Cross species association examination of UCN3 and CRHR2 as potential pharmacological targets for antiobesity drugs.
ABSTRACT Obesity now constitutes a leading global public health problem. Studies have shown that insulin resistance affiliated with obesity is associated with intramyocellular lipid (IMCL) accumulation. Therefore, identification of genes associated with the phenotype would provide a clear target for pharmaceutical intervention and care for the condition. We hypothesized that urocortin 3 (UCN3) and corticotropin-releasing hormone receptor 2 (CRHR2) are associated with IMCL and subcutaneous fat depth (SFD), because the corticotropin-releasing hormone family of peptides are capable of strong anorectic and thermogenic effects.
We annotated both bovine UCN3 and CRHR2 genes and identified 12 genetic mutations in the former gene and 5 genetic markers in the promoter region of the latter gene. Genotyping of these 17 markers on Wagyu times Limousin F(2) progeny revealed significant associations between promoter polymorphisms and SFD (P = 0.0203-0.0685) and between missense mutations of exon 2 and IMCL (P = 0.0055-0.0369) in the bovine UCN3 gene. The SFD associated promoter SNPs caused a gain/loss of 12 potential transcription regulatory binding sites, while the IMCL associated coding SNPs affected the secondary structure of UCN3 mRNA. However, none of five polymorphisms in CRHR2 gene clearly co-segregated with either trait in the population (P>0.6000).
Because UCN3 is located on human chromosome 10p15.1 where quantitative trait loci for obesity have been reported, our cross species study provides further evidence that it could be proposed as a potential target for developing antiobesity drugs. None of the markers in CRHR2 was associated with obesity-type traits in cattle, which is consistent with findings in human. Therefore, CRHR2 does not lend itself to the development of antiobesity drugs.
- [show abstract] [hide abstract]
ABSTRACT: Obesity is not only a health but also an economic phenomenon. This chapter (a) examines underlying economic causes, such as technological advancements, behind the obesity epidemic; (b) describes economic consequences of obesity, including increasing obesity-related medical expenditures; and (c) discusses the role of government in combating the obesity epidemic. Because of the high costs of obesity, and the fact that the majority of these costs are financed by taxpayers, there is a clear motivation for government to try to reduce these costs. However, because obesity may result from poor information and addictive behavior and/or as a result of living in an increasingly obesogenic environment, interventions will need to be multifaceted to ensure the best chance of success.Annual Review of Public Health 02/2005; 26:239-57. · 3.27 Impact Factor
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ABSTRACT: In addition to obesity, many factors, including the distribution of body fat, contribute to the development of insulin resistance and type 2 diabetes mellitus (T2DM). Lipid contained within skeletal muscle as triglyceride is a parameter of regional fat accumulation thought to be an important link among obesity, insulin resistance, and type 2 diabetes, even in the pediatric population. Intramuscular triglycerides can also be a fuel source for healthy muscle during periods of physical activity. Thus, the balance between storage and efficient utilization of muscle triglycerides is likely a key to a better understanding of the interaction between dysregulated fat and glucose metabolism by muscle in both adults and children. This review examines the evidence that muscle lipid accumulation is linked with insulin resistance and type 2 diabetes of both adults and children. In addition, we explore the potential mechanisms for muscle lipid accumulation as well as the effects of weight loss and physical activity on muscle lipid. Further defining the links between muscle lipid accumulation and insulin action should help develop more effective strategies to prevent or treat type 2 diabetes and other obesity-associated disorders.Pediatric Diabetes 01/2005; 5(4):219-26. · 2.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.Proceedings of the National Academy of Sciences 07/2001; 98(13):7570-5. · 9.74 Impact Factor
Cross Species Association Examination of UCN3 and
CRHR2 as Potential Pharmacological Targets for
Zhihua Jiang*, Jennifer J. Michal, Galen A. Williams, Tyler F. Daniels, Tanja Kunej
Department of Animal Sciences, Washington State University, Pullman, Washington, United States of America
Background. Obesity now constitutes a leading global public health problem. Studies have shown that insulin resistance
affiliated with obesity is associated with intramyocellular lipid (IMCL) accumulation. Therefore, identification of genes
associated with the phenotype would provide a clear target for pharmaceutical intervention and care for the condition. We
hypothesized that urocortin 3 (UCN3) and corticotropin-releasing hormone receptor 2 (CRHR2) are associated with IMCL and
subcutaneous fat depth (SFD), because the corticotropin-releasing hormone family of peptides are capable of strong anorectic
and thermogenic effects. Methodology/Principal Findings. We annotated both bovine UCN3 and CRHR2 genes and
identified 12 genetic mutations in the former gene and 5 genetic markers in the promoter region of the latter gene.
