Patent Application: US-201414776318-A

Abstract:
ghrelin is a peptide hormone that binds its receptor , growth hormone secretatgogue receptor 1a , to promote adiposity and obesity in mammals . ghrelin and its receptor are targets for therapeutic intervention to treat obesity - related disease and cancer . a soluble decoy ghs - r1 a receptor is developed that binds ghrelin in the periphery , preventing ghrelin from binding ghs - r1 on cells , thereby antagonizing ghrelin to treat obesity - related pathological conditions and cancer . ghs - r1 a is a transmembrane protein comprising an n - terminal extracellular domain , seven transmembrane regions and three extracellular loops . the nt , ec1 and ec2 are linked together , in the absence of the transmembrane regions , and fused to a fc from an immunoglobulin , to create the decoy ghs - r1 a fusion protein , ghsr - fc . the ghsr - fc inhibits adiposity and weight gain in mice on a high fat diet , while the nt and ecs on their own have no significant effect .

Description:
the present inventors developed ghs - r1a - fusion constructs of a decoy protein containing the ligand - binding domains of the ghrelin receptor . intramuscular injection of the ghsr / fc plasmid decreased circulating levels of acylated - ghrelin . upon challenge with high fat diet ( hfd ), treated mice displayed reduced weight gain compared to controls , which is associated with reduced fat accumulation in the peritoneum but not lean mass . quantitative rt - pcr showed increased pparγ and hormone sensitive lipase transcripts levels in adipose tissue of treated animals , illustrating a preference for increased fat utilization . intraperitoneal glucose tolerance and insulin tolerance tests showed improved glucose clearance and insulin sensitivity in ghsr / fc - treated animals . thus , in vivo expression of the ghsr - based fusion protein prevents diet - induced weight gain , altering adipose gene expression and improving glucose tolerance . the present inventors have shown that in vivo expression of ghs - r1a fusion construct containing the extracellular binding loops 1 and 2 ( ghsr / fc ) caused a reduction in acylated ghrelin ( ag ) but , interestingly , not unacylated ghrelin ( uag ) levels , and protected mice from high fat diet - induced weight gain , which was associated with altered adipose gene expression profile and improved glucose clearance and insulin sensitivity . these observations suggest that the ghsr / fc fusion construct may find clinical use in treating obesity and obesity - related conditions . accordingly , in one aspect , the present disclosure provides a soluble fusion molecule comprising ( a ) at least one of i ) the n - terminal , ii ) extracellular loop 1 and iii ) extracellular loop 2 , of the growth hormone secretagogue receptor ( ghs - r1a ); linked to ( b ) a fusion partner . the term “ growth hormone secretagogue receptor ” or “ ghs - r1a ” refers to the full length growth hormone secretagogue receptor from any species or source , optionally mammalian , such as human or mouse and isoforms and homologs thereof . such receptors have both extracellular and intracellular domains including when folded properly as in fig1 a an n - terminal domain ( ntd ), three extracellular loops ( ec1 , ec2 and ec3 ) and transmembrane domains . the nucleotide sequence of human ghs - r1a is as shown in seq id no : 18 and encodes the human ghs - r1a protein under genbank accession no . q92847 the nucleotide sequence of murine ghs - r1a is as shown in seq id no : 9 and encodes the murine ghs - r1a protein under genbank accession no . q99p50 . the term “ extracellular domain of ghs - r1a ” refers to a domain of the ghs - r1a receptor that lacks the transmembrane and intracellular domains of the receptor , and includes , without limitation , the n - terminal domain , the extracellular loop 1 and the extracellular loop 2 . the term “ n - terminal ” as used herein refers to an isolated peptide corresponding to the amino acid sequence coding for the n - terminal extracellular region of ghs - r1a . in an embodiment , the n - terminal domain extends to the last amino acid of the extracellular domain preceding the transmembrane domain amino acid sequence . the term “ extracellular loop ” as used herein refers to an isolated protein comprising the sequence of a portion of the ghs - r1a that forms a loop on the extracellular side of the cell membrane . ghs - r1a has a first extracellular loop from 102 to 127 amino acids of human ghs - r1a and from 101 to 126 amino acids of mouse ghs - r1a , termed extracellular loop 1 , and a second extracellular loop from 182 to 207 amino acids of human ghs - r1a and from 181 to 206 amino acids of mouse ghs - r1a , termed extracellular loop 2 . in an embodiment , the fusion molecule comprises at least two of i ) the n - terminal , ii ) the extracellular loop 1 and ii ) the extracellular loop 2 of the ghs - r1a , linked to a fusion partner . in another embodiment , the fusion molecule comprises i ) the n - terminal , ii ) the extracellular loop 1 and iii ) the extracellular loop 2 of the ghs - r1a , linked to a fusion partner . in yet a further embodiment , the fusion molecule consists of i ) the n - terminal , ii ) the extracellular loop 1 and iii ) the extracellular loop 2 of the ghs - r1a , linked to a fusion partner . in one embodiment , the n - terminal region of the ghs - r1a has the amino acid sequence as shown in seq id no : 4 or 13 or a variant thereof or is encoded by the nucleic acid sequence as shown in seq id no : 3 or 12 or a variant thereof ; the extracellular loop 1 has the amino acid sequence as shown in seq id no : 6 or 15 or a variant thereof or is encoded by the nucleic acid sequence as shown in seq id no : 5 or 14 or a variant thereof ; and / or the extracellular loop 2 has the amino acid sequence as shown in seq id no : 8 or 17 or a variant thereof or is encoded by the nucleic acid sequence as shown in seq id no : 7 or 16 or a variant thereof . the term “ a fusion molecule ” refers to the linking of a peptide sequence derived from the extracellular domain or domains of ghs - r1a to a fusion partner and can be a direct or indirect linkage via a covalent or non - covalent linkage . the fusion partner may be linked to either the n - terminus or the c - terminus of the peptide sequence derived from ghs - r1a . the term “ soluble fusion molecule ” as used herein refers to the fusion molecule as lacking any transmembrane or intracellular domains and if expressed from a nucleotide sequence in a cell , would be a secreted protein . in an embodiment , the fusion partner is a stabilizing molecule , such as a stabilizing protein or a polymer . the term “ stabilizing molecule ” as used herein refers to a molecule that when linked to the extracellular domain ( s ) provides an increased half - life and / or reduced immunogenicity compared to the extracellular domain ( s ) without such molecule . in one embodiment , the stabilizing protein is an immunoglobulin constant region ( fc ) or an albumin protein . in an embodiment , the fc region is an igg fc domain , optionally mouse igg1 or human igg2 or igg4 . in another embodiment , the stabilizing polymer is a polyethylene glycol or the like . in another embodiment , the soluble fusion molecule comprises the amino acid sequence as shown in seq id no : 2 or 11 or a variant thereof or is encoded by the nucleic acid sequence as shown in seq id no : 1 or 10 or a variant thereof . the domains or regions of the soluble fusion molecule of the present disclosure can be linked directly or can comprise a linker between each region , optionally a peptide linker . the peptide linker can be any size provided it does not interfere with the function of the individual linked regions . in one embodiment , the peptide linker is from about 1 to about 15 amino acids in length , more specifically from about 1 to about 10 amino acids , and most specifically from about 1 to about 6 amino acids . where the soluble fusion molecule is made solely of fused peptides , it may be part of a continuous sequence and as such can be translated as