LECTURE
Functional genetic dissection of nuclear receptor signallingin obesity, diabetes and liver regeneration using spatiotemporally controlled somatic mutagenesis in the mouse
Takeshi Imai
Department of Physiological Genetics of Nuclear Signalling, IGBMC, Illkirch, France(Received for publication on February 13, 2003)

Abstract. The mouse is an excellent animal model for deﬁning human diseases. The null allele
mutations (knockouts, KO) have already provided valuable information about their functions, but havealso revealed major complications and difﬁculties: (1) an early embryonic lethality, (2) temporal effect(developmental stage or adult stage), (3) functional redundancy and (4) spatio-effect (cell-autonomousor non-autonomous). To overcome these limitations, spatio-temporally controlled somatic mutagenesis, Cre-ERT /LoxP system, was established. The nuclear receptors (NRs) play central roles in development, organogenesis, metabolism and energy homeostasis through their ability to transduce hormonal signals into modulation of gene activity. Obesity, excess energy storage in adipose tissue, has astrong link to diabetes. Among NRs, retinoid X receptor a (RXRa)-peroxisome proliferator-activatedreceptor g (PPARg) heterodimers can mediate adipocyte differentiation and obesity which has been
demonstrated with in vitro cell culture systems and RXR- and PPARg-speciﬁc ligand studies. Therefore an adipocyte-speciﬁc temporally controlled somatic mutagenesis system was established and analysed. Furthermore, the functional roles of NRs to control liver regeneration were also studied withsimilar system in hepatocytes. (Keio J Med 52 (3): 198–203, September 2003)
Key words: conditional somatic mutagenesis, nuclear receptor, obesity, diabetes, liver regeneration

Conditional Somatic Mutagenesis in the Mouse
The mouse, the genome of which is similar in size tothe human genome, is an excellent animal model for
deﬁning the functions of mammalian genes as it exhibits
genetic, immunological, reproductive, physiologic andpathologic similarities to man. The existence of techniques allowing the introduction of targeted mutationsin the mouse genome should considerably help in thedetermination of the function of mammalian gene(s)and the characterisation of pathogenetic factors involvedin numerous diseases, as well as produce good animalmodels for human pathologies.
The germ line mutations (knock-out, KO) in themouse have yielded remarkable advances in understanding the roles played by speciﬁc gene products in
mammalian development and adult physiopathology.
However, this strategy has some inherent limitations,which are due to the introduction of the mutation in
the germ line, as follows: (i) The lack of a protein thatserves essential functions in embryogenesis can result inearly lethality, thus precluding analysis of its possiblefunctions at subsequent stages (lethality). (ii) A numberof genes exert multiple functions in distinct cell types
during ontogeny and postnatally (pleiotropy). (iii) Pleiotropic abnormality may result in complex phenotypes
and in difﬁculties in distinguishing cell autonomousfrom more complex origins of abnormalities (cell autonomous or nonautonomous). (iv) The effect of germline mutation may also be compensated during development, thus preventing the appearance of an abnormal phenotype in the adult animal. In the case of
closely related genes belonging to multigene families,
it may be necessary to mutate several members of thefamily which precludes identiﬁcation of the function ofa given member of the family. Deﬁning the function ofPresented at the 1302nd Meeting of the Keio Medical Society in Tokyo, December 9, 2002.
Reprint requests to: Dr. Takeshi Imai, IGBMC, BP 10142, 1, rue Laurent Fries, 67404 Illkirch cedex, France, e-mail: imai@igbmc.u-strasbg.fr
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The strategies of conditional gene targeting in miceis based on the Cre/LoxP system, and makes use of cell/tissue-speciﬁc expression of chimeric Cre recombinases(Cre-ERT s), whose activity is induced by anti-estrogens
such as Tamoxifen (Tam). It was shown that Tam
administration to Cre-ERT transgenic mice efﬁciently
induced the excision of a chromosomally-integrated
LoxP-ﬂanked (ﬂoxed) DNA segment in most if not all
the cells expressing Cre-ERT , whereas no excision was
detected in the absence of ligand treatment. Using theprinciple of this technology, conditional gene ablationin adipocytes and hepatocytes are established (Fig. 3).1
The Nuclear Receptor Superfamily
Fig. 1 Germ line mutation and its limitation. Schematic ﬁgures ofwild type (WT) and germ line mutation in mice are shown. White andgrey represent wild type and germ line mutation, respectively. Problems related to germ line mutation are also listed.

this member may become even more difﬁcult when thefamily is involved in highly pleiotropic signalling pathways (functional redundancy). (v) Other potential effects confounding conventional KOs include the risk of
impaired fertility and generalized, systemic disorders.
In many instances, these limitations prevent the determination of the function of a given gene product in adeﬁned subset of cells at any given time during life
(Fig. 1). Thus, methods to achieve conditional gene inactivation are highly desired (Fig. 2).

