
An emerging model is that CNS fuel sensors, such as AMP kinase (AMPK) and
the mammalian target of rapamycin (mTOR), integrate signals from stored and
immediately available fuels, and in turn regulate food intake. The experiments described in this dissertation focus on novel CNS fuel sensing mechanisms by which fatty acid derivatives and compounds that affect fatty acid metabolism modulate food intake.
Oleoylethanolamide (OEA), a derivative of oleic acid synthesized in the intestine following refeeding, reduces food intake. OEA shares similarities with other nutrient- derived hormones that signal energy status to the CNS, but its mechanisms of action remain unclear. We tested whether OEA-induced anorexia occurs through specific interactions with hormones that modulate food intake through CNS pathways involved in energy homeostasis, or is rather due to unspecific behaviors. Our results indicate that
OEA suppresses feeding without causing visceral illness, and that neither ghrelin, PYY,
GLP-1, apo A-IV nor CCK play a critical role in this effect. OEA is not the only fatty acid metabolism related compound that suppresses food intake. C75 is a fatty acid synthase inhibitor that inhibits food intake via direct actions in the CNS. MTOR, a member of the phosphatidylinositol kinase-related protein kinase family, plays a crucial role in nutrient sensing and the control of protein synthesis. Its inhibition stimulates food intake in rats. We hypothesized that C75-induced anorexia depends on its ability to activate the mTOR pathway in the hypothalamus. Consistent with this hypothesis, C75 increases the phosphorylation of key components of the mTOR pathway and inhibitors of mTOR reverse C75-induced anorexia. Previous work showed that C75 is ineffective when rats are on a ketogenic diet. Consistent with a role for mTOR in mediating the
- I - effects of C75, C75-induced anorexia and activation of the mTOR pathway were abolished in rats maintained on a ketogenic diet. Together, these data argue that neuronal nutrient metabolism is monitored by CNS fuel sensors and contribute to the regulation of food intake.
- II - - III - ACKNOWLEDGEMENTS
Randy: Maybe one of the reasons you always say that you trust my scientific judgment is
that you know you are the one who made me build one over the last four years! As
challenging as those conversations in your office might have been sometimes, I can
honestly say that they are what made me the young scientist I am today. You taught me
how to develop a critical sense, stand up for my ideas and, most importantly, build some
confidence throughout the whole process. I now leave this lab feeling strong and ready to
move to the next step. Thank you also to you and Steve, for showing us what “work hard-
play hard” really means, allowing the passion for science to remain even in tougher
times!
Steve: I remember the very first question Steve asked me during my interview: “Why
would someone coming from such a beautiful city as Montreal (as he said it with his classic Woodsilian French accent!) would want to live in Cincinnati?”. While such a
broad question could have been intimidating coming from someone like Steve Woods, I
exactly knew the answer. My motivation obviously didn’t come from the attractions of
the city (no offense, but that has never been a secret to anyone!). After a few mornings
that I popped my head in his office for questions, he suggested we schedule a weekly
meeting, where we would talk about the philosophy of research. Every time we met from
then until now confirmed that I made the right decision in coming to Cincinnati, and that
I have found exactly the training environment I was looking for. In addition to be a great
mentor, you and Nancy have also been a great second family by making me feel home
during celebrations like Thanksgiving and Easter. Thank you!
- IV - Committee members: Drs. Herman, Jandacek and Williams, I learned so much from our discussions during and outside of our official committee meetings. I truly felt that you were supportive of my research and cared about my career. Our program is blessed to have faculty members like you, who despite their busy schedules maintain an “open- door” policy. You have always made me feel welcome when I needed help for either technical issues or last minute recommendation letters and I want to thank you for that.
Beth: Where should I start, how can I put all those feelings into words? We’ve started together and are finishing it together. We shared so much! You were always there for me, in good as well as bad/ “life threatening” times (I will always remember our infamous
New Orleans episode and how you helped me keep my hot temper low after that guy crashed my car!). I knew I could rely on you whether for intellectual, moral or
“technical” support. I am sure you’ll be a great and successful scientist. I’ll be there to celebrate your tenure-track position on your thirtieth birthday! I wish you the best of luck for your post-doc and I am looking forward to your visit in the UK!
April and Torsten: Thank you for taking me under your wings and getting me started in the lab. You guys were there for my first rat bite, as well as those crazy tail-bleeding sessions at 7h00 AM, after a night of too much celebration at Randall’s house, or at 1h00
AM, because of a light-dark cycle that we chose to accommodate my classes! We can only laugh about it now!
Daniela: You have been not only an awesome colleague, but also a great friend. You were there to celebrate every happy moment, and to support me through all the troubles of this thesis (despite what the ratio between those two might have been, I don’t care anymore!). I want to say how much it meant to me to be able to share ideas and have
- V - scientific conversations that always remained authentic and free of any competition. I say goodbye for now, but I have the feeling it is only temporary. I will probably be meeting
Prof. Cota for a cappuccino in some European café pretty soon!
The Tschoep’s lab: Matthias, Tamara, Paul and Diego: You guys made the OEA
paper happened. I still think that we shouldn’t have had to spend so much time figuring
out how something with a name like “The Clever System” works! Despite how ridiculous
it was to play with the red light until we got the proper “Chinese shadows” to detect the
rat in its cage, I have to admit we had fun! I’ll keep great memories of the time we spent
together in and outside of GRI. Like I said for Daniela, I am sure that our roads will cross
again in the near future my dear European friends.
My non-official committee members: Probably the result of working in such a collaborative environment as the one within the Obesity Research Center and the GRI:
Randall Sakai: Thank you for professional and personal support throughout my time at
UC. Among the many things I will remember from my interactions with you are when
you were there to get me coffee and calm me down the morning of my first big talk at
SFN, your “PR” and networking qualities and, of course, the parties and wine at your
house!
David D’Alessio: Although not an official member of my committee, you surely behaved
like one! You contributed to solving so many issues from the ADX, to OEA to C75
project. I really appreciated you keeping your door opened for my spontaneous questions,
I learned a lot from you.
Patrick Tso: Your enthusiasm for science is contagious and your approach to scientific
questions very unique. I learned a lot from those drawings on the board next to your
- VI - office! Thank you for contributing to my OEA project, and also to regularly check on my
future post-doc career.
Kay Ellis: It was always fun to go drop those plasma samples to you Kay. It was the
occasion to get a little girlie chat, and laugh at how many tubes there were in those boxes!
Matt and Debbie: Although you guys had already left by the time I’ve started working
on C75, you generously offered your help. Sorry for making you go through messy lab
books (mine are not any better!) or trying to find on which paper towel you wrote the
design of the experiment on that day! Thanks for sharing your knowledge of the FAS
system and your insightful discussions.
Past and present members of the “team room” ( Beth, Daniela, Claire, Lynda,
Dong-Hoon, Jason, Darleen, April, Ryuichi, Debbie D., Brad, Debbie C., Stephen,
Matt and Koroh): Despite what Randy says about its lack of light and its small size, this
“team-room” has probably been the most enriching environment I have ever been in. I
grew up so much in that room over the last 4 years, not only as a better scientist, but also
as a better individual. I remember when Koroh and I used to be the only people with an
accent in the first few months I was here, and when all the American fellows could make
fun of how I pronounced “buffet” during my first lab meeting… Not so much anymore!
This room soon became the U.N. as Randy says, bringing people with different cultures
and training backgrounds. I have always been so amazed how such a big group of people, with so many different personalities, can get along and work so well together. It is pretty
rare to find such a big lab free of competition. We always stand up together as a group
and support each other in good and bad times. I couldn’t have wished for better
colleagues and friends, and can only hope to find such an amazing training environment
- VII - during my post-doc. You are all wonderful persons! I’m looking forward to seeing you
again at meetings, and hopefully collaborate when we all become independent researchers.
Neuroscience friends (Beth, Mary, Dennis, Nicole, Susan, Miyuki, Kellie, Alier,
Rick, John D., Derrick, and Brian M.): I couldn’t have made it throughout graduate school without you guys! I felt that we were all on the same boat and there to support each other. Despite what I said earlier about Cincinnati and its attractions, you are the ones who made the difference. You can be in the most entertaining city in the world, without great friends, it doesn’t mean anything. I was glad to be there for those of you who defended already. While I probably won’t be here to celebrate everyone else’s defense, I’ll definitely make sure I’ll have a drink “à votre santé”. Good luck and see you soon!
Deb: You are certainly among the most dedicated and efficient person I met here. From
my first day at UC, when you showed me around Clifton, to the time when you called the
guy who screwed up my application for a US social security number…nothing stops you!
Thanks Deb!
Family and friends: Thank you for supporting my decision in coming to the US. I remember how afraid you were I would not want to come back. You can be reassured, both Randy and Steve would tell you that I have never lost an ounce of my culture
“québécoise” or my French in four years, which by the way, Randy kindly reminded me
when he first read my dissertation! As much as I loved my experience here, I am looking
forward to move back to Canada soon and be closer to you. Merci d’avoir supporté ma
décision de venir étudier aux Etats-Unis. J’ai tellement appris ici en quatre ans mais
- VIII - n’ayez crainte, je n’ai jamais perdu une once de ma culture québécoise! Je vous aime très fort et j’ai hâte de me retrouver parmi vous!
Ian: A ta demande, et pour célébrer le fait que je t’ai officiellement déclaré bilingue, je vais écrire le message en Français mon chéri. Merci de m’avoir compris et encouragé tout au long de ce processus. Merci aussi de m’avoir donné un coup de pied quand je commençais à me laisser aller. Tu avais raison, peu importe ce qui arrive, tout fini
éventuellement par entrer dans l’ordre. Aujourd’hui, je suis fière d’être restée forte malgré les difficultés, et ça c’est grâce à toi. J’ai hâte de débuter notre nouvelle aventure ensemble, enfin fini la distance! Je t’aime.
- IX - TABLE OF CONTENTS
ABSTRACT……………………………………………………………………….………I
ACKNOWLEDGEMENTS……………………………………………………………...IV
TABLE OF CONTENTS…………………………………………………….…………...1
LIST OF TABLES AND FIGURES……….………………..…………………………....2
LIST OF ABBREVIATIONS………………………………………………………...... 4
CHAPTER 1: INTRODUCTORY LITERATURE REVIEW……………………………6
CHAPTER 2: MECHANISMS OF OLEOYLETHANOLAMIDE (OEA)-INDUCED CHANGES IN FEEDING BEHAVIOR AND MOTOR ACTIVITY...……36 I. Abstract……………………………………………………...37
II. Introduction……………..…………………………………..38
III. Materials and Methods……………………………………...41
IV. Results……………..………………………………………..49
V. Discussion……….……………………………………….…53
CHAPTER 3: C75 REGULATES FOOD INTAKE VIA ACTIVATION OF HYPOTHALAMIC MTOR SIGNALING………………….………………71 I. Abstract……………………………………………………...72
II. Introduction……………..…………………………………...74
III. Materials and Methods………………………………………78
IV. Results……………..………………………………………...84
V. Discussion……….………………………………………...... 87
CHAPTER 4: GENERAL DISCUSSION……...…………………………...100
REFERENCES……………………………………………………………...112
APPENDICES.……………………………………………………………...126
- 1 - LIST OF TABLES AND FIGURES
Chapter 2:
Figure 1. Mean + SEM food intake at different time points (A), and 24 h (B) after an ip
injection of vehicle (VEH) or three doses of OEA in 24-h fasted rats…………………..61
Figure 2. Mean + SEM preference ratio of flavors previously paired with an ip injection of saline (SAL), lithium chloride (LiCl), VEH or OEA (20 mg/kg)…………………….62
Figure 3. Mean + SEM 2-h sodium intake after an ip injection of SAL, LiCl, VEH or
OEA (20 mg/kg) in sodium-depleted rats………..………………………………… …...63
Figure 4. Mean + SEM time the rats spent in the side previously paired with an ip
injection of SAL, LiCl, VEH or OEA (20 mg/kg)………………………………………64
Figure 5. Mean + SEM heat expenditure at different time points after an ip injection of
either VEH or OEA (20 mg/kg) (A), and mean + SEM heat expenditure per hour during
the 12-h dark phase (B) in fasted rats…………………………………………………...65
Figure 6. Mean + SEM time the rats displayed an extended posture in the 2 h following
an ip injection of either VEH or OEA (20 mg/kg) (A), and either VEH or capsaicin
(CAPS) (1 mg/kg) (B)…………………………………………………………………..66
Figure 7. Mean + SEM time the rats spent in inactivity (A), number of times the rats crossed a square (B), and number of times the rats either reared or groomed (spontaneous activity) (C) after an ip injection of OEA (20 mg/kg), CAPS (1 mg/kg), Wy-14643 (40 mg/kg) or their respective VEH………………………………………………………...67
Figure 8. Mean + SEM plasma concentrations of ghrelin (A), PYY (B), GLP-1 (C), and
apo A-IV (D) at different time points after an ip injection of either VEH or OEA (20
mg/kg)…………………………………………………………………………………..68
- 2 - Figure 9. Mean + SEM food intake at different time points after an ip injection of either
SAL or lorglumide (LORGL) (1mg/kg), followed by an ip injection of either VEH or
OEA (20 mg/kg) (A), and either SAL or CCK-8 (4 μg/kg) (B) in fasted rats…………...69
Chapter 3:
Table 1. Rat Q-PCR Primer Sequences …………………………………………………93
Figure 1. I3vt administration of rapamycin (25 µg in 1 µl DMSO) prevents the effects of
C75 (50 µg in 3 µl RPMI) on food intake (A) and on body weight (B)…………………95
Figure 2. C75 (30 µg in 2 µl RPMI, i3vt) increases hypothalamic mTOR signaling:
Representative Western blots from RPMI- or C75-treated rats and quantification by
image analysis of hypothalamic phosphorylation of mTOR (A), S6K1 (B) and S6 (C)..96
Figure 3. C75 (30 µg in 2 µl RPMI) does not reduce caloric intake in rats given access to
saccharin alongside with the ketogenic diet, but do so with sucrose……………………97
Figure 4. C75 (30 µg in 2 µl RPMI) does not increase hypothalamic S6K1 and S6 phosphorylation in rats given access to saccharin while maintained on a ketogenic diet, but do so in rats receiving sucrose alongside with the diet. Representative Western blots from RPMI- or C75-treated rats from the saccharin and sucrose groups and quantification by image analysis of hypothalamic phosphorylation S6K1 (A) and S6 (B)…………….98
Figure 5. C75 significantly reduced NPY mRNA levels in the hypothalamus as
compared to RPMI controls, as did rapamycin………………………………………….99
- 3 - LIST OF ABBREVIATIONS
α-MSH Alpha-melanocyte stimulating hormone ACC Acetyl-CoA carboxylase AgRP Agouti related protein AICAR Aminoimidazole-4-carboxamide ribonucleoside AMPK AMP-activated protein kinase ANOVA Analysis of variance Apo A-IV Apolipoprotein A-IV ATP Adenosine triphosphate BMI Body mass index CAPS Capsaicin CART Cocaine and amphetamine related transcript CB1 and 2 Cannabinoid receptors 1 and 2 CCK Cholecystokinin CEACAM-1 Cell adhesion molecule 1 CNS Central nervous system CPA Conditioned place aversion CPT-1 Carnitine palmitoyl-CoA transferase-1 CTA Conditioned taste aversion DMSO Dimethyl sulfoxide FAAH Fatty acid amide hydrolase FAE Fatty acid ethanolamide FAS Fatty acid synthase FRB FKB12-rapamycin binding GE Glucose-excited GI Glucose-inhibited GLP-1 Glucagon-like peptide 1 i3vt Intra-3rd-cerebroventricular LCFA-CoA Long-chain fatty acid-CoA LiCl Lithium chloride
- 4 - LORGL Lorglumide MC4-R Melanocortin receptor-4 MCD Malonyl-CoA decarboxylase mTOR Mammalian target of rapamycin NPY Neuropeptide Y NPY-Y1 NPY receptor 1 NPY-Y5 NPY receptor 5 OEA Oleoylethanolamide POMC Proopiomelanocortin PPAR-α Peroxisome proliferator-activated receptor-alpha PVN Paraventricular nucleus of the hypothalamus PYY peptide YY Q-PCR Quantitative polymerase chain reaction RAPA Rapamycin S6 S6 ribosomal protein S6K1 S6 kinase 1 SAL Saline SCD-1 Stearoyl-CoA desaturase-1 SEM Standard error of the mean TRPV1 Capsaicin receptor UCP Uncoupling protein VEH Vehicle WAT White adipose tissue
- 5 - CHAPTER 1: INTRODUCTORY LITERATURE REVIEW
- 6 - 1. Obesity: general overview.
Obesity is associated with substantially increased mortality from cardiovascular and cerebrovascular disease, diabetes and certain cancers. It also results in severe morbidity from musculoskeletal, gastrointestinal, psychiatric and reproductive diseases and is associated with lowered quality of life, self-esteem and socio-economic performance (1).
The prevalence of obesity is increasing rapidly throughout the world and the disorder is now recognized as a major global public health threat. While these changes in the prevalence of obesity in the last 10-50 years are clearly driven by secular changes in physical activity and the inexpensive availability of high-caloric foods, it is also clear that inherited factors play a major role in determining adiposity. Family, twin and adoption studies all indicate that adiposity is highly heritable and the estimated genetic contribution to Body Mass Index (BMI) ranges between 50 and 90 percent (2). Thus, the environmental factors that are driving the increase in obesity prevalence are operating against the background of a biological system whose susceptibility or resistance to such environmental stressors has a strong inherited component (2). This might be the result of evolution, which could have selected over the millennia for genes and redundant mechanisms that allowed the body to efficiently store energy in periods of “feast” for survival during times of “famine” (3). Understanding the mechanisms by which these genes regulate body adiposity is fundamental to developing new therapeutic strategies for the treatment of obesity and its related metabolic disorders. The following paragraphs will review the current knowledge of the homeostatic mechanisms that regulate food intake and body weight in mammals.
- 7 - 2. Energy homeostasis.
