US 20090220973A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0220973 A1 Gesta et al. (43) Pub. Date: Sep. 3, 2009

(54) OBESITY AND BODY FAT DISTRIBUTION (86). PCT No.: PCT/US2007/065853 (75) Inventors: Stephane Gesta, Boston, MA (US); S 371 (c)(1), C. Ronald Kahn, West Newton, (2), (4) Date: Mar. 13, 2009 MA (US) Related U.S. Application Data (60) Provisional application No. 60/788,955, filed on Apr. Correspondence Address: 3, 2006, provisional application No. 60/790,422, filed FSH & RICHARDSON PC on Apr. 7, 2006. P.O. BOX 1022 O O MINNEAPOLIS, MN 55440-1022 (US) Publication Classification (51) Int. Cl. (73) Assignee: JOSLIN DIABETES CENTER, CI2O I/68 (2006.01) INC., Boston, MA (US) (52) U.S. Cl...... 435/6 (57) ABSTRACT (21) Appl. No.: 12/295,710 Described are methods for predicting and diagnosing geneti cally-based obesity and body fat distribution, and for identi (22) PCT Filed: Apr. 3, 2007 fying compounds for the treatment and prevention of obesity. Hypothetical Scheme of Adipocyte Development

FGC Embryonic RF-4 Stem Cell els PPAR Developmental & cEEPs BEOW Patterning Brown Adipocytes C WisceralType Preadipocytes -1 PPARYCEEPs Subcutaneous N O Type Mesenchymal 1. Retroperitoneal Stem Cels N Developmental & Type Patterning Genes

common White E. 2. Perigonadal Preadipocyte Type Specialized White Specialized White Preadipocytes Adipocytes Patent Application Publication Sep. 3, 2009 Sheet 1 of 7 US 2009/0220973 A1

Epididymal Subcutaneous adipose tissue adipose tissue

Epididymal isolated adipocytes Subcutaneous A. isolated adipocytes (A) RNA extraction Epididymal stroma Y-N Subcutaeous stroma Wascular fractor Wascular fraction (S) (S) expression comparison by Affymetrix Arrays and q-RT-PCR

FIG. 1B U74Aw2 - 12,488 Total Probesets 8,017 Annotated = 674 Genes

SESSC AEASC SO9 770 at p-value < 0.05)

198 Developmental or Patterning Genes Patent Application Publication Sep. 3, 2009 Sheet 2 of 7 US 2009/0220973 A1

FG. 2B FIG. 2C

18 SO 15 1. 20 35 Tox15 3O) 2 SO 25) 2O) 50 00 20

120 3. O 35 3. O) Shox2 25 SO 5 46 ts 2) 1)

s 120 E 1. 2 :

0. S. B 6 Sfro2 St.

3. 2 tal 35. 3. SO HOXC9 s roc 50 OO 5 talEpi S. Epi S.C. Epi ISC Whsie Fatad Adipo After Culture Patent Application Publication Sep. 3, 2009 Sheet 3 of 7 US 2009/0220973 A1

90 ES 8 t 2 SE 250 5 N2f 20 (coup-TFE) . 150 3.

s 35 S. d 3 5 250 O r 3. Gpc4 r : : O 150 a 1.

1 B S. S

2 Thbd 10st 8 E.

s

s SO 300 s 3. HoxA5 25 I s: 5 2 20

35 ESO 300 Fo s 250 HoxC8 r 200 1st 3. : 40 5. Epi SC Episc Episc Ep SC YWhote Fat paid Aips SWF At cle Patent Application Publication Sep. 3, 2009 Sheet 4 of 7 US 2009/0220973 A1

Shox2 . N2 GOO 16 1400 1400 s 1D 12 g OOOO FIG. 4F FIG 4A 25 BOOD e EO 5 SO O

30000 Er g 25000 20000 FG. 4G FG. 4B S 15000 OOE) 5000 2 f

HxC9 SO HxA5 5 F.G. 4C S 4. FIG. 4H 3)

BO Sfro2 2500 HoxC8 SO 40 OD FG. 4D 100 SOD FIG. 4 O O 3. t : 3. : s 2 20 O O Tx15 GpcA. s 14 AEO O FIG. 4E O) F.G. 4

