© 1999 Nature America Inc. • http://genetics.nature.com

correspondence© 1999 Nature America Inc. • http://genetics.nature.com

the appropriate X-only hybrid, mapping
the HNRPG-homologous structural geneto human and mouse X chromosomes andthe retroposons to an autosome (Fig. 1a).
We confirmed localization to the mouse X
chromosome by isolating and FISH mapping a BAC clone (b185) containing the350-bp fragment amplified by oMJ506/508. This further localized the gene to
XA3−XA5 (Fig. 1b), consistent with theXq26 map position for RBMX in human15.
We have named the mouse X gene Rbmx.
We have shown that Hnrpg (ref. 11) is aretroposon derived from Rbmx, and it has
therefore been renamed Rbmxrt (Rbmxretrogene). We have localized Rbmxrt tomouse chromosome 14 (data not shown).
We isolated and sequenced two mouse
Rbmx cDNA clones with distinct 3´ ends,
but 100% identity to the exonic sequencesof the oMJ506/508 genomic product. The
first, phnR8c3, was isolated from an adulttestis cDNA library using the oMJ506/508
genomic product as probe (Fig. 2a, Rbmx2). The second, an adult liver cDNA clone(IMAGE 1891274), was identified via anEST (GenBank AI225991) with 100% identity to the 5´ UTR of Rbmx-2 (Fig. 2a,Rbmx-1). The cDNAs have 100% identity
over 1,005 bp, but Rbmx-2 lacks the 102
carboxy-terminal amino acids encoded by
the 3´ exon of Rbmx-1 and has a distinct 3´UTR. Rbmx-1 has the same structure as
described Hnrpg transcripts. It has 89%

nucleotide identity with the publishedHnrpg sequence11 and 96% amino acididentity, suggesting that the Rbmxrt retrogene has been functionally conserved. We
verified the presence of both 3´ ends at theRbmx locus by PCR with DNA from themouse X-only hybrid and b185 using
primer oMJ586 with either oMJ582 (3´UTR of Rbmx-1), which amplifies a 1.4-kb
genomic fragment, or oMJ587 (3´ UTR ofRbmx-2), which amplifies a 3-kb genomic
fragment (data not shown). RT-PCR withthe same primer pairs showed that bothtranscripts are ubiquitously expressed, andthis, together with the proposed structureof the mouse Rbmx locus, is shown (Fig. 2).
We conclude that RBMY is an X-NRYgene. Our results show that the NRY
multi-copy testis-specific gene families areof two types. First, those that have beenacquired directly by the NRY from anautosomal gene, as exemplified by DAZ,
and second, those that are X-NRY genes
whose transcription has become limited tothe testis. The many other X-NRY genesthat are still ubiquitously transcribed may
therefore function in spermatogenesis
only and cannot be excluded as candidate
spermatogenesis genes.
Acknowledgements

We thank N. Levy and J. Belouque for sequencing;
P. Avner for DNA from the clone 8, 6/6 and CAK
cell lines; N. Affara for DNA from the MOG cell

Absence of Cd36 mutation in theoriginal spontaneously hypertensive
rats with insulin resistanceT

he spontaneously hypertensive rat(SHR) is a model of human insulinresistance syndromes due to the presenceof essential hypertension, hyperinsulinaemia, glucose intolerance, hypertriglyceridaemia and visceral obesity1−5. In
experimental crosses, quantitative traitloci (QTLs) for SHR defects in glucose andfatty acid metabolism in adipocytes5, as
well as hypertension6 and hypertriglyceridaemia7, have been mapped to an overlapping region of rat chromosome 4. Upon
analysis of SHR strains derived from acolony at the NIH, a deletional mutationin Cd36 (formerly Fat) was proposed as
the underlying cause of these chromosome 4 QTLs and the clustering of insulin

226

resistance phenotypes in SHR (ref. 8).
Here we show that the Cd36 mutation isabsent in the original SHR strains, maintained since their development in Japan9(SHR/Izm), and question the aetiologicalrelevance of the Cd36 mutation to insulin
resistance in SHR.
We initially searched for genes that weredifferentially expressed between SHR andits genetic control, the Wistar Kyoto(WKY) rat. Of the 500 independent clones
obtained by subtraction analysis ofadipocyte cDNA from NIH-derived
strains (SHR/NCrj and WKY/NCrj), 2
contained partial cDNA sequences of
Cd36 (ref. 10), the rat homologue ofhuman CD36. Northern-blot analysis

line and comments on the manuscript; and M.A.
Mitchell for proofreading the manuscript. This
work was supported in part by a grant from theGroupement de recherches et d’études sur lesgénomes. S.M. was supported by a study grantfrom the Association pour la recherche sur lecancer and N.S. was supported by a studentshipfrom the Ministère de l’éducation nationale, del’enseignement supérieur et de la recherche.
Sophie Mazeyrat, Noëmie Saut, MarieGeneviève Mattei & Michael J. Mitchell
INSERM U.491, Faculté de médecine, 27 bd.
Jean Moulin, 13385 Marseille Cedex 05, France.
Correspondence should be addressed to M.J.M.
(e-mail: mitchell@ibdm.univ-mrs.fr).
1.
2.
3.