The interaction of genetic and environmental factors dictates an individualized
level of body adiposity that is defended by a powerful process known as energy
homeostasis. Obesity arises as the result of a sustained mismatch where caloric intake
exceeds caloric expenditure. However, despite what the growing obesity epidemic might
argue, under most circumstances the balance between caloric intake and expenditure is
regulated with tremendous precision since mammals defend a very stable body weight
over time. For example, an average weight male who consumes 900,000 calories in a
year would need to consume 4000 more calories than he burns to gain one pound in that
year, the equivalent of 11 calories (or one potato chip!) a day (4). Statistics indicate that the average increase of weight within the adult U.S. population is actually less than one pound per year (5). Back to our example, this means the organism, and especially the central nervous system (CNS), is accurate at more than 99.5% in matching energy intake to energy expenditure (4).
This example highlights two points with regard to energy balance. First, the
amount of inter-individual variability is relatively large, and becomes obvious when
comparing the average yearly weight gain (5) with the actual number of obese individuals
within the population (6). Second, this would suggest the existence of a regulatory system
where deviations from the defended body adiposity level trigger a signal that can be
monitored by a biochemical sensor. This sensor would then induce adaptive responses in
food intake and metabolic processes that affect fuel oxidation, in order to ensure that body weight remains relatively stable over time (7). Assuming this is true, the question
- 8 - then becomes what is this signal and how is it detected? More importantly, what is the
purpose of this complex regulatory system?
3. Fuel sensing within the central nervous system (CNS).
3. 1. Signals of stored and immediately available fuels.
Not surprisingly, the current data indicate that there is not a single signal. Rather,
growing lists of hormones and nutrients have been proposed to indicate the status of fuels
available to cells for the generation of energy/ATP (8). These fuels can either be stored in
adipose tissue or be derived from recently ingested food, and therefore be immediately available. Among the best studied and characterized signals of stored fuels are leptin and insulin. These hormones, synthesized in adipose tissue and pancreas respectively, circulate in proportion to body fat (9). They inform the CNS that adipose stores are increased and that the organism is therefore in a state of positive energy balance (10, 11).
In addition to negative feedback, the CNS also receives positive feedback when energy stores are low. This role is accomplished by ghrelin, a stomach-derived hormone whose circulating levels are inversely correlated to those of leptin (12). Ghrelin increases food intake and adiposity when directly injected into the brain (13), whereas leptin (14) and insulin (15) do the opposite. In addition to monitoring the status of stored energy, the
CNS also keeps track of immediately available food-derived nutrients such as glucose, fatty acids and amino acids. Infusion of any of the above mentioned substrates in the vicinity of the hypothalamus reduces food consumption and expression of the orexigenic neuropeptide Y (NPY) (16-19), suggesting that they can be sensed by the brain.
- 9 - 3. 2. POMC/CART and NPY/AgRP neurons: a site of integration.
Signals reflecting stored and currently available fuels are detected for the most
part in the arcuate nucleus, where receptors for leptin (20), insulin (21, 22) and ghrelin
(23) are highly expressed, as well as transporters for glucose (24) and amino acids (A.
Sweatt, personal communications). At least two distinct populations of neurons are reciprocally regulated by these signals. One population synthesizes the large precursor peptide proopiomelanocortin (POMC), which can be cleaved into a number of biologically active peptides, including α-melanocyte stimulating hormone (α-MSH).
Central administration of this peptide or its synthetic analogues potently reduces food
consumption and causes body weight loss in rodents (25, 26), presumably through
activation of the melanocortin receptor 4 (MC4-R). It is interesting to note that mutation
in the MC4-R gene is the most common monogenic cause of obesity in humans (27). The
second set of neurons in the arcuate nucleus synthesizes both Agouti-Related Protein
(AgRP) and NPY (28, 29). In contrast to α-MSH, central injection of AgRP or NPY
induces profound hyperphagia and weight gain due to inverse agonism of MC4-R (30,
31) in the case of AgRP, and activation of the NPY-Y1 and NPY-Y5 receptors in the case
of NPY (32).
As will be discussed later in this review, signals of both stored and currently
available fuels appear to be integrated within common intracellular signaling cascades in
NPY/AgRP and POMC/CART neurons (33, 34). These neurons in turn send feedback
signals to modify food intake and metabolic processes so that energy balance is defended.
This concept of common integration is a relatively novel one. For a long time, this area of
- 10 - neuroscience research was dominated by two opposing views, each stating that the
hypothalamus monitors the levels of one signal and adjusts food intake accordingly. For
J. Mayer, the father of the glucostatic theory (35), this signal was the storage and
metabolism of glucose, whereas for G. Kennedy and the proponents of the lipostatic
theory, the signal had to be lipid storage and use (36).
Studies have demonstrated over the last 50 years that each of these hypotheses
have their limitations and caveats (7, 33, 37). Novel concepts have emerged concerning
the neuronal mechanisms that regulate food intake. This led to the working hypothesis behind the experiments of this thesis, which is that signals from both stored and immediately available fuels converge in the same intracellular signaling cascades in the
CNS, where they are monitored in parallel by biochemical fuel sensors that trigger adaptive feeding responses (7). The experiments described in this thesis focus on novel
CNS fuel sensing mechanisms by which fatty acids and fatty acid metabolism regulate food intake.
4. AMPK and mTOR: CNS fuel sensors.
Recent studies from Kahn’s laboratory and our own have identified two protein
kinases, AMP kinase (AMPK) (34) and the mammalian target of rapamycin (mTOR)
(19), that play a crucial role in integrating both types of signals. These proteins, commonly referred to as “fuel sensors”, were known to monitor the energy status of
peripheral cells, and regulate metabolic pathways in such a way to restore cellular energy
homeostasis (38-40). The observation that the these fuel sensors are phosphorylated at
- 11 - specific residues, an index of their activity, is regulated by the feeding status of the
animal such fuel sensors in hypothalamic neurons that have been linked to the control of
food intake and body weight, together with that their suggest an important function in the
regulation of energy balance at the whole organism level (19, 34, 41).
4. 1. AMPK.
4. 1. 1. AMPK structure and regulation.
AMPK, a cytosolic serine/threonine protein kinase, is comprised of three
subunits: the α-subunit contains the catalytic domain; the β-subunit, a glycogen-binding
domain; and the γ-subunit, the AMP-binding site. Decreases in the energy state of a cell,
as reflected by a decrease in the ratio of ATP/AMP, activate AMPK via several
mechanisms. This includes phosphorylation of its catalytic subunit on Thr172 by an
AMPK kinase (AMPKK), which is itself activated by AMP. AMP binding also inhibits
AMPK dephosphorylation by protein phosphatases. Importantly, all these effects are
antagonized by high concentrations of ATP and thus allowing the system to precisely
monitor changes in the ratio of ATP:AMP (38, 42).
4. 1. 2. AMPK as a peripheral fuel sensor.
AMPK serves as a cellular fuel sensor, and its activation is crucial to protect the viability of the cell in response to ATP depletion (38). During metabolic stress, such as
exercise or fasting, phosphorylated AMPK (Thr172) inactivates a number of regulatory
enzymes and inhibits the expression of several genes and proteins involved in
biosynthetic (anabolic) pathways (43). Consequently, energy-consuming (anabolic)
- 12 - processes are turned off, while ATP-producing (catabolic) ones are switched on. In peripheral tissues, this translates to an activation of signaling cascades that increase fatty acid and glucose oxidation, in order to restore ATP levels (44). In the brain, recent evidence suggests that activation of AMPK stimulates hypothalamic circuits that promote food intake (34, 41).
4. 1. 3. AMPK as a CNS fuel sensor.
In 2004, two independent research groups published data supporting a role for
AMPK as a CNS fuel sensor. They demonstrated that hypothalamic AMPK is regulated by the feeding status of the animal, as well as several hormonal and nutrient-derived signals (34, 41). For instance, fasting or AgRP, the endogenous inverse agonist of MC3-
R and MC4-R, increases AMPK activity in the same regions where refeeding, glucose and the MC3-R and MC4-R agonist MT-II reduce it (34). Furthermore, the potent orexigens ghrelin and cannabinoids (41, 45), in contrast to leptin and insulin (34), stimulate hypothalamic AMPK levels.
The response of neurons to energy depletion is similar to peripheral cells: switching on ATP-producing pathways. For a hypothalamic neuron whose primary role is to regulate energy balance, activation of AMPK translates into stimulation of food intake.
Consistent with this, genetic (34) or pharmacological (41) activation of AMPK using 5- aminoimidazole-4-carboxamide ribonucleoside (AICAR), in the CNS stimulates the expression of the orexigenic neuropeptides AgRP and NPY, which lead to increased feeding and body weight gain. Moreover, the anorectic effect of leptin is completely
- 13 - abolished, and its ability to reduce weight gain is significantly attenuated under those conditions (34). This indicates that suppression of hypothalamic AMPK is required for the catabolic effects of leptin.
Another action of AMPK relevant to the regulation of energy balance is stimulation of fatty acid oxidation in peripheral tissues. AMPK is an inhibitor of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme for the conversion of acetyl-CoA to malonyl-CoA, and causes a reduction in malonyl-CoA concentration (46). This in turn releases the inhibition exerted by malonyl-CoA on carnitine palmitoyl-CoA transferase-1
(CPT-1), the enzyme responsible for the transport of long chain fatty acyl-CoAs into the mitochondria, and therefore promotes fatty acid oxidation (43). As will be discussed later, these enzymes are also present in hypothalamic neurons (47), where our lab and others have demonstrated their involvement CNS fuel sensing (48-51). Leptin-induced inhibition of AMPK regulates this pathway in the hypothalamus. Of interest to the present discussion is the recent report of Moran and colleagues, who proposed that leptin- induced modulation of intermediates of this pathway, specifically malonyl-CoA, is necessary for its anorectic action (52). In muscle and liver, leptin increases fatty acid oxidation through this pathway as well, via activation of the hypothalamic-sympathetic nervous system axis (53). The case of leptin-AMPK provides a perfect illustration of the role of “CNS fuel sensing”. Hypothalamic AMPK integrates signals of energy status, and regulate a cascade of biochemical events that affect both sides of the energy balance equation, in a way that ensures body adiposity remain within the defended range.
- 14 - 4. 2. mTOR.
4. 2. 1. mTOR structure and regulation.
Cells grow to a certain size before they divide (54). So for a cell and, therefore, a whole organism to grow requires the integration of both genetics and environmental cues including fuel availability. Thus, a complex system ensures that growth is tightly coordinated with the nutritional status of the organism. In addition to AMPK, the target of rapamycin (mTOR in mammals and dTOR in Drosophila) is crucial to this system.
MTOR, a member of the phosphatidylinositol kinase-related protein kinase (PIKK) family, plays a crucial role in nutrient sensing and the control of protein synthesis, angiogenesis, and cell cycle progression (55). It was originally identified and cloned in
1994, shortly after the discovery of the two yeast genes, TOR1 and TOR2 during a screen for resistance to the immunosuppressant drug rapamycin (56-58). MTOR is a 2549 amino acid long protein comprised of several domains, including the FRB (FKB12-rapamycin binding) to which rapamycin binds (59).
MTOR integrates signals from growth factors and nutrients, and in turn regulates the cell’s translational machinery in order to regulate cell growth, proliferation and death.
Insulin and amino acids have been shown to induce mTOR phosphorylation at Ser2448
(60), which correlates with activation of the mTOR pathway signaling, including phosphorylation of its downstream effectors S6 kinase (S6K) at Thr 389 and S6 ribosomal protein (S6) at Ser 240/244 (61).
- 15 - MTOR exists in two distinct multi-protein complexes: one containing a protein called raptor and another containing the protein rictor (62). The former complex is involved in nutrient sensing and therefore, the focus of the following section. During nutrient deprivation, the raptor-mTOR complex is stabilized in a manner that inhibits mTOR kinase activity, whereas raptor binds to the N-terminal domain of mTOR with high affinity and serves as a positive regulator of its activity in the presence of nutrients
(59). Raptor also serves as an adaptor protein that recruits mTOR substrates, such as the
40S ribosomal proteins S6 kinases 1 and 2 (S6K1 and S6K2), and is necessary for their phosphorylation by mTOR (63). The action of mTOR on its multiphosphorylated effectors is two-fold: activation of kinases and inhibition of the phosphatases that dephosphorylate them, phosphatase 2A (PP2A) in the case of S6K (64, 65), therefore ensuring a rapid and coordinated response. The mTOR-raptor complex is the best studied and understood of the two, mainly because it alone is inhibited by rapamycin (62).
Rapamycin, also known as sirolimus or RAPA, is a lipophilic macrolide produced by the soil bacterium Streptomyces hygroscopicus (66). This drug does not prevent the association of raptor to mTOR, but rather strongly destabilizes their interaction and inhibits mTOR kinase activity, ultimately causing dephosphorylation and deactivation of downstream components of the mTOR pathway (67). Rapamycin binds to its intracellular receptor FKBP12, which in turn binds the FRB (FKB12-rapamycin binding) domain of mTOR, and thereby inhibits it (59). In the clinic, rapamycin is prescribed as an anti- cancer drug, since it inhibits the growth of tumors, as well as an immunosuppressant (68).
- 16 - In the laboratory, this drug has been widely used to demonstrate the implication of
mTOR in multiple cellular processes.
4. 2. 2. mTOR as a peripheral fuel sensor.
Like AMPK, mTOR plays the role of fuel sensor. In contrast to AMPK, the
mTOR pathway is activated in energy-rich conditions, when the ratio ATP:AMP
increases (69). Consistent with its role of nutrient sensor, it therefore makes intuitive
sense for this kinase to regulate metabolism in a direction opposite to that of AMPK:
inhibiting energy-producing pathway and stimulating energy-consuming ones, such as
protein synthesis. This way, mTOR participates in a nutrient “checkpoint”, coupling
nutrient availability to downstream translation effectors, including S6K and S6 (59). This
way, amino acid levels can act as a feed-forward activator of proteins that activate protein synthesis and cellular growth.
Complete ablation of TOR is lethal (70). However, mice and Drosophila lacking
S6K1 survive. They have reduced cell size and are smaller than wild-type (71, 72). This
shows that TOR signaling, by controlling cell size, also controls the overall body size.
This effect applies to all cell types, so it is also reflected in pancreatic beta cell mass,
which could explain why S6K1-/- mice have reduced circulating insulin levels and are
glucose intolerant (73).
An interesting finding is that S6K1-/- mice are also protected against diet- and age-
induced insulin resistance and obesity, presumably due to increased fatty acid oxidation
- 17 - and mitochondrial density in muscle and adipose tissue (74). This phenotype supports the
fuel sensor role of mTOR signaling in peripheral tissue. It implies that under conditions
of fuel surplus, S6K1 signaling inhibits ATP-producing pathways like fatty acid
oxidation. From an energy homeostasis perspective, this would be the appropriate cellular
response under normal conditions. In conditions of chronic nutrient excess like under
high fat diet, however, the mTOR-S6K pathway may mediate maladaptive responses and
promote obesity (74). This is an important point that will be further emphasized in
chapter 4.
4.2.3. mTOR as a CNS fuel sensor.
The mTOR pathway is present in virtually all cell types. The case of peripheral
cells has been discussed above, and it appears that activation of mTOR-S6K1 signaling in
conditions of nutrient abundance favors energy storage. The pathway is also activated in
the CNS under such conditions. We have recently demonstrated in rats that mTOR plays
a similar role in the hypothalamus, where its phosphorylated form is found both in the
paraventricular (PVN) and the arcuate nucleus of the hypothalamus (19). In the arcuate,
which appears to be the only region where pmTOR levels are controlled by feeding
status, 90% of NPY neurons and 45% of POMC neurons express pS6K1, a downstream
target activated by pmTOR (reference). Interestingly, compounds that activate
hypothalamic S6K1, such as leptin and leucine, have also been reported to reduce NPY expression in this region (19, 75). Intra-third ventricular (i3vt) injection of rapamycin mimics the effects of fasting by inhibiting pS6K1 and causes short-term stimulation of food intake. Moreover, when administered at a sub-threshold dose that does not affect
- 18 - food intake on its own, it prevents the anorexigenic effects of centrally administered
leptin and leucine (19). These data make a case for hypothalamic mTOR signaling to play
a key role in the regulation of food intake. This novel hypothesis is also supported by
studies in Drosophila. Indeed, Drosophila expressing a dominant negative form of S6K
in the CNS eat more food than controls under ad libitum conditions, whereas the normal
hyperphagic response to fasting is reduced in mutants that express a constitutively active
form of the kinase (76).
5. A role for C75 in the regulation of energy balance.
Signals of stored fuels such as leptin, insulin and ghrelin regulate food intake
through modulation of CNS fuel sensors (19, 34, 41, 45). There is little evidence that
physiological fluctuation in the circulating levels of nutrients, including glucose and fatty
acid, controls meal initiation or termination (33, 77). Nonetheless, CNS fuel sensors
integrate these signals among the same intracellular cascade as those of stored fuels (19,
34). Moreover, recent studies indicate that C75, a fatty acid synthase (FAS) inhibitor that
alters neuronal glucose and fatty acid metabolism (78), inhibits hypothalamic AMPK
(79). Of note, this effect is necessary for C75 to reduce food intake (79). Together, these
data argue that neuronal nutrient metabolism is monitored by CNS fuel sensors and
contribute to the regulation of food intake.
5. 1. C75: a FAS inhibitor.
Originally used in cancer biology for its anti-tumor properties, cerulenin (2,3- epoxy-4-oxo-6-dodecadienoylamide), a natural antibiotic from Cephalosporium
- 19 - ceruleans and a FAS inhibitor, was found to cause profound weight loss in vivo (80).
Thus, it was a serendipitous discovery that modulation of the FAS system, a pathway involved in de novo fatty acid synthesis, can regulate energy balance. Interested in further
studying the mechanisms underlying this effect, but concerned by the toxicity and low
solubility of cerulenin, a group of researchers at the John Hopkins University developed
the synthetic analogue C75 (81). C75 is a potent selective inhibitor of FAS that
inactivates its β-ketoacyl-acyl synthase, enoyl reductase and thioesterase activities (82,
83). In vivo, this translates into a reduction of long chain fatty acids synthesis in the liver,
together with an increase in malonyl-CoA, a substrate of FAS. Intraperitoneal
administration of C75 causes profound, dose-dependent, anorexia and weight loss in rats
and mice (48, 49, 84). These effects are also observed with intraventricular (icv)
administration of much lower doses, suggesting a CNS action (48).