Genotyping of these 17 markers on Wagyu6Limousin F2 progeny revealed significant associations between promoter
polymorphisms and SFD (P=0.020320.0685) and between missense mutations of exon 2 and IMCL (P=0.005520.0369) in the
bovine UCN3 gene. The SFD associated promoter SNPs caused a gain/loss of 12 potential transcription regulatory binding sites,
while the IMCL associated coding SNPs affected the secondary structure of UCN3 mRNA. However, none of five polymorphisms
in CRHR2 gene clearly co-segregated with either trait in the population (P.0.6000). Conclusions/Significance. Because UCN3
is located on human chromosome 10p15.1 where quantitative trait loci for obesity have been reported, our cross species study
provides further evidence that it could be proposed as a potential target for developing antiobesity drugs. None of the
markers in CRHR2 was associated with obesity-type traits in cattle, which is consistent with findings in human. Therefore,
CRHR2 does not lend itself to the development of antiobesity drugs.
Citation: Jiang Z, Michal JJ, Williams GA, Daniels TF, Kunej T (2006) Cross Species Association Examination of UCN3 and CRHR2 as Potential
Pharmacological Targets for Antiobesity Drugs. PLoS ONE 1(1): e80. doi:10.1371/journal.pone.0000080
Obesity has increased at a fast rate in recent years and is now
a worldwide public health problem. The major consequence of
overweight and obesity is that they are associated with more than
30 medical conditions, which cause approximately 300,000 deaths
and total medical expenditures (direct and indirect) of $139 billion
annually in the USA alone . Insulin resistance, a characteristic
of obesity, prevents insulin from taking the sugar from food and
distributing it throughout the body for energy. Many studies have
clearly indicated that intramyocellular accumulation of triglycerides
is a major contributor to insulin resistance . Therefore, identifi-
cation of genes associated with intramyocellular lipid accumulation
would provide a clear target for pharmaceutical intervention and
care for obesity and its related conditions, such as high blood
pressure, type 2 diabetes, coronary heart disease, some types of
Urocortin 3 (UCN3) and corticotropin-releasing hormone
receptor 2 (CRHR2) are members of the corticotropin-releasing
hormone (CRH) family of peptides. UCN3 binds selectively to
CRHR2  and both are co-expressed throughout the central
nervous system, such as in the ventromedial hypothalamic nucleus,
lateral septum and bed nucleus of the stria terminalis , as well as
in the gastrointestinal tract . Both UCN3 and CRHR2 are,
therefore, thought to play a central role in appetite and gastro-
intestinal motor regulation. Indeed, when exposed to a high fat
diet, CRHR2-mutant mice consumed significantly more food while
maintaining the same body weight as their wild-type littermates
. Intracerebroventricular injections of UCN3 were found to
reduce appetite by suppressing food intake in the freely-fed rat .
