1. Enhanced brown adipose tissue differentiation in PTP1B knockout
samples analyzed with Oil Red O staining
Aroldo M. Trejo
ABSTRACT
Background: PTP1B has been associated with inhibitory effects on insulin signaling and brown
adipose tissue (BAT) differentiation.
Objective: The objective of our study was to determine whether a lack of PTP1B would enhance
BAT differentiation. We hypothesized that the knockout treatment (hPTP1B KO) and the
catalytically impaired treatment (hPTP1B D/A) would display greater differentiability compared
to the WT treatment (hPTP1B WT).
Design: PTP1B in BAT was knocked out in rats and various treatments of human PTP1B were
reconstituted in the cells, resulting in hPTP1B WT, hPTP1B D/A, and hPTP1B KO conditions.
After eight days of differentiation, differentiated cells for each treatments (n=2) were stained
with Oil Red O. The Oil Red O was eluted and quantified using spectrophotometry, using
triglyceride (TG) quantification as a marker of differentiation. Differentiated and
undifferentiated cells of each conditioned were lysed and immunoblotted for determination of
PTP1B presence.
Results: The hPTP1B KO and hPTP1B D/A treatments yielded greater differentiation (152%
and 154% respectively) compared to the hPTP1B WT condition (136%). However, there was no
significant difference in differentiation patterns among the three treatments (p>0.05).
Conclusion: PTP1B plays a crucial role in inhibiting BAT differentiation. The results from our
study add to the growing evidence that PTP1B inhibition can play an important role in the
management of the obesity and diabetes epidemic, providing a foreground for PTP1B-targeted
therapeutic advancements in the near future.

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INTRODUCTION
Protein tyrosine phosphatase 1B (PTP1B) is an inhibitory protein that has been analyzed
for its mediatory role in brown adipose tissue (BAT) differentiation (1, 2). BAT has been
characterized by increased mitochondrial concentration and increased energy expenditure
compared to white adipose tissue (WAT) (3), making it an interest in PTP1B studies. Studies
have found that PTP1B can be knocked out in mice BAT, and human PTP1B (hPTP1B) can be
reconstituted into the knockouts for research on how PTP1B innervates BAT differentiation (1,
2). The targets of PTP1B can be analyzed utilizing a substrate-trapping PTP1B (hPTP1B D/A),
which acts similar to the KO due to its catalytic inactivation (1, 2). PTP1B differentiation can be
studied using Oil Red O stain in order to stain triglycerides (TG) and quantify the amount of
differentiation in the cells (1, 4). Presence for hPTP1B presence in reconstituted cells can be
determined utilizing SDS-PAGE protein separation and immunoblotting utilizing a PTP1B
antibody (5).
Previous studies have determined PTP1B’s role in inhibiting BAT differentiation and
decreasing energy expenditure in rat models (6). It has also been determined that a lack a PTP1B
improves insulin signaling, providing a gateway for treatment against insulin resistance (7). The
objective of our study was to add to the growing evidence that PTP1B is a crucial inhibitory
protein in BAT differentiation in order to provide a foreground for possible therapeutic studies
targeting PTP1B, thus providing and effective treatment against obesity and diabetes. We studied
this using three treatments of reconstituted PTP1B: hPTP1B knockout (KO), a substrate-binding
hPTP1B (D/A), and a wild type hPTP1B (WT). We hypothesized that hPTP1B KO and D/A
treatments would yield enhanced BAT differentiation compared to hPTP1B WT cells.