8 5

: O is Sc is Sc W. S. Wis Sk Male Fetake Male Female Patent Application Publication Sep. 3, 2009 Sheet 5 of 7 US 2009/0220973 A1

Subcutaneous

35 300 25 OHO SO OO

BM BM Patent Application Publication Sep. 3, 2009 Sheet 6 of 7 US 2009/0220973 A1

F.G. 5B

Wiscera Subcutaneous

5 2500 35 E. 30 5 2000 50 1500 O s 100 5

5 90 1. SO

18O 3DO 15 sr 5. EO

g O 5 8 s 60 T 50

5 0.50 C 1.1 130 SO

Patent Application Publication Sep. 3, 2009 Sheet 7 of 7 US 2009/0220973 A1

Hypothetical Scheme of Adipocyte Development PGC Embryonic RF-4 Stem Cell el Developmental & BFOW Patterning Genes Brown Adipocytes C WisceralType

Vic. -1PPAR Subcutaneous S CEBPs TWe /32 NS s Na O y

Stemis Cels N. Developmental1. & RetroperitonealType

Patterning Genes

Common White Preadipocyte 2.E. Perie.g. dal Specialized White Specialized White PreadEpocytes Adipocytes FIG. 6 US 2009/0220973 A1 Sep. 3, 2009