5. 2. Fatty acid metabolism in the CNS.
It has long been established that the FAS system is regulated by the nutritional
status of animals in lipogenic tissues such as liver and adipose (85). However, the CNS is
not lipogenic and derives most of its ATP from the oxidation of glucose rather than lipids
(86). Consequently, there was little reason to think that FAS played an important role in
the CNS. So, the discovery that FAS and the other enzymes involved in de novo fatty
acid synthesis are present in areas critical for the regulation of energy balance was quite
surprising (47, 87). Studies using C75, and other pharmacological as well as genetic tools
that modulate the activity of different enzymes of the FAS pathway in the CNS, suggest
that this system senses and integrates signals about the whole body nutrient status (48, 49,
- 20 - 84, 88, 89). This triggers adaptive responses in food intake and energy expenditure that ensures adiposity remains within the defended range over time.
5. 3. Potential mechanisms for the effects of C75 on food intake and energy expenditure.
The effects of C75 on the FAS system are thought to mimic a state of nutrient surplus, like if the animal would be refed (84). Modulation of fatty acid and glucose metabolism in the CNS play a crucial role in the regulation of energy homeostasis (33,
77). Evidence suggests that C75 reduces food intake by modulating these processes, and changing the expression of orexigenic and anorexigenic neuropeptides in the arcuate nucleus (51). There are a number of intracellular signals that have been proposed to mediate the anorexigenic effect of C75 in the CNS. These include increase in the levels of malonyl-CoA (84), long-chain fatty acid-CoA (LCFA-CoA) (88) and ATP (79), and the ratio of (glucose/fatty acid) utilization (49). In addition, some of these mechanisms might also be involved in the effects of C75 on energy expenditure (90, 91), although the latter is a controversial issue with reports of actual decrease after ip C75 (48). Each of these is discussed in the following paragraphs.
5. 3. 1. NPY/AgRP and POMC/CART neurons.
A striking aspect of C75 is its ability to block the normal hypothalamic response to fasting. A single injection of C75 (30 mg/kg, ip), despite suppressing 96% of food intake and causing 45% more weight loss than fasted controls, prevents the stimulation of orexigenic NPY/AgRP neurons (84) and the inhibition of POMC/CART neurons (92).
- 21 - C75-treated rats display hyperphagia in response to NPY injection, suggesting that the
NPY signaling pathway is intact in these animals (84). In addition to regulating CNS
pathways, C75 has also been reported to inhibit the secretion of ghrelin from the stomach
(93). Together, these data suggest that C75 stimulates a signal within the FAS pathway
that mimics the fed state. The organism is fooled into believing that it is in conditions of
energy surplus, and initiates the appropriate responses to restore energy balance: reducing
caloric intake (84) and possibly increasing caloric expenditure (90).
5. 3. 2. C75 and lipid metabolism.
5. 3. 2. 1. Malonyl-CoA.
Since the discovery of leptin, it became increasingly clear that NPY/AgRP- and
POMC/CART-secreting neurons integrate signals about the status of stored and readily
available energy (94). What remain poorly understood are the intracellular signals that inform these neurons about the energy status and influence their activity. Loftus et al. proposed that increased malonyl-CoA and reduced fatty acid oxidation, two features of a positive energy balance, mimic the fed state and underlie the changes observed in NPY and POMC mRNA expression (84, 92).
In most cells, the levels of malonyl-CoA depend on the relative activities of the
enzymes that regulate its synthesis and degradation: acetyl-CoA carboxylase (ACC) and malonyl-CoA decarboxylase (MCD), respectively. Increased fuels elevate cytosolic
malonyl-CoA levels, by increasing acetyl-CoA as well as citrate, which is an activator of
ACC. Thus, in peripheral cells malonyl-CoA is an indicator of cellular energy status (95).
- 22 - A rise in malonyl-CoA results in the inhibition of carnitine palmitoyl CoA transferase 1
(CPT-1), the rate-limiting enzyme for the translocation of LCFA-CoA into the
mitochondria where they undergo β-oxidation favoring lipid storage into triglycerides.
The hypothalamus expresses both isoforms of ACC (ACC-1 and ACC-2) and CPT-1
(CPT-1A and CPT-1B) (96). This is important since ACC-2 is thought to be the isoform responsible for generating the malonyl-CoA that in turn inhibits the CPT-1B isoform.
This mechanism controls the switch from fatty acid to glucose oxidation, and therefore determines the cytoplasmic levels of LCFA-CoA (97).
Loftus et al. reasoned that a signal produced by C75 must mimic the fed state (84).
It was hypothesized that increased hypothalamic malonyl-CoA should be a feature
common to both conditions and so they tested whether it could be a candidate for
mediating the catabolic actions of C75. Indeed, C75 is no longer effective at reducing
food intake and body weight when malonyl-CoA accumulation is prevented. This has
now been demonstrated using both direct (84) and indirect inhibition of ACC (79), as
well as constitutive overexpression of malonyl-CoA decarboxylase (89). To date, there is
still no consensus as to whether or not malonyl-CoA is the signal that makes C75-treated
animals act as if they are in a state of positive energy balance.
Nonetheless, growing evidence supports a role for hypothalamic malonyl-CoA in
the regulation of food intake. Several studies have demonstrated that manipulations that
modulate the activity of ACC, MCD or CPT-1 specifically in the hypothalamus, in a
direction that elevates malonyl-CoA levels, potently inhibit feeding and weight gain, and
- 23 - vice versa (89, 96, 98). Furthermore, hypothalamic malonyl-CoA appears to be a downstream target of leptin signaling, as blocking its elevation with ACC inhibitor also prevents leptin-induced anorexia (52). Recently, Rossetti and colleagues found that rats given ad libitum access to a high fat diet, for a period as short as 3 days, showed reduced hypothalamic levels of malonyl-CoA under basal conditions or following an intravenous infusion of lipids, as compared to rats maintained on standard chow diet (96). While malonyl-CoA is undeniably a nutritionally regulated parameter in the CNS, it is still unclear whether or not it constitutes the metabolic signal that is monitored by neurons and leads to the regulation food intake. An alternative possibility could be that it is regulated in parallel with another factor which actually plays that role.
5. 3. 2. 2. CPT-1 and LCFA-CoA.
Evidence also suggests that C75’s regulation of CPT-1 activity could be involved in causing hypophagia. There are at least two schools of thought regarding the mechanisms that could underlie this effect. One of them refers to the initial inhibitory action of C75 on
CPT-1 (78). While the contribution of this effect to C75-induced anorexia has not been specifically tested, the “LCFA-CoA fuel sensing” model argues that it is a crucial component. According to this model, circulating lipids such as LCFA-CoA regulate feeding by generating an increase in the cellular LCFA-CoA pool in the hypothalamus.
LCFA-CoA, in turn are believed to signal nutrient surplus within the hypothalamus, which activates neural pathways designed to restrict further nutrient intake (88). This hypothesis would predict that the initial inhibitory effect of C75 on CPT-1 may account for the anorexia of C75 by causing LCFA-CoA to accumulate in the cytosol (99). In fact,
- 24 - raising the levels of hypothalamic LCFA through pharmacological (100) or genetic (96)
inhibition of hypothalamic CPT-1A has consequences similar to C75 administration in
terms of food intake and hypothalamic neuropeptide profile (84), and so does icv
administration of oleic acid (18). Furthermore, increased plasma lipid concentration is
indeed accompanied by increased hypothalamic LCFA-CoA levels (96). Intravenous (iv)
infusion of a lipid emulsion (96), or central administration of the LCFA oleic acid (18)
both inhibit food intake supporting the hypothesis that circulating LCFA can signal the
nutritional status of the body to the CNS.
5. 3. 2. 3. Biphasic regulation of CPT-1.
Another school of thought concerns the possible stimulatory effect of C75 on
CPT-1, and its correlation with increased neuronal ATP levels (78, 79). C75 and cerulenin can increase CPT-1 activity in muscle and liver, as well as in cultured adipocytes, hepatocytes and neurons (78, 90, 101). However, these findings have to be interpreted with caution since the stimulatory action of FAS inhibitors is in fact preceded by an initial inhibition of CPT-1 activity, and is therefore characterized as a “biphasic” modulation (101). Nonetheless, according to Ronnett and colleagues, who found this delayed effect in primary cortical neurons (78), C75 could impact both sides of the energy balance equation through this mechanism.
- 25 - 5. 3. 2. 4. CPT-1 and energy expenditure.
There is evidence that systemic administration of C75 (102) or cerulenin (103)
stimulates energy expenditure, a conclusion supported by pair-feeding experiments (104).
This is accompanied by an increase in lipid oxidation, which is unexpected from a compound that elevates cytosolic malonyl-CoA and would be predicted to actually
reduce lipid oxidation through inhibition of CPT-1 (105). This is where the proposed
direct action of C75, i.e. independent of its inhibitory effect on FAS, comes into play
(78). It is difficult to isolate the relative contribution of the inhibitory versus stimulatory
effects of C75 on CPT-1 in regards to caloric expenditure and intake. Nonetheless, the
CPT-1 inhibitor Etomoxir abolishes the thermogenic effect of C75, supporting a role for
CPT-1 activation in C75’s effects (90).
5. 3. 2. 5. CPT-1, ATP and AMPK.
C75 has been reported to modulate neuronal ATP levels in a pattern that correlates
with its biphasic action on CPT-1 activity. In primary cortical neurons, C75 initially
lowers ATP levels, but baseline levels are recovered within an hour after which, ATP
levels rise for at least 15 hours (78). Ronnett and colleagues proposed that a similar
phenomenon could be going on in hypothalamic neurons in vivo, where changes in cellular energy balance may underlie the catabolic actions of C75. This increase in ATP concentration could be a direct effect of CPT-1 activation, or an indirect consequence of the initial decrease of the ATP/AMP ratio (78). This signal might then be integrated by fuel sensors, including AMPK. Such a model has been proposed based on the observation
- 26 - that C75 dephosphorylates and inactivates hypothalamic AMPK, an effect that is required
for its anorectic action (79).
5. 3. 3. C75 and glucose metabolism.
The mechanisms discussed so far are related to the effects of C75 on fatty acid oxidation, and they suggest that the signal sensed by neurons as a state of positive energy
balance lies within fatty acid metabolism. However, C75 also affects neuronal glucose
metabolism. Indeed, both fatty acid and glucose oxidation are significantly increased by
C75 in neurons in vitro (78). Because both pathways are expected to generate ATP, which seems to be part of the signal monitored by fuel sensors (38, 69), the relative contribution of fatty acid versus glucose metabolism to C75-induced anorexia is a matter of debate.
5. 3. 3. 1. Ratio of glucose/fatty acid utilization.
Wortman and colleagues proposed that it is actually the relative ratio of glucose
utilization to fatty acid utilization that is monitored by neurons and signals the overall
energy balance of the body. Indeed, findings from our laboratory demonstrated that C75-
induced anorexia depends on its ability to increase glucose utilization, as a result of
reduced lipid use (49).
5. 3. 3. 2. Glucose: the primary fuel of the brain.
The brain is different from most other tissues in having specific requirement for
glucose as its primary fuel, and oxidizing few fatty acids (86). Blood glucose
- 27 - concentration is a well regulated parameter in mammals, and it remains within a
relatively narrow range under most circumstances. Clearly, large reductions or elevations
in blood glucose levels that occurs during hypoglycemia or hyperglycemia can drive
rapid and profound changes in food intake (106, 107). However, there is very little
evidence that fluctuations within physiological limits play any role in meal initiation or
termination (108-110), in contrast to what the glucostatic hypothesis proposes (35). One
hypothesis is that basal fluctuation in blood glucose concentration participates in a “state-
dependent” regulation of food intake, as one of the several signals that appear to be
integrated by fuel sensors in metabolic neurons such as NPY/AgRP and POMC/CART
neurons of the hypothalamus (33).
While for most neurons glucose serves as a fuel, specific populations of neurons,
referred to as “glucosensing” neurons, also use glucose as a signal to regulate their
activity (111). In contrast to most neurons, glucosensing neurons are localized next to
areas with fenestrated capillaries and ependymal and tanycyte lining cells, including the arcuate and the ventromedial nucleus of the hypothalamus. Glucosensing neurons are therefore likely to be exposed to fluctuations in glucose concentration that are reflective of those occurring in the brain, CSF and blood. They are believed to monitor and integrate the quantitative and temporal changes in glucose concentration, and in turn regulate their activity and neurotransmitter release accordingly (33). They are subdivided in two categories that refer to their response in the presence of increased glucose concentration: glucose-excited (GE) and glucose-inhibited (GI) neurons. Relevant to the present discussion is that GE neurons in the arcuate secrete POMC (112) and GI neurons
- 28 - secrete NPY (113) , and despite their names, glucosensing neurons actually integrate a
variety of metabolic signals (33). This includes signals of stored fuels such as leptin and
insulin, as well as signals of currently available fuels such as free fatty acids (114).
Moreover, selective ablation of glucosensing neurons causes an obese phenotype (115).
Together, these data strongly argue for glucose metabolism as being an important factor
in CNS fuel sensing.
5. 3. 3. 3. Glucose and alternate sources of fuels.
The brain has glucose transporters that maintain a relatively high glucose concentration in the extracellular space, even when circulating glucose levels are at their
lowest levels (116). This system ensures that glucose availability is sufficient for energy
production under most circumstances. Astrocytes also play a crucial role in maintaining
the brain in a “protected” environment, by providing neurons with fuels, such as lactate, glutamine and ketones (117, 118), which can be used instead of glucose to generate ATP.
This relationship ensures that the brain always has proper energy supplies, including
during fasting or intense neuronal activity. Hence, elevation of any of these factors in the
CNS is presumed to decrease glycolysis and oxidative glucose metabolism.
5. 3. 3. 4. Effects of C75 in conditions of reduced neuronal glucose utilization.
As mentioned earlier, the levels of malonyl-CoA are determined by the levels of
acetyl-CoA, and the relative activity of ACC and MCD. There is no evidence that any of
these factors are affected by the presence of lactate, glutamine or elevated ketones, and
therefore increasing the levels of any of these alternate fuels is not expected to prevent
- 29 - malonyl-CoA accumulation in the cytosol. Thus, CPT-1 should still be inhibited and lipid oxidation should still be reduced. According to the “malonyl-CoA hypothesis”, C75 would be predicted to reduce food intake under these conditions, regardless of reduced glucose utilization (84). This was the rationale behind the experiments of Wortman et al., who demonstrated that providing rats with any of these alternative fuels abolished the anorectic action of C75 (49). These data imply that the anorectic signal of C75 depends on increased glucose utilization which occur secondary to malonyl-CoA-induced inhibition of fatty acid oxidation. An additional implication is that the signal perceived by neurons as a state of energy surplus must be generated during glycolysis since glutamine and lactate, two post-glycolytic intermediates, also attenuate the anorexigenic effect of
C75 (49). In summary, increased malonyl-CoA and reduced lipid use would appear to be insufficient per se to elicit the anorexigenic action of C75.
5. 4. Rationale for the research.
C75 alters neuronal glucose and fatty acid metabolism (78) and inhibits
hypothalamic AMPK, and this effect is necessary for C75 to reduce food intake (79). In
contrast to AMPK, hypothalamic mTOR signaling is activated in conditions of positive
energy balance, and its inhibition stimulates food intake in rats (19). These data, together
with the observation that AMPK inhibits mTOR in vitro (119), lead to the hypothesis that
C75-induced anorexia depends on its ability to activate hypothalamic mTOR signaling.
Inhibitors of glycolysis reduce the phosphorylation and activation of the mTOR pathway
in vitro (39). The anorexigenic action of C75 is abolished in conditions of decreased neuronal glucose utilization. Therefore, it is likely that hypothalamic mTOR signaling is
- 30 - reduced under conditions of reduced neuronal glucose metabolism, such as during
ketosis. Chapter 3 describes experiments testing this hypothesis.
6. A role for oleoylethanolamide (OEA) in the regulation of energy balance.
It has been discussed earlier that the long-term maintenance of energy balance is dependent on the coordination and interpretation of signals indicating sufficient long- term energy stores, such as those of leptin and insulin, as well as short-term meal-related
signals. This second category includes hormones secreted from the gastrointestinal tract in response to the mechanical and chemical stimulation of nutrients, a well-characterized example being cholecystokinin (CCK) (120). They mediate their action neurally through vagal afferents, and/or humorally as circulating ligands for receptors located in the
periphery and the CNS (121). Until recently, most of the meal-induced gastrointestinal
signals discovered have been peptides. It was quite unexpected to find that nutrient- derived fatty acids can also be involved in the “meal to meal” regulation of feeding behavior.
6. 1. OEA synthesis and regulation.
OEA is member of the fatty acid ethanolamide (FAE) family, a group of lipid-
derived signaling factors found in animals and plants (122). FAE are characterized by an
ethanolamine residue linked to a long chain fatty acid through an amide bound. The
physiological significance of FAE was first recognized about 50 years ago, with the
demonstration that palmitoylethanolamine is an anti-inflammatory factor (123). However,
these lipids received little attention until the early 90’s, when the endocannabinoid
- 31 - anandamide and its receptors, cannabinoid receptors 1 and 2 (CB1 and 2), were
discovered (124-126). Five years ago, a physiological role has been described for OEA.
Based on the findings that OEA synthesis and secretion is regulated by nutrient ingestion,
and that it reduces food intake and body weight gain when injected to rodents, the
research group of Piomelli proposed the involvement of OEA in the regulation of energy
balance (127).
OEA is synthesized from a phospholipid precursor, which is found in the lipid
bilayer of the cell membrane, through the concerted action of two enzymes:
phospholipase D (PLD) and N-acyltransferase (NAT) (128). Rather than being stored in
vesicles, like classical neurotransmitters and hormones, OEA is produced and released in
a stimulus-dependent manner (129). After release, it is transported back into cells and
rapidly degraded by fatty acid amide hydrolase (FAAH) (130) or palmitoylethanolamide-
preferring acid amidase (PAA) (131), giving rise to oleic acid and ethanolamine. FAAH
is present in all mammalian tissues but highly expressed in liver and brain (132), while
PAA is found predominantly in lung, spleen and small intestine (131).
