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Obesity
INDEX
CONTENTS
SR. NO. TITLE PAGE
NO.
1 Introduction
1.1.Pharmacology
1.2.Location
1.3.Body Function
1.4.Classication
1.!.E"#r$ssion
1.%.&$c$#tor
1.'.Physiology
1-14
2 ()$sity
15-17
3 &$lation *ith ()$sity
18-21
4 +ummary
22-26
5 List (, -))r$.iation
27
6 List (, /a)l$s -nd Figur$s
28
7 &$,$r$nc$
29-33
1
1. INTRODUCTION
Obesity is the most common and rapidly growing nutritional problem in Westernized
countries. By dramatically raising mortality and the risk of morbidity from
hypertension, dyslipidemia, diabetes mellitus, and cardiovascular diseases, this
condition has become one of the major public health concerns for the 2st century.
Obesity is a comple! multifactorial disease caused by the interaction of genetic and
environmental factors. "he potential implication of genetic factors in the
development of human obesity is well demonstrated by the description of five
monogenic forms of human obesity. "he genes implicated in these forms of obesity
encode proteins of the leptin a!is and brain#e!pressed targets of leptin involved in
the melanocortin pathway. "hey include leptin, the leptin receptor, proconvertase ,
pro#opiomelanocortin $%O&'(, and the melanocortin#) receptor $&')#*(. +!cept
for &')#*, mutations in these genes cause rare, recessive, syndromic forms of
obesity, associated with multiple endocrine abnormalities.
&')#* is a ,,2#amino acid protein encoded by a single e!on gene localized on
chromosome -.22. &')#* belongs to the family of seven transmembrane / protein0
coupled receptors $/%'*( and signals through activation of adenylate cyclase. &')#*
&')#* belongs to the family of seven transmembrane / protein0coupled receptors
$/%'*( and signals through activation of adenylate cyclase &')#* belongs to the family
of seven transmembrane / protein0coupled receptors $/%'*( and signals through
activation of adenylate cyclase belongs to the family of seven transmembrane / protein0
coupled receptors $/%'*( and signals through activation of adenylate cyclase &')#*
belongs to the family of seven transmembrane / protein0coupled receptors $/%'*( and
signals through activation of adenylate cyclase "he importance of &')#* in body#weight
regulation is clearly demonstrated by the obese phenotype of the homozygous MC4-R0
deficient mice. 1eterozygous MC4-R
+/
mice present an intermediate average increase in
weight, females being more severely affected than males.
We have reported a case of human obesity associated with a frameshift mutation in MC4-
R. "his case differed from the previously described monogenic forms of obesity by a
dominant transmission of the morbidly obese phenotype and by the absence of associated
endocrine symptoms. "he description of a similar case and the report of several MC4-R
1
missense and nonsense mutations in 2 of two cohorts of e!tremely obese children and
adults suggest that such mutations could be a more fre.uent cause of obesity than the
syndromic forms due to the other genes. 3imited information on the function of most of
the missense mutations as well as incomplete phenotypic characterization of the patients
and their families have not yet allowed a case to be built for the overall importance of
&')#* mutations in human obesity.
1
1.1 PHARMACOLOGY
"he melanocortin#) receptor $&')*( as a molecular target for both indications because
it plays an important role in the regulation of appetite and autonomic function $for details
see 4igure (. Our pharmacological approach consisted in the generation and
characterization of functionally active antibodies against e!tracellular se.uences of the
&')*. Our initial studies in rats evealed an obese phenotype after immunization against
the 5#terminal domain of the &')*. "hese e!periments are described in more detail in
our previous biennial report and have been published in 2667. 8n subse.uent e!periments
we attempted to generate monoclonal anti#&')* antibodies since such molecules can be
produced in unlimited amounts. 8mmunization of mice against the 5#terminal domain
resulted in a monoclonal antibody which blocked the activity of the &')*. 8n further in
vitro studies it was characterised as an inverse agonist with non#competitive antagonistic
activity. 9 priority claim has been filed in collaboration with the "echnology "ransfer
:nit of the :niversity of Basel. "he monoclonal antibody is currently being produced in
larger amounts for acute and chronic studies on its blood#brain penetration after
peripheral administration. "hereafter it will be used as a pharmacological tool to e!plore
the therapeutic potential of &')* blockade in various disease models. 9 simple model
for cache!ia#induced anore!ia, i.e. the loss of appetite in association with inflammatory
diseases, has been established and validated in our laboratory. 8t consists of the
continuous monitoring of food intake in rats after the acute administration of
lipopolysaccharide Our immunization studies on the &')* in rats are being
complemented with several series of immunizations against e!tracellular se.uences of the
&',*. "hese e!periments were initiated because presynaptic &',*s appears to
modulate the release of the &')* agonist alpha#melanocyte stimulating hormone $alpha#
&;1( from nerve terminals. 1owever, the precise function of the &',* is not known
1
because of the lack of suitable e!perimental tools. Based on preliminary results with two
antigens from the first and the third e!tracellular loop a long#term immunization study
has been started in rats which are chronically instrumented with telemetry transmitters.
