Molecular medicine-Differentiation of Adipocytes from Precursor Cells

Together with adipocyte size, the number
of adipocytes in the body is an important
determinant of obesity and of multiple
parameters of energy metabolism. The
number of adipocytes present in an organism
is determined to a large degree
by the adipocyte differentiation process
that generates mature adipocytes from
fibroblast-like preadipocytes. Many of the
molecular details of this process are now
known, and the following section summarizes
our current understanding of the
molecular control of adipogenesis. It is
important to note that our understanding
of how adipocytes are generated from
precursor cells is based primarily on cell
culture models of adipogenesis such as
the mouse 3T3L1 cell line. While these
cell lines are very amenable to experimentation,
they produce adipocytes that are
strikingly different in some respects than
native adipocytes found in adipose tissue
in vivo. For example, fully differentiated
3T3L1 adipocytes are multilocular (contain
multiple lipid droplets), while native
adipocytes in white fat (the predominant
type of adipose tissue in humans) display
a unilocular distribution of lipid (compare
Figs. 2 and 3). While we know that many
of the characteristics of adipocyte differentiation
in cultured cell lines are also
important features of in vivo adipogenesis,
it is important to bear in mind that
some aspects of adipogenesis that have
been learned from cell culture systems,
as described below, may differ from the
process as it occurs in vivo.
When cultured preadipocyte cultures are
grown to confluence and cease cellular
division (growth arrest), they can be induced
to differentiate into adipocytes by
treatment with an adipogenic hormonal
cocktail containing insulin, dexamethasone,
and an inducer of intracellular cAMP
concentration. One of the first steps in
the process of adipogenesis is the reentry
of growth-arrested preadipocytes into
the cell cycle and the completion of several
rounds of clonal expansion. Multiple
genes involved in the cell cycle control are
required for this step to proceed, including
the tumor suppressor retinoblastoma
protein (Rb) and several cyclin-dependent
kinases and their inhibitors (p18, p21, and
p27). This and the subsequent steps of the
program of adipogenesis are controlled,
to a large degree, by a cascade of gene
expression events regulated by a small
set of transcription factors. Two families
of transcription factors have emerged as
the key determinants of this process: the
three CCAAT/enhancer-binding proteins
C/EBP α, β and δ, and the two-peroxisome
proliferator-activated receptors gamma-1
and gamma-2 (PPARγ 1 and PPARγ 2).
One of the initial steps in the transcriptional
cascade in response to adipogenic
signals is the rapid induction of C/EBP
β and δ expression. These transcription
factors orchestrate cell cycle reentry by
stimulating the expression of the CDK inhibitor
p21, which acts to inhibit the Rb
protein and relieve its block on cell cycle
progression. C/EBP δ and β have also been
shown to induce expression of the gene for
the PPARγ transcription factor that plays
12 Adipocytes
Fig. 3 Fluorescence micrograph of a cultured adipocyte expressing EGFP–perilipin
fusion protein. A C3H 10T1/2 mesenchymal cell was differentiated in culture and
transfected with an expression vector encoding perilipin fused to enhanced green
fluorescent protein. Note the localization of the fluorescence to surface of the numerous
lipid droplets (see color plate p. xxi).
a key role in the terminal differentiation
of adipocytes (discussed in more detail below).
The importance of C/EBP β and δ
for adipogenesis was clearly demonstrated
by loss-of-function and gain-of-function
genetic studies in mice. Overexpression
of either C/EBP β or δ in preadipocytes
enhanced adipogenesis, while embryonic
fibroblast cells derived from mice lacking
either C/EBP β or δ had reduced levels of
adipogenesis compared with wild type.
The induction of C/EBP β and δ is
immediately followed by an increase in
PPARγ and C/EBPα expression. PPARγ
is a member of the nuclear hormone
receptor family of ligand-activated transcription
factors. It is absolutely required
for adipocyte differentiation, as a genetic
knockout of the PPARγ gene in mice prevents
the development of all fat tissue.
In addition to its crucial role in adipocyte
differentiation PPARγ is the receptor for
the thiazolidinedione (TZD) class of antidiabetic
drugs, indicating that it is also
important in metabolic regulation in adult
organisms. In mice and humans there
are two isoforms, PPARγ 1 and PPARγ 2,
which are derived from the same gene by
Adipocytes 13
alternative promoter usage and RNA splicing.
While the expression of PPARγ2 is
restricted almost exclusively to adipocytes,
PPARγ 1 has a broader pattern of expression
although it is still most abundant in
adipocytes. Although PPARγ2 is identical
to PPARγ1 except that it contains an additional
28 amino acids on its N-terminus, it
appears that the two proteins have distinct
activities with regard to adipocyte
differentiation. When the expression of the
PPARγ 2 isoform was blocked, adipogenesis
was more strongly inhibited than when
the PPARγ 1 isoform was blocked. In addition,
exogenous delivery of PPARγ 2 into
PPARγ deficient cells was able to completely
restore the adipogenesis, whereas
overexpression of PPARγ 1 had little effect.
It may be that PPARγ 1, which is already
expressed in preadipocytes, behaves as a
priming factor (along with C/EBP β and δ)
for the induction of PPARγ2or for the generation
of endogenous PPARγ ligands that
play a role in later stages of adipogenesis.
As the program of differentiation proceeds,
the expression of C/EBPα rises
immediately after the increase in PPARγ 2
expression. Like PPARγ, C/EBPα also
plays an essential role in adipose development
as targeted gene knockout in mice
results in embryonic lethality and failure
to develop normal adipose tissue. There
has been an intense research effort to understand
the relationship between these
two transcription factors and the role they
play in adipogenesis. Several studies have
demonstrated that PPARγ 2 and C/EBPα
coregulate each other’s expression. Mice
with reduced PPARγ expression due to
heterozygous gene knockout displayed a
drastically reduced level of C/EBPα, and
mice with disrupted C/EBPα expression
showed a reduced level of PPARγ. Introduction
of either PPARγ or C/EBPα
into NIH3T3 cells is sufficient to convert
these normally nonadipogenic cells from
fibroblasts into adipocytes. However, it is
unclear if either of the transcription factors,
completely on its own, could induce
adipogenesis. Taken together, most of the
recent evidence supports the model that
while both of the transcription factors work
coordinately to carry out adipogenesis,
PPARγ 2 probably plays the primary role,
while C/EBPα may act mostly by inducing
and maintaining PPARγ 2 expression.
C/EBPα may also function to regulate
the transcription of genes involved in the
metabolic actions of insulin such as glucose
transporter 4 (Glut 4). Clearly,PPARγ
and C/EBPα are key transcription factors
in adipogenesis, acting synergistically
to generate fully differentiated, insulinresponsive
adipocytes.
Although our understanding of adipocyte
differentiation, as described above, is
derived from work in cultured cell lines, it
is likely that many of the pathways and key
components also play an important role in
generating adipocytes from precursor cells
in vivo and in the remodeling of adipose
tissue as it occurs during certainmetabolic
stress (see Sect. 3, above) or treatment
with specific pharmacological agents (see
Sect. 5, below)