In this study, we Keywords E2F; energy homeostasis; gene trap; high-fat diet; obesity; RMI1 Correspondence A.. Reduced RMI1 expression, lower fasting-blood glucose and a reduced body wei
Trang 1genetic-induced obesity
Akira Suwa1, Masayasu Yoshino2, Chihiro Yamazaki3, Masanori Naitou2, Rie Fujikawa3,
Shun-ichiro Matsumoto2, Takeshi Kurama1, Teruhiko Shimokawa1and Ichiro Aramori2
1 Pharmacology Research Labs, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan
2 Molecular Medicine Labs, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan
3 Trans Genic Inc., Chuo-ku, Tokyo, Japan
Introduction
Obesity is a complex disorder and a major risk factor
for metabolic diseases such as type 2 diabetes mellitus,
hypertension and cardiovascular disease This energy
balance disorder is controlled by multiple pathways
Several genes are known to be responsible for obesity:
the genes obese (ob) [1], fat (fa) [2], agouti (ay) [3],
tubby(tub) [4] and diabetes (db) [5] have been identified
and characterized in genetically obese models
However, other important molecules involved in the
regulation of energy homeostasis have yet to be identified
The exchangeable gene trap method is a powerful strategy that could be used to locate single-gene defects responsible for energy homeostasis disorders [6] With this method, it is possible to mutate the mouse genome randomly on a large scale, and then isolate and identify the mutated gene Several other genes have been identified by this method [7–9] In this study, we
Keywords
E2F; energy homeostasis; gene trap;
high-fat diet; obesity; RMI1
Correspondence
A Suwa, Department of Metabolic
Diseases, Pharmacology Research Labs,
Drug Discovery Research, Astellas Pharma
Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki
305-8585, Japan
Fax: +81 29 852 5391
Tel: +81 29 863 6417
E-mail: akira.suwa@jp.astellas.com
(Received 2 September 2009, revised 19
November 2009, accepted 24 November
2009)
doi:10.1111/j.1742-4658.2009.07513.x
The aim of this study is to discover and characterize novel energy homeo-stasis-related molecules We screened stock mouse embryonic stem cells established using the exchangeable gene trap method, and examined the effects of deficiency of the target gene on diet and genetic-induced obesity The mutant strain 0283, which has an insertion at the recQ-mediated gen-ome instability 1 (RMI1) locus, possesses a number of striking features that allow it to resist metabolic abnormalities Reduced RMI1 expression, lower fasting-blood glucose and a reduced body weight (normal diet) were observed in the mutant mice When fed a high-fat diet, the mutant mice were resistant to obesity, and also showed improved glucose intolerance and reduced abdominal fat tissue mass and food intake In addition, the mutants were also resistant to obesity induced by the lethal yellow agouti (Ay) gene Endogenous RMI1 genes were found to be up-regulated in the liver and adipose tissue of KK-Ay mice RMI1 is a component of the Bloom’s syndrome gene helicase complex that maintains genome integrity and activates cell-cycle checkpoint machinery Interestingly, diet-induced expression of E2F8 mRNA, which is an important cell cycle-related mole-cule, was suppressed in the mutant mice These results suggest that the reg-ulation of energy balance by RMI1 is attributable to the regreg-ulation of food intake and E2F8 expression in adipose tissue Taken together, these find-ings demonstrate that RMI1 is a novel molecule that regulates energy homeostasis
Abbreviations
AUC, area under the curve; A y , lethal yellow agouti; BLM, Bloom syndrome; RMI1, recQ-mediated genome instability 1.