Fig. 2 Spatio-temporally controlled somatic mutagenesis. Schematic
ﬁgures of WT, germ line mutation and spatio-temporally controlled
somatic mutagenesis are shown. Before application of inducer, micedevelop and grow as a wild type. By treatment of inducer (temporally
controlled) somatic mutagenesis occur in speciﬁc cell types (spatiocontrolled). White and grey represent wild type and genetic mutation,
respectively.

The last decade has seen an enormous rise in the interest in nuclear receptors (NRs) because of their central role in the coordination of development and metabolism. NRs can transduce hormonal signals intomodulation of gene activity.2 Thus, NRs are now seen
as integrators of multiple signals and stimuli that participate in the control of all complex processes in livingorganisms. The transcriptional activity of these receptors is controlled by classical hormones, vitamin anddietary nutrient fatty acids derivatives, with the exception of the so-called orphan receptors for which noligands have been identiﬁed.3 The complexity and network nature of hormonal signalling via NRs require
novel approaches and sophisticated tools in the integrated developing and adult organisms.
The Link Between Obesity and Diabetes
Obesity is a major health problem with more than
one billion overweight people in the world. The numberof obese among the Japanese is more than 23 million.
Obesity most likely accompanies adult custom disease,such as hypertension, hyperlipidemia, and especiallydiabetes. In Japan, more than 2 million people are patients and 7 million people are thought to be afﬂictedwithout diagnoses. The disease is rapidly becoming
more common among the Japanese. In patients of diabetes, the risk of heart disease and stroke are elevated
2–4 times. Moreover, diabetes is the leading cause ofend stage nephropathy, neuropathy and retinopathy.
While the human and economic costs of diabetes are
difﬁcult to calculate, the total medical costs incurredannually in the U.S.A. are more than $100 billion.4
Diabetes is deﬁned as a situation in which carbohydrate and lipid metabolism are disordered by insulin.
There are 2 major categories of the disease, types 1 and2. The number of type 1 diabetes patients may accountfor 5–10% of all the diabetes cases. The cause of type1 disease is thought to result from the destruction of
the insulin-producing b cells of the islets of Langerhans,

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Imai T: Conditional mutagenesis in adipocyte & hepatocyte

with rexinoid and TZD indicated that RXR-PPARg
heterodimers could play central roles in adipocyte, particularly in adipocyte differentiation and hypertrophy.5
RXRa and PPARg are highly expressed in adipocyte,
as well as other tissues. The classic loss-of-function
experiments for RXRa and PPARg have proved difﬁcult because homozygous null mice do not survive past
the embryonic stage.9,10 Therefore, mice in which
RXRa and PPARg are selectively ablated in adipocytes
were established to study the link between obesity anddiabetes.
Functional Role of the RXRa in Adipocytes
Fig. 3 The Cre-ERT2 /LoxP system. Chimeric recombinase (CreERT2 ) is a fusion protein between Cre recombinase (empty box) andmutated ligand binding domain of oestrogen receptor a (grey box).
The loxP (ﬁlled triangle) ﬂanked (ﬂoxed) gene is excised by Cre-ERT2
binding to tamoxifen (TAM). No recombination is observed in theabsence of both factors. (DBD: DNA binding domain, dot box).

and type 1 diabetes patients are lead to insufﬁcient of
insulin production. Type 2 diabetes is far more common
and caused from a combination of defects in insulin
action and secretion.
The association of obesity with type 2 diabetes has
been recognized for decades, especially in USA andEurope. The retinoid X receptors (RXRs) and the peroxisome proliferator-activated receptor g (PPARg) inadipocyte may play key roles in both obesity anddiabetes. Therefore, the functional roles or link ofthese receptors to these diseases were analysed byadipocyte-speciﬁc conditional mutagenesis. PPARg is afunctional receptor for an interesting class of insulinsensitizing drugs called thiazolinediones (TZDs), which
are currently used in the treatment of type 2 diabetes
mellitus.5 RXR agonists (rexinoids) also have insulinsensitizing activity in vivo. However, the target cells ofthese compounds are not known.
In vertebrates, the adipose tissue is critical for energy homeostasis.6 Whereas the brown adipose tissue
can dissipate energy through thermogenesis, the white
adipose tissue (WAT) stores excess energy in the formof triglycerides, when caloric intake exceeds expenditure, and release free fatty acids when expenditure exceeds intake. The function of adipose tissue generally
depends on their amount, and adipose tissue amount
can be modulated by the formation of new adipocytes
from precursor cells (adipocyte differentiation) and/or
by increasing in adipocyte size (adipocyte hypertrophy). In the past adipose tissue was only largely regarded as a depot for fuel storage, however, adipocyte
produce a number of enzymes and regulatory proteins
involved in lipogenesis and lipolysis, as well as secreted
paracrine and endocrine factors,7 called adipokines.8
Adipocyte differentiation has mainly been studied
using preadipocyte culture systems.6 In vitro studies