6. 2. OEA: a nutrient-derived lipid mediator.
While a variety of pharmacological agents activate the synthesis of OEA in
neurons in vitro (133), the physiological stimuli in the brain remain unknown. In the intestine (duodenum and jejunum) however, OEA synthesis and release is regulated in a nutrient-dependent pattern: reduced during fasting and increased upon refeeding. The same is true for NAT, the enzyme that synthesizes it (134). This pattern of secretion is
- 32 - consistent with the anorectic action of OEA (134). In addition to inhibition of food
intake, OEA also stimulates lipolysis and fatty acid oxidation, and therefore reduces
adiposity via actions on both sides of the energy balance equation (135).
6. 3. OEA and its receptors.
Although OEA is a structural analogue of the endocannabinoid anandamide, it does
not bind cannabinoid receptors (136). Rather, the activation of peroxisome proliferator-
activated receptor alpha (PPAR-α), a member of the nuclear hormone receptor family of
transcription factors, accounts for its effect on food intake and lipid metabolism (127,
135). In addition, OEA binds with high affinity to the capsaicin receptor (TRPV1), an ion
channel (137) known for its role in pain (138) and locomotion (139). Recent work also
indicates that it is an agonist at GPR119 (140), which is an orphan G protein coupled
receptor (GPCR) expressed predominantly in the brain, pancreas and gastrointestinal tract
(141). Synthetic agonists of this receptor also reduce food intake and body weight (140),
suggesting that it could also be involved in the ability of OEA to regulate energy balance.
6. 4. Hypothetical model for OEA-induced anorexia.
The role of PPAR-α in mitochondrial and peroxisomal fatty acid oxidation has been well characterized, but has never been linked to the control of food intake before
studies involving OEA (127, 142). Because both OEA (143) and its receptors are found
in the brain (144, 145), including in the hypothalamus, it is quite intriguing that OEA
does not affect food intake when injected into either the lateral (134) or third ventricle (K.
Proulx, unpublished data). Nonetheless, systemic administration of OEA stimulates c-
- 33 - FOS in neurons of the PVN, supraoptic nucleus (SON) and nucleus tractus solitarius
(NTS) (134). Very little is known about the relative contribution of these neuronal populations to OEA-induced anorexia. A model has been proposed where OEA activates
PPAR-α located in peripheral tissues, and regulates brain circuits involved in the control of food intake through the vagus nerve (133). This is based on a report that the anorexigenic effect of OEA is abolished in rats in which the subdiaphragmatic vagus nerve has been severed (134). The latter point is a controversial one, since another group found that OEA remains effective at reducing food intake in vagotomized rats (146).
Since it is not clear whether or not OEA mediates its action neurally through vagal afferents, the possibility that OEA alters a peripheral factor, which itself regulates hypothalamic neurons, cannot be ruled out. In fact, an activation of PVN neurons secreting the anorexigenic neuropeptide corticotropin releasing hormone (CRH), or an inhibition of arcuate NPY neurons (such effect would not be expected to be detected by c-FOS, which is a marker of neuronal activation) would be consistent with the CNS c-
FOS pattern induced by OEA (134).
6. 5. Rationale for the research.
It is important to note that OEA reduces food intake, primarily by delaying meal onset and the size of the first meal only (147). This is a crucial observation given that it also reduces motor behavior (134). Although OEA did not cause a conditioned taste aversion per se in the paradigm used in the original report of Piomelli, the drug abolished the preference rats normally display for saccharin over water (134). Therefore, reduced motor activity or illness could each be responsible for the delayed meal initiation and
- 34 - reduced first meal size caused by OEA. Chapter 2 describes experiments that assess
whether the effects of OEA on food intake are due to unspecific mechanisms or whether
they occur through specific actions on other nutrients-derived fuel signals, including
CCK and ghrelin.
- 35 -
CHAPTER 2: MECHANISMS OF OLEOYLETHANOLAMIDE (OEA)-INDUCED
CHANGES IN FEEDING BEHAVIOR AND MOTOR ACTIVITY
Karine Proulx , Daniela Cota , Tamara R. Castañeda , Matthias H. Tschöp, David A.
D’Alessio, Patrick Tso, Stephen C. Woods and Randy J. Seeley.
36
ABSTRACT
Oleoylethanolamide (OEA), a lipid synthesized in the intestine, reduces food intake and stimulates lipolysis through peroxisome proliferator-activated receptor-alpha
(PPAR-α). OEA also activates transient receptor potential vanilloid type 1 (TRPV1) in vitro. Because the anorexigenic effect of OEA is associated with delayed feeding onset and reduced locomotion, we examined whether ip administration of OEA results in non- specific behavioral effects that contribute to the anorexia in rats. Moreover, we determined whether circulating levels of other gut hormones are modulated by OEA, and whether cholecystokinin (CCK) is involved in OEA-induced anorexia. Our results indicate that OEA reduces food intake without causing a conditioned taste aversion or reducing sodium appetite. It also failed to induce a conditioned place aversion.
However, OEA induced changes in posture and reduced spontaneous activity in the open- field. This likely underlies the reduced heat expenditure and sodium consumption observed after OEA injection, which disappeared within 1 hour. The effects of OEA on motor activity were similar to those of the TRPV1 agonist capsaicin, and were also observed with the PPAR-α agonist Wy-14643. Plasma levels of ghrelin, peptide YY
(PYY), glucagon-like peptide 1 (GLP-1) and apolipoprotein A-IV (apo A-IV) were not
changed by OEA. Finally, antagonism of CCK-1 receptors did not affect OEA-induced anorexia. These results suggest that OEA suppresses feeding without causing visceral
illness, and that neither ghrelin, PYY, GLP-1, apo A-IV nor CCK play a critical role in this effect. Despite that OEA-induced anorexia is unlikely to be due to impaired motor activity, our data raise a cautionary note in how specific behavioral and metabolic effects
of OEA should be interpreted.
37
INTRODUCTION
Energy homeostasis in mammals is tightly regulated by complex neuroendocrine
systems matching caloric intake to energy expenditure. In addition to hormones, fatty
acids (18, 28) and fatty acid derivatives (148-150) also signal to central nervous system
(CNS) pathways that control body weight regulation. Recent evidence suggests that
oleoylethanolamide (OEA), the amide of oleic acid and ethanolamine, could play a
significant role in the regulation of energy balance (134). This lipid is synthesized in
astrocytes (151) and neurons (152), as well as in cells of the small intestine where its
levels are reduced by fasting and increased upon refeeding (134).
OEA is a structural analogue of the endocannabinoid anandamide but does not
activate cannabinoid receptors (CB) (136). It is rather an endogenous ligand for the
peroxisome proliferator-activated receptor-alpha (PPAR-α), through which it has been
reported to stimulate lipolysis (135) and inhibit feeding (127). Indeed, OEA reduces food intake when administered peripherally, but appears to be ineffective at doing so when administered centrally (127). In ad libitum fed rats the suppression of food intake is associated with increased latency to initiate a meal without change in meal size or postmeal interval, whereas in fasted rats OEA increases latency to initiate a meal and reduces size of the first meal (147). Furthermore, chronic administration of OEA can reduce the rate of body weight gain in both lean (134) and obese animals (135). All of
these effects are absent in mice that lack PPAR-α, supporting a critical role for these
receptors in the metabolic effects of OEA (127).
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Rodriguez de Fonseca et al. have reported that although intraperitoneal (ip)
administration of OEA reduces food intake without causing a conditioned taste aversion,
it potently reduces locomotor activity in rats (134). While the effects of OEA on feeding
appear to be mediated via PPAR-α activation (127), the mechanisms by which it reduces
locomotion are unknown. It is also unknown whether other PPAR-α agonists have
independent effects on locomotor activity as well. On the other hand, OEA activates the
transient receptor potential vanilloid type 1 (TRPV1) in vitro (137). This receptor is
widely distributed in the nervous system, including on dopaminergic neurons of the substantia nigra (145), a region well known to be involved in the regulation of motor activity. Interestingly, capsaicin-induced activation of the TRPV1 causes a reduction in ambulation as well as other motor behaviors such as rearing and grooming in the open- field test (139).
Although the effects of OEA on feeding behavior are intriguing, consideration of
OEA as a target for pharmacological treatment of obesity requires that several basic
questions be addressed. Thus, the fact that OEA reduces locomotor behavior (134) is
noteworthy given that OEA reduces food intake by delaying meal initiation and reducing
first meal size (147). Given these behavioral effects, we reassessed the possibility that ip
administration of OEA results in non-specific behavioral effects, visceral illness and/or
aversion, which contribute to the reduced food intake. To examine this possibility, we
used three very different paradigms: conditioned taste aversion (CTA), need-induced
sodium appetite and conditioned place aversion (CPA). Furthermore, because of the
potential involvement of reduced motor activity in the anorexigenic effect of OEA, we
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characterized the effects of OEA on motor behavior using computer-based video analysis
and an open-field test. We explored whether TRPV1 receptor activation can alter motor
behavior, and we predicted that a TRPV1 agonist would recapitulate the hypolocomotor effects of OEA while a PPAR-α agonist would not. Assessing the metabolic consequences of OEA administration is also important in studying its role in energy homeostasis. Thus, because OEA up-regulates uncoupling protein-2 (UCP-2) mRNA in both white adipose tissue (WAT) and skeletal muscle (135), we tested whether OEA increases energy expenditure using indirect calorimetry.
OEA shares some similarities with several gut hormones involved in the
regulation of food intake. Like OEA, ghrelin, peptide YY (PYY), glucagon-like peptide
1 (GLP-1), apolipoprotein A-IV (apo A-IV) and cholecystokinin (CCK) are influenced by
the presence of nutrients in the gut (121). Thus, in addition to an interest in the
behavioral mechanisms by which OEA might suppress food intake, we were also
interested in how other gut hormones that have been linked to the control of food intake
might be influenced by OEA. To this end, we measured the plasma levels of ghrelin,
PYY, GLP-1 and apo A-IV at several time points after OEA administration. Given that
CCK (153), like OEA (134), is synthesized in the intestine and mediates its anorexigenic
signal via the vagus nerve, we tested a specific role for endogenous CCK in the effects of
OEA with the use of a pharmacological antagonist of the CCK-1 receptor.
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MATERIALS AND METHODS
Animals: Male Long-Evans rats, weighing 250-300 g at the beginning of the
experiments, were individually housed and maintained on a 12:12-h light-dark cycle. All animal protocols were approved by the University of Cincinnati Institutional Animal
Care and Use Committee.
Drugs: OEA was purchased from Tocris Cookson Inc. (Ellisville, MO). Lorglumide,
CCK-8, Wy-14643 and capsaicin were purchased from Sigma (St. Louis, MO). OEA
was dissolved in 5% Tween-80, 5% propylene glycol and 90% physiological saline.
Capsaicin was first dissolved in 100% ethanol. The stock solution was then dried under
nitrogen after addition of Tween-80, and reconstituted in physiological saline (Tween-
80/saline; 1:16 v/v). The PPAR-α agonist Wy-14643 was dissolved in 70% DMSO and
30% physiological saline. Lithium chloride (LiCl) was dissolved in sterile distilled water
(except in Experiment 2) while lorglumide, CCK-8, and furosemide were dissolved in
physiological saline. For each experiment, vehicle served as the control solution, except
for LiCl where saline was used as a control. All drugs were injected ip in a volume of 1
ml/kg, unless otherwise specified. OEA was administered at 5, 10 and 20 mg/kg (134) in
Experiment 1. Given that 20 mg/kg was the only dose that remained effective at reducing
food intake 1 h after the injection, this dose was used in all subsequent experiments.
Experimental design:
Experiment 1: Effect of OEA on food intake. Rats were assigned to one of four treatment groups (n = 8-9/group). Thirty minutes prior to the onset of the dark phase, 24-h fasted
rats received an injection of either vehicle or OEA (5, 10 or 20 mg/kg, ip) (134) and then
41
received access to food. The food hoppers were weighed at 30 min, 1, 2, 4 and 24 h after
the injection.
Experiment 2: Effect of OEA on CTA. If rats become ill after consumption of a novel
flavor, they will avoid consumption of this flavor at future opportunities (154). An
example of this is reduced consumption of a flavor that has been previously paired with
toxins such as LiCl (155). In order to test whether the anorexigenic effect of OEA is
secondary to illness, rats were assigned to one of two groups (saline-LiCl or vehicle-
OEA, n = 10/group) and preference ratios for novel flavors were determined after pairing
with the drugs. The preference ratio for a drug is calculated as the intake of the drug-
paired flavor over the total intake of both flavors and the critical comparison is whether the ratio for either flavor deviates from 0.5. All animals were trained for 10 days on a water deprivation schedule during which they had access to two water bottles for 1 h/day.
On conditioning day 1, all rats had access to two bottles of saccharin-sweetened Kool-
Aid (5.25 g of saccharin, 3500 ml of water and one packet of either cherry or grape Kool-
Aid; both bottles contained the same flavor, in a counterbalanced manner across rats and
groups) for 1 h. After access to flavor 1, half of the rats from each group received either
saline or vehicle and the other half received either LiCl (0.15 M in a volume equivalent to
2% of the rat’s body weight) or OEA (20 mg/kg, ip). The following day, rats had access
to two water bottles for 1 h, as a rest day. On conditioning day 2, rats had access to two
bottles of flavor 2 for 1 h. Rats that were injected with saline or vehicle on conditioning
day 1 were then injected with LiCl or OEA, and vice versa. Rats then had 2 rest days in
which they had access to two bottles of water for 1 h. On the test day, rats received one
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bottle of each flavor, with the side of flavor presentation being switched at 30 min to avoid any side preference. Fluid intake was measured at 1 h, and water was returned at
the end of the experiment.
Experiment 3: Effect of OEA on need-induced sodium appetite. Another reliable index of
visceral illness is failure to consume hypertonic saline solution in response to sodium
depletion. For instance, when rats rendered sodium-depleted are injected with emetic
and/or toxic agents such as LiCl, they reduce their “need-induced” consumption of
hypertonic saline (156). In order to assess whether OEA is aversive by this definition,
rats were assigned to one of two treatment groups (vehicle or OEA, n = 7/group) and
need-induced sodium appetite was assessed. For 7 days, rats had access to two bottles,
one that contained water and another one that contained a 0.5 M NaCl solution. Twenty-
four hours prior to the experiment, the saline bottle was removed and the food hopper
with regular diet was replaced with one that contained sodium-free rat diet (ICN
Biochemicals, Cleveland, OH). Water remained available at all time. Rats were then
weighed and received two subcutaneous injections of the diuretic furosemide (5mg/kg,
ip), 1 h apart (157). Rats were weighed again 3 h after the first injection, and diuresis
(and presumed sodium depletion) was confirmed by observing at least 18 g of body
weight loss. Twenty-four hours after the first furosemide injection, rats received an ip
injection of isotonic LiCl (0.15 M in a volume equivalent to 2% of the rat’s body weight),
an equal volume of physiological saline, OEA (20 mg/kg) or vehicle. Fifteen minutes
later, rats received two bottles; one that contained distilled water and another one that
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contained a 0.5 M NaCl solution. Intake of both fluids was measured every 30 min for 2
h, and 24 h after the injection.
Experiment 4: Effect of OEA on conditioned place aversion (CPA). The conditioned
place aversion test is a behavioral procedure in which an animal forms an association
between a stimulus of negative value and the location in which it experienced the
stimulus during training. CPA training and testing took place in a 78 x 21-cm box (Med
Associates, Inc.). The box was divided into a black side and a white side (each 25.5 x 21 cm) separated by middle chamber (12 x 21 cm) with two doors. The floor of each side was made of two distinct textures. The training procedure consisted of 6 contiguous days in which rats were placed in alternating sides of the CPA box for 30 min. On one set of alternate days (Days 1, 3, and 5 or Days 2, 4, and 6), rats were injected with either LiCl
(0.15 M in a volume equivalent to 2% of the rat’s body weight) or OEA (20 mg/kg, ip)
and were immediately placed on one side of the CPA box with the door closed. On the
other set of alternate days, rats received either saline or vehicle and were immediately
placed in the alternate side of the box from that paired with the drug on the previous day,
and the door was closed. Conditions were counterbalanced across rats and groups. On
the 7th day (test), rats were placed into the middle chamber and were allowed to explore
the apparatus with both guillotine doors opened for 15 min. Rats were videotaped and
time spent in each chamber was scored by an investigator who was blind to the drug
treatment. An animal was considered to be in a chamber when its head and forepaws were inside it. Results are expressed as the time spent in the drug-paired side.
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Experiment 5: Effect of OEA on heat expenditure. Rats were assigned to one of two
treatment groups (vehicle or OEA, n = 8/group). Four hours before testing, rats were placed in indirect calorimeter chambers for habituation. They were then injected with either vehicle or OEA (20 mg/kg, ip) 30 min before the onset of the dark and returned to the indirect calorimeter chamber immediately after the injection. Food was removed but water remained available throughout the experiment. Heat production was recorded every 24 min for 24 h.
Experiment 6: Comparison of the effects of OEA and capsaicin on behaviors in home
cage. This study examined the effects of OEA on behaviors displayed by rats in their
home cage. Fifteen minutes before the onset of the dark, rats (n = 5-7/group) received an
injection of OEA (20 mg/kg, ip), capsaicin (1 mg/kg, ip) (12) or their respective vehicles,
and were immediately returned to their home cage. Rats were videotaped for 2 h and
behavioral analysis was conducted using a computerized system (HomeCageScan
software, Clever Sys. Inc.), that recognizes, records and quantifies upwards of 22 distinct
rodent behaviors in a home cage environment.
Experiment 7: Comparison of the effects of OEA, capsaicin and Wy-14643 in the open-
field test. This experiment compared the effects of OEA, capsaicin and Wy-14643 on
motor behaviors. Rats (n = 5-7/group) were injected with OEA (20 mg/kg, ip), capsaicin
(1mg/kg, ip) (12), Wy-14643 (40 mg/kg, ip) (13) or their respective vehicles 10 min prior
to testing. The dimensions of the open-field were 36 x 45 cm and the height of the walls
was 30 cm. The floor was divided into 20 squares (9 x 9 cm). Rats were placed in the
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middle of the open-field, and they were videotaped for 10 min. Only the last 5 min of the tape were analyzed, the first 5 min serving as a habituation period. Time spent in inactivity, ambulation (number of squares crossed) and spontaneous activity (number of rearing or grooming events) were measured (139). Rats were considered to have crossed a square when they placed their four paws into an adjacent square. Tapes were scored by an investigator who was blind to the drug treatment.