OBESITY AND BODY FAT DISTRIBUTION is characterized by a marked loss of Subcutaneous adipose tissue in the extremities and trunk, without loss of visceral, FEDERALLY SPONSORED RESEARCHOR neck or facial adipose tissue (Garget al., (1999) J Clin Endo DEVELOPMENT crinol Metab 84, 1704; Shackleton et al., (2000) Nat Genet 0001. This invention was made with Government support 24, 153-6). Some lipodystrophies even appear to have a seg under Grant Nos. ROI DK33201, DK60837, and mental ordermatomal distribution (Shelley and Izumi, (1970) K08DK064906, awarded by the National Institutes of Health. Arch Dermatol 102,326-9). The U.S. Government has certain rights in the invention. SUMMARY TECHNICAL FIELD 0006. At least in pare the present invention is based on the discovery of major differences in expression of multiple 0002 This invention relates to methods of predicting obe genes involved in embryonic development and pattern speci sity and body fat distribution, and methods of identifying fication between adipocytes taken from intra-abdominal and compounds for the treatment of obesity or the manipulation Subcutaneous depots in rodents and humans. Similar differ of body fat distribution. ences were also present in the Stromovascular fraction con taining preadipocytes and that these differences persist in BACKGROUND culture. Some of these developmental genes exhibit changes 0003) Obesity is an epidemic health problem worldwide in expression that are closely correlated with level of obesity that impacts on the risk and prognosis of many diseases, and the pattern of fat distribution. including diabetes, cardiovascular disease, hyperlipidemia, 0007. In one aspect, the invention provides methods for and cancer (Lean, (2000) ProcNutr Soc 59,331-6). However, diagnosing present obesity, e.g., high body mass index not all obese patients have the same risk of developing these (BMI), or of predicting future obesity or undesirable adipose disorders. Individuals with peripheral obesity, i.e., fit distrib tissue distribution, e.g., high waist-hip ratio (WHR), in a uted Subcutaneously in the gluteofemoral region, are at little Subject, e.g., a human. The methods include providing a or no risk of the common medical complications of obesity, sample comprising a tissue or cell, e.g., an adipose tissue or whereas individuals with central obesity, i.e., fat accumulated cell, from the subject; and evaluating the level of mRNA in the in visceral depots, are prone to these complications (Maur cell for one, two, three, four or more of the genes listed in iege et al., (1993) EurJClin Invest 23,729-40; Gillum, (1987) Table 1, e.g., one or more of Tbx 15, ShoX2, En1, Sfrp2. J Chronic Dis 40, 421-8; Kissebah and Krakower, (1994) HoxC9, Nr.2f1, Gpc4, Thbd, HoxA5 or HfoxC8, or a level of Physiol Rev. 74, 761-811; and Abate and Garg, (1995) Prog a encoded thereby. The level of expression, e.g., as Lipid Res 34, 53-70). compared to a predetermined reference level (e.g., as 0004 While differentiation of adipocytes has been exten described herein), indicates whether the subject has, or is at sively characterized (Gregoire, (2001) Exp Biol Med (May risk of developing, obesity or undesirable adipose tissue dis wood) 226,997-1002: Koutnikova and Auwerx, (2001) Ann tribution. Med 33, 556-61; and Tong and Hotamisligil, (2001) Rev 0008. In some embodiments, the methods include deter Endocr Metab Disord 2,349-55) and there have been consid mining a level of expression of at least one mRNA for a gene erable recent insights into the control of appetite and energy selected from the group consisting of Hox57, Gpc4 and expenditure as contributing factors to obesity (Wynne et al., Tbx15 inhuman adipose tissue, or a level of a protein encoded (2005) J Endocrinol 184, 291-318: Ricquier, (2005) Proc thereby, and comparing the levels to a reference, e.g., a ref Nutr Soc 64, 47-52), little is known about the genetic basis for erence that represents a Subject with a selected BMI, e.g., a determination of adipocyte number, differences in body fat normal or near normal BMI. In some embodiments, the meth distribution or their association with metabolic disorders. ods include measuring levels for one or both of Tbx15 in Twin and population studies have revealed that both body visceral fat and Gpc4 in Subcutaneous fat. mass index (BMI) and waist-hip ratio (WHR) are heritable 0009. In some embodiments, the relationship of the levels traits, with genetics accounting for 25-70% of the observed for the mRNA or protein in the human subject and the refer variability (Nelson et al., (2000) Twin Res 3, 43-50; and ence indicates whether the subject has or will develop an Baker et al., (2005) Diabetes 54, 2492-6). In addition, it is unhealthy BMI. The level of the mRNA or protein is used to known that some obese individuals, especially those with select or exclude a Subject for participation in a clinical trial. early onset obesity, have increased numbers of adipocytes, 0010. In some embodiments, the subject is given a treat but how these are distributed and why this occurs is unknown ment or preventive measure for obesity, and the level of the (Hirsch and Batchelor, (1976) Clin Endocrinol Metab 5, 299 mRNA or protein is correlated with the subject's response to 311). Anecdotally, it is also clear that individual humans the treatment or preventive measure for obesity. For example, observe differences in their own body fat distribution as they the level of the protein or mRNA can be determined before, gain or lose weight. during and/or after the treatment, and a change in the level of 0005. The uneven distribution of adipose tissue is extreme the protein or mRNA indicates whether the subject is in Some ethic groups, such as Hottentot women, who have responding or has responded to the treatment. been noted for excessive accumulation offat in the buttocks, 0011. In another aspect, the invention provides methods a condition known as Steatopygia (Ersek et al., (1994) Aes for determining a ratio of intra-abdominal (visceral) accumu thetic Plast Surg 18, 279-82). Striking differences in adipose lation offat versus Subcutaneous (peripheral) fat in a Subject. tissue distribution can also be observed in individuals with The methods include providing a first sample from the subject partial lipodystrophy (Garg and Misra, (2004) Endocrinol comprising visceral adipose cells or tissue; providing a sec Metab Clin North Am 33, 305-31), both in its acquired and ond sample from the Subject comprising peripheral adipose inherited forms. For example, familial partial lipodystrophy cells or tissue; quantifying a level of mRNA in the first and of the Dunnigan type due to mutations in the Lamin A/C gene second samples for one, two, three, four or more of the genes US 2009/0220973 A1 Sep. 3, 2009