a single polypeptide from a coding sequence that codes for both the at least one extracellular domain and the stabilizing protein . in other embodiments , one of skill in the art can appreciate that the fusion molecule can also be formed by linking the at least two protein regions in vitro , for example , using chemical cross - linkers . as used herein , the term “ protein ” or “ polypeptide ” refers to a sequence of amino acid residues encoded by a nucleic acid molecule . within the context of the present application , a polypeptide of the disclosure may in one embodiment include various structural forms of the primary protein . for example , a polypeptide of the disclosure may be in the form of acidic or basic salts or in neutral form . in addition , individual amino acid residues may be modified by oxidation or reduction . the proteins and polypeptides of the present disclosure may also include truncations , analogs and homologs of the proteins and polypeptides as described herein having substantially the same function as the proteins or polypeptides of the present disclosure , such as the ability to decrease acylated ghrelin . analogs of the proteins described herein , may include , but are not limited to an amino acid sequence containing one or more amino acid substitutions , insertions , and / or deletions . amino acid substitutions may be of a conserved or non - conserved nature . conserved amino acid substitutions involve replacing one or more amino acids of the proteins of the disclosure with amino acids of similar charge , size , and / or hydrophobicity characteristics . when only conserved substitutions are made the resulting analog should be functionally equivalent . non - conserved substitutions involve replacing one or more amino acids of the amino acid sequence with one or more amino acids which possess dissimilar charge , size , and / or hydrophobicity characteristics . conservative substitutions are described in the patent literature , as for example , in u . s . pat . no . 5 , 264 , 558 . it is thus expected , for example , that interchange among non - polar aliphatic neutral amino acids , glycine , alanine , proline , valine and isoleucine , would be possible . likewise , substitutions among the polar aliphatic neutral amino acids , serine , threonine , methionine , asparagine and glutamine could possibly be made . substitutions among the charged acidic amino acids , aspartic acid and glutamic acid , could probably be made , as could substitutions among the charged basic amino acids , lysine and arginine . substitutions among the aromatic amino acids , including phenylalanine , histidine , tryptophan and tyrosine would also likely be possible . other substitutions might well be possible . as used herein , the term “ variant thereof ” means a sequence with at least 75 %, 80 %, 85 %, 90 %, 95 % or 99 % sequence identity to a nucleotide or amino acid sequence of interest . the term “ sequence identity ” as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences . to determine the percent identity of two amino acid sequences or of two nucleic acid sequences , the sequences are aligned for optimal comparison purposes ( e . g ., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence ). the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared . when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence , then the molecules are identical at that position . the percent identity between the two sequences is a function of the number of identical positions shared by the sequences ( i . e ., % identity = number of identical overlapping positions / total number of positions . times . 100 %). in one embodiment , the two sequences are the same length . the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm . an optional , non - limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of karlin and altschul , 1990 , proc . natl . acad . sci . u . s . a . 87 : 2264 - 2268 , modified as in karlin and altschul , 1993 , proc . natl . acad . sci . u . s . a . 90 : 5873 - 5877 . such an algorithm is incorporated into the nblast and xblast programs of altschul et al ., 1990 , j . mol . biol . 215 : 403 . blast nucleotide searches can be performed with the nblast nucleotide program parameters set , e . g ., for score = 100 , wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure . blast protein searches can be performed with the xblast program parameters set , e . g ., to score - 50 , wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure . to obtain gapped alignments for comparison purposes , gapped blast can be utilized as described in altschul et al ., 1997 , nucleic acids res . 25 : 3389 - 3402 . alternatively , psi - blast can be used to perform an iterated search , which detects distant relationships between molecules ( id .). when utilizing blast , gapped blast , and psi - blast programs , the default parameters of the respective programs ( e . g ., of xblast and nblast ) can be used ( see , e . g ., the ncbi website ). another optional , non - limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of myers and miller , 1988 , cabios 4 : 11 - 17 . such an algorithm is incorporated in the align program ( version 2 . 0 ) which is part of the gcg sequence alignment software package . when utilizing the align program for comparing amino acid sequences , a pam120 weight residue table , a gap length penalty of 12 , and a gap penalty of 4 can be used . the percent identity between two sequences can be determined using techniques similar to those described above , with or without allowing gaps . in calculating percent identity , typically only exact matches are counted . as used herein , the term “ fragment thereof ” refers to a nucleic acid or amino acid sequence comprising up to 3 , 5 , 10 , 15 , 25 , 50 , 100 , 250 , 500 , 1000 , 2000 or 3000 contiguous residues of a nucleotide or amino acid sequence of interest . within the context of the present disclosure , a protein of the disclosure may include various structural forms of the primary protein which retain biological activity . for example , a protein of the disclosure may be in the form of acidic or basic salts or in neutral form . in addition , individual amino acid residues may be modified by oxidation or reduction . exemplary methods of making the alterations set forth above are disclosed by sambrook et al . ( molecular cloning : a laboratory manual , 2nd ed ., cold spring harbor laboratory press , 1989 ). also provided herein is a nucleic acid molecule encoding the soluble fusion molecule of the present disclosure , wherein the molecule optionally comprise a peptide linker joining the various domains of the fusion molecule . in another embodiment , the present disclosure provides an isolated nucleic acid molecule encoding the n - terminal domain of ghs - r1a . in one embodiment , the isolated nucleic acid comprises the nucleic acid sequence as shown in seq id no : 3 or 12 or encodes the amino acid sequence as shown in seq id no : 4 or 13 . in another embodiment , the present disclosure provides an isolated nucleic acid molecule encoding the extracellular loop 1 of ghs - r1a . in one embodiment , the isolated nucleic acid comprises the nucleic acid sequence as shown in seq id no : 5 or 14 or a variant thereof or encodes the amino acid sequence as shown in seq id no : 6 or 15 or a variant thereof . further provided herein is an isolated nucleic acid molecule encoding the extracellular loop 2 of ghs - r1a . in one embodiment , the isolated nucleic acid comprises the nucleic acid sequence as shown in seq id no : 7 or 16 or a variant thereof or encodes the amino acid sequence as shown in seq id no : 8or 17 or a variant thereof . as used herein , the term “ nucleic acid molecule ” means a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases , sugars and intersugar ( backbone ) linkages . the term also includes modified or substituted sequences comprising non - naturally occurring monomers or portions thereof . the nucleic acid sequences of the present disclosure may be deoxyribonucleic acid sequences ( dna ) or ribonucleic acid sequences ( rna ) and may include naturally occurring bases including adenine , guanine , cytosine , thymidine and uracil . the sequences may also contain modified bases . examples of such modified bases include aza and deaza adenine , guanine , cytosine , thymidine and uracil ; and xanthine and hypoxanthine . the term “ isolated nucleic acid sequences ” as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant dna techniques , or chemical precursors , or other chemicals when chemically synthesized . an isolated nucleic acid is also substantially free of sequences , which naturally flank the nucleic acid ( i . e . sequences located at the 5 ′ and 3 ′ ends of the nucleic acid ) from which the nucleic acid is derived . the term “ nucleic acid ” is intended to include dna and rna and can be either double stranded or single stranded , and represents the sense or antisense strand . further , the term “ nucleic acid ” includes the complementary nucleic acid sequences . the disclosure also provides isolated nucleic acid sequences encoding variants of the cdr sequences and variable region sequences discussed above . variant nucleic acid sequences include nucleic acid sequences that hybridize to the nucleic acid sequences disclosed herein under at least moderately stringent hybridization conditions , or have at least 50 %, 60 %, 70 %, 80 %, 90 %, 95 % or 98 % sequence identity to the nucleic acid sequences that encode the amino acid sequences disclosed herein . the term “ sequence that hybridizes ” means a nucleic acid sequence that can hybridize to a nucleic acid sequence disclosed herein under stringent hybridization conditions . appropriate “ stringent hybridization conditions ” which promote dna hybridization are known to those skilled in the art , or may be found in current protocols in molecular biology , john wiley & amp ; sons , n . y . ( 1989 ), 6 . 3 . 1 - 6 . 3 . 6 . the term “ stringent hybridization conditions ” as used herein means that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution . hybridization may occur to all or a portion of a nucleic acid sequence molecule . the hybridizing portion is at least 50 % the length with respect to one of the polynucleotide sequences encoding a polypeptide . in this regard , the stability of a nucleic acid duplex , or hybrids , is determined by the tm , which in sodium - containing buffers is a function of the sodium ion concentration , g / c content of labeled nucleic acid , length of nucleic acid probe ( l ), and temperature ( tm = 81 . 5 ° c .− 16 . 6 ( log 10 [ na +])+ 0 . 41 (%( g + c )− 600 / l ). accordingly , the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature . in order to identify molecules that are similar , but not identical , to a known nucleic acid molecule a 1 % mismatch may be assumed to result in about a 1 ° c . decrease in tm , for example if nucleic acid molecules are sought that have a greater than 95 % identity , the final wash will be reduced by 5 ° c . based on these considerations stringent hybridization conditions shall be defined as : hybridization at 5 × sodium chloride / sodium citrate ( ssc )/ 5 × denhardt &# 39 ; s solution / 1 . 0 % sds at tm ( based on the above equation ) − 5 ° c ., followed by a wash of 0 . 2 × ssc / 0 . 1 % sds at 60 ° c . one example of a nucleic acid modification to prepare an analog is to replace one of the naturally occurring bases ( i . e . adenine , guanine , cytosine or thymidine ) of the sequence with a modified base such as xanthine , hypoxanthine , 2 - aminoadenine , 6 - methyl , 2 - propyl and other alkyl adenines , 5 - halo uracil , 5 - halo cytosine , 6 - aza uracil , 6 - aza cytosine and 6 - aza thymine , pseudo uracil , 4 - thiouracil , 8 - halo adenine , 8 - aminoadenine , 8 - thiol adenine , 8 - thiolalkyl adenines , 8 - hydroxyl adenine and other 8 - substituted adenines , 8 - halo guanines , 8 amino guanine , 8 - thiol guanine , 8 - thiolalkyl guanines , 8 - hydroxyl guanine and other 8 - substituted guanines , other aza and deaza uracils , thymidines , cytosines , adenines , or guanines , 5 - trifluoromethyl uracil and 5 - trifluoro cytosine . another example of a modification is to include modified phosphorous or oxygen heteroatoms in the phosphate backbone , short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages in the nucleic acid molecules . for example , the nucleic acid sequences may contain phosphorothioates , phosphotriesters , methyl phosphonates , and phosphorodithioates . a further example of an analog of a nucleic acid molecule of the disclosure is a peptide nucleic acid ( pna ) wherein the deoxyribose ( or ribose ) phosphate backbone in the dna ( or rna ), is replaced with a polyamide backbone which is similar to that found in peptides ( p . e . nielsen , et al science 1991 , 254 , 1497 ). pna analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro . pnas also bind stronger to a complimentary dna sequence due to the lack of charge repulsion between the pna strand and the dna strand . other nucleic acid analogs may contain nucleotides containing polymer backbones , cyclic backbones , or acyclic backbones . for example , the nucleotides may have morpholino backbone structures ( u . s . pat . no . 5 , 034 , 506 ). the analogs may also contain groups such as reporter groups , a group for improving the pharmacokinetic or pharmacodynamic properties of nucleic acid sequence . a person skilled in the art will appreciate that the proteins of the disclosure may be prepared in any of several ways , including , without limitation , by using recombinant methods . accordingly , the nucleic acid molecules disclosed herein may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the proteins . possible expression vectors include but are not limited to cosmids , plasmids , or modified viruses ( e . g . replication defective retroviruses , adenoviruses and adeno - associated viruses ), so long as the vector is compatible with the host cell used . the expression vectors are “ suitable for transformation of a host cell ”, which means that the expression vectors contain a nucleic acid molecule of the application and regulatory sequences selected on the basis of the host cells to be used for expression , which is operatively linked to the nucleic acid molecule . operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid . the disclosure therefore contemplates a recombinant expression vector containing a nucleic acid molecule disclosed herein , and the necessary regulatory sequences for the transcription and translation of the inserted protein - sequence . suitable regulatory sequences may be derived from a variety of sources , including bacterial , fungal , viral , mammalian , or insect genes ( for example , see the regulatory sequences described in goeddel , gene expression technology : methods in enzymology 185 , academic press , san diego , calif . ( 1990 )). selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below , and may be readily accomplished by one of ordinary skill in the art . examples of such regulatory sequences include : a transcriptional promoter and enhancer or rna polymerase binding sequence , a ribosomal binding sequence , including a translation initiation signal . additionally , depending on the host cell chosen and the vector employed , other sequences , such as an origin of replication , additional dna restriction sites , enhancers , and sequences conferring inducibility of transcription may be incorporated into the expression vector . the recombinant expression vectors of the disclosure may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the application . examples of selectable marker genes are genes encoding a protein such as g418 and hygromycin which confer resistance to certain drugs , β - galactosidase , chloramphenicol acetyltransferase , firefly luciferase , or an immunoglobulin or portion thereof such as the fc portion of an immunoglobulin , optionally igg . transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as β - galactosidase , chloramphenicol acetyltransferase , or firefly luciferase . if the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with