Experiment 8: Effects of OEA on plasma levels of ghrelin, PYY, GLP-1 and apo A-IV.
Rats were injected with either OEA (20 mg/kg, ip) or vehicle 30 min prior to onset of the
dark and were sacrificed at 15, 30, 90 and 120 min after the injection. An additional
group of rats (n = 8) remained uninjected and served as baseline controls. Trunk blood
samples were collected into tubes that contained an anti-proteolytic cocktail (25 mM
EDTA plus 500 kallikrein inhibitory units aprotinin and 80 units Heparin per ml of
blood). Blood was then centrifuged at 4° C for 10 min at 4000 rpm followed by 2 min at
12,000 rpm. Separated plasma was stored at –20°C until assay. Plasma ghrelin (158)
and PYY (159) concentrations were determined using commercial RIA kits according to
manufacturer’s directions (Phoenix Pharmaceuticals, Inc., Belmont, CA.) as previously
described. The ghrelin assay measures both acylated and des-acetylated ghrelin, and the
PPY assay measures both PYY (1-36) and PYY (3-36). Total plasma GLP-1
concentration was measured by RIA using antiserum 89390 (kindly provided by Dr. Jens
Holst, Paanum Institute, Copenhagen, Denmark) from ethanol extracts of plasma as
previously described (160). This antibody recognizes both the intact hormone GLP-1 (7-
36)-NH2 and the metabolite GLP-1 (9-36)-NH2. The intra- and interassay coefficients
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of variation for these assays were < 5% and < 13%, respectively for ghrelin, < 7 % and
<11% for GLP-1, and < 8.42% and < 14.52% for PYY. Plasma apo A- IV concentration
was measured by an ELISA, as previously described (161).
Experiment 9: Effect of the CCK-1 antagonist lorglumide on OEA-induced reduction of food intake. To test the involvement of CCK-1 receptors in the anorexigenic effect of
OEA, rats were assigned to one of four treatment groups (saline + vehicle, saline + OEA,
lorglumide + vehicle or lorglumide + OEA, n = 6-7/group). Thirty minutes prior to the onset of the dark, 24-h fasted rats received an injection of either saline or lorglumide (1 mg/kg) (162). Fifteen minutes later, rats received a second injection of either vehicle or
OEA (20 mg/kg, ip) and then received access to food. The food hoppers were weighed at
30 min, 1, 2, 4 and 24 h after the last injection. As a positive control, we tested the ability of the same dose of lorglumide to block the anorexigenic effect of CCK-8. Rats were assigned to one of four treatment groups (saline + saline, saline + CCK-8,
lorglumide + saline or lorglumide + CCK-8, n = 8-9/group). Two hours prior to the onset
of the dark, 22-h fasted rats received an injection of either saline or lorglumide (1 mg/kg,
ip) (162). Fifteen minutes later, rats received a second injection of either saline or CCK-
8 (4 μg/kg, ip) (163) and then received access to food. Food hoppers were weighed at 15,
30, 45 min and 1 h after the last injection.
Statistical analysis: Changes in food intake, sodium intake and heat expenditure across time were analyzed by one-way or two-way repeated measures ANOVA, unless otherwise specified in the results. Significant ANOVAs were followed by Least
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Significant Difference post hoc tests. Preference ratios, time spent in the drug-paired
side, hormones levels, and behaviors in the home cage and in the open-field were
analyzed by two-tailed Student’s t-tests. The level of significance was set at P < 0.05.
All values are expressed as the mean ± SEM.
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RESULTS
Experiment 1: Effect of OEA on food intake. OEA significantly reduced 30-min food intake as compared to saline at 10 mg/kg (P < 0.05; see Figure 1A) and 20 mg/kg (P <
0.001; Figure 1A), with the 20 mg/kg suppressing 99.48% of food intake. One hour
following the injection, only the 20 mg/kg dose caused a significant reduction in food
intake (P < 0.01; Figure 1A) and this effect remained significant 24 h after the injection.
(P < 0.05; Figure 1B).
Experiment 2: Effect of OEA on CTA. The positive control LiCl caused a CTA as indicated by the significantly lower preference ratio for the LiCl-paired flavor compared to the saline-paired flavor (P < 0.001; Figure 2). In contrast, there was no significant difference between the preference ratio of the flavor previously paired with OEA as compared to the vehicle-paired flavor (P = 0.746; Figure 2) indicating that rats did not develop a CTA to OEA.
Experiment 3: Effect of OEA on need-induced sodium appetite. LiCl significantly
reduced need-induced sodium appetite as compared to saline at all time points (P <
0.001; Figure 3 for 2 h, data at other time points not shown) for at least 24 h (P < 0.05).
Two-tailed t-test comparisons indicated that sodium intake was significantly suppressed
by OEA relative to vehicle at 30-min (OEA: 4.37 + 1.03 g vs. vehicle: 10.03 + 0.91 g; P
< 0.01), although to a lesser extent than by LiCl (LiCl: 1.67 + 0.12 g; P < 0.05). In
contrast to rats injected with LiCl, OEA-injected rats rapidly compensated for this acute reduction as this effect had disappeared at 1-h (OEA: 8.43 + 1.06 g vs. vehicle: 11.12 +
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2.66 g; P = 0.090). Indeed, one-way repeated measures ANOVA revealed that OEA did not have a significant effect relative to vehicle over 24 h (P = 0.482; Figure 3 for 2 h, data at other time point not shown).
Experiment 4: Effect of OEA on CPA. Rats in the LiCl-saline group spent significantly
less time in the LiCl-paired side as compared to the saline-paired side (P < 0.05; Figure
4), indicating the formation of a CPA to LiCl. In contrast, OEA did not cause a CPA
since rats in the OEA-vehicle group spent a similar amount in both the OEA-paired side
and the vehicle-paired side (P = 0.849). This result together with the lack of a sustained
reduction of sodium appetite and induction of a CTA suggest that acute administration of
OEA does not have a toxic or illness-inducing effect in rats.
Experiment 5: Effect of OEA on heat expenditure. Two-tailed t-test comparison revealed
that OEA significantly reduced heat expenditure relative to vehicle when measured for
the first 48 min following the injection (P < 0.001; Figure 5A). However, this effect
waned by 72 min (P = 0.341; Figure 5A), and one-way repeated measures ANOVA
indicates that there was no significant effect of OEA treatment over the course of either
the 12-h dark (P = 0.367; Figure 5B) or the 12-h light phase (P = 0.403; data not shown).
Experiment 6: Comparison of the effects of OEA and capsaicin on behaviors in home
cage. OEA significantly increased the time rats displayed an extended posture, that is
pushing their abdomen against the floor of the cage with splayed hind limbs, as compared
with vehicle (P < 0.01; Figure 6A), while capsaicin did not cause a significant effect on
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this behavior (P = 0.334; Figure 6B). There was no significant change in the other behaviors analyzed after either OEA or capsaicin administration as compared with their respective vehicles (P > 0.05).
Experiment 7: Comparison of the effects of OEA, capsaicin and Wy-14643 in the open- field test. Rats treated with OEA were significantly more inactive (P < 0.001; Figure 7A) and had significant decreases in ambulation (P < 0.01; Figure 7B) and spontaneous activity (P < 0.001; Figure 7C) as compared to vehicle-treated rats. The effect of OEA on inactivity was recapitulated by Wy-14643 (P < 0.05; Figure 7A), but not by capsaicin
(P = 0.164; Figure 7A). However, neither Wy-14643 (P = 0.420; Figure 7B) nor capsaicin (P = 0.208; Figure 7B) had a significant effect on ambulation. Similar to OEA, both Wy-14643 (P < 0.01; Figure 7C) and capsaicin (P < 0.05; Figure 7C) significantly inhibited spontaneous activity as compared to their respective vehicles.
Experiment 8: Effects of OEA on plasma levels of ghrelin, PYY, GLP-1, and apo A-IV.
OEA did not cause any significant change in plasma levels of ghrelin, PYY, GLP-1 and apo A-IV relative to vehicle, at any of the time points that were examined (P > 0.05;
Figure 8).
Experiment 9: Effect of lorglumide on OEA-induced reduction of food intake. As reported in Experiment 1, OEA significantly reduced food intake relative to vehicle (1-h intake: P < 0.01 and 2-h intake: P < 0.001; Figure 9A). At none of the time points examined was this effect significantly modified by pre-treatment with lorglumide relative
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to saline (P > 0.05; Figure 9A), whereas the same dose of lorglumide was effective at blocking the anorexigenic effect of CCK-8 relative to saline (15- and 30-min intakes: P <
0.05; Figure 9B).
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DISCUSSION
As has been previously reported (127, 134), ip administration of OEA reduces food intake and, at 20 mg/kg, food intake is almost completely suppressed for the first 30 minutes. The anorexigenic effect of OEA is accompanied by a suppression of locomotor activity. In particular, the injection of OEA is followed by a unique behavior in which the rat takes on an extended posture (pushing its abdomen against the floor of the cage) with splayed hind limbs and little ambulatory activity. This behavior resembles the
“lying on belly” that is caused by toxins that produce visceral illness (155). However, on two sensitive assessments of visceral illness OEA had minimal effect, consistent with previous findings (134). Moreover, OEA did not induce the formation of a conditioned place aversion. Based on these results, it is difficult to ascribe the anorectic effects of
OEA to illness or aversion. Therefore, it seems likely that suppression of food intake by
OEA involves specific actions on pathways regulating energy homeostasis.
Illness in rats manifests as a pattern of well-established stereotypic behaviors.
CTA is one of the hallmark behaviors of visceral illness, and CTA tests rely on the ability to learn the association between a flavor and an aversive stimulus (154). In these studies
OEA did not support a CTA. However, other members of the fatty acid ethanolamide family, such as anandamide, have been reported to cause anterograde amnesia (164).
Thus, it is possible that OEA could induce visceral illness, but not produce a CTA because of a separate effect to impair memory. To avoid being misled by this potential confound, we used the additional measure of need-induced sodium appetite (156). While
OEA caused some reduction in sodium consumption as compared to vehicle within the
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first hour following the injection, this effect dissipated more rapidly than the effect on
food intake and was much shorter than the effect of LiCl. Taken together, these data are
consistent with the hypothesis that the effect of OEA to suppress food intake is not
secondary to visceral illness. Moreover, since the ingestion of the NaCl solution involves much of the same motor behavior as ingestion of food, it is unlikely that the anorexia is
simply a result of the reduced motor activity, at least at time points beyond 1 hour.
Given the negative results of the visceral illness and conditioned aversion tests,
the reduced locomotor activity and unusual posturing we observed after OEA administration requires an alternative explanation. We therefore further characterized the
behavior caused by OEA administration using computer-based video analysis. This assay
confirmed that rats display an extended posture with splayed hindlimbs early after an ip
injection of OEA. This behavior is also followed by signs that resemble those of
catalepsy. While the pharmacological effects of OEA are not due to activation of any of
the known cannabinoid receptors (136), it is noteworthy that we have observed
comparable behavior in rats given ip anandamide (unpublished data). Reduced
locomotor activity, ataxia and catalepsy are indeed commonly observed after
administration of cannabinoid agonists (165). Interestingly, stearoylethanolamide is
another member of the fatty acid ethanolamide family that does not activate cannabinoid
receptors but that causes cannabimimetic effects, including catalepsy (166).
In the open field test, OEA not only reduced locomotion but also grooming and
rearing, while increasing time spent in inactivity. We compared these behavioral effects
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of OEA to agonists of either PPAR-α or TRPV1. Agonists of TRPV1 such as capsaicin
have previously been reported to reduce motor activity (139) and our data indicate that
this effect is similar to what occurs with OEA. Like OEA, capsaicin significantly
reduced spontaneous activity. Capsaicin also showed a trend to reduce ambulation, and
increase inactivity and extended posture. Because of this trend, we cannot rule out the
possibility that this is a real effect of capsaicin that would be revealed with higher
numbers of animals.
Given that there have been no previous descriptions of the effects of PPAR-α to
reduce motor behavior, we were surprised to see a potent effect of the PPAR- α agonist
Wy-14643 to reduce motor activity. While these observations are consistent with the distribution of TRPV1 and PPAR-α in areas associated with motor activity such as the substantia nigra compacta and the striatum (144, 145), both receptors are widely distributed and so a firm conclusion about the mechanism for this effect is difficult. Thus it remains unclear how OEA induces postural changes and suppresses motor activity.
Since activation of the PPAR-α is necessary for OEA’s effect on food intake
(127), the current data raise the possibility that the locomotor effects are also the result of
agonism of PPAR-α. Interestingly, it has been reported that capsaicin-induced activation
of TRPV1 leads to OEA production in vitro (139). This raises the hypothesis that PPAR-
α activation, by ligands of either endogenous (capsaicin-induced production of OEA) or
exogenous (administration of either Wy-14643 or OEA) sources could mediate the motor
effects of OEA, capsaicin and Wy-14643. Nonetheless, further experiments making use
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of either highly specific receptor antagonists or targeted genetic disruption of these
receptors will be necessary to conclude whether PPAR-α and/or TRPV1 are involved in
the behavioral effects of OEA on motor activity.
Activation of PPAR-α has been associated with reduced body weight gain due to
effects on lipid catabolism and energy expenditure. For instance, the PPAR-α ligands
Wy-14643 and the fibrate drugs clofibrate, fenofibrate and bezafibrate are all
hypolipemic agents that reduce weight gain in rodent models (167). Moreover, fibrate
treatment has been associated with elevation in UCP-1 mRNA in white adipose tissue
(WAT) and in UCP-3 in WAT and skeletal muscle in rodents (168). The effects of OEA
are consistent with those of fibrates, since OEA up-regulates UCP-2 mRNA in both WAT
and skeletal muscle (135). However, contrary to what we have predicted, OEA did not
increase energy expenditure in rats. We observed a significant reduction in heat during
the first 24 min that followed the OEA administration as compared to vehicle. This effect
is likely the result of the reduced motor activity caused by OEA.
Even though it is unlikely that the effect of OEA on food intake is merely by-
product of its action on other motor behaviors, our results raise a cautionary note as to
how specific behavioral and metabolic data should be interpreted in the light of these potent actions. Recent data continue to point to a critical role in the control of food
intake for a number of gut hormones whose secretion is either increased or decreased by
nutrients being absorbed from the gastrointestinal tract (121). Thus, the possibility that
OEA could influence food intake by altering the secretion of one or more of these
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hormones was necessary to consider. We measured circulating plasma levels of ghrelin,
PYY, GLP-1 and apo A-IV at several time points after the administration of OEA at a dose that reduces food intake. OEA did not have a significant impact on plasma levels of any of these hormones at any time point compared to vehicle-treated rats. These data are consistent with a recent report indicating that OEA failed to modulate either GLP-1 or ghrelin under free-feeding conditions (168). However, in this study OEA did reduce plasma levels of acylated ghrelin in fasted animals. Conclusions from the current data are limited by the fact that rats did not have exposure to nutrients after the administration of OEA. Such a design was used to avoid the possibility that drug-induced change in feeding could influence hormone levels. Consequently, we cannot completely rule out the possibility that OEA modulates the ability of nutrients to alter gut hormone secretion.
Another gut hormone that has been linked to the control of food intake is CCK
(25). However, the critical form (CCK-8) is difficult to measure in plasma and in the rodent several lines of evidence point to paracrine rather than endocrine action of CCK to reduce feeding (169). Thus, to examine whether there may be a role for increased CCK release in the anorexigenic effect of OEA, we took advantage of a well-validated CCK-1 receptor antagonist (162). While pharmacological antagonism of CCK-1 receptors with lorglumide can block the anorexia induced by exogenous CCK-8, it cannot block the anorexia induced by OEA. Thus, it seems unlikely that CCK mediates the effects of
OEA on food intake.
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Perspectives. Taken together, these data argue against the hypothesis that ghrelin,
PYY, GLP-1, apo A-IV or CCK play a critical role in the effect of OEA to suppress food intake, at least under free-feeding conditions. Previous data indicated that neither corticosterone, leptin nor insulin play a role in OEA-induced anorexia (134). While the current experiments were not designed to test this specific hypothesis, it is tempting to speculate that OEA reduces food intake by regulating the expression of genes in the periphery that are involved in lipid metabolism. Ip administration of the fatty acid ethanolamide stearoylethanolamide has been shown to cause anorexia via such mechanism (170). Furthermore, OEA increases the expression of PPAR-α (127), which represses the transcription of lipogenic genes (171) such as hepatic fatty acid synthase
(172, 173). Growing evidence points to a role for lipid metabolism in the regulation of feeding (18, 77). These findings raise the possibility that ongoing modulation of lipid metabolism in the periphery is involved in the anorexigenic effect of OEA.
58
FIGURE LEGENDS
Figure 1. Mean + SEM food intake at different time points (A), and 24 h (B) after an ip
injection of vehicle (VEH) or three doses of OEA in 24-h fasted rats.
* = P < 0.05, ** = P < 0.01 and *** = P < 0.001.
Figure 2. Mean + SEM preference ratio of flavors previously paired with an ip injection of saline (SAL), lithium chloride (LiCl), VEH or OEA (20 mg/kg).
*** = P < 0.001.
Figure 3. Mean + SEM 2-h sodium intake after an ip injection of SAL, LiCl, VEH or
OEA (20 mg/kg) in sodium-depleted rats. *** = P < 0.001.
Figure 4. Mean + SEM time the rats spent in the side previously paired with an ip
injection of SAL, LiCl, VEH or OEA (20 mg/kg). * = P < 0.05.
Figure 5. Mean + SEM heat expenditure at different time points after an ip injection of
either VEH or OEA (20 mg/kg) (A), and mean + SEM heat expenditure per hour during
the 12-h dark phase (B) in fasted rats. *** = P < 0.001.