"his makes it possible to monitor not only endocrine and metabolic parameters but also
blood pressure, heart rate, body temperature and locomotor activity. Our observation that
immunization against the &')* resulted in an obese phenotype in rats prompted us to
e!plore whether comparable findings can be obtained in obese patients. We therefore
initiated a clinical study in collaboration with the :niversity 'linic in ;trasbourg, 4rance,
and the communal hospital in Baden#Baden, /ermany. "hese institutions provided more
than 266 plasma samples from patients with a wide range of body mass indices $B&8(.
"he plasma samples were screened in our laboratory with an +38;9 assay using the 5#
terminal peptide se.uence of the &')*. %ositive samples were further assessed by flow
cytometry $49';( and c9&% measurements in 1+<2=, cells which overe!pressed the
human &')*. "he presence of auto#antibodies against the &')* in humans and the
increased prevalence of such autoantibodies in patients with increased B&8 would
suggest that they might play a pathogenetic role in the development of obesity. By using
such a test in the clinic a subgroup of obese patients with a common pathophysiology
could be identified and possibly causally treated. ;everal options for further clinical
studies are currently being e!plored and e!perimental studies on possible therapeutic
approaches have been initiated in our group. Our studies on the role of brain derived
neurotrophic factor $B>54( as a downstream mediator of the &')* have been
completed and are subject of a recent publication. 8n this paper we reported for the first
time that &')* stimulation in isolated hypothalami in vitro induced a release of B>54
into the incubation medium. 8n e!periments in vivo we have shown that B>54 and a
&')* agonist have comparable effects on food intake. &oreover, the effects of &')*
stimulation could be prevented by the prior administration of a selective anti#B>54
antibody. 8n an additional series of e!periments in rats with telemetry transmitters we
could demonstrate that B>54 and a &')* agonist also showed comparable
cardiovascular effects, i.e. both agents induced an increase in blood pressure and heart
rate which was associated with a rise in body temperature. "hese effects of &')*
stimulation could also be prevented by the anti#B>54 antibody. "hus B>54 appears to
1
mediate not only the anorectic but also the autonomic effects of &')* stimulation. "his
project was performed in collaboration with %rof. ?ves Barde who also supplied the
monoclonal antibody against B>54. <arl /. 1ofbauer contributed to pharmacology
teaching in the &edical 4aculty and the 4aculty for 5atural ;ciences at the :niversity of
Basel. 1e took on responsibility for the planning and coordination of pharmacology
teaching for medical students in the new Bologna system up to the Bachelor level. 1e
contributed to a newly established course for a &aster of 9dvanced ;tudies in
%harmacology degree at the :niversity of Basel. <arl /. 1ofbauer gave also lectures on
drug discovery and development at the :niversity of @uerich. <arl 1ofbauer was
involved in several continuing education activities including the co#organization of and
the active contribution to the annual A'ardiovascular %harmacology ;eminarB in 3uzern.