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METHODS
Oil Red O Staining of Cultured Cells
Intrascapular BAT cells were obtained from PTP1B knockout mice and immortalized.
The immortalized cells were transfected with either human PTP1B (hPTP1B WT), hPTP1B -/-
(hPTP1B KO), or substrate-trapping hPTP1B D/A. Six samples of each condition were cultured
in respective 35mm plates, three samples from each treatment were differentiated for eight days.
Each sample was subject to Oil Red O staining. The plates were washed with 2.0 mL of
PBS, followed by a 2.0mL formalin wash (27.0mL 37% Merck Cat# K36658003 + 63.0mL
ddH20 + 10.0mL 10xPBS), and a 15.0 minute incubation period at room temperature. After
removal of formalin solution, each sample was washed twice with 2.0 mL ddH20, followed by a
5.0 minute washing period with 60% isopropanol (Merck Cat# K36543834). The cells were then
dried for 15.0 minutes, followed by a 15.0 minute incubation at room temperature with working
Oil Red O Solution (Sigma (Cat# O-0625) 0.35g/100mL isopropanol + 4.0mL ddH20). Cells
were washed immediately four times with 2.0mL ddH20, followed by a 5.0 minute drying period.
The Oil Red O was eluted with 1.0mL of 100% isopropanol for 10.0 minutes. The resulting
solution was pipetted into a 1.5mL eppendorf tube and measured with a spectrophotometer at
500nm with a 100% isopropanol blank.
Standard Calibration Curve Preparation
The standard calibration curve was prepared by diluting the standard TG stock solution to
the following concentrations: 1.00, 0.20, 0.10, 0.05, and 0.02 mg/mL. The stock solutions were
measured at 500nm with a spectrophotometer with a 100% isopropanol blank.
Cell Lysis and Protein Extraction
One undifferentiated and one differentiated sample of each treatment was washed with
2.0 mL PBS solution. 1.0 mL of RIPA buffer was added to each plate, cells were scraped into the

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buffer and transferred to a small centrifuge tube. Samples were iced for 20.0 minutes and
vortexed every 3.0 minutes. Samples were centrifuged in Sorvall machine (SS3 4 rotor, 1700
rpm) for 10.0 minutes. The supernatant was used for protein assays, ran with SDS-PAGE, and
transferred to a cellulose sheet for western blot with an hPTP1B antibody as described in
previous protocols (1).
Statistical Analysis
Averages, standard deviations, and two sample paired t-test significant values were
calculated with Microsoft Excel formulas.
RESULTS
Standard Calibration Curve
A standard calibration curve created by averaging the absorption of various TG dilution
standards from the subgroups is depicted in Figure 1. TG concentrations of 0.02, 0.05, 0.10,
0.20, and 1.00 mg/ml yielded average absorption values of 0.04, 0.08, 0.14, 0.34, and 1.60
respectively. The five point calibration curve yielded an R2
value of 0.9996 and a correlation
equation of y=1.5998x.
Figure 1. Five-point standard calibration curve using the average absorption values of various
TG dilutions obtained from the six treatment groups.
y
=
1.5998x
R²
=
0.9996
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
0.00
0.20
0.40
0.60
0.80
1.00
1.20
Absorp'on
Amount
of
TG
(mg/mL)
Standard
Calibra'on
Curve

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Figure 2. hPTP1B protein
western blot detection of hPTP1B
wildtype (WT), knockout (KO),
and substrate (D/A) treatments.
Cell Lysis and hPTP1B Western Blot
Cell lysis and western blot analysis of hPTP1B KO,
hPTP1B D/A, and two hPTP1B WT conditions with the hPTP1B
protein are depicted in Figure 2. The hPTP1B WT and
hPTP1B D/A treatments displayed expression for the hPTP1B
protein, while the hPTP1B KO treatment did not.
hPTP1B WT, KO, & D/A TG Quantification
The hPTP1B KO treatment conditions yielded the highest average differentiated and
undifferentiated cell TG quantifications (1.36 +/- 0.34 and 0.53 +/- 0.09 mg/ml respectively).
The hPTP1B WT condition yielded the least amount of TG compared to all treatments for
differentiated and undifferentiated cells (0.39 +/- 0.07 and 0.19 +/- 0.11 mg/ml respectively).
The hPTP1B D/A treatment yielded an average TG quantification of 0.73 +/- 0.12 and 0.29 +/-
0.06 mg/ml for differentiated and undifferentiated cells respectively.
Table 1. Undifferentiated/differentiated cells absorbance values and triglyceride quantifications
for PTP1B wild type (hPTP1B WT), knockout (hPTP1B), and substrate-trapped (hPTP1B D/A)
treatments. There was no significant difference between differentiated and undifferentiated TG
quantifications in all treatments (p>0.05).
Treatment
Undifferentiated
Cells Absorbance
Undifferentiated
Cells TG (mg/ml)
Average
TG
(mg/ml)
Differentiated
Cells
Absorbance
Differentiated
Cells TG
(mg/ml)
Average
TG
(mg/ml)
P-Value
(Diff. vs.
Undiff.)
hPTP1B
KO
0.958 0.60 0.53 +/-
0.09
2.56 1.60 1.36 +/-
0.34 0.130.753 0.47 1.79 1.12
hPTP1B
WT
0.439 0.27 0.19 +/-
0.11
0.704 0.44 0.39 +/-
0.07 0.100.179 0.11 0.549 0.34
hPTP1B
D/A
0.403 0.25 0.29 +/-
0.06
1.034 0.65 0.73 +/-
0.12 0.070.533 0.33 1.314 0.82
* Indicates significance at alpha=0.05