On the other hand, there is increasing evidence supporting the
involvement of these two peptides in the regulation of energy
homeostasis and in mediating the anorexic effect of CRH at the
adipose level. For example, Seres and colleagues  found that
both UCN3 and CRHR2 are expressed in human visceral and
subcutaneous adipose tissue. Obviously, the local production of
these two peptides within the adipose tissue indicates their direct
involvements in fat cell function in addition to their central effects
on weight regulation. In particular, Doyon and colleagues 
concluded that CRHR2 could be a potential target for the
development of an antiobesity drug. Thus, we hypothesized that
genetic polymorphisms of UCN3 and CRHR2 genes are associated
with intramyocellular lipid accumulation (IMCL) and subcutane-
ous fat depth (SFD) in mammals. In order to test the hypothesis,
we annotated bovine UCN3 and CRHR2 genes and identified
a total of 17 genetic polymorphisms for an association study.
Statistical analysis using the general linear model (GLM) pro-
cedure of SAS and quantitative transmission-disequilibrium test
Academic Editor: Neil Hall, Institute for Genomic Research, United States of
Received October 7, 2006; Accepted November 14, 2006; Published December
Copyright: ? 2006 Jiang et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: Agricultural Research Center, Washington State University, and Merial
Ltd. supported this project.
Competing Interests: The authors have declared that no competing interests
* To whom correspondence should be addressed. E-mail: email@example.com
PLoS ONE | www.plosone.org1December 2006 | Issue 1 | e80
(QTDT) revealed that UCN3 gene, but not its receptor CRHR2
gene, is significantly associated with both intramyocellular and
subcutaneous lipid accumulation in Wagyu6Limousin F2crosses.
MATERIALS AND METHODS
Animals and phenotypic traits
A Wagyu6Limousin reference population was developed, in-
cluding 6 F1bulls, 113 F1dams and ,250 F2progeny . The
Japanese Wagyu breed of cattle has been traditionally selected for
high IMCL accumulation (measured as marbling score with an
average of 8.52), whereas the Limousin breed has been selected for
heavy muscle, which leads to low IMCL accumulation (average
marbling score less than 4.78) . The difference in IMCL
accumulation between these two breeds makes them very unique
for mapping QTLs for the trait. Beef marbling is the term
commonly used to describe the appearance of white flecks or
streaks of fat between the muscle fibers in meat, which is essentially
equivalent to IMCL accumulation measured in humans. Beef
marbling score was a subjective measure of the amount of IMCL
in the longissimus muscle based on USDA standards (http://www.
ams.usda.gov/). Subcutaneous fat depth (SFD) was measured at
the 12–13thrib interface perpendicular to the outside surface
at a point three-fourths the length of the longissimus muscle
from its chine bone end. The marbling scores for IMCL ranged
from 4 to 9.5 and SFD varied from 0.1 to 1.3 inches in the
Sequence annotation and primer design
We determined the genomic organization of bovine UCN3 and
CRHR2 by aligning a bovine cDNA sequence (BC114855) with
a bovine genomic DNA contig (AAFC03043460) for the former
gene, and aligning the human mRNA sequence (NM_001883)
with a bovine genomic DNA contig (AAFC03056271) for the
latter gene. Three pairs of primers were designed to target the
promoter (forward–59GGG GCT GCA CCA AGC AAA TGT
CAA C39 and reverse–59TCT ACC CTT CTT CCT GGA GCC
AAC39), non-coding exon 1 (forward – 59AGG TCT GGG AGA
GAA GGT GGG TAG39 and reverse – 59AAA CAC AGA CAT
TGA CGG TTC AGC39) and coding exon 2 (forward – 59CTG
AAC TTG CAC AAA GCC TGG TAG39 and reverse – 59CCC
AGC CTC CTC CTC TAC TTC TTC39) in the UCN3 gene. An
additional two pairs of primers were designed to amplify products
in the promoter (forward – 59TGA GAC TGG AGC ACA CAA
ACA CAG39 and reverse – 59CAA GTG TGG AGG AGC TGA
AAA CCT39) and exon 1 region (forward – 59TCC TCT CCG
CTA AGG TCC AGA CT39 and reverse – 59 AGG AAC ACT
CAC GGG TCG TGT TAT39) in the bovine CRHR2 gene,
Polymorphism detection and genotyping assay
Approximately 50 ng of genomic DNA each from six F1bulls were
amplified in a final volume of 10 mL that contained 12.5 ng of
each primer, 150 mM dNTPs, 1.5 mM MgCl2, 50 mM KCl,
20 mM Tris-HCl and 0.25 U of Platinum Taq polymerase
(Invitrogen, Carlsbad, CA). The PCR conditions were carried
out as follows: 94uC for 2 min, 32 cycles of 94uC for 30 sec, 63uC
for 30 sec and 72uC for 30 sec, followed by a further 5 min
extension at 72uC. PCR products were then sequenced for
polymorphism detection on an ABI 3730 sequencer in the
Laboratory for Biotechnology and Bioanalysis (Washington State
University) using a standard protocol. The same PCR product
direct sequencing approach was also used to genotype the
polymorphisms on all animals.