Figure 6. Mean + SEM time the rats displayed an extended posture in the 2 h following
an ip injection of either VEH or OEA (20 mg/kg) (A), and either VEH or capsaicin
(CAPS) (1 mg/kg) (B). ** = P < 0.01.
Figure 7. Mean + SEM time the rats spent in inactivity (A), number of times the rats crossed a square (B), and number of times the rats either reared or groomed (spontaneous activity) (C) after an ip injection of OEA (20 mg/kg), CAPS (1 mg/kg), Wy-14643 (40 mg/kg) or their respective VEH. * = P < 0.05, ** = P < 0.01 and *** = P < 0.001.
59
Figure 8. Mean + SEM plasma concentrations of ghrelin (A), PYY (B), GLP-1 (C), and apo A-IV (D) at different time points after an ip injection of either VEH or OEA (20 mg/kg).
Figure 9. Mean + SEM food intake at different time points after an ip injection of either
SAL or lorglumide (LORGL) (1mg/kg), followed by an ip injection of either VEH or
OEA (20 mg/kg) (A), and either SAL or CCK-8 (4 μg/kg) (B) in fasted rats.
* = P < 0.05 and ** = P < 0.01.
60
FIGURES
A VEH 14 OEA (5 mg/kg)
12 OEA (10 mg/kg) OEA (20 mg/kg) 10
8 6 **
Food intake (g) intake Food 4 ** 2 * 0 *** ** 0 1 2 3 4 5 Time (h) B
35
30 * 25
20
15
Food intake (g) 10
5
0 VEH 5 10 20 Dose (mg/kg)
Figure 1
61
1.00
0.75
0.50
*** 0.25 Preference ratio Preference
0.00 SAL LiCl VEH OEA
Figure 2
62
18
16
14
12
10
8
6
4 *** 0.5 intake M NaCl (g) 2
0 SAL LiCl VEH OEA
Figure 3
63
500
400 * 300
200
100
0 SAL LiCl VEH OEA Time spent in the drug-paired side (sec.) side drug-paired the in spent Time
Figure 4
64
A
3.0 VEH OEA
2.5
P = 0.05
Heat (kCal/h) Heat 2.0
1.5 *** 0 24 48 72 96 120 144 168 192 216 240 Time (min)
B 3.0
2.5
2.0
1.5
1.0 12-h Heat (kCal/h) Heat 12-h 0.5
0.0 VEH OEA
Figure 5
65
A
10
8 **
6
4
2 Time in extension in Time (min)
0 VEH OEA
B
10
8
6
4
2 Timeextension in (min)
0 VEH CAPS
Figure 6
66
A
280 *** 280 280
240 240 240 *
200 200 200
160 160 160
120 120 120
80 80 80
40 40 40 Time in inactivityTime in (sec.) (sec.) inactivity in Time Time ininactivity (sec.) 0 0 0 VEH OEA VEH CAPS VEH Wy-14643
B
80 80 80
60 60 60
40 40 40 Ambulation Ambulation Ambulation 20 ** 20 20 (# of squares crossed) (# of squarescrossed) (#of squares crossed) 0 0 0 VEH OEA VEH CAPS VEH Wy-14643
C
50 50 50
40 40 40
30 30 * 30
20 20 20 ** (# of events) (# of events) (# of events) of (# 10 10 10 Spontaneous activity Spontaneous activity *** Spontaneous activity
0 0 0 VEH OEA VEH CAPS VEH Wy-14643
Figure 7
67
A B
VEH 1.50 10.0 OEA
1.25 7.5
1.00 5.0
0.75 2.5 Plasma GLP-1(pg/ml) Plasma ghrelin (ng/ml) Plasma ghrelin
0.50 0.0 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)
C D
100 26
24
75 22
20
50 18
Plasma PYY (pg/ml) PYY Plasma 16 Plasma apo A-IV (mg/dl)
25 14 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)
Figure 8
68
A SAL/VEH 14 LORGL/VEH
12 SAL/OEA LORGL/OEA 10
8
6
Food intake (g) intake Food 4 ** * 2
0 0 1 2 3 4 5 Time (h)
B SAL/SAL 7 LORGL/SAL
6 SAL/CCK-8 LORGL/CCK-8 5
4
3 * 2 Food intake (g) intake Food * 1
0 0 15 30 45 60 75 Time (min)
Figure 9
69
The OEA-induced aversion hypothesis, revisited:
Our attempts at assessing the contribution of TRPV1, a receptor linked with pain
and motor activity, in the effects of OEA were problematic. We did not replicate the
effects of the TRPV1 inhibitor capsazepine at blocking capsaicin-induced
hypolocomotion. Without this positive control, it was difficult to interpret our data. In the
meanwhile, another research group answered the question we aimed to address. In a
series of very elegant studies, Wang et al. have demonstrated that short-term food intake is reduced in response to OEA in wild-type but not in TRPV1 knockout mice (174). They proposed that TRPV1 mediates the initial hypophagic action of OEA, which they
attributed to pain. Our data, together with those of Wang et al., strongly indicate that in
order to realize the therapeutic potential of OEA, it will be important to develop
analogues with high affinity for PPAR-alpha, while limiting activation of TRPV1.
70
CHAPTER 3: C75 REGULATES FOOD INTAKE VIA ACTIVATION OF
HYPOTHALAMIC MTOR SIGNALING.
Karine Proulx, Daniela Cota, Stephen C. Woods and Randy J. Seeley
71
ABSTRACT
C75 is a fatty acid synthase inhibitor that reduces food intake and body weight in rodents when administered either in the central nervous system (CNS) or the periphery.
Such effects are believed to occur via inhibition of the hypothalamic fuel sensor AMP- activated protein kinase (AMPK), as they are prevented by central administration of the
AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). Recent evidence from our laboratory strongly indicates that the mammalian target of rapamycin
(mTOR), a protein kinase that regulates protein synthesis and cell growth, also serves as an energy sensor in the CNS. In contrast to AMPK, hypothalamic mTOR signaling is activated in conditions of positive energy balance and its inhibition with rapamycin stimulates food intake in rats. These data, together with the ability of AMPK to inhibit mTOR signaling in vitro, led us to hypothesize that C75 reduces food intake through activation of the mTOR pathway in the hypothalamus. Using Western blotting, we found that intra-third ventricular (i3vt) administration of C75 (30 μg in 2 μl of RPMI) activates the mTOR pathway as indicated by a significant increase in the phosphorylation of mTOR (P < 0.05) and its downstream effector S6 ribosomal protein (S6) (P < 0.001) in the hypothalamus. As reported before, i3vt administration of C75 (50 μg in 3 μl of
RPMI) caused anorexia and body weight loss (P < 0.05), effects that were absent in rats receiving an i3vt injection of the mTOR inhibitor rapamycin prior to C75 (P < 0.05). We also replicated our previous finding that C75 is ineffective at reducing caloric intake in rats maintained on a ketogenic diet. Interestingly, C75 is also unable to activate the hypothalamic mTOR signaling under these conditions. In conclusion, our results indicate
72
that the effects of C75 on energy balance depend on its ability to activate mTOR signaling in the brain.
73
INTRODUCTION
Energy balance is achieved when caloric intake is accurately matched to caloric
expenditure. A complex neuroendocrine system underlies this process and thus regulates
energy homeostasis in mammals. In addition to sensing hormonal signals of stored fuels,
such as leptin and insulin (9), specific populations of neurons in the central nervous
system (CNS) have the ability to sense the level of available nutrients. In turn, they
regulate their activity to adjust caloric intake and expenditure accordingly. Glucose (33)
and oleic acid (99) inhibit NPY neurons and reduce food intake when injected centrally.
This phenomenon is also observed with intracerebroventricular (icv) administration of
compounds that modify either glucose (175) or fatty acid metabolism (88). One such
compound is the fatty acid synthase (FAS) inhibitor C75.
C75 reduces food intake and body weight in rodents when administered in the
periphery or directly into the CNS (48-50, 84). The C75-induced anorexia is
accompanied by reduced NPY/AgRP and increased POMC/CART mRNA expression
(92). Hypothalamic malonyl-CoA, which accumulates as a result of FAS inhibition, has
been proposed to underlie these changes and thus, to play a key role in mediating the
anorexia of C75 (176). In fact, manipulations that reduce the rate of synthesis of malonyl-
CoA (84) or increase its degradation (89) prevent C75 from reducing food intake.
The serine/threonine protein kinase AMP-activated protein kinase (AMPK) is a
cellular fuel sensor that is activated by signals of energy depletion, specifically a reduction of ATP:AMP, and that exerts rapid regulation of energy-producing and -
74
consuming metabolic pathways (177). In the hypothalamus, AMPK is inhibited by
signals of positive energy status such as leptin, insulin and glucose (34, 41). Genetic or
pharmacological manipulations that increase AMPK activity in the CNS stimulate food
consumption (34, 41) and prevent the anorexigenic effect of leptin (34). C75, which
mimics a state of positive energy balance (84), also reduces hypothalamic AMPK activity
(78, 79). This latter effect has been proposed to mediate C75 anorexigenic action, since
C75-induced anorexia does not occur following central administration of the AMPK
activator AICAR (79). Given that AMPK is an inhibitor of acetyl-CoA carboxylase
(ACC), Hu et al. have proposed a model where the C75-induced disinhibition of ACC
elevates malonyl-CoA levels, which in turn acts as a signal to reduce food intake (89).
Like AMPK, the mammalian target of rapamycin (mTOR) is a serine/threonine
protein kinase that resides at the interface between nutrient sensing and regulation of
energy balance (39). MTOR integrates signals of nutrient availability, including amino
acids and growth factors such as leucine and insulin, and in turn regulates protein
synthesis and cell growth (178). For instance, mice and Drosophila lacking S6 kinase 1
(S6K1), a downstream component of TOR, have smaller cells and consequently smaller bodies than controls (72). In contrast to AMPK, the mTOR pathway is activated in conditions of energy surplus, and is therefore activated when ATP:AMP increases (39).
This occurs in peripheral tissues, and possibly in the brain as well. Our laboratory has recently demonstrated that mTOR signaling is controlled by energy status and co- localizes with neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the arcuate nucleus (19). Leptin and the branched-chain amino acid leucine activate the
75
pathway when administered into the third ventricle (i3vt). This is necessary for leptin and
leucine to reduce food intake, since their anorectic action is abolished by pre-treatment
with the mTOR inhibitor rapamycin (19).
Evidence supports the existence of a regulatory relationship between AMPK and mTOR. Activated AMPK indirectly inhibits mTOR through activation of the TSC2 tumor
suppressor gene (119). These data, together with the inhibitory effect of C75 on AMPK
(79) and the role of hypothalamic mTOR in feeding (19), led us to suggest that C75
reduces food intake through activation of mTOR in the hypothalamus. To test this
hypothesis, we first assessed by Western blots whether C75 increases the phosphorylation
of mTOR and its downstream effectors S6K1 and S6 ribosomal protein (S6). We then
determined whether inhibition of mTOR signaling prevents the inhibitory effects of C75
on food consumption and modulates the mRNA expression of NPY, AgRP and POMC.
Our laboratory has previously reported that the anorectic signal of C75 is derived from its ability to increase CNS glucose metabolism (49). Neurons minimally oxidize
fatty acids, and instead, rely on glucose as their primary fuel under most conditions.
Therefore, we reasoned that the increased glucose utilization relative to the reduced lipid
use would be more likely to serve as the primary biochemical sensor for nutrient excess
in neurons (49); as opposed to the reduction in lipid use per se, caused by the malonyl-
CoA-induced inhibition of carnitine palmitoyl-CoA transferase-1 (CPT-1) (84).
Consistent with this view, we have demonstrated that C75 does not reduce food intake in
various conditions in which its effect on neuronal glucose oxidation is prevented,
76
including when ketone bodies are available as an alternative, and preferred, source of fuel
(49). Here, we explored the possibility that C75 fails to reduce caloric intake under the ketogenic diet because of its inability to activate mTOR signaling in the hypothalamus.
77
MATERIALS AND METHODS
Animals: Adult male Long-Evans rats (250–300 g at the beginning of the study) were housed individually and maintained on a 12:12-h light-dark cycle. Animals had ad libitum access to standard lab chow (15% lipid, 23% protein, and 62% carbohydrate calories)
(Harlan-Teklad, Indianapolis, IN) and water unless specified. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of
Cincinnati.
I3vt cannulation: Rats were implanted with third-ventricular (i3vt) cannulas as previously described (179). Briefly, rats were anaesthetized with ketamine/xylazine
(10: 6.5 solution; 1.0 ml/kg) and placed into a stereotaxic frame. A 22-gauge cannula
(Plastics One, Roanoke, VA, USA) was lowered along the midsagittal plane into the third ventricle (-2.2 mm anteroposterior and -7.5 mm dorsoventral with respect to bregma) and then secured to the skull with screws and dental acrylic. Animals were allowed to recover for a minimum of 7 days. Verification of cannula placement was confirmed by i3vt injection of 10 ng of angiotensin II in 1 µl 0.9% saline. Only animals that consumed at least 5 ml of water within 60 min of injection were included in the studies.
Drugs: All drugs were administered through an i3vt cannula. C75 (Calbiochem, EMD
Bioscience Inc., La Jolla, CA) was dissolved in RPMI (Gibco, Carlsbad, CA) and administered in a 2-µl volume, unless specified otherwise. Rapamycin (RAPA)
(Calbiochem, EMD Bioscience Inc., La Jolla, CA) was dissolved in dimethylsulfoxide
(DMSO) and administered in a 1-µl volume. All vehicle solutions served as controls.
78
Ketogenic diet: Rats were placed on a ketogenic diet (80.7% lipid, 14.3% protein, and
5% carbohydrate calories) (Dyet # 100959 Dyets Inc., Bethlehem, Pennsylvania) for 4
weeks before the experiment. Rats had ad libitum access to the ketogenic diet and water,
and to a second solution containing either 10% sucrose or 0.1 % saccharin, unless stated
otherwise (49).
β-hydroxybutyrate measurement: Fifty μl of blood was taken from the tip of the tail
after 24 h of fasting. Blood samples were collected into heparinised Natelson tubes and
immediately placed on ice in 1.5 ml heparinised Eppendorf tubes. β-hydroxybutyrate
concentration was measured using the KetoSite® test (Stanbio laboratory, Boerne,
Texas). Ketosis was confirmed by a significant elevation of blood β-hydroxybutyrate in
rats that had access to saccharin as compared to those given glucose while maintained on
the ketogenic diet.
Westerns: A wedge of the mediobasal hypothalamus was dissected using as landmarks the mammillary bodies caudally, the optic chiasm rostrally, the optic tract laterally and the apex of the third ventricle. Tissues were homogenized in RIPA lysis buffer (Santa
Cruz Biotechnology, Santa Cruz, CA) with addition of phosphatase and protease
inhibitors (Santa Cruz Biotechnology). Protein concentration was determined by BCA kit
(Pierce, Rockford, IL), following manufacturer’s instructions. Seventy µg of samples
were suspended in Laemmli sample buffer (BioRad, Hercules, CA), with 5% 2-
mercaptoethanol (Fisher Scientific, Hampton, NH) and lysates were denatured at 95°C
for 5 min. Samples were loaded on a 10% SDS-polyacrylamide gel (BioRad). Proteins
were separated by electrophoresis and transferred to nitrocellulose membranes.
Membranes were then incubated 1 h at room temperature in 1x TBS (20 mM Tris base
79
and 136 mM NaCl, pH 7.6) containing 0.1% Tween 20 (TBS-T) and 5% skim milk
powder and washed in TBS-T at room temperature before overnight incubation at 4°C
with one of the following primary antibody: pmTOR at Ser 2448 (Cell Signaling
Technology, Beverly, MA) (1:1000), pS6K1 at Thr 389 (Cell Signaling Technology,
1:500), pS6 at Ser 240/244 (Cell Signaling Technology, 1:500). After washing in TBS-T,
the membranes were incubated for 1 h at room temperature with secondary antibody
conjugated with horseradish peroxidase (goat anti-rabbit, 1:2000, Cell Signaling
Technology). After washes in TBS-T, immunopositive bands were visualized by
chemiluminescence (Lumiglo reagent and peroxide kit, Cell Signaling Technology) using exposure to radiographic films (Denville Scientific, South Plainfield, NJ). After protein detection, membranes were stripped for 15 min at 55 °C with a solution containing 62.5 mM Tris-HCl, 100 mM mercaptoethanol, and 2% SDS before 2 h blocking in TBS-T with
5% skim milk powder at room temperature, and re-blotted with either rabbit anti mTOR
(Cell Signaling Technology, 1:1000), rabbit anti p70 S6K1 (Cell Signaling Technology,
1:250) and rabbit anti S6 (Cell Signaling Technology, 1:500). The optical densitometry
of immunoreactive bands was measured using the NIH program Scion image.
q-PCR: Hypothalami were dissected using as landmarks the mammillary bodies
caudally, the optic chiasm rostrally and the optic tract laterally. Tissues were
homogenized in Tri-Reagent (Molecular Research Center, Cincinnati, OH), and total
RNA was isolated. cDNA was synthesized using SuperScript III kits (Invitrogen Corp.) and verified by L32 amplification products in agarose gel. PCR was performed using
Failsafe PCR kits (EPICENTRE Biotechnologies). For amplification of the reference gene L32, reaction mixture included Buffer I and forward primer 5’-
80
CCTCTGGTGAAGCCCAAGAT-3’ and reverse 5’-CTAGGCAGCATGTGCTTGGT-
3’. Thermocycler conditions were: 3 minutes at 95°C; 30 seconds at 95°C, 30 seconds at
55°C, and 45 seconds at 72°C for 25 cycles; and 5 minutes at 72°C. Q-PCR primer
sequences are shown in TABLE 1. Each primer set was optimized such that the
correlation coefficient was 0.99–1.0 and the PCR efficiency was 90–100%. Q-PCR was
performed in triplicate using an iCycler and the iQ SYBR Green Supermix (Bio-Rad
Laboratories) with 2-step amplification (95°C for 10 seconds, annealing temperature for
30 seconds) for 40 cycles. L32 was amplified from every sample for use as an
endogenous control. For the data analysis, the average threshold cycle (CT) of each set of
triplicates was calculated. To normalize the data, the CT was calculated for each sample
by subtracting the average CT of L32 from the average CT of the gene of interest. For
relative quantitation, the CT was averaged for the defined control group and was then
subtracted from the CT of each experimental sample to generate the CT. The CT was then used to calculate the approximate fold difference, 2 CT.