To perform conditional somatic mutagenesis in adipocytes, we have generated aP2-Cre-ERT2 transgenic
mice expressing Cre-ERT2 selectively in adipocytes. We
have shown that Tam efﬁciently and selectively induces ﬂoxed DNA excision in adipocytes. We havetherefore performed conditional mutagenesis of RXRain adipocytes using aP2-Cre-ERT2 line. The analysisof mice in which RXRa was selectively ablated in adipocytes of adult mice (RXRaad = mice) allowed us
to demonstrate that RXRa plays a key role in adipocyte differentiation, in adipogenesis and in lipolysis.
Indeed RXRaad = mice were resistant to dietary- andchemically-induced obesity, and were impaired in fasting-induced lipolysis.11 Moreover, RXRaad = mice
showed hyperglycemia with insulin resistance underregular diet with normal adiposity. Interestingly among
adipokines, TNFa, leptin and adiponectin expression,
which is disregulated in diabetic animal models andhumans, was also impaired in RXRaad = mice. Using
micro array analysis comparing the adipose transcriptome of RXRaad = and control mice, a gene expression proﬁle was obtained and analysed. Three kind ofgene clusters were identiﬁed, they are insulin signalling,
secreted proteins and lipolysis genes. Apparently WATof mutated mice impaired insulin-stimulated glucose
uptake. Some genes that activate glucose uptake inadipocyte were down regulated in RXRaad = mice,such as GLUT4, CAP, glycogen synthase and so on. It
suggests that RXRa regulates insulin signalling in adipocytes through the regulated expression of some genes
which stimulate insulin signalling. Moreover, insulin
resistance in muscle and liver are also observed in themice with ablated RXRa in their adipocytes. Theseabnormalities are due to adipokines. In addition to theresult of impaired expression of adipokines describedabove, some new candidates of possible adipokines
which are up or down regulated in RXRaad = mice
have been obtained from gene proﬁling analysis. Detailed analyses of their expression patterns are being
performed, and the function of the promising ones will
be analysed in transgenic animals either by overex-Keio J Med 2003; 52 (3): 198–203

pression or conditional mutagenesis. These new adipokines will be the new targets for medical application oftype 2 diabetes patients.
Taken together, RXRa plays a key role in adipocyte
differentiation, adipocyte hypertrophy and lipolysis,
and regulates key genes for glucose homeostasis in
adipocytes. Moreover, RXRaad = mice are a diabetic
mouse model with normal adiposity, suggesting that
RXRa is one of the molecules which dissociate obesityfrom diabetes.
Functional Role of the RXRs and the PPARg
in Adipocytes
As RXRg is upregulated in adipocytes in RXRaad =mice,11 and might therefore partially compensate forthe loss of RXRa function, we disrupted both RXRaand RXRg in adipocytes. Surprisingly RXRa and
RXRg ablated adipocytes (RXRagad = mice) induced
lipodystrophy, thus indicating that RXRs are essential
for the maintenance/survival of adipocytes.
To analyse PPARg function in adipocytes, we selectively ablated PPARg in adipocytes of adult mice using
a similar strategy (PPARgad = mice). As describedabove in RXRa and RXRg ablated adipocytes, PPARg
ablation induced lipodystrophy, indicating that PPARg
is not only involved in adipocyte differentiation, butalso in adipocyte maintenance/survival, and that RXRaand RXRg are its heterodimeric partners.
New Signalling Pathway in Lipodystrophy
Conditional genetic mutation of PPARg or RXRs
alleles in adipocytes induces lipodystrophies. Lipodystrophies are a heterogenous group of adipose tissue
disorders characterized by selective loss of fat fromvarious parts of the body, and associated with insulin
resistance, hypertriglycemia, hepatic steatosis and early
onset of diabetes in humans and mice. Genetic mutation of Seipin, AGPAT2, Lipin and Lamin A/C locilead to lack or reduced number of adipocytes in humans and mice.12–17 Genetic mutation of Seipin or
AGPAT2 lead to one type of human lipodystrophy,
congenital generalized lipodystrophy, and Lamin A/Cmutation leads to another type of lipodystrophy, called
Dunnigan type familial partial lipodystrophy, thus several signalling pathways may lead to a variety of lipodystrophies.
To identify the cascade from PPARg/RXRs function
leading to lipodystrtophy in mice, expression of Seipin,
AGPAT2, Lipin and Lamin A/C were analyzed in
PPARgad = and RXRagad = mice. Surprisingly, theexpression of all the genes are impaired in both mice,thus one signalling pathway (PPARg/RXRs) regulates