The estimates for degrees of Hardy-Weinberg equilibrium within
each mutation and linkage disequilibrium between mutations and
selection of tagging genetic polymorphisms in each of bovine
UCN3 and CRHR2 genes were performed using the HAPLO-
VIEW program . The phenotypic data for both IMCL and
SFD measurements were previously adjusted for year of birth,
sex, age (days), live weight (kilograms), or fat depth (inches), as
appropriate. The adjusted phenotypes were then used in a sub-
sequent association analysis using the GLM (general linear model)
procedure of SAS v9.1 (SAS institute Inc., Cary, NC). Pair-wise
comparisons of least squares means were performed using
a protected t-test. Additionally, quantitative transmission disequi-
librium test (QTDT)  was performed to further examine the
association between the tagging mutations and adjusted obesity-
related phenotype data. P value ,0.05 was considered statistically
significant after Bonferroni correction. For significantly associated
mutations, the MatInspector web server  was used to screen
potential transcriptional regulatory binding site changes caused by
promoter polymorphisms, while the Mfold web server  was
used to predict mRNA secondary structure changes caused by
Genomic organization of the bovine UCN3 and
BLAST searches using the cDNA sequence of the human UCN3
gene (NM_053049) as a reference retrieved three bovine ortholo-
gous cDNA sequences from the GenBank database. The longest
cDNA sequence BC114855 with 1,404 bp was used and retrieved
one genomic DNA sequence (AAFC03043460) of the same gene
from the 7.156 bovine genome sequence database. Alignment
of both cDNA and genomic DNA sequences determined the
genomic organization of the bovine UCN3 gene. Like all four
human CRH paralogs, the bovine UCN3 gene has two exons and
one intron (Figure 1).
For the bovine CRHR2 gene, a BLAST search using the human
mRNA sequence (NM_001883) retrieved three bovine ortholo-
gous ESTs (BI849955, DV873120 and CK774717), but they could
not form a full-length cDNA sequence for the bovine gene.
Fortunately, one bovine genomic DNA contig (AAFC03056271)
from the 7.156 bovine genome sequence database was obtained
using the human cDNA sequence, and alignment of the human
cDNA sequence and the bovine genomic DNA sequence unraveled
the genomic organization of the bovine CRHR2 gene, including
the promoter region. The genomic organization of CRHR2 gene is
also conserved in cattle, which consists of 12 exons and 11 introns
Single and multiple nucleotide polymorphisms
In the bovine UCN3 gene, the promoter region harbors one
multiple nucleotide polymorphism (MNP) and five single nucle-
otide polymorphisms (SNPs), while exons 1 and 2 contain two and
four SNPs, respectively (Figure 1). The MNP has two homozygous
alleles of 10 bp and 5 bp, i.e., AAFC03043460.1:g.8272-
8281AATAATAAAT.GGAGC. The remaining eleven SNPs are
g.8412A.G, g.8426T.A, c.8786C.T, g.9074T.C, c.12609C.T,
UCN3 and CRHR2 for Antiobesity
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Figure 1. Genomic organization and haplotype analysis in the bovine UCN3 gene. Noncoding exon 1, partial non-conding exon 2 and 39untranslated
region are marked by white boxes and coding exon 2 by a black box. Pairwise linkage disequilibrium relationship for 12 mutations is illustrated based
on r2measurements. The mutation g.8272A.B represents AAFC03043460.1:g.8272-8281AATAATAAAT.GGAGC.