Experimental design:
Experiment 1: Effect of C75 on pmTOR, pS6K1 and pS6 levels. Rats were divided in 2
treatment groups based on their baseline food intake and body weight. Food was
removed and rats were weighed 2 h before the experiment. One hour before dark, rats
were injected with RPMI or C75 (30 μg in 2 μl, i3vt) and sacrificed 30 min and 1 h later.
Brains were removed, immediately frozen in isopentane and stored at -80 °C until
performing Western blot analysis.
81
Experiment 2: Effect of rapamycin (RAPA) on C75-induced anorexia. Rats were assigned
to one of four treatment groups (DMSO-RPMI, RAPA-RPMI, DMSO-C75, RAPA-C75) based on their baseline food intake and body weight. On the test day, rats were weighed and food was removed 2 h before the experiment. Three hours before the onset of the dark phase, rats received an injection of either DMSO or RAPA (25 μg in 1 μl, i3vt) followed by an injection of either RPMI or C75 (50 μg in 3 μl, i3vt) 2 h later. Food was immediately returned and weighed 1, 2, 4, 8 and 24 h after the last injection and body
weight was measured at 24 h.
Experiment 3: Effect of the ketogenic diet on C75-induced anorexia. Rats were divided
into 2 groups (saccharin or sucrose) based on their baseline body weight. All rats were
given ad libitum access to a ketogenic diet and to a sucrose or similarly sweet saccharin
solution. Water was available at all time. Four weeks later, 100 μl of blood was collected
into heparinised Natelson from the tip of the rat’s tail and blood β-hydroxybutyrate
concentration was measured using the KetoSite® test according to manufacturer’s
instruction (Stanbio Laboratory, Boerne, Texas). Three days later, the food and the solution of sucrose or saccharin were removed from the cages at the onset of the dark phase. The next day, each group (sucrose or saccharin) was subdivided into 2 treatment groups (RPMI or C75), based on their baseline intake of the ketogenic diet and their body weight, and rats were injected with either RPMI or C75 (30 μg in 2 μl, i3vt) 1 h prior to lights off. Food and the bottles of sucrose or saccharin were immediately returned and
weighed 4 and 24 h later.
82
Experiment 4: Effect of the ketogenic diet on C75-induced changes in pS6K1 and pS6 levels. The protocol was the same as described for Experiment 3, except that food, sucrose and saccharin were not returned after the injection in order to avoid any confounding effect of caloric intake. Rats were sacrificed 1 h later, and brains were taken for Western blot analysis of pS6K1 and pS6.
Experiment 5: Effect of C75 on NPY, AgRP and POMC mRNA expression, and consequence of rapamycin pre-treatment. Twenty-four hour fasted rats were injected with C75 (30 μg in 2 μl, i3vt) or RPMI 1 h before the onset of the dark phase, and sacrificed 2 h and 30 min later (92). Brains were rapidly removed and stored in RNA later (Ambion, Austin, Texas) at 4 °C until processed for RNA extraction and q-PCR.
Statistical analysis: For multiple group designs, the data were analyzed by two-way
ANOVA and Tukey’s post hoc tests. For designs with only two groups, statistical validity was assessed with unpaired t tests. Experiment-wise significance was set at P < 0.05, two- tailed. All values are expressed as the mean ± SEM.
83
RESULTS
Experiment 1: Effect of rapamycin on C75-induced anorexia. C75 significantly reduced
food intake compared with RPMI starting 4 h after the injection (DMSO-C75 = 1.86 +
0.68 vs. DMSO-RPMI = 6.51 + 0.52 g; P < 0.001; data not shown). This effect was still
present at 24 h (DMSO-C75 vs. DMSO-RPMI, P < 0.001) but was prevented by pre- treatment with the mTOR inhibitor RAPA (DMSO-C75 vs. RAPA-C75; P < 0.05, Fig.
1A) at a dose that did not affect food intake by itself (RAPA-RPMI vs. DMSO-RPMI; P
= 0.999, Fig. 1A). RAPA was also effective at preventing the body weight loss induced
by C75 over 24 h (DMSO-RPMI vs. DMSO-C75; P < 0.01 and DMSO-C75 vs. RAPA-
C75; P < 0.05, Fig. 1B).
Experiment 2: Effect of C75 on pmTOR, pS6K1 and pS6 levels. There was no significant
effect of the drug on the phosphorylation of S6K1 (pS6K1/S6K1: RPMI = 100.00 +
8.36% vs. C75 = 86.49 + 11.68 % of RPMI; P = 0.365) or S6 (pS6/S6: RPMI = 100 +
18.36% vs. C75 = 105.59 + 14.45 % of RPMI; P = 0.816) at 30 min post-injection.
However, C75 administration activates mTOR signaling at 1 h, as revealed by a
significant increase in the level of pmTOR (C75 vs. RPMI: P < 0.05; Fig. 2A) and pS6
(C75 vs. RPMI: P < 0.05; Fig. 2C) relative to vehicle. There was also a trend for C75 to
increase the phosphorylation of S6K1 at this time point (P = 0.05, 1-tailed t-test; Fig.
2B).
Experiment 3: Effect of the ketogenic diet on C75-induced anorexia. After 4 weeks on the
ketogenic diet, blood β-hydroxybutyrate concentration was significantly elevated in rats
84
given access to the saccharin solution as compared to sucrose (saccharin = 0.5 + 0.04 mM vs. sucrose = 0.17 + 0.03; P < 0.001). Consistent with its effect on the standard chow diet
(Fig. 1A), C75 significantly reduced caloric intake in rats given access to sucrose while maintained on the ketogenic diet (RPMI vs. C75: P < 0.05, Fig. 3). However, the anorexigenic effect of C75 was absent in rats receiving the saccharin solution (RPMI vs.
C75: P = 0.585; Fig. 3).
Experiment 4: Effect of the ketogenic diet on C75-induced changes in pS6K1 and pS6
levels. The ketogenic diet did not have any significant effect on the basal activation of the
mTOR pathway (levels of pS6K1/S6K1: saccharin-RPMI = 101.92 + 5.17 % of sucrose-
RPMI levels = 100.00 + 4.40 %; P = 0.783; data not shown), although there was a trend
for pS6 to be increased in rats exposed to saccharin as compared with those exposed to sucrose (pS6/S6: saccharin-RPMI = 152.27 + 17.93 % of sucrose-RPMI levels = 100.00
+ 15.17 %; P = 0.09). C75 administration stimulated mTOR signaling, as indicated by a
significant increase in pS6K1 and pS6, only in rats that consumed sucrose along with the
ketogenic diet [saccharin: (RPMI vs. C75; P = 0.729) vs. sucrose: (RPMI vs. C75; P <
0.05), Fig. 4].
Experiment 5: Effect of rapamycin on NPY, AgRP and POMC mRNA expression. The
anorexigenic effect of C75 was also accompanied by a significant reduction in
hypothalamic NPY mRNA levels (DMSO-RPMI vs. DMSO-C75, P < 0.05, Fig.5). There
was a trend for rapamycin to reverse the effect of C75 (DMSO-C75 vs. RAPA-C75, P =
0.07, Fig.5), although significantly reducing NPY levels as compared to DMSO (RAPA-
85
RPMI vs. DMSO-RPMI, P < 0.05, Fig.5). No changes were reported in the levels of
expression of AgRP (DMSO-RPMI = 100.00 + 5.58 % vs. DMSO-C75 = 83.88 + 11.09
%; P = 0.834) and POMC (DMSO-RPMI = 100.00 + 9.97 % vs. DMSO-C75 = 90.69 +
18.92 %; P = 0.856).
86
DISCUSSION
In this series of experiments, we present evidence that the FAS inhibitor C75
activates hypothalamic mTOR signaling in rats. This effect is necessary for the anorectic action of this compound, since pre-treatment with rapamycin, a well-known mTOR inhibitor (180), blocks C75-induced anorexia. We observed a potent effect of C75 at phosphorylating S6 1h after the injection, and a trend for pS6K1 to be increased by C75 as well. Since the phosphorylation of S6K is an event preceding that of S6, perhaps this time point was too late for observing a significant phosphorylation of the kinase. We have also evaluated possible changes in phosphorylation 30 min after the injection, but failed to detect any modification in either protein at this time point. Others have reported examples of conditions under which S6 is phosphorylated without parallel changes in
S6K1. In some cases, S6 activation was attributed to a second S6 kinase, termed S6K2, and the effect was mTOR-dependent (181). Therefore, one interpretation would be that
C75 stimulates S6 phosphorylation mainly via S6K2, which has not been measured in the present study. Another possibility is that C75 directly inhibits the phosphatase that dephosphorylates S6 (182, 183). Thus, the degree of S6 phosphorylation might depend on the balance between S6K and specific phosphatase activities at a specific time point.
In the past, we have proposed that C75’s ability to reduce caloric consumption
depends on its ability to increase glucose use in the brain, rather than reducing fatty acid
use per se (49). Consistent with this view, neuronal exposure to alternative fuels, and
consequent reduction in neuronal demand for glucose, attenuate the anorexigenic effect
of C75 (49). Central administration of glutamine or lactate, which is produced by
87
astrocytes and can be used by neurons as an alternative to glucose for ATP production
(117), significantly reduces the anorectic effect of C75. A similar phenomenon occurs
when rats are maintained on a very low-carbohydrate (ketogenic) diet for a prolonged
period of time (118). The latter condition mimics what happens during starvation, when
blood glucose levels fall and gluconeogenesis is not sufficient to respond to the brain’s
needs for glucose. Metabolic adaptations insure that the brain preferentially uses ketone
bodies over glucose for energy production (184).
In the current study, not only did we replicate our previous findings describing the
inability of C75 to reduce caloric intake in rats maintained on a ketogenic diet (49), but
also we found that C75 is ineffective at activating the mTOR pathway under this
condition. In contrast, when sucrose is provided alongside with the ketogenic diet, rats
remain sensitive to these C75-mediated effects, in a similar fashion to rats exposed to
normal chow. The implication of these findings is that the ability of C75 to activate
mTOR signaling requires increased CNS glucose utilization.
We observed a non-significant increase in the level of hypothalamic pS6 in the vehicle-injected rats exposed to ketogenic-saccharin diet. Interestingly, ketone bodies induce phosphorylation of S6K1 in primary cultured rat hepatocytes (185). Given that leucine is known to activate hypothalamic mTOR signaling (19), and that the levels of
leucine in the brain are increased following exposure to a ketogenic diet (186), these
events could have contributed to the non-significant elevation of pS6 in the vehicle-
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treated rats in our study. Furthermore, this higher baseline could also have contributed to the lack of effect of C75 in the saccharin group.
The implication of our findings is two-fold. First, they demonstrate that activation of mTOR signaling is necessary for the anorexigenic effect of C75. Second, these results suggest that neuronal glucose metabolism is crucial for the effect of C75 on hypothalamic mTOR activation. CNS glucose metabolism is a key biochemical signal of nutrient status monitored by AMPK. Indeed, intracerebroventricular (icv) administration of glucose decreases AMPK activity and food intake, whereas 2-deoxyglucose, a well-known inhibitor of intracellular glucose utilization, does the opposite (34, 187). We have previously demonstrated that hypothalamic mTOR integrates nutrient signals from hormones and amino acids, such as leptin and leucine (19). Although our experiments did not specifically test this possibility, it is tempting to speculate that hypothalamic mTOR, like AMPK, also monitors glucose utilization. In the periphery, glucose-induced activation of S6K requires glucose metabolism, and the signals have therefore been proposed to directly emanate from glycolysis and/or glucose mitochondrial oxidation
(188). Given the established role of mTOR as an ATP sensor (39), an obvious candidate would be ATP.
C75 increases glucose oxidation as well as ATP levels in several cell types, including neurons (78, 90). Specifically, a study found that neuronal ATP levels are affected in a biphasic manner by C75: an initial decrease is then followed by a prolonged increase relative to control (78). Thus, the ability of C75 to stimulate mTOR might
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involve its regulatory action on neuronal ATP levels. Future studies are necessary to
determine whether manipulations that inhibit ATP synthesis impair the ability of C75 to
activate hypothalamic mTOR signaling and to reduce food intake.
An interesting observation in the original report of Loftus et al. was that despite eating very little food, animals treated with C75 had a hypothalamic neuropeptide profile comparable to that of the fed state. Indeed, C75 reduced NPY levels relative to fasting, while suppressing 96% of food intake (84). We have recently reported that the phosphorylated form of mTOR and S6K1 is highly expressed in NPY/AgRP neurons, and to a lesser extent, in POMC/CART neurons. In addition, we found that the mTOR pathway is activated in the arcuate nucleus of the hypothalamus following refeeding
(189). The present data reveals another resemblance between C75 treatment and refeeding (189), since both conditions are able to drive hypothalamic mTOR signaling.
Furthermore, there is a trend for rapamycin to reverse the effect of C75 on NPY.
However, C75’s effect is rather small in comparison to previous reports (47, 92), therefore limiting the interpretation of the effects of rapamycin. Another caveat of our
study is that rapamycin reduced NPY relative to its vehicle DMSO. However, the
direction of this effect would suggest that it is unlikely that it contributed to block the
C75-induced reduction of NPY. Hence, it is still tempting to speculate the possible involvement of mTOR signaling in the effect of C75 at regulating NPY expression. The inhibition of NPY/AgRP neurons is believed to be an important component of the anorexigenic effect of C75 (47, 79, 84), although little is known about the intracellular events leading to this effect. The role of mTOR in regulating protein synthesis is well
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established (55). Thus, an attractive hypothesis is that C75-induced activation of S6, a downstream target of S6K, might be linked to reduced NPY levels. Additional experiments are required to confirm the consequences of rapamycin on the ability of C75 to reduce NPY, and therefore to address the exact role of mTOR in this context.
Our experiments have not addressed whether the effects of C75 on the mTOR pathway are directly mediated, or rather occur indirectly through inhibition of hypothalamic AMPK. mTOR is recognized as a downstream target of AMPK, which inhibits its phosphorylation through activation of the TSC2 (119). Kim et al. have demonstrated the ability of C75 to dephosphorylate and inactivate AMPK. They proposed that this mechanism contributes to the ability of C75 to reduce food intake, since the
AMPK activator AICAR inhibits C75-induced anorexia (79). We cannot rule out the possibility that our observations result from a disinhibition of mTOR, through the inhibitory action of C75 on AMPK, rather than a direct stimulatory effect of the compound on the mTOR pathway. However, the time-course reported for AICAR (79) is very different from the one we observed with rapamycin. AICAR blocks C75’s anorexia exclusively in the first hour following the injection (79), while the effect of rapamycin occurs after 8 h and is still significant at 24 h. While this could be due to differences in experimental design and drug kinetics, it is still a difference that is worth considering.
In conclusion, these data provide evidence that link the CNS FAS system to the hypothalamic mTOR signaling cascade. This connects two separate metabolic pathways that have independently been linked to the regulation of energy balance. Indeed, the
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anorectic action of leptin and the amino acid leucine also depends on its ability to activate hypothalamic mTOR signaling (19). Thus, a hypothetical model would be that C75 increases neuronal glucose metabolism, triggering increase ATP that would be sensed
and integrated by mTOR. The subsequent phosphorylation of this kinase would therefore
activate a cascade of intracellular events aimed at preventing further nutrient intake and
maintaining energy balance.
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TABLE 1
Rat Q-PCR Primer Sequences
Gene Rat Q-PCR primer sequences T (°C)
L32 Forward: 5'-GCC AGG AGA CGA CAA AAA T 61.2
Reverse: 5'-AAT CCT CTT GCC CTG ATC C
AGRP Forward: 5'-TGT GTA AGG CTG CAC GAG TC 61.2
Reverse: 5'-GGC AGT AGC AAA AGG CAT TG
NPY Forward: 5'-AGG CTT GAA GAC CCT TCC AT 61.2
Reverse: 5'-ACA GGC AGA CTG GTT TCA GG
POMC Forward: 5'-CGC CCG TGT TTC CA 58.0
Reverse: 5'-TGA CCC ATG ACG TAC TTC C
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FIGURE LEGEND
Figure 1. I3vt administration of rapamycin (25 µg in 1 µl DMSO) prevents the effects of
C75 (50 µg in 3 µl RPMI) on food intake (A) and on body weight (B). Mean ± SEM of 9
to 15 rats in each treatment group. ** P < 0.01; *** P < 0.001 vs. DMSO/RPMI-treated rats; 1 vs. DMSO/RPMI-treated rats; # P < 0.05 vs. DMSO/C75 rats.
Figure 2. C75 (30 µg in 2 µl RPMI, i3vt) increases hypothalamic mTOR signaling:
Representative Western blots from RPMI- or C75-treated rats and quantification by
image analysis of hypothalamic phosphorylation of mTOR (A), S6K1 (B) and S6 (C).**
P < 0.01; *** P < 0.001 vs. RPMI-treated rats. 7-8 brains examined in each condition.
Figure 3. C75 (30 µg in 2 µl RPMI) does not reduce caloric intake in rats given access to
saccharin alongside with the ketogenic diet, but do so with sucrose. * P < 0.05 vs. RPMI-
treated rats from the same group. Seven to eight rats in each treatment group.
Figure 4. C75 (30 µg in 2 µl RPMI) does not increase hypothalamic S6K1 and S6 phosphorylation in rats given access to saccharin while maintained on a ketogenic diet, but do so in rats receiving sucrose alongside with the diet. Representative Western blots from RPMI- or C75-treated rats from the saccharin and sucrose groups and quantification by image analysis of hypothalamic phosphorylation S6K1 (A) and S6 (B). * P < 0.05; P
< 0.01 vs. RPMI-treated rats from the same group. Five to eight brains were examined in each condition.
Figure 5. C75 significantly reduced NPY mRNA levels in the hypothalamus as
compared to RPMI controls, as did rapamycin. There was a trend for rapamycin to prevent the effect of C75 on NPY expression. * P < 0.05 vs. DMSO/RPMI-treated rats.
Seven to twelve brains were examined in each condition.
