| hai lama altina alta us010024846b2 de atala at mata …

| HAI LAMA ALTINA ALTA DE ATALA AT MATA HATI US010024846B2

( 12 ) United States Patent Kishore et al .

( 10 ) Patent No . : US 10 , 024 , 846 B2 ( 45 ) Date of Patent : Jul . 17 , 2018

( 54 ) METHODS FOR TREATING DIET - INDUCED OBESITY BY DECREASING OR INHIBITING P2Y2 PURINERGIC RECEPTOR EXPRESSION OR ACTIVITY

2310 / 14 ; C12N 2310 / 531 ; A01K 67 / 0276 ; A01K 2267 / 0362 ; A01K 2227 / 105 ; A01K

22177075 ; A01K 2207 / 251 See application file for complete search history .

( 56 ) References Cited E ( 71 ) Applicants : Bellamkonda K . Kishore , Washington , DC ( US ) ; Yue Zhang , Washington , DC ( US ) ; Carolyn M . Ecelbarger , Washington , DC ( US )

U . S . PATENT DOCUMENTS

@ ( 72 ) Inventors : Bellamkonda K . Kishore , Washington , DC ( US ) ; Yue Zhang , Washington , DC ( US ) ; Carolyn M . Ecelbarger , Washington , DC ( US )

6 , 372 , 724 B1 4 / 2002 Pelleg et al . 8 , 697 , 359 B1 * 4 / 2014 Zhang . . . . . . . . . . . . . . . . . . . . C12N 15 / 85

424 / 94 . 1 9 , 072 , 766 B2 7 / 2015 Kahn et al .

2009 / 0297497 AL 12 / 2009 Kishore et al . 2009 / 0306009 A1 * 12 / 2009 Rosenmeier . . . . . . . A61K 31 / 7072

514 / 51 2013 / 0156791 A1 * 6 / 2013 Perfettini . . . . . . . . . . . A61K 31 / 7088

424 / 159 . 1 2015 / 0259692 A1 9 / 2015 Kishore et al .

@ ( 73 ) Assignee : The United States of America as represented by the Department of Veterans Affairs , Washington , DC ( US )

FOREIGN PATENT DOCUMENTS @ ( * ) Notice : Subject to any disclaimer , the term of this

patent is extended or adjusted under 35 U . S . C . 154 ( b ) by 0 days . *

WO wo WO WO

WO2005 - 100990 WO 2006 / 052007 WO2006 - 060079 WO2014 / 066830

10 / 2005 5 / 2006 6 / 2006 1 / 2014

( 21 ) Appl . No . : 14 / 697 , 562 OTHER PUBLICATIONS ( 22 ) Filed : Apr . 27 , 2015

( 65 ) Prior Publication Data US 2016 / 0377598 A1 Dec . 29 , 2016

( 63 ) Related U . S . Application Data

Continuation - in - part of application PCT / US2013 / 066932 , filed on Oct . 25 , 2013 .

No .

( 60 ) Provisional application No . 61 / 795 , 815 , filed on Oct . 25 , 2012 .

Crooke A , et al . Molecular Vision . 15 : 1169 - 1178 . 2009 . Available online at http : / / www . molvis . org / molvis / v15 / a124 . * Alexander SPH , Benson HE , Faccenda E , Pawson AJ , et al . 2013 . “ The concise guide to pharmacology 2013 / 14 : G protein - coupled receptors " . British Journal of Pharmacology 170 : 1459 - 1581 ( Exhibit 6 ) . Chen J , Montamari AM , Widmer WW . 1997 . Two new polymethoxylated flavones , a class of compounds with potential anticancer activity , isolated from cold pressed dancy tangerine peel oil solids . J Agric Food Chem 45 : 364 - 368 ( Exhibit 7 ) . Felix R . A . , Martin S . , Pinion S . , Crawford D . J . 2012 . “ Develop ment of a comprehensive set of P2 receptor pharmacological research compounds ” . Purinergic Signalling S101 - S112 ( Exhibit 8 ) . Greenlee W . J . , American Chemical Society , Division of Medicinal Chemistry , Abstracts , 224th ACS National Meeting , Boston , MA , Aug . 18 - 22 , 2002 ( Exhibit 9 ) . Hsu PD , Lander ES , and Zhang F . 2015 . : " Development and Applications of CRISPR - Cas9 for Genome Engineering . ” Cell 157 ( b ) : 1262 - 1278 ( Exhibit 10 ) . Hwang EI , Kaneko M , Ohnishi Y , Horinouchi S ( May 2003 ) . " Production of plant - specific flavanones by Escherichia coli con taining an artificial gene cluster ” . Appl . Environ . Microbiol . 69 ( 5 ) : 2699 - 706 ( Exhibit 11 ) . Jacobson KA , Jayasekara , MPS , Costanzi S . 2012 . “ Molecular structure of P2Y receptors : mutagenesis , modeling , and chemical probes ” . WIREs Membr Transp Signal 1 : 815 - 827 ( Exhibit 12 ) ( 2012 ) .

( Continued )

( 51 ) Int . Ci . GOIN 33 / 50 ( 2006 . 01 ) A01K 67 / 027 ( 2006 . 01 ) COOK 16 / 28 ( 2006 . 01 ) C12N 15 / 113 ( 2010 . 01 ) A61K 38 / 46 ( 2006 . 01 ) A61K 47 / 26 ( 2006 . 01 )

( 52 ) U . S . CI . CPC . . . . . GOIN 33 / 5044 ( 2013 . 01 ) ; ADIK 6770276

( 2013 . 01 ) ; A61K 38 / 465 ( 2013 . 01 ) ; A61K 47 / 26 ( 2013 . 01 ) ; COZK 16 / 28 ( 2013 . 01 ) ; C12N

15 / 1138 ( 2013 . 01 ) ; C12Y 301 / 00 ( 2013 . 01 ) ; A01K 2207 / 25 ( 2013 . 01 ) ; AOIK 2217 / 075

( 2013 . 01 ) ; AOIK 2227 / 105 ( 2013 . 01 ) ; AOIK 2267 / 0362 ( 2013 . 01 ) ; C07K 2317 / 76

( 2013 . 01 ) ; C12N 2310 / 11 ( 2013 . 01 ) ; C12N 2310 / 12 ( 2013 . 01 ) ; C12N 2310 / 14 ( 2013 . 01 ) ;

C12N 2310 / 531 ( 2013 . 01 ) ; C12N 2320 / 30 ( 2013 . 01 )

( 58 ) Field of Classification Search CPC . . GOIN 33 / 5044 ; C12Y 301 / 00 ; A61K 47 / 26 ;

A61K 38 / 465 ; CO7K 16 / 28 ; COOK 2317 / 76 ; C12N 15 / 1138 ; C12N 2320 / 30 ;

C12N 2310 / 11 ; C12N 2310 / 12 ; C12N

Primary Examiner - Robert S Landsman ( 74 ) Attorney , Agent , or Firm — Adriano & Associates ( 57 ) ABSTRACT The invention provides a method for treating or preventing diet - induced obesity in a subject comprising administering an agent in an effective amount so that expression or activity of the P2Y2 receptor is decreased or inhibited in the subject thereby treating or preventing diet - induced obesity in the subject .

29 Claims , 19 Drawing Sheets

US 10 , 024 , 846 B2

( 56 ) References Cited

OTHER PUBLICATIONS Jacobson KA . “ P2X and P2Y receptors ” . Tocris Bioscience Scien tific Review Series , pp . 1 - 15 ( Exhibit 13 ) . Jacobson KA , Ivanov AA , de Castro S , Harden TK , Ko H . 2009 . “ Development of selective agonists and antagonists of P2Y recep tors ” . Purinergic Signalling 5 : 75 - 89 ( Exhibit 14 ) . Kaulich M , Streicher F , Mayer R , Muller I , Muller CE . 2003 . “ Flavonoids - novel lead compounds for the development of P2Y , receptor antagonists ” . Drug Development Research 59 : 72 - 81 ( Exhibit 15 ) . Kemp PA et al . ( 2004 ) Am J Respir Cell Mol Biol 31 : 446 - 455 ( Exhibit 16 ) . Kishore B . K . et al . , P2Y2 receptors and water transport in the kidney , Purinergic Signalling , DOI 10 . 1007 / s11302 - 009 - 9151 - 5 ; ( Exhibit 17 ) , ( 2009 ) . Kishore B . K . et al . , Targeting renal purinergic signalling for the treatment of lithium - induced nephrogenic diabetes insipidus , Acta Physiol 2015 , 214 , 176 - 188 ( Exhibit 18 ) . Mali P , Esvelt KM , and Church GM . 2013 . “ Cas9 as a versatile tool for engineering biology . ” Nature Methods 10 : 957 - 963 ( Exhibit 19 ) . Mayer R . 1999 . Calycopterones and calyflorenones , novel biflavonoids from Calycopteris floribunda . J Nat Prod 62 : 1274 1278 ] , Leptospermum scoparium Forst . ( Myrtaceae ) [ Mayer R . 1990 ( Exhibit 20 ) . Mayer R . 1990 . Flavonoids from Leptospermum scoparium . Phytochemistry 29 : 1340 - 1342 ( Exhibit 21 ) . Ralevic V , Burnstock G . 1998 . Receptors for purines and pyrimi dines . Pharmacol Rev 50 : 413 - 492 ( Exhibit 22 ) . Rieg et al . , 2007 , FASEB J 21 : 3717 - 3726 ( Exhibit 23 ) .

Trantas E , Panopoulos N , Ververidis F ( 2009 ) . " Metabolic engi neering of the complete pathway leading to heterologous biosyn thesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae ” . Metabolic Engineering 11 ( 6 ) : 355 - 366 ( Exhibit 24 ) . Ververidis F , Trantas E , Douglas C , Vollmer G , Kretzschmar G , Panopoulos N ( Oct . 2007 ) . “ Biotechnology of flavonoids and other phenylpropanoid - derived natural products . Part I : Chemical diver sity , impacts on plant biology and human health ” . Biotechnology Journal 2 ( 10 ) : 1214 - 34 ( Exhibit 25 ) . Ververidis F , Trantas E , Douglas C , Vollmer G , Kretzschmar G , Panopoulos N ( 2007 ) . “ Biotechnology of flavonoids and other phenylpropanoid - derived natural products . Part II : Reconstruction of multienzyme pathways in plants and microbes ” . Biotechnology Journal 2 ( 10 ) : 1235 - 49 ( Exhibit 26 ) . Zhang Y , et al . , Genetic deletion of the P2Y2 receptor offers significant resistance to development of lithium - induced polyuria accompanied by alterations in PGE2 signaling , Am J Physiol Renal Physiol 302 : F70 - F77 , 2012 ( Exhibit 27 ) . Zhang Y , et al . , Attenuation of lithium - induced natriuresis and kaliuresis in P2Y2 receptor knockout mice , Am J Physiol Renal Physiol 305 : F407 - F416 , 2013 ( Exhibit 28 ) . ISR of No . PCT / US13 / 066932 , Jan . 28 , 2014 ( Exhibit 29 ) . Partial manual translation of WO2006052007 dated May 18 , 2006 ( Exhibit 30 ) of the PTO in connection with U . S . Appl . No . 14 / 438 , 854 and full WO2006052007 in Japanese language ( Exhibit 30a ) . Geary ( Expert Opin . Drug Metab . Toxicol . ( 2009 ) 5 ( 4 ) : 381 - 391 ) ( 2009 ) ( Exhibit 32 ) . Lundquist ( Islet Lysosomal enzyme activities and plasma insulin levels in obese hyperglycemic mice following the injection of the lysosomotropic drug suramin , Diabetes Res . 2 : 207 - 211 , 1985 ) ( Exhibit 33 ) .

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METHODS FOR TREATING DIET - INDUCED between 7 % and 14 % of certain cancer burdens are attrib OBESITY BY DECREASING OR INHIBITING utable to overweight and obesity .

P2Y2 PURINERGIC RECEPTOR The global economic burden of overweight and obesity EXPRESSION OR ACTIVITY are not well established . But the numbers are more accu

5 rately available for the United States . Over the past 50 years This application is a continuation - in - part of ( under 35 the prevalence of obesity in the USA has almost tripled from

U . S . C . § 111 ( a ) ) , and claims the priority to ( under 35 U . S . C . 13 % to 34 % , with the percentage of Americans who are $ $ 120 and 365 ( c ) ) , PCT application PCT / US2013 / 066932 , extremely or “ morbidly ” obese rising from 0 . 9 to 6 . The with international filing date Oct . 25 , 2013 , which claims the estimated annual medical costs due to obesity are about benefit of the filing date of U . S . Ser . No . 61 / 795 , 815 , filed filed 10 $ 190 billion . The indirect costs due to overweight and Oct . 25 , 2012 , the entireties of which are hereby incorpo obesity are also sky rocketing . For example , it is estimated

rated by reference into the present patent application . that airlines incur about $ 5 billion annually for additional jet fuel needed to fly obese or heavier people as compared to the The present invention was made with government support fuel needed to fly the same number of people based on the under Department of Veterans Affairs Merit Review Pro 15 body weights prevalent in 1960s . Automobiles also consume gram , Grant No . BLRD 000591 , and the facilities and extra gasoline amounting to about $ 4 billion to transport

resources at the VA Salt Lake City Health Care System . The heavier people in the USA . The annual costs due to absen Government has certain rights to this invention . teeism among over weight and obese people are also very high . In fact , according to a study , the costs of obesity FIELD OF THE INVENTION 20 exceed those of smoking .