Figure 2. Genomic organization and haplotype analysis in the bovine CRHR2 gene. Coding exons are marked by black boxes. Pairwise linkage
disequilibrium relationship for 5 mutations is illustrated based on r2measurements. The mutation g.2072A.B represents AAFC03056271.1:g.33947-
UCN3 and CRHR2 for Antiobesity
PLoS ONE | www.plosone.org3December 2006 | Issue 1 | e80
(Figure 1). Among these five coding SNPs, two (c.12667T.G and
c.12669C.A) are missense mutations and both occur in one codon
(codon 59), changing phenylalanine (TTC) to valine (GTA) at the
preprohormone level of the UCN3 peptide. One MNP and four
SNPs were detected in the promoter region of bovine CRHR2
gene (Figure 2). The MNP possesses two homozygous alleles
CTTTGTCTTGAG with 18 bp in one allele and 12 bp in
other allele. Four SNPs include AAFC03056271.1:g.33704A.G,
g.33803C.T, g.34007C.A and g.34017G.C, respectively. No poly-
morphism was detected in the exon 1 region of bovine CRHR2 gene.
Haplotype analysis and selection of tagging
In the bovine UCN3 gene, sequencing of 6 F1sires indicated that
four SNPs in the promoter region: g.8208C.T, g.8287T.C,
g.8412A.G and g.8426T.A form two haplotypes: CTAT and
TCGA. Two SNPs in the exon 1 and flanking regions c.8784C.T
and g.9072T.C also appear in two haplotypes: CC and TT in the
population, while all four SNPs (c.12609C.T, c.12621T.C,
c.12667T.G and c.12669C.A) in coding exon 2 region have no
historical recombination in either CTTC or TCGA haplotypes. The
lack of historical recombination among SNPs in each of these
regions described above was further confirmed by the HAPLO-
VIEW program on genotype data of all F2progeny (Figure 1).
AAT.GGAGC, c.8784C.T, and c.12669C.A were chosen as
tagging mutations for association analysis. Among five mutations
in the promoter region of bovine CRHR2 gene, HAPLOVIEW
g.34017G.C have no-historical recombination by forming two
haplotypes of GAC and ACG (Figure 2), and thus three mutations
were selected as tagging mutations for association analysis.
Association analysis of UCN3 and CRHR2 genes with
IMCL and SFD
Two statistical approaches–the general linear model (GLM) and
the quantitative transmission disequilibrium test (QTDT) were
used to detect associations between genetic polymorphisms in
both bovine UCN3 and CRHR2 genes with IMCL and SFD in
a reference population of Wagyu6Limousin F2 cross cattle
(Table 1). Overall, the reference population had an average SFD
of 0.394 inches with a standard deviation of 0.18 inches. In the
bovine UCN3 gene, GLM analysis indicated a suggestive associ-
ation between genotype at g.8208C.T and SFD (P=0.0685),
while the QTDT test indicated a significant association between
the genotype and SFD (P=0.0203; Table 1). Animals with TT
genotypes had 0.086 (P=0.0045) and 0.056 inches (P=0.0259)
Table 1. Associations of UCN3 and CRHR2 genes with IMCL and SFD
IMCL (in scores)SFD (in inches)
8208C.T CC52 5.92560.135a
0.6180 0.7053 0.43360.021a
8265C.TCC 158 5.93160.077a
CC 38 6.21460.159b
AG 127 5.97860.089a
33803C.T CC196 5.90160.072a
CT 39 6.04460.159a
*See legends in Figure 1 and Figure 2.
UCN3 and CRHR2 for Antiobesity
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less subcutaneous fat than animals with CC and CT genotypes,
which account for 0.48 and 0.31 standard deviations for the trait,
respectively (Table 1).
