Genetics of obesity: advances from rodent studies

C O M M E N T

Genetics of obesity: advances from rodent studies YVON C. CHAGNON AND CLAUDEBOUCHARD [email protected] [email protected] Ph%tC~LAown'~"SCIENC~LABORATORY, LA'¢ALUNqVERSITY,STE-FOY,QHMEC,CANADAG1K 7P4. Maior advances have occurred over the past four years, but particularly in the past 18 months, concerning the genetic hasis of obesity in rodent models. The cloning of the five genes in which mutations are known to t.'ause obesity in mouse and rat, and the identification of the gene products of all but one of these genes, has fostered a series of innovative studies in human subjecLs. Similarly, the uncovering of se~'e:ral quantitative trait loci (QTL) infloencing body mass, body fat content or fat topography frortl cros.ses nf informative strains of mice and rats has provided a new

impetus to the study of polygenic obesity in rodents and humans. These recent advances have generated a great deal of optimism regarding the possibility of identil~ing the genes responsible for the snsceptibility to the common forms of obesity in humarls and their co-morbidities, such a~ ~ardiovascular diseases, insulin resistance and non-insuUn-dependent diabetes mellims, hypertension and others. This article addresses the major features of the advances in single-gene-mutation rodent models, QTL in mouse and rat, and their impact on human obesity research,

Slngle-gene-mmation models The single-gene-mutation rodent models of obesity are identified and some of their genetic characteristics are summarized in Table 1. The obese (oh) and diabetes (db) mutations have an almost identical phenotype when bred on the same genetic background: juvenile onset of severe obesity adsing from an excess fat in all depots, excess food intake or hyperphagia, insulin resistance, g|ucose intolerance and diabetes. A similar phenotype is observed in the Agoutiyellow(A v) mice, except for an adult onset of a more moderare

TAI~ 1. Loci a n d omrt~polldlng genes I z p o r t e d for mouse alml rat melldellan o r polygenic models o f obesity" Chn~n~ome b

Mouse QTL AKR × SWR Mouse dfabetes/ratfatty-corpulent Rat QTL GK × BN Mouse QTL AKR X SWR Rat QI'L GK × BN Mouse Jin Mouse QTL BSB Mouse Q'IZ BSB Mouse obese Rat QTL GK × BN Mouse QTL AKR X SWR Rat QTL GK × F Mouse tubby Mouse QTL BSB Rat QTL GK X BN Mouse adu/t Rat Q'rL GK × F Mouse QTL BSB

Rat QTL GK × F Mouse Agoutiyellow Mouse QTL NZB Mouse QTL ASB, HSB

~

~

Do/

NA Recessive NA NA NA Recessive HA NA Recessive NA NA Recessive Recessive NA NA Dominant Dominam NA Recessive Dominant NA HA

db/fa-cp Ntdd/gk6 /)o2 N/daYgk..5

fat Mob4

Mob2 ob bw/gkl /3o3

Niddml tub Mobl Nfdd/gkl ad

Welghtl Mob3 Nlddm3 A t" Mob5

BWI3

Mouse/]~

4 4/5 17 9 8 8 15 6 6 7 15 1 7 7 l 7 7 12 10 2 2 X

Huma~

Cmdidsm j~s

lp35-p32 lp31-p21 lq41-q44 3p22-q2.! 3p 4q32 5p15-p12 7q22-q36 7q51,3 8q 8q23-q24,13 t0q24-q26 llp15.1 llpter-pl4 I lp NA 12q22-q2~ 14qt3-q32 17pter-q22 20q13 20pll-q13 X