According to the World Health Organization , the funda Blunting the activity of the P2Y2 receptor results in a mental cause of obesity and overweight is energy imbalance

resistance to diet - induced obesity , an increased metabolic between calories consumed and calories expended . In recent activity , and a better glucose tolerance . Therefore , com - decades globally there has been an increase in caloric intake pounds that inhibit the purinergic P2Y2 receptor are useful 25 and a decrease in the physical activity , driving the incidence for treating diet - induced obesity and metabolic disorders of obesity high . Although supportive environments and associated therewith , such as diabetes mellitus . communities resulting in consumption of healthy foods and

regular physical activity are the best to prevent overweight BACKGROUND OF THE INVENTION and obesity , these do not happen often , in part because of the

30 complex nature of modern day living . Obesity is one of the major public health problems of the Currently used modalities as well as the pipeline drugs for

21st century with significant consequences on health of the the prevention and / or treatment of obesity fall into the individuals and impact on the national economies . The most following three categories : ( 1 ) drugs that suppress appetite common cause of obesity is diet - induced , i . e . , consumption by acting on the brain ( e . g . , sibutramine ( i . e . , Reductil® of more calories than one can metabolize . In view of the 35 and / or Meridia® ) ; ( 2 ) drugs that boost body ' s metabolic rate rapidly growing number of obese people throughout the ( e . g . , cannaboid receptor antagonists and Metformin ) ; and world , the global market for anti - obesity drugs is estimated ( 3 ) drugs that interfere with the body ' s ability to absorb to reach $ 3 . 4 billion by 2016 . However , currently available specific nutrients in food , such as fat ( e . g . , Orlistat® , Xeni drugs for the treatment of obesity have significant limita - cal® , and / or Alii® ) . tions in terms of safety , efficacy or both . Thus , there is a high 40 Drugs that suppress the appetite by acting on the brain unmet need for safe and efficacious medications for the have severe side effects which are neurological and / or prevention or treatment of obesity . This invention discloses psychological in nature . For example , these drugs carry a a potential drug target for the treatment of diet - induced risk of high blood pressure , faster heart rate , palpitation , overweight or obesity . This approach involves increasing the restlessness , agitation , insomnia etc . Due to these limita metabolism of consumed excess calories , which may prove 45 tions , drugs that suppress appetite have been withdrawn to be safer or tolerable than the other two approaches , from the market by the FDA and approval of newer drugs namely suppression of the appetite or interfering with the has been made more stringent . Drugs that increase the absorption of fat in the intestines . It is possible to develop body ' s metabolic rate have their own limitations , such as orally effective chemical compounds that selectively act at lack of consistent effect resulting in sustained loss of weight . the disclosed target as potential anti - obesity drugs . 50 Drugs that block absorption of dietary fat , cause oily stools

According to the World Health Organization , a Body ( steatorrhea ) , abdominal pain and flatulence . Mass Index ( BMI ) > 25 is overweight , and a BMI > 30 is In view of the side effects with these therapies , combi obesity . Overweight and obesity are no more the problems nation therapies that target more than one pathway have of high - income countries of the world . They are rapidly been developed . However , these also have severe side rising in low - and middle - income countries . 55 effects and limitations , despite their efficacy in short - term

In 2008 the World Health Organization estimated that treatment . Accordingly , there is a high unmet need for safe globally more than 1 . 4 billion adults were overweight ; and and effective medications for the prevention or treatment of the 2010 estimates showed that more than 40 million chil overweight and obesity , which is expected to drive the dren under five years are overweight . Overweight and obe - anti - obesity market growth in the future . The invention sity are the fifth leading risk for global deaths , accounting 60 described herein seeks to meet this need . for 2 . 8 million deaths each year . In fact , overweight and obesity are linked to more deaths worldwide than under SUMMARY OF THE INVENTION weight .

Raised BMI is a major risk factor for cardiovascular and Provided herein are methods for selectively blocking the musculoskeletal disorders , and certain types of cancers , such 65 activity of the P2Y2 purinergic receptor for the prevention as breast and colon cancers . In fact , 44 % of the diabetic and / or treatment of diet - induced overweight or obesity . It burden , 23 % of the ischemic heart disease burden and was discovered that the genetic deletion of P2Y2 receptor

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results in resistance to the development of high - fat diet - biochemical reactions leading to synthesis or modification of induced obesity and the associated physical and molecular a flavonoid . In an embodiment , the non - competitive P2Y2 alterations in mice . The results described also suggest that receptor antagonist has an half maximal inhibitory concen the observed resistance in the P2Y2 receptor knockout mice tration ( IC50 ) of below 100 uM , preferably below 35 UM , was not due to reduced consumption of high - fat diet or lack 5 more preferably below 25 UM , even more preferably below of absorption of fat in the intestines , but may be due to the 10 UM , even , even more preferably below 5 uM and most ability of the knockout mice to metabolize or “ burn ” the preferably below 1 uM . In one embodiment , the non extra calories consumed in the diet more efficiently than the competitive P2Y2 receptor antagonist has an IC50 of 35 - 100 wild type mice . Accordingly , targeting P2Y2 receptor with UM . In a separate embodiment , the non - competitive P2Y , specific pharmacological agents or drugs is a safe approach 10 receptor antagonist has an IC50 of 25 - 35 uM . In another to prevent or treat diet - induced overweight or obesity . embodiment , the non - competitive P2Y2 receptor antagonist

In various embodiments , provided herein are methods for has an IC50 of 10 - 25 uM . In another embodiment , the treating or preventing diet - induced obesity in a subject by non - competitive P2Y , receptor antagonist has an IC50 of administering an agent that blunts the expression or activity 5 - 10 uM . In a more preferred embodiment , the non - com of the P2Y , receptor in the subject . 15 petitive P2Y2 receptor antagonist has an IC50 of 1 - 5 uM . In

In some specific embodiments , the agent is a fusion a most preferred embodiment , the non - competitive P2Y , protein or an agent that inhibits the P2Y receptor . In specific receptor antagonist has an IC50 of less than 1 uM . In yet embodiments , the agent is a selective P2Y , receptor antago - another preferred embodiment , the non - competitive P2Y , nist , including but not limited to a non - nucleic acid small receptor antagonist has an IC50 between 0 . 1 - 1 UM . organic molecule . In one embodiment , the P2Y , receptor 20 Examples of non - competitive P2Y , receptor antagonist antagonist is a non - competitive P2Y2 receptor antagonist include strobopinin , strobopinin - 7 - methyl ether , 2 ' - B - Dihy The non - competitive P2Y2 receptor antagonist may be a droxy - 4 ' , 6 - dimethoxy - 3 ' - methylchalcone ( or Oxo - Auren non - selective non - competitive P2Y2 receptor which may tiacin ) , kaempferol , heptamethoxylflavone , and tangeretin inhibit other members of the P2Y receptor family ( Ralevic with chemical structures and additional flavonoids described V , Burnstock G . 1998 . Receptors for purines and pyrimi - 25 in Kaulich M , Streicher F , Mayer R , Muller I , Muller CE . dines . Pharmacol Rev 50 : 413 - 492 ] with no selectivity for 2003 . " Flavonoids — novel lead compounds for the devel P2Y2 receptor , a selective non - competitive P2Y2 receptor opment of P2Y2 receptor antagonists ” . Drug Development an antagonist with a selectivity for P2Y2 receptor or a specific Research 59 : 72 - 81 . non - competitive P2Y , receptor antagonist inhibiting the In one embodiment , the P2Y , receptor antagonist is a activity of only P2Y2 receptor . Preferably , the non - competi - 30 competitive P2Y2 receptor antagonist . In one embodiment , tive P2Y , receptor antagonist is a selective non - competitive the competitive P2Y , receptor antagonist may be a non P2Y2 receptor antagonist . More preferably , the non - com - selective or slightly selective competitive P2Y2 receptor petitive P2Y2 receptor antagonist is a specific non - competi - antagonist . Examples of non - selective or slightly selective tive P2Y2 receptor antagonist . Non - competitive P2Y2 recep competitive P2Y2 receptor antagonists include suramin tor antagonist may be flavonoids . Useful flavonoids may be 35 ( 8 , 8 ' - [ Carbonylbis [ imino - 3 , l - phenylenecarbonylimino ( 4 isolated from plant sources [ Ververidis F , Trantas E , Douglas methyl - 3 , 1 - phenylene ) carbonylimino ] ] bis - 1 , 3 , 5 naphthale C , Vollmer G , Kretzschmar G , Panopoulos N ( October netrisulphonic acid ) and Reactive Blue 2 ( RB2 ) with an 2007 ) . “ Biotechnology of flavonoids and other phenylpro IC50 of between 5 uM and 35 UM [ Kaulich M , Streicher F , panoid - derived natural products . Part I : Chemical diversity , Mayer R , Muller I , Muller C E . 2003 . “ Flavonoids — novel impacts on plant biology and human health ” . Biotechnology 40 lead compounds for the development of P2Y2 receptor Journal 2 ( 10 ) : 1214 - 34 ] , such as Calycopteris floribunda antagonists ” . Drug Development Research 59 : 72 - 81 ; Jacob Lamk . ( Combretaceae ) [ Mayer R . 1999 . Calycopterones and son K A , Jayasekara , MPS , Costanzi S . 2012 . “ Molecular calyflorenones , novel biflavonoids from Calycopteris flori structure of P2Y receptors : mutagenesis , modeling , and bunda . J Nat Prod 62 : 1274 - 1278 ] , Leptospermum sco - chemical probes ” . WIREs Membr Transp Signal 1 : 815 - 827 ; parium Forst . ( Myrtaceae ) [ Mayer R . 1990 . Flavonoids from 45 Jacobson KA . “ P2X and P2Y receptors ” . Tocris Bioscience Leptospermum scoparium . Phytochemistry 29 : 1340 - 1342 ; Scientific Review Series , pp . 1 - 15 ] . In one embodiment , the Mayer R . 1993 . A B - hydroxychalcone from Leptospermum competitive P2Y , receptor antagonist is a selective competi scoparium . Planta Med 59 : 269 - 271 ] , and tangerine peels tive P2Y , receptor antagonist . Examples of selective com from Citrus reticulate ssp . , Rutaceae ( Chen J , Montamari A p etitive P2Y , receptor antagonists include AR - C126313 and M , Widmer W W . 1997 . Two new polymethoxylated fla - 50 AR - C118925 comprising an IC50 of about 6 uM Jacobson vones , a class of compounds with potential anticancer activ - KA , Ivanov A A , de Castro S , Harden T K , Ko H . 2009 . ity , isolated from cold pressed dancy tangerine peel oil “ Development of selective agonists and antagonists of P2Y solids . J Agric Food Chem 45 : 364 - 368 ] , microorganisms receptors ” . Purinergic Signalling 5 : 75 - 89 ; Meghani P . 2002 . genetically engineered to produce flavonoids [ Hwang EI , “ The design of P2Y , antagonists for the treatment of inflam Kaneko M , Ohnishi Y , Horinouchi S ( May 2003 ) . “ Produc - 55 matory diseases ” . 224th ACS National meeting , Abstracts , tion of plant - specific flavanones by Escherichia coli con Division of medicinal chemistry 12 ; Kindon N , Meghani P , taining an artificial gene cluster ” . Appl . Environ . Microbiol . Thom S ( 1998 ) World Pat . 98 / 54180 ; Kemp PA et al . ( 2004 ) 69 ( 5 ) : 2699 - 706 ; Trantas E , Panopoulos N , Ververidis F Am J Respir Cell Mol Biol 31 : 446 - 455 ; Alexander S PH , ( 2009 ) . “ Metabolic engineering of the complete pathway Benson H E , Faccenda E , Pawson AJ , et al . 2013 . “ The leading to heterologous biosynthesis of various flavonoids 60 concise guide to pharmacology 2013 / 14 : G protein - coupled and stilbenoids in Saccharomyces cerevisiae ” . Metabolic receptors ” . British Journal of Pharmacology 170 : 1459 Engineering 11 ( 6 ) : 355 - 366 ; Ververidis F , Trantas E , Doug - 1581 ] . las C , Vollmer G , Kretzschmar G , Panopoulos N ( 2007 ) . In an embodiment , the competitive P2Y2 receptor antago “ Biotechnology of flavonoids and other phenylpropanoid - nist has an half maximal inhibitory concentration ( IC50 ) of derived natural products . Part 11 : Reconstruction of multi - 65 below 100 UM , preferably below 35 uM , more preferably enzyme pathways in plants and microbes ” . Biotechnology below 25 UM , even more preferably below 10 UM , even , Journal 2 ( 10 ) : 1235 - 491 , and products of chemical or even more preferably below 5 uM and most preferably

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below 1 uM . In one embodiment , the competitive P2Y small inorganic molecule or an oligonucleotide , including receptor antagonist has an IC50 of 35 - 100 UM . In a separate but not limited to an antisense oligonucleotide , a ribozyme , embodiment , the competitive P2Y2 receptor antagonist has or an inhibitory RNA , including but not limited to a small an IC50 of 25 - 35 uM . In another embodiment , the competi - inhibitory RNA ( siRNA ) or a short hairpin RNA ( shRNA ) . tive P2Y2 receptor antagonist has an IC50 of 10 - 25 uM . In 5 Also provided herein are methods for increasing energy another embodiment , the competitive P2Y , receptor antago - metabolism in an adipocyte cell in a subject that is at risk for nist has an IC50 of 5 - 10 uM . In a more preferred embodi or suffering from a disorder related to glucose metabolism , ment , the competitive P2Y , receptor antagonist has an IC50 the method comprising administering to the subject an agent of 1 - 5 uM . In a most preferred embodiment , the competitive that inhibits expression or activity of a P2Y , receptor , P2Y , receptor antagonist has an IC50 of less than 1 uM . 10 thereby increasing the metabolism in the cell . Preferably , the competitive P2Y2 receptor antagonist has an In various specific embodiments , the agent is a polypep IC50 between 0 . 1 - 1 uM . tide , a small non - nucleic acid organic molecule , a small