Overall, IMCL accumulation, described by marbling scores, for
all F2progeny averaged 5.916 with a standard deviation of 1
marbling score. Interestingly, the genotype effects on IMCL
accumulation increased in significance with mutations closer to the
coding regions of the UCN3 gene (Table 1). The 12669C.A
marker was significantly associated with IMCL (P=0.0369 for the
GLM analysis and P=0.0055 for the QTDT test, respectively). AA
animals were much leaner, with 0.549 and 0.340 lower marbling
scores, respectively than CC animals (P=0.0045) and CT
heterozygotes (P=0.0164) (Table 1). Unfortunately, none of the
markers in the bovine CRHR2 gene were associated with either
IMCL or (P.0.60) (Table 1).
Functional characterization of promoter and coding
polymorphisms associated with SFD and IMCL
As indicated above, only mutations in the bovine UCN3 gene were
significantly associated with either trait in the reference popula-
tion. Therefore, it might be interesting to characterize how the
promoter polymorphisms affect transcriptional regulatory binding
sites and how coding polymorphisms have an impact on the
mRNA secondary structure. In the promoter region of the bovine
UCN3 gene, four polymorphisms, AAFC03043460.1:g.8208C.T,
g.8287T.C, g.8412A.G and g.8426T.A form two haplotypes:
CTAT and TCGA. MatInspector  detected a remarkable
difference in the number of potential transcriptional regulatory
binding sites between these haplotypes: ten for the former
haplotype, while only two for the latter haplotype (Figure 3).
These twelve transcriptional binding sites were for TFCP2
(transcription factor CP2), NFAT5 (nuclear factor of activated
T-cells 5, tonicity-responsive), NKX3-1 (NK3 transcription factor
related, locus 1), FOXD1 (forkhead box D1), BAPX1 (bagpipe
homeobox homolog 1), ISL1 (ISL1 transcription factor, islet-1),
DBP (D site of albumin promoter binding protein), EGR2 (early
growth response 2), CART1 (cartilage paired-class homeoprotein
1), POU4F1 (POU domain, class 4, transcription factor 1),
ARID3A (AT rich interactive domain 3A) and MSX1 (msh
homeobox homolog 1)/MSX2 (msh homeobox homolog 2),
respectively (Figure 3).
Figure 3. Nucleotide sequence of the proximal promoter region of the bovine UCN3 gene. Primer and partial non-coding exon 1 sequences are
shadowed by pink and bright green color, respectively. The putative transcription start site is numbered as +1. Four polymorphic sites that were
associated with SFD are bold and shadowed by turquoise color. Potential transcription regulatory binding sites for TFCP2, NFAT5, NKX3-1, FOXD1,
ISL1, DBP, CART1, POU4F1, ARID3A and MSX1/MSX2 are associated with haplotype CTAT, while only binding sites for BAPX1 and EGR2 are linked to
UCN3 and CRHR2 for Antiobesity
PLoS ONE | www.plosone.org5December 2006 | Issue 1 | e80
Four coding SNPs in exon 2 of bovine UCN3 gene:
AAFC03043460.1:c.12609C.T, c.12621T.C, c.12667T.G and
c.12669C.A also form two haplotypes: CTTC or TCGA. We used
the Mfold program  to predict how these two haplotypes affect
mRNA secondary structure. In the first run, a complete coding
sequence of 501 bp for the preprohormone was used in the
analysis. The sequences with both haplotypes were folded with
Mfold in a locally automated manner. The complete coding
sequence containing CTTC haplotype yielded a total of 13
secondary structures, while the sequence with the TCGA haplotype
produced a total of 16 secondary structures. However, there was
a difference in single-strandedness counts (ss-counts) between two
haplotypes. The ss-counts measure the number of times each
nucleotide is unpaired across all predicted secondary structures.
For the former haplotype, 122 of 501 bp had zero ss-counts; while
for the latter haplotype, 100 of 501 had zero ss-counts across all
predicted secondary structures (Fisher’s exact test, P=0.0150). In
the second run, we selected 81 bp of sequence surrounding the
SNPs for a more localized structure analysis. As showed in
Figure 4, both haplotypes had a strong effect on the secondary
structure of bovine UCN3 mRNA.