"ogether with several clinical colleagues <arl /. 1ofbauer continued to co#organize a
lecture series at the :niversity of Basel with focus on obesity, metabolism and nutrition
$O&e5(. <arl /. 1ofbauer and members of his group presented their findings at several
national and international scientific meetings including an invited lecture at the Coint
&eeting of the ;wiss and 'anadian %harmacological ;ocieties in Banff, 'anada, in early
2667. 8n addition to original papers and book chapters two reviews from our group
covered the recent developments in the field of anti#obesity drugs.
Figure 1: In the uer !ne" ! s#he$!ti# i""ustr!ti%n %& the stru#ture %& the MC'R is
gi(en.
1
"he activation of the &')* mediates a decrease in food intake and an increase in energy
e!penditure. "hus &')* agonists could be useful drugs for the treatment of obesity.
1owever, the &')* has not only effects on energy balance but also influences
autonomic function. &')* activation results for instance in an increase in blood pressure
and heart rate. 8n order to generate agonists with a selective effect on energy balance it is
essential to identify downstream mechanisms which may mediate the &')* induced
effects separately. B>54 appears to be such a downstream mechanism of the &')* but
it shows no selectivity for the metabolic and autonomic effects. "he subse.uent steps in
the &')* downstream pathway are still unknown.
)*+
1.). LOCATION
"he melanocortin#) receptor $&')*( is e!pressed in the hypothalamus and regulates
energy intake and body weight. In silico screening of the canine chromosome se.uence
and a comparison with the porcine MC4R se.uence by B39;" were performed. "he
nucleotide se.uence of the whole coding region and ,D# and ED#flanking regions of the dog
$2) bp( and red fo! $77 bp( MC4R gene was established and high conservation of the
nucleotide se.uences was revealed $==2(. 4ive sets of %'* primers were designed and a
search for polymorphism was performed by the ;;'% techni.ue in a group of , dogs
representing nineteen breeds and ,E farm red fo!es. ;e.uencing of >59 fragments,
representing the identified ;;'% patterns, revealed three single nucleotide
polymorphisms $including a missense one( in dogs and four silent ;5%s in red fo!es. 9n
average ;5% fre.uency was appro!. F)66 bp in the dog and F,66 bp in the red fo!. We
mapped the MC4R gene by 48;1 to the canine chromosome $'49..( and to the red
fo! chromosome E $GG:Ep.2(.
1.).1. Chr%$%s%$e L%#!ti%n
,
L%#!ti%n* &')* is located on chromosome -.22
P%siti%n* chr-HEI=6EI2#EI-=EI)
Figure sh%-s the "%#!ti%n %& MC'R in #%nte.t %n #hr%$%s%$e 1+.
1
1.).). Pr%tein L%#!ti%n
&')* m*59 has been found in multiple sites in virtually every region of the brain. 8ts
major e!pression sites are thought to be the corte!, thalamus, hypothalamus, brainstem
and spinal cord.
Figure sh%-s the re/i#te/ stru#ture %& the MC'R r%tein. This r%tein !"s% h!s the
syn%ny$ MC'*R
1.).0. Me"!n%#%rtin ' re#et%r, also known as MC'R, is a human gene.
11
it encodes
the MC' protein, a /#protein coupled receptor that binds J#melanocyte stimulating
hormone $J#&;1(. 8n murine models &') receptors have been found to be involved in
feeding behaviour, the regulation of metabolism, se!ual behaviour, and male erectile
function
11*10
8n ==-, it was reported that &')* mutations were associated with inherited
human obesity. "hey were found in heterozygotes, suggesting an autosomal dominant
inheritance pattern. 1owever, based off other research and observations, these mutations
seem to have an incomplete penetrance and some degree of codominance. 8t has a
prevalence of #2.E2 in people with B&8s of greater than ,6, making it the most
commonly known genetic defect predisposing people to obesity.
1'
1.0. 2ODY FUNCTION
>efects in signaling by leptin, a hormone produced primarily by adipose tissue that
informs the brain of the bodyKs energy reserves, result in obesity in mice and humans.
1owever, the majority of obese humans do not have abnormalities in leptin or its receptor
but instead e!hibit leptin resistance that could result from defects in downstream
1
mediators of leptin action. *ecently, two potential downstream mediators, agouti#related
protein $9grp( and its receptor, the melanocortin#) receptor $&c)r(, have been identified.