Trang 2screened gene-trapped mice to identify novel energy
balance-related genes We describe here the phenotype
of mutant mouse strain 0283 This strain exhibited a
phenotype indicative of resistance to diet-induced and
genetic obesity The mutation of the 0283 strain is in
the RMI1 gene
RecQ-mediated genome instability 1 (RMI1) has
recently been identified as a member of the Bloom
syndrome (BLM)–topoisomerase complex [10] This
complex is essential for the maintenance of genome
integrity, and can activate the cell-cycle checkpoint
machinery [11,12] Depletion of RMI1 by siRNA leads
to reduced cell proliferation [13] In addition,
uncon-trolled cell-cycle management in adipose tissue is
asso-ciated with obesity [14,15] It has been shown that
several cell cycle-related molecules play an important
role in the development of obesity [16–21] Therefore,
we hypothesize that RMI1 might modulate energy
homeostasis via regulation of cell-cycle progression in
metabolic tissues In this study, we describe the
associ-ation between RMI1 and energy homeostasis as well
as the contribution of RMI1 to the regulation of E2F
expression, which is a well-documented cell
cycle-related molecule
Results
In vivo phenotype-driven screening
We used a phenotype-driven in vivo approach to
iden-tify novel molecules involved in the regulation of
energy homeostasis Using the gene trap vector
pU-Ha-chi, we performed random insertional mutagenesis, and
then replaced the b-geo gene with any gene of interest
through Cre-mediated integration We isolated 100
trap mouse strains in this study One of these lines was
the 0283 mutant strain, which exhibits a remarkable
obesity-resistant phenotype All homozygous embryos
died; therefore heterozygous mice (RMI1+⁄)) were
used for this study (RMI1 was identified as the target
gene of this mutant strain as described below) Body
and organ weights as well as plasma parameters (Tables S2 and S3) were measured, and learning, mem-ory and behavioral tests (Table S4) as well as histo-pathological analysis (Table S5) were performed for 8-week-old RMI1+⁄) mice fed normal laboratory chow Although RMI1+⁄) mice had a phenotype almost equivalent to that of the wild-type (RMI1+⁄ +), body weight and fasting-plasma glucose were significantly lower in RMI1+⁄) mice (Table 1)
Resistance to diet-induced obesity in RMI1+/) mice
Wild-type (RMI1+⁄ +) and heterozygous (RMI1+ ⁄)) littermate mice were created via in vitro fertilization using a single RMI1+⁄) male At 4 weeks of age, the individually housed littermates were fed either a normal diet or one in which 60% of the calories were from fat (high-fat diet) These mice were kept for 14 weeks, and monitored for body weight changes and food intake Initially, the male RMI1+⁄) mice weighed less than their male RMI1+⁄ + littermates, and those fed a nor-mal diet consistently weighed less than their RMI1+⁄ + littermates during the entire 14 weeks (Fig 1A) The rate of weight gain was equivalent for both genotypes fed a normal diet (Fig 1B) In contrast, RMI1+⁄) mice were more resistant to weight gain than RMI1+⁄ + control mice under high-fat diet conditions (18.3% ver-sus 13.7% at 14 weeks, P = 0.005, Fig 1B) Food intake was significantly lower for RMI1+⁄) mice than RMI1+⁄ + mice on the high-fat diet only, indicative of selective weight control (Fig 1C,D) The female RMI1+⁄) mice exhibited the same phenotype described above (data not shown) These results suggest that the regulation of energy homeostasis was altered in the RMI1+⁄) mice
The RMI1+⁄) also gained less intra-abdominal fat (gonadal fat volumes measured as intra-abdominal fat)
as a result of high-fat feeding compared to the wild-type (Fig 2B) In contrast, liver weights were unaltered
in the RMI1+⁄) mice compared to the wild-type, and
Table 1 Metabolic parameters for RMI1+ ⁄ + and RMI1+ ⁄ ) mice Data for 10-week-old mice (n = 6 per genotype) fasted for 16 h are shown Plasma values are the means ± SEM of the measurements obtained Asterisks indicate statistically significant differences compared with RMI1+ ⁄ + mice (*P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test).