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FIGURES
A
30 DMSO/RPMI 25 RAPA/RPMI 20 DMSO/C75 (50 μg) # RAPA/C75 (50 μg) 15 *** 10
5 24-h food intake (g)
0
B
-5
-15
-25 ** # -35 24-h weight body (g) change
Figure 1 Figure 1
95
A 150 RPMI ** 125 C75 (30 μg)
100
75
50 (% of RPMI) of (% pmTOR/mTOR 25
0
RPMI C75
pmTOR B mTOR 175 150 pS6K1 125 S6K1 100 75 (% of RPMI) of (% pS6 pS6K1/S6K1 50 25 S6 0
C 1200 *** 1000
800
600
pS6/S6 400 (% of RPMI) of (% 200 0 Figure 2
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RPMI 140 C75 (30 μg) 120 100 80 * 60 40 20
24-h energy intake (kcal) intake energy 24-h 0 sucrose saccharin
Figure 3
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RPMI C75 RPMI C75
pS6K1
S6K1
pS6
S6
sucrose saccharin
A B 200 200 RPMI C75 (30 μg) ** 150 * 150
100 100 pS6/S6 (% of RPMI) of (% (% of RPMI) of (% pS6K1/S6K1 50 50
0 0 sucrose saccharin sucrose saccharin
Figure 4
98
120 DMSO/RPMI 100 # * RAPA/RPMI 80 DMSO/C75 (30 μg) RAPA/C75 (30 μg) 60
40
20
0 NPY mRNA (% control DMSO/RPMI)
Figure 5
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CHAPTER 4: GENERAL DISCUSSION
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OEA as a derivative of oleic acid.
Very little is known about the intracellular mechanisms by which OEA-induced stimulation of PPAR-α leads to activation of brain regions involved in the control of energy balance (127). Oleic acid, the precursor of OEA, reduces food intake when directly administered into the CNS (18). As reviewed in the introduction, this causes an increase in the cytoplasmic concentration of long chain fatty acyl-CoAs (LCFA-CoAs), which is believed to mimic conditions of energy surplus and act as a signal to reduce food intake (88). Thus, an attractive hypothesis would be to attribute the anorectic effects of
OEA to the action of its catabolite oleic acid in the CNS. However, OEA does not affect food intake when delivered directly into the brain or when peripheral sensory fibres are removed by treatment with capsaicin (127). These findings suggest that OEA-induced anorexia is vagally mediated rather than a consequence of increased oleic acid levels
(127).
Our laboratory has collected data indicating that the susceptibility to develop obesity on a high fat diet depends on the fatty acid composition of the diet (Seeley R.J. and Woods S.C., unpublished data). Studies suggest that the “satiating” effect of ingested lipids depends not only on their quantity, but also on their properties (chain length, and degree of saturation and conjugation) (77). These properties have been proposed to influence the release of meal-related gastrointestinal signals that inhibit feeding, such as
CCK (190, 191). As such, it is interesting that rats maintained on a high-fat diet rich in monounsaturated fatty acids, such as oleic acid, are protected against diet-induced obesity
(Seeley R.J. and Woods SC., unpublished data). Because oleic acid is a precursor for
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OEA and because it can reduce food intake (18), it is tempting to speculate that the lipid
composition of the diet modulates the synthesis of OEA, and that OEA consequently
contributes to the reduced weight gain and caloric consumption observed under these conditions.
OEA as an inhibitor of AMPK and FAS?
An interesting parallel can be drawn between OEA and its structural analogue
anandamide. Although they do not share the same receptor target, it is possible that
anandamide and OEA, two nutrient-derived signals whose pattern of secretion and
regulatory action on food intake are inversely related (192), share a common target within
the CNS. This is true for leptin and ghrelin. The secretion of these two adiposity signals
is also inversely related (193), and their opposite actions on hypothalamic AMPK are
required for their respective anorexigenic and orexigenic effects (34, 45).
OEA (127), anandamide (149) and stearoylethanolamide (SEA) (170) have all been
reported to control energy balance. An interesting point is that although these lipids are
all part of the same FAE family, and therefore have some degree of structural homology,
they do not regulate food intake through a common receptor. Anandamide appears to
mediate its effect on CNS feeding circuits via both a peripheral and central actions (149,
192), while OEA signal indirectly through the vagus nerve (127). The anorexigenic
action of SEA is accompanied by a reduction of stearoyl-CoA desaturase-1 (SCD-1)
mRNA expression in the liver, but its effects in the CNS have not been evaluated (170).
Previous work has linked the orexigenic effect of anandamide to activation of AMPK and
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FAS in the hypothalamus (45, 194). OEA-induced anorexia occurs through stimulation of
the PPAR-alpha (127). Of interest, this nuclear receptor is involved in the regulation of
several aspects of lipid metabolism in the liver, partly through its ability to inhibit the
transcription of FAS (195). One might speculate that nutrient-derived lipids such as OEA
and anandamide have redundant signaling pathways or could share a common, yet
unidentified, target in the CNS. Given that anandamide is inversely related to OEA, and
that it mediates its orexigenic effect through activation of FAS and AMPK in the hypothalamus, an attractive hypothesis is that OEA and anandamide both converge on
FAS to regulate food intake in an opposite direction.
FAS inhibitors and mTOR.
In addition to AMPK, mTOR is an important CNS biochemical fuel sensor that
participates in the recognition of the energy status of the body and the regulation of
feedback mechanisms to restore energy balance. Hypothalamic mTOR integrates signals
from leptin and amino acids, and its phosphorylation is necessary for their anorexigenic
actions (19). The findings described in chapter 3 suggest that mTOR also integrates
metabolic signals derived from changes in neuronal glucose and fatty acid metabolism
induced by the FAS inhibitor C75.
To investigate the possible role of mTOR in the effects of C75, we assessed
whether rapamycin, a well-known inhibitor of the mTOR-raptor complex, would impair
the anorexia and weight loss produced by C75. Our results demonstrate that C75 increases the phosphorylation of mTOR and its downstream effectors S6K1 and S6 on
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amino acids residues that are specifically related to activation of the mTOR pathway, and
we demonstrate that increased mTOR signaling is necessary for C75-induced anorexia.
These results provide a novel mechanism for the potent action of C75 on the regulation of
energy balance.
Neurons derive their energy primarily from glucose, and oxidize very small
amounts of fatty acids. In the original report of Loftus et al, increased malonyl-CoA and
reduced fatty acid oxidation were believed to mediate C75-induced anorexia (84). M.
Wortman rather proposed that it is the increase in the ratio of glucose to fatty acid
oxidation (glucose: fatty acid oxidation) that is monitored by neurons and interpreted as a
signal of energy surplus (49). Because neurons oxidize relatively more glucose than fatty
acids (86), the former was predicted to contribute to a larger extent to changes in the ratio
of glucose: fatty acid oxidation. Increased glucose utilization was hypothesized to be
necessary for C75 to signal a state of positive energy balance and induce anorexia. CNS
glucose utilization is at its nadir during ketosis, forcing neurons to use ketones as an
alternate source of energy (117). Consistent with this, the anorexigenic effect of C75 was
abolished in conditions that prevent it from increasing glucose use, including when rats
were in ketosis. Providing rats with a sucrose solution alongside with the ketogenic diet, which is presumed to reverse ketosis, restored the anorexigenic effect of C75. Given that malonyl-CoA levels are determined by the presence of acetyl-CoA, as well as the relative activity of ACC and MCD, the effects of C75 at reducing fatty acid oxidation would therefore not expected to be altered under this condition. The implication of these studies
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is that increased glucose use is the primary signal necessary for the anorexigenic action of
C75 (49).
The lack of anorexigenic effect of C75 during ketosis is associated with changes in
the ability of C75 to increase mTOR signaling. This model is also an indirect way to
assess whether the increased neuronal glucose metabolism caused by C75 could be
sensed and integrated by mTOR, as measured by increased mTOR signaling. Our results
indicate the existence of a relationship between CNS glucose metabolism and the mTOR
pathway. Consistent with this view, the hypothalamic mTOR signaling pathway might not only be a target of the action of C75, but also an integrator of the status of glucose
and fatty acid metabolism in the CNS. One way to further confirm this hypothesis, would
be to examine whether inhibitors of glycolysis such as 2-deoxyglucose, recapitulate the
effects of ketosis on the mTOR pathway.
Relative contribution of AMPK and mTOR to C75-induced anorexia.
Activating AMPK or inhibiting mTOR signaling in the CNS both stimulate feeding
(19, 34, 41). Either one of these manipulations also reverses the phenotype induced by
C75 in terms of food intake, and possibly neuropeptide expression, making it difficult to
delineate the relative contribution of AMPK and mTOR to the metabolic effects of C75.
Redundancy is a critical feature of the neurocircuitry regulating food intake. While it is unlikely that a single pathway accounts for the effects of any one compound, an alternative possibility is that different pathways are more or less important to sense and integrate the signal of different fuels. Interestingly, the inhibitory effect of AMPK
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activation on C75-induced anorexia occurs in the first hour following treatment and has
dissipated 3 hours later (79), whereas the effect of mTOR inhibition is revealed after 8
hours and is still significant 24 hours after injection of the drug. These observations
would suggest that inhibition of AMPK is predominantly involved in the acute anorectic
effect of C75, whereas activation of mTOR participates in the delayed and long-lasting
effects of C75.
Hypothetical model of C75-induced anorexia.
Chapter 3 describes experiments where rapamycin tends to prevent the reduction in
NPY levels that normally accompany C75. The fact that the effect of C75 on its own was relatively modest as compared to previous reports (47, 84) limited our interpretation of the data. Nonetheless, a point worth considering here is the similarities that C75 shares with leptin and leucine. All three factors activate the mTOR pathway, suppress food intake in an mTOR-dependent fashion and reduce NPY expression (19, 75). Furthermore, previous studies have demonstrated that this inhibitory action of leptin (196) and C75
(84) on NPY neurons is necessary for their anorectic effect, while this remains unknown in the case of leucine. This is consistent with a model in which mTOR acts as a convergence point within metabolic sensing neurons, through which signals reflecting the levels of stored and currently available fuels affect the synthesis of proteins involved in triggering the appropriate changes in terms of caloric intake and expenditure, so that energy homeostasis is maintained (see hypothetical model 1, Appendix 1). Further experiments are warranted to confirm this model, especially the effect of mTOR inhibition on leptin-, leucine- and C75-induced changes in neuropeptide expression.
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Nevertheless, the data described in this thesis, together with those linking leptin and leucine to the mTOR pathway (19), support this model.
Based on the observation that reduced hypothalamic AMPK activity is required for
the anorexigenic action of both leptin (34) and C75 (79), Moran and colleagues have
proposed that leptin may affect food intake through interactions with the hypothalamic
fatty acid synthesis pathway. They found that leptin, like C75 (176), elevates malonyl-
CoA and that this is necessary for leptin to reduce food intake (52). Therefore it is
reasonable to propose that leptin may modulate mTOR through interaction with the FAS
pathway through the mechanism illustrated in “hypothetical model 2” (Appendix 2).
What is the purpose of this complex regulatory system?
From the perspective of food intake and energy balance regulation, it makes
intuitive sense for CNS fuel sensors to monitor the circulating levels of adiposity signals.
In contrast, meal-induced increase of circulating nutrients such as glucose and fatty acids
is poorly correlated to meal termination (33). Hence, the reasons for which signals of
immediately available fuels are integrated among the same circuits as those of stored
fuels are less clear. At the cellular level, however, this system might be in place to
prevent disruption of energy homeostasis, since this would impact on the ability of the
cell to grow and to survive. The control of cell size impacts on the size of organs, limbs
and the entire organism itself, which can somehow be compared to a large cell. One
difference, for example, is the extent of energy depletion necessary to induce cell versus
organism death, given that most mammals can survive for a relatively long time without
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food. From a philosophical point of view, could these be reasons why the CNS monitors
current fuel status, in addition to stored energy? CNS fuel sensing systems might be
designed in a way to ensure that both the cell and the entire organism engage in the
appropriate anabolic and catabolic responses to maintain energy homeostasis, in order to
maintain their functions and preserve their viability.
Clinical perspectives.
The importance of understanding the molecular pathways underlying CNS fuel
sensing is highlighted by recent evidence indicating that these elegant mechanisms can
become disrupted under conditions of nutrient excess. Indeed, reduced sensitivity to the
anorexigenic effects of leptin, insulin and long chain fatty acids occurs relatively rapidly
after exposure to high fat diet (88, 197). Animals are therefore more susceptible to gain
weight and eventually, to become obese. Another point that is worth considering from a
clinical perspective is the rather large contribution these systems have to the lack of
success achieved so far by behavioral and pharmacological approaches for treating obesity. They counteract for decreased adiposity by driving appetite and hyperphagia,
and reducing energy expenditure. While these compensatory responses might have
evolved to favor animals survival in times of famine (3), they render weight loss difficult
to achieve and even more difficult to maintain in an environment of nutrient abundance
(198).
Thus, the redundancy of intracellular mechanisms that are in place to achieve such
energy homeostasis is undeniable. A example of this is that NPY knock-out mice eat and
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gain weight similarly to wild-type animals, despite its presumed cardinal role as an
orexigenic neuropeptide (199). There is compelling evidence that hypothalamic AMPK
and mTOR are important integrators of fuel signals, but it is likely that other molecules
will emerge as CNS fuel sensors in the future as well. A large portion of the current
knowledge of CNS fuel sensing comes from hypotheses generated from what was already
known about fuel sensing in peripheral cells, and adapted to take into account the unique
environment of the brain. Such approach has been successful in identifying novel
mechanisms by which FAS inhibitors regulate food intake (49) and more recently, in identifying AMPK (34) and mTOR (19) as a CNS fuel sensors.
In that regard, it has recently been reported that carcinoembryonic antigen-related
cell adhesion molecule 1 (CEACAM-1), a glycoprotein downstream of insulin signaling,
is an endogenous inhibitor of FAS in the liver (200). In the fed state, a condition under
which ACC would be expected to be active and its substrate acetyl-CoA elevated, there is
an elevation of hypothalamic malonyl-CoA (176). This suggests that either: 1) FAS
activity is relatively too low to compensate, therefore allowing malonyl-CoA to
accumulate, or 2) there is a simultaneous inhibition of FAS. While Kim et al. have not
observed any change in hypothalamic FAS mRNA, protein or enzymatic activity in free
fed rats relative to animals that been fasted for 24 h (47), we found that refeeding after a
24-h fast acutely reduces FAS activity in the hypothalamus, supporting the latter view (K.
Proulx, unpublished data). Given that refeeding acutely reduces hepatic FAS activity
through activation of CEACAM-1 (200), and that the temporal pattern of this effect is
comparable to the one we observed in the hypothalamus (K. Proulx, unpublished data), it
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is tempting to hypothesize that CEACAM-1 is an endogenous inhibitor of FAS in the hypothalamus as well. In preliminary experiments, we found that CEACAM-1 mRNA is regulated by feeding and insulin in the hypothalamus (K. Proulx, unpublished data). The potent anorexia observed with pharmacological inhibitors of FAS supports the clinical potential of agents that could increase the action of such putative endogenous FAS inhibitor in the brain.
A drug that would affect both sides of the energy balance equation, in a way to reduce food intake and increase fat oxidation in the periphery, would have therapeutic potential. The problem in considering mTOR and AMPK as targets for drug therapy is that their peripheral and central actions oppose each other in terms of energy balance regulation. For instance, pharmacological inhibition of the mTOR pathway in the CNS
(19), as well as total knockout of the S6K1 gene (74) are associated with increased food intake in rodents, supporting that CNS mTOR promotes a negative energy balance by regulating feeding. Despite this, total S6K1 knockout mice maintained on a high fat diet are leaner than controls due to increased fatty acid oxidation (74). These effects might appear to oppose each other in terms of body weight regulation. However, they are consistent with the concept of a fuel sensor. In fact, endogenous factors such as leptin
(34) and α-lipoic acid (187) inhibit AMPK in the CNS while activating it in the periphery, therefore promoting a negative energy balance. The effect of therapeutic agents is more problematic. Insulin therapy is an example. Type II diabetes patients can gain weight as a result of insulin treatment, probably because the anorexigenic action of insulin in the brain is counteracted by its potent stimulatory effect on fatty acid synthesis
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in the periphery. So from a CNS perspective, one could predict that the potency of mTOR activator or AMPK inhibitor to induce weight loss would be determined by the relative balance between their inhibitory action on food intake and fatty acid oxidation. This dual action complicates the development of therapeutic strategies. On a more optimistic note, understanding the mechanisms by which endogenous factors modulate the activity of fuel sensors in an opposite direction in the CNS versus the periphery might lead to the development of “intelligent” drugs, which would do the same. Again, answers to this dilemma might come from identifying differences between cells of the CNS and those of the periphery in terms of their metabolism and the environment in which they operate.
Such an approach might lead to the identification of features that are specific to each cell type, and that could be used as targets to increase the specificity of novel drugs. Ideally, such treatments would activate mTOR in the CNS while inhibiting it in the periphery, and vice versa for AMPK.
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Appendix 1
Hypothalamic neuron
NPY - NPY - pS6- pS6 + Food intake pS6K1+ pS6K1+ pmTOR+ pmTOR
+ +
Leptin Glucose/leucine
Stored fuels (adipose tissue) current fuels (nutrients)
Hypothetical model 1: mTOR may integrate signals from both stored and currently available fuels. MTOR might be a convergence point within metabolic sensing neurons, through which signals reflecting the levels of stored and currently available fuels affect the synthesis of proteins involved in triggering the appropriate changes in terms of caloric intake and expenditure, so that energy homeostasis is maintained.
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Appendix 2
Hypothalamic neuron
C75 NPY - - pS6 FAS Food intake acetyl-CoA Malonyl-CoA LCFA + ACC pS6K1 CPT-1 + AMPK glucose pmTOR
- +
Leptin
Stored fuels (adipose tissue)
Hypothetical model 2: C75 and leptin may modulate mTOR through interaction with the FAS pathway. C75 and leptin might increase glucose oxidation through interaction with the FAS pathway. This would increase the phosphorylation and activation of mTOR signaling, which might in turn reduce NPY levels and, consequently, food intake.
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