Pre-screened
Human
Adipocytes
Adipocytes,
which vary enormously in size (20-200 µm in
diameter), are embedded in a connective tissue
matrix and are uniquely adapted to store
and release energy. There are two types of adipose
tissue: white adipose tissue (WAT) and brown adipose
tissue (BAT). These two tissues are
made up of two types of fat cells. WAT is the major
energy reserve in higher eukaryotes, storing
triacylglycerol in periods of energy excess
and mobilizing during energy deprivation.
Until recently, the adipocyte has
been considered only a passive tissue for the
storage of excess energy in the form of
fat. However, there is now compelling
evidence that adipocytes act as endocrine secretory
cells. It has been shown that several
hormones, growth factors, and cytokines
are actually expressed in white adipose tissue. Through a network of local and systemic signals,
which interact with neuroendocrine regulators,
adipose tissue signaling pathways,
arranged in a hierarchical fashion, constitute
one of the voices of the body that enable the
organism to adapt to a range of different
metabolic challenges such as starvation,
stress, and infection, as well as periods of gross
energy excess.
For
the last 20 years, the cellular and molecular
mechanisms of adipocyte differentiation
have been extensively studied using preadipocyte
culture systems,
as human fat
adipocytes die within 24 hours after
isolation. Committed preadipocytes undergo growth
arrest and subsequent terminal differentiation into
adipocytes. This is accompanied by a
dramatic increase in expression of adipocyte genes
including adipocyte fatty acid binding protein and
lipid-metabolizing enzymes.
At Cell Applications, Inc., primary cultures of
adipocytes were prepared by inducing differentiation
of human primary pre-adipocytes and
were pre-screened for expression of these
important adipocyte functional proteins.
Peroxisome
proliferator-activated receptor-g
(PPAR-
g)is an adipocyte-specific
nuclear hormone receptor, is expressed at a high level in adipose tissue, and
plays an important role in adipocyte
differentiation.
PPAR-g is a specific marker for
adipocytes. CCAAT/enhancer binding
protein a (C/EBPa
) is another transcription factor involved in
creating and maintaining the adipocyte phenotype. C/EBPa
was found to be expressed at high levels in
both white and brown fat of rodents and observed to
accumulate during adipocyte conversion with temporal
kinetics concordant with acquisition of
the differentiated phenotype. It has shown that C/EBPa
regulates terminal adipocyte differentiation,
turning on the battery of
fat-specific genes required for the
synthesis, uptake, and storage of long chain fatty
acids. Both high
level expression of PPAR-g
and C/EBPa
proteins was detected in our pre-screened human
adipocytes but not in the non-differentiated
pre-adipocytes (Fig. 1A and B respectively). Insulin plays a critical role in induction of adipocyte
differentiation as well as its functional
regulation. It was shown that insulin receptor
expression was dramatically increased in the
pre-screened human adipocytes as compared to that in
non-differentiated pre-adipocytes (Fig. 1C).
However, there is no change of Akt expression
between pre-adipocytes and adipocytes (Fig. 1D).
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| 1A |
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PPAR-g
Antibody
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| 1B |
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C/EBPa
Antibody
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| 1C |
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Insulin
Receptor Antibody
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| 1D |
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Akt
Antibody
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Fig.
1. Both undifferentiated human pre-adipocyte (lane
1) and differentiated pre-screened adipocyte (lane
2) lysates were prepared and subjected to Western
blot analysis. Two PPARg
isoforms (53 kDa g1
and 57 kDa g2)were
highly expressed in pre-screened adipocytes.
In pre-adipocytes only very low level
53 kDa g1
was detected (A).
C/EBPa (p42 and p30) protein expression was
significantly induced in differentiated pre-screened
adipocyte (B). Insulin receptor expression was also
increased in differentiated pre-screened
adipocytes (C). The Akt protein levels in
both cells were almost same (D).
The
key enzymes for metabolic regulation in adipocytes
were also screened in our adipocyte preparations.
Acetyl-CoA carboxylase-alpha (ACC-a)
plays a central role in lipogenic capability
of adipocytes. It is a key enzyme for co-ordinating
de novo fatty acid synthesis. Our results showed
that ACC-a
was highly expressed in in our
pre-screened human adipocytes, and
its level was low in the non-differentiated
pre-adipocytes (Fig. 2B).
Glycogen synthase kinase-3 (GSK-3) is a
ubiquitous kinase implicated in both
insulin action and adipogenesis. It not only
regulates glycogen synthesis, but also plays an
important role in regulation of other adipocyte gene
expression as well as adipocyte cell signaling. There are high levels of GSK-3b proteins
detected in our pre-screened human adipocytes and
its protein level is comparably lower in the
undifferentiated pre-adipocytes (Fig. 2A).
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| 2A |
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GSK
Antibody
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| 2B |
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ACC-a
Antibody
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Fig.
2.
Two enzymes, Acetyl-CoA carboxylase-alpha
(ACC-a)
(A) and Glycogen synthase kinase-3 b
(GSK-3b)
(B) were detected in both undifferentiated human
pre-adipocyte (lane 1) and differentiated
pre-screened human adipocyte (lane 2) lysates by
Western blot analysis. Both enzyme expressions were
significantly increased in the pre-screened
adipocytes.
Adipocytes
are known to express and secrete a variety of
bioactive adipokines. Adiponectin is a adipokine
that plays a critical role in metabolic regulation. It
is highly and specifically expressed in
differentiated adipocytes. Our results showed that
a 30-kDa adiponectin protein was detected in
our pre-screen human adipocyte lysate, but not in
the undifferentiated pre-adipocyte lysate (Fig.
3).
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3A |
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Adiponectin
Antibody |
Fig.
3. Adiponectin expression was detected by Western
blot in the differentiated pre-screened human
adipocyte (lane 2), but not in the pre-adipocytes
(lane 1).
Adipocytes
are major insulin response cells.
Insulin is not only critical to adipocyte
differention, but also is a key metabolic regulator
for adipocytes. Thus, insulin response will be a
characteristic assay for adipocyte function.
Our results showed that the pre-screened
adipocytes respond well to insulin stimulation. As
shown in Fig. 4A, after starvation, insulin
stimulates significant tyrosine phosphorylation of
insulin receptors in pre-screened human adipocytes.
The auto-phosphorylation resulted in high activation
of insulin receptor kinase activity and down-stream
signaling.
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4A |
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Phospho-Insulin
Receptor (T1165/6) Antibody |
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4B
Insulin(100nM,
5 min)
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Insulin
Receptor Antibody
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Fig.
4. The differentiated pre-screened human adipocytes
were stimulated by insulin. The cell lysates were
subjected to Western blot analysis.
The tyrosines 1165/6 of insulin receptors
were detected in the insulin stimulated, but not
un-treated cells using phosphor-insulin receptor
(Y1165/6) antibody (A). the insulin receptor protein
levels did not change before and after insulin
stimulated , detected by using Insulin receptor
antibody (B).
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