In other embodiments , the agent is an oligonucleotide . In inorganic molecule , an antibody , an antisense oligonucle specific embodiments , the oligonucleotide is an antisense otide , a ribozyme or an inhibitory RNA , including but not oligonucleotide , a ribozyme or an inhibitory RNA , including 15 limited to a small inhibitory RNA ( siRNA ) or a short hairpin but not limited to a small inhibitory RNA ( siRNA ) or short RNA ( shRNA ) . hairpin RNA ( shRNA ) . Provided herein is also a method for identifying a candi

In other embodiments , the agent is a gene knockout or date agent that modulates expression or activity of the P2Y , gene editing system directed against P2Y2 receptor gene receptor , comprising : ( a ) providing a sample comprising the comprising Cas9 nuclease and guide RNA ( GRNA ) linked to 20 P2Y , polypeptide or a nucleic acid encoding the polypep a P2Y2 receptor sequence to target Cas9 nuclease to P2Y , tide ; ( b ) contacting the sample with a test compound under gene so as to introduce a site - specific double - stranded break conditions in which the polypeptide is active , the nucleic in the P2Y2 genomic DNA and produce a P2Y2 receptor acid is expressed , or both ; ( c ) evaluating expression or knockout or edited gene . Cas9 nuclease and ORNA linked to activity of the P2Y , polypeptide in the sample ; and ( d ) a P2Y2 receptor sequence may be introduced directly into 25 comparing the expression or activity of the P2Y2 polypep desired cells or alternatively by expression from expression tide of ( c ) to the expression or activity of the P2Y2 poly plasmid or vector systems , including lentiviral system , peptide in a control sample lacking the test compound , adenoviral system or adeno - associated viral system . Design wherein a change in the P2Y2 polypeptide expression or and use of such a gene knockout system ( called CRISPR activity indicates that the test compound is a candidate agent Cas9 genome editing system ) is known in the art ( Mali P , 30 that can modulate the expression or activity of the P2Y2 Esvelt KM , and Church G M . 2013 . “ Cas9 as a versatile tool polypeptide . for engineering biology " . Nature Methods 10 : 957 - 963 ; Hsu In various specific embodiments , the candidate agent P D , Lander E S , and Zhang F . 2015 . “ Development and selectively inhibits the P2Y , receptor or is a fusion protein . Applications of CRISPR - Cas9 for Genome Engineering ” . Other features and advantages of the present invention Cell 157 ( 6 ) ; 1262 - 1278 ; as well as , see described references 35 will become more readily apparent to those of ordinary skill in the publications ) . Based on sequence for human P2Y2 in the art after reviewing the following detailed description receptor gene ( NCBI Reference Sequence : NC _ 000011 . 10 and accompanying drawings . for NCBI Gene ID 5029 ) and mRNA transcripts ( NCBI Reference Sequence : NM _ 176072 . 2 , NM 176071 and BRIEF DESCRIPTION OF THE FIGURES NM _ 002564 ) , knockout or mutagenesis that reduces human 40 P2Y2 receptor gene expression or activity may be obtained The details of the present invention may be gleaned in part using publically available sequences and CRISPR / cas9 by study of the accompanying drawings , in which : genome editing system to target specific region of the human FIGS . 1A and 1B illustrate the changes in the body P2Y2 receptor gene with appropriate gRNA linked to 20 - nt weights of P2Y2 knockout and wild - type mice fed regular or so sequence from the P2Y2 receptor gene . Commercial 45 diet or high - fat diet for 16 weeks . Specifically , the line graph sources providing CRISPR / Cas9 genome editing system shows the change in body weight of wild type ( WT ) and targeting the P2Y2 receptor gene include OriGene Tech - P2Y2 knockout ( KO ) mice fed regular diet ( CNT ) or high nologies , Rockville , Md . ( catalog number : KN204159 ) , fat diet ( HFD ) for 16 weeks with ad libitum access to food Santa Cruz Biotechnology , Santa Cruz , Calif . ( catalog num - and drinking water . The * indicates data that is significantly ber : SC - 401301 , SC - 401303 - HDR , SC - 401303 - NIC , 50 different from the corresponding KO - HFD data point , as SC - 401303 - NIC - 2 , SC - 401303 - ACT , SC - 401303 - ACT - 2 , assessed by unpaired t test . SC - 401303 - LAC and Sc - 401303 - LAC - 2 ) . Using known FIG . 2 is a photograph that shows the size of one of the P2Y2 receptor nucleic acid sequences available in public highest weighing wild - type mouse with an average weighing database such as public database housed at National Center P2Y2 knockout mouse from the high - fat diet groups . Spe for Biotechnology Institute ( NCBI , Bethesda , Md . ) , P2Y2 55 cifically , the photograph provides a visual comparison of the receptor gene in other species may also be knocked - out , size of one of the highest weighing wildtype mice from the knocked - down , or mutagenized using the CRISPR / Cas9 high - fat diet group ( WT - HFD ) with an average weighing genome editing system as well as other genetic tools known knockout mouse from the high - fat diet fed group ( KO - FD ) . in the art , such as anti - sense oligonucleotide , ribozyme , FIG . 3A illustrates the food intake ( g / day or g / g body siRNA , shRNA , and miRNA among others . 60 weight ) of the mice after 4 weeks of the Experimental

In other embodiments , provided herein are methods for Period . Specifically , the bar graph shows that food intake of increasing energy metabolism in a cell by contacting the cell WT and P2Y , KO mice on regular diet ( CNT ) or HFD with an agent that blunts the expression or activity of a P2Y determined after 4 weeks of start of the experimental period receptor , thereby increasing the metabolism in the cell . In ( expressed as g / day ) . specific embodiments , the cell is an adipocyte cell . 65 FIG . 3B illustrates the food intake ( g / day or g / g body

In specific embodiments , the agent is a fusion protein , weight ) of the mice after 12 weeks of the Experimental polypeptide , a small non - nucleic acid organic molecule , a Period . Specifically , the bar graphs show that food intake of

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WT and P2Y , KO mice fed HFD for 12 weeks ( expressed FIG . 11 provides the results of a Serum Insulin assay ( left ) as g / day or g / g body weight ) . and a Serum Leptin assay ( right ) after 16 weeks of the

FIG . 4A illustrates the water intake ( ml / day or ml / g body Experimental Period . Specifically , the graphs show serum weight ) of the mice after 4 weeks of the Experimental insulin and leptin levels in WT and P2Y , KO mice fed a Period . Specifically , the bar graph shows the water intake by 5 regular diet ( CNT ) or HFD with ad libitum access to food WT and P2Y , KO mice fed regular diet ( CNT ) or HFD for and drinking water for 16 weeks . 4 weeks with ad libitum access to food and drinking water . FIG . 12 provides the results of a Serum Adiponectin assay

FIG . 4B illustrates the water intake ( ml / day or ml / g body ( left ) and a Serum Free Fatty Acid assay ( right ) after 16 weight ) of the mice after 12 weeks of the Experimental weeks of the Experimental Period . Specifically , the graphs Period . Specifically , the bar graphs show the water intake by 10 show serum adiponectin and Free Fatty Acid ( FFA ) levels in WT and P2Y , KO mice fed HFD for 12 weeks . WT and P2Y , KO mice fed a regular diet ( CNT ) or HFD FIG . 5A illustrates the urine output ( ml / g body weight ) with ad libitum access to food and drinking water for 16 and urine osmolality ( mOsm / Kg ) after 4 weeks of the weeks . Experimental Period . Specifically , the bar graphs show the urine output and urine osmolality in WT and P2Y , KO mice 15 ce 15 FIG . 13 provides the results of a Serum Triglycerides fed regular diet ( CNT ) or HFD for 4 weeks with ad libitum assay ( left ) and a Serum Glycerol assay ( right ) after 16 access to food and drinking water . weeks of the Experimental Period . Specifically , the graphs

FIG . 5B illustrates the urine output ( ml / g body weight ) show serum triglycerides ( TG ) and glycerol levels in WT and urine osmolality ( mOsm / Kg ) after 12 weeks of the and P2Y , KO mice fed a regular diet ( CNT ) or HFD with ad Experimental Period . Specifically , the bar graphs show the 20 libitum access to food and drinking water for 16 weeks . urine output and urine osmolality in WT and P2Y , KO mice FIG . 14 provides Messenger RNA Expression level of fed HFD for 12 weeks with ad libitum access to food and PPAR in the Brown Fat ( left ) and the Messenger RNA drinking water . Expression level of UCP in the Brown Fat ( right ) . Specifi

FIG . 6A ( a , b and c ) are photographs that illustrate the cally , the graphs show messenger RNA Expression of PPAR appearance of abdominal fat in a ( a ) wild type mouse on a 25 ( left panel ) and UCP ( right panel ) in the brown adipose regular diet , ( b ) wild type mouse on a high - fat diet , and ( c ) tissue ( Fat ) of WT and P2Y , KO mice fed a regular diet P2Y2 knockout mouse on a high - fat diet , at the time of ( CNT ) . euthanasia . Specifically , the photographs show the appear - FIG . 15 provides the Messenger RNA Expression level of ance of abdominal fat at the time of euthanasia ( terminal ) . Inflammatory Cytokines and Cell Surface Molecules ( TNF FIG . 6B are representative pictures of white fat from a 30 A , IL - 6 , MCP - 1 , CCR2 , CD68 , F4 / 80 ) in the White Fat ( left ) high - fat diet wild type mouse ( left ) and knockout mouse and the Messenger RNA Expression level of Insulin Recep ( right ) . Specifically , the photographs show white fat tor Substrate Isoforms ( IRS 1 , IRS 2 ) in the White Fat obtained from HFD fed WT mouse ( left panel ) and KO mouse ( right panel ) . ( right ) . Specifically , the graphs show messenger RNA

FIG . 6C are representative pictures of brown fat from a 35 expression of inflammatory cytokines and cell surface mol high - fat diet wild type mouse ( left ) and knockout mouse ecules ( left panel ) and insulin receptor substrate ( IRS ) ( right ) . Specifically , the photographs show brown fat isoforms in the white adipose tissue ( Fat ) of WT and P2Y2 obtained from HFD fed WT mouse ( left panel ) and KO KO mice fed HFD for 16 weeks . mouse ( right panel ) . FIG . 16 provides mRNA expression of P2Y2 receptor in

FIG . 7A illustrates the terminal weight of white adipose 40 the organs involved in the development of obesity and tissue ( mg and mg / 20 g body weight ) . Specifically , the diabetes mellitus . Data obtained by real - time RT - PCR on graphs show the terminal weight of white adipose tissue in RNA extracted from different organs of wild type mice . WT and P2Y , KO Mice fed regular diet ( CNT ) or HFD with Specifically , the graph shows mRNA expression of P2Y , ad libitum access to food and drinking water . receptor in the organs involved in the development of

FIG . 7B illustrates the terminal weight of brown adipose 45 obesity and diabetes mellitus . tissue ( mg and mg / 20 g body weight ) . Specifically , the FIG . 17 provides mRNA expression of P2Y2 receptor in graphs show the terminal weight of brown adipose tissue in WHITE FAT of wild type mice after feeding high - fat diet WT and P2Y , KO mice fed HFD with ad libitum access to feeding for 16 weeks . Specifically , the graph shows mRNA food and drinking water . expression of P2Y , receptor in white fat of wild type mice

FIG . 8 illustrates the terminal weight of the liver and 50 after feeding high - fat diet feeding for 16 weeks . kidney ( mg / 20 g body weight ) in the mice after 16 weeks of FIG . 18 provides morphology of adipocytes in the the Experimental Period . Specifically , the graphs show the WHITE FAT in wild type ( WT ) or P2Y2 receptor knockout terminal weight of liver and kidney in WT and P2Y , KO ( KO ) mice after feeding control ( CNT ) or high - fat ( HFD ) mice fed regular diet ( CNT ) or HFD with ad libitum access diets for 16 weeks . Specifically , the photographs show the to food and drinking water for 16 weeks . 55 morphology of adipocytes in the white fat in WT or P2Y ,

FIG . 9 provides illustrative samples of the fecal matter in receptor KO mice after feeding control ( CNT ) or high - fat high - fat diet fed wild type mice ( left ) and knockout mice ( HFD ) diets for 16 weeks . ( right ) during the 16 week Experimental Period . Specifi FIG . 19 provides morphology of adipocytes in the cally , the photograph shows the appearance of the fecal BROWN FAT in wild type ( WT ) or P2Y2 receptor knockout matter in high - fat diet fed wild type ( WT - HFD ) and knock - 60 ( KO ) mice after feeding control ( CNT ) or high - fat ( HFD ) out ( KO - HFD ) mice during the 16th week of feeding . diets for 16 weeks . Specifically , the photographs show the

FIG . 10 provides the results of a Glucose Tolerance Test morphology of adipocytes in the brown fat in WT or P2Y , during the 14th week of the Experimental Period . Specifi - receptor KO mice after feeding control ( CNT ) or high - fat cally , the graphs provide an evaluation of glucose tolerance ( HFD ) diets for 16 weeks . in WT and P2Y , KO mice fed HFD with ad libitum access 65 FIG . 20 provides whole body insulin sensitivity in wild to food and drinking water . The GTT test was done during type ( WT ) or P2Y2 receptor knockout ( KO ) mice after 14th week . feeding control ( CNT ) or high - fat ( HFD ) diets . Specifically ,

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the graph shows whole body insulin sensitivity in WT or P2Y2 receptor antagonist is considered to be non P2Y2 receptor KO mice after feeding control ( CNT ) or selective or slightly selective if the antagonist is bound by high - fat ( HFD ) diets . two or more members of the P2Y receptor family and / or

FIG . 21 provides relative mRNA expression of insulin P2X receptor family with no or little preferential binding by receptor ( left panel ) or glucose transporter type 4 ( GLUT4 ) 5 the P2Y2 receptor within a given concentration range and ( right panel ) in the white adipocytes of wild type ( WT ) or affects the activity of other members of the P2Y receptor P2Y2 receptor knockout ( KO ) mice after feeding control family and / or P2X receptor family with no or little prefer ( CNT ) or high - fat ( HFD ) diets for 16 weeks . Specifically , ential effect on the P2Y2 receptor .