There are four paralogous corticotropin-releasing hormone genes
in mammalian genomes: corticotropin-releasing hormone, uro-
cortin, urocortin 2 and urocortin 3 . In the present study, we
revealed several interesting features about urocortin 3 in the
bovine genome. First, the bovine UCN3 gene region seems highly
polymorphic. We designed three pairs of primers that amplified
a total of 1,679 bp. A total of 12 mutations (one approximately
every 140 bp of sequence) were detected in this region. Second,
a multiple nucleotide polymorphism was detected in the promoter
region of the bovine UCN3 gene. One allele has 10 nucleotides of
AATAATAAAT, while another has only five nucleotides of GGAGC.
No significant similarity could be determined between these two
alleles. Third, two SNPs (c.12667T.G and c.12669C.A) occurred
in one codon (codon 59), leading to a change from phenylalanine
(TTC) to valine (GTA) at the preprohormone level of UCN3
peptide. Both SNPs only form two haplotypes–TC and GA,
showing no historical recombination in the population. Lastly,
HAPLOVIEW analysis revealed that three amplified regions hold
three haplotype blocks (Figure 1); although the amplified promoter
region and exon 1 region are just 147 bp apart and exon 1 and
exon 2 regions are just 3,209 bp apart. These data might provide
a foundation for further investigation on formation and evolution
of CRH paralogs in mammals.
More importantly, we found that the UCN3 gene is significantly
associated with IMCL accumulation and SFD (Table 1). However,
four promoter SNPs organized into two haplotyes had a strong
association with SFD, while four SNPs that also formed two
haplotypes in exon 2 yielded a strong association with IMCL
accumulation. In the SFD analysis, animals with TT genotypes of
g.8208C.T had 0.086 (P=0.0045) and 0.056 inches (P=0.0259)
less subcutaneous fat than animals with CC and CT genotypes,
which account for 0.48 and 0.31 standard deviations for the trait.
In the IMCL analysis, AA animals at position c.12669C.A had
0.549 and 0.340 lower marbling scores than CC animals
(P=0.0045) and CT heterozygotes (P=0.0164). On the other
hand, the AA animals tended to be leaner with 0.047 and 0.046
less inches of SFD compared to the CC and CA genotypes (Table 1),
which approached the significance level (P=0.0982 for the GLM
analysis and P=0.0522 for the QTDT test when the P values were
uncorrected). These data indicate that increasing SFD with
a promoter polymorphism does not necessarily result in an
Figure 4. UCN3 mRNA secondary structure predicted by Mfold on a partial sequence of 81 bp surrounding four coding SNPs. A: mRNA secondary
structure for haplotype CTTC. B: mRNA secondary structure for haplotype TCGA.
UCN3 and CRHR2 for Antiobesity
PLoS ONE | www.plosone.org6 December 2006 | Issue 1 | e80
increase of IMCL accumulation. However, it is very likely that
increasing IMCL with the exon 2 polymorphisms would also
stimulate high accumulation of SFD, and thus lead to an overall
increase of whole body fat deposition. As intramyocellular lipid
accumulation in muscle is a major contributor to both insulin
resistance and whole body fat deposition, inhibiting IMCL gain
should be a long-term goal for preventing obesity in human.
In the human genome, UCN3 is placed at position 5.40 Mb on
10p15.1, where two independent studies suggested quantitative
trait loci (QTL) for body mass index (BMI) in Pima Indians 
and in Caucasians . Interestingly, both groups used the same
2.23 Mb to 6.76 Mb on human chromosome 10. Obviously, the
UCN3 gene should be a strong candidate gene for the human BMI
QTL detected in the region, as our current study provided strong
evidence supporting its involvement in regulation of lipogenesis. In
the study, we developed five genetic markers in the promoter
region of the bovine CRHR2 gene, but none were significantly
associated with either IMCL accumulation or SFD in Wagyu6
Limousin F2cross cattle (Table 1).