Genotype
Body weight (g)
Glucose (mgÆdL)1)
Insulin (ngÆmL)1)
Triglycerides (mgÆdL)1)
HDL cholesterol (mgÆdL)1)
LDL cholesterol (mgÆdL)1)
RMI1+ ⁄ ) 15.5 ± 0.1 *** 80.3 ± 3.0*** 0.85 ± 0.25 26.5 ± 2.5 34.0 ± 1.7* 59.3 ± 3.4
Trang 3did not differ between the two feeding conditions
(Fig 2A) The blood glucose and plasma insulin
con-centrations in the fasted or fed state did not differ
sig-nificantly between RMI1+⁄) and RMI1+ ⁄ + mice at
14 weeks (Table 2) However, an oral glucose tolerance
test showed that diet-induced glucose intolerance
improved significantly in RMI1+⁄) mice (Fig 2C,D)
Insulin levels did not differ between RMI1+⁄ + and
RMI1+⁄) mice in the oral glucose tolerance test
(Fig 2E,F)
Resistance to KK- and KK-Ay-induced genetic
induced obesity in RMI1+/) mice
To explore resistance to the development of obesity
under other conditions, we generated KK-a⁄ a and
KK-Ay⁄ a RMI1-deficient mice KK mice are known
to be spontaneously hyperinsulinemic and
hyperglyce-mic Introduction of the lethal yellow agouti gene (Ay)
into KK mice resulted in a congenitally lethal yellow
obese KK mouse strain (KK-Ay), which exhibits both
hyperphagia and severe features of type 2 diabetes
Both the KK and KK-Ay strains are useful for
study-ing therapies for the prevention of diabetes and
obes-ity We thus crossed RMI1+⁄) mice with KK-Ay to
obtain F1 heterozygous mice (RMI1+⁄) x KK or
KK-Ay gives RMI1+⁄) a ⁄ a or RMI1+ ⁄ ) Ay⁄ a,
respectively)
Between 7 and 14 weeks of age, both RMI1+⁄) a ⁄ a
and RMI1+⁄) Ay⁄ a mice experienced a consistent and
significant reduction of body weight compared to their wild-type littermates (Fig 3A) Hyperphagia induced
by the Ay mutation was significantly less in RMI1+⁄) mice than RMI1+/+ However, KK-crossed RMI1+⁄) mice did not show altered food intake, even though their body weight was reduced (Fig 3B) The intra-abdominal fat found in KK-crossed mice was not present in RMI1+⁄) mice Similarly, the fat found in the KK-Ay-crossed RMI1+/) mice was a tendency towards reduction compared to KK-Ay F1 mice (Fig 3D) Additionally, the enlargement of the liver observed in KK- and KK-Ay crossed mice was signifi-cantly reduced in RMI1+⁄) mice (Fig 3C)
The fasted blood glucose concentration in RMI1+⁄) a ⁄ a and RMI1+ ⁄ ) Ay⁄ a mice was signifi-cantly lower than in their RMI1+⁄ + littermates The non-fasted glucose did increase slightly in RMI1+⁄)
a⁄ a mice, and this increase was statistically significant (Table 3) The oral glucose tolerance test indicated that glucose tolerance improved in both RMI1+⁄) a ⁄ a and RMI1+⁄) Ay
⁄ a mice (Fig 3E,F)
RMI1 as the target gene of the mutant strain
We analyzed the insertion site of the trap vector to identify the trapped gene Genomic DNA fragments flanking both the 5¢ and 3¢ ends of the integrated vector were obtained using the plasmid rescue method Sequence analysis of this flanking genomic DNA (Appendix S2) revealed that the trap vector was
20
16
18 RMI1+/+ ND
RMI1+/– ND
RMI1+/+ HF
** **
10 12
14 RMI1+/– HF
* *
** **
6 8
2 4
0
Number of days fed diet
5.0
4.5
RMI1+/+ ND RMI1+/– ND
3.5 4.0
3.0
**
2.0 2.5
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Number of days fed diet
45 40
RMI1+/+ ND RMI1+/– ND RMI1+/+ HF
30
35 RMI1+/– HF
**
**
**
**
**
**
**
**
**
20
**
**
**
** ** * * * *
* * * *
15
**
** ** ** **
**
10
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Number of days fed diet
5.0
4.5
RMI1+/+ HF RMI1+/– HF
3.5 4.0
*
*
3.0
**
**
** ***
*
* * * *
*
2.5
2.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Number of days fed diet
**
Fig 1 RMI1 heterozygous (RMI1+ ⁄ )) mice
fed a high-fat diet are resistant to weight
gain and are hypophagic Male wild-type
(RMI1+ ⁄ +) and mutant (RMI1+ ⁄ )) mice
(n = 6 per group) were fed a normal diet
(ND; 10% of total kcal from fat) or a high-fat
diet (HF; 60% of total kcal from fat) for
14 weeks (A) Body weight and (B) body
weight gain for RMI1+ ⁄ + and RMI1+ ⁄ )