A P2Y2 receptor antagonist is said to compete non the graphs show relative mRNA expression of insulin recep tor ( left panel ) or glucose transporter type 4 ( GLUT4 ) ( right UTA ( richt 10 competitively if the antagonist binds to the P2Y2 receptor to

reduce its activity but does not prevent binding of UTP or panel ) in the white adipocytes of WT or P2Y2 receptor KO ATP to the P2Y2 receptor . mice after feeding control ( CNT ) or high - fat ( HFD ) diets for A P2Y2 receptor antagonist is said to compete competi 16 weeks . tively if the antagonist binds to the same site as the site

15 bound by UTP or ATP in the P2Y2 receptor . DETAILED DESCRIPTION OF ILLUSTRATIVE As used herein , activity of P2Y2 receptor or P2Y2 recep EMBODIMENTS tor signaling may be used interchangeably and refers to increase in intracellular free Ca2 + or increased production of After reading this description it will become apparent to prostaglandin E2 or activation of protein kinase C .

one skilled in the art how to implement the invention in 20 In one embodiment , a P2Y2 receptor antagonist may be a various alternative embodiments and alternative applica pure antagonist in which the P2Y2 receptor antagonist tions . However , although various embodiments of the pres - inhibits P2Y2 receptor signaling independent of a cell type ent invention will be described herein , it is understood that or tissue type either in vitro or in a subject . Inhibition of these embodiments are presented by way of example only , P2Y2 receptor activity occurs in all cells . In the absence of and not limitation . As such , this detailed description of 25 its native ligand , such as UTP or ATP , contacting with a pure various alternative embodiments should not be construed to P2Y2 receptor antagonist will not activate P2Y2 receptor limit the scope or breadth of the present invention as set signaling . forth in the appended claims . In one embodiment , a P2Y2 receptor antagonist may be a P2Y2 receptor belongs to a family of purinergic G - protein mixed P2Y2 receptor antagonist and agonist ( antagonist /

coupled receptors numbering eight P2Y receptors in 30 agonist ) , the mixed P2Y2 receptor antagonist / agonist may humans , which are stimulated by nucleotides , such as ATP , act as an antagonist in some tissues and an agonist in others . ADP , UTP , UDP and UDP - glucose . The eight P2Y receptors Such mixed P2Y2 receptor antagonist / agonist are contem cloned in human are : P2Y1 , P2Y2 , P2Y4 , P2Y6 , P2Y1 , plated within the invention so long as the mixed P2Y2 P2Y12 , P2Y13 and P2Y14 ( see for a review of P2Y receptor receptor antagonist / agonist antagonizes or inhibits P2Y2 family , including P2Y2 receptor , its agonist and antagonist 35 receptor signaling in an organ or a tissue involved in the ligands as well as molecular mechanisms and function by development of obesity and / or diabetes . Such an organ or a Abbracchio M P , Burnstock G , Boeynaems J - M , Barnard E tissue comprises skeletal muscle , intestine , brown fat , white A , Boyer JL , Kennedy C , Knight GE , Fumagalli M , Gachet fat , liver , pancreas or kidney . C , Jacobson KA , and Weisman G A . 2006 . “ International In one embodiment , a P2Y2 receptor antagonist or a Union of Pharmacology LVIII : Update on the P2Y G Pro - 40 mixed P2Y2 receptor antagonist / agonist inhibits P2Y2 tein - Coupled Nucleotide Receptors : From Molecular receptor signaling in an organ or a tissue comprising skeletal Mechanisms and Pathophysiology to Therapy ” . Pharmacol . muscle , intestine , brown fat , white fat , liver , pancreas , Rev . 58 : 281 - 341 ) . Relatedness in the structures of the P2Y kidney , or other organ or tissue involved in obesity or receptor family members result in similarities as well as diabetes . In one embodiment , obesity is diet - induced obe differences in their affinity and activation by the different 45 sity . In one embodiment , diabetes is type II diabetes . purine and pyrimidine nucleotides . P2Y2 receptor may be In one embodiment , a mixed P2Y2 receptor antagonist / activated by UTP or ATP , both of which may also activate a agonist may activate P2Y2 receptor signaling in an organ or related P2Y4 receptor in a species dependent manner . In a tissue not involved in obesity or diabetes . One such organ addition to the P2Y receptor family members , other receptor or tissue not involved in obesity or diabetes may be lung in families , such as P2X receptor family , may be stimulated by 50 which P2Y2 receptor agonist may be beneficial to warding nucleotides , raising the issue of specificity and selectivity off lung infection and in the treatment of cystic fibrosis . In for any ligand , either agonist or antagonist , directed to a one embodiment , obesity is diet - induced obesity . In one P2Y2 receptor . embodiment , diabetes is type II diabetes . As used herein , a P2Y2 receptor antagonist is defined as In one embodiment , a P2Y2 receptor antagonist may be a

a ligand which upon binding to a P2Y2 receptor decreases 55 mixed P2Y2 receptor antagonist / agonist that inhibits P2Y2 the activity of the P2Y2 receptor , thereby decreasing P2Y2 receptor signaling in an organ or tissue comprising skeletal receptor signaling . muscle , intestine , brown fat , white fat , liver , pancreas , AP2Y2 receptor antagonist is considered to be specific if kidney , or other organ or tissue involved in obesity or

the antagonist is bound only by P2Y2 receptor at the diabetes , but not in a lung tissue . concentration used and decreases the activity of the P2Y2 60 In one embodiment , a P2Y2 receptor antagonist may be receptor . an antagonist in one species but may be an agonist in another

A P2Y2 receptor antagonist is considered to be selective species . For example , ATP is a potent competitive antagonist if the antagonist is bound preferentially by P2Y2 receptor at the human P2Y4 receptor , but is a potent full agonist at the over a wide range of concentration , for example , 2 - fold to rat P2Y4 receptor ( Herold C I , Qi A D , Harden T K , and 30 - FOLD of the IC50 value of the compound , and decreases 65 Nicholas R A . 2004 . “ Agonist versus antagonist action of preferentially the activity of the P2Y2 receptor over other ATP at the P2Y4 receptor is determined by the second receptors extracellular loop " . J Biol . Chem . 279 ( 12 ) : 11456 - 64 ) .

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In addition to a role for P2Y2 receptor in obesity and As used herein , insulin resistance is a condition in which diabetes , P2Y2 receptor in combination with its G protein cells in a subject or cells cultured in vitro do not respond to effector , G , G , or G12 , and signaling through these receptors insulin effectively , resulting in lowered cellular uptake of in different cell types may bring about different biological glucose from the serum or cultured media . In vitro , insulin and physiological responses as for example listed in Table 1 5 resistance may be induced by chronic exposure to insulin of Erb L and Weisman G A . 2012 . “ Coupling of P2Y and / or glucose . A cell is said to have greater insulin sensi receptors to G proteins and other signaling pathways ” . tivity if the cell is better able to respond to insulin effectively WIREs Membr Transp Signal 1 : 789 - 803 . The different than a less insulin sensitive or insulin resistant cell . Markers

associated with glucose transport or metabolism as well as biological and physiological responses include inhibition of 10 markers associated with insulin signaling may be used to bone formation and mineralization , immune cell recruitment assess insulin sensitivity or resistance of a cell , or alterna and phagocytosis , vascular tone , inflammation , thrombosis , tively , relative insulin sensitivity or resistance of a cell . blood pressure regulation , intraocular pressure regulation , Provided herein are methods for selectively blocking the mechanical and thermal nociception , HIV infection of T activity of the P2Y2 purinergic receptor for the prevention cells , epithelial K + / Cl - secretion , pancreatic and renal func and renal Tunc - 15 and / or treatment of diet - induced overweight or obesity . It tions , liver regeneration , and wound healing . was discovered that the genetic deletion of P2Y2 receptor In one embodiment , a P2Y2 receptor antagonist may results in resistance to the development of high - fat diet preferentially affect obesity and / or diabetes without affect induced obesity and the associated physical and molecular ing one or more selected from bone formation and miner alterations in mice . The results described also suggest that alization , immune cell recruitment and phagocytosis , vas - 20 the observed resistance of the P2Y2 receptor in knockout cular tone , thrombosis , blood pressure regulation , mice was not due to reduced consumption of high - fat diet or intraocular pressure regulation , mechanical and thermal lack of absorption of fat in the intestines , but may be due to nociception , HIV infection of T cells , epithelial K + / C1 - the ability of the knockout mice to metabolize or “ burn ” the secretion , pancreatic and renal functions , liver regeneration , extra calories consumed in the diet more efficiently than the and wound healing . In one embodiment , obesity is diet - 25 wild type mice . Accordingly , targeting P2Y2 receptor with induced obesity . In one embodiment , diabetes is type II specific pharmacological agents or drugs is a safe approach diabetes to prevent or treat diet - induced overweight or obesity .

In one embodiment , a P2Y2 receptor antagonist may In various embodiments , provided herein are methods for affect obesity and / or diabetes and affect one or more selected treating or preventing diet - induced obesity in a subject by from bone formation and mineralization , immune cell 30 administering an agent that blunts the expression or activity recruitment and phagocytosis , vascular tone , thrombosis , of the P2Y , receptor in the subject . blood pressure regulation , intraocular pressure regulation , In some specific embodiments , the agent is a fusion mechanical and thermal nociception , HIV infection of T protein or an agent that inhibits the P2Y , receptor . In specific cells , epithelial K + / Cl - secretion , pancreatic and renal func - embodiments , the agent is a selective P2Y2 receptor antago tions , liver regeneration , and wound healing . In one embodi - 35 nist , including but not limited to a non - nucleic acid small ment , obesity is diet - induced obesity . In one embodiment , organic molecule . In one embodiment , the P2Y2 receptor diabetes is type II diabetes . antagonist is a non - competitive P2Y2 receptor antagonist .

In one embodiment , a mixed P2Y2 receptor antagonist The non - competitive P2Y , receptor antagonist may be a may preferentially affect obesity and / or diabetes without non - selective non - competitive P2Y2 receptor which may affecting one or more selected from bone formation and 40 inhibit other members of the P2Y receptor family [ Ralevic mineralization , immune cell recruitment and phagocytosis , V , Burnstock G . 1998 . Receptors for purines and pyrimi vascular tone , thrombosis , blood pressure regulation , dines . Pharmacol Rev 50 : 413 - 492 ] with no selectivity for intraocular pressure regulation , mechanical and thermal P2Y , receptor , a selective non - competitive P2Y , receptor nociception , HIV infection of T cells , epithelial K + C1 antagonist with a selectivity for P2Y2 receptor or a specific secretion , pancreatic and renal functions , liver regeneration , 45 non - competitive P2Y , receptor antagonist inhibiting the and wound healing . In one embodiment , obesity is diet - activity of only P2Y , receptor . Preferably , the non - competi induced obesity . In one embodiment , diabetes is type II tive P2Y , receptor antagonist is a selective non - competitive diabetes . P2Y , receptor antagonist . More preferably , the non - com

In one embodiment , a mixed P2Y2 receptor antagonist petitive P2Y , receptor antagonist is a specific non - competi may affect obesity and / or diabetes and affect one or more 50 tive P2Y2 receptor antagonist . Non - competitive P2Y2 recep selected from bone formation and mineralization , immune tor antagonist may be flavonoids . Useful flavonoids may be cell recruitment and phagocytosis , vascular tone , thrombo isolated from plant sources [ Ververidis F , Trantas E . Douglas sis , blood pressure regulation , intraocular pressure regula - C , Vollmer G , Kretzschmar G , Panopoulos N ( October tion , mechanical and thermal nociception , HIV infection of 2007 ) . “ Biotechnology of flavonoids and other phenylpro T cells , epithelial K + / Cl - secretion , pancreatic and renal 55 panoid - derived natural products , Part I : Chemical diversity , functions , liver regeneration , and wound healing . In one impacts on plant biology and human health ” . Biotechnology embodiment , obesity is diet - induced obesity . In one embodi Journal 2 ( 10 ) : 1214 - 34 ] , such as Calycopteris floribunda ment , diabetes is type II diabetes . Lamk . ( Combretaceae ) [ Mayer R . 1999 . Calycopterones and

In one embodiment , a P2Y2 receptor antagonist may calyflorenones , novel biflavonoids from Calycopteris flori affect obesity or diabetes through inhibition of adipogenesis , 60 bunda . J Nat Prod 62 : 1274 - 1278 ] , Leptospermum sco increasing energy metabolism , promotion of lipolysis , parium Forst . ( Myrtaceae ) [ Mayer R . 1990 . Flavonoids from stimulation of catabolism , maintaining insulin sensitivity Leptospermum scoparium . Phytochemistry 29 : 1340 - 1342 ; and / or inflammation . In one embodiment , maintaining insu lin sensitivity comprises prevention of insulin resistance scoparium . Planta Med 59 : 269 - 271 ] , and tangerine peels and / or reversing insulin resistance . In one embodiment , 65 from Citrus reticulate ssp . , Rutaceae ( Chen J , Montamari A obesity is diet - induced obesity . In one embodiment , diabetes M , Widmer W W . 1997 . Two new polymethoxylated fla is type II diabetes . vones , a class of compounds with potential anticancer activ