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A visual representation comparing the undifferentiated and differentiated TG
quantifications for the three treatment groups is depicted in Figure 3. The hPTP1B KO and
hPTP1B D/A treatments yielded greater BAT proliferation (152% and 154% respectively)
compared to the hPTP1B WT condition (136%). There was no significant difference between
differentiated and undifferentiated TG quantifications in all treatments (p>0.05). No significant
differences were found between hPTP1B KO and hPTP1B WT (p=0.88), hPTP1B KO and
hPTP1B D/A (p=0.87), or PTP1B WT and hPTP1B D/A BAT proliferation (p=0.86) at an alpha
level of 0.05.
Figure 3. Average undifferentiated vs. differentiated cells triglyceride quantifications for PTP1B
wild type (hPTP1B WT), knockout (hPTP1B), and substrate-trapped (hPTP1B D/A) treatments.
DISCUSSION
Cell Lysis and hPTP1B Western Blot
Cell lysis utilizing the RIPA buffer allowed for the degradation of the cellular membranes
of the triglycerides and the ability to access the proteins in each cell (Figure 2) (8). SDS-page
and western blot allowed us to separate the proteins by size and analyze the presence of hPTP1B
0
0.5
1
1.5
hPTP1B
KO
(n=2)
hPTP1B
WT
(n=2)
hPTP1B
D/A
(n=2)
Amount
of
TG
(mg/ml)
Treatment
TG
Quan'fica'on:
Undifferen'ated
vs.
Differen'ated
Cells
UndifferenCated
Cells
DifferenCated
Cells

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proteins in all three treatments by running an antibody for hPTP1B on the resulting blots (5).
After visualization of the antibody presence, we determined the hPTP1B protein was present in
the WT, but not the KO. This result indicates that successful incorporation of human PTP1B in
the WT conditions, allowing us to allocate the differences observed in our study to the lack of
PTP1B. The hPTP1B D/A condition yielded a positive presence for the hPTP1B, as expected and
shown in previous studies (1). However, the glutamic acid residue in the protein was exchanged
for alanine, yielding a protein that allows for binding of the substrate, but is catalytically
impaired (1, 9). Thus, this allowed us to quantify the effects of the inactive protein, further
allowing us to allocate differences between the conditions to a lack of functioning PTP1B.
BAT Differentiation among Treatments
In all three conditions, differentiated cells yielded higher absorbance values than their
respective undifferentiated cells (Table 1). Elution of Oil Red O from the stained TG in each
condition allowed us to quantify TG concentrations via absorption capability (1, 4); the increased
absorption values in our study correlate with increased TG presence, ultimately depicting
successful differentiation among the treatment groups. The data indicates a large standard
deviation for the average absorption values of hPTP1B WT undifferentiated cells. This may have
been due to experimental error; the Oil Red O strain was not completely eluted in the 100%
isopropanol solution in one sample, ultimately resulting in a decreased absorption value. This
consequently decreased the average differentiation observed in the hPTP1B WT treatment.
Nonetheless, our study yielded no significant differences in differentiation among the
three PTP1B treatment groups (p=0.88 WT vs. KO, p=0.87 KO vs. D/A, p=0.86 WT vs. D/A).
The results of our study fall in line with the results of a previous study conducted by Matsuo et
al., which found an insignificant trend among hPTP1B KO and D/A treatments compared to the