mice over the feeding period (C) The food
intake of the RMI1+ ⁄ ) mice does not
change when fed a normal diet (D) The
food intake for the RMI1+⁄ ) mice is lower
than that for the RMI1+ ⁄ + mice when both
are fed a high-fat diet Values are means ±
SEM Asterisks indicate significant
differences: *P < 0.05, **P < 0.01,
***P < 0.001 versus RMI1+ ⁄ +.
Trang 4inserted into the first exon of the RMI1 gene
(Gen-bank accession number NM_028904) We attempted to
confirm that RMI1 is the target gene of this mutant
mice using quantitative PCR The RMI1 mRNA level
in the skeletal muscle, fat, hypothalamus and liver of
RMI1+⁄) mice was approximately half that in
RMI1+⁄ + mice (Fig 4A), which indicates that RMI1
is the responsible gene for this mutant mouse strain
Next we compared the expression levels of RMI1 in
various tissues from normal mice RMI1 mRNA was
expressed ubiquitously in most tissues (Fig S1) To
clarify the association between RMI1 and the
develop-ment of obesity, we examined the RMI1 mRNA levels
in KK-Ay mice Five-week-old KK-Ay mice did not
exhibit the obese phenotype Therefore, we compared the RMI1 mRNA levels of KK-Ay mice before (5 weeks) and after (15 weeks) obesity was observable Interestingly, the RMI1 mRNA level increased signifi-cantly in the liver and intra-abdominal fat of the obese phenotype mice; however, that in the skeletal muscle did not increase, and that in the subcutaneous fat actu-ally decreased (Fig 4B) These results suggested that the level of RMI1 expression in the fat and liver is associated with development of obesity
RMI1 is the component of the BLM helicase com-plex that maintains comcom-plex stability and aids in the maintenance of genome integrity RMI1 is also known
to regulate the cell-cycle checkpoint machinery In fact,
400
300 350
** *
200 250
100 150
0 50 Blood glucose AUC 0–2 h (mg·dL
–1 )
4
RMI1+/– ND RMI1+/+ HF
2.5
3
3.5
RMI1+/– HF
1.5
2
0.5
1
–1 )
0
Time (h)
350
400
450
250
300
RMI1+/– HF RMI1+/– ND RMI1+/+ ND RMI1+/+ HF
*
150
200
50
100
–1 )
0
Time (h)
2
2.5
1.5
0.5
1
0
2.5
3
1.5 2
1
0
0.5
1.40 1.60
1.80
**
1.00 1.20
0.60 0.80
0.20
0.40
0.00
+/+ +/– +/+ +/–
Fig 2 RMI1 heterozygous (RMI1+ ⁄ )) mice had less visceral adipose tissue and lower glucose tolerance than wild-type (RMI1+/+)
on a high-fat diet Male wild-type (RMI1+ ⁄ +) and mutant (RMI1+ ⁄ )) mice (n = 6 per group) were fed a normal diet (ND) or a high-fat diet (HF) for 14 weeks (A) RMI1+ ⁄ ) mice do not differ from RMI1+ ⁄ + mice in terms of liver weight (B) The amount of intra-abdominal fat was signifi-cantly less in RMI1+ ⁄ ) than RMI1+/+ fed a high-fat diet than RMI1+/+ (C) The blood glucose concentration during the oral glu-cose tolerance test was significantly lower
in RMI1+ ⁄ ) mice than RMI1+/+ at 1 and
2 h after glucose injection (D) The RMI1+ ⁄ ) mice fed a high-fat diet had a lower area under the curve (AUC) than RMI1+/+ for the plasma glucose concentra-tion than RMI1+/+ between 0 and 2 h after glucose injection (E) Plasma insulin concen-trations during the oral glucose tolerance test (F) AUC for plasma insulin levels between 0 and 0.5 h after glucose injection Values are means ± SEM Asterisks indicate significant differences: *P < 0.05,
**P < 0.01, ***P < 0.001 versus RMI1+ ⁄ +.
Trang 5it has been reported that using siRNA to deplete
RMI1 could reduce cell proliferation Therefore, we
speculated that the RMI1 mechanism is important for
the regulation of energy balance and the quantitative
management of metabolic tissues For this reason,
we investigated the change in expression of cell cycle-related molecules in RMI1-deficient mice (Table 4)
We did not detect any changes in the expression of
Table 2 Metabolic parameters in RMI1+ ⁄ + and RMI1+ ⁄ ) mice fed a normal or high-fat (60% fat) diet for 14 weeks Plasma levels are the means ± SEM of measurements obtained Asterisks indicate statistically significant differences compared with RMI1+⁄ + mice (*P < 0.05;