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ity , isolated from cold pressed dancy tangerine peel oil “ Development of selective agonists and antagonists of P2Y solids . J Agric Food Chem 45 : 364 - 368 ] , microorganisms receptors ” . Purinergic Signalling 5 : 75 - 89 ; Meghani P . 2002 . genetically engineered to produce flavonoids [ Hwang E I , “ The design of P2Y2 antagonists for the treatment of Kaneko M , Ohnishi Y , Horinouchi S ( May 2003 ) . “ Produc - inflammatory diseases ” . 224th ACS National meeting , tion of plant - specific flavanones by Escherichia coli con - 5 Abstracts , Division of medicinal chemistry 12 ; Kindon N , taining an artificial gene cluster ” . Appl . Environ . Microbiol . Meghani P , Thorn S ( 1998 ) World Pat . 98 / 54180 ; Kemp PA 69 ( 5 ) : 2699 - 706 ; Trantas E , Panopoulos N , Ververidis F et al . ( 2004 ) Am J Respir Cell Mol Biol 31 : 446 - 455 ; ( 2009 ) . “ Metabolic engineering of the complete pathway Alexander SPH , Benson H E , Faccenda E , Pawson A J , et leading to heterologous biosynthesis of various flavonoids al . 2013 . “ The concise guide to pharmacology 2013 / 14 : G and stilbenoids in Saccharomyces cerevisiae ” . Metabolic 10 protein - coupled receptors ” . British Journal of Pharmacology Engineering 11 ( 6 ) : 355 - 366 ; Ververidis F , Trantas E , Doug 170 : 1459 - 1581 ) . las C , Vollmer G , Kretzschmar G , Panopoulos N ( 2007 ) . In an embodiment , the competitive P2Y , receptor antago “ Biotechnology of flavonoids and other phenylpropanoid - nist has an half maximal inhibitory concentration ( IC50 ) of derived natural products . Part II : Reconstruction of multi - below 100 uM , preferably below 35 uM , more preferably enzyme pathways in plants and microbes ” . Biotechnology 15 below 25 uM , even more preferably below 10 UM , even , Journal 2 ( 10 ) : 1235 - 49 ] , and products of chemical or even more preferably below 5 uM and most preferably biochemical reactions leading to synthesis or modification of below 1 uM . In one embodiment , the competitive P2Y a flavonoid . In an embodiment , the non - competitive P2Y2 receptor antagonist has an IC50 of 35 - 100 UM . In a separate receptor antagonist has an half maximal inhibitory concen - embodiment , the competitive P2Y2 receptor antagonist has tration ( IC50 ) of below 100 uM , preferably below 35 UM , 20 an IC50 of 25 - 35 °M . In another embodiment , the competi more preferably below 25 uM , even more preferably below tive P2Y2 receptor antagonist has an IC50 of 10 - 25 uM . In 10 UM , even , even more preferably below 5 uM and most another embodiment , the competitive P2Y , receptor antago preferably below 1 uM . In one embodiment , the non nist has an IC50 of 5 - 10 uM . In a more preferred embodi competitive P2Y , receptor antagonist has an IC50 of 35 - 100 ment , the competitive P2Y , receptor antagonist has an IC50 uM . In a separate embodiment , the non - competitive P2Y , 25 of 1 - 5 uM . In a most preferred embodiment , the competitive receptor antagonist has an IC50 of 25 - 35 uM . In another P2Y2 receptor antagonist has an IC50 of less than 1 uM . embodiment , the non - competitive P2Y , receptor antagonist Preferably , the competitive P2Y2 receptor antagonist has an has an IC50 of 10 - 25 uM . In another embodiment , the IC50 between 0 . 1 - 1 uM . non - competitive P2Y , receptor antagonist has an IC50 of In other embodiments , the agent is an oligonucleotide . In 5 - 10 UM . In a more preferred embodiment , the non - com - 30 specific embodiments , the oligonucleotide is an antisense petitive P2Y , receptor antagonist has an IC50 of 1 - 5 uM . In oligonucleotide , a ribozyme or an inhibitory RNA , including a most preferred embodiment , the non - competitive P2Y , but not limited to a small inhibitory RNA ( siRNA ) or short receptor antagonist has an IC50 of less than 1 uM . In yet hairpin RNA ( shRNA ) . another preferred embodiment , the non - competitive P2Y2 In other embodiments , the agent is a gene knockout receptor antagonist has an IC50 between 0 . 1 - 1 M . Examples 35 system directed against P2Y2 receptor gene comprising of non - competitive P2Y , receptor antagonist include strobo - Cas9 nuclease and guide RNA ( GRNA ) linked to a P2Y2 pinin , strobopinin - 7 - methyl ether , 2 - B - Dihydroxy - 4 ' , 6 - di receptor sequence to target Cas9 nuclease to P2Y2 gene so methoxy - 3 ' - methylchalcone ( or Oxo - Aurentiacin ) , kaemp - as to introduce a site - specific double - stranded break in the ferol , heptamethoxylflavone , and tangeretin with chemical P2Y2 genomic DNA and produce a P2Y2 receptor knock structures and additional flavonoids described in Kaulich M , 40 out . Cas9 nuclease and ORNA linked to a P2Y2 receptor Streicher F , Mayer R , Muller 1 , Muller C E . 2003 . “ Fla - sequence may be introduced directly into desired cells or vonoids — novel lead compounds for the development of alternatively by expression from expression plasmid or P2Y , receptor antagonists ” . Drug Development Research vector systems , including lentiviral system , adenoviral sys 59 : 72 - 81 . tem or adeno - associated viral system .

In one embodiment , the P2Y , receptor antagonist is a 45 In other embodiments , provided herein are methods for competitive P2Y , receptor antagonist . In one embodiment , increasing energy metabolism in a cell by contacting the cell the competitive P2Y2 receptor antagonist may be a non with an agent that blunts the expression or activity of a P2Y2 selective or slightly selective competitive P2Y , receptor receptor , thereby increasing the metabolism in the cell . In antagonist . Examples of non - selective or slightly selective specific embodiments , the cell is an adipocyte cell . competitive P2Y2 receptor antagonists include suramin 50 In specific embodiments , the agent is a fusion protein , ( 8 , 8 ' - [ Carbonylbis [ imino - 3 , 1 - phenylenecarbonylimino ( 4 - polypeptide , a small non - nucleic acid organic molecule , a methyl - 3 , 1 - phenylene ) carbonylimino ] ] bis - 1 , 3 , 5 naphthale - small inorganic molecule or an oligonucleotide , including netrisulphonic acid ) and Reactive Blue 2 ( RB2 ) with an but not limited to an antisense oligonucleotide , a ribozyme , IC50 of between 5 uM and 35 uM [ Kaulich M , Streicher F , or an inhibitory RNA , including but not limited to a small Mayer R , Muller I , Muller C E . 2003 . “ Flavonoids novel 55 inhibitory RNA ( siRNA ) or a short hairpin RNA ( shRNA ) . lead compounds for the development of P2Y2 receptor Also provided herein are methods for increasing energy antagonists ” . Drug Development Research 59 : 72 - 81 ; Jacob - metabolism in an adipocyte cell in a subject that is at risk for son KA , Jayasekara , M PS , Costanzi S . 2012 . “ Molecular or suffering from a disorder related to glucose metabolism , structure of P2Y receptors : mutagenesis , modeling , and the method comprising administering to the subject an agent chemical probes ” . WIREs Membr Transp Signal 1 : 815 - 827 ; 60 that inhibits expression or activity of a P2Y2 receptor , Jacobson KA . “ P2X and P2Y receptors ” . Tocris Bioscience thereby increasing the metabolism in the cell . Scientific Review Series , pp . 1 - 15 ] . In one embodiment , the In various specific embodiments , the agent is a polypep competitive P2Y2 receptor antagonist is a selective competi - tide , a small non - nucleic acid organic molecule , a small tive P2Y , receptor antagonist . Examples of selective com - inorganic molecule , an antibody , an antisense oligonucle petitive P2Y , receptor antagonists include AR - C126313 and 65 otide , a ribozyme or an inhibitory RNA , including but not AR - C118925 comprising an IC50 of about 6 uM [ Jacobson limited to a small inhibitory RNA ( siRNA ) or a short hairpin KA , Ivanov A A , de Castro S , Harden T K , Ko H . 2009 . RNA ( shRNA ) .

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Provided herein is also a method for identifying a candi ently do not suffer from diseases , have no pathological date agent that modulates expression or activity of the P2Y2 lesions and live healthy . Although one study showed that receptor , comprising : ( a ) providing a sample comprising the these mice develop salt - resistant hypertension , the rise in P2Y2 polypeptide or a nucleic acid encoding the polypep blood pressure was modest , and these mice do not develop tide ; ( b ) contacting the sample with a test compound under 5 complications of hypertension , such as kidney disease . See . conditions in which the polypeptide is active , the nucleic Rieg et al . , 2007 , FASEB J 21 : 3717 - 3726 . acid is expressed , or both ; ( c ) evaluating expression or activity of P2Y , polypeptide in the sample ; and ( d ) com Hence , targeting purinergic signaling for the treatment of paring the expression or activity of the P2Y , polypeptide of obesity is relatively safe as compared to the current state ( c ) to the expression or activity of the P2Y , polypeptide in of - the - art . Any potential increase in blood pressure can be a control sample lacking the test compound , wherein a effectively controlled with combination therapy . change in the P2Y2 polypeptide expression or activity indi cates that the test compound is a candidate agent that can Example 2 : Wild Type and P2Y2 Receptor modulate the expression or activity of the P2Y , polypeptide . Knockout Study

In various specific embodiments , the candidate agent selectively inhibits the P2Y , receptor or is a fusion protein . 15 A study was performed on wild type and P2Y2 receptor As shown herein , it was discovered that mice with genetic knockout mice ( both in B6D2 genetic background ) . Breed deletion of P2Y2 receptor consume the same amount of

high - fat diet as their syngeneic wild type mice do , and do not ers of these mice were originally obtained from Dr . Beverly excrete fatty stools ( steatorrhea ) , yet do not become obese . Koller of the University of North Carolina at Chapel Hill , This suggests that P2Y2 receptor is a potential target for the 20 Chapel Hill , N . C . . . ential target for the 20 Chapel Hill , N . C . development of efficacious and safer anti - obesity drugs . One Obesity is associated with deranged sodium homeostasis possible reason for this phenomenon is that purinergic P2Y2 leading to hypertension among other pathologies . Hence , receptor is involved in energy metabolism and so its genetic this high - fat diet study was conceived to examine the role of deletion significantly protects against high - fat diet - induced P2Y2 receptor in obesity - induced alterations in water and obesity . Accordingly , purinergic antagonism may prove to o sodium handling by the kidney , leading to hypertension . be a very advantageous invention over the current state - of - Since kidney is one of the vital organs affected by obesity , the - art . it was posited that any new information obtained may help

This is further supported by the fact that one promising to address more complex questions , such as the course of line of future anti - obesity drugs is based on the development lithium - induced nephrogenic diabetes insipidus in obese of ribonucleic acid interference ( RNAi ) to silence RIP140 patients and the effect of lack of purinergic signaling . gene , a nuclear co - repressor that regulates fat accumulation . ” However , during the study it was observed that P2Y2 This line of drug development was prompted by the obser receptor knockout mice were resistant to the diet - induced vation that mice with genetic deletion of RIP140 exhibit a obesity , which forms the basis for this invention . lean profile throughout their life , and are resistant to diet induced obesity , and show an enhanced metabolic rate . Experimental Study Parameters CytRx Corporation is currently developing RNAi therapeu - 35 tics against this drug target ( RIP140 ) for the treatment of Breeding colonies were established in the Veterinary obesity and type 2 diabetes . Conversely , disruption of the Medical Unit ( VMU ) of the VA Salt Lake City Health Care molecular interaction between SMRT , a nuclear hormone System . Mice bred were identified by Polymerase Chain receptor co - repressor and its receptor partner leads to

Reaction ( “ PCR ” ) on DNA extracted from the tail clippings , increased adiposity and decreased metabolic rate . The novel findings described herein open the possibility and all mice were tracked by implanting microchip tran

to develop an entirely new line of drugs that target the sponders under the skin . Age matched adult wild type and molecular interactions between C protein - coupled receptors P2Y2 receptor knockout ( KO ) mice were randomly divided and their downstream regulatory effectors to prevent or treat into two sub - groups for each genotype and were fed regular diet - induced obesity . Through the availability of gene 45 rodent chow ( 10 % of total energy as fat ) or high - fat diet knockout mice that carried these molecular disruptions in ( 60 % or total energy as fat ) for 16 weeks as shown below . vive throughout their lifespan , researchers are able to assess The high - fat diet was purchased from the Research Diets , the efficacy and safety of this promising approach in live Inc . ( New Brunswick , N . J . ) . mice .

As discussed in detail herein , it was discovered that genetic deletion of P2Y2 receptor causes resistance to 30 Wild Type Mice P2Y , Receptor Knockout Mice diet - induced obesity . The data indicates that this effect is likely related to increased metabolic rate and the effect is Regular Diet High - Fat Diet Regular Diet High - Fat Diet associated with better glucose tolerance as well as a sign of N = 6 N = 14 N = 6 N = 14 better balance between energy intake and expenditure .

The invention described herein has potential practical and 55 commercial applications to develop a new line of anti All mice had free access to drinking water throughout the obesity drugs that are safe and efficacious . This can be experimental period . Food intake and body weights were achieved by either designing and developing P2Y2 receptor monitored on regular basis . Twenty - four hour urine samples selective antagonists ( chemical molecules ) or RNAi to were collected periodically by placing the mice in metabolic silence the P2Y2 receptor gene . 60 cages with free access to food and drinking water . Feces

EXAMPLES samples of all groups of mice were collected periodically . During the 14 " week , tolerance of the mice to an acute load

Example 1 : Mice that are Genetically Deficient in of glucose was assessed by carrying out glucose tolerance P2Y2 Receptor test ( “ GTT ' ) . All mice were euthanized at the end of the

65 experimental period and samples of blood , liver , kidney , A bench prototype was generated in mice that are geneti - pancreas , spleen and whole white and brown adipose tissues

cally deficient in P2Y2 receptor . These mutant mice appar were collected for analysis in the laboratory .

Um

17

01 .