9grp and &c)r are e!cellent candidates for human disorders of body weight regulation
and represent promising targets for pharmacological intervention in the treatment of these
disorders.
13
"he peptide products of the pro#opiomelanocortin $%O&'( gene have established roles in
the control of physiological processes as diverse as adrenal steroidogenesis, skin
pigmentation, analgesia and inflammation. 8n the last E years, evidence accumulated from
murine and human genetic models of disrupted melanocortin signalling has firmly
established a central role for a population of hypothalamic neurons e!pressing %O&' in
the control of appetite and body weight. Of the five known melanocortin receptors, the
&')* has been most closely linked to body weight regulation. While a#&;1 is active at
this receptor and suppresses appetite after central injection, important roles for other
%O&'#derived products have not been e!cluded. "he development of pharmacological
agonists acting on, or mimicking, the hypothalamic melanocortinergic pathway may
provide e!citing opportunities for the therapy of human obesity.
14
1
1.'. CLA55IFICATION
16*)1
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1.3. 78PR755ION
1
*egulation of endogenous melanocortin#) receptor e!pression and signaling by gluco
corticoids
)1
9 the melanocortin#) $&')( receptor plays a pivotal role in regulating
food intake and energy e!penditure, and obesity results from mutations that interfere
with the &') receptor pathway. We investigated the effect of gluco corticoids on
endogenous &') receptors e!pressed in /"# cells, an immortalized hypothalamic
neuronal cell line. >e!amethasone $>e!( caused a E# to 6#fold increase in the
c9&% response to the &') receptor agonist, 5>%#alpha &;1. "he stimulatory
effect of >e! reached a ma!imum within 2) h and was blocked by the gluco
corticoid antagonist *:)-I. "his gluco corticoid effect was specific for the &')
receptor and not a result of up#regulation of another component of the c9&%
cascade, because the response to endogenous beta#adrenergic receptor stimulation
was not altered by >e!. >e! also potentiated 5>%#alpha &;1#mediated +*<F2
activation. 9fter 2 h, >e! caused a ,# to E#fold increase in M2E8N5>%#alpha &;1
binding, which was maintained for at least )- h and prevented by *:)-I. >e!
withdrawal caused a rapid return of &') receptor concentration to the basal level.
>e!#mediated increases in &') receptor concentration resulted from a rapid but
transient increase in &') receptor m*59. "his regulation apparently re.uires
genomic regulatory se.uences because >e! did not increase &') receptor
e!pression or signaling in '1O cells e!pressing the &') receptor under the control
of a cytomegalovirus promoter. We conclude that in /"# hypothalamic neurons,
gluco corticoids increase the amplitude of &') receptor signaling. "his regulation
may serve as a control to limit the effects of gluco corticoids on food intake.
+!pression of the human melanocortin#) receptor gene is controlled by several
members of the ;p transcription factor family
))
,the melanocortin#) receptor $&')#
*( plays a key role in the
hypothalamic control of food intake, lending importance to
the
understanding of the mechanisms that regulate its e!pression.
"o identify factors
controlling the e!pression of the human
$h( &')#* gene, a fragment containing 2E,
bp of the EK#flanking
region of the h&')#* gene was isolated. 9 series of h&')#*
luciferase
constructs were developed and used to transiently transfect
1+<2=, and
/"07 cell lines, both e!pressing endogenous
&')#* m*59, >eletion analysis of
the 2E, bp fragment showed
that the basal promoter activity is mainly restricted to
1
the
7= bp upstream of the transcription start site in both cell
types. &utation of a
putative ;p#binding site located at position
07I bp resulted in a dramatic reduction
of the luciferase
activity in 1+<2=, and /"07 cells by -7 and -62 respectively.
Both in vitro and in vivo studies $gel shift and chromatin immunoprecipitation
analyses( revealed binding of both ;p and ;p, to this site
in 1+<2=, cells,
cotransfection with an ;p e!pression vector
in >rosophila cells that do not e!press
;p, in conjunction
with treatment of 1+<2=, cells with mithramycin 9, a specific
inhibitor of ;p, confirmed the role of ;p. 4or the first time,
we have demonstrated
that the constitutive activity of the h&')#*
promoter is dependent upon ;p
transcription factors.