Graves, J. Bioessays 17, 311–321 (1995).
Lahn, B.T. & Page, D.C. Science 278, 675–680 (1997).
Mazeyrat, S. et al. Hum. Mol. Genet. 7, 1713–1724(1998).
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(1997).
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9. Agulnik, A.I. et al. Hum. Mol. Genet. 7, 1371–1377(1998).
10. Delbridge, M.L. et al. Nature Genet. 15, 131–136(1997).
11. Delbridge, M.L. et al. Mamm. Genome 9, 168–170(1998).
12. Chai, N.N. et al. Genomics 49, 283–289 (1998).
13. Ehrmann, I. et al. Hum. Mol. Genet. 7, 1725–1737(1998).
14. Soulard, M. et al. Nucleic Acids Res. 21, 4210–4217(1993).
15. Delbridge, M.L., Lingenfelter, P.A., Diseteche, C.M.
& Graves, J.A.M. Nature Genet. 22, 223–224 (1999).
16. Oulad-Abdelghani, M. et al. Int. J. Dev. Biol. 42,2–32 (1998).

showed the presence of aberrant Cd36
mRNA in SHR/NCrj, but not in SHR/Izm(Fig. 1a). Cd36 cDNA from SHR/NCrj
contained multiple sequence variants asreported8, whereas Cd36 cDNA fromSHR/Izm was normal. Genotype analysis
demonstrated that the Cd36 mutation was
not present in any of the original SHR/Izm
strains, but was found in all NIH-derived
strains examined (Fig. 1b).
SHR was established as an animalmodel of genetic hypertension1 and wassent to the NIH Animal Genetic
Resource at the F13 generation. Several
strains separated from the NIH colony
thereafter and were distributed worldwide (Fig. 1c). The observed discrepancy
with respect to Cd36 might therefore
result from the occurrence of a de novo
mutation or acquisition of a foreign
chromosomal fragment during breedingat the NIH. To explore these possibilities,
we compared allele distribution patternsof chromosome 4 markers among SHR
strains (Fig. 1d). Both the three originalnature genetics • volume 22 • july 1999© 1999 Nature America Inc. • http://genetics.nature.com© 1999 Nature America Inc. • http://genetics.nature.com

abcdcorrespondence

Fig. 1 Cd36 mRNA, Cd36 genotypes, genealogical relationships and comparison of chromosomal haplotypes. a, Northern-blot analysis of total RNA (10 µg per lane)
isolated from rat adipose tissue with a rat Cd36 cDNA probe10 (nt 247−1,570). b, Genotype analysis of PCR products derived from Cd36 reveals the T1450C variant
by digestion with BspHI (top) and an 8-bp deletion polymorphism (bottom). Rat Cd36 is at least duplicated, but only one copy is transcribed. In certain substrains ofSHR, the functional copy of Cd36 has a gross deletional rearrangement resulting in the aberrant mRNA splicing8. Sequence polymorphisms in the duplicated genes
can be exploited to distinguish animals that are homozygous with respect to the Cd36 mutation; these give rise to a single band on agarose gels. The 8-bp deletionoccurs in the putative intron 14 splice-acceptor site of the duplicated copy of the gene that would underlie the aberrant mRNA splicing. Most of the aberrantly
spliced mRNAs retained the 5´ sequence of intron 14, possibly due to ‘read-through’ at the intron 14 splice-donor site (data not shown). M, molecular size marker(øx174/HaeIII); NCrj, rats from Charles River (Japan); Izm, Funabashi Farm; N, NIH Animal Genetic Resource; NCrl, Charles River (UK); Tac, Taconic; Ola, Prague6; A1sb−CL, substrains from the original Kyoto colony9. SHR/NCrl and SHR/Ola were analysed in studies by Aitman et al.5,8. c, Schematic representation of the origin ofSHR and the relationship between substrains. SHR(SP), stroke-prone SHR; SHR(SR), stroke-resistant SHR. d, Comparison of allele distribution pattern among 10 SHRstrains. We analysed 32 microsatellite markers surrounding the Cd36 locus. Only the allele distribution pattern of 8 markers is shown because the other 24 markers
provided no additional information. We also analysed 3 markers at the Cd36 locus, including the novel Arg262Gln and 8-bp deletion polymorphisms. The deleted
alleles in SHR/NIH strains are indicated (–). RH, radiation hybrid; SHR/NIH, SHR derived from the NIH colony; cM, centiMorgans; cR, centiRay3,000.