Glutl

d

OB-R NA NA /)o2 Cpe

Gbr Ob OB NA NA NA

~ , lgflr, AaJ3 /gJ/L, Ins, SUR NA

TshP NA ASP

& Ada~Mc3r NA

=Adapted from Refs 7,16,18-20, 21 and 25. bGenes are presented by human chromosomal location and putative identity. cO/tological locations in human are proposed for some QTL according to sy'ateny of flanking markers between ~ oc rat and human. d Human (uppercase) or rodent (lowercase) candidate genes as proposed from original publicatitms. Abbreviatluns: Ada, adenosine deaminase; Ath3~ susceptibility locus for athatcsclerosis; Gbr, growth hormone r ~ , Glutl, glucose transporter 1; /.grit, insulin-like growth factor 1 receptor;/gf/L insulin ~t,ov~h factor II; /.gf/, ~.~Min growth factor 1; lng insulin; SUR,sulfonylurea receptor;, NA, not available;, see text for other abbreviations. TIG NOVEMBER1996 VOL, 12 NO, 11 C~'~ht $ l ~ EIg%iffScit*g~I~d Allrl.gltt~~'~ffl~l.()ffkq9525,Wx~$15 iX) p[I:$0168-952S(90~010~-0

441

C O M M E N T

obesity. The fatty (fat) and tubby (rob) mutations both show moderate maturity-onset obesiW with an axial/ inguinal fat deposition panem, but without hyperphagia and without insulin resistance for the fatmutation.

gene s, followed one year later by the cloning of the diabetes (db) gnne 6. They had been shown eadier to be dilliarent mutations by pambiosis experiments in which the circulatory systems of two animals, one from each strain, were connected to each Agouti other. In these experiments, it was The first gene cloned, Agoutt shown that a circulating factor from a (A locus), was olxained by probing normal or from a db mouse could a mouse eDNA or genomic library restore normal weight in an obmouse, with a DNA from a radiation-induced whereas when this factor came from inveeaion mutation mapping to the a normal or from an ob mouse, it A locus t. The human homolog of failed to do so in a db mouse7, These Agouti was cloned using the mouse early resnhs strongly suggested that eDNA and renamed agouti signaling both genes are in the same metabolic protein or ASP (Red 2). The mouse pathway and that db could be the Agouti mRNA was shown to be ex- receptor of ob (ReL 7). A positional pressed exclusively in the skin of neo- cloning strategy led to the isolation natal mice l, whetr~ it showed a more of ob in the mouse, whereas to done general expression in humans z. In the human gene (OB) an adipocyte mouse, more than 25 mutations are eDNA library was probed with the known in Agouti, five of which are mouse eDNA (Ref. 5). dominant mutations caused by an inTwo different mouse mutations sertion of an intracistemal A-particle were observed in oh, one in each of (lAP) element (AtY, AbO', A~;V,A~aPY the two obmouse strains analyzed: a alleles), or by a deletion of genomic nonsense mutation resulting in the DNA in the proximal gene Raly (A~' ah~nce of the ob protein and the allele), which puts Agnuti under the absence of transcript probably due to control of heterologous promoters an alteration in the promoter of the resulting in its ectoplc and ubiquitous ob gene s. [n human obese subjects, overexpressinrt3. This ectopic expres- mutations in OB have not been sion of Agouti produtvs the obesity observed, but an increase in the OB phenotype and this is recapitulated in protein, named leptin, was observed transgenic mice by the expression of in mast, hut not all, obese persons, the normal Agouti cDNA under the which suggests a reduced sensitivity control of a ubiquitous promoter 4. In to leptin in some obesity cases 8, Lephumans, the expression of ASPin all tin was shown to be a secreted tissues precludes a mechanism simi- adipocyte-specific satiety factor actlar to the mouse in the etiology of ing at the hypothalamic level "~ith a obesity. Up to now, no mutation in strong anorexic effect resuhing in a ASP related to obesity has been decreased fo~l tvansumption, but found. Furthermore, the ASP gene also an increased energy expemlihas been located to human 20q11.2, rare when injected to obe.~ mite and bur no significant linkage or associ- rots peripherally or centrallyg. ation has been reported yet between Tarmglia et al.6 used an elegant the ASPIocus and obesity. functional approach to clone the db it has been hypothesized that the gene. They produced a eDNA exeffect of Agouticould be mediated via presslon library from me hypothalathe regulation of intrat'ellular Ca2÷lev- n'tic cortex. The libmty was mmsd s in skdetal muscle, whereby an in- fected into COS cells and the cells crease in Caz* would lead to insulin were probed with the leptin protein. resistance and hyperinsulinemia, or a From the strongest positive COS depressed basal lipolytic rate in adipo- cells, eDNA clones were purified and cyies resulting from altered intmcellumapped on radiation-hybrid cell lar cAMP levels, or by a decreased panels. One of the clones located at adrenergic tone from antagonizing the same cytological location as the melanocotlth receptors in the brain, dbgene and was indeed shown to Ire which might trigger hyperphagia and the db gene I°. The db protein is a increased efficiency of foed utilizatinn4. single membrane-spanning receptor showing many features of the class 1 Obese and diabetes cytokine receptor family 6, Long and A major breakthrough was the short forms of the protein are cloning in late 1994 of the obese(oh) detected with at least six alternatively "FIG NOVEMBER1996 VOL. 12 NO. 11 442