listed in Table 1, e.g., one or more of Tbx15, ShoX2, En1. possible carryover of residual total RNA, was prepared and Sfrp2, HoxC9, Nr.2f1, Gpc4, Thbd, HoxA5 or HoxC8, or a hybridized to mouse Affymetrix U74AV2 chips. level of a protein encoded thereby; and determining a ratio of 0017 FIG. 1B is a diagram illustrating some of the results the level of mRNA or protein in the first sample to the level of described herein. Among the 12,488 probesets present on the mRNA in the second sample. The ratio of the level of mRNA U74AV2 chip, 8,017 probesets representing 6174 are anno or protein in the first sample to the level of mRNA in the tated for Biological Process. Significant second sample indicates the ratio of visceral accumulation of genes with differential expression in both depots were iden fat versus peripheral fat in the subject. These methods can tified by selecting genes that passed two independent filters of also be used to predict future undesirable distribution of significance (p-value Student's t-test <0.05 and pFDR <0.05) weight. (see Methods). The first filter p-value Student's t-test <0.05) 0012. In a her aspect, the invention provides methods for selected 1,276 genes differentially expressed in the stro identifying a candidate compound, e.g., for the treatment of movascular fraction, 537 genes differentially expressed in obesity. The methods include providing a sample comprising isolated adipocytes and 233 genes differentially expressed in an adipose cell or tissue expressing one, two, three, four or both cell fractions. Of these 233 genes, 197 genes passed the more of the genes listed in Table 1, e.g., one or more of Tbx15, second filter of significance (PFDR <0.05) and were assessed Shox2, En1, Sfrp2, HoxC9, Nr.2f1, Gpc4, Thbd, HoxA5 or against an a priori set of 198 annotated genes involved in HoXC8; contacting the cell or tissue with a test compound, embryonic development and pattern specification (see Meth e.g., a Small organic or inorganic molecule, an inhibitory or ods). Twelve genes from this set were found among the dif stimulatory nucleic acid, or a polypeptide; and evaluating the ferentially expressed genes. expressing of the one, two, three, four or more of the genes 0018 FIGS. 2A-C are bar graphs illustrating the results of listed in Table 1, e.g., one or more of Tbx15, ShoX2, En1. a comparison of Tbx 15, ShoX2, En1, Sfrp2 and HoxC9 gene Sfrp2, HoxC9, Nr.2f1, Gpc4, Thbd, HoxA5 or HoxC8, in the expression between intra-abdominal (Epi; opened bars) and cell. A test compound that appropriately-modulates the subcutaneous (SC; closed bars) adipose tissue of C57316 expression of the gene or genes is a candidate compound for mice performed using real time PCR. These genes had a the treatment of obesity. higher level of expression in Subcutaneous in whole adipose 0013 Further, the invention provides additional methods tissue (2A) (Epi versus Sc: * p-value <0.05), isolated adipo for identifying a candidate compound, e.g., for the treatment cytes and stromovascular fraction (2B) (Epi versus Sc: * of obesity. The methods include providing a sample compris p-value <0.05). These differences of expression were main ing one, two, three, four or more expressed by a gene tained when stromovascular fraction taken from intra-ab listed in Table 1, e.g., one or more of Tbx15, ShoX2, En1. dominal (epididymal) or Subcutaneous adipose were placed Sfrp2, HoxC9, Nr.2f1, Gpc4, Thbd, HoxA5 or HoxC8, or a in culture in a defined serum free medium and Subjected to in cell or tissue expressing the proteins; contacting the sample vitro differentiation (2C) suggesting these differences are with a test compound, e.g., a small organic or inorganic mol independent of extrinsic factors (Epi versus Sc: * p-value ecule, an inhibitory or stimulatory nucleic acid, or a polypep <0.05) tide; and evaluating the level or activity of the protein in the (0019 FIGS. 3A-3C are bar graphs illustrating the results sample. A test compound that appropriately modulates, e.g., of a comparison of Nr.2f1, Gpc4, Thbd, HoxA5 and HoxC8 increases or decreases, the level or activity of the protein is a between intra-abdominal (Epi; opened bars) candidate compound for the treatment of obesity. and subcutaneous (SC; closed bars) adipose tissue of C57B16 0014. Unless otherwise defined, all technical and scien mice performed using real time PCR. These genes had a tific terms used herein have the same meaning as commonly higher level of expression in intra-abdominal (epidydimal) understood by one of ordinary skill in the art to which this whole adipose tissue (3A) (Epi versus Sc; * p-value <0.05), invention belongs. Exemplary methods and materials are isolated adipocytes and stromovascular fraction (3B) (Bpi described herein for use in the present invention; other, suit versus Sc: * p-value <0.05). These differences of expression able methods and materials known in the art can also be used. were maintained when stromovascular fraction taken from The materials, methods, and examples are illustrative only intra-abdominal (epididymal) or Subcutaneous adipose were and not intended to