g418 . cells that have incorporated the selectable marker gene will survive , while the other cells die . this makes it possible to visualize and assay for expression of recombinant expression vectors of the application and in particular to determine the effect of a mutation on expression and phenotype . it will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest . the recombinant expression vectors may also contain genes which encode a fusion moiety which provides increased expression of the recombinant protein ; increased solubility of the recombinant protein ; and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification . for example , a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein . typical fusion expression vectors include pgex ( amrad corp ., melbourne , australia ), pmal ( new england biolabs , beverly , mass .) and prit5 ( pharmacia , piscataway , n . j .) which fuse glutathione s - transferase ( gst ), maltose e binding protein , or protein a , respectively , to the recombinant protein . recombinant expression vectors can be introduced into host cells to produce a transformed host cell . the terms “ transformed with ”, “ transfected with ”, “ transformation ” and “ transfection ” are intended to encompass introduction of nucleic acid ( e . g . a vector ) into a cell by one of many possible techniques known in the art . the term “ transformed host cell ” as used herein is intended to also include cells capable of glycosylation that have been transformed with a recombinant expression vector of the invention . prokaryotic cells can be transformed with nucleic acid by , for example , electroporation or calcium chloride - mediated transformation . for example , nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co - precipitation , deae - dextran mediated transfection , lipofectin , electroporation or microinjection . suitable methods for transforming and transfecting host cells can be found in sambrook et al . ( molecular cloning : a laboratory manual , 3rd edition , cold spring harbor laboratory press , 2001 ), and other laboratory textbooks . suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells . for example , the proteins of the disclosure may be expressed in yeast cells or mammalian cells . other suitable host cells can be found in goeddel , gene expression technology : methods in enzymology 185 , academic press , san diego , calif . ( 1991 ). in addition , the proteins of the disclosure may be expressed in prokaryotic cells , such as escherichia coli ( zhang et al ., science 303 ( 5656 ): 371 - 3 ( 2004 )). in addition , a pseudomonas - based expression system such as pseudomonas fluorescens can be used ( us patent application publication no . us 2005 / 0186666 , schneider , jane c et al .). accordingly , also provided herein is a host cell comprising a nucleic acid molecule of the disclosure . even further provided is a pharmaceutical composition comprising a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure or a host cell of the present disclosure , and a pharmaceutically acceptable carrier . the soluble fusion molecule is optionally present in an amount effective for reducing excessive adiposity in a mammal in need thereof . the term “ effective amount ” as used herein means an amount sufficient to achieve the desired result and accordingly will depend on the ingredient and its desired result . nonetheless , once the desired effect is known , determining the effective amount is within the skill of a person skilled in the art . suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic materials that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition . suitable vehicles are described , for example , in remington &# 39 ; s pharmaceutical sciences ( remington &# 39 ; s pharmaceutical sciences , 20th ed ., mack publishing company , easton , pa ., usa , 2000 ). examples of suitable pharmaceutical carriers include , but are not limited to , water , saline solutions , glycerol solutions , ethanol , n -( 1 ( 2 , 3 - dioleyloxy ) propyl ) n , n , n - trimethylammonium chloride ( dotma ), diolesylphosphotidyl - ethanolamine ( dope ), and liposomes . such compositions contain a therapeutically effective amount of the agent , together with a suitable amount of carrier so as to provide the form for direct administration to the patient . in some embodiments , the composition is formulated for administration to a subject such as a human . in particular embodiments , the composition is formulated for intravenous or oral administration . optionally , the composition is formulated for inhalative , rectal or parenteral administration , including dermal , intradermal , intragastral , intracutan , intravasal , intravenous , intramuscular , intraperitoneal , intranasal , intravaginal , intrabuccal , percutaneous , subcutaneous , sublingual , topical or transdermal administration . in another aspect , the present disclosure provides a method of reducing excessive adiposity in an animal comprising administering a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) to the animal in need thereof . also provided is a use of a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) for reducing excessive adiposity in an animal in need thereof . further provided is a use of a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) in the manufacture of a medicament for reducing excessive adiposity in an animal in need thereof . even further provided is a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) for use in reducing excessive adiposity in an animal in need thereof . the phrase “ reducing excessive adiposity ” as used herein refers to a reduction of at least 1 %, 2 %, 5 %, 10 %, 15 %, 20 % or more in the amount of fat tissue in a treated subject compared to a control that is on the same or similar diet . in one embodiment , the present disclosure provide a method of reducing weight gain in an animal comprising administering a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) to the animal in need thereof . also provided is use of a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) for reducing weight gain in an animal in need thereof . further provided is use of a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) in the manufacture of a medicament for reducing weight gain in an animal in need thereof . even further provided is a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) for use in reducing weight gain in an animal in need thereof . the phrase “ reducing weight gain ” as used herein refers to a reduction of at least 1 %, 2 %, 5 %, 10 %, 15 %, 20 % or more in the mass of a treated subject compared to a control that is on the same or similar diet . in another embodiment , the present disclosure provides a method of treating obesity or an obesity - related condition in an animal comprising administering a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) to the animal in need thereof . also provided is a use of a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) for treating obesity or an obesity related condition to an animal in need thereof . further provided is a use of a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) in the manufacture of a medicament for treating obesity or an obesity related condition to an animal in need thereof . even further provided is a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) for use in treating obesity or an obesity related condition to an animal in need thereof . the term “ obesity ” as used herein refers to a condition in a subject having an accumulation of body fat and includes , without limitation , pre - obesity typically defined as having a body mass index greater than 25 , and obesity typically defined having a body mass index greater than 30 ( often called obese ). the phrase “ treating obesity ” includes a reduction of at least 5 %, 10 %, 15 %, 20 % or more in weight compared to a non - treated subject on a similar or same diet regimen or a reduction of 5 %, 10 %, 15 %, 20 % or more in the ratio of fat mass to lean mass of the subject . the term “ treating ” or “ treatment ” as