**P < 0.01, Student’s t test).
Body weight (g)
Glucose (mgÆdL)1) Insulin (ngÆmL)1)
1.4
1.0 1.2
0.6 0.8
0.0 0.2
0.4
+/–
+/+ +/+ +/–
300 350
RMI1+/– A y /a
*
200 250
**
100 150
*
*
*
**
0 50
–1 )
4.5 *** **
3.0 3.5
4.0
*
1.5 2.0 2.5
0.0 0.5 1.0
+/+ +/– +/+ +/–
7.0
7.5 RMI1+/+ a/a
RMI1+/– a/a RMI1+/+ A y /a
5.5 6.0 6.5 RMI1+/– A y /a
**
**
*
4.5 5.0
*
3.0 3.5 4.0
Age (weeks)
55
60 RMI1+/+ a/a
RMI1+/– a/a RMI1+/+ A y /a
45
50 RMI1+/– A y /a
**
**
**
**
**
**
30 35 40
**
**
**
** ** **
** **
** **
20 25
7 8 9 10 11 12 13 14
Age (weeks)
500 600
**
300 400
100
200
Blood glucose AUC 0–2 h (mg·dL –1 )
0
+/+ +/– +/+ +/–
Fig 3 RMI1 heterozygous (RMI1+ ⁄ )) mice
were resistant to the obesity, hyperphagia
and improved glucose intolerance induced
by the Aymutation (A) RMI1 heterozygotes
(RMI1+ ⁄ ) a ⁄ a and RMI1+ ⁄ ) A y ⁄ a) had
lower body weights than the KK or KK-A y
mice (n = 12 per group) (B) RMI1+ ⁄ ) A y ⁄ a
mice showed a significant reduction in the
hyperphagia induced by the Ay mutation.
At 14 weeks of age, the (C) liver weights
and (D) intra-abdominal fat weights for the
RMI1+ ⁄ ) mice (RMI1+ ⁄ ) a ⁄ a, RMI1+ ⁄ )
A y ⁄ a) were less than those for the KK and
KK-AyF 1 mice (E) The blood glucose
concentration during the oral glucose
toler-ance test was significantly lower in
RMI1+ ⁄ ) mice than the KK or KK-Ay F1
mice (F) The RMI1+ ⁄ ) mice had a lower
AUC for the plasma glucose concentration
between 0 and 2 h after glucose
administra-tion than the KK or KK-A y F1mice Values
are means ± SEM Asterisks indicate
signifi-cant differences: *P < 0.05, **P < 0.01
versus RMI1+ ⁄ +.
Trang 6E2F1, 4 or 5 mRNA in mice fed a high-fat diet In
contrast, E2F8 mRNA was strongly induced by
high-fat feeding (7.1-fold increase over mice fed a normal
diet) Interestingly, the expression of E2F8 mRNA
induced in RMI1+⁄) mice was much less (60%
sup-pression) than that in RMI1+⁄ + mice Recent reports
have indicated that the E2F family quantitatively
regu-lates adipose cells and thus plays an important role in
the development of obesity [21] These results suggest
that E2F8 is associated with development of obesity
via cell-cycle regulation in the metabolic tissues, and,
in this study, regulation of E2F8 was found to be
med-iated by RMI1
Given that RMI1-deficient mice have been found to
eat significantly less food under conditions of excessive
energy diets than under normal conditions, we com-pared levels of RMI1 mRNA in the hypothalamus between normal and high-fat feeding conditions The results showed that RMI1 mRNA levels were signifi-cantly higher in the hypothalamus under high-fat feed-ing conditions than under normal feedfeed-ing (Fig 4C)
In contrast, RMI1 expression was reduced under fast-ing conditions These results suggested that RMI1 might be associated with feeding behavior and energy balance regulation We then investigated whether or not these changes were related to modulation of cen-tral nervous system pathways We compared expres-sion levels of well-documented hypothalamic signaling factors (namely neuropeptide Y, pro-opiomelanocortin, cocaine- and amphetamine-regulated transcript),
Table 3 Metabolic parameters for RMI1+ ⁄ + and RMI1+ ⁄ ) mice crossed with KK or KK-A y mice Data for 14-week-old mice are shown Plasma levels are the means ± SEM of the measurements obtained Asterisks indicate statistically significant differences compared with RMI1+ ⁄ + mice (*P < 0.05; **P < 0.01, Student’s t test) NEFA, non-esterified fatty acids.