+!pression of &c#) *eceptor 8n 1ypothalamuic /t# 'ell
3ine9 mutations in the melanocortin#) receptor $&')#*( cause obesity in both mice
and humans, and the receptor is presumed to have an important role in the regulation
of energy homeostasis. "he &')#* is e!pressed in discrete sets of neurons in the
central nervous system, and thus it has been technically difficult to study the
regulation of e!pression and the signaling mechanisms of this receptor. We report
here a neuronal cell line that e!hibits endogenous functional e!pression for the &')#
*. 8nitially, *"#%'* analysis showed the presence of &')#* *59 in the
hypothalamic /"# and /"#7 cells.
)0*)3
8n addition, /"#7 cells e!pressed
melanocortin#, receptor while the /"# subclone specifically e!pressed
predominantly the &')#* *59. 1igh#affinity binding sites were demonstrated in
the /"# and /"#7 cells for 5>%# melanocyte#stimulating hormone $&;1P <i Q
. R 6
#6
and .- R 6
#6
&( and agouti#related protein $9/*%P <i Q .E)- R 6
#=
and
.II,
#=
&(. #&;1#stimulated c9&% production in /"# cells with an +'E6 of 2.2
R 6
#-
&, and c9&% production was inhibited in the presence of 9/*%, an
endogenous antagonist of the &')#*. ;timulation of gonadotropin#releasing
hormone $/n*1( secretion was achieved with n& to S& concentrations of 5>%#
#&;1 while no /n*1 secretion was observed when the /"# cells were treated
with 9/*%. "he data presented here show that /"# cells specifically e!press a
functional &')#* that couples to /n*1 release.
1.4. R7C7PTOR
1
9cetylcholine, 9ngiotensin, 9pelin , Bombesi n, Bradykinin, 'al ci t oni n,
amyl i n, '/*% and adrenomedul l i n, 'alcium#sensing, 'annabinoid,
'hemoki ne, 'hemot act i c pept i de, 'holecystokini, >opami ne, +ndothelin,
/9B9B, /hrelin, /l ut amat e, met abot ropi c, /lutamate, /onadotrophin#
releasing 1ormone $/n*1(, 1i st ami ne, E#1" $E#1ydro!ytryptamine(,
3eukotriene, 3i po!i n, 3ysophosphatidic acid, &el ani n#concent rat i ng
hormone, &elanocortin, &el at oni n, 5europeptide ?, 5eurot ensi n, Opioid
and opioid#like, Ore!in, % 2 ?, %latelet#activating factor $%94(, %rost anoi d,
%rostanoid, %roteinase#activat ed, *ela!in family peptide, ;omat ost at i n,
;phingosine##phosphate, "achyki ni n, "hyrotropin#releasing hormone,
Gasopressi n and o!yt oci n. 9mong these melanocortin receptors are as under.
Me" !n%#%rti n re#et%r
&elanocortin receptors $provisional nomenclature( are activated by members of the
melanocortin family $&;1 0 a, b, and g forms 0 d form is not found in mammals( and
adrenocorticotrophin $9'"1(. +ndogenous antagonists include agouti and agouti#
related protein $9/*%(.
T!b"e n% ). Me"!n%#%rtin Re#et%r
N%$en#"!t
ure
MC1 MC) MC0 MC' MC3
Other
n!$es
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receptor
T T T
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et al., ==-(,
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al.,
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&;1
%olymorphisms of the &' receptor have been linked to variations in skin
pigmentation. >efects of the &'2 receptor underlie familial glucocorticoid
deficiency. %olymorphisms of the &') and &'E receptors have been linked to
obesity $'hagnon et al., ==7(.
1.6.PHY5IOLOGY
)4*01
Obesity is caused by a dysregulation in energy homeostasis that
promotes an increase in
body fat stores. "he neuroendocrine
system regulates energy balance by controlling
appetite, as
well as food intake and utilization. 3eptin, a cytokine#like
peptide produced
by adipose tissue in proportion to adipose
mass, acts on the melanocortin system,
inducing proopio melanocortin
$%O&'(