used herein and as is well understood in the art , means an approach for obtaining beneficial or desired results , including clinical results . beneficial or desired clinical results can include , but are not limited to , alleviation or amelioration of one or more symptoms or conditions , diminishment of extent of disease , stabilizing ( i . e . not worsening ) the state of disease , prevention of disease spread , delaying or slowing of disease progression , amelioration or palliation of the disease state , diminishment of the reoccurrence of disease , and remission ( whether partial or total ), whether detectable or undetectable . “ treating ” and “ treatment ” can also mean prolonging survival as compared to expected survival if not receiving treatment . “ treating ” and “ treatment ” as used herein also include prophylactic treatment . treatment methods comprise administering to a subject a therapeutically effective amount of an active agent and optionally consists of a single administration , or alternatively comprises a series of applications . the length of the treatment period depends on a variety of factors , such as the severity of the condition , the age of the patient , the concentration of active ingredient or agent , the activity of the compositions described herein , and / or a combination thereof . it will also be appreciated that the effective dosage of the agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime . changes in dosage may result and become apparent by standard diagnostic assays known in the art . in some instances , chronic administration may be required . for example , the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient . in one embodiment , the obesity - related condition is type 2 diabetes , cardiovascular disease or cancer . in an embodiment , the cancer is cancer of the esophagus , breast , endometrium , colorectal or pancreas . in another embodiment , the obesity - related condition is high cholesterol , high triglycerides , high blood pressure metabolic syndrome ( a combination of high blood sugar , high blood pressure , high triglycerides and high cholesterol ) or polycystic ovarian syndrome ( pcos ). in yet a further embodiment , the present disclosure provides a method of improving glucose tolerance and / or insulin sensitivity in an animal comprising administering a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) to the animal in need thereof . also provided is a use of a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) for improving glucose tolerance and / or insulin sensitivity in an animal in need thereof . further provided is use of a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) in the manufacture of a medicament for improving glucose tolerance and / or insulin sensitivity . even further provided is a soluble fusion molecule of the present disclosure , a nucleic acid molecule of the present disclosure , a host cell of the present disclosure , a pharmaceutical composition of the present disclosure , or an extracellular domain of the growth hormone secretagogue receptor ( ghs - r1a ) for use in improving glucose tolerance and / or insulin sensitivity in an animal in need thereof . the phrase “ improving glucose tolerance and / or insulin sensitivity ” as used herein refers to an improved metabolic status including reduced blood glucose levels as a result of enhanced insulin secretion and insulin action in the body . this is also exemplified by enhanced glucose uptake or utilization in the insulin - responsible tissues such as muscle , fat and liver . the term “ subject ” or “ animal ” as used herein includes all members of the animal kingdom , including mammals , and suitably refers to humans . in one embodiment , the animal has type 2 diabetes . the term “ administering the proteins disclosed herein ” includes both the administration of protein as well as the administration of a nucleic acid sequence encoding the protein to an animal or to a cell in vitro or in vivo . the term “ administering ” also includes the administration of a cell that expresses the antibody or antibody fragment thereof . the term “ a cell ” includes a single cell as well as a plurality or population of cells . administering to a cell includes administering in vitro ( or ex vivo ) as well as in vivo . the active agents or compositions of the present disclosure may be used alone or in combination with other known agents useful for reducing excessive adiposity or weight gain , for example , for treating obesity or an obesity - related condition in a subject . when used in combination with other agents , it is an embodiment that the compositions or active agents of the present disclosure are administered contemporaneously with those agents . as used herein , “ contemporaneous administration ” of two substances to a subject means providing each of the two substances so that they are both biologically active in the individual at the same time . the exact details of the administration will depend on the pharmacokinetics of the two substances in the presence of each other , and can include administering the two substances within a few hours of each other , or even administering one substance within 24 hours of administration of the other , if the pharmacokinetics are suitable . design of suitable dosing regimens is routine for one skilled in the art . in particular embodiments , two substances will be administered substantially simultaneously , i . e ., within minutes of each other , or in a single composition that contains both substances . it is a further embodiment of the present disclosure that the composition or active agent of the present disclosure and the other agent ( s ) is administered to a subject in a non - contemporaneous fashion . the dosage of compositions or active agents of the present disclosure can vary depending on many factors such as the pharmacodynamic properties of the composition , the mode of administration , the age , health and weight of the recipient , the nature and extent of the symptoms , the frequency of the treatment and the type of concurrent treatment , if any , and the clearance rate of the composition in the subject to be treated . one of skill in the art can determine the appropriate dosage based on , for example the above factors . compositions of the present disclosure may be administered initially in a suitable dosage that may be adjusted as required , depending on the clinical response , and may be administered in a single daily dose or the total daily dose may be divided into two , three or four daily doses . the above disclosure generally describes the present application . a more complete understanding can be obtained by reference to the following specific examples . these examples are described solely for the purpose of illustration and are not intended to limit the scope of the disclosure . changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient . although specific terms have been employed herein , such terms are intended in a descriptive sense and not for purposes of limitation . depleting circulating ghrelin holds the potential to reduce caloric intake and promote fat energy utilization . as such , mammalian expression plasmid vectors encoding the ligand binding domains of the ghs - r1a were constructed , specifically the n - terminal ( nt ), and / or the first , second extracellular domain ( ec1 , ec2 ) and were fused with a mouse igg constant regions ( fc ), forming ghsr / fc ( fig1 a ). in vivo expression of these fusion proteins was achieved through plasmid - intramuscular injection and subsequent electroporation in gastrocnemius muscle of mice . the fusion constructs omitted the sequence motif corresponding to the transmembrane domains of the receptor , allowing the production of ghsr / fc that is secretable . the expression and secretion of the fusion proteins were first examined under in vitro conditions by the transient transfection of the plasmid vectors ( fig1 a ) into l6 rat skeletal muscle cell line . at 48 h after transfection , the medium and the cells were harvested separately and extracted proteins