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The GTT tests were performed as described by Takahashi are striking when analyzed as weights of individual mice et al ( 2011 ) . Briefly , after a fasting period of about 12 hours , using scattergraph ( FIG . 1B , left ) or as box plots ( FIG . 1B , the mice received an intraperitoneal injection of sterile right ) . glucose solution ( 1 . 5 g / kg body wt . ) . Tiny droplets of blood FIG . 2 shows a picture of mice to compare the size of one ( ~ 5 ul ) were obtained in conscious mice by gently pricking 5 of the highest weighing WT mice from the high - fat diet the dorsal pedal vein with a fine needle . Using these drop - group ( WT - HFD ) with an average weighing KO mouse from lets , blood glucose levels were determined with a clinical the high - fat diet group ( KO - HFD ) . In order to exclude the glucometer . Blood samples were collected prior to ( 0 min ) , possibility that the differences observed in body weights of and 30 , 60 , 90 and 120 min after intraperitoneal injection of the WT and KO mice was due to differences in their food glucose solution . 10 and / or water intake , the food and water intake of the mice at

Twenty - four hour urine output was recorded , and the two different time points was determined ( see below ) . osmolalities of clear urine samples were determined by FIG . 3A shows food intake as g / day / mouse after 4 weeks . vapor pressure method on an osmometer ( Wescor , Logan , As shown in this figure , the high - fat diet fed WT mice Utah ) . Insulin , leptin , adiponectin , triglycerides and glycerol consumed significantly less amount of food as compared to levels in the serum samples were determined by using 15 the regular diet fed WT mice . However , there was no commercial ELISA or EIA kits ( Crystal Chern , Inc . , Down difference in the food intake between the WT or KO mice ers Grove , 111 . or Cayman Chemical Co . , Ann Arbor , Mich . ) . when high - fat diet was fed to them . Weights of total brown or white adipose tissues were deter FIG . 3B shows food intake in high - fat diet fed WT or KO mined for each mouse . Total liver and kidney weights were mice after 12 weeks . The left panel presents the data as also determined . 20 g / day / mouse , whereas the right panel shows the data as g / g

Liver tissue samples were analyzed for metabolic change body weight per day . Notably , at this time point the KO mice in the University of Utah Metabolomic Core Facility using were consuming significantly higher amount of food , despite gas chromatography - mass spectrometry ( GC - MS ) . For this the fact that their body weights were significantly lower as analysis , about 20 mg of liver tissue from each mouse ( N = 6 compared to the body weights of the high - fat diet fed WT mice / group ) was used . The liver tissue was flash frozen in 25 mice ( see , FIG . 1 ) . Thus , the observed lack of gain in the liquid nitrogen at the time of euthanasia and then stored at body weight in high - fat diet fed KO mice was not due to - 80° C . In the core facility , the tissue was mechanically consumption of lesser amount of food . In fact , food con disrupted using a bead mill , and metabolites were extracted sumption in these mice is relatively higher when normalized by a solvent . After evaporating the solvent , the dried to the body weight . samples were suspended in O - methoxylamine hydrochloride 30 FIG . 4A shows water intake as ml / day / mouse after 4 ( MOX ) and further processed for GC - MS . GC - MS data was weeks . Although there were no significant differences collected using MassLynx 4 . 1 software ( Waters ) . between the genotypes , high - fat diet significantly reduced

A two - step process was employed for data analysis , a water intake in both genotypes . FIG . 4B shows water intake targeted followed by non - targeted analysis . For the targeted in high - fat diet fed W and KO mice after 12 weeks . The left approach , known metabolites were identified and their peak 35 panel presents data as ml / day / mouse , whereas the right areas were recorded using QuanLynx . For the non - targeted panel shows data as ml / g body weight per day . Similar to the approach peak picking and analysis was performed using food consumption , the KO mice were drinking significantly MarkerLynx . Principle components analysis ( PCA ) and par - higher amount of water relative to their body weight , despite tial least squares - discriminate analysis ( PLS - DA ) was per the fact that their body weights were significantly low as formed using SIMCA - P 12 . 0 ( Umetrics , Kinnelon , N . J . ) . 40 compared to the body weights of the high - fat diet fed WT Metabolite identity was established using a combination of mice ( see , FIG . 1 ) . an in - house metabolite library developed using pure stan - FIG . 5A shows urine output and urine osmolality after 4 dards and the commercially available NIST library . Not all weeks in WT and KO mice fed regular or high - fat diets . As metabolites were observed using GC - MS . shown in the left panel , high - fat diet caused significant

Quantitative data are presented as mean sem . Compari - 45 decrease in urine output in both genotypes . However , high sons among the means of multiple groups were made by fat diet did not have any effect on the urine osmolality in WT one - way analysis of variance ( ANOVA ) , followed by the or KO mice . FIG . 5B shows urine output and urine osmo assessment of statistical significance by Tukey - Kramer Mul lality in high - fat diet fed WT and KO mice after 12 weeks . tiple Comparison test or Boneferroni test for selected pairs . Interestingly at this time point the urine output when Differences between the means of two groups were deter - 50 expressed as ml / g body wt / day was modest , but significantly mined by unpaired t - test or Mann - Whitney nonparametric higher in the KO mice ( left panel ) . However , no differences method . P values less than 0 . 05 were considered significant . were noted in urine osmolality between these two groups GraphPad Instat® software ( GraphPad Software , Inc . , La after 12 weeks . Jolla , Calif . ) was used for statistical analysis . FIG . 6A shows representative pictures of the appearance Study Results 55 of abdominal fat at the time of euthanasia . Panel 6Aa shows

FIG . 1 illustrates the changes in the body weights in ( WT ) the appearance of normal content and disposition of abdomi and P2Y2 knockout ( KO ) mice fed regular diet or high - fat nal fat in a WT mouse fed regular diet , which is similar to diet for 16 weeks . As shown in FIG . 1A , the WT mice had KO mice fed a regular diet ( not shown here ) . In contrast , modestly , but significantly higher body weights even on day high - fat diet fed WT mice have much higher amounts of O as compared to the age - matched KO mice . Following 60 abdominal fat ( FIG . 6Ab ) as compared to the high - fat diet high - fat diet ( HFD ) feeding , the body weight or WT mice fed KO mice ( FIG . 6Ac ) . steadily increased starting at 2 weeks . In contrast , body In mammals , fat or adipose tissue exists in two distinctive weights of high - fat diet fed KO mice essentially remained forms — the white and brown adipose tissues . White adipose similar to the regular diet ( CNT ) - fed KO mice throughout tissue is a storage form of energy and accumulates excessive the study period . FIG . 1B and FIG . 1C show the terminal ( 16 65 calories consumed as fat droplets , usually found around the weeks ) body weights of the high - fat diet fed WT vs . high - fat abdominal wall or the belly . White adipose tissue burns diet fed KO mice . The differences between these two groups slowly , and is linked to the risk for diabetes and heart

19 US 10 , 024 , 846 B2

20 diseases . Conversely , the brown fat , which is rich in mito - fed WT and KO mice , the differences in blood glucose levels chondria and capillaries ( hence the color ) , burns calories to between the regular diet fed WT and KO mice were also generate heat . Brown fat is abundant in newborns and wide apart at 30 min time point , indicating that KO mice are hibernating mammals , whereas the white fat is abundant in inherently efficient in metabolizing a load of glucose faster . adults . FIG . 6B and FIG . 6C are representative pictures of 5 Insulin , released from the beta - cells of islets of Langer white or brown fat obtained from the high - fat diet fed WT hans in the pancreas in response to increasing blood glucose or KO mice . The difference in the masses of these two types levels ( e . g . , following a meal ) , is the key hormone in of tissues between the genotypes was quite obvious . regulating the blood glucose levels by inducing uptake of

FIG . 7A shows the terminal weights of white adipose glucose by cells and its subsequent metabolism . In type II tissue in the WT and KO mice fed regular or high - fat diet . 10 diabetes , the cells develop resistance to the action of insulin As shown , the WT mice fed high - fat diet had a 2 to 3 - fold with the result the subjects have both high blood glucose and higher amount of white fat as compared to the regular diet high blood insulin levels . The left panel of FIG . 11 shows fed WT mice , depending on whether the fat content was terminal serum insulin levels in the blood samples collected expressed as mg / mouse or mg / 20 g body weight . On the at the time of euthanasia . As illustrated in this figure , other hand , the high - fat diet feeding had no significant effect 15 following high - fat diet feeding for 16 weeks , the WT mice on the white adipose tissue in the KO mice . FIG . 7B shows had more than 2 - fold increase in serum insulin . The insulin the terminal weights of brown adipose tissue in WT and KO levels in the KO mice were not affected by high - fat diet mice fed high - fat diet . Similar to the white adipose tissue , feeding . These results correlate well with the pattern of GTT high - fat diet feeding had a significant effect on brown results obtained . adipose tissue only in the WT mice . 20 Leptin is a peptide hormone elaborated mainly by white

Since the liver and kidney are two vital organs affected by adipose tissue , although brown adipose tissue and other obesity , their weights were recorded as a function of body organs also produce leptin . The levels of circulating leptin weight at the time of euthanasia . As shown in FIG . 8 , in are proportional to the total amount of the fat in the body . In high - fat diet fed WT mice the weights of both liver and the general , leptin is considered to play a key role in regulating kidney were significantly low when adjusted for the body 25 energy intake and energy expenditure , including appetite weight . In contrast , a high - fat diet did not have any effect on and metabolism . Leptin acts on the hypothalamus and inhib the weights of the liver and kidney in KO mice . its appetite . Hence , the absence of leptin leads to uncon

If the apparent resistance of the KO mice to high - fat diet trolled food intake resulting in obesity , e . g . , ob / ob mutant induced increase in body weight and accumulation of body mice that lack leptin . fat is due to defective absorption of fat in the intestines , then 30 When these mutant mice are treated with leptin injections , one would expect steatorrhea in these mice . Steatorrhea is a they lose excess body fat and return to normal body weight . condition where the feces contain partially digested fat as However , despite these properties of leptin , obese subjects oil . The feces are not well formed , and have an oily often develop resistance to leptin so their appetite is not appearance and foul smell . Hence , the fecal matter in the reduced by high circulating leptin levels . This is because of high - fat diet fed WT and KO mice was monitored . FIG . 9 35 leptin desensitization due to high circulating leptin levels in shows representative profiles of the fecal matter in high - fat obese subjects . In view of this , serum levels of leptin in the diet fed wild type ( WT - HFD ) and knockout ( KO - HFD ) mice mice were determined . As shown in the right panel of FIG . collected during the last week of experimental period ( 16 11 , high - fat diet feeding for 16 weeks caused more than a weeks ) . As illustrated in this figure , both WT and KO mice 3 - fold increase in the serum leptin levels in WT mice . The fed high - fat diet have well - formed , and non - oily fecal mat - 40 increase in serum leptin levels in high - fat diet fed KO mice ter . These observations rule out the possibility of lack of was modest . absorption of fat in the intestines in KO mice . Adiponectin is a protein hormone produced exclusively Glucose tolerance or the ability to metabolize a load of from adipose tissue ( and also from placenta during preg

glucose is impaired in obese subjects , And obesity is a nancy ) . Adiponectin modulates a number of metabolic func known risk factor for the development of type II diabetes , 45 tions , such as glucose metabolism and fatty acid catabolism . where the subjects develop resistance to insulin and thus Adiponectin also plays roles in the suppression of metabolic cannot metabolize glucose efficiently , despite high blood derangements , such as those occurring due to type II dia insulin levels . To examine whether the high - fat diet induced betes mellitus , obesity , atherosclerosis , as well as others . In increase in body weight in the wild type mice is associated general , adiponectin is considered as an independent risk with glucose intolerance , a Glucose Tolerance Test ( GTT ) 50 factor for metabolic syndrome . Similar to leptin , the weight using a standard protocol was performed . reducing effects of adiponectin are mediated via brain . In

FIG . 10 shows the results of the GTT in different groups view of this , the serum adiponectin levels in the blood of mice . As shown in the left panel of FIG . 10 , at 30 min samples collected was determined at the time of euthanasia , after an acute load of glucose , the high - fat diet fed WT mice i . e . , after 16 weeks of feeding the respective diets . showed the highest blood level of glucose , about 400 mg / dl . 55 As shown in FIG . 12 left panel , the serum adiponectin The corresponding blood glucose level in high - fat diet fed levels are modestly , but significantly elevated in high - fat diet KO mice was significantly low . In fact at all the time points fed WT mice , but not in KO mice . The increase in serum following an acute glucose load , the high - fat diet fed KO adiponectin in the WT mice may be due to higher fat content mice had significantly lower blood glucose levels as com - in the body , and also in response to the increase in the body pared to the high - fat diet fed WT mice ( FIG . 10 , right panel ) . 60 weight . However , similar to higher serum leptin levels in

Notably , the baseline ( 0 min ) blood glucose levels in the these mice , the higher adiponectin levels did not counter the WT mice were modest , but significantly elevated as com - high - fat diet - induced increase in body weight in WT mice . pared to the KO mice ( FIG . 10 , right panel ) . Furthermore , at Since adiponectin is involved in fatty acid catabolism , the 30 min time point following an acute load of glucose , the serum free fatty acids ( FFA ; FIG . 12 , right panel ) , serum differences among the four groups were very obvious , with 65 triglycerides ( TG ; FIG . 13 , left panel ) and serum glycerol the two WT groups having higher blood glucose levels as ( FIG . 13 , right panel ) were determined . As shown , the serum compared to the two KO groups . Similar to the high - fat diet FFA levels were not significantly different among the four

US 10 , 024 , 846 B2 21 22

groups of mice , although high - fat diet had a tendency for Further insights into the metabolic profiles of the WT and higher levels of serum FFA in both genotypes . Interestingly , zby , KO KO mice and the effect of high - fat diet feeding , were serum TG levels were significantly higher in KO mice as compared to the WT mice , irrespective of the dietary regi - obtained by performing a metabolomics analysis of the liver men , and dietary regimen itself did not have any effect in 5 tissue . Table 1 below provides the data obtained for 23 either genotype . In contrast , a high - fat diet triggered sig nificantly higher ( about 2 - fold ) serum glycerol levels in both carbohydrate metabolites , 20 protein metabolites , 22 lipid genotypes . metabolites and 5 nucleic acid metabolites .