spliced transcripts t0. In db mice, a G to T tons'version results in file replacement of the functional long form by the sbort form, whicl~ probably prevents the signal of satiety from the leptin to be received TM.The mt obesi W mutants fatty (fa) and corpulent (cp) have been shown m be allefic mutants originating, respectively, from the Zucker (13M) and the JCR:LA strains. The f a mutation had been shown to be a putative homolog to the mouse db by genetic mapping u . The cloning of the ob and db genes has provided exciting new opportunities to study the physiological and behavioral mechanisms associated with energy balance and its disregulation as well as to identify the molecular basis of food intake, energy expondimre and the regulation of body mass. An enormous amount of research has been reported on mice, rats and humans since the recent cloning of the oband dbgenes with more than 40 papers published.

FarO' The mouse fat(v (far) gene was identified by positional candidate gene determination. The carboxFpcpt[dase E (cpe) gene, which was shown to have the same cytological and physical location as fat, is involved in the maturation of the proinsulin hormone and was a masonahle candidate gene for fat (heft 12). A direct search for mutations in this gene identilied a Ser202 to Pro mutation, which suppressed ahnost all the activity of tpe in pancreas and pituinrty of mutant ,fat mice 12. A.s a result, proinsulin, which has a lowered capacity to trigger a physiological ~'esponae, is poorly processed to the active form of insulin, thus, producing a hyperproinsulinemia but without insulin resistance, as shown by the normal regulation of gluct~se when porcine insulin is injected into fat mice. Obesity in fat mice could come from this anomaly or might involve defects in the processing of other prohomlofles 12. In humans, it remains to be shown if a similar mutation exists,

Tabby The last of the murine obesiW genes cloned, tubby (tub), was obtained lollowing a joint venture between The Jackson Laboratory and Sequana Therapeutics t:¢, and by the

C O M M E N T

Millermium group in, The tub gene was found by positional doning. Both groups reportgd a G to T transversion in a splice donor site in tub mutant mice, which resulted in a longer transcript without the last exon but including the last unspliced intron. [t was inferred that the truncated abnormal protein produced impaired biochemical pathways controlling appetite and processing of food 13.14.