This was not surprising because no association of CRHR2 gene
has been observed with obesity in humans. Challis and associates
 screened 51 severely obese children (body mass index
(BMI).4 kg/m2standard deviations above the age-related mean),
a UK Caucasian population-based cohort for genetic polymorph-
isms in the human CRHR2 gene. In subjects with extreme early-
onset obesity, three missense mutations were found in CRHR2
(Glu220Asp, Val240Ile and Val411Met). However, none of these
missense mutations clearly cosegregated with obesity in family
studies. A common single-nucleotide polymorphism G1047A
(Ser349Ser) was also detected in CRHR2, but it was not associated
with any obesity-related phenotype. The authors concluded that
mutations in the coding sequence of the CRHR2 gene are unlikely
to be a common monogenic cause of early-onset obesity.
Therefore, the association studies conducted in cattle and in
human failed to provide any evidence supporting CRHR2 as
a potential target for the development of an antiobesity drug, as
proposed by Doyon and colleagues .
The remarkably different expression patterns between UCN3
and CRHR2 genes in adipocyte tissue and skeletal muscle might
provide some hints on why the former, not the latter gene, is
associated with SFD and IMCL accumulation observed in the
present study. In the human subcutaneous fat tissue, quantitative
expression analysis revealed that UCN3 mRNA is expressed
approximately four fold higher than its receptor, CRHR2 mRNA
. UCN3 mRNA is expressed in the skeletal muscle of adult
mammals and Xenopus laevis, but no one has detected any
expression of CRHR2 mRNA in the tissue of any species .
Therefore, the lower expression or no expression of CRHR2
mRNA in these tissues might lead to its limited effects on fat cell
function and muscle thermogenesis. On the other hand, evidence
has shown that UCN3 is directly involved in regulation of
glucagons and insulin secretion . Injection of murine synthetic
UcnIII into male rats significantly increased both blood and
insulin levels. UCN3 also stimulated glucagons and insulin release
from the isolated rat islets. In the present study, the high SFD
associated haplotype CTAT in the promoter region gained 10 new
transcriptional regulatory binding sites in comparison with the
other haplotype of TCAT in the bovine UCN3 gene. Among these
10 transcriptional regulatory binding sites, three are for TFCP2
(transcription factor CP2), NKX3-1 (NK3 transcription factor
related, locus 1) and NFAT5 (nuclear factor of activated T-cells 5,
tonicity-responsive). Studies have shown that these three genes
may affect the risk of Alzheimer’s disease , prostate cancer
 and diabetic nephropathy , conditions often associated
with obesity. All these data clearly support that the UCN3 gene
plays an important role in regulation of adipocyte metabolism
through a broad pathway.
In conclusion, we annotated the bovine UCN3 and CRHR2
genes using a comparative approach and developed a total of 17
genetic markers in both genes. Genotyping these markers on ,250
Wagyu6Limousin F2crosses revealed that the bovine UCN3, but
not its receptor CRHR2 gene, is significantly associated with
intramyocellular lipid accumulation and subcutaneous fat depth in
cattle. The promoter polymorphisms of the bovine UCN3 gene
alter 12 potential transcription regulatory binding sites, some of
which are associated with obesity-related conditions. The coding
polymorphisms of the gene affect the secondary structure of UCN3
mRNA remarkably. Therefore, we propose UCN3 as a strong
target for developing antiobesity drugs. However, the candidacy of
CRHR2 for the purpose needs to be further evaluated.
The authors appreciate the assistance of Dr. Michael MacNeil, USDA-
ARS, Miles City, MT, in providing DNA and data for this research. We
also thank Dr. Xio-Lin Wu, The University of Wisconsin-Madison, for his
assistance in statistical analysis.
Conceived and designed the experiments: ZJ. Performed the experiments:
JM GW TK. Analyzed the data: ZJ JM TD. Wrote the paper: ZJ. Other:
Edited the paper: JM.
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