were subjected to western blot analysis using anti - myc tag antibodies . as shown , the expression of the fusion constructs ( nt -, nt - ec1 , and nt - ec1 - ec2 , denoted as ghsr / fc ) was consistently detected in both the cell lysate and culture media whereas the wt / fc , which contains the transmembrane domains , was only found in the cell lysate ( 45 kd ) ( fig1 b ). similar results were also obtained in the culture media or cell lysate in cho cells transfected with the fusion constructs ( fig1 c ). immunocytochemistry experiments using anti igg - fc antibodies showed that nt - ec1 - ec2 ( ghsr / fc ), nt / fc , ec1 / fc , and wt / fc were detected in the transfected l6 cells ( fig1 d ), suggesting that these fusion proteins can be produced in the mammalian expression system in vitro . to examine the impact of ghsr gene therapy on energy intake and weight gain , food consumption and body weight was measured in mice injected with the ghsr - based constructs and control mice ( injected with the fc empty vector ) fed on a high fat diet ( hfd ). mice treated with the ghsr / fc construct gained significantly less body weight compared to the control animals ( fig2 a ). the weight differences began at 30 days post injection ( 21 . 0 ± 0 . 195 g vs 23 . 8 ± 0 . 433 g in control , p & lt ; 0 . 05 ), and continued until the termination of the experiment at 54 days post the first injection ( 23 . 5 ± 1 . 36 g vs 29 . 6 ± 0 . 434 g in control , p & lt ; 0 . 001 ), ( fig2 a ). mice that received nt / fc , nt - ec1 / fc , or wt / fc injections all showed a modest reduction in weight gain that did not reach statistical significance . the ghsr / fc having the n - terminal as well as both extracellular loop 1 and extracellular loop 2 were the focus of the remaining analysis . since ghrelin was previously shown to have a potent orexigenic effect , the daily food and water consumption was examined . as shown in fig2 b , there was no difference in food intake between control and ghsr / fc treated mice . however , treatment with ghsr / fc increased water intake and urine . in vivo expression of the ghsr / fc was verified by western blot analysis . as shown , western blotting of whole gastrocnemius muscle lysates detected a 55 kda band corresponding to the ghsr / fc fusion protein in each of the ghsr / fc - treated mice ( fig3 a ). furthermore , western blot analysis of plasma showed the presence of the 55 kda band in ghsr / fc treated mice but not in vehicle - injected control mice ( fig3 b ). to confirm the ghrelin neutralizing effect of this treatment , the levels of ag and total ghrelin ( primarily uag ) were measured in circulation . as shown in fig3 c , expression of ghsr / fc did not alter the levels of total ghrelin ( fig2 d 140 . 9 ± 30 . 2 pg / ml vs 140 . 8 ± 37 . 94 pg / ml in control ) but significantly reduced the levels of ag ( fig2 d , 1 . 41 ± 0 . 160 pg / ml vs 2 . 25 ± 0 . 240 pg / ml in control , p & lt ; 0 . 05 ). finally , to address if reduced ag levels had an effect on the growth hormone pathway , plasma levels of the gh - dependent insulin - like growth factor - 1 ( igf - 1 ) were examined . no difference was found for igf - 1 levels between the ghsr / fc treated and control mice ( fig3 d ). to determine the source of the reduced weight gain in the treated animals , fat pad and lean tissue ( gastrocnemius and soleus muscles ) were investigated . visually , the amount of white adipose tissue ( wat ) found in the peritoneum of treated mice was less than control ( fig4 a ). this difference was quantified by the weighing of various fat pads . several fat stores were significantly smaller in the treated mice including retroperitoneal ( 0 . 476 ± 0 . 12 g vs 0 . 948 ± 0 . 119 g in control , p & lt ; 0 . 001 ), peri - renal ( 0 . 44 ± 0 . 19 g vs 0 . 734 ± 0 . 011 g in control , p & lt ; 0 . 05 ) and inguinal fat pads ( 0 . 396 ± 0 . 067 g vs 0 . 722 ± 0 . 56 g in control , p & lt ; 0 . 05 ) ( fig4 b ). lean mass was not affected by treatment as determined by the equal gastrocnemius muscle weight ( 0 . 131 ± 0 . 016 g vs 0 . 109 ± 0 . 011 g in control ) ( fig4 c ). since reduced caloric consumption was not responsible for the reduction in wat , alterations in wat mrna expression in the context of several metabolic genes were examined . visceral wat was examined for the adipokine leptin ( a hormone that signals in response to fat cell anabolism ), hormone sensitive lipase ( hsl ; an enzyme catalyzing the breakdown of triglycerides to fatty acids ) and pparγ ( a nuclear receptor that promotes adipogenesis ) mrna using quantitative rt - pcr . treated animals had lower levels of leptin gene expression in wat ( 0 . 10 ± 0 . 14 fold of control , p & lt ; 0 . 05 ) while hsl mrna ( 1 . 76 ± 0 . 07 fold of control , p & lt ; 0 . 05 ) and pparγ mrna ( 3 . 91 ± 0 . 378 fold of control , p & lt ; 0 . 05 ) expression were elevated ( fig4 d ). in addition , mrna levels of adipose - derived proinflammatory cytokines known to be elevated in obesity were examined in wat . both interleukin - 1 ( il - 1 ) and tumor necrosis factor α ( tnfα ) were reduced in ghsr / fc compared to control ( 2 . 9 ± 1 . 6 % of control for il - 1 , and 15 ± 7 % of control for tnfα , p & lt ; 0 . 05 ). ghrelin has been shown to affect glucose homeostasis ( broglio , arvat et al . 2001 ). to determine the effect of ghsr / fc treatment on glucose metabolism , intra - peritoneal glucose tolerance tests ( ipgtt ) and insulin tolerance tests ( itt ) were performed on control and treated mice before and at the end of the study . treated mice displayed improved glucose tolerance with significantly lower blood glucose at 10 minutes ( 14 . 5 ± 1 . 52 mmol / l vs 18 . 9 ± 1 . 48 mmol / l in control , p & lt ; 0 . 01 ) and 20 minutes ( 10 . 1 ± 0 . 968 mmol / l vs 14 . 2 ± 0 . 989 mmol / l in control , p & lt ; 0 . 05 ) post glucose injection ( fig5 a ). the area under the curve in the ipgtt was also significantly decreased in the treated mice ( 516 ± 32 . 2 vs 668 ± 22 . 1 in control , p & lt ; 0 . 05 ) ( fig5 b ). in itt , area under the glucose curve was lower in treated animals ( 180 ± 19 . 1 vs 230 ± 20 . 3 in control , p & lt ; 0 . 05 ) indicating increased insulin sensitivity ( fig5 c and 5d ). these results suggest that reduced circulating ag improves insulin sensitivity and glucose tolerance in mice on a high fat diet . in this study , an in vivo gene transfer method was used to administer ghsr1a - based fusion proteins as a ‘ decoy receptor ’ for circulating ag in mice . the fusion constructs were designed to incorporate the extracellular domains of the ghsr1a that interact with the acylated portion of ghrelin , fused with igg fc to improve the stability and half - life of the complex in circulation ( soltani , kumar et al . 2007 ). initially , expression and secretion of the construct in vitro was confirmed by plasmid transfection in l6 muscle and cho cell lines . as expected , the full length wt / fc was not detected in culture media as it possesses the hydrophobic transmembrane domains that would retain it in the plasma membrane . the expression was further confirmed in both the muscle and plasma from ghsr / fc plasmid injected animals . to verify that the treatment led to altered ghrelin levels both total ( ag + uag ) and acylated ( ag ) levels were measured in the circulation . while there was no significant difference in the levels of total ghrelin , there was a significant reduction of ag in treated animals . any differences in ag ( 5 % of total ghrelin in circulation ) occurring in the total ghrelin assay would likely be undetectable ( kojima , hosoda et al . 1999 ; ariyasu , takaya et al . 2001 ). the lack of difference in the total ghrelin assay confirms the specificity of the ghsr / fc for binding only ag . the impact of neutralizing ghrelin on energy homeostasis was examined by placing animals on a hfd . thirty days after the hfd feeding , animals treated with ghsr / fc had gained significantly less weight compared to empty vector treated mice on high fat diet . the reduced weight gain in the ghsr / fc group was maintained until the end of the study at 74 days post gene transfer with a final weight gain reduction of over 20 %. these observations of reduced weight gain and reduced circulating ag in ghsr / fc mice is in agreement with previous work examining the effects of ghrelin immunoneutralization ( zorrilla , iwasaki et al . 