Body weight (g)
Glucose (mgÆdL)1) NEFA (mEqÆL)1)
Ketone bodies (mgÆdL)1)
Triglycerides (mgÆdL)1)
a ⁄ a RMI1+ ⁄ + 37.2 ± 0.7 147 ± 1.7 126 ± 4 0.34 ± 0.03 0.58 ± 0.07 53 ± 27 1405 ± 189 268 ± 40 161 ± 15 RMI1+ ⁄ ) 31.8 ± 0.4*** 165 ± 2.7*** 113 ± 4* 0.34 ± 0.02 0.85 ± 0.1* 56 ± 11 1699 ± 103 257 ± 39 139 ± 13
Ay ⁄ a RMI1+ ⁄ + 51.0 ± 0.9 440 ± 11 175 ± 9 0.44 ± 0.03 0.32 ± 0.02 164 ± 17 1987 ± 241 460 ± 61 182 ± 21 RMI1+ ⁄ ) 44.3 ± 0.4*** 431 ± 9.6 150 ± 6* 0.37 ± 0.04 0.38 ± 0.03 191 ± 29 2545 ± 269 525 ± 39 153 ± 7
140
120
RMI1+/+
RMI1+/–
80
100
60
20
40
0
Muscle Fat Hypo Liver
300 250
*
*
200
*
100 150
50 0 Muscle
5 15 5 15 5 15 5 15 Age (week)
Sub-fat Abd-fat Liver
120
130 * **
*
100
110
70
80
90
50
60
Fed Fast Fed Fast
A
C
B
Fig 4 Identification of RMI1 as the trapped gene in RMI1+ ⁄ ) mice (A) Expression of RMI1 mRNA in wild-type (RMI1+ ⁄ +) and RMI1 heterozygous (RMI1+ ⁄ )) mice Hypo, hypothalamus (B) Expression of RMI1 in normal (5 weeks of age) and obese (15 weeks of age) KK-Aymice (n = 6 per group) Sub, subcutaneous; Abd, intra-abdominal (C) Expression of RMI1 in the hypothalamus under normal diet (ND) and high-fat diet (HF) conditions (n = 8 per group) Fast, 16 h fasted Values are means ± SEM Asterisks indicate significant differences: *P < 0.05, **P < 0.01.
Trang 7Agouti-related protein, pro-melanin-concentrating
hor-mone and CPT1c) in the hypothalamus of
RMI1-defi-cient mice No changes were noted in the expression
levels of these factors (Table S1)
Discussion
Using a random mutagenesis approach based on the
exchangeable gene trap method, we identified RMI1 as
a novel regulator of energy homeostasis The attributes
of RMI1 heterozygous mice, which exhibited a typical
lean phenotype, observed in this study are as follows:
first, RMI1-deficient mice were resistant to obesity
resulting from a high-fat diet or genetics Second,
RMI1-deficient mice fed a high-fat diet gained less
abdominal fat Third, the RMI1-deficient mice ate
sig-nificantly less food under the excess energy feeding
conditions Fourth, impaired glucose tolerance induced
by high-fat diet or genetic obesity was improved in the
RMI1-deficient mice In addition, levels of RMI1
expression were higher in the abdominal fat, liver and
hypothalamus of obese model mice than normal mice
We could not find any abnormalities in the
RMI1-deficient mice under normal conditions, except the
reduced body weight and lower fasting glucose Of
note is the fact that the deficient mice showed a rate of
weight gain and amount of food intake equivalent to
those of wild-type mice under normal diet conditions
These results indicate that deficient mice can grow
nor-mally despite development of basal abnormalities,
sug-gesting that resistance to developing obesity under
high-fat feeding conditions is directly due to the RMI1
deficiency However, we could not exclude the
possibil-ity that these slight basal changes and as yet unidenti-fied abnormalities can affect the energy balance indirectly
RMI1, an enzyme-binding protein, has previously been reported to mediate DNA recombination, chro-mosome organization and biogenesis, as well as regu-lating the cell-cycle checkpoint machinery [10] However, no evidence has linked it to energy homeo-stasis RMI1 is also a member of the BLM–topoisom-erase complex Mice with a targeted mutation of BLM are developmentally delayed and die by embryonic day 13.5 [22,23] Bloom’s syndrome is a rare recessive genetic disorder characterized by dwarfism, telangiec-tatic erythema, immune deficiency and a predisposition toward cancer [13,24] Recently, RMI1 was reported to
be an essential component of BLM protein complexes [25] This BLM phenotype may explain the lethality seen in RMI1 homozygous mice Although we did not explore such phenotypes in this study, birth weight reduction might show one aspect of the BLM pheno-type, dwarfism Further studies will be needed to clarify whether the RMI1-deficient mice exhibit a BLM-like phenotype
Obesity develops as the result of an imbalance between energy intake and expenditure The reduction
of energy expenditure leads to an increase in fat mass, ultimately resulting in obesity The increase in cell number (preadipocyte proliferation) and cell size (adi-pocyte hypertrophy) is thought to be responsible for the increase in the fat mass [14,15] The cell cycle plays
an important role in preadipocyte proliferation, and is regulated by several cell cycle-related proteins RMI1