TABLE 1

Metabolomic Analysis of Liver Tissue by GC - MS GC - MS data presented here are in arbitrary units and expressed as mean - sem

Metabolites shown with an asterisk on the right end of the line have interesting or significant trends with respect to genotype or dietary regimen or both .

WT - CNT WT - HFD KO - CNT KO - HFD Carbohydrate Metabolites

Lactic Acid Pyruvic Acid Glycerol Glyceric Acid Citric Acid Aconitic Acid Isocitric Acid 2 - Ketoglutaric Acid Succinic Acid Fumaric Acid Malic Acid 2 - Hydroxyglutarate 2 - Aminoadipic Acid Glucose Fructose Ribose Galactose or isomer Glucose - 1 - Phosphate Glucose - 6 - Phosphate Sorbitol Galacitol Disaccharide Xylulose - 5 - monophosphate Protein Metabolites

687 89 159 34 1 . 26 = 1 . 00 558 – 44 35 . 3 + 3 . 1 7 . 08 = 1 . 38 6 . 03 = 1 . 25 1 . 99 + 0 . 57 2463 + 291 355 + 14 709 + 49 4 . 06 + 0 . 34 5 . 70 + 1 . 02

21885 853 466 + 62

0 . 287 + 0 . 019 2605 + 283 204 + 22

1009 † 106 19 . 9 + 3 . 6 289 + 55

5126 + 1454 24 . 3 + 1 . 9

556 59 129 + 9 0 . 69 + 0 . 06 763 79 34 . 2 + 4 . 4 3 . 08 = 0 . 74 9 . 65 3 . 94 1 . 97 + 0 . 31 2657 293 327 = 45 753 + 110 4 . 29 + 0 . 48 9 . 11 + 1 . 54

22570 + 1575 536 + 60

0 . 395 + 0 . 079 1942 + 112 194 + 18 1025 + 161 12 . 5 + 1 . 1 337 + 15 5581 + 743 20 . 1 + 3 . 5

559 59 110 + 9

3 . 82 + 0 . 24 475 = 32 34 . 4 + 3 . 8 3 . 12 = 0 . 21 5 . 06 + 0 . 21 1 . 08 = 0 . 37 2402 + 332 279 + 50 558 + 73 1 . 72 + 0 . 44 7 . 84 1 . 85

20706 + 587 4 42 47 0 . 222 + 0 . 011 1994 166 338 27

1922 - 142 22 . 2 + 4 . 0 245 40

3866 – 859 22 . 5 + 1 . 5

585 64 118 + 6

2 . 85 + 0 . 78 * 941 + 49 * 37 . 0 + 5 . 9 2 . 85 + 0 . 43 5 . 58 + 0 . 67 1 . 61 + 0 . 24 3130 + 340 330 + 36 819 + 100 3 . 09 = 0 . 39

14 . 72 + 4 . 25 * 20302 + 253

535 = 45 0 . 321 + 0 . 094 * 1571 + 49 * 214 = 25 * 1285 – 149 *

6 . 5 = 1 . 0 * 348 - 52

1474 + 314 * 23 . 2 + 1 . 6

Lysine Valine Leucine Isoleucine Threonine Glycine Serine Alanine Glutamic Acid Glutamine Proline Aspartic Acid Aspargine Methionine Cysteine Phenylalanine Tyrosine Tryptophan Histidine Ornithine Lipid Metabolites

40 . 3 + 16 . 3 417 = 61 1798 + 301 1047 = 152 166 = 29 582 + 60 51 + 9

1037 + 94 104 + 18

2891 – 208 683 + 116 24 . 7 3 . 3

6 . 0 + 1 . 1 81 . 0 E 16 . 5 18 . 6 + 6 . 1 296 = 60 14 . 7 + 5 . 8 33 . 2 + 15 . 4 1 . 99 + 0 . 76 11 . 7 + 2 . 7

41 . 2 + 13 . 2 422 + 41 1749 + 154 1049 + 92 167 + 11 217 33 34 + 4

826 = 128 123 + 12 2470 = 318 776 + 104 29 . 0 + 3 . 6

8 . 4 = 3 . 4 65 . 9 + 8 . 2 27 . 0 + 15 . 1 283 = 30 14 . 3 + 4 . 0 33 . 2 = 12 . 6 2 . 32 = 0 . 96 10 . 0 + 2 . 0

54 . 9 31 . 6 412 + 92 1659 + 401 1002 = 247 141 + 25 486 + 74

39 = 5 989 + 179 92 + 14

1835 + 258 728 174 25 . 0 + 3 . 7

3 . 7 + 1 . 2 53 . 5 = 6 . 2 28 . 1 + 9 . 7 271 = 36 12 . 9 = 3 . 4 24 . 4 + 8 . 4 2 . 95 + 1 . 1 10 . 7 2 . 1

50 . 0 + 8 . 6 414 + 28 1761 134 997 123 211 + 14 * 171 + 18 * 36 + 5

1230 = 100 187 + 32 *

2783 + 86 * 1835 261 * 36 . 1 + 2 . 6 8 . 8 + 1 . 8

75 . 2 + 7 . 1 26 . 7 + 10 . 0 279 + 18 17 . 1 + 3 . 2 16 . 2 6 . 1 1 . 5 + 0 . 3

11 . 4 + 2 . 0

+

Phosphate Diphosphate Phosphoglycerol 3 - Phosphoglycerate 2 - Phosphoglycerate DHAP Inositol Myo - inositol Phosphate Lauric Acid Myristic Acid Palmitaladic Acid Palmitic Acid Linoelic Acid

14724 + 801 1968 + 402

11517 + 299 471 + 84 27 . 4 + 4 . 3 7 . 47 + 1 . 05 2964 266 345 + 70 37 . 5 + 3 . 0 87 . 8 + 10 . 3 73 . 3 + 17 . 0 6714 + 1091 667 + 143

15003 1007 5183 + 2133

10969 + 790 438 + 87 23 . 4 2 . 9 6 . 78 + 0 . 46 2030 + 119 465 + 109 37 . 2 + 3 . 6 73 . 5 + 5 . 5 40 . 4 + 6 . 9 7911 + 1181 759 + 159

13232 = 640 1636 + 161 9647 + 334 205 + 27 15 . 0 = 1 . 5 5 . 53 + 0 . 53 2283 175 258 13 32 . 6 + 1 . 5 87 . 1 + 8 . 7 89 . 3 = 23 . 5 6285 + 967 560 + 145

13590 + 270 2930 608 *

11729 395 204 + 24 * 13 . 0 + 1 . 3 * 5 . 87 + 0 . 60 1940 + 165 363 + 74 * 35 . 4 2 . 0 63 . 0 + 5 . 5 * 24 . 4 5 . 7 * 7838 + 1014 572 + 106

US 10 , 024 , 846 B2 23 24 TABLE 1 - continued

Metabolomic Analysis of Liver Tissue by GC - MS GC - MS data presented here are in arbitrary units and expressed as mean I sem

Metabolites shown with an asterisk on the right end of the line have interesting or significant trends with respect to genotype or dietary regimen or both .

WT - CNT WT - HFD KO - CNT KO - HFD

Oleic Acid Elaidic Acid Stearic Acid Arachidonic Acid 1 - Monooleoylglycerol 1 - monostearylglycerol 1 - Monopalmitoylglycerol 1 - Monopalmitoylylglycerol Cholesterol Nucleic Acid Metabolites

670 + 138 120 25

2662 462 56 . 0 10 . 6 368 + 85 1727 = 367 3808 + 741 26 . 1 + 6 . 0 1316 + 113

851 + 141 126 + 28

4471 + 682 62 . 5 + 15 . 4 498 + 2879 + 502 4606 880 23 . 6 + 6 . 7 1416 + 163

108 + 113

635 + 171 175 + 33

2206 + 344 46 . 4 + 2 . 1 377 + 129

1428 = 337 3645 + 736 36 . 3 + 10 . 8 1146 + 126

662 + 117 87 + 18 *

3892 = 559 * 54 . 8 + 6 . 8 475 + 53

2696 = 289 * 5011 609 16 . 5 = 2 . 6 * 1367 + 109

Xanthine Adenosine Inosine Adenine Uracil

1133 + 129 1 . 87 + 0 . 33 394 + 50 146 + 13 42 . 7 + 11 . 9

1183 † 167 1 . 82 + 0 . 31 295 + 50 153 54 . 3 + 9 . 0

972 + 83 2 . 45 + 0 . 26 276 + 30 127 + 4 21 . 6 + 2 . 8

940 + 150 2 . 16 + 0 . 24 217 + 52 * 167 + 17 *

292 . 8 + 98 12

Significant differences and interesting trends have been energy as heat , instead of converting into storage form of noted in several of these metabolites as shown with respect 25 energy . UCPs play a role in the development of obesity and to genotype ( WT vs . KO ) or the diet ( regular diet vs . high - fat type II diabetes mellitus . As shown in the right panel of FIG . diet ) or both variables . 14 , the basal expression levels of all three UCPs in the

Based on the data disclosed herein , it appears that the KO brown fat of KO mice are significantly higher as compared mice are resistant to high - fat diet - induced obesity . There are to the expression levels in WT mice . This advocates for the three possible explanations for this . The first possibility is a 30 conclusion that KO mice are inherently capable of burning decreased appetite leading to less consumption of food . A extra calories consumed in the food . second possibility is a lack of absorption of fat in the The enlarged fat cells in the white adipose tissue in digestive system . Finally , the third possibility is increased obesity recruit macrophages ( inflammatory cells ) and pro ability to burn more calories due to higher energy metabo - mote inflammation , leading to elaboration of various cytok lism . 35 ines ( inflammatory molecules ) and chemical mediators .

In the studies presented , the first two possibilities were Accordingly , the inflammatory mediators in the white fat of eliminated , i . e , the KO mice were not eating less food than the WT and KO mice were examined after feeding high - fat the WT mice , and the KO mice had well - formed stools with diet for 16 weeks . As shown in the left panel of FIG . 15 , the no evidence of steatorrhea . This leaves the third possibility , expression levels of IL - 6 ( interleukin - 6 , an inflammatory i . e . , increased ability to burn more calories or higher energy 40 cytokine ) , MCP - 1 ( monocyte chemoattractant protein - 1 or metabolism . To confirm this , the expression of key genes in CCL2 ) and its receptor ( CRR2 ) , CD68 ( monocyte / macro the WT and KO mice under basal or normal conditions , i . e . , phage cluster differentiation 68 protein ) , as well as F4 / 80 ( a without feeding high - fat diet , were examined . defining marker of murine macrophages ) were significantly

FIG . 14 shows the expression of two groups of key lower in the KO mice as compared to the WT mice . This molecules involved in energy metabolism in the brown fat , 45 suggests a lesser degree of inflammation in the white adi which is rich in mitochondria . These are the PPARs ( per - pose tissue of KO mice versus WT mice after feeding oxisome proliferator - activated receptor ) , the nuclear recep - high - fat diet . tors , involved in the regulation of inflammation and energy During the study , the expression of insulin receptor sub homeostasis and thus represent important targets for obesity strate ( IRS ) 1 and 2 was examined . These receptors transmit and metabolic syndrome . 50 signals from the insulin receptor and insulin - like growth PPAR - a knockout mice are known to gain more adipose factor - 1 ( IGF - 1 ) , and thus play an important role in meta

tissue mass as compared to the wild type mice . PPAR - Y b olic pathways . Mice lacking IRS1 develop a mild diabetic plays a role in adipocyte differentiation , and reduces inflam - phenotype , whereas the mice lacking IRS2 have a diabetic matory gene expression . Less is known about the role of phenotype . As shown in the right panel of FIG . 15 , the IRS1 PPAR - 8 in obesity , but it stimulates fatty acid oxidation . 55 expression was markedly higher in the KO mice fed high - fat As shown in the left panel of FIG . 14 , PPAR - y and diet versus WT mice fed high - fat diet , whereas the expres

PPAR - d expression levels in KO mice are significantly sion levels of IRS2 did not differ between these two geno higher as compared to the WT mice , while the expression of types . This finding suggests that IRS1 contributes to the PPAR - a did not differ . Thus , the observed resistance of the observed resistance of the KO mice to diet - induced obesity . KO mice to diet - induced obesity is partly related to the 60 differences in baseline expression of the PPAR isoforms in Example 3 : Disruption of P2Y2 Receptor Gene is these mice as compared to the WT mice . Protective Against Diet - Induced Obesity and

Another potential group of molecules considered were the Insulin Resistance mitochondrial uncoupling proteins ( UCPs ) . UCPs are the mogenic proteins and regulate energy expenditure . They 65 Development of insulin resistance ( IR ) in obesity is uncouple mitochondrial respiration for oxidative phospho - attributed to inflammation and lipotoxicity in white adipose rylation ( formation of ATP ) , resulting in dissipation of tissue ( WAT ) , among other factors . We observed that genetic

25 US 10 , 024 , 846 B2

26 deletion of P2Y 2 - R confers significant resistance to devel were significantly higher in HFD fed KO mice vs . WT mice , opment of high - fat diet ( HFD ) - induced obesity and consistent with greater insulin sensitivity of HFD fed KO . improves glucose tolerance . P2Y2 - R is expressed in all the FIG . 21 shows Relative mRNA expression of insulin key organs involved or potentially contributing to JR ( white receptor ( left panel ) or glucose transporter type 4 ( GLUT4 ) fat ( epididymal , EP ) , white fat ( subcutaneous , SC ) , brown 5 ( right panel ) in the white adipocytes of wild type ( WT ) or fat , liver , skeletal muscle , kidney , intestines , and pancreas ; P2Y2 receptor knockout ( KO ) mice after feeding control FIG . 16 ) . We investigated whether P2Y2 - R may facilitate ( CNT ) or high - fat ( HFD ) diets for 16 weeks . mRNA expres diet - induced IR , insulin signaling and inflammation in WAT sion was determined by real - time RT - PCR . As shown , of wild type ( WT ) and P2Y2 - R knockout ( KO ) mice fed HFD - fed KO mice have significantly higher levels of HFD . 10 expression of insulin receptor and GLUT4 compared to