the F l generation interbred or backcrossed to the parental strains. Three different QTL (Dietary obese 1-3; I9ol-3) have been reported in this model so far, all related to mtsl adiposity ts. In a more recent study, three loci (Body weigbt 1-3; Bw l-3) linked to body weight, and all located on the X Chromosome, were also detected by QTL analysis of HSB and ASB strains, which were obtained by genetic crosses between C57BL/6J Adult males and F t females t?om the cr~ses Finally, a less well characterized of female C3H/He and female A/J adult (ad) mouse mutation, some- with male Mus sprett~ respectively 19. times imsclassified as adipose, which Finally, the latest reported QTL is allelic to db (dbUdallele), shows a for body mass and/or adiposity were similar phenotype to fat and tub, found in crosses involving the nonexcept that the increase in adiposity insulin-dependent diabetes mellitus Goto-Kakizaki (GK) rat strain. In a occurs in all fat depots in contrast to the axial/inguinal fat deposition cross between GK and tile nonpattern seen in fat and tub The a d diabetic Fisdler-344 (F) strain, seven mouse strain is apparendy nov,' ex- independendy segregating loci tinct and the cloning of the a d gene (A~21d/gkl-6 bw/gkl) were detected has nol been achieved. from which one (bu/gkl) exhibited a linkage exclusively with adiposity Polygenic models index and body weight whereas There is a lxr.ly of data supporting three other QTL (NideFgkl, -5, -6) tile concept that the collmlon totals influenced insulin secretion and of human obesity have a muhigenic glucose regulation also 2°. In a differorigin15. This observation has pro- ent cross between GK and the nonvided the necessary impetus for the diabetic Brown-Norway (BN) strain, development of polygenic mouse a a QTL related to body w~ip,ht nd rat models (Table 1). The sponta- (Weigbtl) and two QTL related to neously obese BSB strain was devel- Ixx:ly weight and to glucose and oped from the backcro~s of males insulin control (A'iddml, "3) were Mtts musctthrs dmnesticus C57BL/6J detected 21, As shown in Table 1, strain and the F 1fen'tales from a cross some of the QTL are chameterized by between C57BI./6J and Mus spretus. a similar cytological location as that Four loci (Multigenic obesity 1-4; of a single-gene mutation model or Mobl-4) located on four different of other QTL The different loci listed chromosomes have been identified, in Table 1 covering a wide range of so far, by QTL analysis. Each of these rodent chromosomes strongly sugloci differs with respect to its effects gest that the regulation of body mass on percentage body fat, specific fat and body fat content is in2uenced by depots and plasma lipoprotein lev- a wide away of molecules exhibiting els 16. Another cross between NZB DNA sequence variation in rodents and SM/J strains yielded two other and whose functions are paaly moduloci contrihuting to Ixxly fat and lated By the genetic background of body weight, one of which (MobS) is the animal. loc'ated in tile same chromosomal region as the Agouti locus. Attmctive The human obesity gene map candidate genes tbr this QTL are The present status of the human A~outL and the genes for the adeno- obesity gene map was recently sine deaminase, a marker that was reviewed 2z. At that time, no evidence ¢adier shown to be in linkage with was available to support the conbody fat 1~, and the melanocortin tention that the lot:i listed in Table 1 receptor 3, which might be one of were contributing in any way to the Agottti receptors. body mass dysregulation and obesity A dietat3,-induced mouse model in humans. All linkage studies underof obesity was developed from taken with surrogate markers of the the cross between the dietary-lhtanimal model n,utations had yielded sensitive mouse AKR/J with the negative results. More recently, howdietary-fat-resistant strain SWR/J, and ever, two papers have independently TIG NOVEMBER1996 VOL 12 NO. 11 443

reported weak evidence for linkage between markers flanking the human ob gene on 7o.31.3 and severe obesity 23,2~. These results are concordant with an early finding based on the data from the Quebec Family Study in which a linkage with body fat phenotypes was observed with the Ken blood group, which also maps in the same regkm 17. Even though the search for mutations in human subjects in the homologs of oh, db, fat, tub, Agoutf and others has been negative up to now, the advances in the Human Genuine Project, in the single-gene rodent models c,f obesity and in the QTL contributing to polygenic forms of obesity have stimulated a surge of interest in genes for human obesity. A number of industrial and academic laboratories have established new progmms designed to identify the molecular and genetic basis of human obesity and other body mass dysregulatinn characteristics. The task is complex as suggested by the number of loci identified so far in rodent models, but it is commonly believed in the obesity re~arcb community that major progress will occur over the next few years. There is great expectation that these advances in the genetic and n'mlecular causes of human obesity will eventually provide better tools to prevent and treat one of the most prevalent diseases in the Western world.