is known to be a cell cycle-related molecule with the ability to activate the cell-cycle checkpoint machinery [10], and siRNA depletion of RMI1 results in the suppression of cell proliferation [13]
Sakai et al have shown that a deficiency in the Skp2 gene, which encodes a cell cycle-related molecule, results in resistance to obesity due to inhibition of preadipocyte proliferation without causing adipocyte hypertrophy [17] This was found to be the case in both the high-fat diet and Ay-induced obesity models Interestingly, the Skp2 knockout phenotype is very similar to that of RMI1+⁄); however, Skp2 mRNA levels were not altered in RMI1+⁄) mice Fajas et al demonstrated that the E2F protein family also plays a central role in preadipocyte proliferation, and that E2F1-deficient mice are resistant to obesity induced by
a high-fat diet (due to the suppression of fat mass accumulation) [21] In this study, we found that the high-fat diet upregulated E2F8 expression, but not that
of E2F1, E2F3 or E2F5 Interestingly, E2F8 upregula-tion was suppressed in RMI1+⁄) mice Although the
Table 4 Gene expression analysis in the adipose tissue of
RMI1+ ⁄ + and RMI1+ ⁄ ) mice fed a normal or high-fat (60% fat)
diet for 14 weeks The relative amounts of mRNA are the means ±
SEM of the measurements obtained Asterisks indicate statistically
significant differences compared with RMI1+ ⁄ + mice (*P < 0.05,
Student’s t test) E2F1, E2F transcription factor 1; E2F4, E2F
scription factor 4; E2F5, E2F transcription factor 5; E2F8, E2F
tran-scription factor 8; MKP-1, MAP kinase phosphatase1; SKP2,
S-phase kinase-associated protein 2; p27, p27/Kip1 cyclin-dependent
kinase inhibitor.
Gene
RMI1+ ⁄ + RMI1+ ⁄ ) RMI1+ ⁄ + RMI1+ ⁄ )
Trang 8precise molecular mechanism underlying RMI1’s
regu-lation of E2F8 and its downstream targets has yet to
be clarified, our data indicate that RMI1 may be
essential for the E2F8-mediated proliferation of
prea-dipocytes In fact, a deficiency in RMI1 could lead to
decreased adiposity due to deficits in E2F-driven
prea-dipocyte proliferation However, other reports have
found that E2F8 reduces rather than induces cell
pro-liferation [26,27] Recently, Hagemann et al reported
that E2F8 has a novel function as a guanine nucleotide
exchange factor for heterotrimeric G proteins [28]
Given the disparity of these reports, elucidation of
E2F8’s functions and contribution to the regulation of
cell proliferation will require further experiments
Increased energy intake also leads to an increase in
the fat mass, which ultimately results in obesity The
deficiency in RMI1 significantly decreased the food
intake only under conditions of excessive energy diet
These results suggest that regulation of the energy
bal-ance by RMI1 is due to changes in the food intake
Peripheral secreted adipocytokines, such as leptin, can
regulate food intake via the central nervous system in
response to changes in body fat content [29] It is well
established that hypothalamic neurocircuits and signal
transductions modulate feeding behavior, thereby
regu-lating energy homeostasis [30] First, we investigated
the expression levels of RMI1 in the hypothalamus
The results showed that RMI1 expression was
signifi-cantly increased in the hypothalamus under high-fat
feeding conditions, and decreased under fasting
condi-tions Next we examined whether these changes in
feeding behavior were based on modulation of central
nervous system pathways Previous studies have shown
that several hypothalamic signaling factors, such as
neuropeptide Y and pro-opiomelanocortin, affect
feed-ing behavior via central nervous system pathways [30]
In the present study, we did not find any changes in
the expression levels of these factors; however, the
pos-sibility that RMI1 regulates other hypothalamic
signal-ing molecules cannot be ruled out
In summary, we have shown that RMI1 is a novel
regulator of energy homeostasis This suggests the
exciting possibility that an RMI1 modulator may
improve several disorders linked to energy
homeosta-sis, such as obesity
Experimental procedures
Establishment of mutant mice
The 0283 gene trap strain was isolated using a previously
described gene-trap method [31] The gene trap vector
pU_Hachi comprises a splice acceptor region (SA) from the
mouse En-2 gene, lox71, an internal ribosomal entry site, a
(b-geo), loxP, the SV40 polyadenylation sequence and pUC19 The vector was electroporated into embryonic stem cells