(B1, CH, CL) and the NIH-derived SHRstrains were shown to share an identicalchromosomal haplotype on either side ofCd36, supporting the possibility that theCd36 mutation was introduced as a denovo mutation.
We next studied insulin-stimulated
glucose uptake and catecholamine-

induced lipolysis in isolated adipocytes,
phenotypes used initially to identify thechromosome 4 QTLs (ref. 5). We compared these in vitro phenotypes inFig. 2 Comparison of insulin and catecholamine action in adipocytes and in vivo
phenotypes. a, Basal glucose uptake
before insulin stimulation (50,000 pmol/l).
b, Maximal glucose uptake after insulin
stimulation. c, Insulin-mediated glucose
uptake (maximal minus basal). d, Secretionof non-esterified fatty acids (NEFA)
induced by the β-adrenergic agonist isoproterenol (200 nmol/l) in isolated
adipocytes from WKY, SHR/NCrj andSHR/Izm (n=10 in each group). Results fromWKY/NCrj (n=5) and WKY/Izm (n=5) werecombined because they showed no significant differences. Data represent themean±s.e.m.; cpm, counts per minute. We
carried out experiments as described5, butadipocytes were isolated from epididymal
fat pads; these gave results similar to thoseof retroperitoneal fat pads. Given the postulated function of Cd36 as a fatty acidtransporter10,12, the increased basal glucose uptake observed in adipocytes fromSHR/NCrj may be explained by theglucose−fatty acid cycle theory13. In fact,
impaired use of fatty acids and reciprocal upregulation of glucose uptake have been demonstrated in heart of both SHR/NCrl (ref. 14) and human subjects with
CD36 deficiency15. e, Comparison of in vivo phenotypes. Data are from 12-week-old male rats after an overnight fast. Values are means (s.d.). aP values are given
for the comparison between SHR/NCrj and SHR/Izm. bResults are from ten WKY/NCrj and ten WKY/Izm.

nature genetics • volume 22 • july 1999

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correspondence© 1999 Nature America Inc. • http://genetics.nature.com

SHR/NCrj and SHR/Izm, which allowed
us to examine the biological effects of theCd36 defect in SHR (Fig. 2a–d). Weobserved no significant differences ineither phenotype between the two SHR
strains (Fig. 2c,d). We offer two possible
explanations for this observation. First,
there may be another gene mutationcommon to both SHR strains, tightly
linked to Cd36. In this case, the Cd36
mutation is not the basis of the defective
cellular phenotypes observed in SHR.
Second, there may be another unlinked
genetic difference between the two SHR
strains accounting for the cellular phenotypes in SHR/Izm, whereas the Cd36
defect is the cause of these phenotypes inSHR/NCrj. Linkage studies in theSHR/Izm strain will distinguish between
these two possibilities.
We also compared the in vivo phenotypes associated with insulin-resistancesyndrome (Fig. 2e). Despite similar bodyweights and blood pressure levels,
SHR/Izm had significantly higher levels of
fasting blood glucose, triglycerides andepididymal fat pad weight, which favours
SHR/Izm rather than SHR/NCrj as amodel of human insulin-resistance syn-228

dromes. Supportive in vivo evidence
obtained by insulin suppression test has
been reported11.
We therefore conclude that the Cd36
defect is unlikely to be a major cause of
insulin-resistance phenotypes in SHR. The
present study demonstrates the genetic andphenotypic heterogeneity of SHR thatshould be considered when investigating
this important animal model.
Acknowledgements

We thank T. Kashiwa, A. Paku and E. Miyazaki
for assistance, and O. Ezaki, A. Hasty andT. Suzuki for advice. This work was supported by
the Promotion of Fundamental Studies in Health
Science of The Organization for Pharmaceutical
Safety and Research (OPPSR) and by Health
Sciences Research Grants (Research on Human
Genome and Gene Therapy) from the Ministry ofHealth and Welfare of Japan.
Takanari Gotoda1, Yoko Iizuka1,
Norihiro Kato2, Jun-ichi Osuga1,Marie-Therese Bihoreau3,Toru Murakami4, Yukio Yamori5,
Hitoshi Shimano1, Shun Ishibashi1& Nobuhiro Yamada1
1Department of Metabolic Diseases, Faculty ofMedicine, University of Tokyo, Tokyo 113-8655,

Japan. 2Department of Internal Medicine,
Teikyo University School of Medicine, Tokyo,
Japan. 3The Wellcome Trust Centre for HumanGenetics, Oxford, UK. 4Institute of MedicalScience, St. Marianna University School ofMedicine, Kanagawa, Japan. 5Graduate Schoolof Human and Environmental Studies, Kyoto
University, Kyoto, Japan. Correspondence
should be addressed to T.G.
(e-mail: tknrgtd-tky@umin.ac.jp).
1.

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Hypertension 18, 12−16 (1991).
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Lopez, E. & Grimaldi, P.A. J. Biol. Chem. 268, 17665−17668 (1993).
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