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88, 7806-7809 12 Naggen, J.K. er al. (1995) NaE GeneE 10.135-142

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//

16 Warden, C.H. et aL (19951.L Clin. l n ~ s t 95,1545-1552 17 Borecki, I.B. et al. (1%14) Obes. Res.

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LETTER

ALASDAmJ.E, GORDON [email protected]

p53 null mice, nothing to lose? ._*~ Support for the 'two-hit' hypothesis of Knudson t, the functional inactivation of both alleles of turnout suppressor genes in turnout formation, is now commonly reduced to 'it is always the wild-type...aUele that is lost in lomota's '2, However, with respect to tmnour suppressor gene systems that are vulnerable to a dominant-negative mode of self-inactivation, that statement might be a bit tautological, because it is only the presence of the mutant allele that Ls important :s. Recendy, a missense mutant p53 trarLsgene has been sttown to exert a dominant-negative phenotype in a beterozygous p53 (+/null) mouse system'i, and a model for p53 functional inactivation and turnout progression has bc~,n discussed';. In this scheme, genetic instability can he a corksequence of a single dominating p53 mutation that results in decrease or loss of cellular p53 funetinn. Therefore, it remains a formal possibility that the observed p53 allele loss, invariably the wild-type allele in turnouts, is a consequence of this genetic instability. This leaves the key questinn of whether loss of one p53 allele is a frequent occurrence in tumour pro~ession in the absem:e of p53 function. Three strains of p53 knockout mice have now been independently created li-~, each bearing a discrete null constrttct. Heterozygous p53 (+/null) mice succumb to cancer more frcquendy (and rapidly) than do wild-type mice. The majoriW of turnouts arising in these p53

heterozygotes show loss of the wild-type p53 allele (indicated by a loss of heterozygosity for restriction-fragment length polymorphisms). Nullizygous p53 mice present an even more dramatic cancer phenotype, Because all p53 nullizygous mice to date have been obtained by crossing p53 heterozygote littermates, they are homozygous for all markers on chromosome 11 and homonullizygous with respect to the p53 locus. Therefore, the question of a loss of heterozygosity at the Io53 locus in turnouts in these mice has, in a formal sense, no genetic nteaning, and, indeed, has not been asked, However, by crossing heterozygnte mice bearing dittbrent p53 n'all alleles, p53 heteronullizygous mice can be olxained. In this informative situation, loss of heterozygosity has genetic meaning and can be readily assessed :it die p53 Iocns in turnouts in the absence of p53 function, mutant or otherwise. Such analysis n'tight provide a test of the two-hit hypothesis, namely, that information: at the p53 locus is simply prone to loss in a genetically unstable cell (due to an alxsence of p53 function). This, in ram, might have implit~ations for the observed loss of wild-type allele in mmours that contain a dominant-negative p53 allele. One can now test the dictum that it is 'always the wild-type allele lost in tumours' because a situatinn can be created in which there is no wild-type allele to lose. TIG NOVEMBER1996 VOI., 12 NO. 11

O~pytighl © 15'X,Elset,ler Su~et~eLid All lights ~ c ' d PH~ SnlC~ ~sz~ox,~2t~n 2 5

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References I Knudson, A.G. (19851 CancerRes. 45, 1437-1443 2 Editorial (19941 Nat. GeneE 8, 319 3 Herskowitz, t. 0987) Nature329, 219-222 4 Harvey, M. el aL (19951 Nat. Genet. 9, 305-311 5 Harm, B.C. and Line, D.P. 0995) Nat Genet, 9, 221-222 6 Donehower, L,A. etaL(1992) Nature356, 215-221 7 Lowe, S.W el aL (19931 Nature 362, 847-849 8 Clarke, A.R. et al, (19931 NaRire 362, 849-852

Letters to the Editor We welcome letters on any topic of interest to geneticists and developmental hlologists. Wdte to: Mark Patteraon [email protected],uk Fax 01223 464430 Trends in Genetics,

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