2006 ). constructs encoding for the n - terminal region of the ghsr1a ( nt / fc ) or the n - terminal and the first extracellular loop ( nt - ec1 / fc ) were also designed . these constructs , while exerting some weight sparing effects , had only a moderate effect compared with ghsr / fc . the ghsr / fc was the one construct that incorporated all 3 of the extracellular domains of the ghsr1a ( nt , ec1 and ec2 ). without wishing to be bound by theory , of potential importance was the incorporation of both the extracellular loop domains ( ec1 and ec2 ) as these extracellular loops are thought to be the binding sites for ag to the ghs - r1a ( pedretti , villa et al . 2006 ). despite the difference in weight gain , no difference was observed throughout the study in food consumed between the ghsr / fc treatment and control groups . interestingly , the ghsr / fc mice showed increased water consumption and urine output . ghrelin &# 39 ; s action on appetite occurs through binding with the ghsr1a in neurons within the arcuate nucleus of the hypothalamus ( cowley , smith et al . 2003 ). while peripheral ghrelin administration has been shown to cross the blood brain barrier ( bbb ) and stimulate appetite ( banks , tschop et al . 2002 ), a population of ghrelin - producing neurons also exists within the arcuate nucleus ( kageyama , kitamura et al . 2008 ). in the present study , due to the size of the ghsr / fc protein , it is unlikely that it was able to cross the bbb and neutralize hypothalamic ghrelin . thus , without wishing to be bound by theory , the present strategy may only be targeting peripheral ghrelin and its actions . in agreement with this study , ghrelin immunoneutralization studies also had no effect on food intake ( zorrilla , iwasaki et al . 2006 ). furthermore , studies that examined both ghrelin and ghsr1a embryonic knockout mice also had no effect on feeding behavior but instead , when challenged with a high fat diet , were more likely to utilize fat as their energy substrate ( wortley , anderson et al . 2004 ; zigman , nakano et al . 2005 ). consistent with these previous observations , reduced fat pads primarily in the peritoneum were found in the present study . given that the food consumption was unchanged in ghsr / fc - treated mice , the reduced fat mass is likely the consequence of the reduction in ag which affected fat tissue metabolism . indeed , a recent study indicated that ghrelin acts directly on adipocytes to prevent lipolysis ( davies , kotokorpi et al . 2009 ). to determine the effect of reduced circulating ghrelin levels on adipose tissue , the mrna expression of fat metabolism genes in wat was measured . not surprisingly , leptin , which is produced during fat accumulation and adipocyte differentiation ( houseknecht , baile et al . 1998 ), was lower in the ghsr / fc - treated animals . of particular interest , hsl , a key enzyme in the catabolism of triglycerides to fatty acids ( yeaman 2004 ), was found to be elevated in the treated animals . this increased lipase expression is indicative of increased mobilization of fatty acids for energy substrate , which supports the finding that the treated animals were protected from adiposity while eating a hfd . this finding is also in good agreement with a previous indirect calorimetry study on ghrelin ko mice that suggested preferential use of fat as their energy substrate ( wortley , anderson et al . 2004 ). pparγ mrna levels were also found to be elevated in the treated animals . while activation of this nuclear receptor typically leads to the differentiation and growth of adipose tissue , some evidence suggests that increased pparγ may partition fat away from visceral stores ( laplante , sell et al . 2003 ). indeed , the present study shows that the most significant reduction on fat pad weight , in ghsr / fc - treated mice was in the visceral depots . these findings are supported by other &# 39 ; s studies that showed that low dose ghrelin administration caused increased fat pad weight and altered adipocyte gene expression without an effect on feeding in mice ( tsubone , masaki et al . 2005 ). taken together , the present data suggest that reducing circulating ag with ghsr - 1a / fc treatment leads to reduced fat stores and altered adipocyte gene expression . reducing ag with ghsr / fc treatment significantly improved glucose tolerance and insulin sensitivity in mice on hfd feeding . these findings are in agreement with several studies suggesting that ghrelin promotes glucose homeostasis through inhibiting insulin release from the pancreas ( dezaki , sone et al . 2006 ; dezaki , sone et al . 2008 ). since no differences in circulating insulin levels were observed , the improved glucose clearance may be a consequence of reduced hepatic glucose production in the ghsr / fc treated animals . this is , at least in part , supported by previous investigation that demonstrated that ghrelin promotes glucose production in hepatocytes ( gauna , delhanty et al . 2005 ). moreover , the improved glucose tolerance may be a secondary effect to the reduced visceral adiposity in ghsr / fc - mice . it is known that increased adiposity can lead to insulin resistance which is brought on by proinflammatory factors released from inflamed fat stores ( oliver , mcgillicuddy et al . 2010 ). to determine the possible involvement of pro - inflammatory cytokines , the mrna expression of both il - 1 and tnfα was examined in visceral adipose tissue . both these genes were expressed at significantly lower levels in ghsr / fc - treated animals , suggesting that improved insulin sensitivity in ghsr / fc - treated mice may be partially conferred by reduced pro - inflammatory cytokines in these mice . therefore , the strategy involving neutralization of circulating ghrelin , exemplified by the ghsr / fc treatment , prevents weight gain and improves glucose tolerance , which may provide beneficial therapies for t2dm . the present inventors examined if the ghsr / fc treatment had any impact on other metabolic hormones . none of the hormones examined ( glp - 1 , insulin , pyy , pancreatic polypeptide and glucose insulinotropic peptide ) were significantly altered by the expression of ghsr / fc ( data not shown ). as ghrelin is a known gh secretagogue ( kojima , hosoda et al . 1999 ) and gh has effects on glucose metabolism and insulin sensitivity ( moller and jorgensen 2009 ), the effects of ghrelin depletion on gh were examined . since gh varies throughout the day in a pulsatile fashion a more stable measurement of gh levels can be obtained by measuring circulating igf - 1 . interestingly , circulating igf - 1 levels were not affected by ghrelin neutralization and likely were not responsible for the reduced weight and improved glucose parameters . this lack of effect is consistent with a previous report indicating that gh levels are unchanged in ghsr1a ko mice on hfd ( zigman , nakano et al . 