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various types of lipodystrophies. This master regulatorof lipodystrophies is PPARg and RXRs heterodimers.
It may suggest linkages between different types of lipodystrophies with PPARg/RXRs signalling.
NR Signalling in Obesity, Diabetes and Lipodystrophy
NR signalling pathways regulate a number of adipocyte functions. RXRa is a key player in the link between obesity and diabetes. Using conditional ablationof RXRa in adult adipocyte leads to diabetes without obesity, thus diabetes and obesity are distinguishedby RXRa function in adipocytes. Moreover, PPARg/
RXRs are master regulators for a variety of lipodystrophies.
Functional Roles of NRs in Liver Regeneration
The liver is a vital organ which performs critical
functions including energy homeostasis, protein synthesis, bile secretion and detoxiﬁcation. It has an optimal functional mass that is set in relationship to thebody size. The highly differentiated adult hepatocytes
have a half-life of several months. However, they canvery rapidly begin to replicate in response to conditions
that induce cell loss by physical, infectious or toxic injury, until the liver regains its functional mass, andreturns to a non-proliferative state. The exact signalsthat control the functions and the size of the liver are
not clearly deﬁned.
To perform conditional somatic mutagenesis in hepatocytes, we generated transgenic mice that express
Cre-ERT under the control of the a-anti-trypsin promoter (aAT-Cre-ERT ). We selected one line which
expresses selectively the tamoxifen-inducible Cre-ERT
recombinase in more than 50% of the hepatocytes, andefﬁciently induced recombination of ﬂoxed DNA segments after Tam treatment.18
Functional Role of RXRa in Hepatocytes
Many members of the NR superfamily including
RXRs are expressed in the liver, as well as a wealth ofgenes that contain response elements for RXR-NR
heterodimers. As RXRa is the most abundant of thethree RXR isotypes in the liver, it may play a prominent role in hepatic growth, regeneration and homeostatic functions. The possible postnatal role of RXRa
could however not be analysed in knock-out mice lacking RXRa (RXRaKO), as RXRaKO fetuses die at
E13.5–E16.5.9
Using aAT-Cre-ERT mice, as well as transgenic miceexpressing Cre under the control of the albumin promoter selectively in hepatocytes (Alb-Cre; gift from A.

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Imai T: Conditional mutagenesis in adipocyte & hepatocyte

Magnusson), RXRa was efﬁciently and selectively
ablated in hepatocytes either after Tam treatment, orin a constitutive manner during development, respectively. We have demonstrated that RXRa plays important cell-autonomous functions in the mechanism(s) involved in the physiological lifespan of hepatocytes andduring liver regeneration after partial hepatectomy(PH). Our results show that the lifespan of adult hepatocytes lacking RXRa is shorter than that of theirwild-type counterparts. Moreover, proliferative hepatocytes of a regenerating liver lacking RXRa exhibitedan even shorter lifespan. This shortening of the lifespan
was accompanied by increased polyploidy and multinuclearity.19
Estrogen Signalling Triggers Liver Regeneration
Several lines of evidence indicate that the liver is anoestrogen target tissue in vertebrates. Indeed, the levelof a number of liver proteins, including coagulation
factors and ﬁbrolytic proteins, sex hormone binding
globulin are regulated by oestrogens in mammals.20
Moreover, in vitro studies suggest a supportive role for
estrogens in hepatocyte proliferation, and estrogens
have been shown to be involved in the control of liver
growth in mammalian neonate, and to stimulate hepatocyte proliferation and liver weight recovery after PHor liver transplantation.21 Most of the actions of oestrogens are mediated by two NRs, the oestrogen receptor a (ERa) and b (ERb). However, in the liver,
only ERa has been shown to be expressed.
To investigate the oestradiol signalling pathway inhepatocytes, we analysed ovariectomized and E2treated WT and ERa-null mice,22 as well as mice inwhich ERa is selectively ablated in hepatocytes(ERahep = mice) obtained with Alb-Cre mice. The
study demonstrated that E2 is an early signal, upstreamof TNFa, IL-1a, IL-1b and NF-kB that is released after
PH and stimulates hepatocyte proliferation, and that
hepatocyte ERa plays a central role for transducing theE2 proliferation signal in hepatocyte as well as Kupffer
cells before and after PH, in both males and females.
Moreover short oestrogen treatments might possibly be
used in the clinic to improve liver regeneration.
Conclusion
The results demonstrate the feasibility of targeted
spatio-temporally-controlled somatic mutagenesis in
hepatocytes and adipocytes. Importantly, the established systems are not only valuable to study NR function, but also to investigate the function of any gene inthese cell types. The studies should provide animalmodels for a number of human diseases, in addition tothat we have already established.

Acknowledgments: I would like to thank Drs Pierre Chambon andDaniel Metzger for their support of work and discussion and Dr
Nicole Clark for critical reading of this manuscript.

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