Groups of age - matched adult WT and KO mice were fed HFD - fed WT mice . These may contribute to better glucose regular diet ( 10 % calories as fat ; n = 6 ) or HFD ( 60 % calories tolerance and higher insulin sensitivity in KO mice . as fat ; n = 14 ) with free access to food and water for 16 weeks On the contrary , the expression of pro - inflammatory mol and euthanized . An insulin sensitivity test ( IST ) was con - ecules IL - 6 , MCP - 1 , CCR2 , CD68 and F4 / 80 were signifi ducted . 15 cantly higher in WAT of HFD fed WT vs . KO mice . Our

Briefly for insulin sensitivity test , mice were fasted for 4 results demonstrate that KO of the P2Y 2 - R is protective hours with tree access to drinking water . Baseline ( 0 min ) against diet - induced obesity and IR . The mechanisms may blood samples ( 3 to 5 ul ) were collected by gently pricking involve higher expression of insulin receptor signaling pro the foot , and blood glucose was determined by the use of teins , as well as reduced inflammation and lipotoxicity in the Ascensia Contour Blood Glucose Meter ( Bayer Health - 20 WAT of the KO mice . Care ) . The mice were then intraperitoneally injected with human insulin ( Humalin® R - 100 , Eli Lilly ) at a dose of 1 Example 4 : P2Y2 Receptor Knockdown U / kg body weight . Blood glucose measurements were repeated at every 20 min interval for 2 hours , using alternate In addition to the use of agents such as a fusion protein , hind limbs . During the intervals between blood samplings 25 polypeptide , a small non - nucleic acid organic molecule , and the mice are kept warm in regular cages . a small inorganic molecule among other agents , that blunts

Following insulin sensitivity test ( IST ) , mice were eutha the activity of a P2Y2 receptor , the expression of P2Y2 nized . receptor may also be blunted through the use of nucleic acid WAT was collected and processed for the mRNA expres based agents , including an antisense oligonucleotide , a

sion of key molecules involved in inflammation and insulin 30 ribozyme or an inhibitory RNA , including but not limited to sensitivity . a small inhibitory RNA ( siRNA ) or a short hairpin RNA

The expression of P2Y 2 - R in WAT was increased by ( shRNA ) . For example , P2Y2 receptor expression may be 2 - fold in HFD - fed WT mice ( FIG . 17 ) . Morphology of blunted or knocked down in adipocytes as well as other adipocytes in the white fat in wild - type and P2Y2 receptor P2Y2 receptor - expressing cells of interest through delivery , knockout mice after feeding control ( CNT ) or high - fat 35 including targeted delivery , of P2Y2 receptor siRNA ( s ) or ( HFD ) diets for 16 weeks is shown in FIG . 18 . Feeding HFD alternatively P2Y2 receptor shRNA ( s ) . Such methods are to WT mice resulted in marked increase in the size and known in the art . For example , P2Y2 siRNAs and vectors / topology of adipocytes due to storage of excess fat . On the delivery system for expression of P2Y2 shRNAs may be other hand , the increase in size of the adipocytes of KO mice purchased from commercial vendors to knockdown P2Y2 is very low . Furthermore , the adipocytes in KO mice , 40 receptor expression ( Invitrogen , Carlsbad , Calif . , Santa Cruz whether fed control or HFD diet , had a tendency to accu - Biotechnology , Santa Cruz , Calif . ; OriGene Technologies , mulate low fat as evidenced by the collapsed balloon - like ” Rockville , Md . ( e . g . , catalog number : TR302717 , appearance vs . “ inflated balloon - like ” appearance in the WT T G302717 , TL302717 , TF302717 , SR303327 , TR501567 , mice . TG501567 , TF501567 , TL501567 , SR412590 , TR710051 ,

FIG . 19 shows morphology of adipocytes in the BROWN 45 TG710051 , TL710051 , TF710051 , and SR514520 ; see also , FAT in wild type ( WT ) or P2Y2 receptor knockout ( KO ) Li W - H , Qui Y , Zhang H - Q et al . 2013 . “ P2Y2 receptor mice after feeding control ( CNT ) or high - fat ( HFD ) diets for promotes cell invasion and metastasis in prostate cancer 16 weeks . As shown , feeding HFD to WT mice resulted in cells ” . British Journal of Cancer 109 : 1666 - 1675 ) . the accumulation of fat in the cells , as evidenced by Based on sequence for human P2Y2 receptor gene ( NCBI increased number and size of vacuolar structures . On the 50 Reference Sequence : NC _ 000011 . 10 for NCBI Gene ID other hand , the increase in size or number of the similar 5029 ) and mRNA transcripts ( NCBI Reference Sequence : vacuolar structures is strikingly low in the HFD fed KO NM _ 176072 . 2 , NM 176071 and NM 002564 ) , additional mice . siRNA and shRNA may be designed to knockdown the

Whole - body insulin sensitivity were significantly higher P2Y2 receptor gene expression , and hence reduce P2Y2 in HFD fed KO mice vs . WT mice . FIG . 20 shows whole 55 receptor activity in a cell . Other agents that may be designed body insulin sensitivity in wild type ( WT ) or P2Y2 receptor and used to knockdown P2Y2 receptor gene expression knockout ( KO ) mice after feeding control ( CNT ) or high - fat include anti - sense oligonucleotide , ribozyme , and miRNA ( HFD ) diets . The mice were challenged with a measured among others . In addition , based on P2Y2 receptor gene dose of insulin at O time point , and the fall in blood glucose sequences at publically accessible databases such as data was monitored every 20 min up to 2 hours . As shown , at 20 , 60 bases at the National Center for Biotechnology Information 40 and 60 min the HFD - fed KO mice showed significantly ( Bethesda , Md . ) , agents that knockdown gene expression higher insulin sensitivity as compared to the HFD - fed WT may be designed and used to target P2Y2 receptor gene mice . Furthermore , the mean basal levels of blood glucose expression in other mammalian species , including as mouse ( 0 min ) were also significantly lower in HFD - fed KO mice and rat . For example , GE Healthcare Dharmacon Inc . ( La vs . HFD - fed WT mice . 65 fayette , Colo . ) provides 63 siRNA products and 103 shRNA

Similarly , the mRNA expression of the insulin receptor , products as reagents directed to knockdown activity of GLUT4 and IRS - 1 ( insulin receptor substrate - 1 ) in WAT species specific P2Y2 receptor , such as human , mouse and

US 10 , 024 , 846 B2 27 28

rat ( e . g . , Catalog Number : E - 003688 - 00 - 0005 ; EU - 003688 11 . The method of claim 5 , wherein the isolated organic 00 - 0002 ; EQ - 003688 - 00 - 0002 ; E - 040991 - 00 - 0005 ; molecule has a molecule weight of less than 1 , 500 daltons . EU - 040991 - 00 - 0002 ; EQ - 040991 - 00 - 0002 ; E - 091224 - 00 - 12 . The method of claim 1 , wherein the agent comprises 0005 ; EU - 091224 - 00 - 0002 ; EQ - 091224 - 00 - 0002 ; a polypeptide . VGH5526 - EG5029 ; RHS5086 - EG5029 ; RHS4531 - 5 13 . The method of claim 12 , wherein the polypeptide is a EG5029 ; VGH5518 - 200220583 ; VGH5518 - 200224821 ; fusion protein , an antibody , a polyclonal antibody , a mono VGH5526 - EG18442 ; RHS4287 - EG18442 ; RMM4532 clonal antibody , an antigen - binding fragment of an antibody , EG18442 ; RHS5089 - EG18442 ; V3SM7677 - 01EG18422 a Fab antibody fragment , a F ( ab ' ) 2 antibody fragment , a Fv V3SM7671 - 231571660 ; V3SR7679 - 01EG29597 ; V3SR7673 - 238013458 ; V3SR7673 - 238450774 ) . Agents 10 antibody fragment , a single chain variable fragment ( scFv ) ,

or a bispecific antibody . that knockdown gene expression include anti - sense oligo 14 . The method of claim 13 , wherein the antibody , the nucleotide , ribozyme , siRNA , shRNA and miRNA among others . polyclonal antibody , the monoclonal antibody , the antigen

The above description of the disclosed embodiments is binding fragment of an antibody , the Fab antibody fragment , provided to enable any person skilled in the art to make or 15 the F ( ab ' ) , antibody fragment , the Fv antibody fragment , the use the invention . Various modifications to these embodi single chain variable fragment ( scFv ) , or the bispecific

antibody inhibits the activity of P2Y2 receptor . ments will be readily apparent to those skilled in the art , and the generic principles described herein can be applied to 15 . The method of claim 1 , wherein the agent comprises other embodiments without departing from the spirit or a nucleic acid . scope of the invention . Thus , it is to be understood that the 20 16 . The method of claim 1 , wherein the agent comprises description and drawings presented herein represent a pres a system for targeted disruption or knockdown of the P2Y2 ently preferred embodiment of the invention and are there receptor gene in a living cell . fore representative of the subject matter which is broadly 17 . The method of claim 16 , wherein the system com contemplated by the present invention . It is further under prises a delivery system or expression system for antisense stood that the scope of the present invention fully encom - 25 0 om . 25 oligonucleotide , ribozyme or an inhibitory RNA . passes other embodiments that may become obvious to those 18 . The method of claim 17 , wherein the inhibitory RNA skilled in the art and that the scope of the present invention is selected from the group consisting of an siRNA , shRNA is accordingly not limited . and miRNA . What is claimed is : 19 . The method of claim 1 , wherein the agent increases 1 . A method for treating diet - induced obesity in an obese 30 expression or activity of one or more peroxisome prolifera

subject or a subject at risk of diet - induced obesity compris tor - activated receptor ( PPAR ) genes in brown fat . ing administering an agent in an effective amount so that 20 . The method of claim 19 , wherein the PPAR gene is expression or activity of the P2Y2 purinergic receptor is selected from the group consisting of PPAR - O and PPAR - y .

21 . The method of claim 19 , wherein the PPAR gene is decreased or inhibited in the subject , thereby treating diet induced obesity in the obese subject or the subject at risk of 35 26 PPAR - y . diet - induced obesity . 22 . The method of claim 1 , wherein the agent increases

2 . The method of claim 1 , wherein the agent comprises a expression or activity of one or more uncoupling protein P2Y2 receptor antagonist . ( UCP ) genes in brown fat .

23 . The method of claim 22 , wherein the UCP gene is 3 . The method of claim 2 , wherein the P2Y2 receptor antagonist is a non - selective or slightly selective P2Y2 40 selected from the group consisting of UCP - 1 , UCP - 2 and

UCP - 3 . receptor antagonist . 4 . The method of claim 2 , wherein the P2Y2 receptor 24 . The method of claim 22 , wherein the UCP gene is

UCP - 1 . antagonist is a selective or specific P2Y2 receptor antago nist . 25 . The method of claim 1 , wherein the agent decreases

5 . The method of claim 3 , wherein the non - selective or 45 ex ! ve or as expression or activity of pro - inflammatory genes in white slightly selective P2Y2 receptor antagonist is an isolated adipose tissue . organic molecule or isolated inorganic molecule . 26 . The method of claim 25 , wherein the pro - inflamma

6 . The method of claim 5 , wherein the isolated organic tory genes are selected from the group consisting of inter molecule is an isolated organic molecule from a plant or an leukin - 6 ( IL - 6 ) , monocyte chemoattractant protein - 1 isolated flavonoid . ( MCP - 1 or CCL - 2 ) , monocyte chemoattractant protein - 1

7 . The method of claim 6 , wherein the isolated organic receptor ( CRR2 ) , monocyte / macrophage cluster differentia molecule from a plant or an isolated flavonoid is strobopinin , tion 68 protein ( CD68 ) , a defining marker of murine mac strobopinin - 7 - methyl ether , 2 ' - B - Dihydroxy - 4 ' , 6 ' - dime rophage F4 / 80 and combination thereof . thoxy - 3 ' - methylchalcone ( or Oxo - Aurentiacin ) , kaempferol , 27 . The method of claim 1 , wherein the agent increases heptamethoxylflavone , or tangeretin . se expression or activity of insulin receptor substrate - 1 ( IRS1 )

8 . The method of claim 5 , wherein the isolated organic in white fat . 28 . The method of claim 1 , wherein the agent increases or molecule is an isolated organic molecule produced by syn

thetic chemistry , biochemistry and / or an enzymatic method . maintains expression or activity of insulin receptor , glucose 9 . The method of claim 8 , wherein the enzymatic method transporter type 4 ( GLUT4 ) or a combination thereof .

is performed in vitro or in a genetically engineered micro - 60 29 . A method for increasing energy metabolism in an organism . adipocyte cell in a subject that is at risk for or suffering from

10 . The method of claim 3 , wherein the non - selective or a disorder related to glucose metabolism , the method com slightly specific P2Y2 receptor antagonist is AR - C126313 , prising administering to the subject an agent that inhibits or AR - C118925 ( 5 - [ [ 5 - { 2 , 8 - dimethyl - 5H - dibenzo [ a , d ] cy expression or activity of a P2Y2 receptor , thereby increasing clohepten - 5 - yl } - 3 , 4 - dihydro - 2 - oxo - 4 - thioxo - 1 ( 2H ) - pyrim - 65 " e as the metabolism in the cell . idinyl ] methyl ] - N - [ 1H - tetrazol - 5 - yl ] - 2 - furancarboxamide ) . * * * * *

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