trapped clones were isolated The chimeric male mice were
to obtain F1 heterozygotes In this study, we used mice from
male (CLEA Japan) The genetic effects of the KK strain
exami-nation of the effects of high-fat feeding, 4-week-old mice were fed a diet in which 60% of the calories were from fat The components of this high-fat diet were determined using the method described by Ikemoto [32] Briefly, the high-fat diet contains 32% safflower oil, 33.1% casein, 17.6% sucrose, 1.4% vitamin mixture, 9.8% mineral mixture, 5.6% cellulose powder and 0.5% dl-methionine Casein, sucrose and the vitamin and mineral mixtures were purchased from Oriental Yeast Co Ltd (Tokyo, Japan), while the safflower oil was purchased from Benibana Food (Tokyo, Japan) and the dl-methionine from Wako Pure Chemical Industries Ltd (Tokyo, Japan) The caloric density of this diet is
490 kcal per 100 g, with fat energy of 294.7 kcal per 100 g (60.2%) The gonadal depots (representing intra-abdominal fat) and liver tissues of of killed mice were removed and weighed All animal procedures were performed in accor-dance with the international guidelines for biomedical research involving animals (Council for International Orga-nizations of Medical Science) and were approved by the Animal Ethical Committee of Astellas Pharma Inc
Characterization of the trapped gene The previously described plasmid rescue method was used
to obtain the genomic DNA fragment flanking the insertion site [31] DNA samples for genotyping were isolated from the severed tips of the mice tails Genotyping was performed
by PCR using tail genomic DNA as the template
Analysis of plasma constituents Plasma samples were taken from the severed tail tips Plasma glucose, triglycerides, HDL cholesterol and LDL cholesterol levels were determined using an enzyme assay method and Hitachi Autoanalyzer model 7170 (Hitachi Seisakusho, Hit-achi, Japan) The plasma insulin level was measured using an
experiment, levels of glucose, triglycerides, non-esterified
Trang 9fatty acids and ketone bodies were measured using the
glu-cose CII-test reagent, triglyceride G-test reagent, NEFA
C-test reagent and Autokit total ketone bodies reagent,
respec-tively (all from Wako, Osaka, Japan)
Glucose tolerance test
Oral glucose tolerance tests were performed using the
fol-lowing procedures After 16 h of fasting, the mice received
study), at time 0 Plasma samples for glucose
measure-ment were taken from the severed tail tips at 0.1, 0.5, 1.0
and 2 h
Expression analysis of mRNA
The tissue samples were pulverized in liquid nitrogen, and
the total RNA was extracted using an Isogen kit (Nippon
Gene, Tokyo, Japan) according to the manufacturer’s
instructions cDNAs were synthesized using SuperScript III
(Invitrogen, Carlsbad, CA, USA) Target mRNAs were
quantified via RT-PCR and the SYBR green method using
a PRISM 7900 sequence detector according to the
manu-facturer’s instructions (Perkin-Elmer Applied Biosystems,
Foster City, CA, USA) The level of mouse ribosomal
pro-tein (P0) was measured as an internal control The primers
for each target gene are listed in Appendix S1
Statistical analysis
The data represent the means ± SEM The statistical
sig-nificance of the difference between groups was determined
using Student’s t test P values < 0.05 were considered
sig-nificant Statistical and data analyses were performed using
the sas 8.2 software package (SAS Institute Japan Ltd,
Tokyo, Japan)
Acknowledgements
We thank Drs Kiyoshi Furuichi, Masao Kato,
Mas-ayuki Shibasaki, Hitoshi Matsushime, Masato Kobori,
Jiro Hirosumi and Mr Tsutomu Higashiya at Astellas
Pharma Inc., and Junko Kawano and Akemi
Mats-uoka at Trans Genic Inc for their helpful advice and
support
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Supporting information
The following supplementary material is available: Fig S1 Distribution of RMI1 mRNA in adult mouse tissues
Table S1 Gene expression analysis in the hypothala-mus of RMI1+⁄ + and RMI1+ ⁄) mice fed a normal
or high-fat diet for 14 weeks
Table S2 Biochemical findings
Table S3 Hematological findings and absolute organ weights
Table S4 Water field multiple T-maze test for lear-nings and open field test for behavior
Table S5 Histopathological findings
Appendix S1 Primers for each target gene
Appendix S2 Genomic DNA fragments obtained by plasmid rescue
This supplementary material can be found in the online version of this article
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