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WT control (1). However, the lack of significance among the treatments groups observed in our
study may also be due to the small sample size utilized for each treatment. Furthermore,
differentiation among the KO and D/A conditions were almost identical in our study (152% and
154% respectively), indicating similar differentiation patterns between both conditions. This
result falls in line with our hypothesis; in both conditions PTP1B was either not present or
catalytically inactive, thus similar differentiation patterns were expected.
The results from our study indicate increased differentiation patterns among the hPTP1B
KO treatment groups compared to the WT (Figure 3). Previous studies have depicted PTP1B’s
role in differentiation among BAT; a study conducted by Matsuo et al., found enhanced
differentiation among hPTP1B KO and D/A conditions, but a substantial decrease in
differentiation among sumoylation-resistant hPTP1B (1). Another study conducted by Song et al.
determined that adipocytes with an overexpressed version of PTP1B yielded a blunt decrease in
differentiation by 70%, while adipocytes with a knocked out version of PTP1B depicted higher
absorbance of Oil Red O in comparison to the control, indicating greater differentiation (10). The
results from these studies agree with the results determined in our study; PTP1B acts as a
negative regulator of adipocyte differentiation. A proposed mechanism for this observation was
determined in a study conducted by Soledad et al., which found that a lack of PTP1B acts as a
protective mechanism to decrease apoptosis in the induction phase of adipogenesis, ultimately
resulting in greater BAT differentiation (2).
Increased BAT proliferation has metabolically favorable effects, indicating possible
health benefits associated with inhibited PTP1B. BAT is characterized by increased
mitochondria and less TG storage compared to WAT; this morphology induces greater energy
expenditure among BAT in comparison to WAT (3). An analysis of 3,640 BAT compositions in

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humans found an inverse association between increased BAT and BMI, ultimately providing a
possible mechanism to control human metabolism (11). PTP1B KO studies in mice fed a high fat
diet found that the KO produced leaner mice with greater energy expenditure and decreased fat
stores (6); this depicts the health benefits of increased BAT differentiation and provides a
foreground for future research in human models. This research can be applied to humans in order
to increase BAT differentiation, increase energy expenditure, and increase weight loss- providing
a possible mechanism to combat obesity in the population.
hPTP1B: Possible Therapeutic Target
In addition to the previously described health benefits of PTP1B inhibition, the lack of
PTP1B has also been associated with increased induction of the insulin signaling pathway via
decreased inhibition of IR and IRS-1 (1). PTP1B inhibition has also been associated with
increased leptin sensitivity, providing a possible mechanism for increased energy expenditure.
(7). Thus, the exploitation of this pathway can possibly provide benefits for those suffering from
obesity and type II diabetes, making it a probable target for future therapeutic drugs. A problem
in the production of PTP1B inhibitory drugs lies in selectivity, as all PTP active sites are similar
in structure, thus making it difficult to specifically target PTP1B (7). Advancements have been
made in determining alternate binding mechanisms and increasing the potency of proposed
PTP1B inhibitors, solidifying the development of PTP1B inhibitors in the near future (7). Thus,
the results from our study solidify the findings that PTP1B acts as an inhibitor of BAT
differentiation, propagating future research in exploiting its pathways in order to create a market
for PTP1B inhibitors.
CONCLUSION
The results of our study indicate an increase in BAT differentiation among those cells
treated with hPTP1B KO and D/A treatments, as supported by previous studies. This supports

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our initial hypothesis that PTP1B plays a crucial role in inhibiting BAT differentiation. The
results from our study add to the growing evidence that PTP1B inhibition can play an important
role in the management of the obesity and diabetes epidemic in the population, however research
in human models needs to be conducted in order to gain validation. A weakness of our study
includes the small samples size utilized and the lack of analysis of PTP1B metabolic pathways;
we simply quantified TG in order to determine differentiation efficiency. This research has
propagated future studies that look into the effects of PTP1B in humans, in an attempt to further
clarify possible PTP1B therapeutic targets and provide a possible treatment for diabetes and
obesity.
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