2005 ). in summary , a novel strategy has been developed using secretable ghsr1a - based fusion proteins to neutralize the circulating active ghrelin and hence reduce hfd - induced weight gain . among those fusion constructs , the ghsr / fc which contains the nt , ec1 , and ec2 domains of ghsr1a was the most effective in reducing weight gain , improving insulin resistance , and improving glucose tolerance in mice fed with hfd . of particular benefit , this approach reduced body weight and adiposity , without affecting the appetite and , more particularly , lean mass . the treatment also altered adipose gene expression , exemplified by increased fat catabolism and reduced pro - inflammatory cytokines genes , suggesting a shift to fat usage rather than storage in mice . this yields a secondary beneficial outcome exemplified by improved insulin sensitivity and improved glucose clearance . thus , ghsr / fc fusion proteins may have clinical application for the treatment and management of obesity and t2dm . all ghrelin receptor constructs were designed based on the mouse growth hormone secretagogue receptor sequence ( gene id : 208188 ). ghsr regions and mouse igg fc fragment were produced by pcr amplification . primers used for amplification of the n - terminal region were ( 6mntf ) gcg ggg tac cat gtg gaa cgc gac gcc a ( seq id no : 19 ) and ( 6mntr ) gcg agt act cgc ggg gaa cag tgg cag cag ttc ( seq id no : 20 ), the first extracellular loop ( 6mec1f ) gcg aag ctt ttc cag ttt gtc agc gag agc tgc acc tac gcc ccc agc gag acc gtc acc tgc ( seq id no : 21 ) and ( 6mec1r ) cga agc ttg cag agc agg tcg ccg aag ttc cag ggc cga tac tgc cag agg cgc gcg ggg aac agt ggc agc agt tc ( seq id no : 22 ), the second extracellular loop ( 6mec2f ) gcg acg gat ccc cgg gac acc aac gag tgc cgc gcc acc gag ttc gct gtg cgc tct ccc agc gag acc gtc acc tgc aac ( seq id no : 23 ) and ( 6mec2r ) gcggggatccgtg ccg ttc tcg tgc tcc acg ccc acc agc acg gcg tag gtg cag ctc tcg ctg ac ( seq id no : 24 ), and mouse igg fc fragment ( miggf ) gcg agt act tgg ccc agc gag acc gtc acc tgc aac ( seq id no : 25 ) and ( miggr ) gcg ctc gag cag gga aga agt ctg tta tca tgc a ( seq id no : 26 ). each extracellular domain ghsr pcr product was cleaved with kpni and scai while the mouse igg fc fragment was cleaved with scai and xhoi . this was ligated into the psectag2b vector ( invitrogen , on canada ) at kpn1 and xhoi using t4 dna ligase . similar cloning strategy can be utilized for creating fusion molecules comprising the n - terminal , extracellular loop 1 and / or extracellular loop 2 of the mouse or human ghs - r1a . to establish the expression and secretion of ghsr constructs , the rat skeletal muscle cell line l6 and the chinese hamster ovary cho cells were transfected with the designed plasmids . 40 μg of cell extract and media were collected using standard methods and run out on 12 % sds page gels . following the end of experiments , the gastrocnemius muscle was collected and extracted from treated mice to examine the in vivo expression . proteins were transferred to pvdf membrane and probed using anti - myc antibody ( millipore ) at 1 : 3000 at 4 ° c . followed by secondary rabbit ant - mouse hrp at 1 : 5000 at room temp for 1 hr . to determine the localization and expression of transfected ghsr proteins , cell lines were immunostained with anti - igg fc antibodies which only detect the f chain of the transfected protein . l6 cells were grown on coverslips and were fixed for 1 hr at rt in 4 % paraformaldehyde . they were then washed 3 times in pbs followed by 15 minutes of blocking in 5 % normal horse serum . cells were incubated with primary biotinylated anti - mouse fc in blocking solution for 2 hours at room temperature ( rt ). cells were then washed and treated with avidin - conjugated cy3 in blocking solution for 45 minutes in dark at rt . coverslips were mounted on slides and visualized on a zeiss axioplan ii microscope . c57 / bi6 mice were purchased from jackson laboratories ( bar harbor , me ., usa ). mice were housed under controlled light ( 12 h light / 12 h dark ) and temperature conditions , and had free access to food ( normal rodent chow , or high fat - diet where indicated ) and water . all procedures were conducted in accordance with the guidelines of the canadian council on animal care and were approved by the st . michael &# 39 ; s hospital animal care committee . in vivo expression of ghsr / fc was achieved by intramuscular plasmid injection followed by an electroporation as previously described ( soltani , kumar et al . 2007 ). briefly , a total of 50 μg of plasmid dna was injected ( 25 μg per leg ) intramuscularly into gastrocnemius of 8 week old male c57 / bi6 mice and an electrical current was applied using caliper electrodes ( btx , ma ) on the muscle as follows ; 8 pulses ( pulse length 20 ms ) with 1 second intervals at 200v / cm . a conductive ( aquasonic 100 ) gel was used to facilitate current delivery . plasmid injection and electroporation were conducted once weekly for the first 3 weeks of the study course . high fat diet ( research diets , north brunswick , n . j ., usa , containing 60 % of kcal as fat ) began on the day of the first dna injection and continued until the end of the experiment 54 days later . food consumption was measured by weighing of food basket in each cage every 3 days . animals from each group were weighed individually every 3 days . intraperitoneal ( ip ) glucose and insulin tolerance tests were completed after 54 days of high fat diet . animals were fasted overnight for 12 hours prior to tests . for the ipgtt , a single bolus injection of glucose at 1 . 5 mg / kg of mouse weight was administered ip . for the itt , a single bolus injection of insulin at 0 . 75 u / kg was injected ip . tails tips were treated with topical anesthetic ( emla , on canada ) and blood samples were drawn from tail vein at 0 , 10 , 20 , 30 , and 60 minutes post injection . blood samples ( 4 - 5 μl ) were analyzed by the glucose oxidase method using the bayer acensia elite xl glucometer ( bayer , on canada ). post mortem analysis of fat tissues weight was completed by bi - lateral harvesting of fat pads and immediate weighing . the peritoneal fat tissue was snap frozen in liquid nitrogen for later rna extraction . visceral white adipose tissue was collected at the end of the experiment . total rna was extracted using the trizol ® ( invitrogen , ca usa ) extraction phenol chloroform precipitation method as per manufacturer &# 39 ; s protocol . samples were treated with dnaase ( invitrogen ) and cdna was produced using random hexamers under standard methods . realtime pcr was conducted on the bio - rad cfx instrument ( bio - rad , ca usa ) using primers for leptin ( forward : ccaaaaccctcatcaagacc ( seq id no : 27 ), reverse : tgtctccaccaccgaaactc ( seq id no : 28 )), hormone sensitive lipase ( forward : tgtctccaccaccgaaactc ( seq id no : 29 ), reverse tctccagttgaaccaagcaggtca ( seq id no : 30 )), pparγ ( forward : ggaaagacaacggacaaatcac ( seq id no : 31 ), reverse : atccttggccctctgagatg ( seq id no : 32 )), il - 1 ( forward : tgtctgaagcagctatggcaa ( seq id no : 33 , reverse : tgctgcgagatttgaagctg ( seq id no : 34 )) and tnfα ( forward : tgatcggtccccaaagggat ( seq id no : 35 ), reverse : ttgctacgacgtgggctac ( seq id no : 36 )). data was analyzed using bio - rad cfx manager software and relative expression was determined using the standard curve method with 18s as the normalization gene . blood was collected at the end of the experiment in capillary tubes containing edta ( sarstedt , pq ) from ad - libitum fed mice . plasma level of hormones involved in the regulation of energy metabolism was analyzed using the milliplex hormone assay panel ( millipore , ma ) including ; active glp - 1 , insulin , pyy , pancreatic polypeptide , and gip ( millipore ). acylated ghrelin and igf - 1 plasma levels were analyzed by enzyme link immunoassays ( cayman chemical , on and millipore , ma respectively ) and total ghrelin levels were measured using a radioimmunoassay ( phoenix pharmaceuticals , ca ). the relative changes in weight gain over time were analyzed using the two - way anova with bonferroni post test to compare each group to the control group . multiplex hormone assays analyzing each group were compared with the one - way anova . time points during ipgtt and itt were examined by two - way anova . all other comparisons between the control and ghsr / fc group were analyzed with the student &# 39 ; s t - test . while the present disclosure has been described with reference to what are presently considered to be the examples , it is to be understood that the disclosure is not limited to the disclosed examples . to the contrary , the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims . all publications , patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication , patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety . ariyasu , h ., k . takaya , et al . 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